SETD2 Loss Research Articles

Differencesin mutations across tumour sizes in clear-cell renal cell carcinoma.

To assess the distribution of key mutations across tumour sizes in clear-cell renal cell carcinoma (ccRCC), and secondarily to examine the prognostic impact of aggressive mutations in smaller ccRCCs. The distribution of mutations (VHL, PBRM1, SETD2, BAP1 and CDKN2A loss) across tumour sizes was assessed in 1039 ccRCCs treated with nephrectomy in cohorts obtained from the Tracking Cancer Evolution (TRACERx), The Cancer Genome Atlas (TCGA) and the Cancer Genomics of the Kidney (CAGEKID) projects. Logistic regression was used to model the presence of each mutation against size. In our secondary analysis, we assessed a subset of ccRCCs ≤7 cm for associations of key aggressive mutations (SETD2, BAP1, and CDKN2A loss) with metastasis, invasive disease and overall survival, while controlling for size. A subset of localised tumours ≤7 cm was also used to assess associations with recurrence after nephrectomy. On logistic regression, each 1-cm increase in tumour size was associated with aggressive mutations, SETD2, BAP1, and CDKN2A loss, at odds ratios (ORs) of 1.09, 1.10 and 1.19 (P < 0.001), whereas no significant association was observed between tumour size and PBRM1 (OR 1.02; P = 0.23). VHL was mildly negatively associated with a 1-cm increase in size (OR 0.95; P = 0.01). Among tumours ≤7 cm, SETD2 and CDKN2A loss were associated with metastatic disease at ORs of 3.86 and 3.84 (P < 0.05) while controlling for tumour size. CDKN2A loss was associated with worse overall survival, with a hazard ratio (HR) of 2.19 (P = 0.03). Among localised tumours ≤7 cm, SETD2 was associated with worse recurrence-free survival (HR 2.00; P = 0.03). Large and small ccRCCs are genomically different. Aggressive mutations, namely, SETD2, BAP1, and CDKN2A loss, are rarely observed in small ccRCCs and are observed more frequently in larger tumours. However, when present in tumours ≤7 cm, SETD2 mutations and CDKN2A loss were still independently associated with invasive disease, metastasis, worse survival, and recurrence after resection, after controlling for size.

CML-266 SETD2 Loss Contributes to Chronic Myeloid Leukemia Progression Affecting DNA Integrity and Epigenetic Landscape

CML-266 SETD2 Loss Contributes to Chronic Myeloid Leukemia Progression Affecting DNA Integrity and Epigenetic Landscape

SETD2 Loss Contributes to Chronic Myeloid Leukemia Progression Affecting DNA Integrity and Epigenetic Landscape

SETD2 Loss Contributes to Chronic Myeloid Leukemia Progression Affecting DNA Integrity and Epigenetic Landscape

A Phase II Trial of the WEE1 Inhibitor Adavosertib in SETD2-Altered Advanced Solid Tumor Malignancies (NCI 10170).

WEE1 inhibition with adavosertib monotherapy demonstrated limited clinical activity in patients with SETD2-altered solid tumors despite compelling preclinical data indicating a synthetic lethal effect, which did not translate into robust tumor regression. Loss of the H3K36me3 trimethylation mark caused by SETD2-deficiency was confirmed in the majority of evaluable tumors. A subset of patients derived clinical benefit as manifested by minor tumor regressions and prolonged SD.

Tumor cell-intrinsic epigenetic dysregulation shapes cancer-associated fibroblasts heterogeneity to metabolically support pancreatic cancer

The tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDAC) involves a significant accumulation of cancer-associated fibroblasts (CAFs) as part of the host response to tumor cells. The origins and functions of transcriptionally diverse CAF populations in PDAC remain poorly understood. Tumor cell-intrinsic genetic mutations and epigenetic dysregulation may reshape the TME; however, their impacts on CAF heterogeneity remain elusive. SETD2, a histone H3K36 trimethyl-transferase, functions as a tumor suppressor. Through single-cell RNA sequencing, we identify a lipid-laden CAF subpopulation marked by ABCA8a in Setd2-deficient pancreatic tumors. Our findings reveal that tumor-intrinsic SETD2 loss unleashes BMP2 signaling via ectopic gain of H3K27Ac, leading to CAFs differentiation toward lipid-rich phenotype. Lipid-laden CAFs then enhance tumor progression by providing lipids for mitochondrial oxidative phosphorylation via ABCA8a transporter. Together, our study links CAF heterogeneity to epigenetic dysregulation in tumor cells, highlighting a previously unappreciated metabolic interaction between CAFs and pancreatic tumor cells.

