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Abstract Introduction Blast transformation (BT) in CMML occurs in 15-30% of patients (pts) with a median overall survival (OS) <6 months (mo). While clonal evolution can be observed, biological mechanisms of BT remain unknown for many pts. Using paired samples from chronic phase (CP) and at BT, this study defines the cytogenetic and molecular landscape of BT-CMML. Methods BT-CMML pts from Mayo Clinic (n=153, 31%), MD Anderson (n=195, 40%), and 7 centers of the Groupe Francophone des Myélodysplasies (n=145, 29%) were included after IRB approval. ICC 2022 criteria defined CMML and AML. Pt data were collected at initial diagnosis or referral at CP and again at BT. Clonal dominance was inferred from a natural log-fold difference of mutation variant allele fractions (VAF). OS was censored at allogeneic stem cell transplant (alloSCT). Single-cell multiomic analyses used the Tapestri platform (Mission Bio). Statistics used R. Results Among 1,883 cases of CMML with a median follow up of 96 mo, 493 (26%) underwent BT. The median age at CP and BT was 69 and 71 years, respectively. BT-CMML pts were predominantly male (69%) and had proliferative CMML (53%) or CMML-1 (69%) in CP; 39% and 33% were considered high-risk by CPSS-Mol and BLAST-Mol criteria, respectively. The median time to BT was 16 mo (interquartile range 7-35 mo) and was not different in males vs females (p=0.60). Compared to their CP parameters (p<0.001 each), pts at BT had lower median hemoglobin (10.8 vs 9.2 g/dL) and platelets (100 vs 44 x109/L) but higher leukocytes (13.9 vs 16.3 x109/L), peripheral blasts (0% vs 14%), and bone marrow blasts (6% vs 32%). Blasts exhibited mostly monocytic (57%) or immature myeloid (40%) differentiation. Abnormal karyotypes (AK) were more prevalent at BT (25% vs 62%, p<0.001). Among 318 pts with paired karyotype data, 221 (69%) had a normal karyotype (NK) in CP, of which 123 (56%) remained normal at BT and 98 (44%) acquired new abnormalities. Conversely, 97 (31%) pts had an AK in CP, of which 67 (69%) acquired abnormalities or increased clonal metaphases (6%), 23 (24%) remained stable, and 7 (7%) reverted to a NK at BT. Overall, 46% acquired and 8% lost a clonal cytogenetic abnormality, while 46% remained unchanged. Numeric (37%) and structural (30%) changes were most common. Complex (12% vs 3%) and monosomal (5% vs 2%) karyotypes were more frequent at BT than in CP. Only 6 (2%) pts acquired an AML-defining rearrangement at BT. Mutational data were available for 248 paired cases. The number of somatic mutations was higher at BT (median 3 vs 4, p<0.001). Mutations in RUNX1 (27% vs 39%), NRAS (23% vs 33%), KRAS (11% vs 17%), FLT3 (6% vs 15%), NPM1 (0% vs 11%), IDH2 (5% vs 11%), and CEBPA (3% vs 10%) were more frequent at BT (all p<0.05). At BT, the average VAF of the dominant clone increased in 32%, decreased in 35%, and remained unchanged (delta ≤5%) in 33%. The most frequent dominant clones both in CP and at BT had mutations in TET2 (8% vs 4%) or co-dominant mutations involving SRSF2-TET2 (7% vs 3%). Mutational clonal evolution was observed in 72%, with mutations gained in 45% and lost from the dominant clone in 19%. The most gained mutations at BT were in NRAS (15%) and RUNX1 (13%). Single cell proteogenomic analyses in 4 selected CP and BT pts shed light on the impact of NRAS and RUNX1 mutations on clonal hierarchies and differentiation. Among 196 fully evaluable cases, a new cytogenetic and/or molecular aberration was uniquely gained in 106 (54%) cases and lost in 9 (5%), with 40 (20%) both gaining and losing a genetic alteration. Forty-one (21%) patients had no identifiable genetic change between CP and BT, suggesting driver alterations outside of that detected by routine analyses. Median OS was 31 mo from CMML diagnosis and 5.6 mo from BT. Compared to supportive care, treatment in CP did not prolong median time to BT (14 vs 17 mo, p=0.91) or OS (35 vs 29 mo, p=0.05), regardless of intensity. After BT, median OS was 1.2 mo with supportive care (n=83), 8.7 mo with a hypomethylating agent (HMA, n=39), 10 mo with HMA plus venetoclax (n=37), 7.8 mo with chemotherapy (n=91), and 31 mo in pts who underwent alloSCT (n=57). Conclusions In this large BT-CMML cohort, cytogenetic (46% gained, 8% lost) or mutational (45% gained, 19% lost) driver events were identified in 79% of pts, with 21% having no identifiable changes by conventional testing. Whole genome/transcriptome analyses are underway in this subgroup to identify novel drivers of BT.

