Pediatric acute myeloid leukemias (AMLs) are driven by transcription factor (TF) fusions that reprogram the epigenome, creating fusion-specific subtypes with distinct biomarkers, prognoses, and vulnerabilities. Pediatric AMLs driven by the CBFA2T3::GLIS2 (C/G) fusion are among the highest-risk subtypes, with fewer than 15% patients surviving 60 months post-diagnosis. These dismal outcomes stem from an incomplete understanding of how C/G rewires chromatin to establish subtype-specific regulatory dependencies, limiting development of targeted therapies. Comprehensive, multi-omic dissection of oncofusion-driven gene regulation is essential to reveal druggable mechanistic vulnerabilities. To detect C/G-interacting factors, we performed unbiased immunoprecipitation mass spectrometry (IP-MS) profiling of C/G in patient-derived AML cell lines. This revealed a novel interaction with mammalian SWI/SNF (SWItch/Sucrose Non-Fermentable) chromatin remodeling complexes, which normally guide hematopoietic differentiation. CRISPR-based viability assays showed that C/G cell survival relies on BRG1-containing mSWI/SNF, revealing a fusion-specific chromatin remodeling dependency. To investigate how this interaction reprograms myeloid cells, we compared C/G-transformed hematopoietic stem and progenitor cells (HSPCs) to non-transformed counterparts. CUT&RUN profiling of C/G-mSWI/SNF occupancy and histone modifications, integrated with RNA-seq, showed that C/G retargets mSWI/SNF to oncogenic regulatory elements, sustaining overexpression of cell cycle genes CCND2 and CDK6. This C/G-directed chromatin remodeling creates a clinically targetable mSWI/SNF-G1 regulatory axis. Pharmacologic inhibition of this axis using palbociclib, an FDA-approved CDK4/6 kinase inhibitor, reduced leukemic burden and extended survival by ∼56% in an aggressive C/G AML mouse model, highlighting a clinically actionable vulnerability. These findings reveal how the C/G oncofusion repurposes BRG1-containing mSWI/SNF complexes to drive cell cycle gene activation, establishing a chromatin–cell cycle regulatory circuit that is selectively targetable through G1 restriction. This mechanistic insight provides a rationale for repurposing CDK4/6 inhibitors for children with this otherwise lethal AML subtype. Gabriel E. Boyle, Alexander W. Ying, Lara Yao, Chao-Jen Wong, Liam Caven, Morgan Merrill, Farzane Sivandzade, Matthew Wither, Soheil Meshinchi, Rachel Rau, Shan Lin, Cigall Kadoch, Jay F. Sarthy. Targeting Chromatin–Cell Cycle Vulnerabilities in High-Risk Pediatric Acute Myeloid Leukemia [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Fusion-Positive Cancer: From Discovery to Therapy; 2026 Jan 13-15; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86(1_Suppl):Abstract nr PR005.

Differential regulation metrics and values for select features Sheet 1: Log2 fold-change, p-value, and q-value between ARPC and NEPC lines for NPS in the 47 phenotype defining gene bodies (two tailed Mann-Whitney U test, Benjamini-Hochberg adjusted). Sheet 2: Differentially expressed list of 514 transcription factor (TF). All statistical comparison and fold change estimation was done against ARPCs. Sheet 3: Log2 fold-change, p-value, and q-value between ARPC and NEPC lines for central mean coverage in 108 TFs overlapping RNA-Seq up/down regulated TFs (two tailed Mann-Whitney U test, Benjamini-Hochberg adjusted). Sheet 4: Paralogous transcription factors (TFs) for each of the 38 TFs with differential RNA expression between ARPC and NEPC and differential TFBS accessibility. Paralogous TFs that are also differentially expressed by RNAseq analysis of PDX tumors are shown in red text. Paralogs were obtained from ensembl biomart human genes GRCh38.p13 (http://uswest.ensembl.org/biomart/martview/64d8bd7fe9851a2501aece9b74b03631) Sheet 5: Mean values in ARPC, NEPC, and HD lines for central and window means for ARPC and NEPC specific open chromatin regions.

