Abstract Keloids are benign fibroproliferative disorders majorly characterized by excessive extracellular matrix deposition, with recurrence rates exceeding 80% following conventional therapy. Although epigenetic dysregulation has been implicated in keloid pathogenesis, whether genome-wide DNA methylation actively drives pathological cellular reprogramming, and whether this state is therapeutically reversible, remains unclear. We performed genome-wide DNA methylation profiling on keloid tissues, matched primary keloid fibroblasts, and normal controls. Our analysis revealed a shared DNA hypermethylation pattern between keloid tissues and fibroblasts, which was validated by three independent public cohorts. By integrating DNA methylome and transcriptome, we demonstrated that DNA methylation-regulated genes were enriched in osteochondrogenesis-related pathways, such as cartilage and bone development pathways. Furthermore, pharmacologic inhibition of DNA hypermethylation by DNA demethylating agent decitabine reduced the expression of osteochondrogenic markers and inhibited collagen deposition and keloid growth in primary keloid fibroblasts and patient-derived xenograft (PDX) model, offering a potential therapeutic strategy of keloid.

ONECUT2 is a lineage plasticity driver and therapeutic target in aggressive prostate cancer (PCa). This study investigated whether ONECUT2 gene-body DNA methylation regulates its expression and assessed its potential as a biomarker in clinical specimens. We analyzed associations between ONECUT2 gene-body methylation, expression, and patient survival across multiple datasets. The effect of DNA methylation on ONECUT2 expression was tested in prostate cancer cell lines using a DNA methyltransferase inhibitor (DNMTi). Validation was further performed in needle biopsy samples by targeted bisulfite sequencing for DNA methylation and RT-PCR for gene expression. ONECUT2 expression strongly correlated with gene-body DNA methylation and patient survival in multiple datasets. DNMTi treatment confirmed this relationship in prostate cancer cells. In 208 biopsies from prostate cancer patients, hypermethylation of gene-body of ONECUT2 was linked to higher ONECUT2 expression and effectively distinguished tumor from adjacent normal tissue (p < 0.001 and AUC = 0.86). It also predicted aggressive features, including higher Gleason score (p = 0.01 and AUC = 0.68), advanced T stage (p = 0.04 and AUC = 0.65), seminal vesicle invasion (p = 0.0024 and AUC = 0.76), and lymph node involvement (p = 0.0005 and AUC = 0.80). Assessing ONECUT2 gene-body methylation in biopsies may serve as a surrogate for ONECUT2 expression and provide predictive insights into disease progression before surgery. Furthermore, suppressing ONECUT2 through DNMTi treatment represents a potential therapeutic strategy for aggressive PCa.

Background: Neurodevelopmental disorders (NDDs) vary widely in their clinical features and genetic causes, and the molecular picture shifts with population structure. Where consanguineous marriage is common, a heavier load of homozygous, recessive variation is expected, yet how this plays out in South Asian patients remains poorly described. Methods: We screened 500 individuals from Pakistan with a clinical NDD diagnosis and performed whole-exome sequencing (WES) on the 263 who met our inclusion criteria. Variants were annotated, filtered against population and clinical databases, and classified with ACMG/AMP criteria against fixed denominators of 263 individuals and 248 candidate variants. Results: WES returned 248 candidate variants in 242 of 263 individuals, a candidate detection rate of 92.0%. After review, 132 of 263 individuals carried at least one pathogenic or likely pathogenic variant, a molecular diagnostic yield of 50.2%. Homozygous variants and autosomal recessive inheritance each made up 51.6% of candidates, pointing to a recessive-predominant architecture; missense changes were most common at 62.1%. In all, 197 genes were implicated, reflecting marked allelic heterogeneity. Conclusions: This pattern suggests comprehensive exome or genome testing, rather than panel-based screening, is the better first-line approach here. VUS-class findings are best treated as candidate associations that still need validation.

