Hypomethylating Agents Decitabine Research Articles

DIPG-87. TARGETING GENE-REPRESSIVE EPIGENETIC REGULATION TO INDUCE INTERFERON SIGNALING IN DIFFUSE MIDLINE GLIOMA

Abstract BACKGROUND Diffuse intrinsic pontine glioma (DIPG) is a fatal pediatric brainstem tumor with no known effective treatment. DIPG is driven by the H3K27M mutant histone, which promotes epigenetic dysregulation, leading to loss of gene-repressive H3K27me3 marks, global DNA hypomethylation, and a distinct gene expression profile. DIPG cells retain histone methylation at histone H3 lysine 9 (H3K9me2/3), suggesting that the tumor may rely on these marks to repress unwanted expression. Our prior work has demonstrated that treatment with the DNA hypomethylating agent decitabine (DAC) elicits expression of cancer-specific antigens and immune activation, including activation of interferon signaling, but modulating gene-repressive H3K9 methylation in DIPG has not been extensively investigated. METHODS We treated four primary patient-derived DIPG neurosphere cell lines with BIX01294 and UNC0642, inhibitors of H3K9 methyltransferases G9a/GLP, and analyzed gene expression changes by RNA-sequencing and Western blot. We assessed the effect of combination treatments on these four cell lines with both DAC and either BIX01294 or UNC0642 and evaluated impact on gene expression, innate immune signaling, and interferon pathway activation. RESULTS We found that treatment with either of the G9a/GLP inhibitors resulted in the upregulation of genes involved in immune signaling. Given the similarities in the immune signaling response elicited from treatment with DAC and the G9a/GLP inhibitors, we evaluated the effect of combination treatments with both DAC and either BIX01294 or UNC0642. These combinations resulted in an increased induction of genes involved in immune signaling, including STING. CONCLUSIONS Pharmacologic inhibition of DNA methylation and histone H3K9 methylation elicits immune activation in DIPG cells. Upregulation of interferon and STING signaling with this combination treatment may be significant in enhancing the immunogenicity of DIPG cells to allow effective immunotherapy.

Abstract 4413: Identifying genome-wide histone profiles in patient derived models of AML for predictive biomarker development

Abstract Acute myeloid leukemia (AML) is a complex disease with highly individualized treatment plans for patients. Mostly these are based on genetic and phenotypic markers of the disease, but the patient’s overall condition is playing a significant role as well. To develop innovative drugs for a broad patient cohort, it is essential to understand the biological implications of these differences and to model them preclinically. In this study, we are assessing the utility of EpiCypher’s CUT&Tag-FFPE platform to characterize the epigenome of AML PDX samples. CUT&Tag-FFPE is an epigenome profiling assay that is compatible with Formalin-Fixed Paraffin-Embedded (FFPE) samples, enabling robust genome-wide profiling of histone modifications from limited clinical samples. We derived PDX model LEXFAM 2799 (NPM1 wt; FLT3 wt, t(8;21)) and LEXFAM 2966 (NPM1 mut; FLT3 wt) from treatment naïve patients and established in immunocompromised NSG mice. LEXFAM 2799 is a fast-growing model with passaging times of 33 days, whereas LEXFAM 2966 displays a passaging time of 140 days. We next treated with Standard of Care (SoC) agents Decitabine, Cytarabine and all-trans-retinoic acid (ATRA): LEXF 2966 was highly sensitive towards Decitabine leading to complete remission. Cytarabine induced a partial remission and ATRA had only minor effect on tumor growth. In contrast LEXFAM 2799 was resistant towards treatment with ATRA and showed only partial remission under treatment with Cytarabine or Decitabine. Using the CUT&Tag-FFPE platform we are analyzing FFPE slides from the above mentioned two AML PDX models to investigate differences in the genome-wide histone profile that relate to the different growth behavior as well as sensitivity towards SoC treatment. The goal of these studies is to identify tumor model specific profiles that correlated specifically with the sensitivity towards the hypomethylating agent Decitabine. In addition, we plan to analyze additional untreated as well as treated AML PD models to validate the identified signatures confirming them as predictive biomarkers for sensitivity towards current standard of care and prospectively as well innovative compounds. Citation Format: Alysha Simmons, Eva Oswald, Georg Kuales, Michael Keogh, Vishnu U. Kumary, Eva Brill, Martis W. Cowles, Bryan Venters, Julia B. Schueler. Identifying genome-wide histone profiles in patient derived models of AML for predictive biomarker development [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 4413.

DNA methylation landscapes in DIPG reveal methylome variability that can be modified pharmacologically.

Diffuse intrinsic pontine glioma (DIPG) is a uniformly lethal brainstem tumor of childhood, driven by histone H3 K27M mutation and resultant epigenetic dysregulation. Epigenomic analyses of DIPG have shown global loss of repressive chromatin marks accompanied by DNA hypomethylation. However, studies providing a static view of the epigenome do not adequately capture the regulatory underpinnings of DIPG cellular heterogeneity and plasticity. To address this, we performed whole-genome bisulfite sequencing on a large panel of primary DIPG specimens and applied a novel framework for analysis of DNA methylation variability, permitting the derivation of comprehensive genome-wide DNA methylation potential energy landscapes that capture intrinsic epigenetic variation. We show that DIPG has a markedly disordered epigenome with increasingly stochastic DNA methylation at genes regulating pluripotency and developmental identity, potentially enabling cells to sample diverse transcriptional programs and differentiation states. The DIPG epigenetic landscape was responsive to treatment with the hypomethylating agent decitabine, which produced genome-wide demethylation and reduced the stochasticity of DNA methylation at active enhancers and bivalent promoters. Decitabine treatment elicited changes in gene expression, including upregulation of immune signaling such as the interferon response, STING, and MHC class I expression, and sensitized cells to the effects of histone deacetylase inhibition. This study provides a resource for understanding the epigenetic instability that underlies DIPG heterogeneity. It suggests the application of epigenetic therapies to constrain the range of epigenetic states available to DIPG cells, as well as the use of decitabine in priming for immune-based therapies.