Abstract Background: VEXAS (Vacuoles, E1 enzyme, X-linked, Autoinflammatory, Somatic) syndrome is a hemato-inflammatory syndrome due to somatic mutations in UBA1 gene. Recently, DNA methyltransferase inhibitors (specifically, 5-azacitidine) and allogeneic hematopoietic stem cell transplantation (alloHSCT) have shown success in eradication of UBA1 clones and clinical remission in some patients. This study aims to provide a comparative efficacy analysis between DNA methyltransferase inhibitors (hypomethylating agents/HMA) versus alloHSCT in VEXAS syndrome patients. Methods: This is a multi-center, retrospective cohort study. UBA1 mutations were detected either through next generation sequencing [NGS, limit of detection (LOD) 2% variant allele fraction (VAF)] or quantitative droplet digital polymerase chain reaction testing (ddPCR, LOD 0.5% VAF). Outcomes of interest included overall survival (OS), glucocorticoid (GC) discontinuation/reduction in dose (prednisone < 10 mg/day), and complete molecular remission (CMR, defined as an undetectable UBA1 mutation). OS was calculated from the time of diagnosis to death or last follow up. Cumulative incidence of reduction in GC dose was calculated using competing risk analysis after accounting for death. Results: Between years 2020-2024, a total of thirty-nine male patients (median age 67, range 45-82 years) with VEXAS syndrome, necessity for therapy (GC-refractory inflammation and/or progressive marrow failure/myeloid neoplasm) and confirmed UBA1 pathogenic mutation were included. Of these, 18 (46%) received HMA therapy (5-azacitidine 72% and Decitabine 28%) with a median of 4 (range 1-36) cycles. and 21 (54%) underwent alloHSCT (MSD=4, MUD=12, MMUD=2, and haploidentical=3), all received reduced intensity conditioning (Flu/Mel =11, Flu/Bu=5, Flu/Cy/TBI=2, and TBF=3) and 91% received post-transplant cyclophosphamide-based graft-versus-host disease (GVHD) prophylaxis (only 2 patients in alloHSCT group received prior HMA without benefit). Baseline characteristics were well-balanced between HMA and alloHSCT groups; age at treatment (median 70 vs 65 years, p=0.2), and UBA1 M41V subtype (25% vs 40%, p=0.3), cytogenetic and non-UBA1 mutation abnormalities (p=0.4), prior lines of therapy (median 4 in both, p=0.5), time from diagnosis to therapy (median 7 vs 13 months, p=0.15), indication for therapy (p=0.12), Charlson CI scores (median 4 in both, p=0.4) and HCI-CI scores (median 4 in both, p=0.7). At a median follow-up of 45 (range 7-57) months, 11 (28%) deaths (alloHSCT=3 and HMA=8) were documented. Causes of death included infections (n=6), cardiac disease (n=2), poor graft function (n=2) and pulmonary GVHD (n=1). Compared to HMA therapy, alloHSCT group demonstrated higher rates of GC discontinuation (52% vs 6%, p<0.01) and CMR (evaluable= 22, 100% vs 50%, p<0.01). In the HMA group, 50% (n=9) discontinued therapy after a median of 4 months due to infectious complications or lack of benefit. In the alloHSCT group, 3 (14%) patients developed grade 2 acute GVHD (1-skin, 2-gut, no grade 3-4 acute GVHD) and 4 (19%) experienced moderate to severe chronic GvHD (2-lung, 2-mucocutaneous). In multivariable analysis (MVA) adjusted for age and HCT-CI, alloHSCT was identified as the strongest favorable predictor for OS (HR 0.18 95% CI 0.04-0.8) with similar results when analysis was restricted to transplant-eligible patients < 75 years of age (HR 0.1, 95% CI 0.03-0.6). In a 6-month post-treatment landmark analysis (n=31 evaluable), patients who underwent alloHSCT (n=17) exhibited numerically superior GC discontiuation rate (29% vs 14%, p=0.049) and subsequent OS compared to HMA (2-years OS: 84% vs. 61%, median not reached vs 25 months, 95% CI: 0.06–1.7). In a post-treatment competing risk [GC dose reduction (death)] analysis alloHSCT was associated with superior 2-, and 3-year rates of 57% (10%), and 76% (10%), compared to HMA 22% (24%), and 31% (42%) [p<0.01 (0.04)]. AlloHSCT was also associated with higher rate of CMR (evaluable= 21, alloHSCT N=16 and HMA N=5, 100% vs 60%, p=0.01). At the last follow up, none of the patients who achieved CMR had re-emergence of the UBA1 clone (despite early mixed chimerism). Conclusions: This study represents the largest comparative analysis of alloHSCT versus HMA therapy in VEXAS syndrome, and confirms superiority of alloHSCT in achieving molecular remission, GC discontinuation/dose reduction, and overall survival. Prospective clinical trial confirmation is warranted.

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 > 13,000/mm3 and adequate organ function (estimated GFR > 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 < 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 >100% platelet response, one (14%) with normalization of WBC count (baseline WBC count >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: DDX41 (DEAD-box helicase 41) mutations (mt) represent the most prevalent germline predisposition syndrome (GPS) in adult patients (pts) with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). This molecular subtype is typically associated with late-onset disease, runs an indolent clinical course, with a relative absence of high-risk co-mutations. While several retrospective studies have reported favorable responses to venetoclax (VEN)-based therapies, anecdotal observations suggest variable outcomes. We aimed to evaluate the clinical characteristics and treatment outcomes of patients with DDX41-mt MDS/AML treated at the Mayo Clinic. Methods: We conducted a retrospective study of pts diagnosed with DDX41-mt germline predisposition syndrome with concurrent myeloid neoplasms at our institution. Germline testing was conducted under an IRB approved protocol (Mayo Clinic IRB# 16-004173; clinical-trials gov. Identifier: NCT02958462). Of 198 pts with DDX41-mt myeloid neoplasms, 77 had high-risk disease—43 with MDS with increased blasts (MDS-IB1, n = 11; IB2, n = 32) and 34 with AML (WHO 2022). We assessed how clinical, and disease features influenced outcomes in this subgroup. Among these pts, 69 (90%) pts had likely pathogenic/pathogenic variant (DDX41path) variants and 8 (10%) pts had variant of undetermined significance (DDX41VUS) according to variant classification criteria from the American College of Medical Genetics/ the Association of Medical Genetics/ the Association for Molecular Pathology (ACMG/AMP) guidelines. Results: The median age was 68 years (yrs) (range [R],34-92), 70% were male and the median bone marrow blast % was 15 (R,5-60). The most common DDX41 germlinevariants (> 5% cases) included p.D140fs (26%), p.M1I (22%), and p.P258L (6.5%). 