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Abstract: Menin inhibitors that disrupt the menin-MLL interaction hold promise for treating specific acute myeloid leukemia (AML) subtypes, including those with KMT2A rearrangements (KMT2A-r), yet resistance remains a challenge. Here, through systematic chromatin-focused CRISPR screens, along with genetic, epigenetic, and pharmacologic studies in a variety of human and mouse KMT2A-r AML models, we uncovered a potential resistance mechanism independent of canonical menin-MLL targets. We show that a group of noncanonical menin targets, which are bivalently cooccupied by active menin and repressive H2AK119ub marks, are typically downregulated after menin inhibition. Loss of polycomb repressive complex 1.1 (PRC1.1) subunits, such as polycomb group ring finger 1 (PCGF1) or BCL6 corepressor (BCOR), leads to menin inhibitor resistance by epigenetic reactivation of these noncanonical targets, including MYC. Genetic and pharmacological inhibition of MYC can resensitize PRC1.1-deficient leukemia cells to menin inhibition. Moreover, we demonstrate that leukemia cells with the loss of PRC1.1 subunits exhibit reduced monocytic gene signatures and are susceptible to BCL2 inhibition, and that combinational treatment with venetoclax overcomes the resistance to menin inhibition in PRC1.1-deficient leukemia cells. These findings highlight the important roles of PRC1.1 and its regulated noncanonical menin targets in modulating the menin inhibitor response and provide potential strategies to treat leukemia with compromised PRC1.1 function., (© 2024 American Society of Hematology. Published by Elsevier Inc. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Abstract: The interaction between menin and histone-lysine N-methyltransferase 2A (KMT2A) is a critical dependency for KMT2A- or nucleophosmin 1 (NPM1)-altered leukemias and an emerging opportunity for therapeutic development. JNJ-75276617 (bleximenib) is a novel, orally bioavailable, potent, and selective protein-protein interaction inhibitor of the binding between menin and KMT2A. In KMT2A-rearranged (KMT2A-r) and NPM1-mutant (NPM1c) acute myeloid leukemia (AML) cells, JNJ-75276617 inhibited the association of the menin-KMT2A complex with chromatin at target gene promoters, resulting in reduced expression of several menin-KMT2A target genes, including MEIS1 and FLT3. JNJ-75276617 displayed potent antiproliferative activity across several AML and acute lymphoblastic leukemia (ALL) cell lines and patient samples harboring KMT2A or NPM1 alterations in vitro. In xenograft models of AML and ALL, JNJ-75276617 reduced leukemic burden and provided a significant dose-dependent survival benefit accompanied by expression changes of menin-KMT2A target genes. JNJ-75276617 demonstrated synergistic effects with gilteritinib in vitro in AML cells harboring KMT2A-r. JNJ-75276617 further exhibited synergistic effects with venetoclax and azacitidine in AML cells bearing KMT2A-r in vitro, and significantly increased survival in mice. Interestingly, JNJ-75276617 showed potent antiproliferative activity in cell lines engineered with recently discovered mutations (MEN1M327I or MEN1T349M) that developed in patients refractory to the menin-KMT2A inhibitor revumenib. A cocrystal structure of menin in complex with JNJ-75276617 indicates a unique binding mode distinct from other menin-KMT2A inhibitors, including revumenib. JNJ-75276617 is being clinically investigated for acute leukemias harboring KMT2A or NPM1 alterations, as a monotherapy for relapsed/refractory acute leukemia (NCT04811560), or in combination with AML-directed therapies (NCT05453903)., (© 2024 American Society of Hematology. Published by Elsevier Inc. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Abstract: The interplay between T-cell states of differentiation, dysfunction, and treatment response in acute myeloid leukemia (AML) remains unclear. Here, we leveraged a multimodal approach encompassing high-dimensional flow cytometry and single-cell transcriptomics and found that early memory CD8+ T cells are associated with therapy response and exhibit a bifurcation into 2 distinct terminal end states. One state is enriched for markers of activation, whereas the other expresses natural killer (NK)-like and senescence markers. The skewed clonal differentiation trajectory toward CD8+ senescence was also a hallmark indicative of therapy resistance. We validated these findings by generating an AML CD8+ single-cell atlas integrating our data and other independent data sets. Finally, our analysis revealed that an imbalance between CD8+ early memory and senescent-like cells is linked to AML treatment refractoriness and poor survival. Our study provides crucial insights into the dynamics of CD8+ T-cell differentiation and advances our understanding of CD8+ T-cell dysfunction in AML., (© 2024 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Abstract: In acute myeloid leukemia (AML), leukemia stem cells (LSCs) and leukemia progenitor cells (LPCs) interact