Molecular Profiling of Response and Resistance to Venetoclax-Decitabine Therapy in Acute Myeloid Leukemia

The hypomethylating agent decitabine and the BCL-2 inhibitor venetoclax combination (DEC-VEN) is now standard of care in AML, yet a subset of patients remains refractory, and factors that reduce efficacy remain to be fully elucidated. This study aimed to systematically identify molecular determinants of resistance via single-cell (sc)RNA-seq and scATAC-seq analysis of bone marrow (BM) samples from patients participating in a prospective clinical trial of DEC-VEN (NCT03404193). For scRNA-seq, 17 BM samples (total 39,607 cells) from 12 AML patients (median age 67; 8 responders; 4 non-responders; 5 pre- and post-treatment pairs) and 3 healthy BM controls were integrated and annotated into 23 transcriptional cell clusters. In the Uniform Manifold Approximation and Projection (UMAP) plot, C1 overlapped with healthy hematopoietic stem cells (HSCs), C2 appeared only in AML patients, and C3 overlapped with progenitor cells. C1 was characteristic of refractory AML and persisted after DEC-VEN treatment. (Figure 1) C1 cells showed significantly higher expression of FLT3, BCL2, MCL1 and transcription factors MYB, ETV6, RUNX1, LMO2, JUND compared to HSCs from healthy donors. DEC-VEN treatment markedly increased ribosomal protein ( RP ) genes and mitochondrial electron transfer COX family genes and increased expression of the leukemia stem cell (LSC) marker CD74 in C1 of non-responders. In C2 cells, high expression of LSC marker genes FLT3, PIM1, MYC and transforming master regulator genes MYB, ETV6, RUNX2, LMO2, STAT3, NOTCH1, BCL11A was observed. DEC-VEN significantly upregulated RP genes and CD74 in C2 in non-responders, as in C1. In responders, hemoglobin family genes were significantly increased in C2. C3 cells were characterized by highly expressed iron metabolism genes, which were further increased by DEC-VEN in responders. GSEA analysis demonstrated that DEC-VEN upregulated ATP metabolic processes and ribosomal biosynthesis and downregulated stem cell differentiation genes in refractory C1 and C2. In responders' AML clusters, DEC-VEN upregulated cellular oxidant detoxification and iron ion homeostasis and downregulated myeloid leukocyte activation. Next, we extended the AML transcriptome analysis by profiling accessible chromatin by scATAC-seq using BM from pre- and post- DEC -VEN treatment of 2 AML patients (1 refractory, 1 relapsed after remission with incomplete count recovery [CRi]), and 2 healthy controls (total 41,062 cells). Leukemia-like cluster (LLC) cells showed significantly higher chromatin accessibility of CD74, RUNX1, RUNX2, NOTCH1, LMO2, ETV6, JUND, STAT3, BCL2, BCL11A and enriched binding motifs of RUNX2, ETV6, MYB, JUN, JUND, STAT3 compared to other clusters, which was consistent with gene expression findings of C1 and C2 by scRNA-seq. DEC-VEN treatment induced further increased motif enrichment of STAT3 in LLC in CRi AML, and STAT3 RUNX2, IKZF1 in refractory AML. To interpret T cell kinetics in response to DEC-VEN in AML, T cells from scRNA-seq profiling were subclassified based on canonical gene expression. The results showed that the CD3+ and CD3+CD8+ cell signatures were significantly lower in non-responders compared to responders post-treatment (p<0.005). Notably, the ratio of exhausted CD8+ cell clusters was significantly higher in non-responders than in responders in DEC-VEN post-treatment (p<0.05), with downregulation of CD8+ effector marker GZMK, and regulator of CD8+ cytotoxic T cell development TOX. In summary, we identified a subpopulation of AML cells that overlap with HSCs in UMAP plots and are associated with DEC-VEN resistance. LSC-related genes were enriched in resistant AML clusters, while hemoglobin-related genes were increased in sensitive clusters. DEC-VEN induced mitochondrial metabolism genes in resistant cells and further enhanced erythrocytic differentiation in sensitive cells. In addition, scATAC-seq revealed LSC-specific chromatin accessibility patterns, and depletion of CD8+ T cells was observed in non-responders. These findings shed light on the molecular mechanisms of DEC-VEN resistance.

The Impact of Delayed Venetoclax Initiation on Overall Survival in Acute Myeloid Leukemia

