DNA Methyltransferase Inhibitors Treatment Research Articles

Methylation-regulated tumor suppressor gene PDE7B promotes HCC invasion and metastasis through the PI3K/AKT signaling pathway.

Hepatocellular carcinoma (HCC) has a high mortality rate, and the mechanisms underlying tumor development and progression remain unclear. However, inactivated tumor suppressor genes might play key roles. DNA methylation is a critical regulatory mechanism for inactivating tumor suppressor genes in HCC. Therefore, this study investigated methylation-related tumor suppressors in HCC to identify potential biomarkers and therapeutic targets. We assessed genome-wide DNA methylation in HCC using whole genome bisulfite sequencing (WGBS) and RNA sequencing, respectively, and identified the differential expression of methylation-related genes, and finally screened phosphodiesterase 7B (PDE7B) for the study. The correlation between PDE7B expression and clinical features was then assessed. We then analyzed the changes of PDE7B expression in HCC cells before and after DNA methyltransferase inhibitor treatment by MassArray nucleic acid mass spectrometry. Furthermore, HCC cell lines overexpressing PDE7B were constructed to investigate its effect on HCC cell function. Finally, GO and KEGG were applied for the enrichment analysis of PDE7B-related pathways, and their effects on the expression of pathway proteins and EMT-related factors in HCC cells were preliminarily explored. HCC exhibited a genome-wide hypomethylation pattern. We screened 713 hypomethylated and 362 hypermethylated mCG regions in HCC and adjacent normal tissues. GO analysis showed that the main molecular functions of hypermethylation and hypomethylation were "DNA-binding transcriptional activator activity" and "structural component of ribosomes", respectively, whereas KEGG analysis showed that they were enriched in "bile secretion" and "Ras-associated protein-1 (Rap1) signaling pathway", respectively. PDE7B expression was significantly down-regulated in HCC tissues, and this low expression was negatively correlated with recurrence and prognosis of HCC. In addition, DNA methylation regulates PDE7B expression in HCC. On the contrary, overexpression of PDE7B inhibited tumor proliferation and metastasis in vitro. In addition, PDE7B-related genes were mainly enriched in the PI3K/ATK signaling pathway, and PDE7B overexpression inhibited the progression of PI3K/ATK signaling pathway-related proteins and EMT. PDE7B expression in HCC may be regulated by promoter methylation. PDE7B can regulate the EMT process in HCC cells through the PI3K/AKT pathway, which in turn affects HCC metastasis and invasion.

Select EZH2 inhibitors enhance viral mimicry effects of DNMT inhibition through a mechanism involving NFAT:AP-1 signaling.

DNA methyltransferase inhibitor (DNMTi) efficacy in solid tumors is limited. Colon cancer cells exposed to DNMTi accumulate lysine-27 trimethylation on histone H3 (H3K27me3). We propose this Enhancer of Zeste Homolog 2 (EZH2)-dependent repressive modification limits DNMTi efficacy. Here, we show that low-dose DNMTi treatment sensitizes colon cancer cells to select EZH2 inhibitors (EZH2is). Integrative epigenomic analysis reveals that DNMTi-induced H3K27me3 accumulates at genomic regions poised with EZH2. Notably, combined EZH2i and DNMTi alters the epigenomic landscape to transcriptionally up-regulate the calcium-induced nuclear factor of activated T cells (NFAT):activating protein 1 (AP-1) signaling pathway. Blocking this pathway limits transcriptional activating effects of these drugs, including transposable element and innate immune response gene expression involved in viral defense. Analysis of primary human colon cancer specimens reveals positive correlations between DNMTi-, innate immune response-, and calcium signaling-associated transcription profiles. Collectively, we show that compensatory EZH2 activity limits DNMTi efficacy in colon cancer and link NFAT:AP-1 signaling to epigenetic therapy-induced viral mimicry.

Abstract 5881: Upregulation of TET2 expression by DNA methyltransferase (DNMT) inhibitors is associated with resistance in DNMT1 knockout colorectal cancer cells

Abstract Ten-eleven-translocation 2 (TET2) is a member of the TET family proteins (TET1-3) that convert 5-methylcytosine to 5-hydroxymethylcytosine (5-hmC) in DNA and causes site-specific DNA demethylation. The effects of DNMT1 on TET2 phenotype following DNMT inhibitor treatment is unclear in human cancer. In colorectal cancer HCT116 cells with DNMT1 gene intact (DNMT1+/+) and deletion (DNMT1−/−) status, we found that TET2 expression was robustly increased in DNMT1−/− cells after treatment with 0.5 µM and 5 µM decitabine for 72 h, 96 h, and 120 h. The augmentation in TET2 expression was accompanied by a dose-dependent re-expression of p16ink4A in DNMT1−/− cells. Particularly, TET2 upregulation was associated with resistance to DNMT inhibitors, showing a lack of induction of DNA damage, cell-cycle arrest and apoptosis after drug exposure in DNMT1−/− compared to DNMT1+/+ cells. The treatment with 4’-thio-5-aza-2’-deoxycytidine (aza-T-dCyd) only up-regulated TET2 and re-expressed p16ink4A at the lower dose of 0.5 µM, which was consistent with our previous findings that high dose of aza-T-dCyd at 5 µM predominantly eliminates the cancer cells with severe DNA damage by undergoing apoptosis and cell death (Scientific Reports, 13:5964, 2023). Moreover, both decitabine and aza-T-dCyd had minimal effects on TET2 upregulation and p16ink4A re-expression in cells with intact DNMT1 gene. The data suggest that the absence of DNMT1 gene made the cells prone to TET2 upregulation upon DNMT inhibitor challenges. Future study is needed to investigate the interactive relationship between DNMT1 and TET2 as well as demethylation, and its impact on cancer treatment with DNMT inhibitors. Citation Format: Angelo B. Laranjeira, Dat Nguyen, Lorraine C. Pelosof, James H. Doroshow, Sherry X. Yang. Upregulation of TET2 expression by DNA methyltransferase (DNMT) inhibitors is associated with resistance in DNMT1 knockout colorectal cancer cells [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 5881.

