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Lay Abstract

Desmoid-type fibromatosis (DTF) tumors are soft tissue tumors arising from the fibroblast populations of connective tissue resulting in locally aggressive fibromatosis. While noninvasive, chances of relapse are high. Currently FDA approved strategies for treating DTF are scarce. The Wnt-β-catenin pathway is the major driver of DTF. However, targeting this pathway could severely damage normal cellular functions. Our group has pioneered studies on the AP-1 transcription factor aka JUN driving DTF by interacting with β-catenin. Our recent studies demonstrate that JUN transcriptionally modulates the expression of a novel pro-fibrogenic molecule called CD63. CD63 is a cell surface protein that belongs to the tetraspanin family. Molecular investigations in DTF show that JUN increases CD63 expression thereby promoting cell growth and survival of primary patient derived DTF cells. Furthermore, genetic knock down of CD63 in DTF cells significantly inhibits growth both in-vitro and in animals. Using next generation sequencing and cell-based assays we show that CD63 is high in the fibroblasts of DTF patients correlating with JUN and β-catenin expression. Our studies also reveal that JUN in concert with β-catenin transcriptionally modulates CD63. Finally, we aim to use novel 3-D cell-based assays and mouse models to show that JUN, CD63 and β-catenin all work in concert to promote the fibrotic phenotype in long term assays. This proposal is aimed at the hypothesis that the JUN- β- catenin-CD63 link is essential for the onset and progression of desmoid lesions in patients. Altogether, this study focuses on key mechanisms underlying JUN regulation of CD63 in DTF and explores the promising potential of targeting CD63 as an immunotherapeutic target to treat DTF.

Scientific Abstract

The proposed research aims to elucidate the molecular mechanisms underlying desmoid tumor formation (DTF) and how this impacts the reversion of fibrosis observed in DTF patients. DTF tumors arise from fibrotic connective tissue which drive the onset of aggressive fibromatosis.

Current therapeutic modalities for DTF have solely focused on tumor monitoring, surgery, chemotherapy, and a few targeted therapeutics like sorafenib, pazopanib and nirogacestat but research on their mechanisms are still underway. However, targeting key signaling pathways like the WNT signaling pathway could have significant off-target and on-target effects that could be detrimental. This proposal investigates how the AP-1 transcription factor (aka JUN) works in concert with another key transcription factor β- catenin to drive the expression of a novel profibrogenic molecule called CD63 that is significantly elevated in desmoid tumor patients. Others and we have previously shown that JUN can promote the DTF phenotype via tumor survival and proliferation. Also, we have identified a novel molecule called CD63 to be significantly elevated in the cells of primary desmoid patients. Moreover, CD63 can be efficiently targeted using monoclonal antibody therapy with minimal to no side effects. As such, we will evaluate how JUN along with β- catenin mechanistically drives CD63 overexpression in DTF patients, how CD63 in turn promotes aggressive fibromatosis and what would be the most effective strategy to target CD63 for fibrosis reversal after onset of DTF. We expect that this work will provide knowledge pertinent towards understanding the underlying pathological mechanisms that promote fibrosis and contribute to aggressive DTF tumors ultimately providing a new avenue to develop a promising therapeutic to block tumor progression.

Lay Update

Dr. De Souza and her team at Stanford, with the support and partnership of the Desmoid Tumor Research Foundation (DTRF), identified previously unrecognized interactions between immune cells and fibroblasts in tumors from patients with desmoid disease. By analyzing patient-derived samples, the team uncovered immune–stromal signaling pathways that appear to drive fibroblast activation and tumor persistence. These findings provide important insight into how the immune microenvironment contributes to desmoid tumor behavior and highlight new opportunities for mechanism-based therapeutic strategies aimed at improving outcomes for patients with desmoid tumors.

Lay Abstract

Sorafenib is one of the most commonly used drugs in people with desmoid. Other than desmoid, it is used in metastatic (widespread) cancers like kidney cancer and thyroid cancers. The duration of treatment in these cancers is limited and also given the short life expectancy in these cancers, long term side effects of sorafenib on different body functions is not well known.

Other than this the current duration of treatment in desmoid is not known and is continued indefinitely. In smaller studies, sorafenib has been shown to affect thinking, heart functioning, fertility even with limited duration. Through this study we want to see how sorafenib affects these organs systematically. We will assess cardiac functions, vascular functions, cognitive functions and hormonal function and see the trend during the first year. It will be a stepping stone for any intervention that can reduce the chances of long term complications.

We will try to continue this study for 3 years.