Ewing sarcoma (EwS) is a fusion-driven malignancy, peaking in adolescence. Although EwS tumors are driven uniquely by EWS::FLI1 and related fusions, patient outcomes vary greatly. If and how tumor plasticity of EWS::FLI1-regulated transcriptional signatures contribute to disease progression is not known. To address this, we utilized a single-cell co-assay of RNA and chromatin accessibility (ATAC) sequencing to identify gene regulatory networks in EwS. By comprehensively characterizing regulatory elements across cell lines, we identified multiple unique modules of gene regulation. Differential usage and prevalence of these modules was evident across cell lines, associated with distinct epigenetic and transcriptomic signatures, and in specific cases, modifiable by exogenous TGF-β. When we examined primary EwS patient tumors, we observed these same regulatory modules were variably enriched both across and within tumors, highlighting the existence of intratumoral heterogeneity in gene regulatory networks. Our findings demonstrate that multiple, co-existing transcriptional programs shape the phenotypic diversity of EwS and suggest that the balance between these networks may have important implications for clinical outcomes and targeted therapy development.

Resistance to chemotherapy and subsequent relapse remain the primary challenge in pediatric acute myeloid leukemia (pAML), particularly in CBFA2T3-GLIS2 (C/G) fusion-positive acute megakaryoblastic leukemia. Here we demonstrate that the C/G fusion drives extensive DNA methylation changes and oncogenic enhancer activation at cis-regulatory elements (CREs), reshaping gene expression. This multi-omics analysis reveals a distinct hypermethylation pattern at promoters of up-regulated genes in C/G+ pAML across patient samples (n = 24) and representative cell lines, notably enriched in adhesion-related, TGFβ, or Wnt signaling pathways. Hypermethylated regions adjacent to transcription start sites (TSS) maintain open chromatin with H3K27ac enrichment, supporting a mechanism of de novo chromatin looping and active transcription in a non-canonical manner. Additionally, C/G fusion binding near the DNA methyltransferase 3B (DNMT3B) promoter correlates with elevated DNMT3B expression, implicating its role in aberrant DNA methylation changes at CREs. This study elucidates the epigenetic mechanisms driving C/G+ pAML, showing how the fusion reshapes chromatin and DNA methylation landscapes by impacting the expression (and likely activity) of epigenetic modifiers like DNMT3B. Functionally, DNMT3B inhibition enhances apoptotic sensitivity to BCL2 blockade, indicating that targeting DNMT3B may overcome apoptotic resistance in C/G+ leukemic cells and offer a therapeutic strategy for this high-risk subtype.

Unsupervised model predictions and patient/validation cohort sequencing metrics Sheet 1: DFCI Cohort I: Tumor phenotype by histology and estimates of tumor fraction, subtyping score, and inferred subtype calls using ctdPheno. Sheet 2: DFCI cohort I: Tumor phenotype by histology and estimates of tumor fraction, subtyping score, and inferred phenotypic subtype calls using different ATAC-seq site restricted analysis using ctdPheno. Sheet 3: DFCI cohort I: Tumor phenotype by histology and estimates of tumor fraction, subtyping score, and inferred phenotypic subtype calls using different ATAC-seq site restricted analysis using Keraon. Sheet 4: DFCI Cohort II: Tumor phenotype by histology, summary of clinical correlatives and estimates of tumor fraction, subtyping score, and inferred subtype calls using ctdPheno. Sheet 5: Histology, tumor fraction, subtype score, NEPC fraction, and subtype calls for WGS and ULP patient samples (UW cohort). Sheet 6: Complete sequencing metrics for DFCI cohort I. Sheet 7: Complete sequencing metrics for DFCI cohort II (ULP). Sheet 8: Complete sequencing metrics for DFCI cohort II (deep WGS) Sheet 9: Complete sequencing metrics and ichorCNA estimated tumor fractions for the UW cohort (ULP). Sheet 10: Complete sequencing metrics for the UW cohort (deep WGS) Sheet 11: Complete sequencing metrics for the healthy donor cohort. Sheet 12: Clinical data summary for UW cohort.

PDX sequencing metrics Phenotype and sequencing metrics for all PDX LuCaP lines.

Supplementary Figures S1-S18 and Supplementary Table legends.

