DNA-demethylating Agent Research Articles

Abstract 1735: DNA demethylating agents have cell type-specific effects on viability and BRCA2 alternative splicing

Abstract Introduction: BRCA2 is one of two principal tumor suppressors responsible for hereditary breast/ovarian cancer syndrome (HBOC). Patients with strong family histories of breast/ovarian cancer may carry BRCA2 DNA sequence variants of unknown clinical significance (VUSs). To evaluate which of these VUSs may contribute to cancer risk by altering splicing patterns, naturally occurring BRCA2 mRNA alternate splicing events have been extensively characterized, but it is not yet known which if any of these alternate splicing events plays a role in BRCA2 function. One variant lacking exon 3 (∆3), which maintains the full length translational reading frame, lacks sequence encoding EMSY and PALB binding domains as well as transactivation function. Previous work has shown that, while some VUSs associated with increased levels of ∆3 in lymphoblastoid cell lines are not pathogenic, germline deletions that eliminate BRCA2 exon 3 are associated with increased breast cancer risk. It is therefore of interest to characterize the factors that influence the frequency of naturally occurring BRCA2 exon 3-skipping, including cell types and therapies. DNA demethylating agents are used as cancer therapy to promote expression of tumor suppressor genes that may be epigenetically silenced in tumors. However, DNA methylation has been shown to be more frequent in exonic than intronic regions of protein-coding genes, suggesting that methylation may play a role in recruiting spliceosomal components to nascent pre-mRNA. It is therefore of interest to determine whether genomic demethylation affects alternative splicing patterns. Here, we explore whether BRCA2 exon 3 alternative splicing patterns are altered by demethylating agents. Methods: To determine whether DNA demethylating agents can alter the levels of ∆3, MCF7 and/or the non-cancer breast cell line MCF 10A was treated with 5-aza 2’-deoxycytidine (5-AzadC) or 5-Azacytidine (5-AzaC), and isoform-specific RT-PCR was used to compare relative levels of splice junctions containing or skipping exon 3. Results and Conclusions: 1) While the IC50 for both 5-AzadC and 5-AzaC in MCF 10A was similar (less than 1 μM), MCF7 showed considerably more resistance to 5-AzaC than 5-AzadC (IC50 of ≥ 5 μM vs ≤ 0.2 μM). 2) 5-AzaC treatment reduces the proportion of MCF 10A cultures in apoptosis but does not increase the proportion of viable cells, indicating it causes cell death by some other means. 3) While both 5-AzaC and 5-AzadC reduce relative levels of ∆3 in MCF7, 5-azadC does not reduce relative levels of ∆3 in MCF 10A, suggesting there may be a cell type-specific splicing response to genomic demethylation therapies. Citation Format: Jordan Werner, Hiba Siddiqui, Samiha Syed, Osman Khan, Servando Casillas Garabito, James D. Fackenthal. DNA demethylating agents have cell type-specific effects on viability and BRCA2 alternative splicing [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 1735.

Hypermethylation in the promoter regions of flavonoid pathway genes is associated with skin color fading during 'Daihong' apple fruit development.

Apple fruit skin color fading is not well understood although the molecular mechanism of skin color formation is well known. The red-fleshed apple cultivar 'Daihong' (DH) exhibited fading skin color during fruit development despite having a heterozygous R6 allele but lacking Red-TE for red fruit skin. In this study, transcriptomic analysis revealed the expression level of MdMYB10 increased with fruit development whereas reduced expression levels of MdMYBPA1, MdCHS, MdANS, MdUFGT, MdLAR, and MdANR were observed, consistent with decreased levels of chalcone, anthocyanin, catechin, epicatechin, and procyanidin B2. Whole-genome bisulfite sequencing (WGBS) indicated a global gain in cytosine methylation levels and increased methylation in 5' and 3' flanking regions of genes and transposable elements (TEs), and in TE bodies in all CG, CHG and CHH contexts, especially the mCHH context, during fruit development. The increased DNA methylation was attributed to reduced expression levels of DNA demethylase genes, including MdDME1, MdROS1, and MdROS2. Association analysis revealed a significant negative correlation between promoter methylation levels of MdCHS, MdCHI, MdMYBPA1, and their respective transcript levels, as well as a negative correlation between promoter methylation levels of MdCHS, MdCHI, MdANR, and MdFLS, and the content of chalcones, naringenin-7-glucoside, epicatechin, and quercetin. Treatment with the DNA demethylation agent 5-aza-2'-deoxycytidine verified the negative correlation between DNA methylation and gene expression within the flavonoid pathway. These findings suggest that hypermethylation in promoter regions of genes of the flavonoid biosynthesis pathway is associated with the reduction of gene expression and flavonoid content, and fruit skin color fading during DH apple development.

Abstract B091: Overexpression of Muscleblind Like Splicing Regulator 2 (MBNL2) enhances cisplatin resistance in ovarian cancer

