Citations

Abstract Background Cholangiocarcinoma (CCA) is an aggressive primary liver cancer with remarkable molecular heterogeneity. Mutations in SWI/SNF chromatin remodeling components occur in > 20% of CCA cases, with ARID1A and PBRM1 being significantly co-mutated. However, how the dual loss of these subunits reshapes chromatin architecture and drives oncogenic programs remains unclear that we aimed to address by profiling SWI/SNF redistribution, chromatin accessibility, 3D chromatin structure, and transcriptome in a dual Arid1a/Pbrm1 knockout mouse model. Methods CCA was induced in Cas9 knock-in mice via biliary instillation of myr-AKT along with sgRNAs targeting Arid1a and Pbrm1, generating APAC cell line. AC cell line was derived from mice receiving myr-AKT alone. Isogenic dual knockout (AC-sgAP) and control (AC-NT) lines were then generated from AC cells. Multi-omics profiling included ChIP-seq for SMARCA4/BRG1 and DPF2, ATAC-seq, RNA-seq, and Hi-C. Results ChIP-seq revealed that AC-sgAP cells showed decreased BRG1 binding (9,217 vs. 20,769 peaks) but increased DPF2 binding (7,527 vs. 2,068 peaks) and co-occupancy (5,037 vs. 1,888 peaks) compared to AC-NT. In contrast, APAC cells exhibited depletion of both BRG1 (12,994 peaks) and particularly DPF2 (146 peaks) with minimal overlap (68 shared peaks). Genome-wide accessibility profiling demonstrated that APAC cells had 78,582 differentially accessible regions compared to AC-NT, the majority (>70%) representing loss of accessibility, whereas AC-sgAP cells showed only 6,587 differentially accessible regions. Transcriptional profiling identified both unique and common significantly differentially expressed genes in APAC and AC-sgAP relative to AC-NT controls. Interestingly, topologically associating domains (TADs) calling revealed that AC-sgAP cells displayed approximately three-fold more differential TADs compared to AC-NT than APAC cells relative to controls. Conclusions Concurrent Arid1a/Pbrm1 loss drives extensive chromatin and transcriptional reprogramming through SWI/SNF redistribution. APAC cells achieving greater transcriptional dysregulation with fewer TAD disruptions might suggest that intra-TAD regulatory changes are critical events in SWI/SNF-mutant cholangiocarcinoma.

Abstract Background SWI/SNF chromatin remodeling complexes are frequently mutated across human cancers, including biliary tract cancers (BTC). Within the SWI/SNF complexes, loss-of-function (LOF) mutations in ARID1A and PBRM1 are the most common and frequently co-occur alongside PI3K/AKT pathway activation. Yet, how those mutations affect the intracellular signaling landscape and transcriptomic programs is underexplored and may yield novel therapeutic opportunities. Methods Clinicodemographic parameters and mutation profiles of 250 patients with BTC were retrospectively collected through electronic medical records. Advanced overall survival (OS) was assessed in a multivariable analysis. Transcriptomic data were obtained and differentially expressed genes (DEGs) and pathways associated with SWI/SNF mutation status were analyzed. Changes in the transcriptomic landscape were compared to a murine syngeneic model of intrahepatic cholangiocarcinoma (CCA) initiated by loss of ARID1A and/or PBRM1 for cross-species validation. Results After adjusting for age, sex, and anatomical subtype, patients with SWI/SNF LOF mutations were found to have a 30% reduction in mortality risk (HR = 0.70, 95% CI: 0.50-0.98, p = 0.037). The transcriptomic cohort of 233 BTC patients included 71 (30%) with SWI/SNF LOF mutations, 21 (9%) with variants of uncertain significance, and 141 (61%) with SWI/SNF wild-type tumors. Differential gene expression and pathway analysis revealed changes in adaptive immune response, extracellular matrix, and cancer-associated proteoglycans. RNA-sequencing of murine tumors revealed the dominant effect of ARID1A loss in single and double knock-out tumors and conserved pathway alterations between mouse and human tumors. Conclusion Both murine and human primary tumors with either ARID1A alone or ARID1A/PBRM1 co-mutations exhibited altered expression of critical genes and pathways associated with proteoglycan synthesis and extracellular matrix. Ongoing research is investigating how CCA cells depend on those pathways and their canonical regulators for tumor formation and progression.

