SUMMARY:  Immunotherapy is the process of activating a person’s own immune system to attack their cancer. It has been successful in some types of cancers, like melanoma and lung cancer, but little has been done in desmoid tumors. Dr. Gerlinde Wernig will investigate whether desmoid tumors express certain proteins that help it avoid the immune system. These proteins that help tumor cells avoid the immune system are called immune checkpoints and include CD47, PDL1, and CTLA4 (among others). If Dr. Wernig’s group finds these immune checkpoints are expressed on desmoid tumors cells, they will determine if desmoid tumors respond to certain drugs that block these proteins, called immune checkpoint inhibitors. This may lead the way for further research in immunotherapy in desmoid tumors, including possible trials with immunotherapies – several of which are already approved for other cancers.

SCIENTIFIC ABSTRACT:  Desmoid-type fibromatosis (DTF) is a rare, low-grade, soft-tissue tumor affecting the extremities and the trunc. As DTF does not progress after diagnosis in most cases, no specific initial treatment is recommend. However, a minority of patients develops progressive DTF with debilitating and and life-threatining complications. In these cases, therapies include chemotherapy, surgery, radiation, or targeted therapy. Immunotherapy either targets immune checkpoints, thereby increasing the efficiency of the adaptive immune system, or don’t-eat-me signals, thereby enhancing the phagocytosis of tumor cells through the innate immune system. Though immunotherapy has significantly increased survival in a variety of cancers, it has not been explored in DTF yet. This is why, we propose to investigate whether immunotherapy, more specifically blocking CD47 as a don’t-eat-me-signal, as a potential therapy for DTF. To this end, we will first determine through ATAC Seq if CD47 and immune
checkpoint regulatory proteins such as PDL1 are differentiately regulated in DTF. Then, we will use a unique adaptive transfer model to explore whether CD47 blockade eliminates ectopic human DTF grafts under the kidney
capsule of immunocompromised mice. These results will not only expand knowledge how don’t-eat-me-signals and immune checkpoints are regulated in DTF but will also explore CD47 blockade as a new therapy for DTF.

SUMMARY:  In previous research funded by DTRF, Dr. Kris Vleminckx has developed a desmoid tumor model in the frog Xenopus tropicalis. Desmoid tumors can suppress the ability of the immune system to attack it and the current project will investigate if tazemetostat can reverse this suppressive ability in their frog model. Tazemetostat is currently approved for use in epithelial sarcomas, but the mechanism in desmoid tumors is unknown. Dr. Vleminckx has found that the drug affects the WNT/β-catenin pathway, but that mechanism does not kill or decrease the tumor cells. However, it is possible that this drug is affecting the immune system and allowing the immune system to attack the tumor. If successful, this research may lead to combinations of therapies or new therapies that involve activating the immune system to attack tumor cells.

SCIENTIFIC ABSTRACT:  Using a genetic CRISPR/Cas9 based desmoid tumor model in the frog Xenopus tropicalis we have identified the gene EZH2, which encodes a member of the polycomb repressive complex 2 and is thereby involved in epigenetic regulation, as a dependency factor for desmoid tumors. Furthermore, a four-week treatment of Xenopus tropicalis carrying established desmoid tumors with the EZH2 inhibitor Tazemetostat caused a significant reduction in desmoid tumor volume. At the moment the mode of action of Tazemetostat in this anti-tumor response is unknown. Interestingly, we found that Tazemetostat reduces Wnt pathway activity in human desmoid cell cultures but does not have an overt effect on cell proliferation or cell death in vitro. Therefore, given the well-established fact that solid tumors in which the Wnt/β-catenin pathway is activated are immunologically cold and thereby insensitive to immune checkpoint inhibition, we postulate that the regression in desmoid tumor volume observed in the Xenopus model upon Tazemetostat treatment counters this immune suppressive environment and allows the engagement of a natural anti-tumor immune response. We hypothesize that the reduced Wnt signaling activity induced by Tazemetostat alleviates the immune checkpoint controls and allows the occurrence of a T-cell mediated immune response towards the desmoid tumor. Using a range of genetic experiments in Xenopus tropicalis this hypothesis will be investigated.

SUMMARY:  High-intensity focused ultrasound (HIFU) is a new way of treating desmoid tumors. It uses ultrasound waves to increase temperature and ablate tumors without surgery. If you have ever used a magnifying glass to burn a hole in a leaf, you have an idea of how this works. Instead of focusing light to heat up the leaf, HIFU focuses sound to heat up the tumor. Magnetic resonance imaging (MRI) is used with HIFU to target just the tumor cells, without affecting the surrounding normal tissues. There have been a few reports and studies that suggest HIFU is safe and effective. This study is the first prospective HiFU trial focused solely on desmoid tumors. Dr. Braat and team will be conducting a clinical trial based in The Netherlands to determine if HIFU is both safe and effective to treat desmoid tumors and leads to improved patient satisfaction. Over 2 years they will assess if HIFU-treated tumors shrink in response to HIFU in addition to several patient reported outcomes. If successful, this trial can lead the way for larger clinical trials in HIFU so that HIFU becomes more available for more patients who would benefit from this therapy.

SCIENTIFIC ABSTRACT:

Background: Recently magnetic resonance image-guided high intensity focused ultrasound (MR-HIFU) has emerged as a safe and effective treatment for control of desmoid tumors. MR-HIFU is a promising minimally invasive technique that uses focused ultrasound waves to thermally ablate tumors, while minimizing side effects to surrounding healthy tissues.

Aim/hypothesis: We hypothesize that desmoid tumor (DT) patients could benefit from treatment with MR-HIFU. The aim of this study is to assess efficacy, in terms of a newly developed personalized patient satisfaction score, of MR-HIFU as treatment modality for DT.

Plan of investigation: With this purpose, the plan is to set up a two-stage, open-label, single-arm, phase 2 prospective study. Adult patients with DT with failure of active surveillance for their current desmoid tumor considered eligible for the study. Patients will undergo a MR-HIFU treatment procedure. Primary outcome of the trial will be the patient satisfaction rate 12 months after the completion of the MR-HIFU procedure(s). Secondary outcomes include the presence of non-perfused volume on MRI after the MR-HIFU procedure, change in tumor volume, the response rate according to the Response Evaluation Criteria in Solid Tumors version 1.1. (RECIST v.1.1), the number of patients who need a re-intervention, adverse events, pain scores, and health-related quality of life (HRQoL) in the first 12 months after treatment.

Expected outcome: The expected outcome is to have determined that it is both safe and effective to treat patients with DT with MR-HIFU, leading to improved patient satisfaction and thereby offering a new treatment strategy for desmoid patients. In addition, the expected outcome is to have determined that MR-HIFU will decrease tumor volume, thereby providing an insight into the effect of MR-HIFU on local tumor control.

LAY ABSTRACT: While some aspects of individual desmoid tumors are shared between patients, each tumor has unique molecular and clinical characteristics. This means that selecting the right therapies for future patients with desmoid tumors requires a special type of map. This map, which we call a “dependency map,” would enable a clinician in the future to know therapy(ies) to prescribe based on the genetic characterization of that individual patient’s tumor. In our project, we aim to produce freely available foundational resources and develop a pilot version of a desmoid tumor dependency map that the entire scientific community can use. We will first test a large number (>6,000) of existing drugs that might be repurposed for desmoid tumors. But, we hypothesize that the most promising future drugs for desmoid tumors likely don’t yet exist. So, in our project we will focus on using new genetic tools, including a special type of molecular “scissors” called CRISPR/Cas9 to disable every gene in the genome in a systematic fashion and look for evidence of desmoid tumor cell killing in the laboratory. These experiments will prioritize the best targets to ensure drug companies know which targets to work on for desmoid tumors and ensure they include desmoid tumors in future drug discovery programs that might be focused otherwise on other tumor types. At the end of our project, we will complete (1) an inventory of nearly 20 desmoid tumor laboratory models that will be fully genomically characterized and available at a third-party distributor for all scientists to access, and (2) an initial prioritization of existing drugs and drug targets for desmoid tumors. This effort will help us understand what size and scale of a full Desmoid Tumor Dependency Map will ultimately be needed until we can make accurate dependency predictions for each patient.

SCIENTIFIC ABSTRACT: The success of cancer precision medicine is contingent on the ability to determine an optimal therapeutic strategy given the molecular classification of a patient’s tumor. While desmoid tumors are relatively homogenous at the genomic level, we do not yet know to what extent this homogeneity induces dependency on a limited or an expansive number of cellular targets for survival and whether such targets are shared with other common cancers. Here, we hypothesize that desmoid tumors have a finite number of cellular dependencies and that a fraction of these are shared with other common cancers with active b-catenin signaling. We propose creating a pilot version of an experimental map of such dependencies in a manner that can be fully integrated with data emerging from common cancers to provide sufficient statistical power to prioritize dependencies driven by b-catenin mutations in desmoid tumors. To do this, we will perform genome-wide CRISPR/Cas9 viability screens and test over 6,100 existing therapeutics that have been developed for any human disease for evidence of desmoid tumor cell killing, with all data going into the public domain pre-publication to serve the entire desmoid tumor research community. This resource project will yield a reference map to nominate high priority targets for drug discovery, and may support the inclusion of desmoid tumors as inclusion criteria for ongoing clinical studies.

LAY ABSTRACT: We previously developed a model for desmoid tumors in the frog Xenopus tropicalis by using CRISPR/Cas9 mediated inactivation in the apc tumor suppressor gene, thereby mimicking desmoid tumor formation associated with Familial Adenomatous Polyposis. However, in order to also model spontaneous desmoid tumors, we now want to generate a complementary model where desmoid tumor formation is driven by activating point mutations in the ctnnb1 gene, using a novel modification of the CRISPR/Cas9 system. We want to use our models to validate novel genes as targets for future therapy. In addition we want to investigate whether genes that are centrally involved in collagen secretion, a typical hallmark of desmoid tumors, are associated with tumor induction or proliferation. We believe our studies in this frog model can form a nice and relevant bridge between studies in cell culture and future use of the genetic mouse model that is being established.

SCIENTIFIC ABSTRACT: This project aims to further exploit a fast, semi-high throughput and cheap animal model for identifying and/or characterizing promising drug targets for treating desmoid tumors. In addition the platform allows pre-clinical assessment of novel candidate therapeutic compounds. The project builds on a recently developed genetic desmoid tumor model in the frog Xenopus tropicalis, This model, which induces mosaic loss-of-function mutation in the tumor suppressor gene apc, presents a unique and novel experimental platform that (i) allows the rapid screening and evaluation of genes that contribute to the growth of the tumor, (ii) can serve to assess the clinical relevance of novel drug targets for treating desmoid tumors and (iii) can be used as a preclinical drug screening/assessment. A new methodology was developed called CRISPR/Cas9-mediated Negative Selection Identification of genetic Dependencies (CRISPR-NSID) that allows in vivo elucidation of cancer cell vulnerabilities in genetic cancer models. The methodology hinges on the fact that for a genetic dependency there is an incapability for recovering tumors carrying biallelic frameshift mutations in this gene. We will now further expand our model by the inducing the activating S45F point mutation in the ctnnb1 gene using Cas9 Base Editors. In addition we want to use the model for validating novel dependency factors coming out of an in vitro screen and to further investigate the role of the transcription factor CREB3L1 and its link to collagen synthesis and secretion in the process of desmoid tumor formation.

LAY ABSTRACT: While some aspects of individual desmoid tumors are shared between patients, each tumor has unique molecular and clinical characteristics. This means that selecting the right therapies for future patients with desmoid tumors requires a special type of map. This map, which we call a “dependency map,” would enable a clinician in the future to know therapy(ies) to prescribe based on the genetic characterization of that individual patient’s tumor. In our project, we aim to produce freely available foundational resources and develop a pilot version of a desmoid tumor dependency map that the entire scientific community can use. We will first test a large number (>6,000) of existing drugs that might be repurposed for desmoid tumors. But, we hypothesize that the most promising future drugs for desmoid tumors likely don’t yet exist. So, in our project we will focus on using new genetic tools, including a special type of molecular “scissors” called CRISPR/Cas9 to disable every gene in the genome in a systematic fashion and look for evidence of desmoid tumor cell killing in the laboratory. These experiments will prioritize the best targets to ensure drug companies know which targets to work on for desmoid tumors and ensure they include desmoid tumors in future drug discovery programs that might be focused otherwise on other tumor types. At the end of our project, we will complete (1) an inventory of nearly 20 desmoid tumor laboratory models that will be fully genomically characterized and available at a third-party distributor for all scientists to access, and (2) an initial prioritization of existing drugs and drug targets for desmoid tumors. This effort will help us understand what size and scale of a full Desmoid Tumor Dependency Map will ultimately be needed until we can make accurate dependency predictions for each patient.

SCIENTIFIC ABSTRACT: The success of cancer precision medicine is contingent on the ability to determine an optimal therapeutic strategy given the molecular classification of a patient’s tumor. While desmoid tumors are relatively homogenous at the genomic level, we do not yet know to what extent this homogeneity induces dependency on a limited or an expansive number of cellular targets for survival and whether such targets are shared with other common cancers. Here, we hypothesize that desmoid tumors have a finite number of cellular dependencies and that a fraction of these are shared with other common cancers with active b-catenin signaling. We propose creating a pilot version of an experimental map of such dependencies in a manner that can be fully integrated with data emerging from common cancers to provide sufficient statistical power to prioritize dependencies driven by b-catenin mutations in desmoid tumors. To do this, we will perform genome-wide CRISPR/Cas9 viability screens and test over 6,100 existing therapeutics that have been developed for any human disease for evidence of desmoid tumor cell killing, with all data going into the public domain pre-publication to serve the entire desmoid tumor research community. This resource project will yield a reference map to nominate high priority targets for drug discovery, and may support the inclusion of desmoid tumors as inclusion criteria for ongoing clinical studies.

LAY ABSTRACT: The matrisome is the “glue” that exists between our cells, which provides supportive scaffolding and defines the structure of tissues. It has been shown that components of the matrisome provide signals to guide the behaviour of nearby cells. A number of studies indicate that these signals can also determine whether tumour cells become aggressive or respond to 3 anti-cancer drugs, and thus variations in the makeup of the matrisome surrounding tumours may explain why some patients have progressive disease or fail to respond to treatment. Desmoid tumours undergo an unpredictable and variable trajectory where some tumours grow aggressively while others remain unchanged for long periods of time or spontaneously shrink. There are currently no good tests to predict which patients are likely to go on to have aggressive or indolent forms of the disease. Our research is aimed at discovering the matrisomal
components that drive aggressiveness in desmoid tumours and develop biomarkers for predicting tumour outcomes. In this project, we will employ protein profiling of desmoid tumour specimens to establish which components of the matrisome are enriched in patients with aggressive tumours. Once identified, these molecular components will be assessed for their ability to predict the clinical course of desmoid tumours and in doing so, allow us to study the complex biological processes that regulate tumour growth. Our ultimate goals are to develop new ways of identifying which patients are most likely to harbour aggressive tumours and how best to treat these patients. If successful, our work will provide the basis for new clinical trials that will help doctors to
personalise treatment of individual desmoid tumour patients whilst providing important new insights into the molecular drivers of tumour growth in this disease.

SCIENTIFIC ABSTRACT: Desmoid tumours are characterised by an unpredictable and variable natural history with periods of tumour growth, stable disease and even spontaneous regression. While genomic analyses of desmoid tumours have deepened our understanding of the molecular pathology of the disease, there remains a large gap in translating this knowledge into robust prognostic biomarkers and effective treatments. In contrast to the tumour cell compartment, the biology of the tumour microenvironment in desmoid tumours, specifically the microenvironmental component comprising the extracellular matrix and associated proteins (collectively known as the matrisome), remains largely unknown. Notably, the matrisome has been shown in other cancer types to be a rich source of therapeutic targets and prognostic biomarkers. The objective of this proposal is to harness the power of proteomics to characterise, in unprecedented detail, the desmoid tumour matrisome in clinical specimens from both aggressive and indolent forms of the disease. By defining the dynamic matrisomal remodelling associated with aggressive tumours, our novel approach seeks to identify prognostic biomarkers for the prospective stratification of patients with tumours that are likely to follow an indolent versus an aggressive course, as well as candidate drug targets and therapies for this disease. We anticipate that this research will ultimately lead to the discovery of innovative biomarkers and therapies to achieve the goal of improved clinical management of these patients.

