6521 Background: Intensive chemotherapy (IC; cytarabine plus an anthracycline) remains a standard first-line therapy for fit patients with acute myeloid leukemia (AML), a heterogeneous hematologic malignancy with poor long-term survival. IC induces composite complete remission (cCR) in ~60–70% of cases, and IC response variability persists within genetic/ELN risk strata. As emerging regimens increasingly challenge IC as a default therapy, biology-informed predictors are needed to identify patients unlikely to benefit from IC to avoid unnecessary toxicity. Mass spectrometry (MS)-based phosphoproteomics enables quantitative profiling of phosphopeptides (PPs) from patient samples, providing a functional readout of tumor biology. Thus, we sought to develop a PP-based clinically-deployable assay, orthogonal to genetic risk stratification, to predict response to IC in AML. Methods: 261 retrospective samples were collected from patients with newly-diagnosed AML (Table) treated with standard “7+3” IC in 9 centers in Europe, Australia, and North America. Response was assessed at the end of induction by the treating physician. Samples underwent phosphoproteomics using: 1) global MS for biomarker discovery or 2) targeted, clinically-compatible MS for validation. Predictive model performance was evaluated using rebalanced leave-one-out cross-validation. Results: During discovery, 3205 PPs were detected. Using Bayesian approaches, we identified an 80-PP multi-analyte signature of IC response (refractory vs cCR), and achieved an AUROC of 0.68 (95% CI 0.54-0.83). The signature was enriched for DNA damage signalling and repair (DNA-PK-S2612, ATM/cohesin, nucleotide excision and double-strand break repair proteins), and cellular stress pathways (phospho-p38, IL-16, AP-1/c-Jun). In an independent validation cohort, the targeted assay reliably detected the biomarker set and preserved its association with outcome (AUROC 0.69; 95% CI 0.53-0.84). Biomarker-based classification was associated with improved event-free survival (HR 0.45; 95% CI 0.26-0.78). Conclusions: We identified and validated a signature of response to IC in AML, and translated it into a targeted, clinically-deployable assay. This approach captures signaling states relevant to IC mechanisms of action that are not directly inferred from standard clinical or genetic variables. Ongoing analyses are evaluating its relationship to established genetic and ELN risk stratification. These findings support diagnostic phosphoproteomics and suggest functional biomarkers may complement existing approaches for treatment selection in AML. Cohort characteristics. Cohort Discovery Validation Median age at diagnosis (years, quartiles) 54 (23, 65) 59 (47, 68) Median diagnosis year (range) 2014 (1999, 2023) 2013 (2001, 2024) No of patients/samples 135/165 102/106 No of cCR/Refractory 94/41 79/23 PPs 3205 80

Abstract We have used synthetic biology recombination methods in yeast to build herpes simplex virus type-1 (HSV-1) and human cytomegalovirus (HCMV) genomes from multiple fragments. The genomes were built using transformation-associated recombination (TAR) in yeast, by virtue of overlapping sequences between the different fragments. This study demonstrates the successful assembly of the Epstein-Barr virus (EBV) genome. We used as the model genome, the Akata Burkitt’s lymphoma genome, specifically the BX1 genome which encodes a neomycin selectable marker and a GFP expression cassette in the BXLF1 region. The 171.3 kb genome was first deconstructed into 11 fragments in silico , each having 80 bp overlapping sequence between the fragments. The 11 fragments (TAR 1 to TAR 11) were cloned using TAR in yeast, analyzed by restriction enzyme analyses and Nanopore sequencing to validate the cloned fragment. The EBV genome was built in two stages: TAR fragments 1 to 6 and TAR fragments 7 to 11 were assembled to generate two half-genomes. The whole genome (TAR 1-11) was then assembled by joining TAR 1-6 with TAR 7-11. Complete EBV genomes were examined by PCR assays and restriction enzyme analyses and then transfected into HEK-293 cells to generate virus producer cell lines. The HEK-293 cell clones were tested for virus production following lytic induction using baculovirus transduction of Zta, Rta and glycoprotein B ( BALF4 ). The supernatants from these induced cells were harvested and used to infect Raji cells. This analysis revealed a significant number of cells displaying strong GFP fluorescence indicative of infectious virus. We used this supernatant virus to infect primary B cells and were able to derive lymphoblastoid cell lines (LCL) indicative of the ability of this virus to transform B cells. We tested this method for engineering different mutations. Two mutations were made, one in Zta and the other in the small capsid protein ( BFRF3 ). Mutations were engineered in the TAR plasmid in which the genes reside and after sequence validation, assembled into the TAR 1-6 half genome and then the TAR 1-11 genome, which was used to generate HEK-293 cell clones. For the ΔZta cell lines, we could detect virus in the supernatants only if baculovirus expressing Zta in trans was included, this ΔZta EBV virus could transform B cells. The small capsid protein (BFRF3) decorates the capsid shell and is required for capsid assembly in a self-assembly system. When the HEK-293 cell clones were induced using co-expression of Zta, Rta and gB, no virus was detected in the culture supernatants. However, if we provided BFRF3 in trans using baculovirus expressing this protein, virus was detected in the supernatants. This provides the first report of the essential role of the small capsid protein in EBV-infected cells.

