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.”

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 lower responder 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 populations shown 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-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 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.”