with various cell types in the bone marrow (BM) microenvironment, regulating their expansion and differentiation. To study the interaction of CD4+ and CD8+ T cells in the BM with LSCs and LPCs, we analyzed their transcriptome and predicted cell-cell interactions by unbiased high-throughput correlation network analysis. We found that CD4+ T cells in the BM of patients with AML were activated and skewed toward T-helper (Th)1 polarization, whereas interleukin-9 (IL-9)-producing (Th9) CD4+ T cells were absent. In contrast to normal hematopoietic stem cells, LSCs produced IL-9, and the correlation modeling predicted IL9 in LSCs as a main hub gene that activates CD4+ T cells in AML. Functional validation revealed that IL-9 receptor signaling in CD4+ T cells leads to activation of the JAK-STAT pathway that induces the upregulation of KMT2A and KMT2C genes, resulting in methylation on histone H3 at lysine 4 to promote genome accessibility and transcriptional activation. This induced Th1-skewing, proliferation, and effector cytokine secretion, including interferon gamma (IFN-γ) and tumor necrosis factor α (TNF-α). IFN-γ and, to a lesser extent, TNF-α produced by activated CD4+ T cells induced the expansion of LSCs. In accordance with our findings, high IL9 expression in LSCs and high IL9R, TNF, and IFNG expression in BM-infiltrating CD4+ T cells correlated with worse overall survival in AML. Thus, IL-9 secreted by AML LSCs shapes a Th1-skewed immune environment that promotes their expansion by secreting IFN-γ and TNF-α., (© 2024 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Abstract: Molecular measurable residual disease can persist in core-binding factor acute myeloid leukemia in otherwise disease-free patients. Utilizing cell sorting followed by fluorescent in situ hybridization, we show that detection is due to mast cells., (© 2024 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Abstract: The overall prognosis of acute myeloid leukemia (AML) remains dismal, largely because of the inability of current therapies to kill leukemia stem cells (LSCs) with intrinsic resistance. Loss of the stress sensor growth arrest and DNA damage-inducible 45 alpha (GADD45A) is implicated in poor clinical outcomes, but its role in LSCs and AML pathogenesis is unknown. Here, we define GADD45A as a key downstream target of G protein-coupled receptor (LGR)4 pathway and discover a regulatory role for GADD45A loss in promoting leukemia-initiating activity and oxidative resistance in LGR4/HOXA9-dependent AML, a poor prognosis subset of leukemia. Knockout of GADD45A enhances AML progression in murine and patient-derived xenograft (PDX) mouse models. Deletion of GADD45A induces substantial mutations, increases LSC self-renewal and stemness in vivo, and reduces levels of reactive oxygen species (ROS), accompanied by a decreased response to ROS-associated genotoxic agents (eg, ferroptosis inducer RSL3) and acquisition of an increasingly aggressive phenotype on serial transplantation in mice. Our single-cell cellular indexing of transcriptomes and epitopes by sequencing analysis on patient-derived LSCs in PDX mice and subsequent functional studies in murine LSCs and primary AML patient cells show that loss of GADD45A is associated with resistance to ferroptosis (an iron-dependent oxidative cell death caused by ROS accumulation) through aberrant activation of antioxidant pathways related to iron and ROS detoxification, such as FTH1 and PRDX1, upregulation of which correlates with unfavorable outcomes in patients with AML. These results reveal a therapy resistance mechanism contributing to poor prognosis and support a role for GADD45A loss as a critical step for leukemia-initiating activity and as a target to overcome resistance in aggressive leukemia., (© 2024 American Society of Hematology. Published by Elsevier Inc. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Abstract: Identifying and targeting microenvironment-driven pathways that are active across acute myeloid leukemia (AML) genetic subtypes should allow the development of more broadly effective therapies. The proinflammatory cytokine interleukin-1β (IL-1β) is abundant in the AML microenvironment and promotes leukemic growth. Through RNA-sequencing analysis, we identify that IL-1β-upregulated ASF1B (antisilencing function-1B), a histone chaperone, in AML progenitors compared with healthy progenitors. ASF1B, along with its paralogous protein ASF1A, recruits H3-H4 histones onto the replication fork during S-phase, a process regulated by Tousled-like kinase 1 and 2 (TLKs). Although ASF1s and TLKs are known to be overexpressed in multiple solid tumors and associated with poor prognosis, their functional roles in hematopoiesis and inflammation-driven leukemia remain unexplored. In this study, we identify that ASF1s and TLKs are overexpressed in multiple genetic subtypes of AML. We demonstrate that depletion of ASF1s significantly reduces leukemic cell growth in both in vitro and in vivo models using human cells. Using a murine model, we show that overexpression of ASF1B accelerates leukemia progression. Moreover, Asf1b or Tlk2 deletion delayed leukemia progression, whereas these proteins are dispensable for normal hematopoiesis. Through proteomics and phosphoproteomics analyses, we uncover that the TLK-ASF1 pathway promotes leukemogenesis by affecting the cell cycle and DNA damage pathways. Collectively, our findings identify the TLK1-ASF1 pathway as a novel mediator of inflammatory signaling and a promising therapeutic target for AML treatment across diverse genetic subtypes. Selective inhibition of this pathway offers potential opportunities to intervene effectively, address intratumoral heterogeneity, and ultimately improve clinical outcomes in AML.