Abstract 1387: SETD2 loss promotes macrophage infiltration and metastasis in RCC

Abstract Across human cancer, tumors that are high grade, poorly differentiated, and have undergone epithelial-to-mesenchymal transition (EMT) carry a worse prognosis with a high likelihood of metastasizing to distant organs. Despite significant research efforts, metastatic cancers and relapse remain the primary cause of cancer related deaths. In renal cell carcinoma (RCC), metastasis is present in one-third of patients at the time of diagnosis, and one-third of patients who initially present with locally advanced disease eventually develop local or distant recurrence following resection of the primary tumor, highlighting the need for new RCC therapies and further insight into the metastatic process. Using RCC patient biopsies, we found that patients with more epithelial tumors have a significantly lower risk of recurrence. Additionally, tumors that had a mesenchymal protein signature had significantly increased macrophage infiltration, which was significantly associated with a higher stage at diagnosis and worse overall survival, suggesting that macrophages may play an important role in EMT and metastasis in ccRCC. Whole exome sequencing of these patient samples revealed that mutational loss of SETD2, a histone methyltransferase that is lost in about 20% of RCC patients, correlated strongly with increased macrophage infiltration within the primary tumor and was necessary for the correlation between macrophages and EMT. SETD2 loss in syngeneic kidney cancer mouse models confirmed that SETD2 loss within tumor cells resulted in increased EMT, macrophages within the primary tumor microenvironment (TME), and spontaneous metastasis, further supporting the role of macrophages in promoting metastasis in the absence of SETD2. Further characterization of the gene signatures and immune cell infiltrate in RCC will lead to a better understanding of metastasis in an oncogene- and tissue-specific manner and thus lead to more efficacious therapies for metastatic patients. Citation Format: Emily N. Arner, Melissa M. Wolf, Logan Vlach, Jeffrey C. Rathmell, W. Kimryn Rathmell. SETD2 loss promotes macrophage infiltration and metastasis in RCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1387.

The distribution of mutations across tumor size in ccRCC and their prognostic importance in small masses.

479 Background: Substantial efforts have been made to delineate mutational pathways to progression in ccRCC and the prognostic importance of key mutations, yet the distribution of these mutations across tumor size remains unknown. We hypothesized that VHL and PBRM, as more truncal mutations, would be roughly equally prevalent across tumor sizes, while mutations associated with aggressive disease - SETD2, BAP1, and CDKN2a copy-number loss - would be predominantly observed in larger tumors. We further hypothesized that SETD2, BAP1, and CDKN2a copy-number loss mutations, when present in smaller (≤7 cm) ccRCC tumors, would portend worse prognosis. Methods: We assessed a combined cohort of 333 ccRCC tumors from TCGA-KIRC and TRACERx Renal for the distribution of mutations across tumor size. Logistic regression was used to model the presence of each mutation against tumor size. We assessed a subset of 194 tumors ≤7cm for associations of key mutations with clinical outcomes while controlling for size. In small masses, logistic regression was used to model the presence of metastatic disease and invasive disease, and Cox proportional hazards was used to model overall survival, against SETD2, BAP1, CDKN2a copy-number loss, and tumor size. Results: On logistic regression an increase in one centimeter of tumor size was associated with SETD2, BAP1, and CDKN2a loss at odds ratios of 1.16, 1.11, 1.19 (p&lt;0.05); whereas no significant association was observed between tumor size and both VHL and PBRM1 (p=0.18, p=0.65). Among 194 tumors ≤7 cm, SETD2 and CDKN2a loss were associated with metastatic disease at odds ratios of 3.86 and 3.84 (p&lt;0.05); CDKN2a loss was associated with worse overall survival at hazard ratio 2.19 (p&lt;0.05), all while controlling for tumor size. Conclusions: SETD2 mutations, BAP1 mutations, and CDKN2a copy-number loss are rare in small ccRCC and are increasingly common in larger tumors, whereas VHL and PBRM1 are fairly evenly distributed across tumor sizes. In tumors≤7 cm, SETD2 mutation and CDKN2a loss were associated with metastatic disease and CDKN2a loss was associated with worse overall survival. SETD2 mutations and CDKN2a loss may help risk stratify ccRCC in biopsied and resected tumors. [Table: see text]

Loss of Function of SETD2 Tumor Suppressor in Chronic Myeloid Leukemia (CML) Progenitors Fosters Genomic Instability and Enhances Clonogenic Potential