The cell division cycle associated 4 (CDCA4) plays a crucial role in various biological processes and is implicated in the progression of several tumors, however, the mechanisms by which it operates in bladder cancer remain unclear. Utilizing data from the TCGA and GEO datasets of bladder cancer patients, we analyzed the expression of CDCA4 and its prognostic significance. We then constructed stable overexpression and knockdown bladder cancer cell lines to investigate the effects of CDCA4 on cell proliferation, migration, and invasion in vitro, employing CCK-8, colony formation, transwell, and wound healing assays. Additionally, we validated the potential downstream pathways of CDCA4 through data analysis and western blot assays. Our study found that CDCA4 expression is elevated in bladder cancer cells and correlates with poor prognosis in patients. Inhibition of CDCA4 expression reduces the proliferation, migration, and invasion of bladder cancer cells, as well as inhibit the epithelial-mesenchymal transition (EMT) process. Conversely, promoting CDCA4 expression enhances the malignancy of bladder cancer cells. Investigation into the mechanism of CDCA4 revealed that it promotes bladder cancer progression by activating the JAK/STAT signaling pathway, and the JAK inhibitor AG490 can reverse the promoting effects of CDCA4. Our findings suggest that CDCA4 enhances the proliferation, migration, and invasion of bladder cancer cells by positively regulating the JAK/STAT signaling pathway, indicating that CDCA4 may serve as a novel molecular target for bladder cancer treatment.

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You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology I (MP05)1 May 2024MP05-09 A NOVEL METHOD TO IDENTIFY AGGRESSIVE PROSTATE CANCER WITH DNA-METHYLATION SIGNATURES FROM PROSTATE NEEDLE BIOPSY Masatomo Kaneko, Yohei Sekino, Hongtao Li, Xinyi Zhou, Mingda Jin, Wei Guo, Xiaojing Yang, Jeffrey Bhasin, Atsuko Fujihara, Tsuyoshi Iwata, Lorenzo Storino Ramacciotti, Divyangi Paralkar, Giovanni E. Cacciamani, Manju Aron, Osamu Ukimura, Inderbir S. Gill, Gangning Liang, and Andre Luis Abreu Masatomo KanekoMasatomo Kaneko , Yohei SekinoYohei Sekino , Hongtao LiHongtao Li , Xinyi ZhouXinyi Zhou , Mingda JinMingda Jin , Wei GuoWei Guo , Xiaojing YangXiaojing Yang , Jeffrey BhasinJeffrey Bhasin , Atsuko FujiharaAtsuko Fujihara , Tsuyoshi IwataTsuyoshi Iwata , Lorenzo Storino RamacciottiLorenzo Storino Ramacciotti , Divyangi ParalkarDivyangi Paralkar , Giovanni E. CacciamaniGiovanni E. Cacciamani , Manju AronManju Aron , Osamu UkimuraOsamu Ukimura , Inderbir S. GillInderbir S. Gill , Gangning LiangGangning Liang , and Andre Luis AbreuAndre Luis Abreu View All Author Informationhttps://doi.org/10.1097/01.JU.0001008740.27639.cc.09AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: To discover DNA-methylation signatures associated with clinically significant prostate cancer (CSPCa, grade group [GG] ≥2) on prostate biopsy (PBx). METHODS: Patients undergoing radical prostatectomy (RP) for prostate cancer (PCa) from April 2019 to July 2022 were prospectively recruited (IRB# HS-16-00404). Patients who underwent previous treatment for PCa were excluded. Two-core PBx fresh tissue was precisely sampled from the same location in the right and left lobes of the ex-vivo RP specimen (total 4-core per-patient). Then, one-core each was histopathologically (H&E) analyzed, and the other core was processed using Simplified Whole-panel Amplification Reaction Method (SWARM®) a robust targeted bisulfite sequencing approach. From 396,020 CpG sites (CpGs) related to PCa, 1254CpGs were selected for the analysis. The ex-vivo PBx histopathology was used as ground truth for paired-cores to identify methylation CpG sites (CpGs) strongly associated with CSPCa. The multi-class (benign vs GG1 vs CSPCa) discriminating performance was analyzed with a random forest. The samples were randomly separated at a 6:4 ratio into training and testing datasets. The area under the receiver operating characteristic curve (AUC) and diagnostic performance at Youden index on the testing dataset were reported. RESULTS: A total of 334 ex-vivo PBx cores (205 training vs 129 testing) acquired from 149 participants were included. The median age, PSA, and PSA density were 65 years, 6.7ng/mL, and 0.14ng/mL2, respectively. The index lesion on RP histology was GG1 (6.0%), 2 (52%), 3 (25%), 4 (4.0%), and 5 (12%). Benign tissue, GG1 and CSPCa were sampled in 230 (69%), 27 (8.1%) and 77 (23%) ex-vivo biopsy cores, respectively. Individual 44 CpGs as a feature in classifying PCa showed an average AUC of 0.80. The combined set of 44 CpGs achieved AUC 0.88 for PCa detection. Among them, the combination of top 12 CpGs showed AUC of 0.87, sensitivity of 78%, and specificity of 90% for CSPCa detection (Figure 1). CONCLUSIONS: Twelve DNA-methylation CpGs associated with CSPCa were successfully identified in PBx. This novel methodology presents the potential to distinguish patients with poor prognoses from PBx. Download PPT Source of Funding: None © 2024 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 211Issue 5SMay 2024Page: e46 Advertisement Copyright & Permissions© 2024 by American Urological Association Education and Research, Inc.Metrics Author Information Masatomo Kaneko More articles by this author Yohei Sekino More articles by this author Hongtao Li More articles by this author Xinyi Zhou More articles by this author Mingda Jin More articles by this author Wei Guo More articles by this author Xiaojing Yang More articles by this author Jeffrey Bhasin More articles by this author Atsuko Fujihara More articles by this author Tsuyoshi Iwata More articles by this author Lorenzo Storino Ramacciotti More articles by this author Divyangi Paralkar More articles by this author Giovanni E. Cacciamani More articles by this author Manju Aron More articles by this author Osamu Ukimura More articles by this author Inderbir S. Gill More articles by this author Gangning Liang More articles by this author Andre Luis Abreu More articles by this author Expand All Advertisement PDF downloadLoading ...