Abstract: Epigenetic modulation of the cell-intrinsic immune response holds promise as a therapeutic approach for leukemia. However, current strategies designed for transcriptional activation of endogenous transposons and subsequent interferon type-I (IFN-I) response, show limited clinical efficacy. Histone lysine methylation is an epigenetic signature in IFN-I response associated with suppression of IFN-I and IFN-stimulated genes, suggesting histone demethylation as key mechanism of reactivation. In this study, we unveil the histone demethylase PHF8 as a direct initiator and regulator of cell-intrinsic immune response in acute myeloid leukemia (AML). Site-specific phosphorylation of PHF8 orchestrates epigenetic changes that upregulate cytosolic RNA sensors, particularly the TRIM25-RIG-I-IFIT5 axis, thereby triggering the cellular IFN-I response-differentiation-apoptosis network. This signaling cascade largely counteracts differentiation block and growth of human AML cells across various disease subtypes in vitro and in vivo. Through proteome analysis of over 200 primary AML bone marrow samples, we identify a distinct PHF8/IFN-I signature in half of the patient population, without significant associations with known clinically or genetically defined AML subgroups. This profile was absent in healthy CD34+ hematopoietic progenitor cells, suggesting therapeutic applicability in a large fraction of patients with AML. Pharmacological support of PHF8 phosphorylation significantly impairs the growth in samples from patients with primary AML. These findings provide novel opportunities for harnessing the cell-intrinsic immune response in the development of immunotherapeutic strategies against AML., (© 2024 American Society of Hematology. Published by Elsevier Inc. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Abstract: The spectrum of myeloid disorders ranges from aplastic bone marrow failure characterized by an empty bone marrow completely lacking in hematopoiesis to acute myeloid leukemia in which the marrow space is replaced by undifferentiated leukemic blasts. Recent advances in the capacity to sequence bulk tumor population as well as at a single-cell level has provided significant insight into the stepwise process of transformation to acute myeloid leukemia. Using models of progression in the context of germ line predisposition (trisomy 21, GATA2 deficiency, and SAMD9/9L syndrome), premalignant states (clonal hematopoiesis and clonal cytopenia of unknown significance), and myelodysplastic syndrome, we review the mechanisms of progression focusing on the hierarchy of clonal mutation and potential roles of transcription factor alterations, splicing factor mutations, and the bone marrow environment in progression to acute myeloid leukemia. Despite major advances in our understanding, preventing the progression of these disorders or treating them at the acute leukemia phase remains a major area of unmet medical need., (© 2024 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Abstract: BRG1 (SMARCA4) and BRM (SMARCA2) are the mutually exclusive core ATPases of the chromatin remodeling BAF (BRG1/BRM-associated factor) complexes. They enable transcription factors/cofactors to access enhancers/promoter and modulate gene expressions responsible for cell growth and differentiation of acute myeloid leukemia (AML) stem/progenitor cells. In AML with MLL1 rearrangement (MLL1r) or mutant NPM1 (mtNPM1), although menin inhibitor (MI) treatment induces clinical remissions, most patients either fail to respond or relapse, some harboring menin mutations. FHD-286 is an orally bioavailable, selective inhibitor of BRG1/BRM under clinical development in AML. Present studies show that FHD-286 induces differentiation and lethality in AML cells with MLL1r or mtNPM1, concomitantly causing perturbed chromatin accessibility and repression of c-Myc, PU.1, and CDK4/6. Cotreatment with FHD-286 and decitabine, BET inhibitor (BETi) or MI, or venetoclax synergistically induced in vitro lethality in AML cells with MLL1r or mtNPM1. In models of xenografts derived from patients with AML with MLL1r or mtNPM1, FHD-286 treatment reduced AML burden, improved survival, and attenuated AML-initiating potential of stem-progenitor cells. Compared with each drug, cotreatment with FHD-286 and BETi, MI, decitabine, or