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ABSTRACT Background Primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC) are rare, chronic cholestatic liver diseases with heterogeneous progression and limited biomarkers of disease biology. Epigenetic age acceleration has been demonstrated in PSC and PBC, suggesting accelerated biological aging. DNA methylation–derived telomere length (DNAmTL) reflects replicative aging and cellular turnover, but its role in PSC and PBC remains unclear. We aimed to evaluate DNAmTL in PSC and PBC and its association with disease severity. Methods We analysed and compared DNAmTL and age‐adjusted DNAmTL among patients with PSC ( n = 474) versus PSC controls ( n = 484) and patients with PBC ( n = 291) versus PBC controls ( n = 291). Associations with clinical outcomes were assessed using nonparametric tests and multivariable logistic regression models. Correlations with primary sclerosing cholangitis Risk Estimate Tool (PREsTO) score and epigenetic age acceleration (AgeAccelGrim) were evaluated using linear regression. Results Patients with PSC demonstrated significantly shorter age‐adjusted DNAmTL compared with controls (median −0.036 vs. 0.016, p < 0.001). Patients with PBC also exhibited reduced DNAmTL relative to controls (median 6.814 vs. 6.885, p < 0.001). Within PSC, shorter DNAmTL was associated with cirrhosis (OR 0.21, 95% CI 0.06–0.69, p = 0.013) and advanced disease (OR 0.20, 95% CI 0.06–0.67, p = 0.009). Shorter DNAmTL was also observed in hepatic decompensation and hepatobiliary malignancy, including cholangiocarcinoma. DNAmTL inversely correlated with PReSTO score ( p < 0.001). DNAmTL was strongly inversely correlated with AgeAccelGrim in both PSC and PBC ( p < 0.001). Conclusion Cholestatic liver diseases are associated with shortened DNAmTL, which independently correlates with disease severity in PSC. Together with epigenetic age acceleration, these findings support a model of accelerated biological aging in PSC and PBC characterized by converging signatures of accelerated biological aging that may contribute to progressive biliary injury and fibrosis.

Abstract While missense somatic mutations in the serine/arginine rich splicing factor 2 (SRSF2) occur early in the clonal development of myeloid neoplasms, they are uniquely enriched in chronic myelomonocytic leukemia (CMML), an MDS/MPN overlap neoplasm, where their estimated prevalence is ∼50%. Short-read RNA-sequencing experiments have demonstrated that missense alterations of the proline 95 residue hotspot (SRSF2P95) globally dysregulate exon inclusion, though the full transcript structures induced by such alternative splicing events remain uncharacterized. Given the striking enrichment of SRSF2 mutations in CMML, we hypothesized that there exist unique splicing alterations contributing to the biology of this disease, and sought to profile the landscape of alternative transcription induced by SRSF2 mutations in CMML using long-read RNA-Seq. We conducted bulk long-read RNA-Seq on bone marrow mononuclear cells from 5 CMML patients with SRSF2P95H (n=3) and SRSF2P95L (n=2) mutations, with VAFs ranging 20-48%, along with 5 CMML splicing factor wildtype patients. Libraries were prepared with the Pacbio Kinnex full-length RNA kit and sequenced on a Revio SPRQ sequencer. Reads were processed and aligned using the IsoSeq pipeline, and isoform collapsing, quality control, reference comparison, and quantification were performed with IsoQuant and SQANTI3. Differential transcript and gene expression was assessed with DESeq2, while differential transcript usage was assessed with the DRIM-Seq and stageR packages. After long-read collapsing and quality control, we identified 255,505 unique transcript isoforms, of which 14,845 (∼5.8%) were novel to the reference transcriptome annotation (GENCODE v39). We identified 23 transcripts differentially