Hypomethylating Agents Research Articles

Dual inhibitors of DNMT and HDAC remodels the immune microenvironment of colorectal cancer and enhances the efficacy of anti-PD-L1 therapy

Colorectal cancer is the second most prevalent and deadly cancer worldwide. The emergence of immune checkpoint therapy has provided a revolutionary strategy for the treatment of solid tumors. However, less than 5 % of colorectal cancer patients respond to immune checkpoint therapy. Thus, it is of great scientific significance to develop “potentiators” for immune checkpoint therapy. In this study, we found that knocking down different DNMT and HDAC isoforms could increase the expression of IFNs in colorectal cancer cells, which can enhance the effectiveness of immune checkpoint therapy. Therefore, the combined inhibition of DNMT and HDAC cloud synergistically enhance the effect of immunotherapy. We found that dual DNMT and HDAC inhibitors C02S could inhibit tumor growth in immunocompetent mice but not in immunocompromised nude mice, which indicates that C02S exerts its antitumor effects through the immune system. Mechanistically, C02S could increase the expression of ERVs, which generated the intracellular levels of dsRNA in tumor cells, and then promotes the expression of IFNs through the RIG-I/MDA5-MAVS signaling pathway. Moreover, C02S increased the immune infiltration of DCs and T cells in microenvironment, and enhanced the efficacy of anti-PD-L1 therapy in MC38 and CT26 mice model. These results confirmed that C02S can activate IFNs through the RIG-I/MDA5-MAVS signaling pathway, remodel the tumor immune microenvironment and enhance the efficacy of immune checkpoint therapy, which provides new evidence and solutions for the development of “potentiator” for colorectal cancer immunotherapy.

Abstract PO-013: Combined epigenetic therapy to induce latency II/III antigen expression in latency I EBV+ lymphoma

Abstract In EBV+ lymphomas the virus exists in one of three latent states. Cellular therapy with EBV cytotoxic T cells (CTLs) is effective in latency II/III tumors that express the viral proteins LMP1/2 and/or EBNA2-6 but not in latency I tumors. Previously, we reported that EBV latency restriction is regulated through DNA methyltransferase 1 (DNMT1, Guo at al, Nat Micrbiol 2020) and that treatment with decitabine (DCB) induces latency II/III antigen expression in latency I Burkitt lymphoma (BL), rendering BL sensitive to EBV-specific CTLs (Dalton et al, Blood 2020). We have also found that a DNMT1 specific inhibitor (DNMT1i) converts BL to latency II/III, highlighting the key role of DNMT1 in latency regulation. One limitation to DNMT1 inhibition as a therapeutic approach in latency I EBV+ lymphoma is that the induction of latency II/III is only observed in a subset of cells. Here we investigated mechanisms of resistance to latency conversion to develop rational combinations to maximize the conversion of latency I tumors to latency II/III thereby sensitizing them to viral-directed immunotherapy. To determine if epigenetic mechanisms are responsible for resistance to latency conversion, we performed ATACseq and ChIP-qPCR on sensitive vs resistant KemI cells. ATACseq revealed open chromatin at Cp, which controls expression of EBNA2-6, in DCB-sensitive cells, but closed-chromatin in DCB-resistant cells, suggesting chromatin compaction may play a role in suppression of EBNA2-6. We observed no difference in chromatin accessibility at the LMP1 promoter, suggesting an independent mechanism may regulate LMP1. We performed ChIP-qPCR to evaluate activating (H3K27ac, H3K4me3 and H3K36me2) and repressive (H2K27me3 and H3K9me3) marks at Cp and LMP1p. We observed an increase in H3K27me3 and H3K9me3 on Cp and LMP1p in resistant cells. H3K27me3 was increased 2-fold on Cp (adj-p=0.0009) and 2.45-fold on LMP1p (adj-p=0.0086) in DNMT1i resistant vs. sensitive cells. H3K9me3 was increased a 2.4-fold (adj-p<0.0001) and 2.3-fold (adj-p<0.0001) on Cp in DCB and DNMT1i resistant cell respectively. H3K9me3 was increased 3.46-fold (adj-p=0.0013) on LMP1p in DCB resistant cells. Given our observation of increased H3K27me3 resistant cells, we next evaluated the effect of the EZH2 inhibitor tazemetostat, which targets H3K27me3, alone and in combination with DCB or DNMT1i in KemI. Tazemetostat treatment alone did not induce expression of latency II/III. However, when combined with DCB or DNMT1i, tazemetostat increases the percentage of LMP1+ EBNA2+ cells compared to DCB or DNMT1i alone (DCB: 32.3% to 44.5% adj-p=0.0006; DNMT1i: 22.2% to 35.6% adj-p=0.0002). Our data suggests that repressive histone marks contribute to resistance to DNMT1 inhibition-induced latency switch and that the addition of tazemetostat to DCB or DNMT1i can improve latency switch, potentially resulting in a more immunogenic tumor. These findings will guide the development of therapies to improve the treatment of otherwise immune refractory EBV+ latency I lymphoma. Citation Format: Isabella Y Kong, Vicenta Trujillo Alonso, Sarah Clark, Suhong Sun, Alicia Alonso, Rebecca Whitehouse, Stephanie Trezise, Roberta Zappasodi, Ethel Cesarman, Lisa Giulino Roth. Combined epigenetic therapy to induce latency II/III antigen expression in latency I EBV+ lymphoma [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-013.