Abstract Patients with myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML) with high-risk features including TP53 mutations have poor outcomes because of the lack of effective therapies. The atypical chemokine surface receptor C-C motif chemokine receptor-like 2 (CCRL2) is overexpressed in MDS and secondary AML compared with healthy hematopoietic cells, and we recently found that TP53-mutated MDS/AML and AML with erythroid features express the highest levels of this receptor across MDS/AML subtypes. To illustrate the therapeutic potential of CCRL2 as a therapeutic target, we developed an anti-CCRL2 antibody-drug conjugate (ADC) by conjugating an anti-CCRL2 antibody with the cytotoxic drug pyrrolobenzodiazepine (PBD), which causes DNA double-strand breaks, leading to cancer cell death. Anti-CCRL2 ADC demonstrated strong CCRL2-selective cytotoxicity associated with DNA damage against cell lines derived from patients with MDS/AML with TP53 mutations and erythroid features, surpassing the cytotoxic effects observed with gemtuzumab and PBD-conjugated anti-CD33 and anti-CD123 ADCs. It also induced apoptosis and suppressed the clonogenicity of primary MDS/AML bone marrow samples without affecting the survival, differentiation, and clonogenicity of healthy hematopoietic stem and progenitor cells. This agent also suppressed the leukemic growth of TP53-mutated MDS/AML cell line xenografts, improving mice survival and decreasing the leukemic burden in patient-derived TP53-mutated MDS/AML xenografts. In conclusion, our study introduces CCRL2 as a potential new therapeutic target in high-risk MDS/AML, including TP53-mutated subsets.

Outcomes for adults with Philadelphia chromosome positive pre-B cell acute lymphoblastic leukemia (Ph + B-ALL) have improved dramatically, but questions remain regarding the optimal induction regimen and role of allogeneic hematopoietic cell transplantation (alloHCT). We analyzed 60 consecutive patients who received reduced-intensity (RII) or hyper-CVAD induction with continuous, second-generation tyrosine kinase inhibitors (TKIs). Reduced hematologic toxicity occurred after RII. Measurable residual disease (MRD) clearance by multicolor flow cytometry (MFC, 61 vs. 94%, p = 0.02) favored hyper-CVAD, but subsequent MRD-directed blinatumomab negated this difference. Four-year relapse-free survival (RFS) was 72.3% (95% confidence interval: 57.4-91.0%) and 79.8% (65.4-97.3%, p = 0.3) in RII and hyper-CVAD groups, respectively. AlloHCT, predominantly using reduced-intensity conditioning, bone marrow grafts, and post-transplant cyclophosphamide, was the only variable associated with improved overall survival on multivariate analysis. Concurrent chemotherapy and TKIs followed by blinatumomab for MRD positivity and alloHCT, all in less intensive forms, yield excellent outcomes for patients with Ph + B-ALL.