expressed in SRSF2-mutant samples, including 9 novel transcripts, the majority of which (n=7) were upregulated in SRSF2-mutant cases. A higher number of transcripts (n=80) and genes (n=65) demonstrated significant changes in proportional transcript usage. Among transcripts with increased expression and usage in SRSF2-mutant cases, we identified a novel transcript of the gene YBX1, which encodes an RNA- and DNA-binding transcription factor required for cell survival and self-renewal in myeloid neoplasms. This novel transcript, which we termed YBX1ΔEx4, has an in-frame loss of exon 4, and is predicted to encode a full-length protein missing a 30 amino acid region of the cold-shock (DNA-binding) domain. Using short read RNA-Seq data from an additional 29 CMML patients, as well as an independent dataset (GSE251806) comprising 43 blast-phase CMML patients, we confirmed increased exclusion of YBX1 exon 4 in SRSF2-mutant cases, with an even greater loss of exon 4 in blast-phase SRSF2-mutant cases. We hypothesize an important role of this isoform in CMML disease progression and are currently performing functional studies to characterize the effect of YBX1ΔEx4. Citation Format: Nickolas Steinauer, Terra Lasho, Christy Finke, Pankaj Pradeep, Jenna Fernandez, Alejandro Ferrer, Moritz Binder, Abhishek Mangaonkar, Mrinal M. Patnaik, . Long-read RNA-Seq reveals a novel isoform landscape of SRSF2-mutant chronic myelomonocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1497.

Abstract BACKGROUND: DNA methyltransferase inhibitors (DNMTi) are US FDA approved for the management of CMML. These agents are associated with an overall response rate (ORR) of 40-50%, with a true complete remission (CR) rate of &lt;20%. TET2 mutations are seen in 60% of CMML, with TET2 being a ferrous and oxoglutarate dependent DNA-dioxygenase that oxidizes 5mC to generate 5hmC, 5fC and 5caC. Ascorbic acid (AA) is critical for TET activity and can enhance TET 1/2/3 dioxygenase function by binding to the catalytic domain and when given in high doses, can result in glutathione depletion and free radical induced cell death. In a pilot study (Xie et al. Blood 2024), we demonstrated that high dose IV AA can be clinically effective and induce changes in differentially methylated regions (DMR) involving hematopoietic enhancers and improve cytopenias in TET2-mutant (mt) patients [CCUS]. We expanded this study to combine high dose IV AA with DNMTi to enhance the demethylation effect and produce durable responses in new diagnosed, treatment requiring, TET2mt CMML patients. METHODS: At Mayo Clinic, the LS1781 (NCT03418038) clinical trial was amended to include Arm E, combing high dose IV AA with standard of care decitabine in newly diagnosed TET2mt CMML patients that needed therapy. IV AA was dosed at 1 g/kg on days 1, 3 and 5 (per 28-day cycle, max dose 100 gm) through a central line, based on demonstrated safety from phase 1 studies. Standard dosing was adopted for decitabine, and patients were then asked to take oral AA, 1 gm daily from days 6-28. Key inclusion criteria included a WHO-defined diagnosis of CMML with at least one detectable TET2 mutation requiring therapy with adequate performance status (ECOG PS &lt;2) and organ function (creatinine clearance &gt; 55 mL/min). Response was evaluated by the MDS/MPN International Working Group Criteria (Savona et al. Blood 2015). We measured pre- and post-therapy (4 cycles) bone marrow variant allele fractions (VAF) through next generation sequencing assays, transcriptomic changes through RNA seq, and DNA methylation changes using the Illumina Methylation EPIC array. RESULTS: Between February 2024 and July 2025, a total of six patients were enrolled. The median age at enrollment was 73 years (range: 65-80); all Caucasian with 3 (50%) males. All patients had a diagnosis of CMML-1 with a median bone marrow (BM) blast percentage