Stem cells have lower facultative heterochromatin as defined by trimethylation of histone H3 lysine 27 (H3K27me3) compared to differentiated cells. However, the mechanisms underlying these differential H3K27me3 levels remain elusive. Because H3K27me3 levels are diluted 2-fold in every round of replication and then restored through the rest of the cell cycle, we reasoned that the cell cycle length could be a key regulator of total H3K27me3 levels. Here, we propose that a short G1 phase restricts H3K27me3 levels in stem cells. To test this model, we determined changes to H3K27me3 levels in mouse embryonic stem cells (mESCs) globally and at specific loci upon G1 phase lengthening - accomplished by thymidine block or growth in the absence of serum (with the "2i medium"). H3K27me3 levels in mESCs increase with G1 arrest when grown in serum and in 2i medium. Additionally, we observed via CUT&RUN and ChIP-seq that regions that gain H3K27me3 in G1 arrest and 2i media overlap, supporting our model of G1 length as a critical regulator of the stem cell epigenome. Furthermore, we demonstrate the inverse effect - that G1 shortening in differentiated human HEK293 cells results in a loss of H3K27me3 levels. Finally, in human tumor cells with extreme H3K27me3 loss, lengthening of the G1 phase leads to H3K27me3 recovery despite the presence of the dominant negative, sub-stoichiometric H3K27M mutation. Our results indicate that G1 length is an essential determinant of H3K27me3 landscapes across diverse cell types.

PTM peak data and phenotype 47 fragment variability Sheet 1: PDX sample representation in 3 histone PTM CUT&RUN nucleosome profiling assays (H3K4me1, H2K27ac and H3K27me3). Sheet 2: Log2 fold-change, p-value, and q-value between ARPC and NEPC lines for coefficient of variation in the 47 phenotype defining gene bodies (two tailed Mann-Whitney U test, Benjamini-Hochberg adjusted). Sheet 3: Log2 fold-change, p-value, and q-value between ARPC and NEPC lines for coefficient of variation in the 47 phenotype defining gene promoters (two tailed Mann-Whitney U test, Benjamini-Hochberg adjusted).

Feature-region combination AUCs and benchmarking Sheet 1: Log2 fold-change, p-value, and q-value between ARPC and NEPC lines for central mean coverage in all queried (338) TFs (two tailed Mann-Whitney U test, Benjamini-Hochberg adjusted). Sheet 2: 100-fold cross-validation AUCs for all region and feature combinations subset by the ‘AR10’ overlapping features (see Methods). Sheet 3: 100-fold cross-validation AUCs for all region and feature combinations subset by the ‘Phenotype 47-defining’ overlapping features. Sheet 4: 100-fold cross-validation AUCs for all region and feature combinations (global). Sheet 5: Predictions scores, tumor fraction, depth of coverage, and subtype for benchmarking admixtures. Sheet 6: AUCs for unsupervised prediction of admixture subtypes grouped by tumor fraction and depth. Sheet 7: NEPC:ARPC ratio, tumor fraction, and ARPC and NEPC fraction predictions for mixed phenotype admixtures using Keraon (see Methods).

BACKGROUND:Anthracyclines are among the most effective chemotherapeutic agents used to treat pediatric malignancies. However, their clinical use is limited by dose-dependent toxicities, particularly cardiotoxicity and secondary malignancies. Aclarubicin (Acla) is an anthracycline derivative that induces chromatin damage while sparing DNA damage, offering potential therapeutic benefit with reduced long-term toxicity. METHODS:We evaluated the anti-tumor efficacy and safety profile of Acla in multiple in vitro pediatric cancer models and in vivo mouse models designed to mimic anthracycline re-treatment following prior doxorubicin (Doxo) exposure. Tumor growth, genotoxic stress, survival, and organ toxicity were assessed. RESULTS:Acla demonstrated robust anti-tumor activity comparable to Doxo across diverse pediatric in vitro models. Unlike Doxo, Acla treatment did not induce significant genotoxic stress. In vivo, mice receiving Acla after Doxo exposure showed no evidence of cumulative cardiotoxicity or end-organ damage. In contrast, a second course of Doxo led to significant toxic mortality but was surprisingly not attributable to classic cardiac injury. CONCLUSION:Our study highlights Acla as a promising anthracycline derivative for pediatric cancers, with potent anti-tumor efficacy and a superior safety profile, even following prior anthracycline exposure. These results support continued investigation of chromatin-damaging anthracyclines that can kill pediatric cancer cells without inducing genotoxic stress. In addition, our studies underscore the need to refine preclinical models to better understand both acute and chronic anthracycline toxicities in pediatric and adolescent populations.