DAC combinations serve as frontline therapies for AML. DAC forms DNA-DNMT1 crosslinks and activates the ATR-CHK1-WEE1 DDR. Combinations of DNA-protein crosslinking agents with ATR, CHK1, or WEE1 inhibitors (i) have shown efficacy in solid tumors. This study evaluates the cytotoxic synergy of DAC plus ATRi, CHK1i, or WEE1i in AML. Public datasets were queried for mRNA expression and dependency. Commercial cell lines (U937, KG1a, Molm13, MV-4-11) were genomically validated. Molm13 TP53-/- clones were generated via CRISPR. Cells were cultured in complete RPMI. To treat, cells were cultured for 48 h in RPMI with drugs or diluent (0.2% v/v DMSO). Protein expression was evaluated by Western blotting (WB). Apoptosis was assayed by flow cytometry with annexin V or propidium iodide. Primary AML samples were obtained at study centers after informed consent. Isolated mononuclear cells (Ficoll) were used in colony forming unit (CFU) assays with DMSO or drug for 12-16 days. Synergy was determined by median-effect (CalcuSyn) where a combination index <1 is synergistic and >1 antagonistic. DNMT1 expression (TCGA) was highest in AML vs all other tumor types (p<0.001). In CRISPR screens (DepMap), AML cell lines were increasingly dependent on DNMT1, ATR, CHK1, and WEE1 (ANOVA p<0.001). WB of AML cell lines treated with DAC (25 - 200 nM) for 24 h showed phosphorylations of H2AX and CHK1 S296/S345 that were most pronounced in TP53MT lines. In apoptosis assays, ATRi, CHK1i, and WEE1i were synergistic with clinical concentrations of DAC (Table 1). In isogenic Molm13 lines, TP53 loss conferred DAC resistance that was overcome with combination treatment. Selective CHK1i were generally more synergistic than CHK1/2i, consistent with a CHK1 DDR. CFU assays of primary AML samples also showed synergy between DAC and ATRi, CHK1i, and especially WEE1i. Table 1. Synergy between decitabine and replication checkpoint inhibitors in preclinical models of AML. Cell line data are derived from subdiploid apoptosis assays. U937 data were replicated using annexin V. Prexasertib showed no activity in KG1a cells (suggesting drug efflux) requiring the use of the alternate CHK1 inhibitor, rabusertib. Abbreviations: ATR, ataxia telangiectasia and Rad3 related; CHK1/2, checkpoint kinase 1 and/or 2; CI, combination index; Conc, concentration; DNMT1, DNA methylatransferase 1; IQR, interquartile range; MT, mutated; ND, not determined; SD, standard deviation; WT, wild type. Drug Target Conc. (nM) Molm13 (TP53WT) Molm13 (TP53-/-) U937 (TP53G187fs*/-) KG1a (TP53V225fs*/-) Primary AML (TP53WT orTP53MT) Units Decitabine DNMT1 6.25 – 200 25 ± 11 350 ± 260 310 ± 180 6.1 ± 1.6 ND EC50, nM (mean, SD) Ceralasertib ATR 250 – 500 0.93 (0.66 – 1.24) 0.90 (0.84 – 0.96) 0.15 (0.08 – 0.21) 0.74 (0.63 – 1.02) 0.66 (0.43 – 2.43) CI with decitabine (median, IQR) Camonsertib ATR 10 – 100 0.27 (0.19 – 0.59) 0.24 (0.22 – 0.34) 0.34 (0.21 – 0.39) ND ND Prexasertib CHK1/2 1.0 – 6.0 0.89 (0.82 – 1.06) 0.78 (0.68 – 0.90) 0.95 (0.93 – 1.08) ND 0.88 (0.29 – 1.28) MK-8776 CHK1 250 – 1, 000 0.57 (0.48 – 0.80) 0.76 (0.73 – 1.19) 0.89 (0.57 – 1.39) ND ND Rabusertib CHK1 600 – 1, 000 ND ND ND 0.51 (0.48 – 0.56) ND Adavosertib WEE1 100 – 400 0.70 (0.63 – 1.11) 0.99 (0.78 – 1.14) 0.67 (0.60 – 0.74) 0.67 (0.37 – 0.93) 0.65 (0.43 – 0.88) DAC synergizes with ATRi, CHK1i, and WEE1i in AML. Future work will test these combinations in vivo and incorporate BH3 mimetics. Clifford M. Csizmar, Antoine N. Saliba, Olivia K. Rossman, Kevin L. Peterson, X. Wei Meng, B. Douglas Smith, Gabriel Ghiaur, Jonathan A. Webster, Mrinal M. Patnaik, Aref Al-Kali, Scott H. Kaufmann. Decitabine (DAC) induces a DNA damage response (DDR) and synergizes with replication checkpoint inhibitors in acute myeloid leukemia (AML) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1170.