of 3 (0-10), median hemoglobin 13 (9.5-14.5) g/dL, median WBC count of 5.7 (2.1-12.6) x 109/L, absolute monocyte count 1.4 (0.7-2.7) x 109/L, and platelet count of 92.5 (32-123) x 109/L. TET2 (100%, 4 patients &gt; 1 TET2 mutation at mean VAF of 36%), SRSF2 (67%), and KRAS (33%) were the most frequent molecular abnormalities. Neutropenia (without infection/sepsis) was the only treatment-associated adverse effect seen in 3 (50%, all grade 3) patients. Response was assessed in patients who completed at least 4 cycles (n=5) of therapy, with all (100%) patients meeting criteria for either a complete response (n=2, 40%), optimal marrow response (n=1, 20%) or clinical benefit (platelet response, n=2, 40%). In five patients who had NGS assessment pre-and post-therapy, there was a &gt;50% reduction in TET2mt VAF, with one patient loosing the KRAS G12D clone in its entirety. Furthermore, we identified significant changes in DNA methylation associated with treatment with &gt;85% of the CpGs being hypomethylated post-treatment. RNA-sequencing on patients pre- and post-treatment, identified key transcriptomic changes, with a decrease in expression of leukemogenic genes. Integration of the differential methylation data with transcriptomic changes identified several genes in which changes in DNA methylation correlated with a change in gene expression. These genes include regulators of blood cell development and inflammatory response including GATA2, BCL2, and HLA-DPA. Several leukemia-associated genes were downregulated including MEIS1, TCL1A, and SETBP1. CONCLUSION: In TET2mt CMML, high dose IV AA acid enhances TET activity and synergizes with DNMTi with encouraging clinical responses in all patients and with no new safety signals. On completion of this study, we plan a randomized phase 2 trial comparing high dose IV AA with decitabine vs decitabine alone in newly diagnosed TET2/IDHmt CMML.

Abstract Introduction: VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) is an adult-onset autoinflammatory disorder defined by somatic UBA1 mutations in hematopoietic stem cells (HSCs). Given the higher median age at onset and prevalent inflammation, approximately 60% of patients have concurrent clonal hematopoiesis (CH) involving epigenetic regulators, DNMT3A and TET2. We have previously demonstrated (Gutierrez-Rodrigues, Blood 2023) that DNMT3A and TET2 mutations in VEXAS are independently associated with increased mortality, without an increased risk of myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). To define the role of CH in VEXAS, we investigated the impact of CH on mortality and related epigenetic dysregulation in primary VEXAS patient samples and validated these findings using an adenine base-editing (ABE) strategy to model cooccurring UBA1 and DNMT3A mutations in CD34+ HSCs. Methods: After IRB approval, 114 VEXAS patients from the Mayo Clinic and the NIH were retrospectively assessed, including 104 screened for CH by error-corrected DNA sequencing (ECS). DNA methylation changes were identified using the Illumina Infinium MethylationEPIC BeadChip and genome-wide using Biomodal 6-base sequencing on peripheral blood samples (PB). Cellular indexing of transcriptomes and epitopes (CITE)-seq was performed using the 10x Chromium 3' GEX platform; 21,114 pooled cells were subjected to UMAP dimensionality reduction, followed by Azimuth based cell identification. HSCs from different donors were base edited for UBA1 M41T mutation with and without cooccurring DNMT3A splice-site mutations. Levels of cytokines in cell culture supernatants were measured using Olink technology, transcriptomic changes were assessed using bulk RNA-seq. Statistical analyses were carried out using R. Results: Patients' median age was 68 (62-74) years; 95% were white and 99% were males. 