DIPG-58. TARGETING DISORDERED DNA METHYLATION IN DIPG TO CONSTRAIN VARIABILITY AND INDUCE IMMUNE SIGNALING

Abstract BACKGROUND Diffuse Intrinsic Pontine Glioma (DIPG) is a universally fatal pediatric brain cancer characterized by the histone H3 K27M mutation. The known downstream consequences of this mutation include loss of repressive chromatin marks, global DNA hypomethylation, and altered gene expression. METHODS We sought to investigate the role of epigenetic variability as a basis of DIPG cellular heterogeneity and plasticity by using Whole Genome Bisulfite Sequencing (WGBS). Existing methods of analysis of WGBS are limited in that they average/smooth methylation data and fail to capture intrinsic variability. We performed (WGBS) on 23 primary patient samples of DIPG and applied an analysis that captures the variability of DNA methylation reads to characterize methylation stochasticity. We then sought to modulate the epigenome pharmacologically using a DNA methyltransferase inhibitor and assessed changes through RNA-Seq and immunopeptidomics. RESULTS We find that DIPG has a marked increase in methylation stochasticity (quantified as methylation entropy). Additionally, while DIPG is globally hypomethylated, it retains DNA methylation at specific regulatory regions, such as bivalent promoters, and key genes, such as FOXG1, CXCR4, and KLF4. We sought to reverse these changes using the DNA methyltransferase inhibitor, decitabine, in 4 DIPG neurosphere cell lines. Treatment with decitabine reversed the entropy seen in DIPG and induced innate immune signaling and interferon signaling in DIPG cells. Using RNA-seq, we found that decitabine treatment dramatically alters gene expression, including activation of endogenous retroviral elements, activation of interferon signaling, de-repression of hypermethylated targets, and expression of putative neoantigens, such as PRAME and DAZL. Immunopeptidomics profiling of MHC Class I bound canonical and non-canonical peptides of DIPG cell lines after decitabine treatment also revealed decitabine-induced differences. CONCLUSIONS Taken together, our study finds that the methylome of DIPG is highly disordered but is also highly responsive to pharmacologic modulation by hypomethylating agents. Furthermore, hypomethylating drugs can increase the immunogenicity of DIPG, thus offering the potential for future combination with immunotherapy.

IMMU-05. TARGETING THE DYSREGULATED DNA METHYLOME DURING CAR T CELL MANUFACTURING IMPROVES THEIR FITNESS IN IMMUNOCOMPETENT BRAIN TUMOR MODELS

Abstract BACKGROUND Epigenetic reprogramming and T-cell exhaustion are key factors impacting the effectiveness of chimeric antigen receptor (CAR) T-cell immunotherapies in brain tumors. These processes are regulated by DNA methylation, an epigenetic modification that is crucial for gene regulation. Here, we investigated whether targeting the dysregulated DNA methylome in engineered immune cells can improve their anti-tumor efficacy. METHODS We optimized a transduction protocol that incorporates adding DNA methyltransferase inhibitors (DNMTi) early during the manufacturing process of murine CAR T-cells targeting B7-H3. We then used standard in vitro assays and immunocompetent glioma models to evaluate whether DNMTi treatment improved CAR T-cell effector functions by regulating activation, memory differentiation, and cytokine responses in adoptive T-cells. RESULTS In repeated-stimulation assays, CAR T-cells generated in the presence of DNMTi exhibited significantly higher levels of expansion and persistence compared to CAR T-cells without DNMTi exposure. Notably, CAR T-cells manufactured in the presence of DNMTi persisted up to 12 repeat stimulations, in contrast to a maximum of 7 stimulations for CAR T-cells generated without DNMTi treatment. Moreover, DNMTi-treated CAR T-cells expanded up to 55-fold higher than T-cells generated without DNMTi treatment upon repeated-stimulation with B7-H3-positive glioma cells. This was also associated with remarkably enhanced secretion of immune-stimulatory cytokines that persisted across repetitive stimulations. Furthermore, DNMTi-treated CAR T-cells exhibited greater cytotoxicity at lower effector-to-target ratios compared to untreated CAR T-cells, with sustained differential cytotoxicity even after the 4th stimulation. Preliminary in vivo data from immunocompetent glioma models further support the potential of DNMTi pre-treatment, showing improved T-cell persistence and reduced dose requirements. Mechanistic studies suggest that DNMT inhibition enhances B7-H3 CAR T-cell anti-tumor activity by regulating memory differentiation of CAR T-cells early in the production phase. CONCLUSION This study proposes that DNMTi pre-treatment may offer a promising approach for optimizing CAR T-cell manufacturing and enhancing performance in brain tumors.

Electro-elution-based purification of covalent DNA-protein cross-links.

Covalent DNA-protein cross-links (DPCs) are pervasive DNA lesions that challenge genome stability and can be induced by metabolic or chemotherapeutic cross-linking agents including reactive aldehydes, topoisomerase poisons and DNMT1 inhibitors. The purification of x-linked proteins (PxP), where DNA-cross-linked proteins are separated from soluble proteins via electro-elution, can be used to identify DPCs. Here we describe a versatile and sensitive strategy for PxP. Mammalian cells are collected following exposure to a DPC-inducing agent, embedded in low-melt agarose plugs and lysed under denaturing conditions. Following lysis, the soluble proteins are extracted from the agarose plug by electro-elution, while genomic DNA and cross-linked proteins are retained in the plug. The cross-linked proteins can then be analyzed by standard analytical techniques such as sodium dodecyl-sulfate-polyacrylamide gel electrophoresis followed by western blotting or fluorescent staining. Alternatively, quantitative mass spectrometry-based proteomics can be used for the unbiased identification of DPCs. The isolation and analysis of DPCs by PxP overcomes the limitations of alternative methods to analyze DPCs that rely on precipitation as the separating principle and can be performed by users trained in molecular or cell biology within 2-3 d. The protocol has been optimized to study DPC induction and repair in mammalian cells but may also be adapted to other sample types including bacteria, yeast and tissue samples.