Among molecular and cellular hallmarks of aging, changes in genetics, the epigenome, and inflammation are now recognized as key mediators of decreased regenerative potential. Such molecular changes compound the impact of myelodysplastic syndrome (MDS) mutations (e.g., SRSF2, RUNX1) and underlie the selection of mutant clones. To understand the mechanistic basis of MDS, leukemic transformation, and therapy resistance, we applied a multimodal single-cell profiling strategy to serial bone biopsies for patients with MDS (cellular indexing of transcriptomes and epitopes by sequencing [CITE-Seq], long-read isoform, and genotyping). The analysis spanned young and aged normal donors to complement MDS at diagnosis, hypomethylating agent (HMA) treatment, and secondary acute myeloid leukemia (sAML). Isoform, splicing, and clonal impacts were determined using a new bioinformatic toolkit, AltAnalyze-LR. In normal donor material, we observed significant age-related differences in specific populations, including the most primitive hematopoietic stem cells (HSCs), where we found significant deregulation of inflammation and ubiquitin pathway gene expression. These changes were accompanied by broad splicing alterations, impacting known regulators of cell survival, clonal hematopoiesis, and inflammation. Comparison of normal aged marrow versus diagnostic biopsies for patients with MDS defined a highly coordinated gene program in MDS hematopoietic stem/progenitor cell (HSPC), largely associated with downregulation of inflammatory, glucocorticoid, and cytokine signaling and splicing deregulation. We find that HMA therapy drives HSC output toward megakaryocyte-erythroid progenitor (MEP) and megakaryocyte progenitor (MkP) regardless of clinical response and induces treatment and genotype-specific changes in splicing (including bona fide SRSF2-P95 targets). The latter was confirmed in both a mouse model of MDS (Srsf2-P95 Runx1−/−) and an SRSF2-P95 human cell line. The role of alternative isoform regulation in aging, MDS progression, and therapy has been largely underestimated, resulting in profound differences in the cell state impacts of MDS oncoproteins.

Myelodysplastic syndromes (MDS) are heterogeneous hematopoietic stem cell disorders defined by ineffective hematopoiesis, multilineage dysplasia, and risk of progression to acute myeloid leukemia. Improvements have been made to identify recurrent genetic mutations and their functional roles, but translating this into preclinical models is still difficult. Traditional murine systems lack the human-specific cytokine support and microenvironmental support that is necessary to reproduce MDS pathophysiology. Humanized mouse models, particularly those incorporating human cytokines (e.g., MISTRG, NSG-SGM3, NOG-EXL), immunodeficient backgrounds, and co-transplantation strategies, have improved the engraftment and differentiation of human hematopoietic stem and progenitor cells. These models allow the study of clonal evolution, mutation-specific disease dynamics, and response to therapies in vivo. However, difficulties persist, such as limited long-term engraftment, incomplete immune reconstruction, and limited possibilities of modeling early-stage or low-risk MDS. This review presents an overview of current humanized and genetically engineered mouse models suitable for studying MDS, evaluating their capacity to replicate disease complexity, preserve clonal architecture, and support translational research. We highlight the need to develop new approaches to improve the actual methodologies and propose future directions for standardization and improved clinical relevance.

Measurable residual disease (MRD) assessment has become a cornerstone in the management of acute myeloid leukemia (AML), offering critical prognostic information and guiding post-remission therapy. Conventional MRD detection methods, including multiparameter flow cytometry (MFC), quantitative PCR (qPCR), and next-generation sequencing (NGS), have demonstrated strong predictive value but are limited by technical complexity, marker specificity, and accessibility. This review explores the current landscape of MRD monitoring in AML, covering cytogenetic, immunophenotypic, and molecular approaches, with particular emphasis on the strengths and limitations of each. We further examine promising emerging technologies—namely DNA methylation profiling and surface-enhanced Raman scattering (SERS)—as non-invasive alternatives. DNA methylation-based assays capitalize on the epigenetic dysregulation characteristic of AML, while proof-of-concept studies indicate SERS as a promising alternative for cancer subtypes, stages or specific mutation detection by analyzing biofluids or extracted DNA from blood. Together, these developments hold the potential to overcome current diagnostic limitations, enabling more universal and precise MRD assessment. Ongoing research and validation will determine their future integration into standard clinical practice.