39% had a pathogenic CH variant, with DNMT3A (18%; median VAF: 42%; range 6-46%) and TET2 (11.5%; median VAF: 11%; range: 2-37%) being most common (2% had both). While the presence of any CH mutation did not impact survival (7.7 vs 11.5yrs, p =0.69), DNMT3Amt CH had an age-independent negative impact on mortality (9.2 vs 5.6yrs, p=0.02), without an increased risk of MDS/AML (p=0.31). Changes in DNA methylation were profiled using the methylation array in 19 VEXAS patients without CH (Median Age: 68 years) and in 9 with DNMT3Amt CH (Median Age: 74 years), respectively. VEXAS samples with DNMT3Amt CH displayed a hypomethylation phenotype compared to VEXAS samples with no CH, with 4,096 unique hypomethylated CpGs, occurring mainly at gene enhancers (Enh) and transcriptional states (Tx, TxWk). This loss of DNA methylation was enriched in inflammatory pathways with extensive hypomethylation observed in the promoters and gene bodies of IL-1 family genes. CITE-seq performed on 7 VEXAS samples (4 DNMT3Amt CH vs 3 no CH) demonstrated an upregulation of gene expression predominantly in T-cell subsets. This increased expression was enriched in inflammatory genes, largely centered around IL-1 and IL-18 family signaling. Pathway analysis revealed an enrichment in respiratory electron transport and oxidative phosphorylation (oxphos), with the VEXAS samples with DNMT3Amt CH demonstrating significantly increased oxphos gene signatures. Base editing of CD34+ cells was done using an adenine base-editing (ABE) strategy to model dual UBA1 and DNMT3A splice-site mutations. We observed that the HSCs base edited for the UBA1 M41T recapitulated the cellular hallmarks of VEXAS demonstrating reduced ubiquitination, unfolded protein response (UPR) activation, and increased inflammation. The addition of cooccurring DNMT3A mutations resulted in decreased DNMT3A expression, further increasing inflammatory markers, confirming altered inflammatory and metabolic pathways. Conclusion Our study validates the age-independent, negative prognostic impact of DNMT3Amt CH on survival in VEXAS syndrome. DNMT3A mutations mediate epigenetic and transcriptional priming associated with upregulation of inflammatory and mitochondrial function pathways. These findings have been validated in a base edited cell model with UBA1 and DNMT3A co-mutations.

Abstract BACKGROUND: DNA methyltransferase inhibition offers palliative benefit but does not alter disease biology. in chronic myelomonocytic leukemia (CMML, Merlevede J et al. Nat Comm 2016). This was conclusively demonstrated in the phase 3 DACOTA study, where decitabine did not improve event-free survival in comparison to hydroxyurea (median 12.1 versus 10.3 months, P=0.27) in advanced myeloproliferative CMML (MP-CMML). Hence, there is in urgent need to develop disease modifying therapies for this dismal condition. We and others have demonstrated a role for RAS mutations in MP-CMML through the activation of the KMT2A-PLK1 axis. Targeting of this axis using onvansertib, an inhibitor of PLK1, has demonstrated its pre-clinical efficacy in MP-CMML patient-derived organoid and xenograft models. (Carr RM et al. Nat Comm 2021). METHODS: We conducted a single center phase 1 study to assess the safety and efficacy of onvansertib in relapsed/refractory MP-CMML (NCT05549661). We used a Bayesian Optimal Interval (BOIN) study design to determine the maximum tolerated dose (MTD). The three dose cohorts included 6, 9 and 12 mg/m2 given orally from days 1 to 21 of each 28-day cycle. Key inclusion criteria included a WHO-defined diagnosis of MP-CMML with a white blood cell (WBC) count &gt; 13,000/mm3 and adequate organ function (estimated GFR &gt; 50 mL/min/m2). Response was evaluated per the MDS/MPN International Working Group Criteria (Savona et al. Blood 2015). Targeting efficacy was assessed by measuring phosphorylation of TCTP (a PLK1 substrate), specifically pTCTP/TCTP ratios by immunoblotting in peripheral blood