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 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.”

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 proof of principle 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 blueprint 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 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.”

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:

• expand our desmoid tumor tissue repository as well as to continue trying to establish a desmoid tumor murine model
• unravel the molecular mechanism underlying the differences in the induction of apoptosis between the CTNNB1 T41A and S45F mutation
• further investigate the molecular mechanism of action of sorafenib, and additional possible therapeutic combinations;
• 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.”

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.”