Molecular Analyses of Endometrial Cancer

National Human Genome Research Institute

Investigators

Linked publications & trials

Abstract

Goals and Objectives The overall goal of this project is to identify, and understand the effects of, molecular abnormalities that contribute to the development of endometrial carcinomas (EC). We are particularly interested in investigating the occurrence and effects of mutations that are present in an individuals endometrial tumor genome but are absent from the genomes of healthy cells from the same individual. Such tumor-specific mutations are referred to as somatic mutations. Our research focuses on clinically aggressive subtypes of endometrial cancer for which new therapeutic options are needed. We and others have systematically identified a myriad of somatic mutations in these cancers. Our current goal is to determine the functional effects of somatic mutations in genes that produce potentially druggable proteins. Our main objectives are to: 1. Determine the functional consequences and potential druggability of FBXW7 mutations in EC 2. Determine the functional consequences and potential druggability of ALK2 mutations in EC 3. Determine the functional consequences and potential druggability of ALK5 and SMAD2 mutations in EC Summary Uterine corpus cancer is the 6th leading cause of cancer death amongst American women. The American Cancer Society predicts that in 2023 there will be 66,200 newly diagnosed cases of uterine corpus cancer and 13,030 deaths from this disease in the United States. The majority (98%) of these cancers are endometrial carcinomas (ECs), which arise from the inner epithelial lining (the endometrium) of the uterus. Although the prognosis for many women with newly diagnosed EC is good, certain tumors including high-grade endometrioid ECs, late-stage endometrioid ECs, and aggressive histological subtypes such as serous EC and uterine carcinosarcoma generally have unfavorable outcomes (recently reviewed in: Urick & Bell, Nature Reviews Cancer, 2019; and Bell & Ellenson, Annual Review of Pathology: Mechanisms of Disease, 2019). In the past decade we used genomic tools to identify somatic mutations in the most clinically aggressive subtypes of E. In recent years we built on those successes to determine: 1) how mutations in candidate driver genes impact the function of the encoded protein, and 2) whether functionally altered mutant proteins can be leveraged as druggable targets in EC cells. This report describes ongoing projects to determine the functional consequences and potential druggability of somatic mutations in FBXW7, ALK2, and ALK5 in EC. 1. Determine the functional consequences and potential druggability of FBXW7 mutations in EC The protein encoded by FBXW7 is a tumor suppressor and a component of a ubiquitin ligase complex that degrades numerous other proteins, many of which are oncogenic. We previously discovered frequent and recurrent somatic mutations of FBXW7 in ECs (Le Gallo et al., Nature Genetics 2012), particularly serous ECs. We hypothesized that the recurrent mutations disrupt the normal function of the FBXW7 protein. In 2018, we reported novel functional effects of the recurrent FBXW7 mutations in serous EC (Urick and Bell, Molecular Carcinogenesis, 2018). Specifically, we showed that recurrent mutations in FBXW7 cause increased levels of phosphorylated Cyclin E1, SRC-3, c-MYC, Rictor, GSK3, P70S6, and AKT proteins in serous EC cells. We also showed that FBXW7-mutant serous EC cells are more sensitive to two drugs, SI-2 (a SRC3 inhibitor-2) and dinaciclib (a cyclin dependent kinase CDK inhibitor), than parental unedited cells (Urick and Bell, Molecular Carcinogenesis, 2018). Many proteins regulated by FBXW7 are transcription factors that regulate the expression of other genes. We hypothesize that FBXW7 mutations alter the levels of a multitude of proteins. Therefore, we sought to determine the repertoire of protein alterations that result from mutation of FBXW7 in EC cells. In previous years, we determined the global proteomic and phosphoproteomic changes caused by FBXW7 mutations in serous EC cells, including increased levels of PADI2, a potentially druggable protein (Urick and Bell, Cancer Medicine, 2020). We also found that FBXW7 mutation increases PADI2 levels in endometrioid EC cells. In addition, we provided the first direct evidence that FBXW7 mutation affects levels of L1CAM, a known clinically relevant biomarker in EC, and of TGM2, a potentially druggable protein (Urick et al, Cancer 2021) in high-grade endometrioid EC cells. In the previous reporting period, we identified several thousand dysregulated proteins and phosphoproteins in association with mutated FBXW7 in intermediate-grade endometrioid EC cells. In the current reporting period: 1) We prioritized a subset of those proteins for orthogonal validation by immunoblotting, and we are preparing a manuscript to report our findings. 2) We initiated molecular studies to determine the mechanism by which FBXW7 mutations result in increased PADI2 levels in EC cells. 3) We initiated analyses of a new ubiquitinated-proteomics dataset generated by our laboratory, with the goal of identifying ubiquitinated-modified proteins that are dysregulated in FBXW7-mutated EC cells. These three avenues of research are ongoing and will extend into the next reporting period. 2. Determine the functional consequences and potential druggability of mutations in ALK2 in EC ALK2 (ACVR1) is a BMP-responsive serine-threonine kinase that signals through the canonical SMAD1/5/9 pathway and SMAD-independent pathways. As a kinase, ALK2 is druggable. ALK2 somatic mutations occur in 5% of non-ultramutated endometrial tumors and are preferentially located in the TGF/GS and kinase domains of the protein. The ALK2-R206H somatic hotspot mutation is recurrent in EC and is identical to a germline pathogenic variant that causes Fibrodysplasia Ossificans Progressiva (FOP) due to a gain-of-function effect. Several non-recurrent ALK2 mutations in EC are identical to germline pathogenic variants that cause variant-FOP. We thus hypothesize that in EC somatic mutations in ALK2 encode gain-of-function proteins that disrupt SMAD-dependent and/or SMAD-independent signaling and are potentially druggable. In previous reporting periods we examined the effects of ALK2 mutations on the canonical SMAD1/5/9 pathway. In the current reporting period, we extended our studies to determine the effect of ALK2 mutations on SMAD1/5/9-independent signal transduction. This work is ongoing and will extend into the incoming reporting period. 3. Determine the functional consequences and potential druggability of ALK5 and SMAD2 mutations in EC ALK5 (TGFBR1) is a TGF-responsive serine-threonine kinase that signals through the canonical SMAD2/3 pathway or through SMAD-independent pathways. TGF has both tumor suppressive and tumor promoting activities that are context-dependent. Somatic missense mutations in ALK5 occur in 3% of human ECs, but their functional effects are currently unknown. Based on in silico predictions and structural modeling results, we hypothesize that a subset of ALK5 mutations found in human endometrial tumors perturb signal transduction via the TGF-ALK5-SMAD2/3 pathway. To test this hypothesis, we are assessing the biochemical and cellular consequences of a subset of ALK5 mutations that occur in EC. In parallel studies we are evaluating the functional effects of a subset of SMAD2 mutations in EC. SMAD2, which encodes a downstream effector of ALK5, is mutated in 2% of ECs. Our work on mutated ALK5 and SMAD2 represents a long-term project that is ongoing and will extend into the next reporting period.