Abstract Ovarian cancer is the second most commonly diagnosed gynecological cancer and has the highest mortality rate of all gynecological cancers worldwide. Although cisplatin is one of the most effective antitumor drugs for ovarian cancer, the emergence of chemoresistance to cisplatin is a major barrier to successful therapy. To identify epigenetically regulated genes directly associated with ovarian cancer cisplatin resistance, we compared the expression and methylation profiles of cisplatin-sensitive and -resistant human ovarian cancer cell lines. We identified Muscleblind Like Splicing Regulator 2 (MBNL2) as one of the key candidate genes for cisplatin drug response. Interestingly, in cisplatin-resistant cell lines, MBNL2 mRNA expression was significantly upregulated, and the MBNL2 promoter was associated with hypomethylation of the specific cytosine-phosphate-guanine (CpG) sites. Reduced MBNL2 expression in cisplatin-sensitive cell lines was restored by treatment with a DNA demethylation agent, suggesting epigenetic regulation of MBNL2 expression by promoter methylation. Furthermore, depletion of MBNL2 in cisplatin-resistant cells induced cytotoxicity in response to cisplatin, whereas overexpression of MBNL2 in cisplatin-sensitive cells increased chemoresistance, indicating that MBNL2 plays a vital role in developing chemoresistance in ovarian cancer cells. Furthermore, we performed meta-analysis using fourteen publicly available datasets to assess the association between expression of MBNL2 and overall survival in patients with serous-type ovarian cancer. The pooled Hazard ratios (HRs) estimate was significantly greater than 1 for overall survival (HR=1.09, p-value=0.0034). The association was more significant (HR=1.12, p-value=0.001) when tumor stage was added to the survival analysis as a covariate. The meta-analysis revealed that high expression of MBNL2 was significantly associated with poor prognosis and a higher risk of death in ovarian cancer patients with serous tumor. Additionally, we further validated the MBNL2 mRNA expression in primary ovarian tumor tissues. The statistically significant upregulation of MBNL2 mRNA expression was observed in cisplatin-resistant patients compared to that of cisplatin-sensitive patients. Our findings suggest MBNL2 may be a potential therapeutic target for the prevention of chemoresistance to cisplatin in ovarian cancer. Citation Format: Woong Ju, Hye Youn Sung, Jung-Hyuck Ahn. Overexpression of Muscleblind Like Splicing Regulator 2 (MBNL2) enhances cisplatin resistance in ovarian cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B091.

A Combination of Alectinib and DNA-Demethylating Agents Synergistically Inhibits Anaplastic-Lymphoma-Kinase-Positive Anaplastic Large-Cell Lymphoma Cell Proliferation.

The recent evolution of molecular targeted therapy has improved clinical outcomes in several human malignancies. The translocation of anaplastic lymphoma kinase (ALK) was originally identified in anaplastic large-cell lymphoma (ALCL) and subsequently in non-small cell lung carcinoma (NSCLC). Since ALK fusion gene products act as a driver of carcinogenesis in both ALCL and NSCLC, several ALK tyrosine kinase inhibitors (TKIs) have been developed. Crizotinib and alectinib are first- and second-generation ALK TKIs, respectively, approved for the treatment of ALK-positive ALCL (ALK+ ALCL) and ALK+ NSCLC. Although most ALK+ NSCLC patients respond to crizotinib and alectinib, they generally relapse after several years of treatment. We previously found that DNA-demethylating agents enhanced the efficacy of ABL TKIs in chronic myeloid leukemia cells. Moreover, aberrant DNA methylation has also been observed in ALCL cells. Thus, to improve the clinical outcomes of ALK+ ALCL therapy, we investigated the synergistic efficacy of the combination of alectinib and the DNA-demethylating agent azacytidine, decitabine, or OR-2100 (an orally bioavailable decitabine derivative). As expected, the combination of alectinib and DNA-demethylating agents synergistically suppressed ALK+ ALCL cell proliferation, concomitant with DNA hypomethylation and a reduction in STAT3 (a downstream target of ALK fusion proteins) phosphorylation. The combination of alectinib and OR-2100 markedly altered gene expression in ALCL cells, including that of genes implicated in apoptotic signaling, which possibly contributed to the synergistic anti-ALCL effects of this drug combination. Therefore, alectinib and OR-2100 combination therapy has the potential to improve the outcomes of patients with ALK+ ALCL.

Epigenetic PPARγ preservation attenuates temporomandibular joint osteoarthritis

ObjectivePrevious studies have demonstrated that PPARγ deficiency is associated with osteoarthritis in the knee joint. However, whether epigenetic PPARγ dysregulation has any effect on temporomandibular joint osteoarthritis (TMJOA) is unknown. This study aims to determine the role and mechanism of epigenetic PPARγ dysregulation in TMJOA. MethodsPartial TMJ discectomy was performed to induce TMJOA in rat. Primary condylar chondrocytes were isolated, and TNF-α-induced inflammatory condition was created in vitro. The expressions of PPARγ and DNA methyltransferase were investigated in vivo and in vitro. The association of PPARγ and DNA methylation was further studied by treating chondrocytes with DNA demethylation agent 5-Aza-2′-deoxycytidine (5Aza) and transfecting with siRNA of DNA methyltransferase (DNMT)1 and DNMT3a, and the methylation level of PPARγ promoter was evaluated by Bisulfite-sequencing PCR. The chondroprotective effects of 5Aza were explored in vitro and in vivo. ResultsPPARγ suppression and upregulated DNMT1/DNMT3a expression exist in TMJOA cartilage in vivo and primary condylar chondrocytes under TNF-α-induced inflammatory conditions in vitro. DNMT1 and DNMT3a elevation contributes to PPARγ-promoter hypermethylation in TMJ chondrocytes under TNF-α-induced inflammation conditions. DNA demethylation intervention by 5Aza protects chondrocytes from inflammation response in vitro. Mechanistically, 5Aza reversed the hypermethylation of the PPARγ promoter and subsequently resulted in PPARγ restoration and decreased expression of cartilage-catabolic factors in chondrocytes. Rat TMJOA model revealed that 5Aza, by reversing PPARγ suppression, effectively attenuated cartilage degeneration and stabilized cartilage homeostasis by balancing anabolic factor and catabolic factor expression. ConclusionEpigenetic PPARγ suppression may play a causal role in TMJOA pathogenesis, which can be alleviated by DNA demethylation with 5Aza treatment. This study provides new insights into the pathogenic mechanism and therapeutic strategy of TMJOA.