mononuclear cell (PBMC) samples pre and post cycle 1 (Days 1, 7, 21) and end of cycle 3. Pharmacokinetic studies and translational correlative studies were conducted per protocol. RESULTS: Between April 2023 and July 2025, a total of 9 patients were enrolled. The median age at enrollment was 68 years (range: 60-83); all caucasian with 4 (44%) males. All patients had a diagnosis of CMML-1 with a median bone marrow (BM) blast percentage of 4 (0-9), median hemoglobin 10 (7.1-13.2) g/dL, median WBC count of 11.1 (3.1-72.7) x 109/L (4 patients had a WBC count &lt; 13 x 109/L at enrollment due to hydroxyurea use but were confirmed to be MP-CMML at diagnosis), absolute monocyte count 2.1 (1.9-19.6) x 109/L, and platelet count of 36 (20-494) x 109/L. TET2 (89%), SRSF2 (78%), ASXL1 (56%), NRAS, KRAS and CBL (22% each) were the most frequent molecular abnormalities. Previous therapies included hydroxyurea (n=8) and decitabine (n=1). Four patients were enrolled at the 6mg/m2dose level, 2 at the 9 mg/m2 dose level and we currently have one patient at the 12 mg/m2 dose level. While there were 2 planned patients at the 6 mg/m2 dose level, due to a case of pericardial effusion that was possibly drug related, an additional two patients were enrolled at the same dose level, without any dose limiting toxicity (DLT). Targeting efficacy was confirmed by pTCTP immunoblotting in PBMC samples from 4 patients (6 and 9 mg/m2 dose levels) showing suppressed pTCTP/TCTP ratios. No confirmed DLT has been identified thus far, and no grade 3 or 4 toxicities attributable to the study drug have been reported. Possible or unrelated grade 3 adverse effects included infections (sepsis/cellulitis, n=2, 22%), acute kidney injury (n=1, 11%), pericardial effusion (n=1, 11%), and atrial fibrillation with rapid ventricular response (n=1, 11%). Median duration on therapy was 124 (range: 15-654) days with most common cause of study discontinuation being disease progression (33%). Response was assessed as per the MDS/MPN IWG criteria in patients who received at least 1 cycle (n=8) with 3 (38%) patients meeting criteria for either hematological or optimal marrow response. Specifically, there was one (14%) patient with &gt;100% platelet response, one (14%) with normalization of WBC count (baseline WBC count &gt;50 x 109/L), one (14%) optimal marrow response with BM blast% reduction from 8 to 2, facilitating candidacy for an allogeneic hematopoietic stem cell transplantation, and three patients (38%) remained as stable disease. CONCLUSION: In MP-CMML, PLK1 inhibition with onvansertib is relatively well tolerated and shows preliminary efficacy in approximately 40% of patients, with one patient having an optimal marrow response at the 9 mg/m2 dose. Future directions include a dose expansion phase and studying upfront combination with DNA methyltransferase inhibitors.

Abstract Introduction: Inherited & acquired bone marrow failure syndromes (BMFS) have “stress erythropoiesis” with macrocytic anemia, elevated fetal hemoglobin (HbF) & erythropoietin (EPO) levels. The degree of HbF elevation & its capacity to discriminate between patients with inherited & acquired BMFS needs to be systemically studied. Genetic testing is often delayed & it is important to establish an early clinical diagnosis to make clinical decisions such as initiation of immunosuppressive therapy. Methods: After institutional review board approval, we compiled a clinically & genetically well-annotated cohort of patients with BMFS evaluated at Mayo Clinic. All patients were required to have at least cytopenia(s) & evidence of low or variable bone marrow cellularity upon a bone marrow evaluation. Patients with pre-clonal or malignant disorders were excluded. Clinical diagnoses were independently verified for accuracy. Detailed clinical & laboratory features were recorded. Germline genetic testing was performed as clinically