Steinauer, Nickolas

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 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.

Abstract Introduction: Somatic mutations in splicing factors (SF) - SF3B1, SRSF2, U2AF1, and ZRSR2 – represent one of the most common drivers of premalignant clonal hematopoesis. While SF mutations are potent drivers of clonal expansion (Fabre et al., Nature 2022), multiple SF mutations are rarely observed in the same individual (negative epistasis). Prior studies have identified rare cases of concurrent SF mutations (<4%) and described specific hotspot combinations that escape negative epistasis (Taylor et al., Blood 2020). We conducted this study to further characterize the frequency and clonal relationship between concurrent SF mutations using a larger patient cohort. Methods: After IRB approval, we assessed 3,369 myeloid next generation sequencing (NGS) results that identified somatic variants in SF3B1, SRSF2, U2AF1, or ZRSR2, in 2,500 adult patients from 08/2015 to 07/2025. Patient demographics, ICD-9/10 codes, and complete blood counts with differentials were obtained, retaining only those datapoints assigned 30 days before or after NGS testing. To allow comparison of clone size between autosomal and the X-linked SF ZRSR2, VAFs were multiplied by two to estimate the cancer cell fraction (CCF), except for ZRSR2 mutations in males. For patients harboring concurrent SF mutations, we categorized them as co-dominant if the difference in respective CCFs was < 10% of the greater CCF. Specific SF variants were categorized into common hotspots (n=13), except for ZRSR2 variants, which do not occur in defined hotspots. Fisher's exact testing was used to assess the enrichment of SF hotspot variants in patients with concurrent SF mutations, as well as combinations of SF variants in patients with co-dominant SF mutations. Results: We identified 2,331 patients with at least 1 pathogenic SF mutation, of which 6.7% (n=156) had > 1 SF mutation (multi-SF MT). The total cohort comprised cases of AML (n=549, 24%), MDS (n=977, 42%), MDS/MPN (n=233, 10%), MPN (n=228,10%), and uncategorized ICD codes (“Other”, n=326), of which the largest category was clonal cytopenia(s). Notably, the prevalence of multi-SF cases was higher than previously described and our cohort included 5 patients with 3 SF mutations and 1 with 4 SF mutations. Of the multi-SF MT patients, we identified 42 (27%) with co-dominant CCFs among their respective SF mutations. Thirty-three multi-SF MT patients had multiple NGS sampling timepoints (median 2, range 2-4) spanning a median of 0.97 years (range 0.06-5.69 years); we found that 17 of these 33 demonstrated correlative SF CCFs over multiple timepoints, suggesting their SF mutations occurred in the same clone (“co-clonal”). Of these 17 patients, 82% had co-dominant CCFs at the single-timepoint level, whereas none of the 16 non-co-clonal demonstrated co-dominance. Thus, we used SF co-dominance as a surrogate for cases likely to be co-clonal. ZRSR2 (p=8.8x10-29), U2AF1Q157 (p=9.4x10-9), SF3B1K666 (p=1.4x10-4) and infrequent SF3B1 (“SF3B1-Other”, p=.0029) mutations were overrepresented in the multi-SF MT cohort compared to patients with a single SF mutation. Within the multi-SF MT cohort, 3 SF mutation combinations showed overrepresentation in codominant cases – SRSF2P95R/H/L+ ZRSR2 (p=0.014), SRSF2P95R/H/L+ SF3B1K666 (p=0.044), and U2AF1S34 + U2AF1Q157 (p=0.024), although not significant after multiple hypothesis correction. Dual ZRSR2 mutations were the most common combination seen in the multi-SF MT cohort but were underrepresented in codominant cases (p=0.025). Although there was no disease subtype uniquely associated with total or co-dominant multi-SF MT cases, occurrence of multiple SF mutations was more common in males (8% vs. 3% in females), an effect driven by ZRSR2 mutations. Hemoglobin and WBC were lower in multi-SF MT cases, with the lower hemoglobin strongly driven by the codominant multi-SF MT cases (median 8.3 g/dL vs 9.4 g/dL, p=0.00046). Conclusions: These findings suggest that SF mutation co-occurrence in myeloid neoplasms is more prevalent than previously estimated (~7%). Furthermore, we identified 6 individuals harboring > 2 SF mutations, which has not been previously described. We validate specific SF variant pairs that appear to exist in the same clone and are working to confirm the clonal architecture and characterize global splicing aberration in selected cases with single-cell studies to fully uncover the mechanism by which negative epistasis is overcome.