74% had DDX41 truncating variants. Thirty-one (40%) acquired a somatic DDX41-mt (R525H [81%]) and 43 (56%) had other somatic mutations. Among these, the most frequent were ASXL1 (n= 9, 21%), TP53 (n= 8, 10%), splicing factor (n= 7, 9%), and DNMT3A (n= 5, 6%). Twenty-six, (34%), 26 (34%), 15 (19%) and 9 (12%) pts were treated with hypomethylating agents (HMA), HMA+VEN, intensive chemotherapy (IC) and active surveillance/supportive care, respectively. Overall, the composite complete remission rate (cCR) in high-risk MDS and AML was 56%. The cCR rates (41%, 59% and 68.7%, p= 0.10) and median duration of response (DOR; 43.7, 44.9 and 28.9 months [mo], p= 0.46) were not significantly different with HMA, HMA+VEN, and IC induction. Thirty-one (40%) pts received allogeneic stem cell transplantation (allo-HCT); 4 (36%), 15 (47%) and 12 (35%) in MDS-IB1, MDS-IB2 and AML groups, respectively (p= 0.56). With a median follow up of 34.7 mo (R, 0.7-136.7), the median overall survival (OS) from the time of diagnosis was 56.9 mo (95% CI, 50.7-63.0) and was not significantly (NS) different between MDS-IB1 (not reached, 70% at 3 yrs), MDS-IB2 (56.9 mo, 86% at 3 yrs) and AML (55.7 mo, 60% at 3 yrs) groups, p= 0.16). The median OS was NS different between DDX41path and DDX41VUS (58.6 vs 55.7 mo, p= 0.50), respectively. We conducted a multivariable analysis for OS using variables that showed significance or trend towards significance in univariable analysis. Concurrent splicing factor mutations (HR; 5.29, 95% CI: 1.29-21.6, p= 0.02) remained negatively prognostic, while there was a trend towards better survival outcomes with allo-HCT (HR; 0.48, 95% CI: 0.21-1.07, p= 0.07). Bone marrow blast ≥ 20% (HR; 1.87, p= 0.12) and TP53-mt (HR; 2.14, p= 0.12) did not retain significance. Conclusion: In our cohort of patients with germline DDX41-mt high-risk MDS and AML, VEN-based regimens were not superior to HMA alone or IC, in terms of response rates and outcomes. On multivariable analysis, the presence of concurrent splicing factor mutations independently and negatively impacted survival with a trend towards better outcomes with allo-HCT. These findings highlight the heterogeneity within DDX41-mt myeloid neoplasms and underscore the need for future studies in larger cohorts to better define predictors of response and survival.

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 >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 (>1%) in 75% versus 83% (p=0.6) patients respectively. On the other hand, minor PNH clone (>0.1% in at least two cell types) was detectable in the acquired group only (0 versus 55%, p<0.0001). Similarly, acquired group had a shorter (<3 months) onset of cytopenias preceding diagnosis (P<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<0.05 for all regions). Four of these preferentially accessible genomic regions (p<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 Germline mutations in the RNA-helicase gene DDX41 cause predisposition to myeloid malignancies (MDS/AML) with a penetrance of 50% by age 90. DDX41 germline mutations are heterozygous and predominantly truncating variants, indicative of loss-of-function. More than half of DDX41-mutated carriers that develop MDS/AML will acquire a second-hit mutation in the other allele of DDX41, frequently causing the amino acid substitution p.R525H. Single-cell analysis from a biallelic DDX41-mutated MDS patient showed that the p.R525H mutation was highly enriched in dendritic cells (DCs) but less frequent in other cell types. We hypothesized that p.R525H causes the hematopoietic stem and progenitor cells (HSPCs) to differentiate preferentially into DC. To precisely model the germline of DDX41-mut patients, we generated human induced pluripotent stem cells (iPSC) with a frameshift mutation in exon 6 of one allele of DDX41, modeling the most common germline mutation. We attempted to modify the other allele of DDX41 to express the p.R525H mutation but could not derive viable