BACKGROUND AND AIMS - Genomic instability is a hallmark of chronic myeloid leukemia (CML) cells since the chronic phase (CP) of the disease, and results in BCR::ABL1 mutations and/or additional genetic and genomic aberrations that may drive resistance to tyrosine kinase inhibitors (TKIs) and progression to blast crisis (BC). Genomic instability is also a feature of CML stem and progenitor cells and may contribute to their persistence. The SETD2 tumor suppressor codes for a protein that trimethylates histone H3 at lysine 36 (H3K36me3). In solid tumors, SETD2 loss of function has been shown to impair H3K36me3-mediated recruitment of DNA damage response components. We have recently reported that non genomic loss of function of SETD2 is a feature of BC CML and results from premature proteasome-mediated degradation of the SETD2 protein triggered by Aurora kinase A phosphorylation and MDM2 ubiquitination. In the present study, we aimed to assess SETD2/H3K36me3 status in CD34+ progenitors of CP CML patients (pts) and whether SETD2/H3K36me3 deficiency may play a role in genomic instability in CML models. METHODS - Western blotting (WB) was used to assess SETD2 protein expression and H3K36me3 as a surrogate marker of SETD2 function in the CD34+ cell fraction isolated from the bone marrow of 20 newly diagnosed CP CML pts and from a pool of healthy donors (HD). SETD2 forced expression in CD34+ progenitors from newly diagnosed CP CML pts and in the SETD2-deficient KCL22 cell line was performed by nucleofection. SETD2 knock-down in the SETD2-proficient LAMA84 cell line was performed by RNAi. Clonogenic capacity was evaluated by clonogenic assays. Chromatin immunoprecipitation sequencing (ChIP-seq) for H3K36me3 was performed on an Illumina HiSeq2000 with a min 50 million 150-bp single-end reads per replicate. High resolution karyotyping wias perfomed with Cytoscan HD arrays. DNA damage and DNA repair activation were assessed in primary samples and cell lines by WB and immunofluorescence (IF) using antibodies specific for phospho-H2AX, mismatch repair (MMR) and homologous recombination repair (HR) proteins. RESULTS - WB demonstrated a marked down-modulation of SETD2 expression, paralleled by H3K36me3 deficiency, in the CD34+ cells of all newly diagnosed CP CML pts as compared to the total mononuclear fraction and to CD34+ cells from HDs. To investigate whether SETD2 loss affects the activation and proficiency of HR and MMR, we used chronic exposure to UV rays or a single exposure to hydrogen peroxide (1mM for 30 and 60 min). Both induced DNA damage in SETD2 siRNA-depleted LAMA84 cells. Compared to parental cells, cells silenced for SETD2 failed to activate the ATM-dependent repair pathway. Moreover, we found that ATM inactivation prevents H2AX foci formation, associated with a loss of RAD51 and RAD54 (HR) and MSH6 (MMR) repair foci. To confirm the hypothesis that SETD2 is a tumor suppressor implicated in maintaining genomic stability in CML, we transfected SETD2-deficient KCL22 cells with an ectopic SETD2 plasmid. SETD2 forced expression was able to restore DNA damage response, as demonstrated by WB and IF detection of ATM, p95 and H2AX phosphorylation, BRCA1, BRCA2 and CtIP expression and finally RAD51 and RAD54 localization on HR repair foci observed after UV or hydrogen peroxide exposure. High-resolution karyotyping after DNA damage showed increased rate of DNA breakpoints in SETD2-deficient cells, preferentially occuring at loci where H3K36me3 marks were lost as assessed by ChiP-seq. In line with the effects observed in cell line models, forced expression of SETD2 in CD34+ cells from 5 CP CML pts was found to restore proliferation control, since a &amp;gt;50% reduction in clonogenic potential was observed after nucleofection. CONCLUSIONS - Our findings demonstrate that SETD2 is a bona fide tumor suppressor in CML progenitors and establish a functional link between SETD2 loss of function and genomic instability. SETD2 inactivation may thus play a pivotal role in the harmful cascade of events that may foster drug resistance and ultimately lead to disease progression. Since SETD2 loss of function in CML is mediated by post-translation mechanisms, hence is reversible, therapeutic strategies aimed at interfering with these mechanisms may restore proliferation control and interrupt this cascade. Supported by AIRC IG 2019 (23001) and by Italian Ministry of Health, “Bando Ricerca Finalizzata 2016”, project GR-2016-02364880.