LAY ABSTRACT: Focus groups and surveys from adult patients with desmoid tumor emphasize that there is a negative impact of the disease and treatment on their daily lives.  Patient-reported outcomes (PROs) assessments are questionnaire measures of patients’ symptoms, functioning, and health-related quality of life. The first PRO Tool in desmoid tumor has identified 11 symptoms and 17 psychosocial parameters as critical to the adult patient experience.  This PRO tool is being tested in adult clinical trials for desmoid tumor.  To our knowledge, there are no studies collecting quality of life or PROs data specifically in children with desmoid tumor.  There is an important need to learn about the challenges facing pediatric patients with desmoid tumors and the effects (good or bad) of treatment on daily living that may not be obvious by routine scans.  In order to address this need, we will evaluate the desmoid-specific adult PRO tool in children as well as a quality of life questionnaire for children called Patient Reported Outcomes Measurement Information System (PROMIS) in the pediatric clinical trial of the gamma secreatase inhibitor nirogracestat. We plan to collect  questionnaires before the study treatment starts, different times during treatment and at the end of treatment.

SCIENTIFIC ABSTRACT:

For some patients, desmoid tumor is a chronic disease associated with functional impairment and pain which negatively impacts psychosocial and emotional well-being.  A better understanding of the effects of the disease and interventions on quality of life in these patients, adults and pediatrics, is needed. Patient-reported outcomes (PROs) assessments are questionnaire measures of patients’ symptoms, functioning, and health-related quality of life.  The first prospectively developed PRO Tool in desmoid tumor for adults has identified 11 symptoms and 17 psychosocial parameters as critical to the patient experience. Prospective validation studies of this tool are ongoing in adult trials for desmoid tumors. There are no studies collecting quality of life or PROs data specifically in children with desmoid tumor.  In order to address this gap in knowledge, in the prospective pediatric clinical trial of the gamma secretase inhibitor nirogacestat, we will utilize at various time points during therapy this tool as well as a validated quality of life assessments for children called Patient Reported Outcomes Measurement Information System (PROMIS), to gain a better understanding of the challenges facing pediatric patients with symptomatic desmoid tumors and to assess whether disease outcomes (i.e., tumor response and PFS) with the study drug correlate with PROs. There is anecdotal evidence that the benefits of systemic therapies may be underestimated by imaging response in this disease. We will explore whether PROs correlate with imaging response assessments and outcome.

LAY ABSTRACT:

Almost all desmoid tumors have a change (a mutation) in either a gene called CTNNB1 or less commonly the APC gene.  These mutations are thought to play a major role in causing the disease and the type of mutation may determine how a patient responds to treatment. Other changes in the genetic material of the tumor have also been described. In this study, we want to understand more about desmoid tumors in pediatric patients by looking at the genetic material in the tumor in patients who are participating in the pediatric clinical trial of the gamma secretase inhibitor nirogacestat.  If available, some leftover tumor tissue, either from the time of diagnosis or prior to study entry and any available tumor tissue collected while taking nirogacestat or within 30 days after the last dose, will be used for these genetic tests. We will also study whether the type of mutation or the pattern of genetic changes can predict response to the study treatment and if or how the genetics of desmoid tumors differ from adult desmoid tumors. The results from this study will be published in a peer-reviewed journal.  In addition, the project will be set up in the Children’s Hospital Los Angeles data sharing platform, Childhood Cancer Knowledge Base, which is cloud based, for investigators to access the data.

SCIENTIFIC ABSTRACT:

Disruptions of the Wnt/β-catenin pathway are believed to play a major role in the pathogenesis of desmoid tumors. Almost all desmoid tumors have a mutation in either CTNNB1 or less commonly the APC gene. Chromosomal aberrations have also been reported in 23-35% of desmoid tumors using array comparative genomic hybridization, karyotyping or fluorescent in-situ hybridization.  With exception of CTNNB1 and APC mutations, most of the molecular characterization of desmoid tumors, including the presence or absence of chromosomal aberrations, has been in tumor tissue derived from adult patients.  Limited data is available on the genomic profile of pediatric desmoid tumor. As part of the pediatric study (ARST1921) through the Children’s Oncology Group of the gamma secretase inhibitor nirogacestat in patients with unresectable, progressive desmoid tumors, we will collect blood and tumor tissue for comprehensive genomic analysis using platforms validated to detected all common alterations in desmoid tumors as well as provide an opportunity for novel discovery.  We will explore correlations between mutational status and genetic signatures and outcome and response. The molecular findings will also be compared to the adult genomic data available in the literature and publicly available databases.  Any leftover samples will be banked for future research. This is a rare opportunity to better understand the pathogenesis of desmoid tumors in a cohort of pediatric patients prospectively treated with a novel drug.  The results from this study will be published in a peer-reviewed journal.  In addition, the project will be set up in the CHLA data sharing platform, Childhood Cancer Knowledge Base, which is cloud based, for investigators to access the data.

LAY ABSTRACT: Desmoid-type fibromatosis (DF) is a rare benign tumor characterized by a fibroblastic features and a variable and often unpredictable clinical course. Despite lacking metastatic potential, DF is locally invasive and with a high local recurrence rate. Desmoid-type fibromatosis is often associated with local and repeated injuries; it shows differences in growth rate, spontaneous regression, and stabilization of disease progression. It occurs both sporadically and in patients with familial adenomatous polyposis (FAP) and it is well known that alterations of the Wnt/b-catenin pathway can promote its onset. However, the wide variability of the growth rate, localization and aggressiveness of the tumor indicate that the desmoid tumor requires a favorable microenvironment to develop and grow. Different desmoid cell samples show a generally shared response to external microenvironmental stimuli, but with a considerable degree of variability and this could at least partially correlate to the clinical heterogeneous outcome of the desmoid tumors. We demonstrated that the inflammatory cytokine, TGF-b, plays a key role in the proliferation and myofibroblast differentiation of DF cells and it regulates cell-adhesion and cell-cell interactions in DF myofibroblast differentiation. However, primary cells from desmoid tumors are difficult to treat and investigate because of the limited biological material and for the variability between samples. For this reason, our aim is to create simple different desmoid-like cell models with single or few molecular alterations that ensure genomic and phenotypic stability and that allow us to perform extensive studies. We will use the CRISPR/Cas9 system to induce efficient, specific, stable and multiplexed gene-editing in the Wnt pathway genes to generate controlled cell models that could in part or more widely mimic DF cells behavior.

SCIENTIFIC ABSTRACT: Desmoid-type fibromatosis (DF) is a mesenchymal tumor unable to metastasize, but locally invasive and with a high-local recurrence rate (Alman et al., 1997; Lewis et al., 1999; Escobar et al., 2012). It occurs both sporadically and in patients with familial adenomatous polyposis (FAP) and it is well known that alterations of the Wnt/b-catenin pathway can promote its onset. The frequent association of this type of tumor with local and repeated injuries, the spontaneous regression and stabilization of disease progression make the clinical course unpredictable and the study of the shared molecular characteristics very puzzling. Different DF cell samples show a generally shared response to external factors, but with a considerable degree of variability and this could at least partially reflect the clinical heterogeneous outcome of the desmoid tumors, with cases of continuous growth, fluctuation, stabilization but also of regression of the tumor. The combination of the intrinsic biological characteristics of the desmoid tumor cells and the host microenvironment appear to be critical and to account for the diversity of clinical outcomes and the recurrence risk of desmoid tumors. One additional aspect of primary cultures from tumor biopsies is the concomitant presence of neoplastic and reactive tissues with a varying degree of the cell populations. Considering the high similarities between the neoplastic DF cells and normal activated fibroblasts it becomes even more difficult to isolate DF cells alone and select their peculiarities. For these reasons we opted to create a more controlled environment where to study the effects of isolated genetic changes in the Wnt pathway and the effect that these changes have on the response to external stimuli. More in particular, we decided to use the CRISPR/Cas9 system to induce efficient, specific, stable and multiplexed gene-editing in the Wnt pathway genes to generate controlled cell models that could in part or more widely mimic DF cells behavior. The project aims, at initially, developing stable cell lines with mutations in the APC and CTNNB genes and to extend the study by altering single Wnt pathway genes expression in these two cell lines together with the non-modified cells. The initial cell lines will be then characterized both by phenotype and genotype and in particular their response to external stimuli. Based on the results of these studies we will attempt to recreate the DF phenotype without altering the CTNNB or APC gene.

LAY ABSTRACT: We previously developed a model for desmoid tumors in the frog Xenopus tropicalis by using CRISPR/Cas9 mediated inactivation in the apc tumor suppressor gene, thereby mimicking desmoid tumor formation associated with Familial Adenomatous Polyposis. However, in order to also model spontaneous desmoid tumors, we now want to generate a complementary model where desmoid tumor formation is driven by activating point mutations in the ctnnb1 gene, using a novel modification of the CRISPR/Cas9 system. We want to use our models to validate novel genes as targets for future therapy. In addition we want to investigate whether genes that are centrally involved in collagen secretion, a typical hallmark of desmoid tumors, are associated with tumor induction or proliferation. We believe our studies in this frog model can form a nice and relevant bridge between studies in cell culture and future use of the genetic mouse model that is being established.

SCIENTIFIC ABSTRACT: This project aims to further exploit a fast, semi-high throughput and cheap animal model for identifying and/or characterizing promising drug targets for treating desmoid tumors. In addition the platform allows pre-clinical assessment of novel candidate therapeutic compounds. The project builds on a recently developed genetic desmoid tumor model in the frog Xenopus tropicalis, This model, which induces mosaic loss-of-function mutation in the tumor suppressor gene apc, presents a unique and novel experimental platform that (i) allows the rapid screening and evaluation of genes that contribute to the growth of the tumor, (ii) can serve to assess the clinical relevance of novel drug targets for treating desmoid tumors and (iii) can be used as a preclinical drug screening/assessment. A new methodology was developed called CRISPR/Cas9-mediated Negative Selection Identification of genetic Dependencies (CRISPR-NSID) that allows in vivo elucidation of cancer cell vulnerabilities in genetic cancer models. The methodology hinges on the fact that for a genetic dependency there is an incapability for recovering tumors carrying biallelic frameshift mutations in this gene. We will now further expand our model by the inducing the activating S45F point mutation in the ctnnb1 gene using Cas9 Base Editors. In addition we want to use the model for validating novel dependency factors coming out of an in vitro screen and to further investigate the role of the transcription factor CREB3L1 and its link to collagen synthesis and secretion in the process of desmoid tumor formation.

LAY ABSTRACT: DTF is a devastating cancer of the soft tissue and more effective treatments are needed. We found that JUN expression is increased in DTF patients by sequencing studies and protein expression measurements and that JUN-inducible mice developed desmoid-type fibromatosis when aged prior to JUN induction. These results suggest that desmoid-type fibromatosis has a pathological mechanism that relies on JUN induction and some other yet unidentified molecular event, e.g., possibly b-catenin activation. Novel treatment strategies could potentially be developed once the pathological mechanism is better understood. The JUN-inducible mice represent a novel mouse model for desmoid-type fibromatosis that, after complete characterization, could be used for preclinical evaluation of novel treatments.

SCIENTIFIC ABSTRACT: Desmoid-type fibromatosis (DTF) is not well understood. It is a rare, devastating, lowgrade, soft-tissue malignancy characterized by severely fibrotic tumors occurring preferentially deep visceral or axial/truncal, currently treated with surgical resection and/or radiation and/or chemotherapy. Despite initial good response, DTF has a high propensity to relapse locally, and repetitive surgical excisions can severely debilitated patients, causing comorbidities with poor quality of life and mortality. One of the most important drawbacks in DTF research is the lack of a detailed understanding of the molecular pathomechanism beyond b-catenin activation. We have demonstrated by sequencing and immunohistochemistry studies that the AP1 transcription factor JUN is upregulated and expressed in DTF tumors in patients. In addition, our preliminary data suggest that very low level of JUN induction in vivo leads to DTF in mice aged between 3–7 months. In the current proposal, we propose to first better characterize this mouse model, with particular attention to the role of b-catenin/wnt and Notch pathways. Since tumors only occur in aged mice, we hypothesize that mutations in other pathways co-operate in a 2- hit model with JUN induction to induce DTF lesions. Additionally, the small molecule PF- 03084014 showed promising effects in a Phase I clinical trial for DTF, suggesting a role for a g-secretase inhibitor-dependent pathway. We therefore propose to evaluate efficacy of PF-03084014 in JUN-inducible mice with regards to prevention of desmoid tumor formation. In addition, we will study effects of JUN and NOTCH pathway inhibition in primary cell lines of human Desmoids, in contrary to our initial proposal we will modify our approach and study the molecular interactions of JUN with the WNT and NOTCH pathways with next generation mass spectrometry and pull-down and immunoprecipitation assays. Our inducible DTF mouse model subsequently could serve as novel preclinical platform to evaluate additional inhibitors to ultimately find better treatments.

Desmoid-type fibromatosis (DTF) is not well understood. It is a rare, devastating, low-grade, soft-tissue malignancy characterized by severely fibrotic tumors occurring preferentially deep visceral or axial/truncal, currently treated with surgical resection and/or radiation and/or chemotherapy. Despite initial good response, DTF has a high propensity to relapse locally, and repetitive surgical excisions can severely debilitate patients, causing co-morbidities with poor quality of life and mortality. One of the most important drawbacks in DTF research is the lack of a detailed understanding of the molecular pathomechanism beyond b-catennin activation. We have demonstrated by sequencing and immunohistochemistry studies that the AP1 transcription factor cJUN is upregulated and expressed in DTF tumors in patients. In addition, our preliminary data suggest that very low level of c-Jun induction in vivo leads to DTF in mice aged between 3–7 months. In the current proposal, we propose to first better characterize this mouse model, with particular attention to the role of b-catenin/wnt and Notch pathways. Since tumors only occur in aged mice, we hypothesize that mutations in other pathways cooperate in a 2-hit model with c-Jun induction to induce DTF lesions. Additionally, the small molecule PF03084014 showed promising effects in a Phase I clinical trial for DTF, suggesting a role for a g-secretase inhibitor-dependent pathway. We therefore propose to evaluate efficacy of PF-03084014 in c-Jun-inducible mice. In addition, we will study effects of c-JUN and NOTCH pathway inhibition in primary cell lines of human Desmoids as well as patient samples from the clinical trial pre- and post treatment with gene expression studies. Our inducible DTF mouse model subsequently could serve as novel pre-clinical platform to evaluate additional inhibitors to ultimately find better treatments.

LAY VERSION OF ABSTRACT- “c-Jun-inducible mice, to study the pathomechanism of desmoid-type fibromatosis in vivo with particular attention to the role of b-catenin/wnt and Notch pathways.”

Desmoid tumors (DTs) are rare mesenchymal lesions with a high rate of local recurrence. Their common feature is a deregulated WNT pathway, mainly caused by gain-of-function mutations in exon 3 of the CTNNB1 gene (encoding for β-catenin), resulting in nuclear accumulation of β-catenin. Sorafenib has emerged as a promising therapeutic strategy based on compassionate use, which has led to an ongoing nationally-denominated phase III clinical trial. Concurrently, we conducted a comprehensive analysis of sorafenib efficacy in a large panel of desmoid cell strains to probe for response mechanisms. While much is already known about the molecular mechanism of sorafenib action in other cancers, little is currently known about sorafenib in desmoid tumors. Our preliminary results have shown distinctive groups of higher- and lower-responder cells. Clustering the lowerresponder group, we observed CTNNB1 mutation was a determinant of outcome. Our results showed that even lower doses of sorafenib were able to inhibit cell viability, migration and invasion of wildtype and T41A-mutated DTs. Apoptosis induction was observed in those cells after treatment with sorafenib. On the other hand, lower doses of sorafenib were not able to inhibit cell viability, migration, and invasion or to induce apoptosis in the S45F-mutated DTs. In the first aim of this grant, we propose to investigate the differences between desmoids harboring different CTNNB1 mutations and their response to sorafenib. Multiple mechanisms contribute to drug resistance, including alterations in the tumor microenvironment (TME). The tumor microenvironment describes stromal and other cells that surround tumor cells. Several studies have shown that TME cells can affect tumor growth, invasion, local recurrence and drug resistance; however, to the best of our knowledge, the role of the microenvironment in desmoid tumor cells resistance to drugs has not been investigated to date. Moreover, in studies conducted within the premise of our previously funded DTRF seed grant, we showed that there was no significant induction of apoptosis in the S45F mutated desmoid cell strains when compared to the T41A mutated cells. Therefore, it is important to investigate if S45F desmoid-associated TME cells differ from TME sorted from T41A-mutated desmoid cells. In the second aim of this grant, we propose to investigate the role of the TME in the resistance of S45F-mutated desmoid tumors to therapy. Our preliminary results have shown that TME sorted from S45F-mutated DTs are more tolerant to doxorubicin when compared to T41A desmoid-associated TME. Interestingly, as opposed to results with mixed population showed previously by our group, pure desmoid populations sorted from S45F-mutated cells did not show tolerance to doxorubicin, suggesting that TME may play a role in the resistance to drugs observed in some desmoid cells. Finally, we have shown that the pure population of T41A-mutated desmoids cultured with media collected from TME sorted from S45F-mutated desmoid tumors are more tolerant to doxorubicin treatment, as oppose to the sensitivity seen in the mixed population, suggesting that the TME releases factors that may play a critical role in the therapeutic resistance of desmoid tumors harboring the S45F mutation. These preliminary results are intriguing; however, these findings are in need of further investigation to identify the molecular mechanisms underlying the role of the TME in desmoid resistance to drugs. With such enhanced understanding, these findings may potentially have an impact on patients in the clinic.