Abstract 3289: APOBEC3A drives tumor evolution through activation of ERVs in non small cell lung cancer

Abstract Acquired drug resistance to even the most effective anti-cancer targeted therapies remains an unsolved clinical problem. Although many drivers of acquired drug resistance have been identified, the underlying molecular mechanisms shaping tumor evolution during treatment are incompletely understood. We recently demonstrated that lung cancer targeted therapies commonly used in the clinic induce the expression of cytidine deaminase APOBEC3A (A3A), leading to sustained mutagenesis in drug-tolerant cancer cells persisting during therapy. Preventing therapy-induced A3A mutagenesis by gene deletion decreased the chromosomal aberration events and delayed the emergence of drug resistance. Thus, inhibition of A3A may represent a potential therapeutic strategy to prevent acquired resistance. Understanding the regulatory mechanisms of A3A may provide alternative approaches to inhibit A3A mutagenesis. Using a panel drug screen, we found that DNA methyltransferase (DNMT) inhibitor remarkably induces A3A in non-small cell lung cancer cells. RNA-seq profiling revealed that targeted therapy as well as DNMT inhibitor treatment activate expression of distinct classes non-coding repetitive RNA elements, including endogenous retrovirus (ERVs), in drug-tolerant persister cells. Activation of intracellular viral sensing pathways by introduction of exogenous nucleic acids mimicking viral elements induced A3A expression. While the transcription factor NFkB was acutely activated by targeted therapy, TBK1-IRF3 signaling was activated at later time points. These findings suggest that reactivation of ERVs may underlie A3A mutagenic activity and tumor evolution during targeted therapy. Citation Format: Hideko Isozaki, Ramin Sakhtemani, Naveed Nikpour, Susanna Monroe, Michael Lawrence, Aaron Hata. APOBEC3A drives tumor evolution through activation of ERVs in non small cell lung cancer [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 3289.

Abstract PR-005: ZNFX1 is a master regulator for epigenetic reprograming of mitochondrial inflammasome signaling and pathogen mimicry in cancer cells

Abstract Immunomodulatory agents represent an important recent advance in cancer therapy, but utility is often limited by tumor immune evasion mechanisms. Strategic therapeutic activation of intracellular antiviral immune responses offers an opportunity to reverse immune evasion mechanisms and improve treatment outcomes. Anti-cancer agents such as, DNA methyltransferase inhibitors (DNMTis) induce re-expression of endogenous retroviral elements (ERVs), leading to cytosolic double-stranded RNA (dsRNA) accumulation that activates interferon/inflammasome signaling. Moreover, poly (ADP ribose) polymerase inhibitors (PARPi) increase cytosolic dsDNA leading to activation of stimulator of interferon genes (STING). We previously reported in triple negative breast cancer (TNBC) and ovarian cancer (OC) that DNMTis in combination with PARPi induce STING-dependent interferon/inflammasome signaling in a process termed pathogen mimicry response (PMR). Mitochondria (mt) are an important gateway for antiviral inflammasome signaling, but upstream activating events in cancer are not understood. We now show NFX1-type zinc finger–containing 1 (ZNFX1), a little-studied innate immune gene, acts as a master nucleic acid (dsRNA/DNA) sensor for this mt gateway function. Importantly, in primary ovarian tumors from TCGA and clinical trial RNAseq datasets, increased ZNFX1 expression tracks with tumor stage and grade but inversely correlates with a mt dysfunction signature. In studies of high grade serous (TYK-nu, OVCAR4) as well as endometrial ovarian (A2780) cancer cells, transfection of dsRNA/DNA mimics or DNMTi azacytidine (AZA) and PARPi (talazoparib) treatments induce increased ZNFX1 expression and binding to mt antiviral protein (MAVs) localized on the mt outer membrane, using immunofluorescence-based proximity ligation assays. In studies of mt dysfunction, we further show that dsRNA/DNA as well as above-described anti-cancer drugs increase mt reactive oxygen species (ROS), using flow cytometry of ROS dye mitoSOX. DNMTi and PARPi treatments also increase fragmented mtDNA and oxidative mtDNA base damage, as measured long range PCR and 8-oxoguanine (8-oxoG) ELISA assays, respectively. Importantly, these drug treatment increase release of mtDNA into the cytosol, resulting in STING-dependent inflammasome signaling and cytokine release. ZNFX1 knockout robustly attenuates these dynamics, thus defining this immune gene as essential for interferon/inflammasome signaling induced by mtDNA damage. Our data indicates a master role for the little-studied dsRNA/DNA sensor ZNFX1 in initiating a pathogen mimicry response to DNMTi and PARPi combination treatment, and further highlights the critical role of the mt in cellular antiviral signaling mechanisms. Importantly, this work suggests a novel avenue for manipulation of inflammasome signaling to improve cancer therapy responses and disease outcomes. Citation Format: Lora Stojanovic, Rachel Abbotts, Kaushlendra Tripathi, Collin M. Coon, Sheng Liu, Jun Wan, Michael J. Topper, Kenneth P. Nephew, Stephen B. Baylin, Feyruz V. Rassool. ZNFX1 is a master regulator for epigenetic reprograming of mitochondrial inflammasome signaling and pathogen mimicry in cancer cells [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr PR-005.

