“Investigating EZH2 as a druggable mediator of immune cell exclusion in desmoid tumors.” Year 2 of 2

Summary

In previous research funded by the 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 immunosuppressive 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 are 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.

Summary

In previous research funded by the 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

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 tumor 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 trunk. As DTF does not progress after diagnosis in most cases, no specific initial treatment is recommended. However, a minority of patients develop progressive DTF with debilitating and life-threatening 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 differentially 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 on 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.

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.

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.