Introduction The use of venetoclax in acute myeloid leukemia (AML) has become routine - following FDA approval, approximately half of patients with AML have received venetoclax. However, the outcomes of the timing of venetoclax initiation remain unknown. Such outcomes are of notable importance, as it is often critical to allow time for patient optimization and to await molecular profiling - particularly as ongoing research contributes to a deeper understanding of the mechanisms of venetoclax sensitivity and resistance. A critical real-world limitation is that the turnaround times of complete molecular profiling via NGS assays are frequently 14 to 21 days. Therefore, we performed the first stratified analysis that details the impact of delays in venetoclax-based regimen initiation on overall survival. Methods We analyzed 185 patients with AML treated with venetoclax and a hypomethylating agent (azacitidine or decitabine; VEN+HMA) from January 1, 2018 to April 18, 2023 at VCU Massey Comprehensive Cancer Center. We recorded baseline patient-related and disease characteristics, including age, ECOG, Charlson comorbidity index (CCI) scores, molecular profiling and ELN 2022 cytogenetic risk, dates of regimen initiation, and survival. We individually analyzed the first-line and relapsed or refractory (RR) groups. In each group, we separated patients into three cohorts: those that started therapy in 0-7 days, 8-14 days, and 15 days and beyond. We used the D'Agostino & Pearson method for normality testing and the Mann-Whitney test for between-group comparisons. We applied the Bonferroni correction if multiple comparisons were made. We analyzed survival by the Kaplan-Meier method, with significance determined by the log-rank test. The event for calculating the overall survival (OS) was the date of death. Patients were otherwise censored at the date of last contact. Results Of the 185 patients treated with venetoclax and a hypomethylating agent, 100 (54.1%) were in the first-line setting, and 85 (45.9%) had RR AML. In the first-line setting, the median time to initiation of VEN+HMA was 5 days (range: 0-69 days). Compared with the first-line setting, VEN+HMA for RR AML was started at a significantly longer time from the date of disease progression at a median of 11.5 days (range: 0-110 days, p < 0.0001). In the first-line setting, the median OS was significantly worse for earlier initiation of VEN+HMA at 5.8 months for the 0-7 day cohort, compared with 8.9 months for the 8-14 day cohort and 12.7 months for 15 days and beyond (p = 0.023, Figure A). Similarly, in RR AML, the median OS was also significantly worse in the 0-7 day cohort at 4.9 months - but significantly better in the 8-14 day cohort at 10.5 months compared with the 15-day and beyond cohort at 7.1 months (p = 0.027, Figure B). Discussion In the first-line setting, short delays in initiating VEN+HMA do not adversely impact survival. This approach may be preferred to optimize comorbidities and disease-related complications while awaiting the results of molecular profiling. In RR AML, delays in therapy initiation also do not appear to decrease survival - although the optimal therapeutic window appears to be shorter. Taken together, these findings are the first to suggest that with lower-intensity venetoclax-based strategies, short delays in treatment for patient optimization and comprehensive molecular profiling are associated with improved survival.

Checkpoint Immunotherapy Is Associated with Preferential Activation of Tumor-Antigen Specific CD4 + T Cells in MDS

Background: Patients with intermediate and higher-risk MDS are generally treated with the DNA hypomethylating agents (HMAs) azacytidine and decitabine. These agents improve survival and cytopenias in ~50% of those treated; but only responders derive clinical benefit, and no pre-treatment response prediction has proven efficacy. There is thus an urgent need to develop new approaches to complement or enhance the response to standard-of-care HMA therapies. Studies suggest that HMAs might act in part via activation of anti-tumor immune mechanisms. Based upon this hypothesis a series of early phase trials combining immune checkpoint inhibitors (ICI: both anti-PD1/PDL1 and CTLA4) with azacitidine in MDS/AML patients were undertaken but clinical and immunological efficacy have proven disappointing. In these studies, the lack of antigen specificity made systematic assessment of the anti-MDS immune response and milieu challenging. Antigen specific assessments are particularly important in MDS patients treated with ICIs because distinct from solid tumors, myeloid stem cell cancers involve the populations of antigen presenting cells that modify the capacity for T-cell response, such as dendritic cells. Our group has shown that MDS/AML patients treated with decitabine show increased expression of NY-ESO-1, a well-characterized tumor antigen whose expression is normally suppressed through dense promoter hypermethylation. In our prior Phase I study, MDS patients received vaccination against NY-ESO-1 in combination with standard decitabine. This combination was not only safe, but it also induced NY-ESO-1 specific CD4 + and CD8 + T cell responses in most patients. We hypothesized that combining anti-NY-ESO-1 vaccination (an antigen specific approach) with decitabine and an ICI would allow us to understand the immune synapse in patients with MDS. Methods: We developed an investigator-initiated Phase 1 trial in transplant-ineligible patients with MDS/low blast count AML adding an anti-PD-1 ICI to our established combination of decitabine + NY-ESO-1 vaccination (#NCT 0335871). This open-label, non-randomized single center Phase 1 study used an HLA unrestricted NY-ESO-1 vaccine (CDX-1401 (1 mg) + poly-ICLC (1.8 mg)) in combination with standard dose decitabine (20mg/m2/d x 5 days) and nivolumab (3 mg/kg every 2 weeks), 4 cycles of combination therapy were planned on study; patients deriving clinical benefit could continue treatment at the discretion of the treating physician. The study was conducted in accordance with the Declaration of Helsinki and approved by the Roswell Park Cancer Institute (RPCI) Internal Review Board. Six patients were enrolled, treated, and completed > 4 cycles of decitabine; serial bone marrow and peripheral blood samples were collected for analysis. The combination was safe. Results: Two patients developed CR and the remainder had stable disease as the best response to therapy (Table 1). The majority of patients mounted NY-ESO-1 specific CD4 + T cell responses associated with up-regulation of anti-PD-1 immunotherapy gene signatures in the CD4 + memory T cell compartment. No responses in CD8 + T cells were seen. In seeking explanation for the disparate findings in CD4 + and CD8 + T-cell compartments, we examined conventional dendritic cell populations associated with activation of CD8 + T cells and identified that our patients had markedly reduced numbers of conventional dendritic cells marked by high expression of CD141 (cDC1). This population is critical for a successful response to immunotherapy. cDC1 from MDS patients also showed reduced expression of genes that are key for optimal T-cell activation and expansion. Conclusions: These results suggest that the use of immunotherapy to induce effective, cytotoxic anti-tumor T cell responses depends upon the myeloid immunologic milieu in MDS patients. The critical importance of cDC1 function has recently been described and appreciated in the context of solid tumor immunotherapy. The observation of both numerical and functional defects of this cell population in patients with myeloid neoplasia provides a possible explanation for the disappointing efficacy of immunotherapies in this disease state. Approaches to augment the number and function of specific cDC1 populations in myeloid disease might overcome this defect and thereby enhance the efficacy of immunotherapy for patients with MDS.