Uncovering the role of TET2-mediated ENPEP activation in trophoblast cell fate determination

Early trophoblast differentiation is crucial for embryo implantation, placentation and fetal development. Dynamic changes in DNA methylation occur during preimplantation development and are critical for cell fate determination. However, the underlying regulatory mechanism remains unclear. Recently, we derived morula-like expanded potential stem cells from human preimplantation embryos (hEPSC-em), providing a valuable tool for studying early trophoblast differentiation. Data analysis on published datasets showed differential expressions of DNA methylation enzymes during early trophoblast differentiation in human embryos and hEPSC-em derived trophoblastic spheroids. We demonstrated downregulation of DNA methyltransferase 3 members (DNMT3s) and upregulation of ten-eleven translocation methylcytosine dioxygenases (TETs) during trophoblast differentiation. While DNMT inhibitor promoted trophoblast differentiation, TET inhibitor hindered the process and reduced implantation potential of trophoblastic spheroids. Further integrative analysis identified that glutamyl aminopeptidase (ENPEP), a trophectoderm progenitor marker, was hypomethylated and highly expressed in trophoblast lineages. Concordantly, progressive loss of DNA methylation in ENPEP promoter and increased ENPEP expression were detected in trophoblast differentiation. Knockout of ENPEP in hEPSC-em compromised trophoblast differentiation potency, reduced adhesion and invasion of trophoblastic spheroids, and impeded trophoblastic stem cell (TSC) derivation. Importantly, TET2 was involved in the loss of DNA methylation and activation of ENPEP expression during trophoblast differentiation. TET2-null hEPSC-em failed to produce TSC properly. Collectively, our results illustrated the crucial roles of ENPEP and TET2 in trophoblast fate commitments and the unprecedented TET2-mediated loss of DNA methylation in ENPEP promoter.

Fetal hemoglobin induction in azacytidine responders enlightens methylation patterns related to blast clearance in higher-risk MDS and CMML

BackgroundAs new treatment options for patients with higher-risk myelodysplastic syndromes are emerging, identification of prognostic markers for hypomethylating agent (HMA) treatment and understanding mechanisms of their delayed and short-term responses are essential. Early fetal hemoglobin (HbF) induction has been suggested as a prognostic indicator for decitabine-treated patients. Although epigenetic mechanisms are assumed, responding patients’ epigenomes have not been thoroughly examined. We aimed to clarify HbF kinetics and prognostic value for azacytidine treated patients, as well as the epigenetic landscape that might influence HbF re-expression and its clinical relevance.ResultsSerial HbF measurements by high-performance liquid chromatography (n = 20) showed induction of HbF only among responders (p = 0.030). Moreover, HbF increase immediately after the first azacytidine cycle demonstrated prognostic value for progression-free survival (PFS) (p = 0.032, HR = 0.19, CI 0.24–1.63). Changes in methylation patterns were revealed with methylated DNA genome-wide sequencing analysis (n = 7) for FOG-1, RCOR-1, ZBTB7A and genes of the NuRD-complex components. Targeted pyrosequencing methodology (n = 28) revealed a strong inverse correlation between the degree of γ-globin gene (HBG2) promoter methylation and baseline HbF levels (p = 0.003, rs = − 0.663). A potential epigenetic mechanism of HbF re-expression in azacytidine responders was enlightened by targeted methylation analysis, through hypomethylation of site -53 of HBG2 promoter (p = 0.039, rs = − 0.504), which corresponds to MBD2-NuRD binding site, and to hypermethylation of the CpG326 island of ZBTB7A (p = 0.05, rs = 0.482), a known HbF repressor. These changes were associated to blast cell clearance (pHBG2 = 0.011, rs = 0.480/pZBTB7A = 0.026, rs = 0.427) and showed prognostic value for PFS (pZBTB7A = 0.037, HR = 1.14, CI 0.34–3.8).ConclusionsEarly HbF induction is featured as an accessible prognostic indicator for HMA treatment and the proposed potential epigenetic mechanism of HbF re-expression in azacytidine responders includes hypomethylation of the γ-globin gene promoter region and hypermethylation of the CpG326 island of ZBTB7A. The association of these methylation patterns with blast clearance and their prognostic value for PFS paves the way to discuss in-depth azacytidine epigenetic mechanism of action.Graphical abstract

Pharmacokinetic assessment of low dose decitabine in combination therapies: Development and validation of a sensitive UHPLC-MS/MS method for murine plasma analysis

Decitabine is a DNA methyltransferase inhibitor used in the treatment of acute myeloid leukemia and myelodysplastic syndrome. The notion that ongoing trials are presently exploring the combined use of decitabine, with or without the cytidine deaminase inhibitor cedazuridine, and other antileukemic drugs necessitates a comprehensive understanding of pharmacokinetic properties and an evaluation of drug-drug interaction liabilities. We report here the development and validation of a sensitive UHPLC-MS/MS method for quantifying decitabine in mouse plasma, which should be useful for such studies. The method involved a one-step protein precipitation extraction, and chromatographic separation on an XBridge HILIC column using gradient elution. The method was found to be robust, accurate, precise, and sufficiently sensitive (lower limit of quantitation, 0.4 ng/mL) to determine decitabine concentrations in microvolumes of plasma from mice receiving the agent orally or intravenously in the presence or absence of cedazuridine.