appropriate on peripheral blood or bone marrow samples through targeted panels. Distribution of continuous variables was statistically compared using nonparametric (Mann-Whitney or Kruskal-Wallis) tests, while nominal variables were compared using the Chi-Square test. Kaplan-Meier estimate of overall survival (OS) was computed from the date of diagnosis to date of death or last follow-up (also censored at the time of allogeneic hematopoietic stem cell transplant). Results: Between years 1998 - 2023, 129 patients met criteria for inclusion in the study. Among these, clinical diagnoses included immune-mediated aplastic anemia (AA, n=56, 43%, 21% severe or very severe as per the Camitta criteria), telomere biology disorders (TBD, n=37, 29%), Fanconi anemia (n=10, 8%), GATA2 haploinsufficiency (n=8, 6%), Diamond-Blackfan anemia (DBA, n=2, 2%), deficiency of adenosine deaminase-2 (DADA2, n=2, 2%), congenital dyserythropoietic anemia (CDA, n=1, 1%), congential amegakaryocytic thrombocytopenia (CAMT, n=1, 1%), Schwachman-Diamond syndrome (n=1, 1%) & undefined (n=11, 9%). Among the inherited group, 63% had an identifiable germline pathogenic variant (most commonly, TERT/TERC 30%, FANC group 25% & GATA2 15%) with the rest diagnosed on a clinical basis. Elevated HbF &gt;1% was noted in 79% of all BMFS patients; 83% AA, 70% TBD, 50% in DADA2 & 67% in undefined patients (no data was available for other inherited subtypes). In patients with an elevated HbF, absolute monocyte count (p=0.04) was significantly higher; however, there were no other significant associations with variables at diagnosis such as hemoglobin (p=0.5), white blood cell count (WBC, p=0.14), absolute neutrophil count (p=0.5), platelet count (p=0.7), mean corpuscular volume (MCV, p=0.2), absolute lymphocyte count (p=0.1), absolute reticulocyte count (p=0.4), EPO level (p=0.3), presence of a PNH clone (p=0.2), or presence of a germline variant (p=0.3). When diseases were grouped into inherited & acquired (AA) subgroups, median HbF was not significantly different (median%, 4.5 versus 2.3, p=0.4), with elevated HbF (&gt;1%) in 75% versus 83% (p=0.6) patients respectively. On the other hand, minor PNH clone (&gt;0.1% in at least two cell types) was detectable in the acquired group only (0 versus 55%, p&lt;0.0001). Similarly, acquired group had a shorter (&lt;3 months) onset of cytopenias preceding diagnosis (P&lt;0.0001), with no significant differences in MCV (p=0.2), marrow dysplasia (p=0.09) or atypia (p=0.6), & splenomegaly (p=0.9). At median follow-up of 7 years, Kaplan-Meier estimate of transplant-censored median OS was lower in inherited versus acquired group (16 years versus median not reached, p=0.02). Elevated HbF did not significantly predict adverse OS in either group. Conclusions: Fetal hemoglobin is elevated in both patients with inherited & acquired (aplastic anemia) BMFS. When compared to established variables, elevated HbF has neither diagnostic utility nor prognostic relevance in predicting adverse outcomes. On the other hand, onset of cytopenias and presence of a minor PNH clone are both useful clinical variables in differentiating patients with inherited from acquired BMFS. Our data offers practical clinical utility in making early treatment decisions for patients with BMFS.

Abstract Introduction: ASXL1-mutant (mt) Chronic Myelomonocytic Leukemia (CMML) is an aggressive myeloid neoplasm characterized by increased proliferation, resistance to epigenetic therapies, and poor survival. Epigenetic remodeling and up-regulation of leukemogenic driver genes are hallmarks of ASXL1mt CMML. Truncating mutations in ASXL1 activate enhancers that drive the expression of key leukemogenic driver genes such as HOXA9 and its co-factor MEIS1. Here, we investigate the chromatin accessibility of these leukemogenic enhancers by ASXL1 genotype at single-cell resolution (GoT-ChA-seq). Methods: After IRB approval, we performed bulk RNA-seq and ChIP-seq (H3K4me1, H3K4me3, H3K27ac, H3K27me3, H2AK119ub, H2BK20ac) on bone marrow (BM) mononuclear cells from 40 patients with CMML (19 ASXL1mt, 21 -wildtype/wt). After quality control, differential analyses of gene expression and histone modification occupancy between ASXL1mt and wt patients were conducted. A consensus peak calling framework was used to define genomic regions with ASXL1mt-specific histone modifications. Canonical enhancers were defined as genomic regions with a co-occupancy of H3K4me1 and H3K27ac. We then added regions marked by H2BK20ac to capture regulatory regions associated with p300 (Narita et al. Nat Genet 2023). We used an in silico prediction strategy to associate regulatory regions marked by strong histone modification occupancy with highly up-regulated leukemogenic driver genes (HOXA6-10, MEIS1). The ReMap database (Hammal et al. Nucleic Acids Res 2022) was queried for transcription factor binding in the genomic regions of interest. We then performed GoT-ChA-seq (Izzo et al. Nature 2024) to investigate chromatin accessibility for ASXL1mt (n=3) and wildtype BM single cells from CMML patients. In vitro colony forming assays were conducted on primary CMML BM cells (11- ASXL1mt and 9 wt CMML), assessing the efficacy of EP31670, a novel, oral, dual inhibitor of p300/BRD4. Results: This study included 40 patients with CMML, 19 ASXL1mt and 21 wt. The variant allele frequencies for ASXL1 were compatible with heterozygosity (median 0.41) and TET2 mutations were balanced between the two groups. We identified 149 candidate regulatory regions correlating with the expression of up-regulated target genes of interest (HOXA6-10, MEIS1) by bulk ChIP- and RNA-seq. Publicly available ChIP-seq data from myeloid cell lines demonstrated enrichment of p300 (in THP-1 cells, enrichment effect size +2.58, p=0.002) and BRD4 (in K-562 cells, enrichment effect size +1.58, p=0.045) in these candidate regulatory regions. Next, we interrogated the chromatin accessibility of ASXL1mt (n=13620) and ASXL1wt (n=24269) single cells from 3 CMML patients using GoT-ChA-seq. Differential accessibility analysis revealed 144 genomic regions, all of which were preferentially accessible in ASXL1mt cells compared to ASXL1wt cells (p&lt;0.05 for all regions). Four of these preferentially accessible genomic regions (p&lt;0.001 for all regions) overlapped with the previously identified candidate regulatory regions for MEIS1. The H3K27ac and H2BK20ac occupancy levels in these regions was strongly associated with the expression of MEIS1 (median Pearson r=0.73, p=1.16x10-7). These candidate regulatory regions were annotated in ENCODE as distal and proximal enhancers. Targeting this biology with the dual p300/BRD4 inhibitor EP31670 in progenitor colony forming assays led to a preferential therapeutic effect in ASXL1mt BM samples (median IC50 = 25nM) compared to in ASXL1wt samples (median IC50 = 74nM, p=0.03). Treatment with EP31670 was also associated with increased differentiation (increased blast forming units-erythroid; BFU-E) and organization of colonies in ASXL1mt vs wt CMML. Conclusions: ASXL1mt CMML is characterized by the up-regulation of several leukemogenic driver genes including HOXA9 and its co-factor MEIS1, secondary to the activation of genotype-specific enhancers. The preferential accessibility of these enhancers can be defined between ASXL1mt and ASXL1wt cells within patients. The enhancers of interest interact with p300 and BRD4 in myeloid cell lines and targeting them with the novel dual p300/BRD4 inhibitor, EP31670, induces therapeutic responses at low nanomolar drug concentrations in vitro, preferentially in ASXL1-mutant cells. These data solidify the biological rationale for targeting p300/BRD4 in ASXL1mt chronic myeloid neoplasms for therapeutic benefit (NCT05488548).