cells. To circumvent potential lethality of the acquired mutation, we inserted a Doxycycline (DOX)-inducible wild-type DDX41 expression cassette in the AAVS1 safe harbor locus. Then we modified the endogenous allele to cause the p.R525H mutation in trans to the existing frameshift mutation. In this manner, we derived iPSCs that would model the most common configuration of DDX41 mutations (DDX41R525H/-) upon the withdrawal of DOX from the culture. In undifferentiated iPSC cultures, we observed profound defects in cell proliferation and survival upon DOX withdrawal. To determine the effect of the p.R525H mutation on hematopoietic cells, we differentiated the iPSCs into CD34+ HSPCs in the presence of DOX, and then withdrew DOX and studied the effect of the DDX41R525H/- genotype on HSPC function. In colony formation assays, DDX41R525H/- cells produced fewer and smaller myeloid and erythroid colonies after DOX withdrawal. In liquid culture, DDX41R525H/- cells showed cell cycle arrest by EdU incorporation assay and protein synthesis deficit by HPG assay. Thus, we conclude that DDX41 is required for proliferation and survival in iPSC and hematopoietic progenitor cells and that p.R525H lacks the essential function. Next, we tracked HSPC differentiation in liquid culture containing Flt3 ligand, stem cell factor and thrombopoietin with or without GM-CSF. Identification of DC subsets by flow cytometry was based on the literature (Sontag S, et al. Stem Cells 2017;35(4)). We observed reduced overall differentiation in DDX41R525H/- cultures, evidenced by maintenance of CD34 positivity. However, among the CD45+CD34- cells, DDX41R525H/- cells were 3-4 fold more likely to be identified as plasmacytoid DC (pDC) compared to control conditions (+DOX or DDX41+/- cells). Notably, in DDX41R525H/- CD45+CD34- cells, overall CD123 and CD304 expression were profoundly upregulated and CD11c was downregulated, indicative of increased pDC character. We used a similar DOX-inducible approach to generate Molm13 human AML cell line with DDX41R525H/- and homozygous knockout (DDX41-/-) genotype and found that these cells had profound survival defects. Both genotypes exhibited DC-like morphological change with extended multiple projections upon DOX withdrawal. In flow cytometry analysis, both genotypes exhibited upregulated Clec9a (conventional DC1 (cDC1) marker) and Clec10a and CD1c (cDC2 marker) upon DOX withdrawal. DDX41R525H/- cells specifically exhibited pDC features shown by upregulated CD123 by flow cytometry and increased IRF7 by western blotting, while DDX41-/- had decreased IRF7. Furthermore, we found that DDX41R525H/- cells specifically showed increased IRF9 and phosphorylated STAT1 and STAT2, which are downstream of interferon receptor activation, indicating that type-I interferon produced by DDX41R525H/- pDC activates interferon signaling in autocrine/paracrine manner. These results indicate that p.R525H promotes differentiation into pDC, which is not observed in DDX41-/- cells. In conclusion, DDX41 p.R525H mutation has bimodal functions in HSPC: loss-of-function for proliferation/survival and gain-of-function for skewed differentiation into pDC. Dysregulated DCs may contribute to the pathogenesis of DDX41-mutated MDS/AML, particularly in light of recent descriptions of AML with pDC differentiation and the broader role of DC dysregulation in MDS/AML.

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 > 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 >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.