Marine Pollutant Tributyltin Affects DNA Methylation and Fitness of Banded Murex (Hexaplex trunculus) Populations

Banded murex, Hexaplex trunculus, is a marine gastropod whose reproductive fitness can be severely affected by very low concentrations of antifouling compound tributyltin (TBT). TBT has strong xenoandrogen impacts on snails, causing the development of imposex (e.g., the superimposition of male sexual characteristic in females), thereby affecting the fitness of entire populations. TBT is also known as a DNA-demethylating agent and an obesogenic factor. The aim of this study was to unravel the interactions between TBT bioaccumulation, phenotypic responses, and epigenetic and genetic endpoints in native populations of H. trunculus. Seven populations inhabiting environments along the pollution gradient were sampled in the coastal eastern Adriatic. These included sites of intense marine traffic and boat maintenance activity and sites with low anthropogenic impact. Populations inhabiting intermediately and highly polluted sites exhibited higher TBT burdens, higher incidences of imposex, and higher wet masses of snails than populations in lowly polluted sites. Other morphometric traits and cellular biomarker responses did not show clear differentiation among populations in relation to marine traffic/pollution intensity. An analysis of methylation sensitive amplification polymorphism (MSAP) revealed environmentally driven population differentiation and higher epigenetics than genetic within-population diversity. Moreover, decreases in genome-wide DNA methylation coincided with the imposex level and snail mass, suggesting an epigenetic background of the animal phenotypic response.

Selenite as a dual apoptotic and ferroptotic agent synergizes with EGFR and KRAS inhibitors with epigenetic interference

BackgroundSelenium, an essential trace element, has previously been investigated as a pro-apoptotic and DNA demethylation agent. It sensitizes the response to chemotherapy in patients who were refractory to cytotoxic agents. Meanwhile, ferroptosis is a novel approach to cancer treatment by triggering cell death and reversing drug resistance. The role of selenium in treating cancer cells harboring druggable oncogenic alterations and its underlying mechanism are largely unknown.ResultsWe treated lung adenocarcinoma cell lines—EGFR-mutant H1975 (H1975 EGFR p.L858R and p.T790M) and KRAS-mutant H358 (H358 KRAS p.G12C), with sodium selenite to examine its effect on cell apoptosis, ferroptosis, and DNA methylation, as well as its interaction with existing targeted therapy, osimertinib, and adagrasib. We observed selenite to be a dual apoptotic and ferroptotic agent on lung cancer cells, associated with the activation of p38-ATF4-DDIT3 axis in the unfolded protein response. Ferroptosis induction was more remarkable in H1975 than H358. Selenite also altered cellular DNA methylation machinery through downregulating DNMT1 and upregulating TET1, though not as a major mechanism of its activity. Low-dose selenite synergized with osimertinib in EGFR-mutant H1975, and with adagrasib in KRAS-mutant H358, with stronger synergism observed in H1975.ConclusionThese results suggest that selenite is a potential apoptotic and ferroptotic drug candidate for the treatment of especially EGFR- and potentially KRAS-mutant lung cancer.

Treatment Outcomes of Childhood Picalm:MLLT10+ Acute Leukemias: An International Retrospective Study