Abstract Introduction Surface proteins in cancer not only reflect tumor–microenvironment interactions but also serve as targets for diagnostic flow cytometry and precision therapies, including monoclonal antibodies and CAR-T cell therapies. A better understanding of plasma cell immunophenotypes could inform flow cytometric prognostic assays and development of targeted therapies. Methods RNA-seq and Seq-FISH data from CD138-enriched bone marrow plasma cells, along with corresponding clinical annotations, were obtained from the Multiple Myeloma Research Foundation-CoMMpass Study. A curated list of surface genes was assembled by integrating three sources: the Human Protein Atlas (plasma membrane and cell junction annotations), the Gene-Set Enrichment Analysis membrane-associated gene set (Ontology:0016020), and the Cell Surface Protein Atlas (validated high-confidence category). A total of 4,579 genes coding for cell surface proteins were identified. Genes below the 25th variance percentile were removed. PCA (≥90% variance) followed by k-means (k=2, silhouette) and confirmed by hierarchical clustering. A Plasma Cell Maturity (PBM) score was computed using singscore on 84 mature plasma cell–associated genes to compare cluster phenotypes. Pathway analysis was done using GSEA software for Hallmark pathways. Clinical biomarkers and survival kinetics were compared between the clusters. Results 761 newly diagnosed patients were included. (Age: 27-93 years, median overall survival/OS: 29 months/mo). Clustering revealed two groups which we termed Atypical/Aggressive Cluster 1 (C1, n=105, 13.8%) and Classical Cluster 2 (C2, n=656, 86.2%), based on the patterns and clinical characteristics. Among non-missing cases, high-risk disease per the International Myeloma Society–International Myeloma Working Group (IMS–IMWG) definition was observed in 27.7% of patients in C2 (n = 552) and 24.7% in C1 (n = 90) (p = 0.4).C1 exhibited enrichment for t(14;16)-MAF (OR = 2.6, 95% CI: 1.1–6.1, p = 0.04) and t(11;14)-CCND1 (OR = 1.7, CI: 1.01–2.8, p = 0.04). In contrast, hyperdiploidy was more common in C2 (OR for C1 = 0.6, CI: 0.4–0.9, p = 0.04). Elevated LDH was observed in C1 (OR = 2.6, CI: 1.6–4, p < 0.001). Median serum Beta-2 microglobulin was lower in C1.(p=0.009) Upregulated genes in C1 in decreasing order of log Fold Change(logFC): AHNAK, MT-ND1, EEF2, EZR, HLA-B, TXNIP, HLA-E, TRIB1, MCL1, RHOB, CD74, CFLAR, HLA-A, HERPUD1, RPSA, ERN1, HSP90AB1, RAPGEF2, ADGRE5, and LAPTM5. Downregulated genes in C1 in descending order of |logFC|: B2M, SDC1 (CD138), HSP90B1, SEL1L, CALR, CANX, PECAM1, SLAMF7, STT3A, TNFRSF17 (BCMA), FCRL5, STT3B, PALM2AKAP2, CD53, PARP1, CCR2, RPN1, CD38, EDNRB, and LMAN2. Upregulated pathways in C1: proliferation (MYC, E2F, PI3K/AKT/mTOR, KRAS, Wnt); evasion of apoptosis (p53, TGF-β, Notch, NF-κB, IL6/JAK/STAT3); Epithelial to Mesenchymal Transition (angiogenesis, apical junction); inflammatory (interferon, TNF-α, complement, IL2/STAT5); metabolic reprogramming (oxidative phosphorylation, lipid/cholesterol); and stress (DNA repair, unfolded protein response). The PBM score for C1 was significantly lower, suggesting a less differentiated phenotype. C1 patients had inferior survival (n = 93, median OS 43.9 mo, p = 0.04) compared to those in the C2 cluster (n = 559, median OS 102.5 mo). Patients in the C1 + IMS–IMWG high-risk status had the shortest survival (20.7 mo), compared to C1+low (50 mo), C2/high (68.3 mo), and C2+low (not reached). In a multivariable analysis, C1 status (HR = 1.93; 95% CI: 1.1–3.4, p = 0.02) was an independent predictor, along with older age (1.05), UAMS70 (1.02), EMC-92 (1.4). In C1, early (<18 mo) progression occurred in 76.3% (n=71) (p < 0.001). In patients receiving first line of treatment(LOT1), median PFS was 39.7 mo in C1vs. 47.3 mo in C2 (p=0.8). Among patients who received stem cell transplant in LOT1, the median survival for the C2 was 102.5 mo, compared to 44.5 mo for C1 (n = 52) (p = 0.01). Among non-SCT patients on Immunomodulator + Proteasome Inhibitor therapy, C1 (n=28) had shorter survival of 50.0 vs. 71.7 C2 (n=137; p =0.8). Conclusion Our transcriptomic analysis identified a higher-risk group of patients (C1) exhibiting downregulation of myeloma specific markers (CD38, CD138, BCMA, SLAMF7, FCRL5) and inferior outcomes. It is important to diagnose and find appropriate therapy for these patients as they don't obtain the same benefit from the current standard of therapy as the remaining patients.

ABSTRACT Two major etiologies of hyperbilirubinemia include hemolysis and cholestasis. Although rare, the former can give rise to the latter through the formation of pigment gallstones and subsequent biliary tree obstruction. We report a case of a 57-year-old woman with systemic lupus erythematosus who presented with dyspnea and right upper quadrant abdominal pain. She was found to have hepatolithiasis and choledocholithiasis secondary to warm autoimmune hemolytic anemia in the setting of COVID-19. In patients with symptomatic anemia secondary to acute hemolysis and concomitant right upper quadrant abdominal pain, elevated hepatocellular laboratory results should prompt a high clinical suspicion for biliary tree pigment stones.