LAY VERSION OF ABSTRACT-   “The role of the tumor microenvironment in S45F desmoid tumor chemotherapeutic resistance.”

Desmoid tumors (DT) are locally invasive soft tissue growths with no directed therapies and high rates of recurrence. Two mutated genes (CTNNB1and APC2) have been linked to patients who develop desmoids, both which translated to proteins in the Wnt canonical pathway. It is our hypothesis that the desmoid tumor microenvironment has altered metabolism, similar to that of pancreatic ductal adenocarcinoma (PDAC). Pancreatic tumors are like desmoid tumors, in that they consist of an abundance of desmoidic stromal tissue, are driven by mutations of K-ras, another Wnt protein, and accumulate β-catenin. In a recent study of the PDAC metabolism, it was found that the activated fibroblasts cells in PDAC release alanine to fuel the TCA cycle, rather than glucose and glutamine-derivatives, which therefore facilitates tumor survival and growth. It is possible that the alanine secretions observed in PDAC represent metabolic reprogramming of tumors driven by the canonical Wnt pathway. We will use stable isotopic resolved metabolomics (SIRM) to better define the desmoid tumor and adjacent normal fibroblast metabolism through the TCA cycle, glycolysis, and the synthesis of non-essential amino acids. By using 13C-labeled tracers in cell media and tail injections, we will be able to use 1 and 2D NMR spectroscopy to follow the metabolism differences of primary cell lines and animal tissue. Matched primary cell lines will be used to determine the metabolic flux of the TCA cycle, glycolysis, and the synthesis of nonessential amino acids. We will also evaluate the cell lines by β-catenin mutation status, as the S45F has been noted to be more aggressive and recur more frequently than T41A. Following the cellbased SIRM, we will evaluate the metabolism of two animal models. The first model is representative of Familial adenomatous polyposis (FAP), Apc1638N, and has been studied extensively by our laboratory. The second animal model is currently being developed and will contain the two CTNNB1 mutations, T41A and S45F. We will determine if the metabolic flux follows the same patterns in the animal models as in the cells. We will compare the two Wnt pathway protein mutations, as this would be first time the metabolism of these two will be evaluated. This study of primary cells and animal models using SIRM will provide valuable insight into the metabolism of desmoid tumors and the surrounding normal tissue to better understand the microenvironment and potentially why these tumors recur so frequently.

LAY VERSION OF ABSTRACT-   “Stable isotope resolved metabolomics to interrogate the interactions between stroma and desmoid tumors.”

This is a proposal to generate a conditional S45F and a T41A knock-in allele of Ctnnb1. These are the two most common Ctnnb1 mutations found in desmoid tumors.   Ozgene will generate the conditional mutant alleles by flanking wt exons 3-6 and an inverted murine cDNA of exons 3-15 with the S45F mutation or the T41A and an IRES_eGFP with loxP and lox2272 sites via gene targeting in mouse ES cells. The IRES_eGFP cassette within the KI will also be floxed with Rox sites.  Breeding to flp will remove the neo cassette. Cre-mediated recombination of the “floxed” regions will delete the wt exons and invert the KI cDNA_IRES_eGFP into the proper orientation for expression. Both Ctnnb1 with the S45F mutation and eGFP will be expressed. Further breeding to Cre will excise the IRES_eGFP. Ctnnb1 with the S45F mutation, but not eGFP, should be expressed.  This approach avoids potentially dominant negative effects of the knock-in mutation.

LAY VERSION OF ABSTRACT- “A genetically targeted mouse model of desmoid tumors.”

Desmoid-type fibromatosis (DTF) are locally aggressive benign clonal fibroblastic/myofibroblastic proliferations that are defined by infiltrative growth in deep soft tissue have an increased propensity for local recurrence but no metastatic potential. They are known to be related to perturbation in the APC/beta-catenin/Wnt-signalling pathway and are characterized by demonstration of frequent beta-catenin nuclear localization on immunohistochemistry. The majority of cases occur sporadically while rare cases arise infrequently as part of a familial syndrome (called familial adenomatous polyposis) due to germline mutation in the adenomatous polyposis gene (APC). There are 3 types of DTF based on anatomic distribution: 1. Intra-abdominal (mesenteric) fibromatosis; 2. Abdominal fibromatosis; 3. Extra-abdominal fibromatosis. These tumors are morphologically identical and essentially indistinguishable from each other. More so, the biology of these tumors is unpredictable and consistent reproducible predictive biomarkers in terms of response to therapy are lacking. Emerging data from recent large clinical studies and molecular profiling seems to indicate significant heterogeneity in tumor biology among the different types of DTF. Specifically, desmoid tumors with CTNNB1 41A gene mutation has been found to be associated with a better statistically significant 5-year recurrence free survival compared to those with S45F mutation and desmoids of abdominal site behave less aggressively than those in extra-abdominal sites. Indeed, the largest prospective outcome data on DTF from the French Sarcoma Group, suggests that anatomic location is the single most important determinant of event free survival.

In spite of the recent strides in the biology of DF, the optimal treatment remains a challenge with unacceptably high recurrence rate with various multi-modality approaches including surgery, chemoradiation, anti-estrogenic and different molecular targeted therapies. There is thus an urgent need for thorough understanding of the molecular biology of DTF with the ultimate goal to developing better treatment strategies. There is paucity of data in the proteomic analysis of desmoid tumors. Using a novel quantitative mass spectrometry based proteomics platform, we propose to elucidate any differences in protein signatures between the different types of desmoid type fibromatosis.

LAY VERSION OF ABSTRACT-  “Quantitative Proteomics Analysis of Desmoid-Type Fibromatosis.”

Desmoid tumors are proliferations of fibroblasts, myofibroblasts, and dense collagen. They are histologically similar to superficial fibromatoses such as palmar (Dupuytren’s disease) and plantar fibromatoses, as well as keloids. Unlike the superficial fibromatoses, desmoid tumors are located in the deep tissue and stratified based on an abdominal or extra-abdominal location. Historically, wide surgical resection was the treatment of choice. This often resulted in a disfiguring appearance and recurrence was common. Additionally, radiation and systemic therapies are performed with an approximate 26% rate of objective response based on RECIST. These therapies are however not without side effects.

Corticosteroids, such as triamcinolone acetonide, have long been used in the treatment of hypertrophic scars and keloids in order to decrease the size of the lesion. Proposed mechanisms of action include a decrease in the production of collagen, dissolution of insoluble collagen, a decrease in the local inflammatory process, and an increased rate of apoptosis of fibroblast and inflammatory cells. Recent reports have also evaluated the use of corticosteroid in palmar fibromatosis with promising results. We therefore aim to perform a pilot study to evaluate the response rate and tolerability of intralesional injections of triamcinolone acetonide into desmoid tumors, using RECIST to evaluate for response.

In addition to evaluating for response of the triamcinolone acetonide we will perform genetic testing for CTNNB1 mutations. New research has shown specific mutations, such as the 45F have been correlated with worse recurrence-free survival following treatment. We will observe the frequency of CTNNB1 mutations in our patient population.

LAY VERSION OF ABSTRACT-  “A Pilot Study of Intralesional Injection of Triamcinolone Acetonide for desmoid tumors.”

There are very few randomized studies providing thorough data and evidence on treatment decisions in desmoids. In this context we need to evaluate existing evidence from publications in order to draw data-based conclusions, particularly if such an approach is intended to find transatlantic acceptance. We will focus on the following aspects of desmoid treatment by answering pico questions for:

1) Pathology and molecular genetics of desmoids and its role in prognosis and prediction of therapeutic results 2) Indications to an active treatment in different populations of desmoid tumor patients (sporadic, FAP-associated, beta-catenin mutated vs wildtype, type of mutation) a) Is watchful waiting the primary approach indicated for all? b) When to switch to active therapy in the different populations? 3) Definition of a hierarchy of available medical therapies in different indications (tumor location, extent of progression, type of desmoid) – Which assessment of treatment effect was used in the studies (no pico question) 4) Role of pain control and physical therapy?

Data will be discussed in a consensus meeting in Milano, Italy at the Istituto Nazionale dei Tumori, on June 17-18, 2018.

LAY VERSION OF ABSTRACT-  “An evidence-based consensus on the treatment of desmoids/aggressive fibromatosis.”

The tumor stroma includes many nonneoplastic cells and the extracellular matrix, making up the microenvironment in which neoplastic cells grow. The non-neoplastic cells are predominantly stromal fibroblasts, sometimes called “tumor associated fibroblasts”. These cells are important for the maintenance and remodeling of the microenvironment, providing the appropriate conditions for neoplastic cell growth and invasion. The role of stromal fibroblasts in promoting tumor invasion has recently been highlighted in a number of cancers such as breast cancer, gastric carcinoma, non-small cell lung carcinoma, and colorectal cancer. This interaction between the stromal fibroblasts and the neoplastic cells can occur indirectly through secreted paracrine factors, or directly by physical cell-cell contact. Desmoid tumors (DTs) are characterized by proliferating and invading fibroblastic cells embedded in depositions of extracellular matrix driven by mutations that activate β-catenin signaling. We hypothesized that desmoid tumor cells interact with the stromal fibroblasts and that this interaction is responsible for maintaining the neoplastic phenotype of tumor cells. To test our hypothesis, we proposed to answer the following questions:

Aim 1: How do the neoplastic and stromal populations of desmoid tumors differ?

Our main goal is to study the composition and potential cross-talk of sub-populations within DTs. However, it is difficult to distinguish the neoplastic, mutant DT cells from the non-neoplastic stromal, “wild-type” fibroblasts due to the invasive nature of the tumor and due to both cellular populations exhibiting a mesenchymal fibroblastic phenotype. In the past year, we utilized a single cell-based method we developed to isolate individual cells from DT patient samples to establish several individual clones in fibroblastic colony forming culture media. We characterized individual clones to study whether it is a mutant neoplastic cell, or a non-mutant stromal cell. We also characterized differences in the expression of cell surface markers. This helped us identify a unique cell surface marker that can accelerate our isolation efforts and can more sensitively quantify tumor composition of heterogeneous samples. In the coming year, we aim to take advantage of our identified markers to establish more mutant and non-mutant pairs from more samples. We also aim to perform gene expression analyses which will be used to further characterize similarities and differences between the different population.

Aim 2: Do desmoid tumor cells interact with the surrounding stromal cells by paracrine signals?

In the past year, we analyzed the differential expression of secreted factors between mutant and nonmutant populations and found several factors that are uniquely expressed by either subpopulation. Modulation of the activity of selected factors altered the growth of DT cultures. We also co-cultured DT cells with normal cells separately using a permeable co-culture system that allows the sharing of released factors but without cell contact. We measured the effect of co-culturing on cells by viability, proliferation and migration assays to study how the two subpopulations can influence cell behavior. This work allowed us to optimize our assays and decide on readouts for subsequent studies. In the coming year, we aim to study gene expression differences to study the underlying changes in molecular biology due to secreted factors. We also aim to further study the role of candidate secreted factors on DT culture growth. The selected pathways will be disrupted by selective drugs or neutralizing antibodies and the effect on the co-culturing experiment will be measured by the same outlined assays that we have established in the last year. Our work so far has identified potentially novel pathways for further studies, and we expect that we will identify more pathways that modulate tumor-stromal interaction which can be targeted for therapy.

Aim 3: Do desmoid tumor cells interact with the surrounding stromal cells by direct cell-cell contact?

In addition to paracrine signals, invading tumor cells can also interact with the microenvironment via direct cell-cell adhesion. Molecules involved in this process include integrins, cadherins, and Notch signaling pathway components. Since β-catenin is known to interact with these molecules, directly or indirectly, we anticipate that direct cell contact will have an effect on the mixed populations. In the past year, we conducted a high throughput cell surface antigen screen to study the expression of proteins that can play a role in cell-cell interaction and how they differ between mutant and non-mutant populations. Our preliminary analysis has identified a number of integrin molecules that exhibit aberrant expression in the mutant populations. We anticipate that these molecules play a role in direct cell-cell contact. In the coming year, we will label DT cells by generating cell lines that stably express a marker, such as GFP (green fluorescence protein). We will then mix DT cells with normal cells and allow the two cell populations to grow together in direct cell contact. We will measure the effect of direct co-culturing on each cell population, taking advantage of our labelling method, by conducting viability and proliferation assays in addition to gene expression analyses. We will take a candidate approach at inhibiting these molecules that are likely to play a role in this process based on our surfaceomics data to date, in addition to any selected pathways from Aim 1, and we expect that we will identify a number of potential therapeutic targets.

By elucidating the composition of desmoid tumors, and how the tumor cells interact with the normal stromal cells, we can greatly enhance our knowledge of how they grow. DTs are invasive tumors characterized by abundant extracellular matrix deposition. Therefore, it is very likely that tumorstromal interactions are important in the way they grow and infiltrate the surrounding tissue. By isolating and characterizing individual cell populations from the tumor mass, we can gain a better understanding of what distinguishes the mutant cells from the normal cells. Our method of separating individual cells will also allow us to study variability between patient samples at the cellular level, without the confounding factor of having a different proportion of normal cells in each tumor sample. Since DTs are known to be heterogeneous, this method is key at understanding the source of this heterogeneity, which can influence treatment decisions. Elucidating what signaling pathways are involved in the process of tumor-stroma interaction will also allow us to potentially identify novel therapeutic targets that we can use to inhibit tumor growth and infiltration. We anticipate that the established single cell-derived clones, and the knowledge gained from this project of desmoid tumor biology, will be the basis for future experiments that build on our knowledge of desmoid tumor

heterogeneity and tumor-stromal interactions.

LAY VERSION OF ABSTRACT- “Single cell-derived clonal analysis of desmoid tumors to investigate tumor-stroma interactions.”

This project aims to provide a fast, semi-high throughput and cheap animal model for identifying and/or characterizing promising drug targets for treating desmoid tumors. We have obtained proofof-principal that, in an approach of mosaic multiplexed gene knockout using CRISPR/Cas9, we can identify genes that are critical for desmoid tumor formation. Using a streamlined experimental pipeline, we will be able to further assess whether additional  genes that are highly expressed in human desmoid tumors play an essential role in tumor proliferation and could serve as anchor points for novel therapeutic drug development. In addition we will further evaluate and optimize the EZH2 inhibitors GSK126 and Tazemetostat (EPZ-6438) on desmoid tumor formation in our experimental Xenopus model.  We will also test inhibitors for membrane cleavage – hence activation – of the transcription factor CREB3L1. Such inhibitors – AEBSF and PF429242 – have recently been described to block CREB3L1 activation and AEBSF has been used in an in vivo context (Feng et al. , Nat. Comm. 2017). We will copy the treatment protocol (intraperitoneal  injection) to our Xenopus model. Evidently more specific inhibitors will have to be developed since AEBSF also interferes with the proteolytic activation of SREBP, a master transcription factor for lipogenesis. We believe that our model offers a unique experimental platform that can foster the research lines of several groups active in the field.

LAY VERSION OF ABSTRACT-  “Identifying targets for therapy in a novel genetic Xenopus model for desmoid tumor formation.”

The tumor stroma includes many nonneoplastic cells and the extracellular matrix, making up the microenvironment in which neoplastic cells grow. The non-neoplastic cells are predominantly stromal fibroblasts, sometimes called “tumor associated fibroblasts”. These cells are important for the maintenance and remodeling of the microenvironment, providing the appropriate conditions for neoplastic cell growth and invasion. The role of stromal fibroblasts in promoting tumor invasion has recently been highlighted in a number of cancers such as breast cancer, gastric carcinoma, non-small cell lung carcinoma, and colorectal cancer. This interaction between the stromal fibroblasts and the neoplastic cells can occur indirectly through secreted paracrine factors, or directly by physical cell-cell contact. Desmoid tumors (DTs) are characterized by proliferating and invading fibroblastic cells embedded in depositions of extracellular matrix driven by mutations that activate β-catenin signaling. We hypothesized that desmoid tumor cells interact with the stromal fibroblasts and that this interaction is responsible for maintaining the neoplastic phenotype of tumor cells. To test our hypothesis, we proposed to answer the following questions:

Aim 1: How do the neoplastic and stromal populations of desmoid tumors differ?