Histones are essential nucleosome components that regulate DNA accessibility and transcription. Mutations in histones, such as H2B-E76K, are prevalent in various cancers, including lung and genitourinary cancers. The H2B-E76K mutation destabilizes nucleosomes by disrupting H2A/H2B dimer binding to H3/H4 tetramers, altering chromatin structure and function. Investigate the effects of the H2B-E76K mutation on gene expression, chromatin interactions, signaling pathways, and cellular phenotypes, focusing on the induction of epithelial-mesenchymal transition (EMT) in lung cancer models. CRISPR-Cas9 created BEAS-2B bronchial epithelial cell lines with heterozygous and homozygous H2B-E76K mutations in the H2BC4 gene. Phenotypic assays assessed cell growth, migration, invasion, and EMT marker expression. Transcriptomic changes were analyzed using scRNA-seq, bulk RNA-seq, and PRO-seq to evaluate pathway deregulation, transcriptional instability, and RNA polymerase activity. The μMap technique mapped protein-chromatin interactions disrupted by the mutation. Chromatin profiling with CUT&RUN and ATAC-seq examined histone incorporation and chromatin accessibility changes. H2B-E76K mutant cells showed enhanced migration and invasion, increased mesenchymal markers (N-cadherin, vimentin), and decreased epithelial markers (E-cadherin), consistent with EMT. These cells also resisted apoptosis upon growth factor withdrawal. Transcriptomic analyses revealed transcriptional instability, with increased cell-to-cell variability in gene expression, altered patterns linked to oncogenic signaling, transcript switching, and disrupted RNA polymerase activity. The μMap technique identified disrupted interactions between chromatin and RNA-processing enzyme RAVER1, correlating with altered splicing, exon skipping, and gene expression variability. ATAC-seq showed enhanced binding of EMT-related transcription factors (e.g., AP1, CEBPB, ZNF384, FOXC2) and increased chromatin accessibility at EMT-related promoters. CUT&RUN revealed diffuse genome-wide deposition of H2B-E76K histones, consistent with replication-coupled incorporation. These chromatin changes promoted transcriptional programs associated with EMT and cancer progression. H2B-E76K histones are broadly distributed throughout the genome, causing transcriptomic instability, altered splicing, and transcription factor binding changes. These alterations drive EMT phenotypes, including enhanced invasion, migration, and resistance to apoptosis, contributing to aggressive cancer behavior. H2B-E76K acts as a key epigenetic driver of oncogenesis by modifying chromatin accessibility, gene regulation, and cellular plasticity. Pierre Priam, Kimberly Espinoza Pereira, Jixiu Shan, Anthony Lamberto, Jay Sarthy, Wuyue Zhou, Ciaran Seath, Richard L. Bennett, Jonathan D. Licht. The H2B-E76K oncohistone alters chromatin accessibility, composition, and gene expression to induce EMT [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 2743.