Background Venetoclax (Ven) in combination with hypomethylating agent (HMA) is the standard treatment for newly diagnosed elderly/unfit patients with acute myeloid leukemia (AML). Treatment options for patients with relapsed/refractory (RR) disease following Ven-HMA are limited in the absence of targetable mutations. In a recent phase 2 study, the addition of Ven to cladribine (CDA) plus low-dose cytarabine (LDAC), in newly-diagnosed elderly/unfit AML, resulted in complete remission with (CR) or without (CRi) count recovery rate of 93% ( J Clin Oncol 20;40(33)). In the current study, we share our experience with this regimen in patients relapsed or refractory to Ven-HMA, treated outside of clinical trials. Methods Under an institutional review board approved minimum risk protocol, the Mayo Clinic (MN, AZ, FL) database was searched to identify patients with AML who failed treatment with Ven-HMA and subsequently received at least one cycle of salvage therapy with Ven-CDA-LDAC: Ven 100-400 mg, dose adjusted, d1-21, CDA 5 mg/m 2 d1-5, LDAC 20 mg twice daily d1-10, outside of clinical trials. Cytogenetic and molecular studies were performed at the time of Ven-CDA-LDAC initiation in most cases, by conventional karyotype and next-generation sequencing, respectively. The 2022 European LeukemiaNet (ELN) criteria were applied to define risk and response. Survival was calculated from the time of Ven-CDA-LDAC treatment start to last follow-up or death. Results Patient characteristics A total of 39 RR AML patients (median age 65 years, 67% male, 64% de novo), with prior exposure to Ven-HMA, received Ven-CDA-LDAC (median 1 cycle; range 1-5). Study patients had received one ( n=16), two ( n=14), three ( n=5), four ( n=2), five ( n=1) or nine ( n=1) prior therapies, including Ven-HMA. Nine (23%) patients had relapsed following allogeneic hematopoietic stem cell transplant (AHSCT). ELN cytogenetic risk included intermediate (46%, n=18), and adverse (54%, n=21). Mutations involved TP53 in 9 patients (23%), K/ NRAS in 8 (21%), RUNX1 in 7 (18%), TET2 in 6 (15%), ASXL1 in 5 (13%), and STAG2 in 5 (13%). Response and toxicity Eleven (28%) patients achieved CR ( n=2; 5%) or CRi ( n=9; 23%); median time to response was 1 month and median response duration 3.4 months. In addition, 2 (5%) patients each experienced partial remission (PR) and bone marrow aplasia without fulfilling criteria for morphologic leukemia free state. Measurable residual disease (MRD) was negative by multiparametric flow cytometry in 2 of 7 (29%) informative CR/CRi cases. CR/CRi rates were higher in females (54% vs 15%; p=0.01), de novo vssecondary AML (40% vs 7%; p=0.02), and absence of adverse karyotype (50% vs 9%; p<0.01), K/NRAS (35% vs 0%, p=0.01), ASXL1 (32% vs 0%, p=0.05), or STAG2 mutations (32% vs 0%, p=0.05) (Table 1). Multivariable analysis confirmed superior response in de novo AML (p<0.01), absence of adverse karyotype, (p<0.01), and absence of K/NRAS mutations (p=0.02). CR/CRi rates were not significantly affected by relapsed vs refractory setting ( p=0.95), number of prior therapies ( p=0.51), prior AHSCT ( p=0.64), or TP53 mutations ( p=0.64) (Table 1). Treatment-emergent toxicities included infections (49%, n=19), comprising bacteremia ( n=11), pneumonia ( n=10), peri-rectal abscess ( n=6), and laboratory evidence of tumor lysis syndrome (3%, n=1). Survival At a median follow-up of 3.9 months, from initiation of Ven-CDA-LDAC, 31 deaths (79%), 6 relapses (15%), and 7 AHSCT (18%; including 5 in CR/CRi, 1 in aplasia) were documented. Median survival following Ven-CDA-LDAC was 4.6 months, and superior in the presence vs absence of CR/CRi (8.1 vs 2.9 months; p=0.01), and in patients receiving AHSCT (10.5 vs 3.4 months; p=0.02). Absence of adverse karyotype was also associated with superior survival (6 vs 3.4 months; p=0.05). Multivariable analysis confirmed the favorable survival impact of CR/CRi ( p=0.03) and AHSCT ( p=0.05); survival impact was not apparent for secondary AML ( p=0.29) or K/NRAS ( p=0.70) or TP53 ( p=0.10) mutations. Conclusions The current study suggests transplant bridging value for Ven-CDA-LDAC in AML patients relapsed or refractory to Ven-HMA, especially in the absence of adverse karyotype or K/NRAS mutation. AHSCT and achievement of CR/CRi appear to be indispensable for favorable post-Ven-CDA-LDAC survival.