The therapeutic landscape in ALL has changed dramatically over the last decade. Allogeneic blood or marrow transplantation (AlloBMT) has also evolved and remains an important option for consolidation. We assessed the interplay between these factors by analyzing the outcomes of 251 adult ALL (214 B and 37 T ALL) patients undergoing alloBMT with post-transplantation cyclophosphamide (PTCy) across two eras: 2008-2014 (ERA1) and 2015-2022 (ERA2). ERA1 patients were younger (median age 45.5 vs. 50, p=0.03), less likely to have an HCT-CI ≥4 (9% vs. 21%, p=0.01), more likely to have MRD by flow cytometry (20% vs. 9%, p=0.01) and receive myeloablative conditioning (56% vs. 3%, p

BACKGROUND:Blinatumomab has proven efficacy in the frontline, consolidation, and relapsed/refractory settings in B cell acute lymphoblastic leukemia. Its efficacy and safety among patients commonly excluded from clinical trials are unknown. PATIENTS AND METHODS:This single center, retrospective cohort study included patients treated for acute leukemia with blinatumomab with the following pre-existing conditions: liver dysfunction, renal impairment, central nervous system (CNS) conditions, autoimmune disease, solid organ transplantation, or uncontrolled infection. Rates of blinatumomab completion, efficacy outcomes, cytokine release syndrome (CRS), neurotoxicity (ICANS), and adverse events specific to the impaired organ leading to inclusion were assessed. RESULTS:Thirty-four patients were included, and 88 % completed at least one cycle of blinatumomab. One patient stopped prematurely due to toxicity and three due to lack of response in the relapsed/refractory setting. Response rates and survival were similar by treatment setting to those treated in clinical trials. The 60-day cumulative incidence of grade ≥ 2 CRS and ICANS were 23.5 % and 20.8 %, respectively. No excess liver injury, ICANS, autoimmune flares, organ rejection, or infections were observed in cohorts defined by impairment of each system. CONCLUSION:Blinatumomab was successfully administered to the vast majority of patients with a baseline condition that would have led to clinical trial exclusion. Adverse events generally occurred at rates similar to prior clinical trials. These data provide the basis to consider removing certain exclusion criteria from future studies involving blinatumomab, allowing more patients to benefit from its efficacy.

Metabolic cues are crucial for regulating haematopoietic stem and progenitor cells (HSPCs). However, the metabolic profile of human HSPCs remains poorly understood due to the limited number of cells and the scarcity of bone marrow samples. Here we present the integrated metabolome, lipidome and transcriptome of human adult HSPCs (lineage-, CD34+, CD38-) upon differentiation, ageing and acute myeloid leukaemia. The combination of low-input targeted metabolomics with our newly optimized low-input untargeted lipidomics workflow allows us to detect up to 193 metabolites and lipids from a starting material of 3,000 and 5,000 HSPCs, respectively. Among other findings, we observe elevated levels of the essential nutrient choline in HSPCs compared with downstream progenitors, which decline upon ageing and further decrease in acute myeloid leukaemia. Functionally, we show that choline supplementation fuels lipid production in HSPCs and enhances stemness. Overall, our study provides a comprehensive resource identifying metabolic changes that can be utilized to promote and enhance human stem cell function.