Real-World Treatment Patterns Among Patients with Myelodysplastic Syndromes Initiating Oral Decitabine and Cedazuridine or Intravenous/Subcutaneous Hypomethylating Agents

Introduction: Hypomethylating agents (HMAs) are recommended standard of care for patients with higher-risk myelodysplastic syndromes (HR-MDS). HMA therapies include intravenous (IV) or subcutaneous (SC) decitabine and azacitidine; however, parenteral administration of HMAs has been associated with increased patient burden. The oral HMA decitabine and cedazuridine (DEC-C) was FDA-approved in July 2020 for the treatment of MDS, and offers an alternative to IV/SC HMAs, with similar pharmacokinetic exposure and response rates compared to IV decitabine. This study examined treatment patterns among patients with MDS receiving oral DEC-C compared with IV/SC HMAs in real-world clinical practice. Methods: This was a retrospective analysis using the US Cerner Enviza claims database; medical and prescription claims data for patients were linked to mortality data from Datavant. Adults aged ≥18 years, who were diagnosed with MDS (ICD-10-CM code D46.x; all risk levels), and who had initiated a HMA (≥1 claim; August 1, 2020─August 31, 2022) were included; index date was defined as the first pharmacy or medical claim for DEC-C or IV/SC HMAs, respectively. Patients had variable follow-up after index and were followed until end of enrollment, death, or end of study. Propensity score matching (PSM; 1:1) was performed to balance for confounding factors (age, sex, acute myeloid leukemia [AML] diagnosis, Charlson Comorbidity Index [CCI] score, and red blood cell [RBC] transfusion); adjusted bivariate analysis was used to compare outcomes between treatment groups. Longitudinal persistence was assessed according to the number of cycles of therapy received during follow-up, where a cycle was defined as either 3-10 days of administration of index IV/SC HMA or 1 claim for oral DEC-C, within a 28-day cycle. Results: Of 1569 patients included, 160 (10.2%) received DEC-C and 1409 (89.8%) received IV/SC HMAs. In the DEC-C cohort, median age was 72.5 years and 66.9% were male; in the IV/SC cohort, median age was 69.0 years and 58.2% were male. Two-thirds of patients newly initiated HMAs during the study period (69.4% for DEC-C and 72.2% for IV/SC HMAs); 30.6% of the DEC-C cohort had switched from prior IV/SC HMA treatment. Both groups had high levels of comorbidity; 55.6% of patients in the DEC-C cohort and 58.4% in the IV/SC HMA cohort had CCI scores of ≥3, and 14.4% and 23.8%, respectively, had an AML diagnosis. During the 6-month pre-index period, half (55.3%) of patients in both groups received RBC transfusions, and one quarter (25.9%) received platelet transfusions. After matching, there were 158 patients in each treatment cohort and all covariates included in the PSM were balanced (standardized mean difference <0.2). Median age was 72.0 years and 74.0 years for the DEC-C and IV/SC HMAs matched cohorts, respectively, and 66.5% and 69.6%, respectively, were male. Mean CCI scores were 3.45 for DEC-C and 3.15 for IV/SC HMA groups and 14.6% and 15.8%, respectively, had an AML diagnosis pre index. Longitudinal persistence was comparable between the matched DEC-C and IV/SC cohorts during the first 6 months post-index, with similar proportions receiving the maximum number of treatment cycles (based on a 28-day cycle) at each month following index (73.8% vs 71.1%, 46.5% vs 48.7%, 28.6% vs 24.8%, for 2, 4, 6 months, respectively; Figure). However, a trend towards improved persistence with DEC-C versus IV/SC HMAs was observed in patients receiving treatment beyond 6 months (25.0% vs 17.0%, 18.5% vs 9.0%, 11.4% vs 7.6%, for 8, 10, 12 months, respectively). Mean time to discontinuation of treatment was also numerically higher for DEC-C compared with the IV/SC HMA group (87.7 vs 82.0 days); however, differences were not statistically significant. Conclusions: This study, which is to our knowledge the largest real-world report of oral DEC-C use to date, reveals similar but high levels of disease burden and comorbidities among patients with MDS receiving oral DEC-C and parenteral HMA therapy. Trends in treatment patterns suggest comparable persistence with oral DEC-C and IV/SC HMAs at early stages of therapy and improved persistence with oral DEC-C beyond 6 months. These data support the consideration of oral DEC-C as an alternative to chronic parenteral HMA therapy, with the potential for oral DEC-C to reduce the treatment burden associated with administration of IV/SC HMAs.