Setd2 deficiency promotes gastric tumorigenesis through inhibiting the SIRT1/FOXO pathway

Gastric cancer (GC) is the fifth most common cancer and the second leading cause of cancer death globally. SETD2 is a histone methyltransferase catalyzing tri-methylation of H3K36 (H3K36me3) and has been shown to participate in diverse biological processes and human tumors. However, the mechanism of SETD2 in GC remains unclear. Here, we reported that Setd2 deficiency predicts poor prognosis of gastric cancer. SETD2 loss facilitated H. felis/MNU and c-Myc-induced gastric tumorigenesis, respectively. The mouse model of stomach-specific Setd2 depletion together with c-MYC overexpression (AMS) developed high-grade epithelial defects, intestinal metaplasia and dysplasia at only 10–12 weeks of age. Mechanistically, Setd2 depletion resulted in impaired epigenetic regulation of Sirt1, thus inhibiting the SIRT1/FOXO pathway. Moreover, the agonists of FOXO signaling or overexpression of SIRT1 significantly rescued the enhanced cell proliferation and migration caused by Setd2 deficiency in SGC7901 cells. Together, our findings highlight an epigenetic mechanism by which SETD2 regulates gastric tumorigenesis through SIRT1/FOXO pathway. It may also pave the way for the development of targeted, patient-tailored therapies for GC patients with Setd2 deficiency.

Comprehensive evaluation of the Infinium human MethylationEPIC v2 BeadChip

Infinium Methylation BeadChips are widely used to profile DNA cytosine modifications in large cohort studies for reasons of cost-effectiveness, accurate quantification, and user-friendly data analysis in characterizing these canonical epigenetic marks. In this work, we conducted a comprehensive evaluation of the updated Infinium MethylationEPIC v2 BeadChip (EPICv2). Our evaluation revealed that EPICv2 offers significant improvements over its predecessors, including expanded enhancer coverage, applicability to diverse ancestry groups, support for low-input DNA down to one nanogram, coverage of existing epigenetic clocks, cell type deconvolution panels, and human trait associations, while maintaining accuracy and reproducibility. Using EPICv2, we were able to identify epigenome and sequence signatures in cell line models of DNMT and SETD2 loss and/or hypomorphism. Furthermore, we provided probe-wise evaluation and annotation to facilitate the use of new features on this array for studying the interplay between somatic mutations and epigenetic landscape in cancer genomics. In conclusion, EPICv2 provides researchers with a valuable tool for studying epigenetic modifications and their role in development and disease.

Abstract B017: SETD2-loss mediated H3K36me3 causes disruptions in topologically associated domains (TADs) to promote oncogenic expression in clear cell renal cell carcinoma

Abstract SETD2 is an RNA Polymerase II (Pol II)-associated histone methyltransferase that catalyzes the co-transcriptional methylation of H3K36me2 to create H3K36me3 marks in gene bodies of actively transcribed genes. SETD2 is one of the most frequently mutated chromatin-modifying genes across different cancer types, with the highest prevalence (13%) in clear cell renal cell carcinoma (ccRCC). ccRCC has been recognized as a VHL loss-driven disease in which VHL is mutated or silenced in up to 80-90% of tumors, and the heterozygous loss of chromosome 3p where VHL resides is identified in &amp;gt;90% of ccRCC. The other three top mutated genes in ccRCC are also located at chromosome 3p, including PBRM1 (~40%), SETD2 (8-13%), and BAP1 (6-10%). Notably, deletion of Vhl alone is insufficient to induce kidney cancer in mice, whereas combined deletion of Vhl and Pbrm1 results in multifocal, transplantable non-metastatic clear cell kidney cancer. Human ccRCC genomics and cancer evolution studies revealed that SETD2 mutations often co-occur with VHL and PBRM1 mutations and SETD2 mutations associate with tumor progression. Previously, using VHLMT PBRM1MT SETD2MT metastatic ccRCC patient-derived cell line and xenograft models we found that H3K36me3 restoration reduced distant metastases of ccRCC in mice, establishing SEDT2 as a metastatic tumor suppressor in kidney cancer. An integrated ATAC-seq, ChIP-seq, and transcriptome analysis uncovered a tumor suppressor model of SETD2 in which loss of SETD2-mediated H3K36me3 creates an epigenetic landscape consisting of widespread open chromatin that amplifies oncogenic transcriptional output through aberrant activation of enhancers. Here, we hypothesize that loss of SETD2-mediated H3K36me3 alters 3D chromosome architecture to increase chromatin accessibility and enhancer-promoter interactions, which in turn activates oncogenic transcription, including pro-metastatic genes such as MMP1, HGF, MYC, and ETV1. Here, we performed chromosome conformation capture (Hi-C) to interrogate 3D chromatin architecture in kidney cancer with defined mutations in VHL, PBRM1, or SETD2 for the first time. Our preliminary global analysis comparing SETD2 mutant and wildtype HiC samples showed disruptions in Topologically Associated Domains (TADs) and increased long-range interaction with loss of SETD2 function. Further understanding of how dysregulated epigenetics conferred by SETD2 loss promotes tumor initiation and metastasis will allow the discovery of novel therapeutic vulnerabilities associated with the loss of SETD2. Citation Format: Amrita M. Nargund, Rui Yang. SETD2-loss mediated H3K36me3 causes disruptions in topologically associated domains (TADs) to promote oncogenic expression in clear cell renal cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr B017.