Abstract Introduction: Protein truncating somatic ASXL1 mutations in CMML are associated with high-risk features and poor outcomes. We have previously demonstrated that ASXL1-mutant (mt) CMML is characterized by permissive chromatin states and overexpression of leukemia stemness genes (Binder et al. Nat Commun; 2022). DLK1 (delta-like homolog 1) is a pseudo-NOTCH like ligand that is maternally imprinted and paternally expressed and is physiologically not expressed beyond embryogenesis. Aberrant DLK1 expression has been associated with poor outcomes in solid tumors and we have shown differential overexpression in ASXL1mt versus wild-type (wt) CMML. Both the promoter and the last exon of DLK1 contain CpG islands that play a role in regulating DLK1. The CpG island within the fifth exon of DLK1, known as the DLK1-differentially methylated region (DMR) [chr14:100,726,892-100,738,224] is completely unmethylated on the maternal allele and partially methylated on the paternal allele. Previous work (Developmental Cell; 2021) has also identified a DLK1 imprinting control region (ICR) known as the IG-DMR [chr14:100,811,001–100,811,037]. DNA methylation state of the IG-DMR has been shown to impact the expression of MEG3 mRNA (lncRNA) which functions as a cofactor to direct PRC2 to the DLK1 promoter, suppressing DLK1 expression. Since the regulation of DLK1 in ASXL1mt CMML is not well understood, we conducted this multiomic analyses to define epigenetic mechanisms of DLK1 overexpression in ASXL1mt CMML. Methods: After IRB approval, we performed bulk RNA-seq, ChIP-seq (H3K4me1/me3, H3K27ac, H3K27me3) and Illumina MethylationEPIC array on bone marrow (BM) mononuclear cells of 40 CMML patients (19 ASXL1mt, 21 wt). scRNA-seq was conducted on 5 ASXL1mt and 3 wt cases. All libraries were sequenced on an Illumina NovaSeq instrument. We assessed these samples for differential gene expression (RNA-seq) and changes in histone modifications (ChIP-seq) between ASXL1mt and wt CMML, specifically at the DLK1-DMR and IG-DMRregulatory loci. We conducted ddPCR (digital droplet PCR) to determine allelic status of DLK1 in ASXL1mtand wt cases. Global and allele/sequence specific DNA methylation changes were determined using Biomodal's duet EvoC methodology (6 base sequencing, including 5mC and 5hmC) in 5 ASXL1mt and 5 wt CMML cases. Results: We validated the overexpression of DLK1 in 19 ASXL1mt versus 21 wt (log2 fold change +4.85, FDR 4.67e-13) CMML BM samples. scRNA-seq on 5 ASXL1mt versus 3 wt samples demonstrated that DLK1 is only expressed in mutant cases, with the expression being limited to HSPCs. ChIP-seq analysis at the DLK1 regulatory loci validated (a) reduced occupancy of H3K27me3, correlating with ASXL1mt associated disruption of the PRC2-complex, and (b) increased co-occupancy of H3K4me1 and H3K27ac in mt cases, validating a permissive chromatin state in ASXL1mt cases at the DLK1 regulatory regions. Through correlation analysis of RNA-seq and ChIP-seq, we defined the promoter for DLK1, characterized by high Pearson correlation coefficients (R &gt; 0.5) and enrichment of H3K27ac and H3K4me3. Differential methylation analysis identified global hypomethylation around the DLK1-DMR and promoter regions. We observed a mean reduction of ~40% in methylation status at the DLK1 promoter in ASXL1mt versus wt cases, suggesting a loss of epigenetic silencing at this locus. This was corroborated by significant enrichment of 5hmC at the fifth exon of DLK1 indicating active DNA demethylation. Conversely, we observed a gain in methylation at the IG-DMR, suggesting loss of imprinting control through MEG3 silencing and DLK1 overexpression. To further determine the impact of imprinting deregulation on overexpression of DLK1 in ASXL1mt CMML, allele-specific methylation was profiled using the Biomodal whole genome methylation data. This demonstrated that the methylation patterns in ASXL1mt CMML cases were significantly deregulated at the DLK1-DMR, with &gt;30% loss of methylation compared to GRCh38 reference alleles. Furthermore, ddPCR was able to confirm bi-allelic expression of DLK1 in ASXL1mt CMML, suggesting that the observed methylation dysregulation leads to loss of imprinting of the maternal allele. Conclusion: In summary, we demonstrate that ASXL1 mutations in CMML disrupt epigenetic pathways, promoting DNA demethylation, resulting in permissive chromatin states and an aberrant and oncogenic re-expression of maternal DLK1, due to loss of imprinting control.