BIOM-35. IDENTIFICATION AND VALIDATION OF POTENTIAL BIOMARKERS FOR DECITABINE-MEDIATED SENSITIZATION TO TEMOZOLOMIDE IN GLIOBLASTOMA

Abstract Glioblastoma (GBM) is an aggressive form of brain cancer with limited treatment options and a poor prognosis. Our previous research demonstrated that the DNA hypomethylating agent decitabine (DAC) enhances the sensitivity to temozolomide (TMZ) of a subset of primary GBM cell lines derived from fresh surgical tissue specimens, and that this effect may be mediated by upregulation of DNA mismatch repair genes including MLH1. In this context, we aim to identify additional biomarkers for DAC-mediated sensitization to TMZ with potential clinical utility through in vitro and in vivo experiments. We performed single cell RNA sequencing on three DAC-sensitized and three DAC-desensitized cell lines and found 4 genes that exhibited significant differential expression: DCN, LUM, CRABP2 and NUPR1. We validated these findings using quantitative immunoblots, RT-qPCR and immunohistochemistry, finding that NUPR1 protein expression levels varied most significantly between DAC-sensitized and desensitized GBM tissues. Long-range direct methylation sequencing of non-amplified genomic DNA enriched using CRISPR/Cas9 demonstrated several CpG sites within the 4 genes that may be predictive of TMZ sensitization response after treatment with DAC. Finally, we performed mouse survival experiments using orthotopic patient-derived xenografts (PDXs) of the six GBM cell lines, with preliminary results showing that in selected PDX survival was significantly increased with combined DAC-TMZ versus TMZ monotherapy as predicted by in vitro assays. In conclusion, our study demonstrates that combining DAC with TMZ may be a potential treatment strategy for GBM in a subset of patients. There likely exist several biomarkers that can be used at the time of diagnosis, alone or in combination, to predict the response to epigenetic preconditioning using DAC for TMZ sensitization, which will be necessary for optimal patient selection in an early phase clinical trial. Further data and validation studies are necessary to strengthen these findings and to confirm their clinical relevance.

Emerging Trends in Frontline Treatment for Acute Myeloid Leukemia (AML): A Survey of NCI-Designated Cancer Centers

Introduction: For the past several decades the standard of care treatment for frontline therapy for acute myeloid leukemia (AML) in individuals that are eligible for intensive induction therapy has been 7 + 3 (7 days of cytarabine (Ara-C) + 3 days of danuorubicin). Older individuals with AML and those that are ineligible for intensive induction tend to be a more difficult population to treat. Since 2018, venetoclax + hypomethylating agents (HMA) (azacitidine or decitabine) has been considered the standard of care frontline treatment for AML in those who are induction ineligible. Over the past decade there have been significant improvements in genetic and molecular testing that have greatly impacted the development of new frontline treatment therapies for individuals with acute myeloid leukemia (AML) with promising treatment outcomes. Methods: In an effort to quantify the impact that these evolving therapies have had on influencing leukemia treatment practices, a questionnaire was sent to every director/chair of Leukemia at each National Cancer Institute (NCI) designated center. Our two-question questionnaire centered around determining the preferred AML treatment for both patients who are not on clinical trials that are eligible and those that are ineligible for intensive induction therapy. Questions were created based on the 2019 NCCN clinical practice guidelines for AML. The survey was limited to 2 questions so as to elicit higher response rate to the questionnaire. Question #1: What is the preferred induction regimen backbone used at your center - In patients eligible for intense chemo, off-study? 7+3FLAG-IDACLAG-IDAVenetoclax w/ 5-azacytidine or decitabineOther Question #2: What is the preferred induction regimen backbone at your center - In patients who are ineligible for intensive chemo, off-study, and having actionable mutations (IDH or FLT3)? Venetoclax + Hypomethylating agentTargeted agent against IDH or FLT3 withoutvenetoclax +/- hypomethylating agentTargeted agent against IDH or FLT3 withvenetoclax +/- hypomethylating agentOther Results: From our study, we received 31/63 (49.2%) responses and saw that for the intensive induction 90.3% (28/31) respondents selected 7 +3 as the preferred treatment with 3.2% (1/31) choosing FLAG-IDA, with the remaining 6.5% (2/31) choosing “other” ((7 +3) w/ FLT3 inhibitor). Comparatively, for the intensive induction ineligible treatment, 61.3% (19/31) respondents preferred venetoclax + HMAs with 38.7% (12/31) preferring targeted agent w/ venetoclax +/- HMA. Of note, four respondents selected venetoclax + HMA as the preferred treatment, but also preferred aza-ivosidenib for IDH1 mutant AML. Conclusion: Preliminary findings from our survey suggest that results from genetic molecular testing may have a greater influence on the evolution in treatment for the intensive induction ineligible population. As more data comes out regarding combining targeted therapy with more broad-based treatments, clarity will emerge on the most appropriate standard-of-care approach. Additionally, improved turnaround time of testing methodology will likely modify treatment approaches. Our next steps include repeat outreach to obtain 100% response rate and sending a follow-up questionnaire to understand the rational for each specific treatment choice.

Long-Term Follow-up and T Cell Characteristics of Patients with ASXL1-Mutated Relapsed or Refractory MDS or CMML Treated with Guadecitabine and Atezolizumab