Our main goal is to study the composition and potential cross-talk of sub-populations within DTs. However, it is difficult to distinguish the neoplastic, mutant DT cells from the non-neoplastic stromal, “wild-type” fibroblasts due to the invasive nature of the tumor and due to both cellular populations exhibiting a mesenchymal fibroblastic phenotype. In the past year, we utilized a single cell-based method we developed to isolate individual cells from DT patient samples to establish several individual clones in fibroblastic colony forming culture media. We characterized individual clones to study whether it is a mutant neoplastic cell, or a non-mutant stromal cell. We also characterized differences in the expression of cell surface markers. This helped us identify a unique cell surface marker that can accelerate our isolation efforts and can more sensitively quantify tumor composition of heterogeneous samples. In the coming year, we aim to take advantage of our identified markers to establish more mutant and non-mutant pairs from more samples. We also aim to perform gene expression analyses which will be used to further characterize similarities and differences between the different population.

Aim 2: Do desmoid tumor cells interact with the surrounding stromal cells by paracrine signals?

In the past year, we analyzed the differential expression of secreted factors between mutant and nonmutant populations and found several factors that are uniquely expressed by either subpopulation. Modulation of the activity of selected factors altered the growth of DT cultures. We also co-cultured DT cells with normal cells separately using a permeable co-culture system that allows the sharing of released factors but without cell contact. We measured the effect of co-culturing on cells by viability, proliferation and migration assays to study how the two subpopulations can influence cell behavior. This work allowed us to optimize our assays and decide on readouts for subsequent studies. In the coming year, we aim to study gene expression differences to study the underlying changes in molecular biology due to secreted factors. We also aim to further study the role of candidate secreted factors on DT culture growth. The selected pathways will be disrupted by selective drugs or neutralizing antibodies and the effect on the co-culturing experiment will be measured by the same outlined assays that we have established in the last year. Our work so far has identified potentially novel pathways for further studies, and we expect that we will identify more pathways that modulate tumor-stromal interaction which can be targeted for therapy.

Aim 3: Do desmoid tumor cells interact with the surrounding stromal cells by direct cell-cell contact?

In addition to paracrine signals, invading tumor cells can also interact with the microenvironment via direct cell-cell adhesion. Molecules involved in this process include integrins, cadherins, and Notch signaling pathway components. Since β-catenin is known to interact with these molecules, directly or indirectly, we anticipate that direct cell contact will have an effect on the mixed populations. In the past year, we conducted a high throughput cell surface antigen screen to study the expression of proteins that can play a role in cell-cell interaction and how they differ between mutant and non-mutant populations. Our preliminary analysis has identified a number of integrin molecules that exhibit aberrant expression in the mutant populations. We anticipate that these molecules play a role in direct cell-cell contact. In the coming year, we will label DT cells by generating cell lines that stably express a marker, such as GFP (green fluorescence protein). We will then mix DT cells with normal cells and allow the two cell populations to grow together in direct cell contact. We will measure the effect of direct co-culturing on each cell population, taking advantage of our labelling method, by conducting viability and proliferation assays in addition to gene expression analyses. We will take a candidate approach at inhibiting these molecules that are likely to play a role in this process based on our surfaceomics data to date, in addition to any selected pathways from Aim 1, and we expect that we will identify a number of potential therapeutic targets.

By elucidating the composition of desmoid tumors, and how the tumor cells interact with the normal stromal cells, we can greatly enhance our knowledge of how they grow. DTs are invasive tumors characterized by abundant extracellular matrix deposition. Therefore, it is very likely that tumorstromal interactions are important in the way they grow and infiltrate the surrounding tissue. By isolating and characterizing individual cell populations from the tumor mass, we can gain a better understanding of what distinguishes the mutant cells from the normal cells. Our method of separating individual cells will also allow us to study variability between patient samples at the cellular level, without the confounding factor of having a different proportion of normal cells in each tumor sample. Since DTs are known to be heterogeneous, this method is key at understanding the source of this heterogeneity, which can influence treatment decisions. Elucidating what signaling pathways are involved in the process of tumor-stroma interaction will also allow us to potentially identify novel therapeutic targets that we can use to inhibit tumor growth and infiltration. We anticipate that the established single cell-derived clones, and the knowledge gained from this project of desmoid tumor biology, will be the basis for future experiments that build on our knowledge of desmoid tumor

heterogeneity and tumor-stromal interactions.

LAY VERSION OF ABSTRACT- “Single cell-derived clonal analysis of desmoid tumors to investigate tumor-stroma interactions.”

Desmoid tumors (DT) are locally invasive lesions that are difficult to treat using conventional therapies. While there is some success with chemotherapy in people who have recurrence of their tumors after surgery and/or radiation, much of the time the results from chemotherapy are temporary, and regrowth of tumor is seen. One approach to treatment is to develop a combination of medications that target different aspects of tumor growth, but have few side effects. Since desmoids are locally invasive but do not metastasize to other locations in the body, it is not necessary to eliminate every tumor cell, but instead to stabilize or shrink the tumor. We assembled a consortium of researchers from different backgrounds, and with complementary skills, whose ultimate goal is to develop a treatment to therapeutically target tumor cells in desmoid patients without causing serious side effects.

To achieve this goal, over the past two years, we screened drugs in the laboratory on cell lines from desmoid tumor patients to see which agents specifically target tumor cell viability and tested a first cohort of drugs in mice that develop desmoid tumors, identifying several agents already in use or close to being in use for patient care that could be repurposed for use in desmoid tumor treatment.

Over the remaining course of the proposal we plan to:

1. Complete rescreening for drug combinations with synergistic effects.

3. Complete testing multi-drug regimen in mice.

Eighteen months ago, we screened desmoid tumor cell lines using the Maybridge HitFinder™ Collection; DIVERSet™ Collection (Chembridge); LOPAC (Sigma); BIOMOL collection; Prestwick collection (Prestwick Chemical); Seminatural compounds (AnalytiCon Discovery); and the NIH Clinical Collection (BioFocusDPI). This screening identified 45 compounds that inhibited cell proliferation in desmoid tumors while not effecting normal fibroblast proliferation, and that also are either in use in patients or that that have the potential to be rapidly developed for patient care. We then tested these agents using a collection of desmoid tumor cell cultures of different genetic etiologies, and based on this, as well as how available the selected agents are for clinical use today, prioritized 18 for testing in mice that develop desmoid tumors. We completed testing of nine agents in mice.

We are currently testing combination drugs in cell cultures and testing positive results in mice.

For the combination testing, we are evaluating the following agents in combinations in mice: FAK inhibitor, AV412; Dasatinib; PF03084014; Y26763 and KBR7943. (cation channel blockers); IKK-16; and dexamethasone.

This data will identify new treatment regiments, which can rapidly be brought to patient care. In addition, it will identify new pathways activated by beta-catenin and new agents not yet in use for patient care that target desmoid tumors, both of which can be developed in future work which might ultimately identify even more effective therapeutic approaches.

LAY VERSION OF ABSTRACT- “Collaboration for a Cure: Identifying new therapeutic targets for desmoid tumors”

Pathogenesis of desmoid-type fibromatosis has been investigated and the CTNNB1 mutation has been identified as a genetic factor for the development of this disease, particularly of sporadic ones. Treatment modality including surgical treatment with tumor-free margin, various conservative drug therapies, radiotherapy, and wait & see policy has been applied to patients of this disease. Although several recent studies demonstrated that CTNNB1 mutation status is significantly associated with the clinical outcomes of the treatment, particularly of surgery, other studies did not. Traditionally, desmoid-type fibromatosis is considered to arise in the area of trauma, such as surgical trauma of Caesarean operation. Many studies revealed that clinical outcomes and/or disease course depend on the site of onset of this disease. Patients with disease in lower extremity were reported to have poorer outcomes. Taking these together, disease behavior and efficacy of various treatment could be affected microenvironmentally. We hypothesize that desmoid cells and tumors are affected by the mechanical forces, and planned to investigate the effects of mechanical forces on the cell proliferation and motility, biochemical and molecular biological responses.

LAY VERSION OF ABSTRACT- “Effects of mechanical forces on the cell and tumor behavior of desmoid-type fibromatosis.”

Desmoid tumors (DTs) are rare mesenchymal lesions with a high rate of local recurrence. Their common feature is a deregulated WNT pathway, mainly caused by gain-of-function mutations in exon 3 of the CTNNB1 gene (encoding for β-catenin), resulting in nuclear accumulation of β-catenin. Even though it is controversial, several studies have shown that the mutation S45F strongly correlates with increased propensity for desmoid recurrence as compared to T41A mutation. Therefore, it is important to investigate the differences between these two genetic alterations in order to identify potential targets for novel molecular therapies. In studies conducted within the premise of our previously funded DTRF seed grant we were able to establish a desmoid tissue and cell strain repository. Furthermore, we showed that the difference between the CTNNB1 T41A and S45F mutations is not due to differential protein expression levels, since β-catenin is equally expressed in both mutations. Our gene array analysis showed that pro-apoptotic genes are downregulated and anti-apoptotic genes are upregulated in the cells with the S45F mutation when compared to the T41A mutation. Moreover, we showed that there is no significant induction of apoptosis in the S45F mutated desmoid cell strains when compared to the T41A mutated cells. Interestingly, the impairment of apoptosis appears to be specific to the CTNNB1 S45F mutation and not to desmoid tumors per se. We also showed that RUNX3 seems to play a role in the cross-talk between the WNT and apoptosis pathway and that RUNX3 overexpression overcomes apoptotic resistance in S45F transfected cells. Interim results are promising, confirming our initial observation. However, these findings are in need of further investigation to better understand the molecular mechanisms driving the differences in the induction of apoptosis between the T41A and S45F mutated tumors. Once we understand these differences, these findings may potentially have an impact on patients in the clinic. Finally, we have also evaluated the effects of Sorafenib as potential alternative therapies for patients harboring the β-catenin S45F mutation. Our results showed that the mechanism of action of sorafenib might not rely on targeting of tyrosine kinases VEGFR2 and PDGFR-β in DTs. Interestingly, we showed that sorafenib inhibited the activation of ERK, but was not able to inhibit activation of MEK. We also showed that sorafenib exhibits significant anti-DT cell activity and elicits apoptosis in a subset of these cell strains based on mutation status. Lastly, our results suggest that autophagy plays a role in the sorafenib response in the S45F mutated DT cells, which seems to be highly dependent on autophagy for survival. These exciting results are in need of further investigation to find the specific kinase that plays a role in the response to sorafenib and to evaluate the impact of sorafenib in the autophagy pathway. This knowledge may lead us to the discovery of new deregulated pathways, which could help the development of new target therapies that will hopefully have an impact on patients in the clinic. In this third year of funding, we propose to:

1. expand our desmoid tumor tissue repository as well as to continue trying to establish a desmoid tumor murine model
2. unravel the molecular mechanism underlying the differences in the induction of apoptosis between the CTNNB1 T41A and S45F mutation
3. further investigate the molecular mechanism of action of sorafenib, and additional possible therapeutic combinations;
4. evaluate autophagy as a survival mechanism in S45F mutated desmoids as well as the impact of sorafenib in this pathway.

LAY VERSION OF ABSTRACT- “Reactivating apoptosis: a potential therapeutic target for desmoid tumors with CTNNB1 S45F mutation.”

Desmoid-type fibromatosis (DF) is a rare benign tumor characterized by a fibroblastic features and a variable and often unpredictable clinical course. Indeed desmoid tumors show differences in growth rate, spontaneous regression, and recurrences. Although it is known that desmoid tumours are associated with Wnt pathway alterations there is no perfect correlation between either betacatenin or APC mutations and disease penetrance or recurrence. Furthermore the frequent association of this type of tumour with local and repeated injuries, the spontaneous regression and stabilization of disease progression are even more difficult to explain, suggesting the involvement of other altered signaling or the aberrant response of the DF cells to external environmental stimuli (Escobar C. et al, 2012). This might explain why efficacy of single pharmaceutical agents is limited, but also suggests that other pathways might represent additional targets for inhibitor therapy in this disease (Mignemi NA et al., 2012). These findings and the peculiar elements of desmoid tumors such as the fibroblast proliferation and the massive amounts of extracellular matrix remind the characteristics of wound healing and fibrosis process. Numerous studies on fibrotic diseases and on wound-healing processes highlighted how the cell spreading and the cell adhesion complexes could play an important role in mediating the TGF-β stimulus by modulating the intracellular signal transduction pathways. In this project we aim to investigate the type and the role of cellular communications with extracellular matrix protein (ECM) in desmoid tumors cells for the translation of matrix-derived signals into cellular response. In particular we will study the role of cell adhesion receptor, and of focal-adhesion formation in the induction of stress-fiber assembly and myofibroblastic differentiation after TGF-β stimulus.

LAY VERSION OF ABSTRACT- “In vitro study of the involvement of cell-cell and cell-matrix and focal adhesion formation in desmoid-type fibromatosis myofibroblastic differentiation under pro-inflammatory stimuli.”

Desmoid tumors are proliferations of relatively benign appearing fibroblasts. Despite their histologic bland appearance, a significant subset of these tumors recurs aggressively and requires often debilitating surgery. Currently there are no molecular markers that predict the behavior of desmoid tumors. The purpose of the study in the third year is to perform a validation of several candidate markers that could be used to address two clinically relevant questions. First, we will validate markers that could distinguish desmoid tumors that can be followed by “watchful waiting” at the time of their initial biopsy from tumors that require aggressive surgical therapy. Second, we will validate overexpression of selected markers in scars as compared with recurrent desmoid tumors to identify markers that could be used in surgical pathology practice to discern these two entities.

This work will build on an the gene expression dataset generated in our laboratory and will benefit from the experience that we have generated in our laboratory for the use of archival formalin-fixed, paraffin-embedded tissue in high throughput studies using next generation sequencing and tissue microarrays. The project greatly benefits from the collaboration with Dr. Kristen Ganjoo at Stanford University Hospital and from the collaboration with Dr. Raffi Avedian, orthopedic surgeon at Stanford. We also plan to continue our collaboration with Drs. Cates and Stricker from Vanderbilt to cross-validate NGS analyses in a range of comparisons.

In the third year of the project we also plan to perform novel functional studies using desmoid cell strains obtained from Dr. Raphael Pollock and Dr. Danielle Braggio from The Ohio State University. It is well characterized that these cell strain cultures are composed of fibroblasts and desmoid cells with mutant β-catenin. It has been shown that fibroblasts exhibit preferential growth in these cultures and eventually outgrowth the desmoid cells. We plan to perform a series of experiments in which we will treat desmoid tumor cell strains with inhibitors of Wnt signaling and γ-secretase to promote growth of desmoid cells and fibroblasts in these cultures, respectively. We anticipate that treatment with Wnt inhibitors will block the endogenous Wnt signaling in fibroblasts and will allow deriving cultures of pure desmoid tumor cells that could be used as an improved model for functional studies, e.g. testing novel drugs in desmoid tumors. This study has been designed and will be performed in collaboration with Dr. Roel Nusse from Stanford University.

LAY VERSION OF ABSTRACT- “Next generation sequencing approach to desmoid tumors.”

This project aims to provide a fast, semi-high throughput and cheap animal model for identifying and/or characterizing promising drug targets for treating desmoid tumors. We have obtained proofof-principal that, in an approach of mosaic multiplexed gene knockout using CRISPR/Cas9, we can
identify genes that are critical for desmoid tumor formation. Using a streamlined experimental pipeline, we will be able to rapidly assess whether a list of other genes that are highly expressed in human desmoid tumors play an essential role in tumor proliferation and could serve as anchor points for novel therapeutic drug development. In addition we will further evaluate and optimize the compound BC-2059 (BetaCat Pharma) on desmoid tumor formation in our experimental Xenopus model. The treatment protocol established with BC-2059 will serve as a blue print for future assessment of additional compounds with the goal of testing toxicity, uptake and efficacy. We believe that our model offers a unique experimental platform that can be easily plugged into the research lines of several groups active in the field.

LAY VERSION OF ABSTRACT- “Identifying targets for therapy in a novel genetic Xenopus model for desmoid tumor formation.”