Abstract SETD2 is frequently mutated or deleted in clear cell renal cell carcinoma (ccRCC). Loss of SETD2 could create synthetic lethal dependencies that confer therapeutic vulnerabilities. In this study, we demonstrated that SETD2 deficiency promotes cytoplasmic mitochondrial DNA (mtDNA) leakage, leading to basal activation of cGAS-STING inflammatory signaling and increased apoptotic priming. This inflammatory state upregulated the BH3-only protein NOXA, constrained myeloid cell leukemia 1 (MCL-1) function, and enforced a synthetic lethal dependency on the antiapoptotic protein BCL-xL. Pharmacologic inhibition of BCL-xL further amplified cGAS-STING signaling in SETD2-deficient cells through sublethal mitochondrial outer membrane permeabilization, resulting in increased mtDNA release and robust NOXA induction. Elevated NOXA neutralized the compensatory MCL-1–mediated survival signaling, triggering apoptosis. In contrast, SETD2-proficient ccRCC cells exhibited minimal cGAS-STING activation and failed to induce NOXA following BCL-xL inhibition, rendering them resistant. Genetic ablation of cGAS, STING, IRF3, or NOXA rescued sensitivity to BCL-xL inhibition, confirming that mtDNA-driven innate immune signaling is required for this dependency. In vivo, BCL-xL inhibition suppressed tumor growth and prolonged survival in SETD2-deficient xenograft models. Collectively, these findings establish a mechanistic link between SETD2 loss, mtDNA-driven innate immune activation, and enforced BCL-xL dependence in ccRCC, revealing a therapeutically targetable vulnerability in SETD2-deficient tumors. Significance: SETD2 deficiency, which is associated with worse prognosis in kidney cancer, promotes mitochondrial DNA-driven cGAS-STING signaling and NOXA-mediated apoptotic priming, creating a therapeutically targetable dependency on BCL-xL in kidney cancer.

ABSTRACT We disclose an efficient transformation of the relatively common pyrroloiminoquinone discorhabdin alkaloids from Alaskan Latrunculia spp. to the complex ring system of aleutianamine by an acid catalyzed process that mimics the proposed endogenous genesis from 3‐dihydrodienyl discorhabdin precursors. The azepine ring system is formed by a concerted 1,2‐alkyl shift of a proposed thiocarbenium‐iminium dication and this mechanism is supported by extensive density functional theory (DFT) analysis. Additional in vitro biological data further supports the aleutianamines as novel and highly potent inhibitors of multidrug resistant cancer lines exemplified further with cytotoxicity toward chemotherapy resistant chromophobe renal cell carcinoma (IC 50 = 130 nM). This report provides a key transformation that allows the incorporation of either synthetic or biosynthetic starting materials leading to the generation of the aleutianamine complex ring system in just a single step from the discorhabdin ring system.

ABSTRACT We disclose an efficient transformation of the relatively common pyrroloiminoquinone discorhabdin alkaloids from Alaskan Latrunculia spp. to the complex ring system of aleutianamine by an acid catalyzed process that mimics the proposed endogenous genesis from 3‐dihydrodienyl discorhabdin precursors. The azepine ring system is formed by a concerted 1,2‐alkyl shift of a proposed thiocarbenium‐iminium dication and this mechanism is supported by extensive density functional theory (DFT) analysis. Additional in vitro biological data further supports the aleutianamines as novel and highly potent inhibitors of multidrug resistant cancer lines exemplified further with cytotoxicity toward chemotherapy resistant chromophobe renal cell carcinoma (IC 50 = 130 nM). This report provides a key transformation that allows the incorporation of either synthetic or biosynthetic starting materials leading to the generation of the aleutianamine complex ring system in just a single step from the discorhabdin ring system.

Abstract SWI/SNF chromatin remodeling complexes are frequently mutated across human cancers, including cholangiocarcinoma (CCA). Dysregulation of this complex due to loss of function mutations in AT-rich interaction domain-1A (ARID1A) and polybromo-1 (PBRM1) occur in up to 18% and 10% of cases respectively. SWI/SNF subunit mutations frequently co-occur alongside PI3K/AKT pathway activation. Preexisting literature suggests altered gene expression and protein synthesis contribute to CCA oncogenesis, but our understanding of these mechanisms remains immature. Genetically accurate preclinical models are needed to define the roles of ARID1A and PBRM1 in CCA and identify therapeutic targets. We developed novel murine models of CCA with either ARID1A, PBRM1 or dual ARID1A/PBRM1 (APAC) knockout in the context of AKT activation, utilizing a surgical biliary transfection method for somatic gene editing with CRISPR/Cas9. Within 8 months, mice formed numerous liver tumors, predominantly CCA and mixed CCA/HCC. Knockout of ARID1A and PBRM1 was confirmed through genomic PCR, qRT-PCR, and immunoblot assays. Histologically, murine APAC tumors displayed phenotypic similarities to human CCA, including dense cancer-associated fibroblast infiltration and variable CD45+ immune cell infiltration. A novel, syngeneic CCA cell line was isolated from a dual ARID1A/PBRM1 knockout tumor (APAC-2). RNA-sequencing of primary tumors and the APAC-2 cell line versus a non-invasive cholangiocyte cell line with AKT activation only (AC), was performed to identify differentially expressed genes (DEGs) of interest in ARID1A and PBRM1-deficient CCA. Periostin (Postn) was among the top upregulated genes in the APAC-2 cell line. POSTN is a secreted glycoprotein that is upregulated in human CCA and may be therapeutically targetable. Knockdown of periostin inhibited the in vitro growth of APAC-2 cells and a human CCA cell line with ARID1A mutation and periostin overexpression (HuH-28). These studies serve to characterize and validate a genetically relevant murine model of epigenetic dysregulation in ARID1A and PBRM1-deficient CCA. This model can be used for identification and validation of potential drug targets. Ongoing work focuses on studying multiple dysregulated pathways and testing dependence on genes of interest, including periostin, as potential treatment strategies for CCA. Citation Format: Jayla Millender, Jennifer Tomlinson, Ryan Watkins, Danielle Carlson, Jack Sample, Nathan Werneburg, Rory Smoot, Keith Robertson, Gregory Gores, Caitlin Conboy. Novel murine models of epigenetic dysregulation in SWI/SNF-mutated cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2741.