ABSTRACT:SY-2101 is a novel oral formulation of arsenic trioxide (ATO). Although IV ATO in combination with all trans retinoic acid is highly efficacious in treating acute promyelocytic leukemia (APL), there remains a significant unmet need due to the treatment burden associated with receiving daily ATO infusions for nearly a year and the risk of complications associated with indwelling central catheters. The pharmacokinetics (PK), safety, and tolerability of SY-2101 and ATO IV after single- and multiple-dose administration and the impact of food on PK for SY-2101 were evaluated in this phase 1 study in 15 participants with APL. SY-2101 in the fasted state demonstrated comparable systemic exposure to ATO IV based on the active metabolite arsenious acid [As(III)], with geometric mean ratios (GMRs) of SY-2101 to ATO IV of 1.00 for area under the plasma concentration (AUC) from 0 hour to last time point (AUC0-last) and from 0 hour to infinity (AUC0-inf). The GMR of SY-2101 to ATO IV maximum concentration (Cmax) was 0.76, as was expected due to the different routes of administration. Comparisons of SY-2101 in fed to fasted states demonstrated similar exposure with GMRs of AUC0-last, AUC0-inf, and Cmax at 1.08, 1.12, and 0.85, respectively, allowing for SY-2101 administration with or without food. SY-2101 was well tolerated. Most adverse events were of low grade. This study provides the first intrapatient PK crossover results directly comparing SY-2101 with ATO IV and supports the likelihood of clinical equivalence between the 2 formulations. This trial was registered at www.ClinicalTrials.gov as #NCT04996030.

A hallmark of cancer biology is resistance to apoptosis. BCL-2 is an anti-apoptotic molecule that is being overexpressed in several myeloid diseases, such as acute myeloid leukemia and myelodysplastic syndromes, but also in several lymphoid cancers, such as acute lymphoblastic leukemia, chronic lymphocytic leukemia, non-Hodgkin lymphomas and multiple myeloma. Venetoclax (VEN) is a BCL-2 small molecule inhibitor. Data about its structure, biochemical characteristics and in vitro efficacy against several blood cancer cell lines were first reported in 2013. Shortly after, the first clinical trials reported that single-agent VEN provides no long-term survival benefits. In contrast, when used in combination, VEN led to significantly improved outcomes and eventually to its first US FDA approvals in 2018. As the modern approach to treating hematological malignancies are the chemotherapy-free regimen, in the current manuscript, we provide a comprehensive view on all available therapies that are considered to be chemotherapy-free, with a special emphasis on acute myeloid leukemia (AML), where phase I-III clinical trials have provided the most data.

Patients with myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML) with high-risk features including TP53 mutations have poor outcomes due to lack of effective therapies. The atypical chemokine surface receptor C-C motif chemokine receptor-like 2 (CCRL2) is overexpressed in MDS and secondary AML (sAML) compared to healthy hematopoietic cells and we recently found that TP53-mutated MDS/AML and AML with erythroid features express the highest levels of this receptor across MDS/AML subtypes. To illustrate the therapeutic potential of CCRL2 as a therapeutic target, we developed an anti-CCRL2 antibody-drug conjugate (ADC) by conjugating an anti-CCRL2 antibody with the cytotoxic drug pyrrolobenzodiazepine (PBD), which causes DNA double-strand breaks leading to cancer cell death. The anti-CCRL2 ADC demonstrated strong CCRL2-selective cytotoxicity against cell lines derived from MDS/AML patients with TP53 mutations and erythroid features, surpassing the cytotoxic effects observed with gemtuzumab and PBD-conjugated anti-CD33 and anti-CD123 ADCs. It also induced apoptosis and suppressed the clonogenicity of primary MDS/AML bone marrow samples without affecting the survival, differentiation and clonogenicity of healthy hematopoietic stem and progenitor cells. This agent also suppressed the leukemic growth of TP53-mutated MDS/AML cell line xenografts, improving mice survival and decreasing the leukemic burden in patient-derived TP53-mutated MDS/AML xenografts. In conclusion, our study introduces CCRL2 as a potential new therapeutic target in high-risk MDS/AML.