Introduction: Acute leukemia with translocation t(10;11)(p12-13;q14-21) results in generation of the PICALM:MLLT10 fusion gene, and is distinct from t(10;11)(p11-13;q23) which gives rise to KMT2A (formerly MLL) gene rearrangement. Defective PICALM function leads to aberrant hematopoiesis, while defective MLLT10 function impairs chromatin-mediated gene regulation. The PICALM:MLLT10 gene has been reported in patients with Acute Myeloid Leukemia (AML), Acute Lymphoblastic Leukemia (ALL) predominantly T-cell, Acute Undifferentiated Leukemia (AUL) and Mixed Phenotype Acute Leukemia (MPAL). MLLT10 interacts directly with DOT1L in a complex that regulates the methylation of H3K79. MLLT10 fusions are thought to misdirect DOT1L to the promoters of HOXA genes, leading to hypermethylation of H3K79. This hypermethylation causes constitutive activation of HOXA activity and prevents cell maturation and differentiation. The clinico-biological characteristics, response to conventional treatments, and survival outcomes of childhood PICALM:MLLT10+ leukemias are poorly understood. Methods: This was an international, multicenter, retrospective cohort study. Data were submitted by ten International-BFM (I-BFM) study Group-AML and ALL committees and by the Children`s Oncology Group (COG)-AML Study committee. For study inclusions, patients were required to be ≤ 21 years of age at diagnosis, diagnosed between January 1995 and December 2019 with de novo ALL, AML, AUL or MPAL and PICALM:MLLT10 fusion-positive. Patients with KMT2A-rearranged leukemia or acute promyelocytic leukemia were excluded. Kaplan Meier curves were employed for survival analysis with Cox proportional hazard modelling to compare effect of AML vs ALL therapy, and receipt of hematopoietic stem cell transplant (HSCT) vs no HSCT, on survival. Pearson's Chi Squared was employed as a non-parametric measure to determine the effect of end of induction-1 (EIO-1) MRD positive vs negative status on incidence of relapse. P value cut-off for data to reach statistical significance was <0.05. Results: Ninety-eight children with acute leukemia were included, 60 were males. Median age at diagnosis was 12 years (IQR:9-14.8 years) and median initial white cell count was 40.5 x 109/L (IQR:13.8-168.9). Fifty-five (55.6%) patients were diagnosed with T-ALL, 39 (39.4%) with AML, while precursor-B ALL, AUL and MPAL were each diagnosed in single patients; 1 subtype diagnosis was unknown. Ten (10.1%) patients presented with CNS disease and 13 (13.1%) presented with non-CNS extramedullary manifestations. Of 66 with cytogenetic/molecular data available, 33 (50%) had additional cytogenetic-molecular aberrations; NOTCH1, WT1 and RAS pathway mutations predominated. Most patients received an initial chemotherapy regimen guided by their disease immunophenotype: 58/98 (58.6%) received an ALL-like chemotherapy regimen upfront, 38 (38.4%) received an AML-like regimen, 1 (1.0%) a hybrid regimen, 1 initial chemotherapy regimen was unknown. Five-year event-free survival (EFS) was 50.4% (CI 39.1-64.9). For AML patients, 5-year EFS was 22.5% (CI 9.75-52.0) and for ALL 67.7% (CI 54.2-84.7), p=<0.001. Five-year overall survival (OS) was 56.3% (CI 46.3-68.5). For AML patients, 5-year OS was 26.7% (CI 13.3-53.6) and for ALL 75.7% (CI 64.6-88.8), p=<0.001. Thirty-eight patients died, 22 (57.9%) deaths were disease-related, 4 (10.5%) treatment-related. Patients who received HSCT in CR1, demonstrated a trend towards an inferior 5-year EFS (23.7% vs 56.5%) and OS (42.6% vs 64.5%) than those who did not; however, analysis was limited by small sample size. Four (22.2%) HSCT recipients had ALL but most, 14 (77.8%), had AML, which may account for the unfavourable outcomes associated with HSCT. There was no statistically significant difference in rate of relapse in the EOI-1 MRD-positive compared with MRD-negative patients (p=0.49). Of those who relapsed, 25 (75.8%) had an isolated bone marrow relapse, 3 (9.1%) isolated CNS, 3 (9.1%) other sites and, for 2 patients, site of relapse was unknown. Conclusions: Children with PICALM:MLLT10+ AML have inferior 5-year EFS and OS outcomes compared to their T-ALL counterparts. MRD status at EOI-1 was not predictive of relapse. Targeted therapies with DOT1L inhibitors or DNA demethylating agents should be explored, particularly for patients with AML.

DNA-Demethylating Agents Combined with Anti-PD-1 Inhibitors Showed Activity in Patients with Relapsed or Refractory Extranodal NK/T Cell Lymphoma Who Have Failed Immunotherapy: An Exploratory Study

Background: Extranodal natural killer (NK)/T-cell lymphoma (ENKTL) is a rare aggressive type of non-Hodgkin's lymphoma associated with Epstein-Barr virus infection. Relapse or refractory (RR)-ENKTL cases are characterized by a high rate of relapse and dismal prognosis with limited therapeutic options. Immune check point inhibitors (ICIs) represent the cornerstone of treatment for RR-ENKTL; however, limited information is available of a rechallenge with ICIs after ICIs are discontinued due to disease progression. Epigenetic aberrations are critical as genetic abnormalities is the hallmarks of ENKTL. One of current promising strategies is that combining epigenetic and ICIs can overcome tumor resistance and enhance immunotherapy responses. Here, we assess the efficacy and safety of DNA methyltransferases inhibitors (DNMTi) plus anti-PD-1 therapy in RR-ENKTL. Methods: This trial enrolled eligible patients with histologically confirmed RR-ENKTL failing from asparaginase-based regimen or ICIs therapy; adequate organ function and bone marrow function; and at least one measurable or evaluable lesion or symptoms. Patients received standard dosage of anti-PD-1 antibodies plus decitabine (10mg/d, d1-5) or azacytidine (100mg/d, d1-5) every 21 days until disease progression, death, intolerable toxicity. Patients accept ASCT as consolidation after they obtained CR. The primary endpoints were objective response rate (ORR) based on Lugano 2016 criteria and clinical benefit rate. Secondary endpoints were progression-free survival (PFS), overall survival (OS) and safety. Adverse events (AEs) were defined according to CTCAE 5.0. Results: From March 2020 to May 2022, 27 eligible patients were enrolled. Median age 40 years (range, 12-77), 15 (55.6%) male, 13 (48.1%) patients with stage IV of disease at screening, 11 (40.7%) patients with PINK-E score ≥ 3 points. Among them, 5(18.5%) patients had no imaging lesions, but all peripheral blood ctDNA were positive, they had high plasma EBV-DNA level(n=3), persistent fever(n=2) associated with ENKTL. The median number of prior systemic therapies was 2 (1-5), 21（77.8%）patients received ≥ 2 lines of prior systemic therapy. Twenty-two (81.5%) patients experienced prior ICIs therapy, 23(85.2%) previously received HDACi. The median time between the last dose of prior ICIs and initiation of this trial was 0.8 months (0.8 -23). Seven (25.9%) patients received decitabine, 20 (74.1%) received azacytidine. Of 22 imaging objective response evaluable patients, 13 (59.1%) achieved response (iORR) including 10 (45.5%) patients with CR (iCR). Clinical benefit analysis of 27 patients showed that clinical response(cORR) was 63.0% (17/27), 14 (59.1%) got CR (cCR). Among 5 patients with positive ctDNA alone, 4 patients achieve clinical improvement ,ctDNA negativity(n=4), EBV clearance(n=2) or fever disappearance (n=2). Among 22 patients previously received ICIs (20 patients previously received anti-PD-1 antibody plus HDACi), of these, 11 (50%) had response. After anti-PD-1 antibodies rechallenge plus DNMTi, iORR and cORR were 58.8% (10/17), 63.6% (14/22), iCR and cCR were 41.2% (7/17), 50.0% (11/22), respectively. Anti-PD-1 antibodies rechallenge plus DNMTi yielded iORR were 72.7% (8/11) and 55.6% (5/9) in patients responded to prior ICIs therapy. The difference of efficacy between decitabine and azacytidine was no significant, iORR and cORR were 60% (3/5), 58.8% (10/17) and 71.4% (5/7), 60.0% (12/20), respectively in this study. The median follow-up time was 12.1 (1.9-28.0) months, 1-year OS rate was 70.4%, median PFS rate was 12.8months. Survival results were similar with decitabine and azacytidine cohort. Sixteen (59.3%) patients reported treatment-related AEs (TRAEs). The most frequently observed (≥10%) TRAEs were lymphopenia (44.4%), neutropenia (37.0%), hypothyroidism (25.9%), nausea (18.5%), hyperglycemia (11.1%). Conclusion: DNMTi combined with anti-PD-1 immunotherapy showed promising efficacy and manageable safety profile in RR-ENKTL for the first time. ICIs rechallenge is still an active and feasible strategy, further prospective studies are needed to clarify the role of rechallenge ICIs in this population. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Role of DNA methylation in ecophysiological responses to salinity in natural populations of Phragmites australis from coastal and inland habitats