Home Time Among Older Adults With Acute Myeloid Leukemia Following Chemotherapy

Patients with acute myeloid leukemia (AML) recognize days spent at home (home time) vs in a hospital or nursing facility as an important factor in treatment decision making. No study has adequately described home time among older adults with AML. To describe home time among older adults with AML (aged ≥66 years) and compare home time between 2 common treatments: anthracycline-based chemotherapy and hypomethylating agents (HMAs). A cohort of adults aged 66 years or older with a new diagnosis of AML from the Surveillance, Epidemiology, and End Results (SEER)-Medicare linked database in 2004 to 2016 was identified. Individuals were stratified into anthracycline-based therapy, HMAs, or chemotherapy, not otherwise specified (NOS) using claims. The primary outcome was home time, quantified by subtracting the total number of person-days spent in hospitals and nursing facilities from the number of person-days survived and dividing by total person-days. A weighted multinomial regression model with stabilized inverse probability of treatment weighting to estimate adjusted home time was used. The cohort included 7946 patients with AML: 2824 (35.5%) received anthracyclines, 2542 (32.0%) HMAs, and 2580 (32.5%) were classified as chemotherapy, NOS. Median (IQR) survival was 11.0 (5.0-27.0) months for those receiving anthracyclines and 8.0 (3.0-17.0) months for those receiving HMAs. Adjusted home time for all patients in the first year was 52.4%. Home time was highest among patients receiving HMAs (60.8%) followed by those receiving anthracyclines (51.9%). Despite having a shorter median survival, patients receiving HMAs had more total days at home and 33 more days at home in the first year on average than patients receiving anthracyclines (222 vs 189). This retrospective study of older adults with AML using SEER-Medicare data and propensity score weighting suggests that the additional survival afforded by receiving anthracycline-based therapy was entirely offset by admission to the hospital or to nursing facilities.

DUSP5 regulated by YTHDF1-mediated m6A modification promotes epithelial-mesenchymal transition and EGFR-TKI resistance via the TGF-β/Smad signaling pathway in lung adenocarcinoma

BackgroundLung adenocarcinoma (LUAD) patients have a dismal survival rate because of cancer metastasis and drug resistance. The study aims to identify the genes that concurrently modulate EMT, metastasis and EGFR-TKI resistance, and to investigate the underlying regulatory mechanisms.MethodsCox regression and Kaplan–Meier analyses were applied to identify prognostic oncogenes in LUAD. Gene set enrichment analysis (GSEA) was used to indicate the biological functions of the gene. Wound-healing and Transwell assays were used to detect migratory and invasive ability. EGFR-TKI sensitivity was evaluated by assessing the proliferation, clonogenic survival and metastatic capability of cancer cells with treatment with gefitinib. Methylated RNA immunoprecipitation (MeRIP) and RNA immunoprecipitation (RIP) analyses established the level of m6A modification present on the target gene and the protein’s capability to interact with RNA, respectively. Single-sample gene set enrichment (ssGSEA) algorithm used to investigate levels of immune cell infiltration.ResultsOur study identified dual-specificity phosphatase 5 (DUSP5) as a novel and powerful predictor of adverse outcomes for LUAD by using public datasets. Functional enrichment analysis found that DUSP5 was positively enriched in EMT and transforming growth factor-beta (TGF-β) signaling pathway, a prevailing pathway involved in the induction of EMT. As expected, DUSP5 knockdown suppressed EMT via inhibiting the canonical TGF-β/Smad signaling pathway in in vitro experiments. Consistently, knockdown of DUSP5 was first found to inhibit migratory ability and invasiveness of LUAD cells in in vitro and prevent lung metastasis in in vivo. DUSP5 knockdown re-sensitized gefitinib-resistant LUAD cells to gefitinib, accompanying reversion of EMT progress. In LUAD tissue samples, we found 14 cytosine-phosphate-guanine (CpG) sites of DUSP5 that were negatively associated with DUSP5 gene expression. Importantly, 5′Azacytidine (AZA), an FDA-approved DNA methyltransferase inhibitor, restored DUSP5 expression. Moreover, RIP experiments confirmed that YTH N6-methyladenosine RNA binding protein 1 (YTHDF1), a m6A reader protein, could bind DUSP5 mRNA. YTHDF1 promoted DUSP5 expression and the malignant phenotype of LUAD cells. In addition, the DUSP5-derived genomic model revealed the two clusters with distinguishable immune features and tumor mutational burden (TMB).ConclusionsBriefly, our study discovered DUSP5 which was regulated by epigenetic modification, might be a potential therapeutic target, especially in LUAD patients with acquired EGFR-TKI resistance.Graphical

Azacitidine as epigenetic priming for chemotherapy is safe and well-tolerated in infants with newly diagnosed KMT2A-rearranged acute lymphoblastic leukemia: Children's Oncology Group trial AALL15P1.