Abstract The abnormal changes in DNA methylation are linked to the early stages of carcinogenesis. Identifying these epigenetic changes in circulating tumor DNA (ctDNA) can reveal potential biomarkers for the early diagnosis of various cancers. However, analyzing such data poses bioinformatics challenges due to the lack of sensitivity in detecting the low abundance ctDNA signals in biopsy samples, which are often overwhelmed by the complexity of libraries containing hundreds of targeted regions. Read-level methylation analysis holds the promise of more in-depth DNA methylation detection due to the wide coverage and high sensitivity of rare signals. However, this approach is hindered by the absence of a standardized workflow capable of generating interpretable reports suitable for both bench scientists and professional bioinformaticians. Here, we present a bioinformatics workflow that examines next-generation sequencing (NGS) data and characterizes the read-level methylation patterns of amplicons. Compared to other currently available tools, our method is designed to work with high-multiplex, large-scale targeted assays. It effectively eliminates the undesired noise derived from sequencing byproducts such as false CpG calls, dimers, and off-target alignments. Additionally, to accommodate the substantial volume of data generated by state-of-the-art NGS platforms, the workflow enables parallel processing of samples compatible with both cloud-based and on-premises computing resources. This workflow provides a comprehensive per-sample visualization of DNA methylation patterns and reports read-level methylation results in a “pattern-as-a-feature” table. In this table, the occurrence of an amplicon epiallelic haplotype (pattern) for every sample is represented as a “feature column” and is aggregated with all patterns discovered in the experiment. These read-level patterns, along with other information, can be used to develop machine learning algorithms to reiteratively harvest true predictive features and penalize confounding signals in predicting cancer diagnosis. Citation Format: Mingda Jin, Masatomo Kaneko, Steven Cen, Hongtao Li, Wei Guo, Xinyi Zhou, Atsuko Fujihara, Tsuyoshi Iwata, Lorenzo Storino Ramacciotti, Divyangi Paralkar, Giovanni E Cacciamani, Manju Aron, Osamu Ukimura, Inderbir S. Gill, Gangning Liang, Andre L. Abreu, Jeffrey Bhasin, Xiaojing Yang, Xi-Yu Jia. Read-level methylation pattern extraction for high-multiplex large-scale targeted NGS assay [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 3493.