There are different environmental pressures in coastal and inland wetlands resulting in phenotypic variation among plant populations, which might be related to epigenetic modifications. Phragmites australis is a widespread plant in coastal and inland wetlands, and the phenotype of the population is selected by salinity, but the reasons for the population differences in salt tolerance and phenotype are unclear. We investigated coastal and inland P. australis populations grown under two salinities and two manipulated DNA methylation levels in common gardens. The plants were sprayed with 5-azacytidine (DNA demethylation agent) regularly, and the physiological and morphological traits of reeds were measured. Plant height, density, and basal stem of reeds from different sources were significantly different and correlated with soil conductivity of sampling sites (P &amp;lt; 0.05). Salinity significantly decreased the biomass (37.04%, P &amp;lt; 0.05) and plant height (24.68%, P &amp;lt; 0.05) of inland reeds but had no significant effect on coastal populations (13.48%). P. australis responds to salt stress through phenotypic plasticity, and inland wetland populations exhibit local adaptation to freshwater. Increased salt tolerance in inland populations following DNA demethylation, particularly biomass, plant height, and basal stems (increased 23.62%; 13.08%; 5.35%, respectively), could provide more opportunities in adverse environments. This study will provide important insights into the highly adaptive mechanisms of the large non-model plant.

Double-stranded RNA induction asa potential dynamic biomarkerfor DNA-demethylating agents

Hypomethylating agents (HMAs), such as azacitidine and decitabine, induce cancer cell death by demethylating DNAs to promote the expression of tumor-suppressor genes. HMAs also reactivate the transcription of endogenous double-stranded RNAs (dsRNAs) that trigger the innate immune response and subsequent apoptosis via viral mimicry. However, the expression patterns of endogenous dsRNAs and their relevance in the efficacy of HMAs remain largely uninvestigated. Here, we employ amidine-conjugated spiropyran (Am-SP) to examine the dynamic expression pattern of total dsRNAs regulated by HMAs. By analyzing the bone-marrow aspirates of myelodysplastic syndrome or acute myeloid leukemia patients who received the HMAs, we find a dramatic increase in total dsRNA levels upon treatment only in patients who later benefited from the therapy. We further apply our approach in solid tumor cell lines and show that the degree of dsRNA induction correlates with the effectiveness of decitabine in most cases. Notably, when dsRNA induction is accompanied by increased expression of nc886 RNA, decitabine becomes ineffective. Collectively, our study establishes the potential application of monitoring the total dsRNA levels by a small molecule as an analytical method and a dynamic marker to predict the clinical outcome of the HMA therapy.

Low-dose selenite synergizes with KRAS inhibitor as a dual apoptotic and ferroptotic agent in lung adenocarcinoma.

e21039 Background: Selenium, an essential trace element, has long been investigated as a pro-apoptotic and DNA demethylation agent. It sensitizes the response to chemotherapy in patients refractory to cytostatic agents. Meanwhile, ferroptosis is a novel approach in cancer treatment by triggering cell death and reversing drug resistance. However, the role of selenite in targeted therapy of lung adenocarcinoma (LUAD) and its underlying mechanism to demethylation and ferroptosis are largely unknown. Methods: EGFR- and KRAS-mutant LUAD cells (H1975 EGFR p.L858R and p.T790M and H358 KRAS p.G12C) were treated with selenite for 72 hours to examine its effect on DNA methylation, apoptosis and ferroptosis by western blot, C-11 BODIPY staining, methylation-specific PCR and transcriptome sequencing validated by qPCR and western blot. Cell proliferation and apoptosis of both cells treated with selenite and osimertinib in H1975 or adagrasib in H358 were assessed by MTS assay and western blot. Results: We confirmed selenite to be a dual apoptotic and ferroptotic agent in LUAD, associated with the activation of the p38-ATF4-CHOP axis in the unfolded protein response. This pathway linked ferroptosis with apoptosis by the upregulation of BH3-only proteins. Selenite also altered the DNA methylation machinery by downregulating DNMT1 and upregulation of TET1. We further found that low-dose selenite synergized with adagrasib in KRAS-mutant H358 but only yielded additive interaction at a high dose. Conclusions: Selenite is a dual apoptotic and ferroptotic agent with dose-dependent, oncogenic driver-dependent therapeutic effects in LUAD. These data suggest that low-dose selenite should be a potential drug candidate for the treatment of KRAS mutation-positive lung cancer.