DNA damage, demethylation and anticancer activity of DNA methyltransferase (DNMT) inhibitors

Role of DNA damage and demethylation on anticancer activity of DNA methyltransferase inhibitors (DNMTi) remains undefined. We report the effects of DNMT1 gene deletion/disruption (DNMT1−/−) on anticancer activity of a class of DNMTi in vitro, in vivo and in human cancers. The gene deletion markedly attenuated cytotoxicity and growth inhibition mediated by decitabine, azacitidine and 5-aza-4′-thio-2′-deoxycytidine (aza-T-dCyd) in colon and breast cancer cells. The drugs induced DNA damage that concurred with DNMT1 inhibition, subsequent G2/M cell-cycle arrest and apoptosis, and upregulated p21 in DNMT1+/+ versus DNMT1−/− status, with aza-T-dCyd the most potent. Tumor growth and DNMT1 were significantly inhibited, and p21 was upmodulated in mice bearing HCT116 DNMT1+/+ xenograft and bladder PDX tumors. DNMT1 gene deletion occurred in ~ 9% human colon cancers and other cancer types at varying degrees. Decitabine and azacitidine demethylated CDKN2A/CDKN2B genes in DNMT1+/+ and DNMT1−/− conditions and increased histone-H3 acetylation with re-expression of p16INK4A/p15INK4B in DNMT1−/− state. Thus, DNMT1 deletion confers resistance to DNMTi, and their anti-cancer activity is determined by DNA damage effects. Patients with DNMT1 gene deletions may not respond to DNMTi treatment.

Abstract 6020: The role of TP53 mutation status in the response to DNA methyltransferase inhibitor treatment

Abstract The purpose of this study was to understand the effect of p53 mutation status in ovarian cancer cells on the expression of repetitive elements (REs) following epigenetic treatment. High-grade serous ovarian carcinoma is the most aggressive subtype of ovarian cancer and the 5 year survival rate has remained unchanged for over two decades. Over 90% of high-grade serous ovarian carcinoma (OC) patients have a mutation in the TP53 gene, which encodes the p53 protein. P53 is a transcription factor primarily involved in maintaining genome integrity, but it can also regulate the expression of REs in the genome. REs account for more than half of the genome and these repetitive genomic elements are epigenetically silenced in healthy cells to prevent genome instability. The expression of some REs, such as long interspersed nuclear elements and endogenous retroviruses, are upregulated in human OC patients and cell lines. The mechanism by which p53 regulates expression of REs is not well-understood; the literature documents some instances of p53 activating RE transcription and others of p53 repressing RE transcription, depending on the target elements. We have previously shown that RE expression can be induced by treating OC cells with low doses of DNA methyltransferase inhibitors (DNMTis). Recently, we showed that OC cell lines with mutant p53 have higher baseline expression of REs compared to wild type (WT) cell lines. Treatment of TP53 mutant OC cell lines with DNMTis had a muted effect on RE expression compared to treatment of WT OC cell lines. To explore the relationship between TP53 mutation status and RE expression after DNMTi treatment, OC cell lines with different p53 mutation statuses were treated with 500 nM of the DNMTi 5-azacytidine. We then analyzed the expression of several REs via RT-qPCR and western blot. We hypothesized that TP53 mutant cell lines would have lower DNMTi upregulation of REs than TP53 WT cell lines. While at the RNA level RE expression was variable, at the protein level, we found that DNMTi treatment increased protein levels of several REs in TP53 WT and mutant, but not null, OC cell lines. Future work will examine the feasibility of utilizing these RE proteins as immunotherapy targets in OC. Citation Format: Khadra K. Omar, Erin E. Grundy, Olivia Cox, Melissa Hadley, Katherine B. Chiappinelli. The role of TP53 mutation status in the response to DNA methyltransferase inhibitor treatment [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 6020.