Infants less than 1 year old diagnosed with KMT2A-rearranged (KMT2A-r) acute lymphoblastic leukemia (ALL) are at high risk of remission failure, relapse, and death due to leukemia, despite intensive therapies. Infant KMT2A-r ALL blasts are characterized by DNA hypermethylation. Epigenetic priming with DNA methyltransferase inhibitors increases the cytotoxicity of chemotherapy in preclinical studies. The Children's Oncology Group trial AALL15P1 tested the safety and tolerability of five days of azacitidine immediately prior to the start of chemotherapy on day six, in four post-induction chemotherapy courses for infants with newly diagnosed KMT2A-r ALL. The treatment was welltolerated, with only two of 31 evaluable patients (6.5%) experiencing dose-limiting toxicity. Whole genome bisulfite sequencing of peripheral blood mononuclear cells (PBMCs) demonstrated decreased DNA methylation in 87% of samples tested following five days of azacitidine. Event-free survival was similar to prior studies of newly diagnosed infant ALL. Azacitidine is safe and results in decreased DNA methylation of PBMCs in infants with KMT2A-r ALL, but the incorporation of azacitidine to enhance cytotoxicity did not impact survival. Clinicaltrials.gov identifier: NCT02828358.

Targeted silencing of SOCS1 by DNMT1 promotes stemness of human liver cancer stem-like cells

BackgroundHuman liver cancer stem-like cells (HLCSLCs) are widely acknowledged as significant factors in the recurrence and eradication of hepatocellular carcinoma (HCC). The sustenance of HLCSLCs’ stemness is hypothesized to be intricately linked to the epigenetic process of DNA methylation modification of genes associated with anticancer properties. The present study aimed to elucidate the stemness-maintaining mechanism of HLCSLCs and provide a novel idea for the clearance of HLCSLCs.MethodsThe clinical relevance of DNMT1 and SOCS1 in hepatocellular carcinoma (HCC) patients was evaluated through the GEO and TCGA databases. Cellular immunofluorescence assay, methylation-specific PCR, chromatin immunoprecipitation were conducted to explore the expression of DNMT1 and SOCS1 and the regulatory relationship between them in HLCSLCs. Spheroid formation, soft agar colony formation, expression of stemness-associated molecules, and tumorigenicity of xenograft in nude mice were used to evaluate the stemness of HLCSLCs.ResultsThe current analysis revealed a significant upregulation of DNMT1 and downregulation of SOCS1 in HCC tumor tissues compared to adjacent normal liver tissues. Furthermore, patients exhibiting an elevated DNMT1 expression or a reduced SOCS1 expression had low survival. This study illustrated the pronounced expression and activity of DNMT1 in HLCSLCs, which effectively targeted the promoter region of SOCS1 and induced hypermethylation, consequently suppressing the expression of SOCS1. Notably, the stemness of HLCSLCs was reduced upon treatment with DNMT1 inhibitors in a concentration-dependent manner. Additionally, the overexpression of SOCS1 in HLCSLCs significantly mitigated their stemness. The knockdown of SOCS1 expression reversed the effect of DNMT1 inhibitor on the stemness of HLCSLCs. DNMT1 directly binds to the SOCS1 promoter. In vivo, DNMT1 inhibitors suppressed SOCS1 expression and inhibited the growth of xenograft.ConclusionDNMT1 targets the promoter region of SOCS1, induces hypermethylation of its CpG islands, and silences its expression, thereby promoting the stemness of HLCSLCs.

A Hydrogen-Bonded Organic Framework-Based Mitochondrion-Targeting Bioorthogonal Platform for the Modulation of Mitochondrial Epigenetics.

Bioorthogonal chemistry represents a powerful tool in chemical biology, which shows great potential in epigenetic modulation. As a proof of concept, the epigenetic modulation model of mitochondrial DNA (mtDNA) is selected because mtDNA establishes a relative hypermethylation stage under oxidative stress, which impairs the mitochondrion-based therapeutic effect during cancer therapy. Herein, we design a new biocompatible hydrogen-bonded organic framework (HOF) for a HOF-based mitochondrion-targeting bioorthogonal platform TPP@P@PHOF-2. PHOF-2 can activate a prodrug (pro-procainamide) in situ, which can specifically inhibit DNA methyltransferase 1 (DNMT1) activity and remodel the epigenetic modification of mtDNA, making it more susceptible to ROS damage. In addition, PHOF-2 can also catalyze artemisinin to produce large amounts of ROS, effectively damaging mtDNA and achieving better chemodynamic therapy demonstrated by both in vitro and in vivo studies. This work provides new insights into developing advanced bioorthogonal therapy and expands the applications of HOF and bioorthogonal catalysis.