Background Treatment of patients with myelodysplastic syndrome (MDS) or chronic myelomonocytic leukemia (CMML) after failure of a hypomethylating agent (HMA) remains an unmet need. We and others demonstrated a modest overall response rate (ORR) to the combination of immune checkpoint inhibition (ICI) and an HMA in such patients; a promising overall survival (OS) compared to historical data (5-6 months) was seen in some, but not all, studies. Our study and that of Daver et al. suggested that patients with ASXL1 mutations, in particular, may benefit from the addition of ICI. We conducted a Phase I/II clinical trial evaluating the addition of a programmed death ligand 1 (PD-L1) inhibitor, atezolizumab, to a subcutaneous prodrug of the HMA decitabine, guadecitabine. We evaluated the presence of common driver mutations in CD3- bone marrow mononuclear cells (BMMC) and CD3+ T lymphocytes. Among 27 tested patients, 12 had the ASXL1 mutations in the myeloid compartment and, of those, 5 patients' T cells carried the exact same mutation at a variant allele frequency (VAF) of >10%. Both patients with complete remission in the study (n=2) carried the mutation in both compartments (co-mutated) and median OS was significantly better for co-mutated patients (not reached) than for those with CD3- BMMC (myeloid) mutations (9.5 months) or wild type ASXL1 (16.5 months). Herein we report long-term follow-up of this cohort of patients, along with T cell programmed death-1 (PD-1) expression analyzed based on mutational status. Methods Details of the trial design and correlative plan are published. In the published study we used a VAF cutoff of 2% (1% for TP53) for BMMC and 10% for CD3+ T lymphocytes for the panel of 40 commonly mutated genes. In the current study we also evaluated patients with a T cell ASXL1 VAF of 5-10%. Patients have been followed for progression and survival every 6 months. Demographic and disease characteristics and outcome data were compared based on ASXL1 mutation status. Previously obtained T lymphocyte PD-1 expression data from samples obtained pre-treatment (PRE), during cycle 2 (EARLY) and after cycle 3 or 4 (LATE) were analyzed based on ASXL1 mutation status. Results Among 27 patients from 4 centers with DNA available for genetic analysis, 15 had no ASXL1 mutation ( ASXL1 WT), 5 had ASXL1 mutations restricted to the myeloid lineage, and 7 had ASXL1 mutations in both the myeloid and T-cell populations ( ASXL1 co-mutated). Two co-mutated patients had T cell VAFs of 5-10% and 5 had VAFs>10% (Figure 1). No baseline characteristic by ASXL1 mutational status was significantly associated with response to study treatment, though 6 of 7 co-mutated patients (vs 3/5 myeloid-only patients) had not responded to first-line HMA (refractory). With a median follow-up time of 64.9 months, median survival times were 48.9 months (15.1, 64.9+) for co-mutated patients with T cell VAF >10%, 34.9 months (8.5, NR) for co-mutated patients including those with T cell VAF of >5%, 16.4 months (3.3, 27.3) for ASXL1 WT patients, and 9.5 months (2.1, 35) for myeloid-only patients (Figure 2). This was statistically significant after adjusting for age and ECOG performance status at study entry, diagnosis, baseline bone marrow blast percentage, and number of prior chemotherapy regimens using the likelihood ratio test from multivariate analysis for overall survival (P = 0.008). Early treatment-induced upregulation of CD8 + T cell PD1 was observed in the ASXL1 WT, before returning to baseline levels. This upregulation was more pronounced in the ASXL1 co-mutated group beginning at a lower baseline and peaking at a higher MFI and was statistically significant (P = 0.0098). No significant treatment-induced change in PD-1 expression was noted in the myeloid-only subset. Discussion In patients with relapsed or refractory MDS or CMML, the presence of an ASXL1 mutation in both myeloid and T lymphocyte compartments, as compared to WT or myeloid-only mutations, was associated with a significantly longer median OS when treated with the combination of ICI and HMA. Significant upregulation of PD-1 was noted in T lymphocytes from both wild-type and co-mutated patients. The effect of mutant ASXL1 on T cell responsiveness to ICI deserves further investigation; patients with ASXL1-mutated HMA-refractory myeloid malignancies may benefit from ICI.

Hypomethylating agent decitabine sensitizes diffuse large B-cell lymphoma to venetoclax.

Despite recent advances in the therapy of diffuse large B-cell lymphoma (DLBCL), many patients are still not cured. Therefore, new therapeutic strategies are needed. The anti-apoptotic B-cell lymphoma 2 (BCL2) gene is commonly dysregulated in DLBCL due to various mechanisms such as chromosomal translocation t(14;18)(q32;q21) and copy number alterations; however, targeting BCL-2 with the selective inhibitor, venetoclax, led to response in only a minority of patients. Thus, we sought to identify a rational combination partner of venetoclax to improve its activity against DLBCL cells. Utilizing a functional assay, dynamic BH3 profiling, we found that the DNA hypomethylating agent decitabine increased mitochondrial apoptotic priming and BCL-2 dependence in DLBCL cells. RNA-sequencing analysis revealed that decitabine suppressed the pro-survival PI3K-AKT pathway and altered the mitochondria membrane composition in DLBCL cell lines. Additionally, it induced a DNA damage response and increased BAX and BAK activities. The combination of decitabine and venetoclax synergistically suppressed proliferation of DLBCL cells both in vitro and in vivo in a DLBCL cell line-derived xenograft mouse model. Our study suggests that decitabine plus venetoclax is a promising combination to explore clinically in DLBCL.

RNautophagic regulation of DNMT3a-dependent DNA methylation by Linc00942 enhances chemoresistance in gastric cancer.

Energy balance has long been known to extend lifespans and inhibit carcinogenesis in multiple species by slowing age-related epigenetic changes while the underlying mechanisms remain largely unknown. Herein, we found that starvation activated autophagy to remodel the DNA methylation profile by inhibiting DNMT3a expression. Illumina Infinium MethylationEPIC BeadChip and dot blot assay were performed to quantify the global DNA methylation level. Protein-RNA interactions were validated through RNA immunoprecipitation and RNA pull-down assay. In vitro and in vivo experiments were carried out to testify the effect of DNMT3a on chemoresistance. Autophagy is impaired in chemoresistance which was associated with differential DNA methylation and could be reversed by DNMT3a inhibition. Autophagy activation decreases the expression of DNMT3a mRNA, accompanied with the downregulation of chemoresistance-related Linc00942. Knockdown of Linc00942 reduces DNMT3a expression and genome-wide DNA methylation while Linc00942 overexpression increased DNMT3a expression and correlated hypermethylation in cancer cells and primary tumour tissues. Mechanistically, Linc00942 recruits RNA methyltransferase METTL3 to stimulate N6-methyladenosine (m6A) deposit on DNMT3a transcripts, triggering IGF2BP3/HuR to recognize modified mRNA for reinforced stability. SQSTM1/p62 recruits Linc00942 for autophagic degradation which can be abrogated after autophagy inhibition by p62 knockdown or chloroquine treatment. Inhibition of autophagy increases Linc00942 expression to promote chemoresistance and autophagy activation or hypomethylating agent decitabine restores chemosensitivity by reducing global DNA methylation. Overall, this study identifies a novel methylation cascade linking impaired RNautophagy to global hypermethylation in chemoresistance, and provides a rationale for repurposing decitabine to overcome chemoresistance in cancer treatment.