Inhibiting DNA methylation and RNA editing upregulates immunogenic RNA to transform the tumor microenvironment and prolong survival in ovarian cancer

BackgroundNovel therapies are urgently needed for ovarian cancer (OC), the fifth deadliest cancer in women. Preclinical work has shown that DNA methyltransferase inhibitors (DNMTis) can reverse the immunosuppressive tumor microenvironment...

DNMT and EZH2 inhibitors synergize to activate therapeutic targets in hepatocellular carcinoma

The development of more effective targeted therapies for hepatocellular carcinoma (HCC) patients due to its aggressiveness is urgently needed. DNA methyltransferase inhibitors (DNMTis) represented the first clinical breakthrough to target aberrant cancer epigenomes. However, their clinical efficacies are still limited, in part due to an “epigenetic switch” in which a large group of genes that are demethylated by DNMTi treatment remain silenced by polycomb repressive complex 2 (PRC2) occupancy. EZH2 is the member of PRC2 that catalyzes the placement of H3K27me3 marks. EZH2 overexpression is correlated with poor HCC patient survival. We tested the combination of a DNMTi (5-aza-2′-deoxycytidine, DAC) and the EZH2 inhibitor (EZH2i) GSK126 in human HCC cell lines on drug sensitivity, DNA methylation, nucleosome accessibility, and gene expression profiles. Compared with single agent treatments, all HCC cell lines studied showed increased sensitivity after receiving both drugs concomitant with prolonged anti-proliferative changes and sustained reactivation of nascently-silenced genes. The increased number of up-regulated genes after combination treatment correlated with prolonged anti-proliferation effects and increased nucleosome accessibility. Combination treatments also activate demethylated promoters that are repressed by PRC2 occupancy. Furthermore, 13–31% of genes down-regulated by DNA methylation in primary HCC tumors were reactivated through this combination treatment scheme in vitro. Finally, the combination treatment also exacerbates anti-tumor immune responses, while most of these genes were downregulated in over 50% of primary HCC tumors. We have linked the anti-tumor effects of DAC and GSK126 combination treatments to detailed epigenetic alterations in HCC cells, identified potential therapeutic targets and provided a rationale for treatment efficacy for HCC patients.

Activating STING1-dependent immune signaling in TP53 mutant and wild-type acute myeloid leukemia

DNA methyltransferase inhibitors (DNMTis) reexpress hypermethylated genes in cancers and leukemias and also activate endogenous retroviruses (ERVs), leading to interferon (IFN) signaling, in a process known as viral mimicry. In the present study we show that in the subset of acute myeloid leukemias (AMLs) with mutations in TP53, associated with poor prognosis, DNMTis, important drugs for treatment of AML, enable expression of ERVs and IFN and inflammasome signaling in a STING-dependent manner. We previously reported that in solid tumors poly ADP ribose polymerase inhibitors (PARPis) combined with DNMTis to induce an IFN/inflammasome response that is dependent on STING1 and is mechanistically linked to generation of a homologous recombination defect (HRD). We now show that STING1 activity is actually increased in TP53 mutant compared with wild-type (WT) TP53 AML. Moreover, in TP53 mutant AML, STING1-dependent IFN/inflammatory signaling is increased by DNMTi treatment, whereas in AMLs with WT TP53, DNMTis alone have no effect. While combining DNMTis with PARPis increases IFN/inflammatory gene expression in WT TP53 AML cells, signaling induced in TP53 mutant AML is still several-fold higher. Notably, induction of HRD in both TP53 mutant and WT AMLs follows the pattern of STING1-dependent IFN and inflammatory signaling that we have observed with drug treatments. These findings increase our understanding of the mechanisms that underlie DNMTi + PARPi treatment, and also DNMTi combinations with immune therapies, suggesting a personalized approach that statifies by TP53 status, for use of such therapies, including potential immune activation of STING1 in AML and other cancers.

Epigenetic Therapies in Ovarian Cancer Alter Repetitive Element Expression in a TP53-Dependent Manner.

Epithelial ovarian carcinomas are particularly deadly due to intratumoral heterogeneity, resistance to standard-of-care therapies, and poor response to alternative treatments such as immunotherapy. Targeting the ovarian carcinoma epigenome with DNA methyltransferase inhibitors (DNMTi) or histone deacetylase inhibitors (HDACi) increases immune signaling and recruits CD8+ T cells and natural killer cells to fight ovarian carcinoma in murine models. This increased immune activity is caused by increased transcription of repetitive elements (RE) that form double-stranded RNA (dsRNA) and trigger an IFN response. To understand which REs are affected by epigenetic therapies in ovarian carcinoma, we assessed the effect of DNMTi and HDACi on ovarian carcinoma cell lines and patient samples. Subfamily-level (TEtranscripts) and individual locus-level (Telescope) analysis of REs showed that DNMTi treatment upregulated more REs than HDACi treatment. Upregulated REs were predominantly LTR and SINE subfamilies, and SINEs exhibited the greatest loss of DNA methylation upon DNMTi treatment. Cell lines with TP53 mutations exhibited significantly fewer upregulated REs with epigenetic therapy than wild-type TP53 cell lines. This observation was validated using isogenic cell lines; the TP53-mutant cell line had significantly higher baseline expression of REs but upregulated fewer upon epigenetic treatment. In addition, p53 activation increased expression of REs in wild-type but not mutant cell lines. These data give a comprehensive, genome-wide picture of RE chromatin and transcription-related changes in ovarian carcinoma after epigenetic treatment and implicate p53 in RE transcriptional regulation. SIGNIFICANCE: This study identifies the repetitive element targets of epigenetic therapies in ovarian carcinoma and indicates a role for p53 in this process.

