Genomic Analyses of Endometrial Cancer

National Human Genome Research Institute

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Abstract

Uterine corpus cancer is the 6th leading cause of cancer death amongst American women. The American Cancer Society predicts that in 2022 there will be 65,950 newly diagnosed cases of uterine corpus cancer and 12,550 deaths from this disease in the United States. The vast 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 disease subgroups including high-grade endometrioid ECs, late-stage endometrioid ECs, and aggressive histological subtypes such as serous EC and uterine carcinosarcoma have generally unfavorable outcomes (recently reviewed in: Urick & Bell, Nature Reviews Cancer, 2019; and Bell & Ellenson, Annual Review of Pathology: Mechanisms of Disease, 2019). During the last decade, this laboratory has successfully used genomic tools to identify somatic mutations in the most clinically aggressive subtypes of EC, to home in on potential pathogenic driver mutations that contribute to tumor development. In the current reporting period, we published a manuscript reporting our identification of KLF3 and PAX6 as statistically significantly mutated genes in late-stage but not early-stage endometrioid EC (Rudd et al., PLoS One 2022). These findings are hypothesis-generating, raising the possibility that KLF3 and PAX6 mutations may contribute to endometrial tumor progression. The focus of the laboratory has now shifted 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 therapeutic targets in EC cells. This report describes ongoing projects in the Bell laboratory, which aim to determine the functional consequences and potential druggability of somatic mutations in the FBXW7, ALK2, ALK5 and SMAD2 genes and related pathways 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 critical component of a ubiquitin ligase complex that degrades numerous substrate proteins involved in tumorigenesis. We previously discovered frequent and recurrent somatic mutations of FBXW7 in ECs (Le Gallo et al., Nature Genetics 2012), particularly among serous ECs. We thus hypothesized that recurrent FBXW7 mutations disrupt the proper function of the encoded protein in EC. In 2018, we reported novel functional effects of 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 cell lines. By performing drug sensitivity assays in a serous EC cell-line that was CRISPR/Cas9-edited to knock in a series of FBXW7 mutations, we found 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). Collectively these studies shed novel insights into the functional consequences of FBXW7 mutations in serous EC. Many proteins known to be regulated by FBXW7 are transcription factors that in turn regulate the expression of other genes. We thus hypothesize that FBXW7 mutations alter the levels of a multitude of proteins within the cell. Therefore, we sought to determine the repertoire of protein alterations that result from mutation of FBXW7 in EC cells. In previous reporting periods, we systematically determined the global proteomic and phosphoproteomic changes in serous EC cells CRISPR/Cas9 edited to knock in FBXW7 mutations, as compared to the parental FBXW7-nonmutated cells. This approach yielded new insights into proteomic changes resulting from FBXW7 mutations in serous EC cells including increased levels of the PADI2 (peptidyl arginine deiminase 2) protein, which is potentially druggable (Urick and Bell, Cancer Medicine, 2020). We also demonstrated an association between FBXW7 mutation and increased PADI2 levels in endometrioid EC cells, which raises the possibility that FBXW7 mutations may have similar effects in divergent histotypes of EC. In addition, we delineated proteomic changes resulting from FBXW7 mutations in a high-grade endometrioid EC cell line and provided the first direct evidence that FBXW7 mutation affects levels of L1CAM (L1 cell adhesion molecule), a known clinically relevant biomarker in EC, and of TGM2 (transglutaminase 2), a potentially druggable protein (Urick et al, Cancer 2021, 127(16):2905-2915. doi: 10.1002/cncr.33567). In the current reporting period, we continued our proteomic and phosphoproteomic analyses of isogenic FBXW7-wildtype and FBXW7-mutated intermediate-grade endometrioid EC cell lines. We identified several thousand dysregulated proteins and phosphoproteins in association with mutated FBXW7. We have prioritized a subset of these proteins for orthogonal validation. This study will extend into the incoming 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 initiated during the current reporting period, 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.