Chromatin modifiers in endometriosis pathogenesis

Introduction. It was revealed that various epigenetic abnormalities may play an important role in the endometriosis pathogenesis. The regulation of chromatin structure is carried out mainly by chromatin modifiers (CMs), which stimulate generation of genomic regions with different functional structures and thus change the patterns or levels of gene expression by exerting expected biological functions and causing epigenetic changes.Aim: to consider СМs role in endometriosis pathogenesis and their regulation mechanism assessing current publications.Materials and Methods. The search was conducted in the databases PubMed, Scopus, Web of Science, Google Scholar and eLibrary. Keywords and phrases in Russian and English related to the research topic were used as follows: "endometriosis", "chromatin modifiers", "histone acetylation", "DNA methylation", "microRNA", "endometriosis", "chromatin modifiers", "histone acetylation", "DNA methylation", "microRNA". The evaluation of articles was carried out in accordance with PRISMA recommendations.Results. Chromatin modifiers control differentiation, growth and development, aging and cell death by interacting with various functional chromatin elements. They can cause abnormal gene expression by regulating chromatin structure affecting emergence and development of endometriosis. DNA methylation determines cell types, controls gene expression and genome stability. Abnormal DNA methylation in gene promoter regions necessary for normal endometrial response affects endometriosis development. DNA methyltransferase (DNMT) inhibitors reduce the methylation of human homeobox A10 (HOXA10) and progesterone receptor (PR) genes and potentiate their expression in endometrial cells, improving endometrial susceptibility and inhibiting cell cycle progression. Abnormal histone modifications in endometrial cells may facilitate or hinder the access of transcription mechanisms to chromatin DNA. Histone deacetylase inhibitors effectively eliminate the effects of abnormal histone modifications in endometriosis cells and prevent endometriosis progression. The expression of non-coding RNAs and chromatin remodeling complexes also alters chromatin structure being involved in arising endometriosis and is associated with infertility by promoting proliferation, invasion and migration of endometriod cells.Conclusion. Chromatin modifiers play a key role in developing endometriosis by controlling gene expression and chromatin structure. Understanding underlying mechanisms provides valuable information for diagnostics and development of new approaches to treat endometriosis.

The DNA Methyltransferase Inhibitor 5-Aza-4'-thio-2'-Deoxycytidine Induces C>G Transversions and Acute Lymphoid Leukemia Development.

DNA methyltransferase inhibitors (DNMTi), most commonly cytidine analogs, are compounds that decrease 5'-cytosine methylation. DNMTi are used clinically based on the hypothesis that cytosine demethylation will lead to re-expression of tumor suppressor genes. 5-Aza-4'-thio-2'-deoxycytidine (Aza-TdCyd or ATC) is a recently described thiol-substituted DNMTi that has been shown to have anti-tumor activity in solid tumor models. In this study, we investigated the therapeutic potential of ATC in a murine transplantation model of myelodysplastic syndrome. ATC treatment led to the transformation of transplanted wild-type bone marrow nucleated cells into lymphoid leukemia, and healthy mice treated with ATC also developed lymphoid leukemia. Whole-exome sequencing revealed 1,000 acquired mutations, almost all of which were C>G transversions in a specific 5'-NCG-3' context. These mutations involved dozens of genes involved in human lymphoid leukemia, such as Notch1, Pten, Pax5, Trp53, and Nf1. Human cells treated in vitro with ATC showed 1,000 acquired C>G transversions in a similar context. Deletion of Dck, the rate-limiting enzyme for the cytidine salvage pathway, eliminated C>G transversions. Taken together, these findings demonstrate a highly penetrant mutagenic and leukemogenic phenotype associated with ATC. Significance: Treatment with a DNA methyltransferase inhibitor generates a distinct mutation signature and triggers leukemic transformation, which has important implications for the research and clinical applications of these inhibitors.

Time toxicity for patients receiving oral versus parenteral hypomethylating agents for myelodysplastic syndromes/neoplasms (MDS).

6568 Background: Patients spend substantial time receiving cancer care. Patients with higher-risk MDS treated with hypomethylating agents (HMAs) are generally older and have a median life expectancy of 11–17 months. Patients receiving parenteral treatment for MDS spend significant time in clinics receiving HMA treatment; as such, information on the time burden of treatments (“time toxicity”) is needed to help clinicians guide patients and caregivers with alternative routes of administration. The development of oral HMA therapies offers a strategy that mitigates the time toxicity associated with MDS treatment by increasing the number of “home days” for patients. This study details the time burden among patients with MDS receiving oral HMA therapy versus those receiving intravenous and subcutaneous (IV/SC) HMAs. Methods: This was a retrospective analysis of adult patients with MDS initiating HMA therapy (oral or IV/SC HMA), using the US Cerner Enviza claims database (08/2020–08/2022). Propensity score matching (1:1) was performed on the treatment groups to balance confounding factors; matched cohorts were analyzed (N=158 each). The total direct healthcare encounter days for oral and IV/SC HMA administered patients were evaluated based on the number of healthcare encounter days spent on parenteral HMA administration (0 days for oral HMAs), outpatient, inpatient, and emergency room (ER) visits, and infusion days. Only distinct encounter days were included in the total count; multiple visits in a day were de-duplicated. Mean (SD) healthcare days for oral versus IV/SC HMA groups were calculated for each type of healthcare encounter and summarized. Results: Patients receiving oral HMA incurred a mean total of 15.2 healthcare encounter days compared with 32.8 days for those receiving IV/SC HMAs (Table). Most encounter days for the oral HMA cohort were in the outpatient setting (34.9%) while most encounters for the IV/SC HMA cohort were attributed to receiving parenteral HMA administration (55.5%), followed by inpatient (16.2%) and outpatient visits (14.9%). Conclusions: This study, which is the first report to date of time toxicity in MDS among patients treated with HMA therapy, revealed that patients receiving oral HMA incurred half the time burden of those receiving IV/SC HMAs. Further research is warranted to validate these results in a larger patient cohort and compare these findings with other cancer therapies. [Table: see text]

Outcomes of blast-phase MPN and JAK2, MPL, and CALRmutated de novo AML: A propensity score-adjusted cohort study.