Abstract Background: DNMT3A and TET2 are epigenetic regulator genes commonly mutated in age-related clonal hematopoiesis (CH). Despite having opposed epigenetic functions, these mutations are associated with increased all-cause mortality and a low risk for hematological neoplasms. In this study, we make use of a natural inflammatory response occurring during COVID-19 to define the impact of these mutations on inflammation and outcomes. Methods: A cohort of 243 community-based patients (Olmsted County, MN) with COVID-19 were identified. CH status was determined on PBMC using error corrected sequencing (lower detection limit 0.5% VAF). In a subset of patients, we use multi-omics interrogation of the genome, transcriptome, and epigenome at single cell (sc) resolution along with DNA methylation, in PBMC, from patients with COVID-19 and CH. Results: We detected 97 CH mutations in 72 (29.6%) of 243 patients with COVID-19. The most frequent being DNMT3A (n=30, 30%) and TET2 (n=26, 28%). The presence of CH negatively impacted overall survival when restricted to TET2 and DNMT3A mutations. However, only DNMT3Amt CH retained age and comorbidity independent prognostication (HR 2.84, p = 0.022). We then assessed for changes in DNA methylation using Illumina MethylationEPIC array on patients with COVID-19 and DNMT3A (n=3) or TET2mt (n=4) CH. Site specific DNA methylation changes revealed significant loss of methylation in DNMT3Amt CH in comparison to TET2mt patients, with 10,944 hypomethylated and 1,160 hypermethylated sites. We found that actively transcribed states (Tx, TxWk) were more commonly hypomethylated in DNMT3Amt CH while enhancers (Enh) and promoters (TssA, TssAFlnk) were more commonly hypomethylated in TET2mt CH. We then conducted scDNA-seq in DNMT3Amt (n=3) and TET2mt (n=1) CH in COVID-19 and observed that while in TET2mt CH, mutations were largely restricted to classical and intermediate monocytes, in DNMT3Amt CH, mutations were seen in myeloid and lymphoid lineage cells. We performed scRNA-seq on patients with COVID-19 and DNMT3A (n=3) or TET2mt (n=6) CH. Differential gene expression analysis identified 1,569 upregulated genes in DNMT3Amt patients and 205 downregulated. Pathway analysis demonstrated an upregulation of genes involved in T cell survival, function, and inflammation in DNMT3Amt patients compared to TET2mt patients with a significant overexpression of GIMAP1, GIMAP4, IRF2, IL32 in multiple cell types. We correlated transcriptomic changes with chromatin accessibility using the 10X Genomics Multiome platform to profile both gene expression and open chromatin from the same samples. Analysis of global distribution of cut sites and differentially accessible peaks showed increased chromatin accessibility in DNMT3Amt CH, especially CD4+ T lymphocytes and NK cells. Conclusion: We demonstrate an age, sex, and comorbidity-independent adverse impact on mortality associated with DNMT3Amt CH in the context of COVID-19 and highlight transcriptomic and epigenetic differences between DNMT3A and TET2mt CH, with single cell resolution. Citation Format: Jenna A Fernandez, Wazim Mohammed Ismail, Moritz Binder, Terra Lasho, Susan M. Geyer, Amelia Mazzone, Christy M. Finke, Abhishek Mangaonkar, Jeong-Heon Lee, Liguo Wang, Vernadette A. Simon, Fariborz Rakhshan Rohakthar, Amik Munankarmy, Jonathan J. Harrington, Melissa R. Snyder, Keith D. Robertson, Alexandre Gaspar-Maia, Mrinal Patnaik. Divergent impacts of DNMT3A and TET2 mutant clonal hematopoiesis on COVID-19 related outcomes [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Methylation, Clonal Hematopoiesis, and Cancer; 2025 Feb 1-4; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2025;85(3 Suppl):Abstract nr A012.