SETD2 loss in renal epithelial cells drives epithelial-to-mesenchymal transition in a TGF-β-independent manner.

Histone-lysine N-methyltransferase SETD2 (SETD2), the sole histone methyltransferase that catalyzes trimethylation of lysine 36 on histone H3 (H3K36me3), is often mutated in clear cell renal cell carcinoma (ccRCC). SETD2 mutation and/or loss of H3K36me3 is linked to metastasis and poor outcome in ccRCC patients. Epithelial-to-mesenchymal transition (EMT) is a major pathway that drives invasion and metastasis in various cancer types. Here, using novel kidney epithelial cell lines isogenic for SETD2, we discovered that SETD2 inactivation drives EMT and promotes migration, invasion, and stemness in a transforming growth factor-beta-independent manner. This newly identified EMT program is triggered in part through secreted factors, including cytokines and growth factors, and through transcriptional reprogramming. RNA-seq and assay for transposase-accessible chromatin sequencing uncovered key transcription factors upregulated upon SETD2 loss, including SOX2, POU2F2 (OCT2), and PRRX1, that could individually drive EMT and stemness phenotypes in SETD2 wild-type (WT) cells. Public expression data from SETD2 WT/mutant ccRCC support the EMT transcriptional signatures derived from cell line models. In summary, our studies reveal that SETD2 is a key regulator of EMT phenotypes through cell-intrinsic and cell-extrinsic mechanisms that help explain the association between SETD2 loss and ccRCC metastasis.

Extensive intratumor regional epigenetic heterogeneity in clear cell renal cell carcinoma targets kidney enhancers and is associated with poor outcome

BackgroundClear cell renal cell cancer (ccRCC), the 8th leading cause of cancer-related death in the US, is challenging to treat due to high level intratumoral heterogeneity (ITH) and the paucity of druggable driver mutations. CcRCC is unusual for its high frequency of epigenetic regulator mutations, such as the SETD2 histone H3 lysine 36 trimethylase (H3K36me3), and low frequency of traditional cancer driver mutations. In this work, we examined epigenetic level ITH and defined its relationships with pathologic features, aspects of tumor biology, and SETD2 mutations.ResultsA multi-region sampling approach coupled with EPIC DNA methylation arrays was conducted on a cohort of normal kidney and ccRCC. ITH was assessed using DNA methylation (5mC) and CNV-based entropy and Euclidian distances. We found elevated 5mC heterogeneity and entropy in ccRCC relative to normal kidney. Variable CpGs are highly enriched in enhancer regions. Using intra-class correlation coefficient analysis, we identified CpGs that segregate tumor regions according to clinical phenotypes related to tumor aggressiveness. SETD2 wild-type tumors overall possess greater 5mC and copy number ITH than SETD2 mutant tumor regions, suggesting SETD2 loss contributes to a distinct epigenome. Finally, coupling our regional data with TCGA, we identified a 5mC signature that links regions within a primary tumor with metastatic potential.ConclusionTaken together, our results reveal marked levels of epigenetic ITH in ccRCC that are linked to clinically relevant tumor phenotypes and could translate into novel epigenetic biomarkers.

