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The Desmoid Tumor Research Foundation

“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.” Year 2 of 2

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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, 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 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 a 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 cooperate 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 a novel preclinical platform to evaluate additional inhibitors to ultimately find better treatments.

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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 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.

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Lay Abstract

Desmoid-type fibromatosis (DF) is a rare benign tumor characterized by  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.

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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.

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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 secretase 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.

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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.