Desmoid tumors are rare, locally aggressive neoplasms with unpredictable behavior. Although some tumors are indolent, resolving spontaneously, with tamoxifen, or with anti-inflammatory medications, others are prone to local recurrence, leading to significant morbidity, impaired function and poor quality of life for patients. For patients with advanced, refractory tumors, intensive chemotherapy regimens such as methotrexate/vinblastine or doxorubicin/dacarbazine can produce significant radiographic responses and symptomatic improvement. However these therapies are associated with both short and long-term toxicities. The ability to personalize therapy based on a predictive marker from an individual patient’s tumor would allow early treatment of aggressive tumors with chemotherapy, while sparing patients with indolent tumors or those more likely to respond to milder interventions.

Recently, desmoid tumors with particular mutations in CTNNB1, including S45F, have been shown tohave a higher risk of recurrence. Additionally, desmoid tumors with S45F mutations were less likely to respond to conservative treatments with imatinib or meloxicam. However, it is unknown whether S45F mutated tumors are more likely to respond to chemotherapy. In this proposal, we will obtain tissue and radiographic imaging for our existing database of desmoid tumor patients, and perform CTNNB1 sequencing to identify specific mutations. Through a collaboration with MD Anderson Cancer Center and Mount Sinai School of Medicine, our combined datasets will then be analyzed to determine whether the presence of S45F mutations correlates with MRI response to various chemotherapy regimens. The ability to use CTNNB1 mutation status to predict behavior of desmoid tumors and to guide therapeutic decisions would have immediate implications for clinical management of these rare yet often aggressive tumors.

LAY VERSION OF ABSTRACT- “Correlation of CTNNB1 mutation status with chemotherapy response by MRI in patients with desmoid tumors.”

The tumor stroma includes many non-neoplastic cells and the extracellular matrix, making up the microenvironment in which neoplastic cells grow. The non- neoplastic cells are predominantly stromal fibroblasts, sometimes called “tumor associated fibroblasts”. These cells are important for the maintenance and remodeling of the microenvironment, providing the appropriate conditions for neoplastic cell growth and invasion. The role of stromal fibroblasts in promoting tumor invasion has recently been highlighted in a number of cancers such as breast cancer, gastric carcinoma, non-small cell lung carcinoma, and colorectal cancer. This interaction between the stromal fibroblasts and the neoplastic cells can occur indirectly through secreted paracrine factors, or directly by physical cell-cell contact. Desmoid tumors (DTs) are characterized by proliferating and invading fibroblastic cells embedded in depositions of extracellular matrix driven by mutations that activate β-catenin signaling. We hypothesize that desmoid tumor cells interact with the stromal fibroblasts and that this interaction is responsible for maintaining the neoplastic phenotype of tumor cells. To test our hypothesis, we propose to answer the following questions:

Aim 1: How do the neoplastic and stromal populations of desmoid tumors differ?

Aim 2: Do desmoid tumor cells interact with the surrounding stromal cells by paracrine signals?

Aim 3: Do desmoid tumor cells interact with the surrounding stromal cells by direct cell-cell contact?

By elucidating the composition of desmoid tumors, and how the tumor cells interact with the normal stromal cells, we can greatly enhance our knowledge of how they grow. DTs are invasive tumors characterized by abundant extracellular matrix deposition. Therefore, it is very likely that tumor- stromal interactions are important in the way they grow and infiltrate the surrounding tissue. By isolating and characterizing individual cell populations from the tumor mass, we can gain a better understanding of what distinguishes the mutant cells from the normal cells. Our method of separating individual cells will also allow us to study variability between patient samples at the cellular level, without the confounding factor of having a different proportion of normal cells in each tumor sample. Since DTs are known to be heterogeneous, this method is key at understanding the source of this heterogeneity, which can influence treatment decisions. Elucidating what signaling pathways are involved in the process of tumor-stroma interaction will also allow us to potentially identify novel therapeutic targets that we can use to inhibit tumor growth and infiltration. We anticipate that the established single cell-derived clones, and the knowledge gained from this project of desmoid tumor biology, will be the basis for future experiments that build on our knowledge of desmoid tumor heterogeneity and tumor-stromal interactions.

______________

In neoplasia, tumor cells interact with the normal stromal cells, such as the surrounding fibroblasts. This makes studying desmoid tumors difficult as both tumor and stromal cells display a mesenchymal phenotype and there are no well-established markers to distinguish the two populations. To elucidate the composition of desmoid tumors, we isolated and expanded single cells derived from patient desmoid tumor samples. Sequencing of individual clones derived from the same patient sample revealed that desmoid tumors do consist of mutant sub-populations carrying the beta-catenin activating mutation, and normal sub-populations lacking the mutation. To improve our isolation methodology, we performed a high throughput flow cytometry surface antigen screen to study the expression pattern of over 300 surface markers on mutant and non-mutant cells. From this analysis, we identified CD142 as a unique positive marker for the mutant population. Having isolated mutant and non-mutant cell populations, we next looked into the expression of secreted factors that could potentially play a role in cell-cell communication. From this study, we found that CTHRC1 is a factor secreted by the mutant cells. Gain- and loss-of-function experiments showed that CTHRC1 can influence the proliferation rate of desmoid tumor primary cultures. Studying desmoid tumors at the clonal level will enhance our understanding of the intratumoral heterogeneity of these tumors. Identifying unique surface markers allows for the rapid isolation of mutant and non-mutant subpopulations while minimizing cell divisions. In addition to studying tumor-stroma communication, measuring tumor composition may also be useful for ongoing drug screening efforts and as a potential post-treatment readout.

LAY VERSION OF ABSTRACT- Single cell-derived clonal analysis of desmoid tumors to investigate tumor-stroma interactions

Aims: To define the natural history and outcomes of patients with desmoid tumors, a rare, locally invasive soft tissue tumor. Methods: Collaborate with Fu Jen University, Taiwan to use a record linkage approach with the Taiwan National Health Insurance Research Data Base (NHIRD) that contains clinical information on 23 million people. Use ICD 10 codes and key words to pull pathology records for review; review 100 records or more to develop an algorithm to ensure specificity; mine the data and develop steps for descriptive epidemiology. Innovation: Data generated from this project will further the understanding of the disease course, impact of available interventions, and the burden of the disease for an individual patient and society as a whole. It will inform the design of future clinical trials for therapeutic interventions in patients with desmoid and could lead to the development of a new treatment for a disease for which there is currently no US Food and Drug Administration (FDA) approved systemic treatment. The pilot project could become the prototype of studying rare diseases using Big Data.

LAY VERSION OF ABSTRACT- Desmoid Tumors for Big Data Linkage

Desmoid tumors (DT) are locally invasive soft tissue growths with no directed therapies currently. While two genes (β-catenin and adenomatous polyposis coli) have been found in patients who develop desmoids, it is unclear how these mutations and other downstream mechanisms lead to desmoid tumorigenesis. Extensive research has been explored in the molecular biology of desmoids; however, the use of metabolomics to understand the how the low molecular weight complements of cells, tissues, and biological fluids are perturbed by this highly localized disease. Additionally, the Desmoid Collaboration for a Cure has identified 45 active drugs against primary cell lines. It is unclear how these therapies perturb the metabolome, outside the Wnt and notch pathways. This proposal will use broad spectrum metabolomics to study the tumorigenesis process of fibroblasts to desmoids by investigating paired desmoid and fibroblast cell lines, in addition to unaffected fibroblast cells. Additionally, this proposal will explore the effects of three of the active drugs identified by Collaboration for a Cure on the desmoid and fibroblast cells. It is our hope that this research project will provide knowledge to understand more about the biochemistry of desmoid formation from normal tissue, and how these drugs will help fight desmoid tumors.

LAY VERSION OF ABSTRACT- A metabolomics pilot study on desmoid tumors and novel drug candidates

Desmoid tumors (DTs) are rare mesnchymal lesions with a high rate of local recurrence. Their common feature is a deregulated WNT pathway, mainly caused by gain-of-function mutations in exon 3 of the CTNNB1 gene (encoding for beta-catenin), resulting in nuclear accumulation of beta-catenin. Even though it is convtroversial, several studies have shown that the mutation S45F strongly correlates with increased propensity for desmoid recurrence as compared to T41A mutation. Therefore, it is imporant to investigage the differences between these 2 genetic alterations in order to identify potential targes for novel moleculat therapies. In studies conducated within the premise of our previously funded DTRF seed grant, we were able to establish a desmoid tissue and cell strain repository. Futhermore, we showed that the difference between the CTNNB1 T41A and S45F mutations is not due to differential protein expression levels, since beta-catenin is equally expressed in both mutations. Our gene array analysis showed that pro-apoptotic genes are downregulated and anti-apoptotic genes are upregulated in the cells with the S45F mutation when compared to the T41A mutation. Moreover, we showed that there is no significant induction of apoptosis in the S45F mutated desmoid cell strains when compared to the T41A mutated cells. Interestingly, the impairment of apoptosis appears to be specific to the CTNNB1 S45F mutation and not to desmoid tumors per se. Interim results are not promising, confirming our initial observation. However, these findings are in need of further investigation to identify the molecular mechanisms driving the differences in the induction of apoptosis between the T41A and S45F mutated tumors. Once we understand these differences, these findings may potentially have an impact on patients in the clinic. Finally, we have also started evaluating the effects of agents commonly used for the treatment of DT such as Sorafenib and Imatinib, as potential alternative therapies for patients harboring the beta-catenin S45F mutation. In the second year, we propose to: 1) continue establishing and characterizing human desmoid tumor cell strains, creating a desmoid tumor tissue repository in our new instituation as well as to continue trying to establish a desmoid tumor mice model; 2) unravel the molecular mechanism behind the differences in the induction of apoptosis between the CTNNB1 T41A and S45F mutation, and 3) evaluate the efficacy of other therapies for patients harboring the beta-catenin S45F mutation, such as Sorafenib and Imatinib.

LAY VERSION OF ABSTRACT- Reactivation of apoptosis: a potential therapeutic target for desmoid tumors with CTNNB1 S45F mutation

This project aims to provide a fast, semi-high throughput and cheap animal model for identifying and/or characterizing promising drug targets for treating desmoid tumors. In addition the platform allows pre-clinical assessment of novel candidate therapeutic compounds. The project builds on the recent introduction of efficient genome editing methods using TALEN and CRISPR/Cas9, which are creating unique and unmatched opportunities in several research fields, including cancer research. For the first time it is now possible to create functional gene knockouts in a number of model organisms. We have recently generated the first genetic tumor model in the organism Xenopus tropicalis. Because of the external development of the Xenopus embryo and its diploid genome, gene targeting experiments using CRISPR/Cas9 or TALEN are extremely efficient and cheap. Interestingly, when locally targeting the tumor suppressor gene APC we generated tadpoles that rapidly (< 1.5 months) and efficiently (>90%) developed desmoid tumors. This model presents a unique and novel experimental platform that (i) allows the rapid screening and evaluation of genes that contribute to the growth of the tumor, (ii) could serve to assess the clinical relevance of novel drug targets for treating desmoid tumors and (iii) can be used as a preclinical drug screening/assessment. We believe that our model offers a unique experimental platform that can be easily plugged into the research lines of several groups active in the field.

LAY VERSION OF ABSTRACT- Identifying targets for therapy in a novel genetic Xenopus model for desmoid tumor formation

Desmoid tumors are rare, locally aggressive neoplasms with unpredictable behavior. Although some tumors are indolent, resolving spontaneously, with tamoxifen, or with anti-inflammatory medications, others are prone to local recurrence, leading to significant morbidity, impaired function and poor quality of life for patients. For patients with advanced, refractory tumors, intensive chemotherapy regimens such as methotrexate/vinblastine or doxorubicin/dacarbazine can produce significant radiographic responses and symptomatic improvement. However these therapies are associated with both short and long-term toxicities. The ability to personalize therapy based on a predictive marker from an individual patient’s tumor would allow early treatment of aggressive tumors with chemotherapy, while sparing patients with indolent tumors or those more likely to respond to milder interventions. 
Recently, desmoid tumors with particular mutations in CTNNB1, including S45F, have been shown to have a higher risk of recurrence. Additionally, desmoid tumors with S45F mutations were less likely to respond to conservative treatments with imatinib or meloxicam. However, it is unknown whether S45F mutated tumors are more likely to respond to chemotherapy. In this proposal, we will obtain tissue and radiographic imaging for our existing database of desmoid tumor patients, and perform CTNNB1 sequencing to identify specific mutations. Through a collaboration with MD Anderson Cancer Center, our combined datasets will then be analyzed to determine whether the presence of S45F mutations correlates with MRI response to various chemotherapy regimens. The ability to use CTNNB1 mutation status to predict behavior of desmoid tumors and to guide therapeutic decisions would have immediate implications for clinical management of these rare yet often aggressive tumors.

LAY VERSION OF ABSTRACT- Correlation of CTNNB1 mutation status with chemotherapy response by MRI in patients with desmoid tumors

Desmoid tumors (DT) are locally invasive lesions that are difficult to treat using conventional therapies. While there is some success with chemotherapy in people who have recurrence of their tumors after surgery and/or radiation, much of the time the results from chemotherapy are temporary, and regrowth of tumor is seen. One approach to treatment is to develop a combination of medications that target different aspects of tumor growth, but have few side effects. Since desmoids are locally invasive but do not metastasize to other locations in the body, it is not necessary to eliminate every tumor cell, but instead to stabilize or shrink the tumor. We assembled a consortium of researchers from different backgrounds, and with complementary skills, whose ultimate goal is to develop a treatment to therapeutically target tumor cells in desmoid patients without causing serious side effects.

To achieve this goal, over the past two years, we screened drugs in the laboratory on cell lines from desmoid tumor patients to see which agents specifically target tumor cell viability and tested a first cohort of drugs in mice that develop desmoid tumors, identifying several agents already in use or close to being in use for patient care that could be repurposed for use in desmoid tumor treatment.

Over the remaining course of the proposal we plan to:

1. Complete the testing of drugs identified in the screen in a mouse model that is engineered to develop desmoids.

2. The agents identified will then be rescreening for drug combinatons with synergistic effects.

3. A pilot multi-drug regimen will be tested in mice.

In the past two years we screened eight cell lines using the Maybridge HitFinderTM Collection; DIVERSetTM Collection (Chembridge); LOPAC (Sigma); BIOMOL collection; Prestwick collection (Prestwick Chemical); Seminatural compounds (AnalytiCon Discovery); and the NIH Clinical Collection (BioFocusDPI). This screening identified 45 compounds that inhibited cell proliferation in desmoid tumors by greater than 50% while not effecting normal fibroblast proliferation, and that also are either in use in patients or that that have the potential to be rapidly developed for patient care. We then tested these agents using a collection of desmoid tumor cell cultures of different genetic etiologies, and based on this, as well as how available the selected agents are for clinical use today, prioritized 18 for testing in mice that develop desmoid tumors. We completed testing of nine agents, and found that five: Dasatinib (an oral Bcr-Abl tyrosine kinase inhibitor); A multi-target PI3K inhibitor; an FAK inhibitor; Letrozole; and a gamma secretase inhibitor. An additional nine drugs are currently being tested in the mice.

In the next year, we will undertake a rescreening of the five drugs that were effective in mice, by undertaking a sensitization test, in which we first treat the cells with the positively identified agents, and then rescreening them using the libraries. Agents that have a synergistic or additive effect will show lower cell viability than either agent alone. This approach will rapidly identify a multi-drug combination. We will then test the drug combinations in mice. Depending on the number of agents identified, we will either test all combinations, or if the number if too large, we will prioritize which to test based on a pharmacokinetic and then undertake analysis prioritizing agents that can be used in patients at the present time.

This data will identify new treatment regiments, which can rapidly be brought to patient care. In addition, it will identify new pathways activated by beta-catenin and new agents not yet in use for patient care that target desmoid tumors, both of which can be developed in future work which might ultimately identify even more effective therapeutic approaches.