Is There a Role for Epigenetic Therapies in Modulating DNA Damage Repair Pathways to Enhance Chemotherapy and Overcome Drug Resistance?

Simple SummaryEpigenetic changes in a cell’s genome can cause the inactivation of genes and affect how tumours respond to chemotherapy. These epigenetic changes do not alter the DNA sequence and small molecule inhibitors of maintenance of the epigenetic state can change the sensitivity of a tumour to chemotherapy. Epigenetic therapies are being clinically evaluated as single agents or in combination with chemotherapies, including those that induce DNA damage. DNA damage induced by chemotherapy may be repaired and the cell either survives or alternatively cell death pathways are engaged and the cell is eliminated. Epigenetic silencing of gene expression during tumour development may lead to sensitivity of the tumour to chemotherapy if DNA repair of the DNA lesion is inactivated, or to chemotherapy resistance if cell death responses are inactivated. This illustrates the clinical challenge of using epigenetic therapies in combination with chemotherapy. Will such epigenetic drugs confer chemosensitivity and increased efficacy of chemotherapy, or induce resistance? Is there an effect of epigenetic therapies on normal cell chemo-sensitivity leading to increased toxicity? This review will illustrate the clinical challenges in combination studies of epigenetic therapies with chemotherapy and discuss future perspectives on addressing these challenges.Epigenetic therapies describe drug molecules such as DNA methyltransferase, histone methyltransferase and histone acetylase/deacetylase inhibitors, which target epigenetic mechanisms such as DNA methylation and histone modifications. Many DNA damage response (DDR) genes are epigenetically regulated in cancer leading to transcriptional silencing and the loss of DNA repair capacity. Epigenetic marks at DDR genes, such as DNA methylation at gene promoters, have the potential to be used as stratification biomarkers, identifying which patients may benefit from particular chemotherapy treatments. For genes such as MGMT and BRCA1, promoter DNA methylation is associated with chemosensitivity to alkylating agents and platinum coordination complexes, respectively, and they have use as biomarkers directing patient treatment options. In contrast to epigenetic change leading to chemosensitivity, DNA methylation of DDR genes involved in engaging cell death responses, such as MLH1, are associated with chemoresistance. This contrasting functional effect of epigenetic modification on chemosensitivity raises challenges in using DNA-demethylating agents, and other epigenetic approaches, to sensitise tumours to DNA-damaging chemotherapies and molecularly targeted agents. Demethylation of MGMT/BRCA1 could lead to drug resistance whereas demethylation of MLH1 could sensitise cells to chemotherapy. Patient selection based on a solid understanding of the disease pathway will be one means to tackle these challenges. The role of epigenetic modification of DDR genes during tumour development, such as causing a mutator phenotype, has different selective pressures and outcomes compared to epigenetic adaptation during treatment. The prevention of epigenetic adaptation during the acquisition of drug resistance will be a potential strategy to improve the treatment of patients using epigenetic therapies.

Combination effect of poly (ADP-ribose) polymerase inhibitor and DNA demethylating agents for treatment of epithelial ovarian cancer

ObjectivePoly (ADP)-ribose polymerase inhibitors (PARPi) are effective clinical agents for treatment of epithelial ovarian cancer (EOC) harboring BRCA mutations as well as those without BRCA mutations. In this study, we evaluate the efficacy of combined PARPi and DNA methyltransferase inhibitor (DNMTi) in EOCs. MethodsExpression levels of DNMT1 and PARP1 proteins in EOC cells were assessed using western blot analysis and immunohistochemistry. To evaluate the effects of co-treatment of PARPi (olaparib) and DNMTi (5-azacitidine, 5-AZA), we performed cell proliferation, apoptosis, and wound-healing assays in EOC cells. In addition, we performed in vivo experiments using both cell-line and patient-derived xenograft (PDX) models of EOC. ResultsThe combination of olaparib and 5-AZA significantly inhibited cell proliferation and migration and induced apoptosis compared with olaparib or 5-AZA alone in EOC cell lines including A2780, HeyA8, A2780-CP20, and HeyA8-MDR. Moreover, in vivo experiments with this combination showed significantly decreased weight and nodule numbers of tumors in cell-line xenograft models with A2780 cells and a PDX model compared with control, olaparib, and 5-AZA groups. As a potential mechanism, the expression of intracellular reactive oxygen species (ROS) and its related proteins, including p-ERK, NRF2, p-p38, HO-1, and γH2AX, was affected in EOC cells. ConclusionsCo-treatment with PARPi and DNMTi had a significant anti-tumor effect in EOC cells. This combination might be a potential therapeutic strategy for EOCs.

DNA Methylation and Detoxification in the Earthworm Lumbricus terrestris Exposed to Cadmium and the DNA Demethylation Agent 5-aza-2'-deoxycytidine.