DNA Demethylating Agents Generate a Brcaness Effect in Multiple Sporadic Tumor Types: Prediction for Sensitivity to PARP Inhibitors in AML

Poly (ADP-ribose) polymerase inhibitors (PARPis) have shown clinical efficacy in breast and ovarian cancers with inherited mutations in BRCA genes that abrogate homologous recombination (HR) DNA double-strand break (DSB) repair activity. However, to date, PARPishave not had success in sporadic breast and ovarian cancers, or other cancers, such as acute myeloid leukemia (AML), with intact BRCA genes. We have recently reported that triple-negative breast cancer (TNBC) and AML are sensitive to low doses of the potent PARPiTalazoparib in combination with DNA methyl transferase inhibitors (DNMTi), decitabine (DAC) or azacitidine (AZA), in vitro and in vivo. In these cancer models, the DNMTi/PARPicombination increases amplitude and retention of PARP1 directly at laser-induced DNA damage sites, increasing DNA DSBs, promoting synergistic tumor cytotoxicity, blunting cell self-renewal, and leading to strong anti-tumor responses. These results have led to a Phase I/II Clinical trial of DNMTi/PARPi combination therapy in patients with relapsed/refractory AML that is underway.In identifying the molecular pathways that may underlie efficacy of this inhibitor combination in sporadic TNBC, ovarian cancer and AML, we find that low doses of DNMTisreprogram the cancer genome, in part by changing gene expression patterns of DSB repair genes, potentially creating a HR defect (HRD) or “BRCAness” effect. Expression array (Agilent Human 44K v2) analysis was performed on RNA extracted from multiple BRCA-intact sporadic cell lines from AML (including, Kasumi-1, KG-1 and MOLM-14), TNBC (including, MDA-MB-231, SUM159) and ovarian cancer (including, A2780) on days 1, 3, 5 and 7 and 10 post DNMTi(DAC 10nM or AZA 500nM) treatment. Hallmark, KEGG and reactome gene ontologies were utilized. Only gene sets with log fold change (FDR) >.250 were considered significant and genes demonstrating 2-fold or greater change with drug treatment were selected. While multiple DNA repair pathway genes were found both up- and down- regulated post DNMTi treatment, one or more HR genes, were consistently downregulated in all cell lines examined. QPCR of mRNA and/or immunoblotting of HR proteins, in particular RAD51, BRCA1, BRCA2, or HR-related genes, FEN1 or FANCD2, showed significantly decreased expression with DNMTi treatment (p<0.05) in TNBC, ovarian and AML cell lines, validating array data. Functional assays in TNBC cell lines, using a stably expressed HR repair reporter (DRGFP) also confirmed decreased HR activity with DNMTi treatment. Additionally, in response to DNA damage with ionizing radiation (IR, 4Gy), RAD51 foci were reduced in AZA-treated TNBC and ovarian cancer cell lines, compared to untreated controls. Significantly increased triradial chromosomes were also observed in TNBC in the presence of AZA when exposed to DNA cross-linking agents mitomycin C (MMC, 50ng/ml) or cisplatin (2μM) treatment, confirming functional down-regulation of the HR-related FANCD2 pathway.The above HRD-like or BRCAness phenotype with DNMTi treatment translated to sensitivity to PARPis in primary bone marrow mononuclear cells (BMMNC) from AML patients. Thus, in 6 of 8 (75%) AML samples, HR gene expression by QPCR of RNA and HR functional activity by immunostaining for RAD51 foci, following DAC treatment (10nM, 72 hrs), DNMTi treatment resulted in a significant decrease in HR or HR-related gene expression, including RAD51, BRCA1, BRCA2, and decreased levels of RAD51 foci (p<0.05). All 6 AML samples were also significantly more sensitive to DAC/talazoparib combination treatment (p<0.05) in colony forming assays, compared with single agent treatments. AML samples included those with MLL translocations that are reported to have high HR activity and resistance to PARPis. Studies are underway to elucidate the molecular characteristics of the minority of AML patient samples (N=2) that did not generate a BRCAness effect with DAC treatment. In conclusion, our data suggest that one of the mechanisms underlying DNMTi/PARPi therapy efficacy in AML is DNMTi-induced BRCAness. In vitro testing for BRCAness with DNMTitreatment could be a biomarker to predict sensitivity to DNMTi/PARPis and inform selection of AML patients for future clinical trials of this inhibitor combination. DisclosuresCivin:ConverGene LLC: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; 3DBioWork Inc: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; BD (Becton Dickinson): Honoraria; GPB Scientific LLC: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Kingsbury:3DBioWorks: Consultancy, Equity Ownership.