LAY VERSION OF ABSTRACT- Collaboration for a Cure: Identifying new therapeutic targets for desmoid tumors

This project is to develop the first validated PRO tool in desmoid tumors. The PRO tool will be developed and validated in partnership between MSKCC and Quintiles, a leading PRO company. Desmoid tumor (DT/DF) is a sarcoma of fibroblastic origin that afflicts children, adolescents and adults and has a median age of onset of 30 years. Desmoid tumors can occur in any anatomic location and threatens vital organs and limbs which often results in significant morbidity from organ failure, mutilating surgeries (and amputations), loss of limb function, pain and death. Surgery is associated with high rates of recurrences (~40%). In advanced disease, systemic therapies range from anti-estrogens to cytotoxic chemotherapies with variable response rates however no systemic therapies has been approved since pivotal trials evaluating survival or quality of life has never been conducted. In the last few years, deeper biological understanding of this disease has led to promising drugs such as sorafenib, mTOR and Notch inhibitors that are currently in clinical trials. These and other drugs cause both tumor reduction and improvement in quality of life. Tumor reduction is measured by standard images and by established criteria (RECIST or WHO). However, the improvement in quality of life described by patients is lost as there are no validated tools to measure this drug effect. The classical regulatory (FDA) pathway to approving new drugs in cancer is demonstration of improvement in overall survival. Surrogates of overall survival include progression free survival and durable tumor responses. An emerging endpoint for regulatory approval is patient reported outcomes (PRO) tools. More recently, the FDA approved JakafiTM, a drug to treat myelodysplastic syndrome based on a PRO tool developed in MDS. We hypothesize that development of a PRO tool in desmoid tumors, will facilitate a new regulatory endpoint that can be used in all future clinical trials and will be a great investment of resources to advance the field for researchers worldwide.”

LAY VERSION OF ABSTRACT- Development of a patient reported outcome (PRO) tool in desmoid tumors

Desmoid tumors are proliferations of relatively benign appearing fibroblasts. Despite their histologic bland appearance, a significant subset of these tumors recurs aggressively and requires often debilitating surgery. Currently there are no molecular markers that predict the behavior of desmoid tumors.

The aims:

AIM 1: To generate an extensive molecular dataset covering gene expression profiling and gene mutation analysis of desmoid tumors and scar samples.

AIM 2: To identify gene changes in expression level (for mRNA and non-coding RNAs) and gene mutations that correlate with risk for recurrence.

AIM 3: To use the same approach to identify genetic markers that can distinguish scar tissue from recurrent desmoid.

AIM 4: To explore the molecular heterogeneity of desmoid tumors based on the analysis of multiple regions sampled from a single tumor.

AIM 5: To search for low frequency CTNNB1/APC mutations in “wild-type” tumors.

The purpose of this study will be to perform a very broad molecular search for markers that can be used to address two clinically highly relevant questions. Using a carefully selected group of cases with known clinical outcome, we will use stranded RNA-Seq data (for mutation and expression level analysis) to generate a dataset that will characterize desmoid tumors in the broadest sense possible. First, this dataset will be searched for markers that can distinguish desmoid tumors that can be followed by “watchful waiting” at the time of their initial biopsy from tumors that require aggressive surgical therapy. When desmoid tumors recur, the distinction between recurrent tumor and scar tissue from prior surgery can be very difficult by histology, and immunohistochemistry for beta-catenin is insufficient. In the second approach that will use the dataset mentioned above, we will compare RNA-Seq findings on scar tissue with those seen in desmoid tumors to identify markers that distinguish scar from desmoid in an effort to find a novel immunohistochemistry marker to discern these two entities and that can be used in surgical pathology practice.

We also plan to perform deep targeted sequencing of CTNNB1 and APC genes in samples with no detectable mutations in Sanger sequencing/RNA-Seq data. According to the recent findings of Crago et al. (Genes Chromosomes Cancer. 2015 Oct;54(10):606-15), many of desmoid tumors initially considered as wild-type appear to carry low frequency mutations in CTNNB1/APC genes that can be detected using next generation sequencing. Our set of samples comprises relatively high portion of wild-type cases (27%) therefore we aim to further investigate this issue in order to better characterize our samples.

This work will build on an already existing large dataset in the van de Rijn laboratory and will benefit from the experience that we have generated in our laboratory for the use of archival formalin-fixed, paraffin-embedded tissue in high throughput studies using next generation sequencing. The project greatly benefits from the collaboration with Dr. Kristen Ganjoo at Stanford University Hospital and from the collaboration with Dr. Raffi Avedian, orthopedic surgeon at Stanford. We have also started a new collaboration with Dr. Justin Cates and Dr. Thomas Stricker from Vanderbilt University, and with Dr. Chiara Colombo from Milan, Italy. We have previously performed quantitative measurement of the expression levels for all know human protein-encoding genes in 9 desmoid tumor samples, 4 scars and 42 non-desmoid fibroblastic lesions. The number of scar samples that we have analyzed was too low but the preliminary results were encouraging. Our current dataset includes 29 primary, 20 recurrent desmoid tumors and 15 scars from 26 patients.

LAY VERSION OF ABSTRACT- Next generation sequencing approach to desmoid tumors

Desmoid tumors are proliferations of relatively benign appearing fibroblasts. Despite their histologic bland appearance, a significant subset of these tumors recurs aggressively and requires often debilitating surgery. Currently there are no molecular markers that predict the behavior of desmoid tumors.

The aims:

AIM 1: To generate an extensive molecular dataset covering gene expression profiling and gene mutation analysis of desmoid tumors and scar samples.

AIM 2: To identify gene changes in expression level (for mRNA and non-coding RNAs) and gene mutations that correlate with risk for recurrence.

AIM 3: To use the same approach to identify genetic markers that can distinguish scar tissue from recurrent desmoid.

AIM 4: To explore the molecular heterogeneity of desmoid tumors based on the analysis of multiple regions sampled from a single tumor.

AIM 5: To search for low frequency CTNNB1/APC mutations in “wild-type” tumors.

The purpose of this study will be to perform a very broad molecular search for markers that can be used to address two clinically highly relevant questions. Using a carefully selected group of cases with known clinical outcome, we will use stranded RNA-Seq data (for mutation and expression level analysis) to generate a dataset that will characterize desmoid tumors in the broadest sense possible. First, this dataset will be searched for markers that can distinguish desmoid tumors that can be followed by “watchful waiting” at the time of their initial biopsy from tumors that require aggressive surgical therapy. When desmoid tumors recur, the distinction between recurrent tumor and scar tissue from prior surgery can be very difficult by histology, and immunohistochemistry for beta-catenin is insufficient. In the second approach that will use the dataset mentioned above, we will compare RNA-Seq findings on scar tissue with those seen in desmoid tumors to identify markers that distinguish scar from desmoid in an effort to find a novel immunohistochemistry marker to discern these two entities and that can be used in surgical pathology practice.

We also plan to perform deep targeted sequencing of CTNNB1 and APC genes in samples with no detectable mutations in Sanger sequencing/RNA-Seq data. According to the recent findings of Crago et al. (Genes Chromosomes Cancer. 2015 Oct;54(10):606-15), many of desmoid tumors initially considered as wild-type appear to carry low frequency mutations in CTNNB1/APC genes that can be detected using next generation sequencing. Our set of samples comprises relatively high portion of wild-type cases (27%) therefore we aim to further investigate this issue in order to better characterize our samples.

This work will build on an already existing large dataset in the van de Rijn laboratory and will benefit from the experience that we have generated in our laboratory for the use of archival formalin-fixed, paraffin-embedded tissue in high throughput studies using next generation sequencing. The project greatly benefits from the collaboration with Dr. Kristen Ganjoo at Stanford University Hospital and from the collaboration with Dr. Raffi Avedian, orthopedic surgeon at Stanford. We have also started a new collaboration with Dr. Justin Cates and Dr. Thomas Stricker from Vanderbilt University, and with Dr. Chiara Colombo from Milan, Italy. We have previously performed quantitative measurement of the expression levels for all know human protein-encoding genes in 9 desmoid tumor samples, 4 scars and 42 non-desmoid fibroblastic lesions. The number of scar samples that we have analyzed was too low but the preliminary results were encouraging. Our current dataset includes 29 primary, 20 recurrent desmoid tumors and 15 scars from 26 patients.

LAY VERSION OF ABSTRACT- Next generation sequencing approach to desmoid tumors

Desmoid tumors (DT) are locally invasive lesions that are difficult to treat using conventional therapies. While there is some success with chemotherapy in people who have recurrence of their tumors after surgery and/or radiation, much of the time the results from chemotherapy are temporary, and regrowth of tumor is seen. One approach to treatment is to develop a combination of medications that target different aspects of tumor growth, but have few side effects. Since desmoids are locally invasive but do not metastasize to other locations in the body, it is not necessary to wipe out every tumor cell, but just to get the tumor to shrink. We assembled a consortium of researchers from different backgrounds, and with complementary skills, whose ultimate goal is to develop a treatment to therapeutically target tumor cells in desmoid patients without causing serious side effects.

To achieve this goal, over the past year, we screened drugs in the laboratory on cell lines from desmoid tumor patients to see which agents specifically target tumor cell viability, and are in the process of testing the drugs in cell lines. Over the remaining course of the proposal we plan to: (1) Complete the testing of drugs identified in the screen using cell lines from a variety of desmoid tumors to determine the mechanism by which they inhibit cell viability. (2) Test drugs that are effective in cell lines in a mouse model that is engineered to develop desmoids. (3) The agents identified in the screen will then be analyzed for the ability to use them in combination, and these combinations will be tested in both desmoid cell cultures and in genetically modified mice.

In the past year we screened eight cell lines using the Maybridge HitFinderTM Collection; DIVERSetTM Collection (Chembridge); LOPAC (Sigma); BIOMOL collection; Prestwick collection (Prestwick Chemical); Seminatural compounds (AnalytiCon Discovery); and the NIH Clinical Collection (BioFocusDPI). This screening identified 45 compounds that inhibited cell proliferation in desmoid tumors by greater than 50% while not affecting normal fibroblast proliferation, and that also are either in use in patients or that that have the potential to be rapidly developed for patient care.

We are currently testing these drugs using a large collection of desmoid tumor cell cultures of different genetic etiologies, and based on this, will test them in mice that develop desmoid tumors. Drugs that inhibit desmoid tumor growth in both cells and mice will then be studied using pharmacokinetic analysis, to identify drugs that work in an additive fashion when used in combination, but will still have an acceptable safety profile. We will then test the drugs already approved for use in patients in a multi drug regimen as informed by pharmacokinetic data. Then these combinations will be tested in multiple human desmoid tumor cultures and in genetically modified mouse models.

New candidate drugs will also be analyzed to identify their underlying biologic mechanism, to identify novel pathways that are activated by beta-catenin in desmoid tumors. These data will be used to prioritize new research into the biology of how beta-catenin causes desmoid tumors. This approach should rapidly identify a multi-drug combination that can then safely be tested for efficacy in patients. In addition, it will identify new pathways activated by beta-catenin and new agents not yet in use for patient care that target desmoid tumors, both of which can be developed in future work which might ultimately identify even more effective therapeutic approaches.

Desmoid-type fibromatosis (DTF) has a propensity for locally aggressive behavior. However, a subset of these tumors does not necessarily follow an aggressive clinical course, as rare cases of spontaneous regression and many more examples of tumor quiescence have been reported. Clinical, histopathologic and molecular predictors of DTF outcome remain elusive. Recently, specific mutations of the CTNNB1 gene have been shown to have prognostic significance. This raises the possibility that there are other driver mutations which, in addition to CTNNB1, determine the biological potential of these tumors. Therefore, we propose to use whole exome sequencing of primary, non-syndromic desmoid tumors and adjacent normal tissues to identify other possible driving mutations in DTF.

This project also includes an internal case-control study design, in which primary DTF cases that subsequently recurred locally will be matched and compared to cases unassociated with local recurrence. Identification of additional driver mutations associated with an increased likelihood of local recurrence might assist clinicians and patients faced with difficult management decisions. These altered proteins or their functional pathways might also represent postential pharmacotherapeutic targets.

Desmoid tumors (DTs) are mesenchymal tumors that occur sporadically or in the context familial adenomatous polyposis (FAP). Although rare in the general population, DTs are a leading cause of death in FAP patients. There is no effective cure for patients with unresectable disease, and little is known about the molecular biology driving desmoid tumorigenesis. Our laboratory seeks to characterize the biological requirements necessary for DT formation and identify targeted therapies for this disease. Currently, we are investigating the role of hyaluronic acid (HA) in driving desmoid proliferation and have preliminary evidence that HA is an abundant constituent of DTs.

Based on our novel observations, we hypothesize that hyaluronic acid plays a critical role in desmoid tumorigenesis and provides a novel target for therapy. Furthermore, we propose that HA promotes DT progression through crosstalk with inflammatory components of the tumor microenvironment such as mesenchymal stem cells (MSCs). We will test this hypothesis through two specific and independent aims:

Aim 1: To evaluate the anti-tumorigenic effects of hyaluronic acid inhibition in human desmoid-derived cells. Aim 2: To evaluate the anti-tumorigenic effects of hyaluronic acid inhibition in the Apc1638N mouse and xenograft models. Our findings will 1) address major gaps in our current understanding of DT pathophysiology, 2) define the role that HA plays in driving desmoid tumorigenesis, and 3) identify novel targets for therapy. As a clinical referral center for patients with DTs, we are uniquely situated to carry out this proposal given our access to rare patient tumors that will sustain our future work and allow us to expeditiously translate benchside discoveries to clinical trials.

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Although desmoid clinical biology has been well characterized, the molecular biology of those tumors tumor remains poorly understood, perhaps because of the global lack of needed research bioresources such as desmoid cells and human desmoid tissue banks needed for the high throughput research necessary to identify mutated genes and abnormal proteins driving desmoid tumor inception, progression, invasion, treatment resistance, and recurrence. In studies conducted within the premise of our DTRF- funded seed grants we were able to establish a large desmoid tissue and cell strain repository in our former lab and have identified WKDW ȕ-catenin mutations are highly prevalent in sporadic desmoid tumors and that a specific mutation, S45F possibly significantly correlates with dismal patient outcome. Moreover, Hamada et al. have shown that CTNNB1 S45F mutation predicts poor efficacy of meloxicam treatment for DT. Those interesting initial observations led further investigation by our group.

We performed a gene array which we included 44 desmoid tumor tissues (14 tumors harboring the 45F mutation, 16 tumors with a 41A mutation and 14 wild type tumors), 14 corresponding normal tissues and 14 desmoid cell strains (8 cells strains with a 41A mutation and 6 cell strains with a 45F mutation). Our initial results showed that that the mutation T41A was enriched with pro apoptosis gene and that negative apoptosis regulator genes were upregulated in the mutation S45F. In the current application we suggest to continue our studies and propose a two-pronged research design consisting of distinct yet interrelated components that will address current knowledge deficits. Our studies will focus on reestablishing and characterizing new human desmoid tumor cell strains, recreating a desmoid tumor tissue repository as well as establishing a desmoid tumor mice model in our new lab at the Ohio State University. Furthermore, understanding that the deregulation of the beta-catenin pathway is potentially a significant contributor to tumorigenesis and progression and the limitation of direct therapeutic targeting of E-catenin we would attempt to identify potential targets that could reactivate the apoptosis which may be related to the aggressive behavior of the desmoid tumors harboring the CTNNB1 S45F mutation. Identifying genes and their cognate proteins whose alteration would explain the aggressive behavior of some desmoid tumors might provide future targets for novel molecular therapies relevant to patients burdened by this devastating disease.

Desmoid-type fibromatosis (DF) is a rare connective tissue disease, characterized by the presence of one or more benign non metastasizing myofibroblastic neoplasms with a high local recurrence rate. DF can be diagnosed at any age in both genders but it is more common in young women.

A minority of DF is inheritable and is associated with mutations of the Adenomatous Polyposis Coli (APC) gene, while most of the DF arises sporadically. On the molecular level, 50-80% of the sporadic DF cases have somatic mutations in the codons encoding serine 41 and threonine 45 in exon 3 of the β-catenin gene, CTNNB1. These sites are required for β-catenin phosphorylation and subsequent degradation. Consequently alterations in these sites lead to the Wnt pathway alteration with β-catenin accumulation and its translocation to the nucleus.

Nuclear β-catenin is detected in almost 90% of the desmoid cells and it is considered a positive marker of DF cells once malignant cell transformation is excluded. In addition to this marker, we recently demonstrated that GSK-3β, also involved in Wnt pathway regulation, translocates in the nucleus of DF cells as well, colocalizing and interacting with β-catenin. This event occurs regardless of the presence of CTNNB1 gene mutations suggesting that the β-catenin/GSK-3β interaction and the nuclear translocation can be unrelated to the genetic alterations of CTNNB1. This led us to formulate the hypothesis that the alteration Wnt/β-catenin signaling pathway in desmoid tumor is modulated by the interaction with microenvironmental factors.

The aim of this project is the identification and the characterization of the microenvironment factors responsible of the proliferation and aggressiveness of DF cells. Since the deregulation of the inflammatory process can lead to aberrant fibroblast activation and accumulation of ECM proteins with subsequent tissue fibrosis that can evolve in fibrotic disease and tumor development, we will focus our attention on the inflammatory and growth factors and their interaction with the Wnt/β-catenin pathway. For this purpose cells derived from desmoid tumor and control cell samples will be incubated with different cytokines, growth factors and hormones. Their effect on the growth rate and aggressiveness will be evaluated by immunofluorescence using specific antibodies for cell cycle markers, for the receptors of the microenvironmental factors, for the proteinases degrading the extracellular matrix, for cysteine proteases and for the markers of the basal membrane, lysosomes and vesicles.

