Lasho, Terra L.

ABSTRACT Hypomethylating agents (HMA) and allogeneic hematopoietic stem cell transplantation (alloHSCT) have both demonstrated remissions in VEXAS; however, comparative data is lacking. We conducted a multicenter, retrospective analysis of 66 patients diagnosed with VEXAS syndrome treated with HMA ( n = 35) or alloHSCT ( n = 31). Baseline characteristics such as genetics, co‐morbidities, and performance status were balanced between the groups, except older age in the HMA group. Median follow‐up from therapy initiation was 18 months (95% CI: 11–26), and 14 (21%) deaths were reported (alloHSCT n = 3; HMA n = 11). Among all evaluable patients within the alloHSCT cohort, all patients achieved molecular remission, and a substantial proportion of patients discontinued glucocorticoids (58%). In contrast, HMA therapy was associated with lower but meaningful rates of molecular remission (22%) and glucocorticoid discontinuation (6%). In a real‐world setting, HMA therapy was associated with a high discontinuation rate related to toxicity or lack of response. On multivariable analysis adjusted for age and Charlson Comorbidity Index, alloHSCT was associated with improved overall survival (HR = 0.20, 95% CI: 0.05–0.81; p = 0.024). This association remained consistent across multiple ancillary sensitivity analyses, including restriction to transplant‐eligible patients, patients aged ≤ 75 years, 1:1 matching, and propensity score‐based weighted analyses. Although limited by retrospective design, these findings suggest that alloHSCT remains an attractive and potentially curative strategy in selected patients with VEXAS. Prospective validation of these findings is warranted.

Abstract Recent updates to monocyte count thresholds recognize oligomonocytic chronic myelomonocytic leukemia (OM-CMML) as an early form of CMML. However, the clinical validity of these changes remains uncertain without incorporating biological and genomic factors. In this study, we analyzed a cohort of 911 patients (249 with OM-CMML, 359 with overt CMML, and 303 with myelodysplastic syndromes) using unsupervised clustering to evaluate the role of genomic determinants in refining CMML diagnosis. Our findings show that CMML molecular signatures (biallelic TET2 mutations or SRSF2-TET2 comutations) are linked to a distinct transcriptome, monocytic bias, classical monocytosis, and a higher risk of progression to overt CMML in OM-CMML cases. We developed a weighted genomic model and diagnostic workflow showing that combining genomic signatures with bone marrow monocyte frequencies in OM-CMML more accurately predicts progression to overt CMML. These findings support integrating genomic determinants and our clinic-ready diagnostic workflow into the CMML diagnostic framework to improve accuracy. Significance: Through comprehensive clinical and genomic profiling of a large patient cohort, alongside immunophenotypic and transcriptional cellular analyses, this study provides evidence that incorporating genomic determinants into the diagnostic criteria for OM-CMML improves diagnostic accuracy and refines the identification of early-stage CMML, thereby preventing misclassification.

Abstract TP53 and PPM1D are key regulators of DNA damage response and repair, and somatic mutations in these genes often co-occur in hematopoietic cells, expanding under genotoxic stress. Unlike TP53 mutations, where mechanisms of progression are defined, pathways underlying clonal fitness and transformation in PPM1D mutant cells remain unclear. In collaboration with 5 academic institutions, we analyzed the clinical and molecular landscape of 337 patients with clonal hematopoiesis (CH) and clonal cytopenia of undetermined significance (CCUS) across 4 genotypes: PPM1Dmt/TP53wt (n = 170 [50%]), PPM1Dmt/TP53mt (n = 25 [7%]), TP53mt/PPM1Dwt (n = 17 [5%]), and TP53wt/PPM1Dwt (n = 125 [38%]). All PPM1D variants were truncating, located in exon 6 of the gene, with a median variant allele frequency (VAF) of 6% (range, 0.3%-64%). The PPM1Dmt/TP53mt genotype was most frequently encountered in therapy-related CH/CCUS (t-CH/t-CCUS; 80%, 66.5%, 76.5%, and 19%; P ≤ .001) and had a shorter time interval to detection from last genotoxic exposure (6.2, 5.9, 11.25, and 24.5 months; P ≤ .001) compared with PPM1Dmt/TP53wt, TP53mt/PPM1Dwt, and TP53wt/PPM1Dwt genotypes, respectively. Acknowledging the short follow-up duration, rates of malignant transformation were lower in the PPM1Dmt/TP53wt (2%) and PPM1Dmt/TP53mt (4%) groups compared with PPM1Dwt/TP53wt (12%) and PPM1Dwt/TP53mt (17%) groups (P ≤ .001), respectively. In summary, PPM1D mutations are frequently observed in t-CH/t-CCUS, with low median VAFs, and are associated with low rates of progression, even when comutated with TP53.

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 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 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 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.