Background: FLT3-ITD mutations are common in AML, occur in a diverse co-mutation landscape, and are generally associated with a poor prognosis. DNMT3A and NPM1 are commonly co-mutated with FLT3. Historically, DNMT3A, NPM1, and FLT3-ITD triple-mutated AML (TM-AML) has been reported to have a higher risk of relapse and worse overall survival in response to treatment with chemotherapy alone. However, recent approval and widespread use of multiple FLT3 inhibitors have significantly improved disease outlook. We evaluated the outcomes of DMNT3A/NPM1/FLT3-ITD triple-mutated AML from a cohort of newly diagnosed AML patients treated with modern therapeutic approaches, including intensive chemotherapy, azacitidine and venetoclax, allogeneic transplant, and FLT3 inhibition, guided by a high-sensitivity FLT3-ITD MRD assay. Methods: The study cohort consists of 431 adult patients with newly diagnosed AML who were admitted and treated in consecutive fashion on the leukemia service at the Johns Hopkins Hospital between January 2020-July 2024. Targeted sequencing of a panel of 60 leukemia-associated genes was carried out using genomic DNA derived from peripheral blood or bone marrow specimens collected at diagnosis. Clinical records were reviewed to determine patients' baseline characteristics, MRD status (using the Invivoscribe FLT3-ITD MRD assay), FLT3 inhibitor use, event free survival, and overall survival. Statistical testing, survival analysis, and Cox regression were performed using R. Results: FLT3 mutations were found in 101/431 (23.4%), 72 with FLT3-ITD (17%), 25 (6%) with FLT3-TKD, and 8 (2%) with non-canonical mutations. Of the 72 FLT3-ITD patients, 17 patients had DNMT3A, NPM1, and FLT3-ITD triple mutations (24% of FLT3-ITD; 4% of all AML patients) and 10/17 (59%) of these received allogeneic transplant. The median white count for the TM subgroup at diagnosis was 78, and the median age was 61 with a wide range (27-85). Karyotype was intermediate risk in 100% of TM subgroup (82% diploid). 15/17 (88%) TM-AML patients received FLT3 inhibitors during their treatment course. Of the 2 patients who did not receive FLT3 inhibitors, both were over the age of 70 and were induced with azacitidine and venetoclax, and one transitioned to hospice during induction. Kaplan-Meier survival analysis revealed patients with TM-AML (n=17) had significantly better overall survival (OS) and event-free survival (EFS) compared to non-TM FLT3-ITD AML (n= 55) (2-year OS: 82% vs. 50%; 2-year EFS: 84% vs. 43%; log-rank, p < 0.05). Patients with TM-AML had a comparable overall survival to patients with APL (n=25) and CBF AML (n=24) treated during the same time period (log-rank, p = 0.94). Subgroup analysis of FLT3-ITD AML demonstrated TM-AML had the longest OS and EFS compared to any other combination of those 3 mutations (log-rank, p < 0.01).Multivariate Cox regression confirmed a significant 3-fold ([1.03-8.41], p<0.05) increased hazard ratio with DNMT3A alone and yet paradoxically improved survival with both DNMT3A and NPM1 (HR 0.08 [0.01-0.55], p < 0.05). FLT3 inhibitor use is additionally associated with decreased mortality (HR 0.35 [0.13-0.97], p < 0.05). We also evaluated FLT3-ITD MRD negativity rates following 2 cycles of treatment among FLT3-ITD patients who received a FLT3 inhibitor during treatment. We found higher MRD negative rates in 8/10 (80%) TM-AML patients vs. 10/25 (40%) non-TM-AML while accounting for FLT3 inhibitor exposure, suggesting that TM-AML is more responsive to FLT3 inhibition (Fisher's exact, p < 0.05). In the non-TM-AML subgroup, 24/55 (44%) harbored MDS-defining molecular abnormalities. The median FLT3-ITD VAF was 27% in the TM-AML group versus 10% in the non-TM-AML group arising from antecedent MDS (Wilcoxon rank-sum, p < 0.05), highlighting dominance of the FLT3-ITD clone at diagnosis in TM-AML versus a sub-clonal event in myelodysplasia-related AML. Conclusions: FLT3 mutations by themselves do not define an AML subtype, but in this new era of FLT3 inhibitors, DNMT3A/NPM1/FLT3-ITD triple-mutated AML appears to represent a unique subgroup of AML with especially favorable outcomes and a prognosis similar to CBF and APL. This subgroup may be uniquely dependent on FLT3 signaling and therefore distinctly responsive to FLT3 inhibition. High sensitivity MRD assays can be used to guide therapeutic decisions for this subset, which may contribute to the overall favorable outcome.