Abstract Introduction Chronic myelomonocytic leukemia (CMML) is a myeloid neoplasm with overlapping features of myelodysplastic syndromes (MDS) and myeloproliferative neoplasms (MPN). Blast transformation (BT) is a frequent complication of CMML with a 3-year risk of approximately 31%. MPN driver mutations (i.e., JAK2, CALR, MPL) are known to occur in CMML but their impact on overall survival (OS) or BT remains uncertain. Methods The current study was conducted under institutional review board approved minimum risk protocols that allowed retrospective collection and analysis of patient data. Diagnostic criteria were according to the International Consensus Classification (Blood 2022; 140:1200). For survival analysis, patients were censored at the time of allogeneic stem cell transplant (ASCT). All statistical analyses were conducted using JMP Pro 17.0.0 software (SAS Institute, Cary, NC, USA) and Bluesky Statistics Software (Worldwide Headquarters, Chicago, IL, USA). Results A total of 612 CMML patients were considered (median age 71 years; 68% males). MPN driver mutations (MPNMUT) were detected in 56 (9%) patients, including 43 (77%) with JAK2, 10 (18%) MPL, and 3 (5%) CALR. The median variant allele frequencies were 44% for JAK2 (range, 0.3–100%), 49% for MPL (range, 0.3–97%), and 50% for CALR (range, 20–80%). Age and sex distributions and CMML-1 vs. CMML-2 representation were similar between CMML patients with and without MPNMUT. On the other hand, CMML patients with MPNMUT were more likely to display higher hemoglobin level (p <0.01), platelet count (p <0.01), absolute neutrophil count (p = 0.02) and leukocytosis (≥13 ×109/L; p <0.01); they were also less likely to be red blood transfusion dependent (p <0.01), display platelet counts of <100 ×109/L (p <0.01) or abnormal karyotype (p= 0.03), present with severe anemia (p= 0.03) or harbor NRAS mutation (p = 0.02). Higher Mutational frequencies of SRSF2 (median 53% vs. 41%; p = 0.01), TP53 (median 8% vs. 2%; p = 0.02) and IDH2 (median 13% vs. 4%; p = 0.01) were noted in CMML patients with MPNMUT. During a median follow-up of 23 months (inter-quantile range 11-51 months) for all 612 CMML patients, 402 (67%) deaths, 118 (19%) BT and 108 (18%) ASCT were documented. Of these, 36 (9%) deaths, 18 (15%) BT and 13 (12%) ASCT occurred in patients with MPNMUT. Median OS for the entire group (N=612) was 32 months with 1-, 2- and 3-year OS rates of 78%, 58% and 47%, respectively. There was no significant difference in OS between CMML patients with or without MPNMUT (median 41 vs 31 months; p = 0.27). Thrombotic events were reported in 53 (9%) patients after diagnosis of CMML, including 48 venous and 5 arterial events. There was no significant difference in arterial (p = 0.10) or venous (p = 0.76) thrombosis-free survival between CMML patients with or without MPNMUT. Among all 612 patients with CMML, followed for a median of 18 months for living patients, 118 (19%) experienced BT, with 1-, 3-, and 5-year BT rates of 10.8%, 23.1%, and 31%, respectively. Cumulative incidence analysis function (CIF) was calculated in order to assess the BT-free survival while accounting for death as a competing risk; 3-year CIF was 30% for CMML patients with vs. 18% for those without MPNMUT (Gray's test; p = 0.01). This observation was confirmed in univariate analysis of time to BT (HR 1.7; p=0.03) as well as the corresponding multivariable analysis involving other mutations and clinical risk factors: MPNMUT (HR 1.8; 0.036), ASXL1MUT (HR 2.3; p<0.01), DNMT3AMUT (HR 2.6; p<0.01), CMML-2 vs. CMML-1 (HR 3.4; p<0.01), and leukocyte count ≥13 ×109/L (HR 2.0; p<0.01). SRSF2, IDH2, and TP53 mutations did not impact BT. Notably, the aforementioned risk factors for BT, including ASXL1MUT and DNMT3AMUT, were no longer significant when analysis was restricted to patients with MPNMUT. Conclusions The current study suggests a higher risk of BT in CMML patients with MPNMUT, independent of other molecular or clinical risk factors for BT. On the other hand, overall and thrombosis-free survival did not appear to be similarly influenced. The differential impact of previously recognized risk factors for BT in CMML (e.g., ASXL1MUT, DNMT3AMUT, CMML-2, CMML-MP), in the presence (lost significance) vs. absence (remained significant) of MPNMUT was intriguingly noted.