Earthworms are well-established model organisms for testing the effects of heavy metal pollution. How DNA methylation affects cadmium (Cd) detoxification processes such as the expression of metallothionein 2 (MT2), however, is largely unknown. We therefore exposed Lumbricus terrestris to 200 mg concentrations of Cd and 5-aza-2′-deoxycytidine (Aza), a demethylating agent, and sampled tissue and coelomocytes, cells of the innate immune system, for 48 h. MT2 transcription significantly increased in the Cd- and Cd-Aza-treated groups. In tissue samples, a significant decrease in MT2 in the Aza-treated group was detected, showing that Aza treatment inhibits basal MT2 gene activity but has no effect on Cd-induced MT2 levels. Although Cd repressed the gene expression of DNA-(cytosine-5)-methyltransferase-1 (DNMT1), which is responsible for maintaining DNA methylation, DNMT activity was unchanged, meaning that methylation maintenance was not affected in coelomocytes. The treatment did not influence DNMT3, which mediates de novo methylation, TET gene expression, which orchestrates demethylation, and global levels of hydroxymethylcytosine (5hmC), a product of the demethylation process. Taken together, this study indicates that Aza inhibits basal gene activity, in contrast to Cd-induced MT2 gene expression, but does not affect global DNA methylation. We therefore conclude that Cd detoxification based on the induction of MT2 does not relate to DNA methylation changes.

Epigenetic Priming with Decitabine Augments the Therapeutic Effect of Cisplatin on Triple-Negative Breast Cancer Cells through Induction of Proapoptotic Factor NOXA.

Simple SummaryTriple-negative breast cancer is a subset of breast cancer that occurs frequently in young women and tends to exhibit aggressive, metastatic behavior. The therapeutic molecular targets found in other types of breast cancer are absent; therefore, this type of cancer has a poorer prognosis. To search for effective treatments for this type of cancer, we analyzed the effect of the DNA-demethylating agent, decitabine, which is commonly used in patients with myelodysplastic syndrome. We found that in triple-negative breast cancer cell subtypes, inhibition of cell death and cell growth in response to cisplatin, which is used to treat metastatic breast cancer, is enhanced when used in combination with decitabine. We also found that in decitabine-refractory cell subtypes, cisplatin alone is effective at inducing cell death. These results indicate the possibility of effective new combination therapies in triple-negative breast cancers.Epigenetic alterations caused by aberrant DNA methylation have a crucial role in cancer development, and the DNA-demethylating agent decitabine, is used to treat hematopoietic malignancy. Triple-negative breast cancers (TNBCs) have shown sensitivity to decitabine; however, the underlying mechanism of its anticancer effect and its effectiveness in treating TNBCs are not fully understood. We analyzed the effects of decitabine on nine TNBC cell lines and examined genes associated with its cytotoxic effects. According to the effect of decitabine, we classified the cell lines into cell death (D)-type, growth inhibition (G)-type, and resistant (R)-type. In D-type cells, decitabine induced the expression of apoptotic regulators and, among them, NOXA was functionally involved in decitabine-induced apoptosis. In G-type cells, induction of the cyclin-dependent kinase inhibitor, p21, and cell cycle arrest were observed. Furthermore, decitabine enhanced the cytotoxic effect of cisplatin mediated by NOXA in D-type and G-type cells. In contrast, the sensitivity to cisplatin was high in R-type cells, and no enhancing effect by decitabine was observed. These results indicate that decitabine enhances the proapoptotic effect of cisplatin on TNBC cell lines that are less sensitive to cisplatin, indicating the potential for combination therapy in TNBC.

Inhibition of DNA methyltransferase aberrations reinstates antioxidant aging suppressors and ameliorates renal aging.

DNA methylation alterations play mechanistic roles in aging; however, the epigenetic regulators/mediators causally involved in renal aging remain elusive. Here, we report that natural and D‐galactose (D‐gal)‐induced aging kidneys display marked suppression of antiaging factor NRF2 (nuclear factor erythroid‐derived 2‐like 2) and KLOTHO, accompanied by upregulations of DNA methyltransferase (DNMT) 1/3a/3b and NRF2/KLOTHO gene promoter hypermethylations. Administration of a DNMT inhibitor SGI‐1072 effectively hypomethylated the promoters, derepressed NRF2/KLOTHO, and mitigated the structural and functional alterations of renal aging in D‐gal mice. Moreover, oleuropein (OLP), an olive‐derived polyphenol, also displayed similar epigenetic modulation and antiaging effects. OLP inhibited the epigenetic NRF2/KLOTHO suppressions in a gain of DNMT‐sensitive manner in cultured renal cells, demonstrating a strong DNA‐demethylating capacity. In NRF2 knockout and KLOTHO knockdown D‐gal mice, OLP exhibited reduced antiaging effects with KLOTHO displaying a prominent gene effect and effect size; consistently in KLOTHO knockdown mice, the antiaging effects of SGI‐1027 were largely abrogated. Therefore, the KLOTHO recovery is critical for the antiaging effects of DNA demethylation. Collectively, our data indicate that aberrant DNMT1/3a/3b elevations and the resultant suppression of antiaging factors contribute significantly to epigenetic renal aging, which might be targeted for epigenetic intervention by synthetic or natural DNA‐demethylating agents.

Antiviral Responses in Cancer: Boosting Antitumor Immunity Through Activation of Interferon Pathway in the Tumor Microenvironment.

In recent years, it became apparent that cancers either associated with viral infections or aberrantly expressing endogenous retroviral elements (EREs) are more immunogenic, exhibiting an intense intra-tumor immune cell infiltration characterized by a robust cytolytic apparatus. On the other hand, epigenetic regulation of EREs is crucial to maintain steady-state conditions and cell homeostasis. In line with this, epigenetic disruptions within steady-state cells can lead to cancer development and trigger the release of EREs into the cytoplasmic compartment. As such, detection of viral molecules by intracellular innate immune sensors leads to the production of type I and type III interferons that act to induce an antiviral state, thus restraining viral replication. This knowledge has recently gained momentum due to the possibility of triggering intratumoral activation of interferon responses, which could be used as an adjuvant to elicit strong anti-tumor immune responses that ultimately lead to a cascade of cytokine production. Accordingly, several therapeutic approaches are currently being tested using this rationale to improve responses to cancer immunotherapies. In this review, we discuss the immune mechanisms operating in viral infections, show evidence that exogenous viruses and endogenous retroviruses in cancer may enhance tumor immunogenicity, dissect the epigenetic control of EREs, and point to interferon pathway activation in the tumor milieu as a promising molecular predictive marker and immunotherapy target. Finally, we briefly discuss current strategies to modulate these responses within tumor tissues, including the clinical use of innate immune receptor agonists and DNA demethylating agents.