Loss of Adar1 increases T cell migration and prolongs survival in ovarian cancer

Abstract Novel therapies are urgently needed for ovarian cancer (OC), the fifth deadliest cancer in women. OC is generally characterized by an immunosuppressive tumor microenvironment (TME) and less than 10% of patients respond to immune checkpoint blockade (ICB) therapy, though better OC prognosis is associated with high tumor-infiltrating CD8 T cells. DNA methyltransferase inhibitors (DNMTis) remove methylation and activate transcription of double-stranded (ds)RNA in OC. These dsRNAs are sensed in the cytoplasm and trigger the induction of type I IFN and also the transcription of interferon-stimulated genes (ISGs). Adenosine deaminase 1 (ADAR1) is an ISG that edits mammalian dsRNA with an A-to-I nucleotide change. These edited dsRNAs cannot be sensed by dsRNA sensors, and thus ADAR1 can inhibit this type I IFN response. Our preliminary RNA-seq analysis from human OC cell lines indicates that RNA editing by ADAR1 is increased after DNMTi treatment. To test the hypothesis that Adar1 loss will amplify the DNMTi-induced IFN response in OC, we implanted mice with syngeneic ID8 murine OC Adar1 knockdown (KD) cells or control cells and treated with DNMTi. We show that DNMTi treatment reduces tumor burden and extends survival in this OC model. Loss of Adar1 significantly prolongs survival in this model, which is further improved with DNMTi treatment. DNMTi treatment of murine OC cells combined with Adar1 KD increases T cell migration in transwell migration assays and also enhances expression of chemokines CCL5, CCL2, and CXCL10. We are currently performing immunophenotyping analyses to interrogate the composition of immune cells in the TME in the mouse model of OC. These studies thus describe a role of Adar1 in the DNMTi-induced immune response in OC.

Epigenetic modulation of immune synaptic-cytoskeletal networks potentiates \u03b3\u03b4 T cell-mediated cytotoxicity in lung cancer

γδ T cells are a distinct subgroup of T cells that bridge the innate and adaptive immune system and can attack cancer cells in an MHC-unrestricted manner. Trials of adoptive γδ T cell transfer in solid tumors have had limited success. Here, we show that DNA methyltransferase inhibitors (DNMTis) upregulate surface molecules on cancer cells related to γδ T cell activation using quantitative surface proteomics. DNMTi treatment of human lung cancer potentiates tumor lysis by ex vivo-expanded Vδ1-enriched γδ T cells. Mechanistically, DNMTi enhances immune synapse formation and mediates cytoskeletal reorganization via coordinated alterations of DNA methylation and chromatin accessibility. Genetic depletion of adhesion molecules or pharmacological inhibition of actin polymerization abolishes the potentiating effect of DNMTi. Clinically, the DNMTi-associated cytoskeleton signature stratifies lung cancer patients prognostically. These results support a combinatorial strategy of DNMTis and γδ T cell-based immunotherapy in lung cancer management.

Transcriptional analysis of multiple ovarian cancer cohorts reveals prognostic and immunomodulatory consequences of ERV expression

BackgroundEndogenous retroviruses (ERVs) play a role in a variety of biological processes, including embryogenesis and cancer. DNA methyltransferase inhibitors (DNMTi)-induced ERV expression triggers interferon responses in ovarian cancer cells via...

Abstract PO-015: Hypermethylated genes in leiomyosarcoma can be targeted through guadecitabine treatment

Abstract Leiomyosarcomas (LMS) are aggressive mesenchymal tumors of smooth muscle origin. Understanding the molecular alterations associated with LMS is needed to identify potential therapeutic targets. Epigenetic alterations such as hypermethylation of gene promoter regions with associated transcriptional silencing is a hallmark of most cancers. Epigenetic reprograming is fundamentally linked to tumor initiation, progression and metastasis. Frequent epigenetic alterations have been observed in LMS. Epigenetic drugs such as DNA methyltransferase inhibitors (DNMTi) have been used to reverse aberrant DNA methylation and restore expression of silenced genes. Our previous studies using DNMTi, including guadecitabine, have shown that DNMTi treatment has anti-proliferative and pro-apoptotic effects in in vitro and in vivo models of LMS. Our goal was to characterize the methylation changes following guadecitabine treatment of the leiomyosarcoma cell line SK-UT1. SK-UT1 cells were treated with guadecitabine (0.05 µM-0.1 µM) and methylation was determined using Illumina 450K platform. We filtered the promoter CpG island regions that respond to guadecitabine treatment and were associated with decreased methylation compared to untreated SK-UT1 control (p≤0.05, Δβ≥0.15). In order to assess the relevance of these differentially methylated regions, we compared them to the methylation and expression profiles of LMS patient samples (GSE68312). We focused on the genes that have decreased expression and are associated with increased methylation of the promoter CpG island regions in the patient LMS samples. In conclusion, treatment of SK-UT1 cell line with guadecitabine resulted in decrease of methylation of a subset of promoter CpG island regions that were differentially hypermethylated in LMS patient samples. Further studies are needed to explore the effect of differential methylation on gene expression in guadecitabine treated SK-UT1 cell line. Understanding the effect of DNMTi on LMS is needed to identify potential therapeutic applications targeting the epigenetic pathways. Funding supported by Astex Pharmaceuticals. Citation Format: Nesrin Hasan, Cynthia De Carvalho Fischer, Anup Sharma, Nita Ahuja. Hypermethylated genes in leiomyosarcoma can be targeted through guadecitabine treatment [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-015.