Introduction: Hypomethylating agent and venetoclax (VEN)–based therapy has transformed the treatment landscape for older adults with AML, offering improved remission rates and survival compared to traditional chemotherapy. However, patients with TP53 mutations (TP53mut) and complex karyotype (CK), continue to experience dismal outcomes characterized by primary resistance, early relapse, and high therapy-related toxicity. In this population, the cytotoxic effect of standard-dose azacitidine and VEN is poorly tolerated, often leads to treatment discontinuation or early mortality. Low-dose, metronomic decitabine (LoDec, 0.1–0.2 mg/m²/WEEK) offers a non-cytotoxic alternative that depletes DNMT1 and retains epigenetic-modifying activity. LoDec has demonstrated efficacy in relapsed AML and in combination with VEN for de novo disease, but its role in the frontline treatment of TP53mut CK MDS/AML has not been defined. Here, we evaluate the safety and efficacy of LoDec/VEN in this high-risk population. Methods: We retrospectively reviewed all patients with newly diagnosed TP53mut CK myeloid neoplasms (MDS and AML) treated at Johns Hopkins between 2018 and 2024.TP53mut were identified by targeted next-generation sequencing, and CK was defined as the presence of ≥3 cytogenetic abnormalities. Patients were grouped by frontline therapy: LoDec plus VEN (400 mg, one dose per WEEK), standard azacitidine (Aza, 75 mg/m² × 7 days) plus VEN (400 mg/day), or standard decitabine (StDec, 20 mg/m² × 5 days). We collected baseline demographic and clinical data at diagnosis and evaluated transfusion independence (TFI, ≥4 weeks without RBC or platelet transfusions), overall survival (OS), 90-day mortality (90dM), and hospitalization burden (total inpatient days, proportion of days hospitalized over total days survived). Analyses were performed using Kaplan-Meier estimates for OS, ANOVA for comparison of continuous variables, and chi-square tests for categorical variables. Results: We identified 94 patients with TP53mut CK myeloid neoplasms who received the above therapies: 20 received first-line LoDec/VEN (4 MDS, 5 AML/MDS, 11 AML), 57 received Aza/VEN (2 MDS, 10 AML/MDS, 45 AML) and 17 received StDec (1 MDS, 8 AML/MDS, 8 AML). Patients in the LoDec/Ven group were similar in age (mean 73 years, p=0.23), gender (50% male, p=0.24), and ECOG (mean 2.05, p=0.2) when compared to those in the Aza/VEN (mean age 69, 70% male, mean ECOG 1.63) and StDec (mean age 68, 70% male, mean ECOG 2.00) groups. Patients in the LoDec/Ven group had on average higher Charlson co-morbidity index (CCI) scores compared to the other groups (LoDec/Ven 7.9, Aza/Ven 6.3, StDec 6.4, p<0.05). A notable feature at presentation for TP53mut CK myeloid neoplasm was the occurrence of “disease-related fever,” defined as T > 38C without clinical, laboratory, or radiographic evidence of infection suggesting a potential association between TP53mut CK disease biology and inflammatory cytokine production. This clinical feature was similarly distributed (p=0.3) in all treatment groups (15% LoDec/VEN, 26% Aza/VEN, 54% StDec). Median OS for patients treated with LoDec/VEN was 203 days, significantly different from Aza/VEN (90 days) and StDec (371 days) (p=0.01). LoDec/Ven group had significantly lower 90dM (15%, p<0.01) compared to StDec (23.5%) and Aza/VEN (51.7%). These results underscore the poor outcomes in this high-risk population and the relative tolerability of LoDec/VEN. Hospitalization burden was significantly lower in the LoDec/Ven cohort, with patients spending in average 19.7 days (p<0.01) in the hospital (15% of days survived, p<0.01), versus 31.9 days (40%) with Aza/VEN and 50 days (40%) with StDec. TFI was achieved in 47% of LoDec/VEN-treated patients, 47% of the StDec patients, and 30% of the Aza/VEN patients (p=0.28). Conclusions: Our data suggest that LoDec/Ven is a reasonable frontline approach for TP53mut CK myeloid neoplasms. Currently, many providers favor Aza/VEN or StDec for patients deemed fit to proceed to BMT. However, only 6/74 patients treated with these regimens ultimately underwent transplant, and none survived beyond 14 months. In contrast, those treated with LoDec/Ven had higher CCI at diagnosis. Nevertheless, many achieved TFI but were not properly assessed for depth of response, raising the possibility that some may have achieved remission levels sufficient for more intensive consolidation. A prospective clinical trial is underway to assess this strategy.