Abstract 1226: SETD2 loss in renal epithelial cells induces epithelial to mesenchymal transition in a TGFβ-independent manner

Abstract Clear cell renal cell carcinoma (ccRCC) is the most common subtype of renal cell carcinoma (RCC) and accounts for majority of the deaths from renal cancer. SETD2, the sole histone methyltransferase that catalyzes trimethylation of lysine 36 on histone H3 (H3K36me3), is frequently mutated in ccRCC. Studies have shown that SETD2 mutations are often subclonal and the mutation rate dramatically increases from 25% in primary ccRCC to more than 60% in metastatic ccRCC, suggesting SETD2 loss plays an important role in renal cancer metastasis. However, mechanisms underpinning how SETD2 mutation drives invasion and metastasis in ccRCC is largely unknown. Here, by performing RNA-seq and ATAC-seq in a non-tumorigenic renal epithelial cell line isogenic for SETD2 loss-of-function, we found that SETD2 inactivation induces dramatic changes in the transcriptome and drastically enhances chromatin accessibility, which allows binding of a variety of transcription factors. We also discovered that SETD2 loss induces a TGFβ-independent EMT program, which is a major pathway linked to metastasis. Correlation of RNA-seq with ATAC-seq identifies SOX2, OCT2 and PRRX1 as key transcription factors promoting the SETD2 loss-driven EMT program. Furthermore, we found that this distinct EMT program is in part triggered by secreted factors, which suggests that SETD2 mutation promotes a metastasis-permissive tumor microenvironment through paracrine signaling. In summary, our findings reveal that SETD2 is a key regulator of EMT phenotypes that may explain the role of SETD2 mutations in ccRCC metastasis and further uncover potential therapeutic targets for SETD2-mutant ccRCC patients. Citation Format: Tianchu Wang, Ryan Wagner, Ryan Hlady, Sungho Kim, Xiaoyu Pan, Thai Ho, Keith Robertson. SETD2 loss in renal epithelial cells induces epithelial to mesenchymal transition in a TGFβ-independent manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1226.

Abstract 6794: SETD2 loss in renal cell carcinoma generates peptides from aberrantly translated retained introns

Abstract Introduction: SETD2, a histone H3-K36 trimethyltransferase, is necessary for regulation of proper intron splicing. SETD2 is frequently inactivated in kidney cancer. We hypothesized that SETD2 deficiency would cause aberrant translation of retained introns (ATaRI) that could serve as potential immunotherapeutic targets in SETD2-mutant states. Methods and Results: We detected increased presence of retained introns in SETD2-mutant vs WT tumors in two publicly available renal cell carcinoma RNA-seq data sets. We hypothesized that if intronic sequences translated into proteins, accumulation of misfolded proteins would activate the Unfolded Protein Response (UPR). The UPR pathway was strongly transcriptionally enriched as measured by GSEA, as were several immunotherapy-relevant pathways, suggesting that tumors are inflamed, possibly related to the antigenic nature of translated introns. To investigate this further, we generated Setd2-isogenic mouse renal cancer cells using CRISPR in the RENCA cell line. RNAseq followed by GSEA confirmed upregulation of UPR pathway transcriptional signature in the Setd2-null condition, supporting our observation in human tumors. Indicators of UPR activation such as cleavage of ATF6 and increase in ATF4 and Xbp1 levels in Setd2 deficient vs. unedited RENCA cells, as well as nuclear translocation of ATF6 in SETD2-mutant human kidney cancer samples validates these findings. We then measured the presence of ATaRI-derived peptides in Setd2-mutant RENCA cells using mass spectrometry, detecting 47 such peptides. Using publicly available proteomics data from one human data set, we preliminarily identified peptides translated from 151 introns that are retained in SETD2-mutant cases. Applying netMHCpan to both the human and murine data, we found that detectable ATaRI peptides were predicted to generate multiple 10-amino acid-long peptides which strongly bind to the 8 murine MHC or to the 20 most common human HLA receptors. We propose that these presentable peptides might activate an immune response to SETD2-mutant renal cell carcinoma. Conclusions: We identified intron retention which may cause activation of the UPR as a feature of SETD2-mutant kidney cancer. ATaRI peptides are detectable and should be presented to the adaptive immune system. These features may represent a new therapeutic vulnerability for exploitation as a rationale for personalized medicine. Citation Format: Marya T. Kozinova, Alexander Metz, Robert Uzzo, Janusz Franco-Barraza, Michael Slifker, Jessica Peskin, Angel Fernández Sanromán, Samra Turajlic, Edna Cukierman, Phillip Phillip Abbosh. SETD2 loss in renal cell carcinoma generates peptides from aberrantly translated retained introns. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6794.