Scientific Abstract

So far, sorafenib has been used in various advanced cancers with limited life expectancy and long term side effects involving cognition, cardiovascular and reproductive functions have rarely been looked at. Currently, the duration of sorafenib therapy in desmoid is not evidence based and it is continued indefinitely. In a study by Mulder et al, comprised of patients with GIST and RCC, the effects of VEGF inhibitors was larger on learning, memory and executive functions as compared to patients not on these drugs. (1). So far, no studies have been published on the effects of the vascular endothelial growth factor receptor (VEGFR) inhibitors on cognitive functioning in desmoid. Once the cognition function is delineated then further research can be done on using neuroprotective strategies.(2)

In a meta analysis by Dai et al, VEGFR inhibitors are also significantly associated with hyperlipidemia. Similarly, it has been recently shown that sorafenib acutely affects cardiac contractility by reducing S16 PLN phosphorylation, leading to reduced SR calcium content, CaT amplitude, and slowed cytosolic calcium removal (3). Coronary artery disease has been also been described in patients on sorafenib especially associated with patients with preexisting cardiovascular comorbidity.(4)

Though menstrual abnormalities are not reported commonly with TKIs but in our unpublished data (ongoing study), it is one of the most common complaints on long term use. In a report by Sanctis et al, an 18 year old girl developed reversible ovarian insufficiency on pazopanib (5). . There is only one study of 17 women with desmoid tumor addressing fertility preservation using IVF, of which only one was on Sorafenib therapy. The study reported very poor yield of oocytes in her first IVF cycle (2.7 (±1.3) which improved significantly after stopping Sorafenib 13.2 (±3.3) (P = 0.02). (6)

We have devised comprehensive screening strategies at various time points as shown in the table below with the budget justification. We want this to be a recurring project for the next 3 years. It will be a prospective study which will be enrolling patients with fibromatosis more than 18 years old and have been started on sorafenib within 2-3 months. A total of 50 patients will be recruited in around 1 year duration.

Methodology

It will be a prospective study which will be enrolling patients with fibromatosis more than 18 years old and are preferably newly diagnosed (drug naïve) or have been started on sorafenib within 2-3 months. A total of 50 patients will be recruited in around 1 year duration.

Lay Abstract

Desmoid tumors consist of both tumor and non-tumor cells growing together. It remains unknown how the proportions and types of cells, or the interactions between them, affect the clinical behavior of a tumor. Advances in technologies called single-nuclei or single-cell RNA sequencing (sn/sc-RNAseq) now enable us to quantify the level of expression of genes in individual cells within the same tissue to compare their biological activity. These technologies have enabled significant advancement in our understanding of the behavior of other cancers. However, in desmoid tumors, we encounter a major challenge: the desmoid cells appear very similar to most of the non-tumor cells in the same area. Still, the most distinguishing feature of desmoid cells is the fact they carry a mutation in either the CTNNB1 gene or the APC gene. In this project, we propose to build a team that will work together to improve upon sn/sc-RNAseq technology by also sequencing the CTNNB1 or APC genes at the single-cell level. We will utilize this improved technology on tissues stored at the Dana-Farber Cancer Institute BioBank for desmoid tumors. We propose to combine this molecular information with clinical histories to understand the differences in gene activities between desmoid and non-desmoid cells in the same tissue and across different patients with different clinical behaviors. If successful, our study’s results will help answer whether gene activity levels can relate to spontaneous regression, and if so, which aspects of unique desmoid biology would be promising to pursue in future validation experiments.

Scientific Abstract

Desmoid tumors are fibroblastic neoplasms with differential clinical behavior often present as CTNNB1 or APC mutant cells intermixed with non-mutant cells. It remains unknown whether the cellular composition, or tumor-stromal interaction, can be used to infer tumor progression, therapeutic response, or spontaneous regression. While single-cell atlases have enabled valuable insights into the biology of many epithelial cancers, mesenchymal tumors, desmoid tumors in particular, are challenging to study because current references cannot distinguish between mutant and non-mutant fibroblasts. Since most patients present with CTNNB1 or APC mutations, we hypothesize that spontaneous regression can be detected by the transcriptional composition of desmoid tumors. We aim to test this hypothesis by concurrent genotyping of single cells to enable in sc/sn-RNAseq experiments. Here we propose to form a team of scientists and clinicians to study the clinical history of our biobank of 75 patients who donated tissues to identify cases of spontaneous regression, stable disease, and progression. Based on clinical history and onco-panel sequencing we will then nominate samples for parallel single-cell/nuclei genotyping and transcriptomics. We will then analyze the results to identify differentially expressed genes and pathways related to mutation status and clinical history. Preliminary data from 3 samples (2 tumors, and 1 adjacent normal) demonstrate the feasibility of our sequencing approach. If successful, expanding to a full project will create the first genotyped, clinically annotated single-cell reference of desmoid tumors.