DIPG-42. MAPPING THE DNA METHYLATION LANDSCAPE OF DIPG REVEALS NOTABLE METHYLOME VARIABILITY RESPONSIVE TO PHARMACOLOGIC MODULATION

Abstract Diffuse intrinsic pontine glioma (DIPG) is an incurable pediatric brainstem tumor, driven by the H3K27M mutation leading to epigenetic dysregulation. Epigenomic analyses of DIPG have shown global loss of repressive chromatin marks accompanied by DNA hypomethylation and altered gene expression. However, most investigations of the epigenetic landscape of DIPG only provide a static view of the epigenome. Such studies are not capable of adequately capturing the regulatory underpinnings of DIPG intratumoral heterogeneity and phenotypic plasticity. To address this, we performed Whole-Genome Bisulfite Sequencing (WGBS) on 23 primary patient DIPG specimens, 4 patient-derived DIPG neurosphere cell lines, and 5 normal controls. To analyze this data set, we applied a novel framework for analysis of DNA methylation variability, called informME analysis, which permits the derivation of comprehensive genome-wide DNA methylation potential energy landscapes that capture intrinsic epigenetic variation. We show that DIPG exhibits a markedly disordered epigenome, with increased stochasticity localizing to key regulatory elements and genes. We demonstrate that epigenetic instability maps onto key genes regulating pluripotency and developmental identity and hypothesize that these changes might enable tumor cells to sample a diverse range of transcriptional programs and phenotypes. Given this, we evaluated the responsiveness of the DIPG epigenetic landscape to pharmacologic modulation. We found that treatment with the DNA hypomethylating agent decitabine produced profound genome-wide demethylation and reduced DNA methylation stochasticity at active enhancers, bivalent promoters, and promoters of key developmental genes. Decitabine treatment also elicited changes in gene expression, including upregulation of genes related to immune signaling. Finally, treatment with DAC sensitized DIPG cells to the effects of histone deacetylase inhibition. This study suggests the application of epigenetic therapies to constrain cellular heterogeneity and plasticity in DIPG.

Combating breast cancer progression through combination therapy with hypomethylating agent and glucocorticoid

Breast cancer is the leading cause of cancer-related death in women. Among breast cancer types, triple-negative breast cancer (TNBC) accounts for 15% of all breast cancers with aggressive tumor behavior. By using bioinformatic approaches, we observed that the microRNA-708 promoter is highly methylated in breast carcinomas, and this methylation is linked to a poor prognosis. Moreover, microRNA-708 expression correlates with better clinical outcomes in TNBC patients. Combination treatment with the hypomethylating agent decitabine and synthetic glucocorticoid significantly increased the expression of microRNA-708, reactivated DNMT-suppressed pathways, and decreased the expression of multiple metastasis-promoting genes such as matrix metalloproteinases (MMPs) and IL-1β, leading to the suppression of breast cancer cell proliferation, migration, and invasion, as well as reduced tumor growth and distant metastasis in the TNBC xenograft mouse model. Overall, our study reveals a therapeutic opportunity in which a combined regimen of decitabine with glucocorticoid may have therapeutic potential in treating TNBC patients.

GSK-3484862 targets DNMT1 for degradation in cells.

Maintenance of genomic methylation patterns at DNA replication forks by DNMT1 is the key to faithful mitotic inheritance. DNMT1 is often overexpressed in cancer cells and the DNA hypomethylating agents azacytidine and decitabine are currently used in the treatment of hematologic malignancies. However, the toxicity of these cytidine analogs and their ineffectiveness in treating solid tumors have limited wider clinical use. GSK-3484862 is a newly-developed, dicyanopyridine containing, non-nucleoside DNMT1-selective inhibitor with low cellular toxicity. Here, we show that GSK-3484862 targets DNMT1 for protein degradation in both cancer cell lines and murine embryonic stem cells (mESCs). DNMT1 depletion was rapid, taking effect within hours following GSK-3484862 treatment, leading to global hypomethylation. Inhibitor-induced DNMT1 degradation was proteasome-dependent, with no discernible loss of DNMT1 mRNA. In mESCs, GSK-3484862-induced Dnmt1 degradation requires the Dnmt1 accessory factor Uhrf1 and its E3 ubiquitin ligase activity. We also show that Dnmt1 depletion and DNA hypomethylation induced by the compound are reversible after its removal. Together, these results indicate that this DNMT1-selective degrader/inhibitor will be a valuable tool for dissecting coordinated events linking DNA methylation to gene expression and identifying downstream effectors that ultimately regulate cellular response to altered DNA methylation patterns in a tissue/cell-specific manner.

Pre-Clinical Evaluation of the Hypomethylating Agent Decitabine for the Treatment of T-Cell Lymphoblastic Lymphoma.