Epigenetic Variation Induced by Gamma Rays, DNA Methyltransferase Inhibitors, and Their Combination in Rice.

DNA methylation plays important roles in the regulation of gene expression and maintenance of genome stability in many organisms, including plants. In this study, we treated rice with gamma rays (GRs) and DNA methyltransferase inhibitors (DNMTis) to induce variations in DNA methylation and evaluated epigenetic diversity using methylation-sensitive amplified polymorphism (MSAP) and transposon methylation display (TMD) marker systems. Comparative and integrated analyses of the data revealed that both GRs and DNMTis alone have epimutagenic effects and that combined treatment enhanced these effects. Calculation of methylation rates based on band scoring suggested that both GRs and DNMTis induce epigenetic diversity by demethylation in a dose-dependent manner, and combined treatment can induce variations more synergistically. The difference in the changes in full and hemi-methylation rates between MSAP and TMD is presumed to be caused by the different genomic contexts of the loci amplified in the two marker systems. Principal coordinate, phylogenic, and population structure analyses commonly yielded two clusters of individuals divided by DNMTi treatment. The clustering pattern was more apparent in TMD, indicating that DNMTis have a stronger effect on hypermethylated repetitive regions. These findings provide a foundation for understanding epigenetic variations induced by GRs and DNMTis and for epigenetic mutation breeding.

Abstract 3892: Targeting Hippo-dependent and Hippo-independent regulation of the YAP1 oncoprotein in childhood rhabdomyosarcoma

Abstract Background: Patients with metastatic or recurrent rhabdomyosarcoma (RMS), the most common childhood soft tissue sarcoma, continue to do poorly and new treatments are urgently needed. Previously, we showed that the Src family tyrosine kinase YES1 is upregulated in RMS and necessary for growth, but clinical trials using single agent Dasatinib (Src family kinase inhibitor) have failed in sarcomas. YAP1 (YES-associated protein) is also highly expressed in RMS, driving growth and survival when the upstream Hippo tumor suppressor pathway is silenced. Thus far, efforts to pharmacologically inhibit YAP1 have been unsuccessful. We hypothesize that a better understanding of the Hippo-dependent and Hippo-independent (through YES1) regulation of YAP1 in RMS may lead to novel therapeutic approaches. Methods: We studied the effect of DNA methyltransferase inhibitors (DNMTi) on cell growth in human RMS cell lines using live cell imaging and analyzed the YES1-YAP1 interaction by proximity ligation assays. We analyzed YES1 and YAP1 signaling using cell fractionation, immunoblots, qPCR, RNAseq, and genetic loss-of-function. We evaluated changes in DNA methylation by methylation-specific PCR and the Illumina EPIC methylation profiling microarray. To evaluate drug treatments in vivo we utilized orthotopic xenografts in immunocompromised mice. Results: We demonstrate that DNMTi treatment decreases RMS cell growth, and increases apoptosis and myogenic differentiation. DNMTi treatment upregulates Hippo-activators RASSF1 and RASSF5 by promoter demethylation, activates canonical Hippo signaling through LATS1/2, and increases inactivation of YAP1 by phosphorylation. The DNMTi-induced growth inhibition is partially rescued by expression of constitutively active YAPS127A, suggesting the effects of DNMTi treatment are in part due to Hippo-dependent inhibition of YAP1. Additionally, YES1 and YAP1 interact in the nucleus of RMS cells and YES1 shRNA knockdown or pharmacologic inhibition results in YAP1 cytoplasmic retention and decreased YAP1 target gene expression, suggesting YES1 can regulate YAP1 in a Hippo-independent manner. Lastly, to target both Hippo-dependent and Hippo-independent regulations of YAP1 we combined DNMTi treatment with Dasatinib. Combination treatment in vitro ablates RMS cell growth and induces more apoptosis than either drug alone. Combination treatment in orthotopic xenografts in mice results in decreased tumor weight, a trend towards decreased tumor growth, and an increase in myogenic differentiation gene expression. Conclusions: In RMS, YAP1 is regulated by Hippo-dependent and Hippo-independent mechanisms and both can be inhibited by combined treatment of a DNMTi with Dasatinib, laying the groundwork for future clinical investigations of this combination. Citation Format: Katherine Slemmons, Choh Yeung, Josh Baumgart, Jhazeel Martinez Juarez, Amy McCalla, Lee Helman. Targeting Hippo-dependent and Hippo-independent regulation of the YAP1 oncoprotein in childhood rhabdomyosarcoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3892.