We will use a custom Ion AmpliSeq RNA panel to analyze the gene expression of DF cells under treatment, in particular focusing on genes of the Wnt pathway and of the other correlated pathways. Modulating the microenvironment we expect to identify potential factors that promote the DF tumor growth and aggressiveness. This is a critical milestone in defining the potential targets for a therapy aimed at reducing the proliferation, local aggressiveness and recurrence rate of DF tumors.

This study is part of a long term multidisciplinary research program that is ongoing since 2011 and it aims to improve the knowledge of the Desmoid-type fibromatosis and to identify a viable treatment. To date the study has led to the identification of a second potential positive marker of desmoid tumor cells, the nuclear translocation of GSK-3β.

Desmoid-type fibromatosis (DF) is a rare connective tissue disease, characterized by the presence of one or more benign non metastasizing myofibroblastic neoplasms with a high local recurrence rate. DF can be diagnosed at any age in both genders but it is more common in young women.

A minority of DF is inheritable and is associated with mutations of the Adenomatous Polyposis Coli (APC) gene, while most of the DF arises sporadically. On the molecular level, 50-80% of the sporadic DF cases have somatic mutations in the codons encoding serine 41 and threonine 45 in exon 3 of the β-catenin gene, CTNNB1. These sites are required for β-catenin phosphorylation and subsequent degradation. Consequently alterations in these sites lead to the Wnt pathway alteration with β-catenin accumulation and its translocation to the nucleus.

Nuclear β-catenin is detected in almost 90% of the desmoid cells and it is considered a positive marker of DF cells once malignant cell transformation is excluded. In addition to this marker, we recently demonstrated that GSK-3β, also involved in Wnt pathway regulation, translocates in the nucleus of DF cells as well, colocalizing and interacting with β-catenin. This event occurs regardless of the presence of CTNNB1 gene mutations suggesting that the β-catenin/GSK-3β interaction and the nuclear translocation can be unrelated to the genetic alterations of CTNNB1. This led us to formulate the hypothesis that the alteration Wnt/β-catenin signaling pathway in desmoid tumor is modulated by the interaction with microenvironmental factors.

The aim of this project is the identification and the characterization of the microenvironment factors responsible of the proliferation and aggressiveness of DF cells. Since the deregulation of the inflammatory process can lead to aberrant fibroblast activation and accumulation of ECM proteins with subsequent tissue fibrosis that can evolve in fibrotic disease and tumor development, we will focus our attention on the inflammatory and growth factors and their interaction with the Wnt/β-catenin pathway. For this purpose cells derived from desmoid tumor and control cell samples will be incubated with different cytokines, growth factors and hormones. Their effect on the growth rate and aggressiveness will be evaluated by immunofluorescence using specific antibodies for cell cycle markers, for the receptors of the microenvironmental factors, for the proteinases degrading the extracellular matrix, for cysteine proteases and for the markers of the basal membrane, lysosomes and vesicles.

We will use a custom Ion AmpliSeq RNA panel to analyze the gene expression of DF cells under treatment, in particular focusing on genes of the Wnt pathway and of the other correlated pathways. Modulating the microenvironment we expect to identify potential factors that promote the DF tumor growth and aggressiveness. This is a critical milestone in defining the potential targets for a therapy aimed at reducing the proliferation, local aggressiveness and recurrence rate of DF tumors.

This study is part of a long term multidisciplinary research program that is ongoing since 2011 and it aims to improve the knowledge of the Desmoid-type fibromatosis and to identify a viable treatment. To date the study has led to the identification of a second potential positive marker of desmoid tumor cells, the nuclear translocation of GSK-3β.

Desmoid tumors (DT) are locally invasive lesions that are difficult to treat using conventional therapies. While there is some success with chemotherapy in people who have recurrence of their tumors after surgery and/or radiation, much of the time the results from chemotherapy are temporary, and regrowth of tumor is seen. One approach to treatment is to develop a combination of medications that target different aspects of tumor growth, but have few side effects. Since desmoids are locally invasive but do not metastasize to other locations in the body, it is not necessary to wipe out every tumor cell, but just to get the tumor to shrink. Dr. Alman has assembled a consortium of researchers from different backgrounds, and with complementary skills, whose ultimate goal is to develop a treatment to therapeutically target tumor cells in desmoid patients without causing serious side effects.

To achieve this goal, the collaborators will:

1. Screen drugs in the laboratory on cells taken from desmoid tumor patients to see which drugs kill the tumor cells.

2. Test successful drugs using cell cultures from a variety of desmoid patients or cell lines.

3. Test drugs that are effective in cell lines in a mouse model that is engineered to develop desmoids.

4. The agents identified in the screen will then be analyzed for the ability to use them in combination, and these combinations will be tested in both desmoid cell cultures and in genetically modified mice.

Dr. Alman previously screened 1,000 agents in desmoids. There are libraries of over 100,000 compounds available for testing. The team of collaborators will use desmoid tumor primary cell cultures and normal fibroblasts to screen a large and comprehensive compound library using a robotic high throughput facility, to identify drugs that inhibit desmoid tumor cell growth but not normal fibroblast cells. The screening process will employ both commercially available drugs as well as otherwise untested agents.

Drugs that yield a positive result from the screen will be tested using a large collection of desmoid tumor cell cultures of different genetic etiologies, and in mice that develop desmoid tumors. Drugs that inhibit desmoids tumor growth in both cells and mice will then be studied in to using pharmacokinetic analysis, to identify drugs that work in an additive fashion when used in combination, but will still have an acceptable safety profile. They will then test the drugs already approved for use in patients in a multi drug regimen as informed by pharmacokinetic data. Then these combinations will be tested in multiple human desmoid tumor cultures and in genetically modified mouse models.

New candidate drugs will also be analyzed to identify their underlying biologic mechanism, to identify novel pathways that are activated by beta-catenin in desmoid tumors. These data will be used to prioritize new research into the biology of how beta-catenin causes desmoid tumors.

This approach should rapidly identify a multi-drug combination that can then safely be tested for efficacy in patients. In addition, it will identify new pathways activated by beta-catenin and new agents not yet in use for patient care that target desmoid tumors, both of which can be developed in future work which might ultimately identify even more effective therapeutic approaches.

This study will collaborate with the below study of Dr. van de Rijn at Stanford. In many patients with desmoid-type fibromatosis (DTF), the tumors recur after surgical excision. A major problem confronting clinicians who treat DTF is the lack of well-established criteria that predict which tumors will recur. Treating physicians do not know which patients are at high risk, and they are sometimes unsure about how aggressively to treat an individual patient. Recently, researchers have suggested that specific genetic mutations in the gene encoding β catenin predict a greater risk of tumor recurrence. While β catenin mutations seem to be useful prognostic markers, it is hypothesized that there are other so-called “driver mutations” that contribute to aggressive behavior in DTF. Therefore, Dr. Cates proposes to sequence the entire coding region of genomic DNA in DTF and adjacent normal tissues to identify additional DNA mutations in these tumors. To discover somatic mutations that predict tumor recurrence, he will also compare cases of DTF that recurred to similar cases that did not recur. Identification of mutations associated with an increased risk of local recurrence might assist doctors and patients faced with difficult management decisions and lead to discovery of potential drug targets.

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Desmoid tumors (DTs) are rare tumors that can be inherited or occur spontaneously. Although they do not metastasize, DTs can invade surrounding structures and cause pain, bowel obstruction, bleeding, and death. Recently, progress has been made in research efforts to understand DT biology. However, there is still no cure or reliable treatment for these tumors. Dr. Cho’s laboratory is interested in understanding how DT cells “talk” to their surrounding environment to promote tumor survival, invasion, and growth. There is data to show that hyaluronic acid (HA) is a prominent component of the cellular matrix surrounding DTs. Dr. Cho believes that HA is critical in recruiting inflammatory cells that are necessary for DT formation and proliferation. This proposal will test the effects of anti-hyaluronic acid drugs in desmoid tumor cell lines obtained from human patients as well as animal models of this disease. It will also investigate whether HA promotes desmoid growth by recruiting specialized bone marrow progenitor cells that contribute to DT proliferation and invasion. This study will gain an understanding of how DTs manipulate their host environment to enhance tumorigenesis and may identify a novel target for therapy. The overall goal is to identify treatments for DT patients and improve survival and quality of life.

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Desmoid tumors (DTs) are rare mesenchymal disease with a high tendency to recur despite an adequate surgical excision. An observational approach has been recently proposed in order to select patients who mostly benefit from medical therapy or surgical intervention because they have a painful or progressive disease or patients having an indolent or stable disease who can just be observed. There is not enough data or information at the beginning of the history that can help in predicting the behavior. Recently, specific β-catenin mutation (45F) has been correlated with a higher risk of recurrence after complete surgery. These results are based on retrospective data and they can be biased. In order to understand the natural history of this challenging disease, Fondazione IRCCS Instituto Nazionale dei Tumori proposed a prospective multicenter observational study for patients with measurable primary extra-abdominal disease who undergo W&S approach. This study has just been approved in Italy and the recruitment is ongoing. The aim of this translational project is to identify genome alterations that guide an aggressive behavior and to look for predictive factors in patients enrolled in the prospective clinical study using a high throughput genome approach. Dr. Colombo expects to observe different pattern of disease behavior that may reflect specific genome alterations. The data will be then validated in a larger cohort of patients surgically treated in the past and for whom FFPE materials are available. The assumption is that progression (in patients “observed”) and recurrence (in patients “operated”) represents different faces of biological aggressiveness. This could be helpful in the future to personalize treatments according to the predicted aggressiveness of the disease in each patient from the beginning.

This is the final year of a study started by Dina Lev, MD, Phd at MD Anderson Cancer Center.

The goal is to investigate the molecular driving forces behind the development and progression of desmoid tumors. Dr. Pollock, et al, are trying to identify potential targets that modify beta-catenin transcriptional activity using an siRNA screen. Identifying genes and their cognate proteins whose alteration would inhibit beta-catenin activity might provide future targets for novel molecular therapies relevant to DT patients.

Aim 1: To validate previous findings of the prognostic power of beta-catenin 45F mutation in predicting the outcome of patients with primary desmoids.

Aim 2: To identify the molecular deregulations contributing to the sensitivity or resistance to the commonly used anti-desmoid therapies, mainly Tamoxifen, NSAIDS and Gleevec.

Aim 3: To identify potential novel anti-desmoid therapeutic targets using a rational siRNA screen. Potentially these targets harbor the capacity to modify the transcriptional effect of beta-catenin.

Note: Dr. Lev has previously identified significant associations between the presence of 45F mutations and an increased rate of of desmoid recurrence as well as shorter time intervals to recurrence. Analysis of a large primary desmoid subset showed that desmoids having a 45 F mutation had an estimated five year recurrence -free survival rate of only 47% and a median time to recurrence of 3.16 years. In sharp contrast, the estimated five year recurrence free survival rate for all other desmoid primary tumors was 83%; median time to recurrence not yet being reached.

Dr. van de Rijn has started to perform gene expression profiling (determining at which level each of >20,000 human genes is expressed) on archival tissue of desmoid tumors, scars from desmoid patients and a large number of fibroblastic lesions using an approach developed in his laboratory. The current proposal is a logical and necessary continuation of this project. This project will use cutting edge next generation sequencing to pursue two major clinical problems in desmoid tumors. First, it is well known that not all desmoid tumors behave in the same manner. Some are very aggressive, others have an indolent behavior. While attempts have been made to develop predictors for the behavior of desmoids, these rely only on clinical parameters such as tumor size, site and age of the patient and no molecular markers for recurrence risk have been identified. At this moment it is impossible to tell with certainty which tumors require aggressive treatment and which can be followed by “watchful waiting”. Through the ability to perform next generation sequencing on paraffin embedded tumor samples he can now perform a very broad search for changes in DNA that can help predict the behavior of desmoids. The data acquired under this proposal will complement the already existing dataset in his laboratory. Through collaboration with the oncology group at Stanford and at the Dana Farber Cancer Institute, there will be access to a more than sufficient number of cases for the study. In addition to identifying markers that can be used to predict the behavior of desmoid tumors, Dr. van de Rijn will also perform an in-depth search for markers that can be used by pathologists to distinguish scar from desmoid recurrence.

This is a pilot study examining the role of sirolimus in the treatment of children and young adults with desmoid tumor that is deemed likely to recur following resection.  Sirolimus directly targets and inhibits a pathway that may be critical for the development and growth of desmoid tumor.  Sirolimus and several related drugs have been used in children and adults to suppress the immune system following transplant and, more recently, as an anti-neoplastic agent in a variety of cancer types.  Sirolimus is attractive as a potential drug for desmoid tumor because it is well-tolerated in children and young adults and it can be given orally.  Sirolimus acts by blocking a growth promoting pathway called the mTOR pathway.  Dr. Weiss will determine whether sirolimus can decrease tumor size, decrease tumor-associated pain, and block the mTOR pathway.  The data obtained in this pilot study will be used to leverage additional resources to support a national collaborative study, such as one run through the Children’s Oncology Group (COG), in children and young adults.

*The above grant awards are subject to a mutually satisfactory grant agreement between the parties.

First year of two-year project for correlative research to define novel biomarkers of response in desmoid tumors treated with sorafenib.

Desmoid tumors (DT/DF) are clonal connective tissue malignancies of fibroblastic origin. DT/DF lack metastatic potential however, can cause significant morbidity, loss of function and pain through mass effects. Mortality from DT/DF results from local infiltration of vital structures. There is no standard of care for the treatment of DT/DF. Surgery is curative however associated with high recurrences. In unresectable patients, systemic therapies range from anti-estrogens to cytotoxic chemotherapies. DT/DF over express c-KIT and PDGFR and a Phase II study of imatinib showed a partial response (PR) of 6%. Sorafenib is a multi-target kinase inhibitor of b-Raf, PDGFR and VEGFR and available through an expanded access program. We treated an index patient with recurrent, unresectable DT/DF with sorafenib. A PR and clinical benefit prompted us to evaluate our experience of sorafenib in 26 patients. We noted a 25% PR by RECIST 1.1 and improvement of symptoms in 70% of patients. We noted a 30% decrease in the MRI T2 signal in 90% of patients with extra-abdominal disease. We hypothesize that sorafenib will lead to improved response rates, symptoms and progression free survival (PFS) in DT/DF patients. This is a multicenter, Phase III, randomized, double-blind, placebo-controlled trial of sorafenib in DT/DF patients. The primary endpoint is to evaluate overall response rates (ORR) for patients treated with sorafenib versus those treated with placebo. This study will enroll 126 patients over 2 years and will have a 90% power to detect a 22% increase in response rates (1-sided a=0.05). The secondary endpoints are 1) PFS and 2) pain palliation by the Brief Pain Inventory scale (BPI). Patients on placebo will cross over to sorafenib. Explorative studies include pre- and post-treatment biopsies in responding patients to determine biomarkers of response and mechanism of action of sorafenib. Decrease in MRI T2 signal may be an early marker of RECIST response and may be a more sensitive imaging marker for clinical benefit than RECIST response. We will conduct an explorative study a novel imaging biomarker by correlating changes in T2 signal to RECIST response and clinical benefit by pain palliation.

A major focus of my research program is to study the molecular pathology of desmoid tumors (DTs), with a long-term goal of developing improved treatments. DTs are locally invasive soft tissue tumors, which we found are caused by mutations resulting in the stabilization of the protein, ß-catenin. Stabilized ß-catenin binds to TCF transcription factors to regulate the expression of genes in a cell type specific manner. We developed mouse models of DT based on this knowledge and used these to study the role of target genes in the DT phenotype. Our ultimate goal is to develop an improved approach to DT therapy.

In this project we will continue our ongoing work funded by the DTRF in which we identified pharmacologic agents that target DT cell viability by screening compound libraries composed of agents which have a high potential to be rapidly translated into patient care. We identified agents that decreased cell viability in DT cultures but not in normal fibroblasts. Over the past two years, we tested several compounds in multiple DT cell lines, tested one in a mouse model of DT, and this work is in press in PLoS One, and found three others that have strong potential to be used as a therapeutic approach to DTs.