Abstract Ewing sarcoma (EwS) is a bone and soft tissue tumor that is driven by the fusion of FET and ETS genes, most commonly resulting in creation of the EWS::FLI1 chimeric oncoprotein. The scaffolding protein Menin regulates transcription through interactions with transcription factors and chromatin modifying enzymes and has both oncogenic and tumor suppressive roles, depending on the cellular context. The half-life of Menin is prolonged in EwS cells leading to high levels of the protein that is dependent on EWS::FLI1 expression. To investigate tumorigenic functions of Menin in EwS, we used CRISPR/Cas9 to generate clonal A673 and TC32 Menin knockout cells. Consistent with our earlier studies using shRNA-mediated knockdown, loss of Menin had little effect on proliferation in 2-D culture but resulted in reduced anchorage-independent growth in soft agar assays. When MEN1-KO cells were injected into NSG mice, either subcutaneously or by tail vein, tumor formation was delayed. Injection of MEN1-KO cells into the renal subcapsule of NSG mice resulted in locally invasive tumors but their capacity to metastasize to the liver was reduced compared to control cells. To elucidate how loss of Menin led to diminished tumorigenic and metastatic potential, we performed RNA-seq of control and MEN1-KO cells. This analysis revealed significant and reproducible down-regulation of MYC target genes and up-regulation of the epithelial to mesenchymal transition pathway and extracellular matrix protein-encoding genes in MEN1-KO cells. To determine if genes altered in MEN1-KO cells were dependent on the role of Menin in Menin/MLL methyltransferase complexes, we analyzed transcriptomes of EwS cells that had been exposed to the Menin/MLL interaction inhibitor, VTP-50469. Only a subset of genes altered by the MEN1-KO were also altered by the Menin/MLL inhibitor, suggesting that Menin regulates gene expression in EwS cells by both MLL-dependent and MLL-independent mechanisms. To test this, we performed CUT&RUN to define Menin binding sites and discovered that Menin binds not only at H3K4me3-marked gene promoters but also at intragenic enhancers marked by H3K27Ac. Importantly, comparison of RNA-seq data revealed substantial overlap between Menin-regulated and established EWS::FLI1 target genes and we identified that a subset of EWS::FLI1-bound intragenic enhancers are also bound by Menin. Significantly, these co-bound, co-regulated loci encode for pro-metastatic genes, including IL1RAP, STEAP1, and CAV1. Finally, we found that Menin and EWS::FLI1 co-immunoprecipitate in EwS nuclear extracts suggesting that they may exist in complex with one another at sites of shared gene regulation. Taken together, these data indicate that Menin promotes EwS metastasis and that this is achieved by its function as a scaffolding protein that augments the transcriptional activity of EWS::FLI1 at intragenic enhancers of pro-metastasis genes. Citation Format: Katherine A. Braun, Aya Miyaki, Nicolas M. Garcia, Darleen Tu, Feinan Wu, Jay F. Sarthy, Elizabeth R. Lawlor. Menin drives oncogenesis in Ewing sarcoma cells by activating transcription of key metastatic factors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6601.

Recent genome-wide analyses identified chromatin modifiers as one of the most frequently mutated classes of genes across all cancers. However, chemotherapies developed for cancers involving DNA damage remain the standard of care for chromatin-deranged malignancies. In this review we address this conundrum by establishing the concept of ‘chromatin damage’: the non-genetic damage to protein–DNA interactions induced by certain small molecules. We highlight anthracyclines, a class of chemotherapeutic agents ubiquitously applied in oncology, as an example of overlooked chromatin-targeting agents. We discuss our current understanding of this phenomenon and explore emerging chromatin-damaging agents as a basis for further studies to maximize their impact in modern cancer treatment.

The interaction of the tumor necrosis factor receptor (TNFR) family member CD27 on naive CD8+ T (Tn) cells with homotrimeric CD70 on antigen -presenting cells (APCs) is necessary for T cell memory fate determination. Here, we examined CD27 signaling during Tn cell activation and differentiation. In conjunction with T cell receptor (TCR) stimulation, ligation of CD27 by a synthetic trimeric CD70 ligand triggered CD27 internalization and degradation, suggesting active regulation of this signaling axis. Internalized CD27 recruited the signaling adaptor TRAF2 and the phosphatase SHP-1, thereby modulating TCR and CD28 signals. CD27mediated modulation of TCR signals promoted transcription factor circuits that induced memory rather than effector associated gene programs, which are induced by CD28 costimulation. CD27-costimulated chimeric antigen receptor (CAR) -engineered T cells exhibited improved tumor control compared with CD28-costimulated CAR -T cells. Thus, CD27 signaling during Tn cell activation promotes memory properties with relevance to T cell immunotherapy.