Abstract 4153: SETD2 loss and ATR inhibition synergistically promote cGAS signaling and immunotherapy response in renal cell carcinoma

Abstract Rationale: Immune checkpoint blockade (ICB) demonstrates durable clinical benefit only in a minority of renal cell carcinoma (RCC) patients. Identifying molecular features that determine response and developing approaches to enhance the response remain an urgent clinical need. Targeting S phase DNA damage repair (S-DDR) network has been reported to activate cGAS signaling and convert a non-immunogenic tumor microenvironment to an immunogenic tumor microenvironment, thus improving immunotherapy response in breast cancer and small cell lung cancer. SETD2 has been known to be required for ATM-mediated DNA damage response, and we hypothesize that SETD2 loss confers greater dependence on ATR activity and provides vulnerability to ATR inhibitors, which may further potentiate the response to immunotherapy. Methods: Applying molecular, cellular, and bioinformatic approaches, we comprehensively investigated the effects of SEDT2/Setd2 loss on the cytosolic DNA damage response pathway, the cGAS-mediated cytosolic DNA sensing pathway, the tumor immune microenvironment, and immunotherapy response in RCC cell lines, in the Renca-BALB/c immune competent murine model, and in RCC patients cohorts with or without ICB therapy. Results: We found that, in multiple RCC cell lines, pharmacological inhibition of the ATR-CHK1 axis activated the cGAS-IRF3-dependent cytosolic DNA sensing pathway, resulting in inflammatory cytokine expression. SETD2 mutated RCC cell lines or tumor samples were associated with preferential ATR-CHK1 activation over ATM-CHK2 activation. SETD2 knockdown promoted the cytosolic DNA sensing pathway and conferred greater sensitivity to ATR inhibition. In murine Renca tumors, Setd2 knockdown and the ATR inhibitor VE822 synergistically activated the cytosolic DNA sensing pathway, immune cell infiltration, and immune checkpoint protein expression. Setd2 deficient Renca tumors demonstrated greater vulnerability to ICB monotherapy or in combination with ATRi than Setd2 proficient tumors. SETD2 mutations were associated with a higher response rate and prolonged overall survival in ICB-treated RCC patients, but not in non-ICB-treated RCC patients. Conclusion: This study reveals that SETD2/Setd2 loss and ATR inhibitor synergize to enhance cGAS signaling and RCC tumor immune responsiveness, and provides mechanism-based guidance to develop more personalized combination therapy regimens for RCC patients with SETD2 mutations. Citation Format: Xiande Liu, Yanting Zhang, Xuesong Zhang, Daniel McGrail, Truong Lam, Anh Hoang, Elshad Hasanov, Ganiraju Manyam, Christine B. Peterson, Haifeng Zhu, Shwetha V. Kumar, Rehan Akbani, Nizar M. Tannir, Guang Peng, Eric Jonasch. SETD2 loss and ATR inhibition synergistically promote cGAS signaling and immunotherapy response in renal cell carcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4153.

SETD2 non genomic loss of function in advanced systemic mastocytosis is mediated by an Aurora kinase A/MDM2 axis and can be therapeutically targeted

BackgroundThe SETD2 tumor suppressor gene encodes a histone methyltransferase that safeguards transcription fidelity and genomic integrity via trimethylation of histone H3 lysine 36 (H3K36Me3). SETD2 loss of function has been observed in solid and hematologic malignancies. We have recently reported that most patients with advanced systemic mastocytosis (AdvSM) and some with indolent or smoldering SM display H3K36Me3 deficiency as a result of a reversible loss of SETD2 due to reduced protein stability.MethodsExperiments were conducted in SETD2-proficient (ROSAKIT D816V) and -deficient (HMC-1.2) cell lines and in primary cells from patients with various SM subtypes. A short interfering RNA approach was used to silence SETD2 (in ROSAKIT D816V cells), MDM2 and AURKA (in HMC-1.2 cells). Protein expression and post-translational modifications were assessed by WB and immunoblotting. Protein interactions were tested by using co-immunoprecipitation. Apoptotic cell death was evaluated by flow cytometry after annexin V and propidium iodide staining, respectively. Drug cytotoxicity in in vitro experiments was evaluated by clonogenic assays.ResultsHere, we show that the proteasome inhibitors suppress cell growth and induce apoptosis in neoplastic mast cells by promoting SETD2/H3K36Me3 re-expression. Moreover, we found that Aurora kinase A and MDM2 are implicated in SETD2 loss of function in AdvSM. In line with this observation, direct or indirect targeting of Aurora kinase A with alisertib or volasertib induced reduction of clonogenic potential and apoptosis in human mast cell lines and primary neoplastic cells from patients with AdvSM. Efficacy of Aurora A or proteasome inhibitors was comparable to that of the KIT inhibitor avapritinib. Moreover, combination of alisertib (Aurora A inhibitor) or bortezomib (proteasome inhibitor) with avapritinib allowed to use lower doses of each drug to achieve comparable cytotoxic effects.ConclusionsOur mechanistic insights into SETD2 non-genomic loss of function in AdvSM highlight the potential value of novel therapeutic targets and agents for the treatment of patients who fail or do not tolerate midostaurin or avapritinib.