Improvements in survival have been made over the past two decades for childhood acute myeloid leukemia (AML), but the approximately 40% of patients who relapse continue to have poor outcomes. A combination of checkpoint-inhibitor nivolumab and azacitidine has demonstrated improvements in median survival in adults with AML. This phase I/II study with nivolumab and azacitidine in children with relapsed/refractory AML (NCT03825367) was conducted through the Therapeutic Advances in Childhood Leukemia & Lymphoma consortium. Thirteen patients, median age 13.7 years, were enrolled. Patients had refractory disease with multiple reinduction attempts. Twelve evaluable patients were treated at the recommended phase II dose (established at dose level 1, 3 mg/kg/dose). Four patients (33%) maintained stable disease. This combination was well tolerated, with no dose-limiting toxicities observed. Grade 3–4 adverse events (AEs) were primarily hematological. Febrile neutropenia was the most common AE ≥ grade 3. A trend to improved quality of life was noted. Increases in CD8+ T cells and reductions in CD4+/CD8+ T cells and demethylation were observed. The combination was well tolerated and had an acceptable safety profile in pediatric patients with relapsed/refractory AML. Future studies might explore this combination for the maintenance of remission in children with AML at high risk of relapse.

CONCLUSIONS: UTUC has a distinct DNA methylation pattern that is distinguishable from normal urothelial tissue. UTUC-speci fi c DNA methylation probes can be used to differentiate muscle-invasive UTUCs.

Abstract SETD2 deficiency alters the epigenetic landscape by causing depletion of H3K36me3 and plays an important role in diverse forms of cancer, most notably in aggressive and metastatic clear-cell renal cell carcinomas (ccRCC). Development of an effective treatment scheme targeting SETD2-compromised cancer is urgently needed. Considering that SETD2 is involved in DNA methylation and DNA repair, a combination treatment approach using DNA hypomethylating agents (HMA) and PARP inhibitors (PARPi) could have strong antitumor activity in SETD2-deficient kidney cancer. We tested the effects of the DNA HMA 5-aza-2′-dexoxydytidine (DAC), the PARPi talazoparib (BMN-673), and both in combination in human ccRCC models with or without SETD2 deficiency. The combination treatment of DAC and BMN-673 synergistically increased cytotoxicity in vitro in SETD2-deficient ccRCC cell lines but not in SETD2-proficient cell lines. DAC and BMN-673 led to apoptotic induction, increased DNA damage, insufficient DNA damage repair, and increased genomic instability. Furthermore, the combination treatment elevated immune responses, upregulated STING, and enhanced viral mimicry by activating transposable elements. Finally, the combination effectively suppressed the growth of SETD2-deficient ccRCC in in vivo mouse models. Together, these findings indicate that combining HMA and PARPi is a promising potential therapeutic strategy for treating SETD2-compromised ccRCC. Significance: SETD2 deficiency creates a vulnerable epigenetic status that is targetable using a DNA hypomethylating agent and PARP inhibitor combination to suppress renal cell carcinoma, identifying a precision medicine–based approach for SETD2-compromised cancers.

Abstract 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.

Summary Tumors with mutations in chromatin regulators present attractive targets for DNA hypomethylating agent 5-aza-2′-deoxycytidine (DAC) therapy, which further disrupts cancer cells' epigenomic fidelity and reactivates transposable element (TE) expression to drive viral mimicry responses. SETD2 encodes a histone methyltransferase (H3K36me3) and is prevalently mutated in advanced kidney cancers. Here, we show that SETD2-mutant kidney cancer cells are especially sensitive in vitro and in vivo to DAC treatment. We find that the viral mimicry response are direct consequences of mis-splicing events, such as exon inclusions or extensions, triggered by DAC treatment in an SETD2-loss context. Comprehensive epigenomic analysis reveals H3K9me3 deposition, rather than DNA methylation dynamics, across intronic TEs might contribute to elevated mis-splicing rates. Through epigenomic and transcriptomic analyses, we show that SETD2-deficient kidney cancers are prone to mis-splicing, which can be therapeutically exacerbated with DAC treatment to increase viral mimicry activation and provide synergy with combinatorial immunotherapy approaches.