Chromosomal translocations involving the mixed-lineage leukemia (MLL) locus generate potent oncogenic fusion proteins (oncoproteins) that disrupt regulation of developmental gene expression. By profiling the oncoprotein-target sites of 36 broadly representative MLL-rearranged leukemia samples, including three samples that underwent a lymphoid-to-myeloid lineage-switching event in response to therapy, we find the genomic enrichment of the oncoprotein is highly variable between samples and subject to dynamic regulation. At high levels of expression, the oncoproteins preferentially activate either an acute lymphoblastic leukemia (ALL) program, enriched for pro-B-cell genes, or an acute myeloid leukemia (AML) program, enriched for hematopoietic-stem-cell genes. The fusion-partner-specific-binding patterns over these gene sets are highly correlated with the prevalence of each mutation in ALL versus AML. In lineage-switching samples the oncoprotein levels are reduced and the oncoproteins preferentially activate granulocyte-monocyte progenitor (GMP) genes. In a sample that lineage switched during treatment with the menin inhibitor revumenib, the oncoprotein and menin are reduced to undetectable levels, but ENL, a transcriptional cofactor of the oncoprotein, persists on numerous oncoprotein-target loci, including genes in the GMP-like lineage-switching program. We propose MLL oncoproteins promote lineage-switching events through dynamic chromatin binding at lineage-specific target genes, and may support resistance to menin inhibitors through similar changes in chromatin occupancy. The effects of chromosomal translocations involving the mixed-lineage leukemia (MLL) locus on gene expression regulation remain to be explored. Here, the authors find that MLL oncoproteins support lineage-switching events through dynamic chromatin binding.

Chromosomal translocations involving the Lysine-Methyl-Transferase-2A ( KMT2A ) locus generate potent oncogenic fusion proteins (oncoproteins) that disrupt regulation of developmental gene expression. By profiling the oncoprotein-target sites of 36 broadly representative KMT2A -rearranged leukemia samples, including three samples that underwent a lymphoid-to-myeloid lineage-switching event in response to therapy, we find the genomic enrichment of the oncoprotein is highly variable between samples and subject to dynamic regulation. At high levels of expression, the oncoproteins preferentially activate either an acute lymphoblastic leukemia (ALL) program, enriched for pro-B-cell genes, or an acute myeloid leukemia (AML) program, enriched for hematopoietic-stem-cell genes. The fusion-partner-specific-binding patterns over these gene sets are highly correlated with the prevalence of each mutation in ALL versus AML. In lineage-switching samples the oncoprotein levels are reduced and the oncoproteins preferentially activate granulocyte-monocyte progenitor (GMP) genes. In a sample that lineage switched during treatment with the menin inhibitor revumenib, the oncoprotein and menin are reduced to undetectable levels, but ENL, a transcriptional cofactor of the oncoprotein, persists on numerous oncoprotein-target loci, including genes in the GMP-like lineage-switching program. We propose KMT2A oncoproteins promote lineage-switching events through dynamic chromatin binding and can induce epigenetic lesions, marked by ENL, that support resistance to targeted therapies.

CBFA2T3-GLIS2 (C/G) fusion is the most frequent chimeric oncogene observed in non-Down syndrome acute megakaryoblastic leukemia (AMKL) in infants. C/G-AMKL has a dismal prognosis due to resistance and relapse following standard AML chemotherapy. Our group previously reported that transduction of C/G fusion followed by coculture with an engineered endothelial cell (EC) niche was sufficient to transform human cord blood hematopoietic stem and progenitor cells (HSPC) into C/G-AMKL, providing a platform to interrogate the biology of this aggressive leukemia. To identify putative leukemic stem cells (LSC) and elucidate their resistance mechanisms, we conducted single-cell RNA sequencing on engineered C/G-AMKL during their transformation from HSPC in the EC niche. We identified a subset of cells co-expressing LSC-associated genes CD96 and CD70 that were enriched in known pediatric LSC gene signatures and signatures of dormancy. Further, a subset of C/G-AMKL cells from both engineered leukemias and patient samples co-expressed CD96 and CD70 by flow cytometry. Notably, ECs protected C/G-AMKL cells from cytarabine treatment, which enriched for the CD96+CD70+ population, suggesting niche-mediated resistance of dormant LSCs. Computational analysis of receptor-ligand interactions between ECs and C/G-AMKL LSCs suggested a prominent role for chemokine/cytokine signaling, integrin signaling, and cell adhesion, which are predicted to support LSC survival within the niche based on enriched downstream target genes. Overall, our study revealed that CD96+CD70+ LSCs may mediate resistance of C/G-AMKL through niche interactions promoting niche-retention and dormancy. Future studies using this platform could facilitate the development of therapeutic strategies to overcome treatment resistance by targeting CD96+CD70+ LSCs in this high-risk subset.