6526 Background: Progression to blast phase (BP) occurs in about 10% of patients with BCR-ABL-negative myeloproliferative neoplasms (MPN) and is associated with an extremely poor prognosis with no defined standard of care. The VIALE-A trial, which showed that venetoclax (VEN) improves overall survival (OS) in patients with acute myeloid leukemia (AML) when combined with azacitidine, excluded patients with MPN-BP. Rarely, patients develop de novo AML with a genetic profile like an MPN, harboring JAK2, MPL, or CALR mutations. However, there are a lack of data on the outcomes of these patients. Methods: We conducted a single-center retrospective cohort study to compare composite complete response (CCR), OS and event-free survival (EFS) of patients with MPN-BP or de novo AML with underlying JAK2, MPL, or CALR mutations (AML-D-MPN). CCR was defined as complete response (CR) or CR with incomplete count recovery. Propensity score (PS) modeling was used to adjust for baseline characteristics at AML diagnosis: age, sex, race, comorbidities, ECOG status, cytogenetics, and underlying mutations. Results: Of 750 patients treated from 2015 to 2023, 61 had MPN-BP and 12 had AML-D-MPN. The table describes baseline characteristics. The median time for MPN to BP transformation was 8 years (IQR: 2.8-13.6). With a median follow-up of 8.2 months (mo) (95CI: 1.6-59.6), the median OS for MPN-BP was 4.8 mo (95CI: 2.7-8) and for AML-D-MPN was 6.1 mo (95CI: 1.6-NC). In the MPN-BP, 40 received therapy, which included: intensive regimen [7+3 (n=13); CPX-351 (n=1)], hypomethylating agents (HMA) (n=11), HMA+VEN (n=11) and others (n=4). All AML-D-MPN received therapy, which included 7+3 (n=4), HMA (n=1), HMA+VEN (n=2), and others (n=5). In the whole treated population (n=52), the unadjusted CCR was 60% in intensive regimen, 20% in HMA, 20% in HMA+VEN and 0% in others (p=0.3). The PS-adjusted median (m) OS in mo (95%CI) was: 2.6 (1.4-NC) in intensive regimen, 6.2 (1.2-NC) in HMA, 15 (10-15) in HMA+VEN and 1.7 (0.46-NC) in others (p=0.1). The adjusted m-EFS in the same treatment groups was 1.5 (0.43-NC), 6.2 (1.2-NC), 15 (4.3-15) and 0.92 (0.46-NC) (p=0.16), respectively. In MPN-BP group, the adjusted m-EFS in mo (95%CI) was: 0.62 (0.43-NC) in the intensive regimen, 6.2 (1.2-NC) in HMA, 15 (4.2-15) in HMA+VEN and 0.92 (0.92-NC) in others (p=0.25). Conclusions: Patients with MPN-BP and AML-D-MPN have poor survival with similar responses to therapy. These patients have variable outcomes with different treatments; however, there is a trend toward improved survival with the use of HMA+VEN. [Table: see text]

Trends on treatment patterns in patients with newly diagnosed AML: Preliminary findings from a real-world analysis.

e18506 Background: In the last two decades, new therapeutic options have emerged to complement traditional intensive chemotherapy (IC) regimens in the treatment of Acute myeloid leukemia (AML). Hypomethylating agents (HMAs), the B-cell lymphoma 2 (BCL-2) protein inhibitor Venetoclax, and other targeted therapies are examples. Guideline-based treatment decisions are intricate, considering both patient fitness to IC and disease characteristics. AML real-word studies on treatment patterns typically focus on specific subpopulations (e.g., elderly or unfit patients), and few studies analyze broader populations. We analyzed an US electronic health records (EHR) database to describe AML treatment patterns. Methods: Study was designed as retrospective, descriptive analysis of deidentified secondary data from COTA database in the US. Data was abstracted for AML patients diagnosed for the first time between January 2017 and September 2022. Follow-up period (FP) was defined as the period starting at the diagnosis until the last information available for: death, or last contact, or the emergence of other malignancies. To improve descriptive analysis, treatment categories were classified accordingly: IC and non-IC. We conducted descriptive statistics (expressed as counts, percentages or means and medians [with SD or IQR]) for the different assessed features. Results: From the 3,500 patients included on the database, 1,436 were eligible for the analysis with a newly diagnosed AML disease after January 2017. Individuals’ age at diagnosis falls between 60 and 74 years old for 41% of the cohort with mean age of 64[15] years and 60% were followed on community centers. Male patients slightly outnumber female patients (53% and 47%, respectively). Primary AML accounted for 41% of the population and secondary for 28%. Mean time to first treatment after diagnosis was 14[42] days for the overall cohort, the main reason for the end of follow-up was death for 58% of individuals and 47% of the patients had a follow-up inferior to one year. While analyzing treatment patterns according to line of therapies (LoT) sequences, IC only was predominant for 30% of the population, followed by non-IC only for 28%. Remaining counts (42%) correspond to association of both categories. Most prevalent regimen on IC only group was High-dose Cytarabine monotherapy (HIDAC) (12%), and HMA (Azacitidine)+Venetoclax (27%) for the non-IC only group. Conclusions: Our preliminary and recent findings demonstrate that incidence of AML remains high in elderly groups. Despite the introduction of novel therapies, IC remains the predominant treatment approach for newly diagnosed AML patients. Our ongoing real-world analysis aims to assess a more detailed description of LoT sequences in AML population.