T-cell lymphoblastic lymphoma (T-LBL) is a rare and aggressive lymphatic cancer, often diagnosed at a young age. Patients are treated with intensive chemotherapy, potentially followed by a hematopoietic stem cell transplantation. Although prognosis of T-LBL has improved with intensified treatment protocols, they are associated with side effects and 10-20% of patients still die from relapsed or refractory disease. Given this, the search toward less toxic anti-lymphoma therapies is ongoing. Here, we targeted the recently described DNA hypermethylated profile in T-LBL with the DNA hypomethylating agent decitabine. We evaluated the anti-lymphoma properties and downstream effects of decitabine, using patient derived xenograft (PDX) models. Decitabine treatment resulted in prolonged lymphoma-free survival in all T-LBL PDX models, which was associated with downregulation of the oncogenic MYC pathway. However, some PDX models showed more benefit of decitabine treatment compared to others. In more sensitive models, differentially methylated CpG regions resulted in more differentially expressed genes in open chromatin regions. This resulted in stronger downregulation of cell cycle genes and upregulation of immune response activating transcripts. Finally, we suggest a gene signature for high decitabine sensitivity in T-LBL. Altogether, we here delivered pre-clinical proof of the potential use of decitabine as a new therapeutic agent in T-LBL.

Absence of early platelet increment in healthy mice during decitabine treatment

Treatment of myelodysplastic syndromes includes the administration of the hypomethylating agent decitabine. An early platelet response in decitabine-treated myelodysplastic syndrome patients is a predictor of overall survival. The effect of decitabine on megakaryocytes and the bone marrow, however, is understudied. We show that an early platelet increment was not detectable in healthy mice during decitabine treatment. Analyses of bone marrow sections revealed vessels with dilated lumina, decreased cellularity, but increased number of red blood cells and the presence of (pro)platelet-like particles. Taken together, decitabine treatment of healthy mice does not induce an early platelet increment, but affects the bone marrow.

Triple combination targeting methyltransferase, BCL-2, and PD-1 facilitates antileukemia responses in acute myeloid leukemia.

A recent breakthrough therapy combining the BCL-2 inhibitor venetoclax with hypomethylating agents (HMAs) targeting DNA methyltransferase has improved outcomes for patients with acute myeloid leukemia (AML), but the responses and long-term survival in older/unfit patients and in patients with relapsed/refractory AML remain suboptimal. Recent studies showed that inhibition of BCL-2 or DNA methyltransferase modulates AML T-cell immunity. By using flow cytometry and time-of-flight mass cytometry, the authors examined the effects of the HMA decitabine combined with the BCL-2 inhibitor venetoclax (DAC/VEN therapy) on leukemia cells and T cells in patients with AML who received DAC/VEN therapy in a clinical trial. The authors investigated the response of programmed cell death protein 1 (PD-1) inhibition in the DAC/VEN-treated samples in vitro and investigated the triple combination of PD-1 inhibition with HMA/venetoclax in the trial patients who had AML. DAC/VEN therapy effectively targeted leukemia cells and upregulated the expression of the immune checkpoint-inhibitory receptor PD-1 in T cells while preserving CD4-positive and CD8-positive memory T cells in a subset of patients with AML who were tested. In vitro PD-1 inhibition potentiated the antileukemia response in DAC/VEN-treated AML samples. The combined use of azacitidine, venetoclax, and nivolumab eliminated circulating blasts and leukemia stem cells/progenitor cells and expanded the percentage of CD8-positive memory T cells in an illustrative patient with relapsed AML who responded to the regimen in an ongoing clinical trial. Immunomodulation by targeting PD-1 enhances the therapeutic effect of combining an HMA and venetoclax in patients with AML.

IMPDH inhibition activates TLR-VCAM1 pathway and suppresses the development of MLL-fusion leukemia.

Inosine monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme in de novo guanine nucleotide synthesis pathway. Although IMPDH inhibitors are widely used as effective immunosuppressants, their antitumor effects have not been proven in the clinical setting. Here, we found that acute myeloid leukemias (AMLs) with MLL-fusions are susceptible to IMPDH inhibitors invitro. We also showed that alternate-day administration of IMPDH inhibitors suppressed the development of MLL-AF9-driven AML invivo without having a devastating effect on immune function. Mechanistically, IMPDH inhibition induced overactivation of Toll-like receptor (TLR)-TRAF6-NF-κB signaling and upregulation of an adhesion molecule VCAM1, which contribute to the antileukemia effect of IMPDH inhibitors. Consequently, combined treatment with IMPDH inhibitors and the TLR1/2 agonist effectively inhibited the development of MLL-fusion AML. These findings provide a rational basis for clinical testing of IMPDH inhibitors against MLL-fusion AMLs and potentially other aggressive tumors with active TLR signaling.

Disturbed Flow-Facilitated Margination and Targeting of Nanodisks Protect against Atherosclerosis.

Disturbed blood flow induces endothelial pro-inflammatory responses that promote atherogenesis. Nanoparticle-based therapeutics aimed at treating endothelial inflammation in vasculature where disturbed flow occurs may provide a promising avenue to prevent atherosclerosis. By using a vertical-step flow apparatus and a microfluidic chip of vascular stenosis, herein, it is found that the disk-shaped versus the spherical nanoparticles exhibit preferential margination (localization and adhesion) to the regions with the pro-atherogenic disturbed flow. By employing a mouse model of carotid partial ligation, superior targeting and higher accumulation of the disk-shaped particles are also demonstrated within disturbed flow areas than that of the spherical particles. In hyperlipidemia mice, administration of disk-shaped particles loaded with hypomethylating agent decitabine (DAC) displays greater anti-inflammatory and anti-atherosclerotic effects compared with that of the spherical counterparts and exhibits reduced toxicity than "naked" DAC. The findings suggest that shaping nanoparticles to disk is an effective strategy for promoting their delivery to atheroprone endothelia.