Evaluation of a DNA demethylating agent in combination with all-trans retinoic acid for IDH1-mutant gliomas.

Isocitrate Dehydrogenase 1/2 (IDH1/2) mutations are diagnostic for Astrocytoma or Oligodendroglioma, IDH-mutant. In these IDH-mutant gliomas, retinoic acid-related gene expression is commonly silenced by DNA hypermethylation. DNA demethylating agents can epigenetically reprogram IDH-mutant cells and reduce proliferation, likely by re-expression of silenced tumor suppressor pathways. We hypothesized that DNA demethylation might restore the retinoic acid pathway and slow tumor growth. This was the rationale for a preclinical evaluation combining the DNA demethylating agent, 5-Azacytidine (5-Aza), and retinoic acid pathway activation with all-trans retinoic acid (atRA) in IDH-mutant glioma. In this study, we evaluated the effect of 5-Aza and atRA combination on cell proliferation, apoptosis, and gene expression in human glioma cells. In addition, the efficacy of this combination was tested in patient-derived xenograft (PDX) bearing the IDH1R132H mutation, utilizing subcutaneous and orthotopic models. 5-Aza reduced the DNA methylation profile and increased the gene expression of retinoic acid-related genes. Combination of 5-Aza and atRA reduced cell growth, increased differentiation marker expression, and apoptosis in IDH1R132H glioma cells. Mechanistically, 5-Aza sensitized IDHIR132H glioma cells to atRA via upregulation of the retinoic acid pathway. Importantly, the drug combination reduced significantly the growth rate of subcutaneous tumors, but in an orthotopic mouse model, the combination did not improve survival and 5-Aza alone provided the best survival benefit. Use of DNA demethylating agent in combination with retinoids shows promise, but further optimization and preclinical studies are required for treatment of intracranial IDH-mutant gliomas.

Abstract LB111: Drug repurposing of niclosamide to regulate Wnt/beta-catenin signaling pathway through upregulating SFRP1 in colorectal cancer

Abstract Aim: This study investigated the inhibition of proliferation and invasiveness of colorectal cancer (CRC) by niclosamide via targeting the secreted Frizzled-related protein 1 (SFRP1) and the Wnt/beta-catenin signaling pathway. Background: CRC is the third most common cancer worldwide. Aberrant activation of various signaling pathways in CRC leads to its poor response to chemotherapy. Hyperactivation of Wnt/beta-catenin pathway is known to drive cell proliferation, invasion, and migration of CRC. Epigenetic silencing of extracellular Wnt inhibitors, such as secreted Frizzled-related proteins (SFRPs), has been shown to stabilize beta-catenin and subsequently activate the Wnt signaling pathway. Niclosamide is a clinically approved drug indicated for the treatment of tapeworm infections. We have recently reported the repurposing of niclosamide to potentiate chemotherapeutic drugs for treating CRC by STAT3 inhibition. Niclosamide was also reported to be a Wnt/beta-catenin inhibitor but the precise mechanism is not clear. Methods: The antiproliferative effect was assessed by MTT assay and colony formation assay. The mRNA level, protein expression, and methylation status of SFRP1, and sets of SFRP1 co-expressed genes in CRC were analyzed by the TCGA data portal. The mRNA expression of SFRP1 and CpG island methylator phenotype (CIMP) marker genes were measured by qRT-PCR. DNA methylation was analyzed by bisulfite genomic sequencing. Inhibition of Wnt/beta-catenin and its downstream targets were examined by Western blot analysis. Cell migration and invasion were investigated by wound healing and transwell chamber migration assay. The intracellular localization of beta-catenin was determined by immunofluorescence assay. Results: Niclosamide inhibited HCT116 cell proliferation and colony formation in a concentration-dependent manner. Compared with normal colon tissues, CRC expressed significantly lower level of SFRP1 mRNA. SFRP1 methylation was inversely correlated with its mRNA expression. The DNA-demethylating agent 5-azacytidine was shown to restore expression of SFRP1 and CIMP panel genes in HCT116 cells after 5-day treatment. However, SFRP1 mRNA level was only upregulated by 1-day niclosamide treatment, but not 3-day niclosamide treatment. The upregulation of SFRP1 by niclosamide was found not relevant to DNA demethylation according to bisulfite genomic sequencing data. Immunofluorescence and Western blotting analysis showed that the increased SFRP1 expression was accompanied by reduced expression and nuclear accumulation of beta-catenin. Moreover, a low concentration of niclosamide that has minimal effect on cell proliferation, was shown to significantly suppress migration and invasion of HCT116 cells. Conclusion: Niclosamide is a promising candidate for drug repurposing. It was shown to upregulate SFRP1 and subsequently suppress Wnt/beta-catenin signaling, thereby impairing CRC cell migration and invasion. Upregulation of SFRP1 by niclosamide was not dependent on DNA demethylation. Citation Format: Mia Mingxia Wu, Christy Wing Sum Tong, Vivi Wei Yan, Kenneth Kin-wah To. Drug repurposing of niclosamide to regulate Wnt/beta-catenin signaling pathway through upregulating SFRP1 in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB111.