Two agents target platelet derived growth factor (PDGF) signaling, a pathway we also found can modulate ß-catenin activity. The other targets Notch signaling. We also developed a new mouse model in which we can regulate expression of conditional stabilized ß-catenin alleles, which can be used to rapidly screen signaling pathway interactions in-vivo. We hypothesize that compound screening will identify agents that can be developed into novel therapies to treat DTs, and rapidly be brought to patient care. In this next phase of our ongoing work, we propose to test this hypothesis by answering the following two questions:

How does Platelet Derived Growth Factor (PDGF) and Notch signaling modulation target DT cell viability? In the initial phase of our DTRF work, we identified three compounds that decreased cell viability in DT cultures but not normal fibroblasts: suntinib and mastinib, both of which block PDGF signaling; and DAPT, a gamma-secretase inhibitor which blocks canonical notch signalling. In our preliminary data, we found that PDGF signaling regulates ß-catenin activity. Cells in culture will be tested for how they alter DT cell behavior, by studying proliferation rate, apoptosis rate, and ß-catenin protein level. A new mouse model we developed will be used to test the interaction of signaling pathways identified in the compound screen.

Can PDGF and Notch pharmacologic blockade be used to treat DTs? Suntinib, Mastinib, and DAPT will be tested using a genetically modified mouse that develops DTs and using xenografted human DT tumors in immunodeficient mice. The mechanistic data generated in aim one will also be verified in-vivo in this aim.

The role of these agents in modulating different cell subpopulations in DTs will also be analyzed. In our previous work, we found that DTs contain a small cell population, which excludes Hoechst dye (called side population, or SP, cells). The SP cells have an enhanced ability to form tumors when implanted into immunodeficient mice, and as such act as tumor initiating cells (TICs), also called cancer stem cells. TICs may be resistant to anti-neoplastic drugs, and as such SP cells may be responsible for the resistance to treatments, and for tumor recurrence. We will also test theses compounds for differential effects on SP and non-SP cell populations.

This work will identify compounds that can rapidly be developed as a novel DT treatment. Such agents will ultimately be translated to clinical trials, resulting in an improved pharmacologic approach to DT management. In addition, the various agents likely target different aspects of controlling DT viability (e.g. proliferation -vs- apoptosis), and as such, this knowledge could be used to develop a rationale multi-drug approach to DT treatment.

Desmoid tumors (DTs) are a locally aggressive tumor type that can cause remarkable morbidity and even mortality in afflicted patients. While desmoid clinical phenotype has been well characterized, the underlying tumor molecular biology is poorly understood. In studies conducted within the premise of our previously funded DTRF seed grant we were able to establish a large desmoid tissue and cell strain repository and have confirmed that β-catenin mutations are highly prevalent in sporadic desmoid tumors. Furthermore, we showed that a specific mutation, 45F possibly significantly correlates with dismal outcome for patients with primary lesions. This initial observation merits further confirmation and in the recent grant period, through an international collaboration, we have been able to assemble a large cohort (>160) of clinically annotated primary DT samples; all were sequenced for β-catenin. Interim results are promising, confirming our initial observation.

We have also evaluated the effects of agents commonly used for the treatment of DT such as tamoxifen, celecoxib, and Gleevec on the growth of a large panel of desmoids primary cultures. Sensitive and resistant cell strains were identified; therapeutic response was independent of β-catenin mutation status or type or the expression level of the presumed drug targets. Finally, we have identified a potential DT gene expression signature enriched for genes regulated by TCF/LEF highlighting the role of β-catenin deregulation in these tumors. Midkine is one of the DT overexpressed targets; our studies further identified a correlation between midkine expression level and primary DT recurrence potential and a role for this protein in DT cell migration and invasion.

In the current, third year of the project, we aim to:

1. Finalize our studies evaluating the role of β-catenin mutations as DT molecular prognosticators.
2. Identify molecular signatures corresponding with sensitivity/resistance to commonly utilized anti-DT systemic agents.
3. Determine the role of the notch pathway in DT and its utility as a node of DT therapeutic vulnerability.
4. Unravel additional gene mutations operative in DT through whole exome sequencing of human samples (including matched specimens). We hope that these studies will result in meaningful observations that will enhance the management of patients burdened by this devastating disease.

Desmoid Tumors are locally invasive soft tissue tumors, which are found to be caused by mutations resulting in the stabilization of the protein, ß-catenin. Stabilized ß-catenin binds to TCF transcription factors to regulate the expression of genes in a cell type specific manner. Mouse models were developed based on this knowledge and used to study the role of target genes in the DT phenotype. The ultimate goal is to develop an improved approach to Desmoid Tumor therapy. Dr. Alman is continuing the work begun last year to screen compounds to identify pharmacologic agents that target Desmoid Tumor cell viability. He screened libraries composed of agents already in use for patient care to identify compounds that decreased cell viability in Desmoid Tumor cultures but not normal fibroblasts. In the past year, three compound libraries were screened, and three potential classes of compounds were identified that have the potential to be used as a therapeutic approach to Desmoid Tumors. Three of the compounds identified are drugs already approved for use by the FDA, and as such this work has the potential to be rapidly brought to patient care.

Dr. Lev proposes a three-pronged research design based on her findings stemming from the previously funded DTRF seed grant that will address these knowledge deficits. Her studies will focus on improving the understanding of the role of ß-catenin, a protein which is highly deregulated in desmoids, on tumor progression and recurrence. Studies will use cell strains and also siRNA screens to identify potential anti-desmoid therapeutic targets.

• Aim 1: To validate the prognostic impact of the specific beta-catenin 45F mutation in predicting the outcome of patients with primary desmoids.
• Aim 2: To identify the molecular deregulations contributing to sensitivity or resistance to the commonly used anti-desmoid therapies mainly tamoxifen, NSAIDs (sulindac), and Gleevec.
• Aim 3: To identify potential novel anti-desmoid therapeutic targets using a rational siRNA screen.

One way to identify new drug therapies is to screen known drugs on cell cultures from tumors to see which drugs will kill the tumor cells. In the past Alman et al had tested 1,000 drugs on desmoid tumor cells and identified one such agent. Unfortunately, this agent is not readily available for use in North America. In this 2010 DTRF-funded study Dr. Alman plans to under take a comprehensive screen of agents to identify drugs that specifically target cell viability in DTs. Building on previous work, Dr. Alman will focus on drugs that inhibit DT cell growth presumably by blocking Beta catenin signaling in mesenchymal cells. The agents identified will be tested for their ability to alter DT cell proliferation, Beta catenin protein level and differential affects on stem cells. Such drugs will be tested using DT cell cultures from patients and also transgenic mice that develop DTs. Identified agents can then be investigated in clinical trials. The exciting part is that the agents found to have clinical benefit will already have been FDA-approved for use in patients and can therefore be rapidly brought to patient care.

This is a pilot study examining the role of Rapamycin in the treatment of children and young adults with DT deemed likely to recur following resection. Rapamycin directly targets and inhibits mTOR, a growth promoting pathway that may be critical in the development and growth of DT. The mTOR cell proliferation/survival pathway was shown to be activated and essential for tumorigenesis when the APC gene is mutated (affecting the Beta catenin signaling pathway). It is an oral drug being evaluated as an anti-neoplastic agent in a variety of cancer types.

Skapek et al plan to determine how effectively rapamycin blocks mTOR activation in desmoid tumors and whether mTOR inhibition blocks desmoid tumor blood vessel growth. Skapek et al plan to determine how effectively rapamycin blocks mTOR activation in desmoid tumors . If Rapamycin is found to be active, they intend to extend this pilot study to a national collaborative study run through the Children’s Oncology Group (COG).

The goal is to investigate the molecular driving forces behind the development and progression of desmoid tumors. Lev et al are trying to identify potential targets that modify beta-catenin transcriptional activity using an siRNA screen. Identifying genes and their cognate proteins whose alteration would inhibit beta-catenin activity might provide future targets for novel molecular therapies relevant to DT patients.

• Aim 1: To validate previous findings of the prognostic power of beta-catenin 45F mutation in predicting the outcome of patients with primary desmoids.
• Aim 2: To identify the molecular deregulations contributing to the sensitivity or resistance to the commonly used anti-desmoid therapies, mainly Tamoxifen, NSAIDS and Gleevec.
• Aim 3: To identify potential novel anti-desmoid therapeutic targets using a rational siRNA screen. Potentially these targets harbor the capacity to modify the transcriptional effect of beta-catenin.

Note: Dr. Lev has previously identified significant associations between the presence of 45F mutations and an increased rate of of desmoid recurrence as well as shorter time intervals to recurrence. Analysis of a large primary desmoid subset showed that desmoids having a 45 F mutation had an estimated five year recurrence -free survival rate of only 47% and a median time to recurrence of 3.16 years. In sharp contrast, the estimated five year recurrence free survival rate for all other desmoid primary tumors was 83%; median time to recurrence not yet being reached.

DTRF-funded investigators at Stanford have developed a novel technique to analyze RNA from paraffin embedded tissue. This is important as many more paraffin embedded samples are available for study than frozen. Currently this technology is being used to study “usual” coding mRNA in desmoids in the Stanford lab. In this study they will be including the study of a new class of “non-coding RNA” and to examine the role of these molecules in the clinical behavior of desmoids. Conventional standard microarray expression profiling has been limited in that it only measures known genes that contain probes on the array platform. The new method being used allows for identification of novel transcripts. The new class of RNA (large intergenic non-coding RNA (lincRNA)) is thought to be a major regulator of gene expression. In the past year it has been shown that over one thousand evolutionarily conserved lincRNA’s are expressed in mammalian cells and that expression of these lincRNA’s correlates significantly with genes important in carcinogenesis. The long term goal is that the characterization of lincRNA’s in DT will lead to new diagnostic markers and treatment strategies for DT.

This is a grant for a three-year project studying Receptor tyrosine (RTK’s) which are proteins that are expressed on the surface of cells and are known to play an important role in cellular differentiation and tumor growth. ROR2 is a RTK which is expressed at high levels in a significant subset of desmoid tumors. It is a receptor for the Wnt family of proteins, proteins that are known to play a role in a pathway in the development of desmoid tumors. They will generate a novel monoclonal antibody against the extracellular part of ROR2 and study its effect on desmoid tumor cell growth in vitro.

This is a three year grant studying the role of hydroxyurea in the treatment of primary and recurrent Desmoid Tumors in adults and children.  Hydroxyurea is a generally well tolerated anti-neoplastic agent used in the treatment of myeloproliferative disease, CML and small group of carcinomas.  This study builds on the  favorable efficacy:toxicity experience in an earlier study in children in which preliminary results showed partial response or stable disease in 79% of patients. This is a non-randomized trial.

This is the third year of a three-year study. These investigators will be studying three molecular factors believed to be important in desmoid tumors: beta catenin, TCF-3 (a transcription factor) and ER-beta (an estrogen receptor). They are using a three-pronged approach. Aim 1 is the establishment of multiple desmoid tumor cell lines.  Aim 2 is the examination of the role of beta catenin, TCF-3 and ERbeta interactions in desmoid proliferation using the cell lines developed in Aim 1. Aim 3 is the identification of desmoid tumor related molecular markers using oligoarrays and tissue microarrays.

This is the third year of a three-year study. A small portion of stem cells in a given tissue give rise to all of the cells that make up that tissue. These cells help regenerate and maintain the tissue over its lifespan. These stem cells have been identified in a variety of tumor types and are called tumor initiating cells (TIC). These investigators will be testing the hypothesis that desmoid tumors contain a subpopulation of TIC and that desmoid tumors are derived from mesenchymal stem cells (MSC) that have a mutation leading to elevated levels of beta catenin, a protein thought to be instrumental in the development of familial adenomatous polyposis along with a mutation in the APC gene. In familial cases, desmoid tumors are associated with an APC mutation. Preliminary data suggest that desmoid tumors may be derived from MSC’s in which beta catenin signaling is misregulated. Dr. Alman and his team currently have 22 desmoid tumor cell lines with which to work. They will be (1) attempting to identify cell lines of TICs in desmoid tumors using established surface markers, and (2) using mice to determine if the tumors arise from misregulated MSC’s.  Their goal is to ultimately develop a novel strategy of targeting desmoid tumor initiating cells.

This is a new grant for a three-year project studying Receptor tyrosine (RTK’s) which are proteins that are expressed on the surface of cells and are known to play an important role in cellular differentiation and tumor growth. ROR2 is a RTK which is expressed at high levels in a significant subset of desmoid tumors. It is a receptor for the Wnt family of proteins, proteins that are known to play a role in a pathway in the development of desmoid tumors. They will generate a novel monoclonal antibody against the extracellular part of ROR2 and study its effect on desmoid tumor cell growth in vitro.

Conclusions:

1. MMP1 (matrix metallopeptidase) expression predominant in highly invasive FAP-related DT while MMP3 more representative of less invasive sporadic DT. (two manuscripts in progress)
2.FAP desmoids are more likely resistant to chemo than sporadic DT.

Dr. David Joyner will continue to contribute to the DTRF collaborative effort that was initiated during the May 2008 Symposium.

This is the second year of a three-year study. These investigators will be studying three molecular factors believed to be important in desmoid tumors: beta catenin, TCF-3 (a transcription factor) and ER-beta (an estrogen receptor). They are using a three-pronged approach. Aim 1 is the establishment of multiple desmoid tumor cell lines.  Aim 2 is the examination of the role of beta catenin, TCF-3 and ERbeta interactions in desmoid proliferation using the cell lines developed in Aim 1. Aim 3 is the identification of desmoid tumor related molecular markers using oligoarrays and tissue microarrays.

This is the second year of a three-year study. A small portion of stem cells in a given tissue give rise to all of the cells that make up that tissue. These cells help regenerate and maintain the tissue over its lifespan. These stem cells have been identified in a variety of tumor types and are called tumor initiating cells (TIC). These investigators will be testing the hypothesis that desmoid tumors contain a subpopulation of TIC and that desmoid tumors are derived from mesenchymal stem cells (MSC) that have a mutation leading to elevated levels of beta catenin, a protein thought to be instrumental in the development of familial adenomatous polyposis along with a mutation in the APC gene. In familial cases, desmoid tumors are associated with an APC mutation. Preliminary data suggest that desmoid tumors may be derived from MSC’s in which beta catenin signaling is misregulated. Dr. Alman and his team currently have 22 desmoid tumor cell lines with which to work. They will be (1) attempting to identify cell lines of TICs in desmoid tumors using established surface markers, and (2) using mice to determine if the tumors arise from misregulated MSC’s.  Their goal is to ultimately develop a novel strategy of targeting desmoid tumor initiating cells.

Desmoid tumors can grow rapidly and be highly invasive or they can remain static for many years. These investigators are studying whether the proteins EGF (epidermal growth factor) and TGF-alpha (transforming growth factor) influence the invasive nature of desmoids and if either factor determines desmoid drug sensitivity. They hypothesize that these factors exert an influence on relevant genes.  They have a three-pronged approach (1) measuring the mRNA and protein levels of relevant genes using polymerase chain reaction and tissue staining, (2) testing cell cultures for the influence of EGF and TGF-alpha on cell invasiveness and doxorubicin toxicity and (3) performing microarrays on lab-altered desmoid cell cultures in order to identify genes responsive to growth factor modulation.  They anticipate that this may provide a list of potential therapeutic targets for use against desmoid tumors.

A small portion of stem cells in a given tissue give rise to all of the cells that make up that tissue. These cells help regenerate and maintain the tissue over its lifespan. These stem cells have been identified in a variety of tumor types and are called tumor initiating cells (TIC). These investigators will be testing the hypothesis that desmoid tumors contain a subpopulation of TIC and that desmoid tumors are derived from mesenchymal stem cells (MSC) that have a mutation leading to elevated levels of beta catenin, a protein thought to be instrumental in the development of familial adenomatous polyposis along with a mutation in the APC gene. In familial cases, desmoid tumors are associated with an APC mutation. Preliminary data suggest that desmoid tumors may be derived from MSC’s in which beta catenin signaling is misregulated. Dr. Alman and his team currently have 22 desmoid tumor cell lines with which to work. They will be (1) attempting to identify cell lines of TICs in desmoid tumors using established surface markers, and (2) using mice to determine if the tumors arise from misregulated MSC’s.  Their goal is to ultimately develop a novel strategy of targeting desmoid tumor initiating cells.

These investigators will be studying three molecular factors believed to be important in desmoid tumors: beta catenin, TCF-3 (a transcription factor) and ER-beta (an estrogen receptor). They are using a three-pronged approach. Aim 1 is the establishment of multiple desmoid tumor cell lines.  Aim 2 is the examination of the role of beta catenin, TCF-3 and ERbeta interactions in desmoid proliferation using the cell lines developed in Aim 1. Aim 3 is the identification of desmoid tumor related molecular markers using oligoarrays and tissue microarrays.