Molecular Analyses of Endometrial Cancer

National Human Genome Research Institute

Investigators

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Abstract

Uterine corpus cancer is the 5th leading cause of cancer death amongst American women. The American Cancer Society predicts that in 2025 there will be ~ 69,120 newly diagnosed cases of uterine corpus cancer and ~13,860 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 (reviewed in: Urick & Bell, Nature Reviews Cancer, 2019; and Bell & Ellenson, Annual Review of Pathology: Mechanisms of Disease, 2019). In our early studies, we used genomic tools to discover somatic mutations in the most clinically aggressive subtypes of EC. In recent years, we strategically pivoted and re-tooled to perform functional studies utilizing biochemical approaches, quantitative proteomics and cellular approaches to determine: 1) how EC mutations in FBXW7, ALK2, and ALK5 impact the biochemical functions of the encoded proteins, and 2) whether functionally altered mutant proteins encoded by these genes can be leveraged as druggable targets in EC cells. 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 EC (Le Gallo et al., Nature Genetics 2012; Le Gallo et al., Cancer 2017; Le Gallo et al., Cancer 2018). We hypothesized that the recurrent mutations disrupt the ability of FBXW7 to bind to protein substrates, thus preventing substrate degradation. To enable functional studies of FBXW7 mutation in EC, we developed a series of FBXW7 mutation-knockin and FBXW7 mutation-corrected EC cell lines. In subsequent functional studies 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 further demonstrated that compared to FBXW7-wildtype EC cells, isogenic FBXW7-mutation knockin serous EC cells exhibit increased sensitivity to SI-2, a SRC3 inhibitor-2, and to dinaciclib, a cyclin dependent kinase inhibitor (Urick and Bell, Molecular Carcinogenesis 2018). Many substrate proteins that are regulated by FBXW7 are transcription factors or signal transduction proteins. We therefore hypothesized that FBXW7 mutations will alter the levels of a multitude of proteins, phosphoproteins, and ubiquitinated-proteins in EC cells. To identify those altered protein levels, our isogenic cell lines were subjected to quantitative global proteomic and phosphoproteomic profiling by LC-MS/MS. By rigorously analyzing those datasets we detected hundreds of proteins and phosphoproteins at significantly different (P<0.05) levels in FBXW7-mutant cells compared to isogenic FBXW7-wildtype cells. Our findings included the identification of increased levels of PADI2, a potentially druggable protein, in FBXW7-mutant EC cells (Urick and Bell, Cancer Medicine 2020). We also reported 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, in high-grade endometrioid EC cells (Urick et al, Cancer 2021). In the past year, we reported the global proteomic and phosphoproteomic alterations induced by endogenous FBXW7 mutations in MSI+ endometrioid EC cells (Urick et al, Cancer Medicine 2025). Major findings of this study include our discovery that endogenous FBXW7 mutations in EC cells result in increased levels of TROP2 and galectin-3. These results are novel and significant because an antibody-drug conjugate (sacituzumab govitecan) that targets TROP2 is showing promising results in clinical trials for the treatment of EC, and galectin-3 is druggable in preclinical studies of EC. In the past year, we have also advanced our ongoing molecular studies to determine the mechanism by which FBXW7 mutations result in increased PADI2 levels in EC cells, and preclinical studies to determine whether FBXW7-mutant EC cells exhibit increased sensitivity to PADI inhibitors. Additionally, we significantly advanced our biochemical studies designed to determine the effects of FBXW7 mutations on the ubiquitinated proteome in EC cells. 2. Determine the functional consequences and potential druggability of ALK2 mutations in EC ALK2 (ACVR1) is a BMP-responsive serine-threonine kinase that signals through the SMAD1/5/9-dependent and SMAD-independent pathways. ALK2 is potentially druggable, and small molecule inhibitors of this kinase have been developed by others. 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 recent reporting periods we initiated biochemical studies to determine the effects of ALK2 mutations on the canonical SMAD1/5/9 pathway and on SMAD1/5/9-independent pathways in EC cells. In the current reporting period, we advanced those studies and made novel discoveries that we are following up on with additional approaches. 3. Determine the functional consequences and potential druggability of ALK5 and SMAD2 mutations in EC ALK5 (TGFBR1) is a TGFbeta-responsive serine-threonine kinase that signals through the canonical SMAD2/3 pathway or through SMAD-independent pathways. TGFbeta 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 TGFbeta-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. During the past year, we reported our findings that the ALK5-A230V mutation, within the ATP-binding site, is a loss-of-function mutation (Yu and Bell, PLoS One 2024). The mutant protein has reduced stability resulting from increased ubiquitin-mediated protein degradation, reduced kinase activity in an SBE reporter assay, and reduced biochemical sensitivity to SB-431542, an ATP-competitive small molecule inhibitor of ALK5. We also significantly advanced our research on an additional ALK5 mutant in EC (manuscript in preparation), and we performed preliminary investigations into the functional effects of SMAD2 mutations in EC.