Background: Myeloid malignancies, including myelodysplastic neoplasms (MDS), chronic myelomonocytic leukemia (CMML), and myeloproliferative neoplasms (MPNs), pose significant morbidity and mortality for affected patients and can progress to acute myeloid leukemia (AML). Additional sex combs-like protein 1 ( ASXL1) is an essential epigenetic regulator with important biological functions. Damaging ASXL1 mutations are prevalent in myeloid neoplasms and are associated with poor prognosis and lower response rates with hypomethylating agents (HMA). In a phase I-II trial published by O'Connell et al. in 2022, patients with MDS and CMML previously exposed to HMA were treated with a combination of atezolizumab, an immune checkpoint inhibitor (ICI), and guadecitabine, an HMA. Bone marrow (BM) CD3+ T cells and CD34+ myeloid cells were tested for a panel of recurrent myeloid mutations. The two patients who achieved complete remission carried the ASXL1 mutation in both cell compartments. Furthermore, the ASXL1 mutation in T cells correlated with a significantly longer overall survival among the 27 patients tested, suggesting that ASXL1 mutations may prime T cells for response to immune checkpoint blockade. In this pilot study, we aim to determine the frequency of ASXL1 mutated T cells and the impact on T cells methylation profiles in patients with untreated, ASXL1 mutated myeloid neoplasms. Methods:We included eight patients with known ASXL1 mutations detected by next-generation sequencing (NGS) in BM mononuclear cells . CD3+ T cells were isolated from the peripheral blood and separated by magnetic bead flow cytometry into CD8+ and CD4+ fractions. Sequencing libraries were generated using ClearSeq (Agilent) panel and were sequenced on MiSeq(Illumina). CD8+ T cell samples were aligned using a BWA aligner on Partek Flow (Partek) pipeline. Somatic calling was performed using the Strelka algorithm on Partek Flow. Each significant variant was confirmed by manual review using Integrative Genomics Viewer (IGV), and here we report the mutations present at a variant allele frequency (VAF) of at least 0.05 that correspond to those detected by NGS in the whole blood. In addition, DNA methylation studies were performed in the sorted T cells. We removed the loci with inadequate quality, and those associated with single nucleotide polymorphisms/age/sex chromatin. We performed methylation studies to identify differentially methylated locus and copy number variations. Results: We obtained peripheral blood samples from eight patients with ASXL1 mutated myeloid neoplasms detected by commercial NGS testing; aspirate was also obtained from a diagnostic BM biopsy in one patient. Table 1 shows patient demographics, characteristics and mutational analyses in both T and myeloid cells. Most patients were treatment-naïve, but one had been treated with HMA until 6 months prior to sample collection, and one patient had received cyclosporine for over 20 years for an undiagnosed autoimmune condition. T cells generated a mean sequencing coverage of 388x (min=341x, max=475x). ASXL1 mutated T cells were present in 50% (4/8) of the patients with myeloid ASXL1 mutations (Table 1). The median ASXL1VAF detected in T cells was 0.112. In 1 patient who also had a BM sample, the ASXL1 VAF in the T cells within the BM for this patient was 0.142, but only 0.082 in the peripheral blood. Methylation studies of peripheral blood T cell DNA did not delineate a clear difference between ASXL1 mutated and wild-type T cells (Figure 1). Discussion: Approximately 50% of patients with ASXL1 mutated myeloid malignancies carry the same mutation in their T lymphocytes, which confirms the findings from a published report in a different cohort of patients, all of whom had been previously exposed to HMA. In that study the T cells were collected from BM aspirate whereas in this study they were from the peripheral blood. Unsurprisingly, the ASXL1 VAFis lower in T cells than in myeloid cells and may be lower in the peripheral blood than in the marrow, but this needs confirmation. Methylation studies did not detect unique profiles in samples with mutated T cells, but we were not able to isolate ASXL1 mutated T cells, so these data are less likely to represent the affected clones. Given the potential benefit of treatment with ICI in these patients, further characterization of ASXL1 mutated T cells including functional studies and receptor profiling is warranted.