SETD2 regulates chromatin accessibility and transcription to suppress lung tumorigenesis.

SETD2, a H3K36 trimethyltransferase, is the most frequently mutated epigenetic modifier in lung adenocarcinoma, with a mutation frequency of approximately 9%. However, how SETD2 loss of function promotes tumorigenesis remains unclear. Using conditional Setd2-KO mice, we demonstrated that Setd2 deficiency accelerated the initiation of KrasG12D-driven lung tumorigenesis, increased tumor burden, and significantly reduced mouse survival. An integrated chromatin accessibility and transcriptome analysis revealed a potentially novel tumor suppressor model of SETD2 in which SETD2 loss activates intronic enhancers to drive oncogenic transcriptional output, including the KRAS transcriptional signature and PRC2-repressed targets, through regulation of chromatin accessibility and histone chaperone recruitment. Importantly, SETD2 loss sensitized KRAS-mutant lung cancer to inhibition of histone chaperones, the FACT complex, or transcriptional elongation both in vitro and in vivo. Overall, our studies not only provide insight into how SETD2 loss shapes the epigenetic and transcriptional landscape to promote tumorigenesis, but they also identify potential therapeutic strategies for SETD2 mutant cancers.

Tumor Cell-Intrinsic SETD2 Deficiency Reprograms Neutrophils to Foster Immune Escape in Pancreatic Tumorigenesis.

Genetic and epigenetic alterations play central roles in shaping the immunosuppressive tumor microenvironment (TME) to evade immune surveillance. The previous study shows that SETD2-H3K36me3 loss promotes KRAS-induced pancreatic tumorigenesis. However, little is known about its role in remodeling the TME and immune evasion. Here, it is shown that SETD2 deficiency can reprogram neutrophils to an immunosuppressive phenotype, thereby promoting immune escape during pancreatic tumor progression. By comprehensive profiling of the intratumoral immune cells, neutrophils are identified as the subset with the most significant changes upon Setd2 loss. Setd2-deficient pancreatic tumor cells directly enhance neutrophil recruitment and reprogramming, thereby inhibiting the cytotoxicity of CD8+ T cells to foster tumor progression. Mechanistically, it is revealed that Setd2-H3K36me3 loss leads to ectopic gain of H3K27me3 to downregulate Cxadr expression, which boosts the PI3K-AKT pathway and excessive expression of CXCL1 and GM-CSF, thereby promoting neutrophil recruitment and reprogramming toward an immunosuppressive phenotype. The study provides mechanistic insights into how tumor cell-intrinsic Setd2 deficiency strengthens the immune escape during pancreatic tumorigenesis, which may offer potential therapeutic implications for pancreatic cancer patients with SETD2 deficiency.

SETD2 transcriptional control of ATG14L/S isoforms regulates autophagosome–lysosome fusion

Macroautophagy/autophagy is an evolutionarily conserved and tightly regulated catabolic process involved in the maintenance of cellular homeostasis whose dysregulation is implicated in several pathological processes. Autophagy begins with the formation of phagophores that engulf cytoplasmic cargo and mature into double-membrane autophagosomes; the latter fuse with lysosomes/vacuoles for cargo degradation and recycling. Here, we report that yeast Set2, a histone lysine methyltransferase, and its mammalian homolog, SETD2, both act as positive transcriptional regulators of autophagy. However, whereas Set2 regulates the expression of several autophagy-related (Atg) genes upon nitrogen starvation, SETD2 effects in mammals were found to be more restricted. In fact, SETD2 appears to primarily regulate the differential expression of protein isoforms encoded by the ATG14 gene. SETD2 promotes the expression of a long ATG14 isoform, ATG14L, that contains an N-terminal cysteine repeats domain, essential for the efficient fusion of the autophagosome with the lysosome, that is absent in the short ATG14 isoform, ATG14S. Accordingly, SETD2 loss of function decreases autophagic flux, as well as the turnover of aggregation-prone proteins such as mutant HTT (huntingtin) leading to increased cellular toxicity. Hence, our findings bring evidence to the emerging concept that the production of autophagy-related protein isoforms can differentially affect core autophagy machinery bringing an additional level of complexity to the regulation of this biological process in more complex organisms.