Abstract A09: Targeting Hippo-dependent and Hippo-independent regulation of the YAP1 oncoprotein in childhood rhabdomyosarcoma

Abstract Background: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood. While outcomes for patients with localized disease have improved, patients with metastatic or recurrent disease continue to do poorly; therefore, new treatments are needed. Previously, we showed that the Src family tyrosine kinase YES1 is upregulated in RMS and necessary for growth, but clinical trials using single-agent dasatinib (Src family kinase inhibitor) have not shown clinical activity in sarcomas. YAP1 (YES-associated protein) is also highly expressed in human RMS tumors, driving cell growth and survival. Thus far, efforts to pharmacologically inhibit YAP1 have been unsuccessful. We hypothesize that a better understanding of the Hippo-dependent and Hippo-independent (through YES1) regulation of YAP1 in RMS may lead to novel therapeutic approaches. Methods: We studied the effect of DNA methyltransferase inhibitors (DNMTi) on cell growth in human RMS cell lines using live cell imaging and analyzed the YES-YAP interaction by proximity ligation assays. We analyzed YES1 and YAP1 signaling with cell fractionation, immunoblots, methylation-specific PCR, qPCR, RNAseq, and genetic loss of function. Results: We demonstrate that DNMTi treatment decreases RMS cell growth and increases apoptosis and differentiation. DNMTi treatment activates canonical Hippo signaling through LATS, shown by a LATS activity biosensor, and increases YAP1 phosphorylation. DNMTi treatment also induces RASSF1 re-expression by promoter demethylation, and the DNMTi-induced growth inhibition can be partially rescued by expression of constitutively active YAPS127A. RNAseq analysis after DNMTi treatment also revealed upregulation of RASSF family members RASSF5 and RASSF6 and downregulation of RASSF4. RASSF4 was previously shown to inhibit MST1 and promote tumorigenesis in the alveolar subtype of RMS. These data suggest the effects of DNMTi treatment are in part due to inhibition of YAP1 in a Hippo-dependent manner. DNMTi treatment also decreases YES1 expression and activation, suggesting DNMTis impact both YES1 and YAP1 activity. Furthermore, YES1 and YAP1 interact in the nucleus of RMS cells and shRNA knockdown of YES1 results in YAP1 cytoplasmic retention and decreased expression of YAP1 target genes, suggesting a role for YES1 in regulating YAP1 cellular localization in a Hippo-independent manner. Lastly, to target both Hippo-dependent and Hippo-independent regulation of YAP1 we combine DNMTi treatment with dasatinib, a YES1 kinase inhibitor. Combination treatment in vitro ablates RMS cell growth and leads to a greater induction of apoptosis than either drug alone. We are currently evaluating this combination strategy in RMS xenografts in mice. Conclusions: Overall, we show that YAP1 is regulated in both Hippo-dependent and Hippo-independent mechanisms in RMS and both mechanisms can be inhibited by a combined treatment of DNMTi and dasatinib, laying the groundwork for future clinical investigations of this novel therapy. Citation Format: Katherine K. Slemmons, Choh Yeung, Josh Baumgart, Amy McCalla, Lee Helman. Targeting Hippo-dependent and Hippo-independent regulation of the YAP1 oncoprotein in childhood rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr A09.

Targeting Hippo-Dependent and Hippo-Independent YAP1 Signaling for the Treatment of Childhood Rhabdomyosarcoma.

Rhabdomyosarcoma is the most common childhood soft-tissue sarcoma, yet patients with metastatic or recurrent disease continue to do poorly, indicating a need for new treatments. The SRC family tyrosine kinase YES1 is upregulated in rhabdomyosarcoma and is necessary for growth, but clinical trials using single agent dasatinib, a SRC family kinase inhibitor, have failed in sarcomas. YAP1 (YES-associated protein) is highly expressed in rhabdomyosarcoma, driving growth and survival when the upstream Hippo tumor suppressor pathway is silenced, but efforts to pharmacologically inhibit YAP1 have been unsuccessful. Here we demonstrate that treatment of rhabdomyosarcoma with DNA methyltransferase inhibitor (DNMTi) upregulates Hippo activators RASSF1 and RASSF5 by promoter demethylation, activating canonical Hippo signaling and increasing inactivation of YAP1 by phosphorylation. Treatment with DNMTi decreased rhabdomyosarcoma cell growth and increased apoptosis and differentiation, an effect partially rescued by expression of constitutively active YAP (S127A), suggesting the effects of DNMTi treatment are, in part, due to Hippo-dependent inhibition of YAP1. In addition, YES1 and YAP1 interacted in the nucleus of rhabdomyosarcoma cells, and genetic or pharmacologic suppression of YES1 resulted in cytoplasmic retention of YAP1 and decreased YAP1 target gene expression, suggesting YES1 regulates YAP1 in a Hippo-independent manner. Combined treatment with DNMTi and dasatinib targeted both Hippo-dependent and Hippo-independent regulation of YAP1, ablating rhabdomyosarcoma cell growth in vitro and trending toward decreased tumor growth in vivo. These results show that the mechanisms regulating YAP1 in rhabdomyosarcoma can be inhibited by combinatorial therapy of DNMTi and dasatinib, laying the groundwork for future clinical investigations. SIGNIFICANCE: This study elucidates the signaling pathways that regulate the oncogenic protein YAP1 and identifies a combination therapy to target these pathways in the childhood tumor rhabdomyosarcoma.