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.

You have accessJournal of UrologyCME1 Apr 2023MP69-05 FEASIBILITY OF DNA METHYLATION MARKERS FOR DIAGNOSIS AND PROGNOSIS OF UPPER TRACT UROTHELIAL CARCINOMA Farshad Sheybaee Moghaddam, Alireza Ghoreifi, Hongtao Li, Yohei Sekino, Kian Asanad, Manju Aron, Gangning Liang, and Hooman Djaladat Farshad Sheybaee MoghaddamFarshad Sheybaee Moghaddam More articles by this author , Alireza GhoreifiAlireza Ghoreifi More articles by this author , Hongtao LiHongtao Li More articles by this author , Yohei SekinoYohei Sekino More articles by this author , Kian AsanadKian Asanad More articles by this author , Manju AronManju Aron More articles by this author , Gangning LiangGangning Liang More articles by this author , and Hooman DjaladatHooman Djaladat More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003332.05AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Upper tract urothelial carcinoma (UTUC) is an uncommon, yet biologically heterogeneous disease that accounts for 5-10% of all urothelial tumors. The diagnosis and risk-stratification of UTUC patients is challenging given the limitations of current diagnostic tools. DNA methylation has emerged as a potential prognostic and diagnostic factor in urothelial carcinoma, yet few studies have examined its role in UTUC. In this study, we aim to identify specific tissue DNA methylation markers in muscle invasive (≥pT2) and non-muscle invasive (0.3, p<0.05) (Figure 2). CONCLUSIONS: UTUC has a distinct DNA methylation pattern that is distinguishable from normal urothelial tissue. UTUC-specific DNA methylation probes can be used to differentiate muscle-invasive UTUCs. Source of Funding: University of Southern California Urology research council © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e965 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Farshad Sheybaee Moghaddam More articles by this author Alireza Ghoreifi More articles by this author Hongtao Li More articles by this author Yohei Sekino More articles by this author Kian Asanad More articles by this author Manju Aron More articles by this author Gangning Liang More articles by this author Hooman Djaladat More articles by this author Expand All Advertisement PDF downloadLoading ...

Large-scale sequencing efforts of human cancers have identified recurrent mutations and deletions in a variety of chromatin-regulating proteins that modulate DNA methylation, histone modifications, and nucleosome positioning. The dysregulation of histone H3 lysine 36 (H3K36) methyltransferase, SETD2, through transcriptional or genetic aberrations is associated with worse clinical outcomes and metastasis in kidney cancer. Limited targeted therapeutic interventions are available for aggressive SETD2-mutant ccRCC tumors. Here, we reveal that kidney cancer cells displaying diminished H3K36 trimethylation levels, a consequence of SETD2 deficiency, show increased sensitivity of anti-tumor effects to DNA hypomethylating agent (HMA). We discovered that HMA treatment induced stronger viral mimicry activation and immune upregulation, which is potentiated by higher transposable element (TE) expression in SETD2-mutant cancer cells. Mechanistically, we provide evidence that substantial number of the HMA-induced TE expression is a consequence of mis-splicing, which is associated deficient in slicing in SETD2-loss content along with rapid gain of H3K9me3 across exons. These all suggested HMA could turn immune-cold tumor to immune-hot tumor and sensitize tumors to immune therapy. Then we performed in vivo assay in immune competent mice. Indeed, SETD2 deficient tumors were extremely sensitive to combination treatment of HMA and immune checkpoint inhibitor. Our finding provides one of the first preclinical and in vivo evidence that demonstrates the SETD2 dysregulation can be an epigenetic therapeutic target in ccRCC, especially in combination with immune checkpoint inhibitors, for future clinical trials. Citation Format: Hong-Tao Li, Hyo Sik Jang, Krizia Rohena-Rivera, Hemant Gujar, Minmin Liu, Justin Kulchycki, Xinyi Zhou, Shuqing Zhao, Peter Jones, Neil Bhowmick, Gangning Liang. Loss of SETD2 sensitizes kidney cancer cells to DNA methylation inhibitors by inducing viral mimicry and RNA mis-splicing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3618.