Venetoclax combined with hypomethylating agents for favorable risk acute myeloid leukemia: A single center experience.

e18514 Background: Favorable risk acute myeloid leukemia (FR-AML) is typically treated with intensive induction chemotherapy followed by high dose consolidative cytarabine. However, a subgroup of FR-AML are not candidates for high intensity induction because of advanced age, comorbidities, or poor performance status. For these patients, hypomethylating agents (HMA) combined with venetoclax (HMA-Ven) is inferred as the standard of care based upon results of the VIALE-A trial; however, that trial enrolled only intermediate or poor risk AML. The efficacy of HMA-Ven in FR-AML is unclear. Here we present a single center experience of this regimen in FR-AML. Methods: In this retrospective single center case series, we collected data from all patients >18 years old with newly diagnosed FR-AML between January 2018 to December 2022 at AdventHealth Orlando who were treated with HMA-Ven. Patient with relapsed/refractory disease or a diagnosis of acute promyelocytic leukemia were excluded. FR-AML was defined using ELN 2022 guidelines based on the results of genomic testing of the initial bone marrow aspirate. The primary endpoints were the complete remission (CR) rate and 1-year overall survival (OS). Statistical analyses were performed using the R survminer package. Results: Newly diagnosed FR-AML who received HMA-Ven were identified (n=12). The median age was 67.5 years. 3 (25%) cases had inversion of chromosome 16 and 9 (75%) had NPM1 mutations without FLT3-ITD mutations. 11 (92%) patients received decitabine and venetoclax. Patients received a median of 2.5 cycles of HMA-Ven, and all 12 patients achieved CR after one cycle of treatment. 6 (50%) patients were consolidated with intermediate-dose cytarabine (IDAC), 3 (25%) continued HMA-Ven, 2 (17%) underwent allogeneic stem cell transplant, and 1 (8%) received enasidenib. The 1-year OS was 80% (CI 0.59-1), and the median OS was not reached. 1 patient died due to chemotherapy toxicity with IDAC, and 1 patient died due to heart complications. 2 patients relapsed, 1 after IDAC and 1 after intolerance to further treatment after 6 cycles of HMA-Ven. Conclusions: Our findings suggest that HMA-Ven is an effective treatment for newly diagnosed favorable risk AML. All patients achieved CR with one cycle of treatment with most surviving more than one year. Large multicenter studies are needed to further validate these promising results.

A phase 1b/2 study of pivekimab sunirine (PVEK, IMGN632) in combination with venetoclax/azacitidine for patients with newly diagnosed CD123-positive acute myeloid leukemia.

TPS6585 Background: Despite improved outcomes with azacitidine (AZA) and venetoclax (VEN) in newly diagnosed (ND) unfit acute myeloid leukemia (AML), only a subset of patients (pts) respond (CR 37%; CR/CRi 66%) and long-term survival remains inadequate (mOS <15m, DiNardo NEJM 2020). The measurable residual disease (MRD)-negative rate was 41% in AZA-VEN treated pts in VIALE-A which was associated with improved survival (Pratz JCO 2022). CD123 is expressed on the majority of AML blasts and leukemic stem cells while minimally expressed on normal hematopoietic stem cells (Kovtun Blood Adv 2018). Pivekimab sunirine (PVEK, IMGN632) is an antibody-drug conjugate comprising a high-affinity CD123 antibody, cleavable linker, and an indolinobenzodiazepine pseudodimer (IGN) payload. The IGN payload alkylates DNA and causes single strand breaks without crosslinking. IGNs are designed to have high potency against tumor cells, while demonstrating less toxicity to normal marrow progenitors than other DNA-targeting payloads. Clinical data from the first 50 pts in the dose expansion cohorts 1 and 2 with ND AML demonstrated a 76% (22/29) MRD negativity rate (by flow cytometry, negativity threshold <0.1%) (Daver ASH 2023), supporting the continued enrollment of the PVEK+AZA+VEN triplet and further evaluation of the regimen’s antileukemia activity and safety in consideration of potential registration-enabling trials. Methods: This is an open-label, multicenter, phase 1b/2 study of PVEK administered in a combination with AZA+VEN in pts with ND CD123-positive (CD123+ by flow cytometry or IHC) AML, with no prior treatment with hypomethylating agents (HMA). Pts will receive the recommended phase 2 dose of PVEK 0.045 mg/kg IV as a < 30-minute outpatient infusion on day 7, AZA 75 mg/m2 SC or IV daily on days 1 to 7, and VEN 400 mg PO daily for up to 28 days in a 28-day cycle. During cycle 1, a bone marrow evaluation at or around day 21 is required to inform VEN dose duration. Current eligibility criteria for continued enrollment of AML patients unfit for intensive chemotherapy include age ≥ 75 years old, or age < 75 years old with ECOG 2-3, or at least one defined comorbidity. The primary study objectives are to assess antileukemia clinical activity (composite CR [complete remission] rate, overall response rate, duration of remission) and MRD-negativity rates. Key secondary objectives are safety and tolerability, pharmacokinetics and immunogenicity. The PVEK+AZA+VEN triplet in pts with ND unfit AML is currently enrolling across sites in France, Germany, Italy, Spain, UK and USA. Clinical trial information: NCT04086264 .