venetoclax significantly reduced AML burden and improved survival, without inducing significant toxicity. These findings highlight the FHD-286-based combinations as a promising therapy for AML with MLL1r or mtNPM1., (© 2024 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Abstract: Acute myeloid leukemia (AML) is an aggressive hematological malignancy originating from transformed hematopoietic stem or progenitor cells. AML prognosis remains poor owing to resistance and relapse driven by leukemia stem cells (LSCs). Targeting molecules essential for LSC function is a promising therapeutic approach. The phosphatidylinositol 3-kinase (PI3K)/AKT pathway is often dysregulated in AML. We found that although PI3Kγ is highly enriched in LSCs and critical for self-renewal, it was dispensable for normal hematopoietic stem cells. Mechanistically, PI3Kγ-AKT signaling promotes nuclear factor erythroid 2-related factor 2 (NRF2) nuclear accumulation, which induces 6-phosphogluconate dehydrogenase (PGD) and the pentose phosphate pathway, thereby maintaining LSC stemness. Importantly, genetic or pharmacological inhibition of PI3Kγ impaired expansion and stemness of murine and human AML cells in vitro and in vivo. Together, our findings reveal a key role for PI3Kγ in selectively maintaining LSC function by regulating AKT-NRF2-PGD metabolic pathway. Targeting the PI3Kγ pathway may, therefore, eliminate LSCs without damaging normal hematopoiesis, providing a promising therapeutic strategy for AML., (© 2024 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Abstract: UBTF tandem duplications (UBTF-TDs) have recently emerged as a recurrent alteration in pediatric and adult acute myeloid leukemia (AML). UBTF-TD leukemias are characterized by a poor response to conventional chemotherapy and a transcriptional signature that mirrors NUP98-rearranged and NPM1-mutant AMLs, including HOX-gene dysregulation. However, the mechanism by which UBTF-TD drives leukemogenesis remains unknown. In this study, we investigated the genomic occupancy of UBTF-TD in transformed cord blood CD34+ cells and patient-derived xenograft models. We found that UBTF-TD protein maintained genomic occupancy at ribosomal DNA loci while also occupying genomic targets commonly dysregulated in UBTF-TD myeloid malignancies, such as the HOXA/HOXB gene clusters and MEIS1. These data suggest that UBTF-TD is a gain-of-function alteration that results in mislocalization to genomic loci dysregulated in UBTF-TD leukemias. UBTF-TD also co-occupies key genomic loci with KMT2A and menin, which are known to be key partners involved in HOX-dysregulated leukemias. Using a protein degradation system, we showed that stemness, proliferation, and transcriptional signatures are dependent on sustained UBTF-TD localization to chromatin. Finally, we demonstrate that primary cells from UBTF-TD leukemias are sensitive to the menin inhibitor SNDX-5613, resulting in markedly reduced in vitro and in vivo tumor growth, myeloid differentiation, and abrogation of the UBTF-TD leukemic expression signature. These findings provide a viable therapeutic strategy for patients with this high-risk AML subtype., (© 2024 American Society of Hematology. Published by Elsevier Inc. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Abstract: Chimeric antigen receptor T-cell (CAR T) therapy has produced remarkable clinical responses in B-cell neoplasms. However, many challenges limit this class of agents for the treatment of other cancer types, in particular the lack of tumor-selective antigens for solid tumors and other hematological malignancies, such as acute myeloid leukemia (AML), which may be addressed without significant risk of severe toxicities while providing sufficient abundance for efficient tumor suppression. One approach to overcome this hurdle is dual targeting by an antibody-T-cell receptor (AbTCR) and a chimeric costimulatory signaling receptor (CSR) to 2 different antigens, in which both antigens are found together on the cancer cells but not together on normal cells. To explore this proof of concept in AML, we engineered a new T-cell format targeting Wilms tumor 1 protein (WT1) and CD33; both are highly expressed on most AML cells. Using an AbTCR comprising a newly developed TCR-mimic monoclonal antibody against the WT1 RMFPNAPYL (RMF) epitope/HLA-A2 complex, ESK2, and a secondary CSR comprising a single-chain variable fragment directed to CD33 linked to a truncated CD28 costimulatory fragment, this unique platform confers specific T-cell cytotoxicity to the AML cells while sparing healthy hematopoietic cells, including CD33+ myelomonocytic normal cells. These data suggest that this new platform, named AbTCR-CSR, through the combination of a AbTCR CAR and CSR could be an effective strategy to reduce toxicity and improve specificity and clinical outcomes in adoptive T-cell therapy in AML., (© 2024 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Abstract: Myelodysplastic syndromes (MDSs) are neoplastic myeloid proliferations characterized by ineffective hematopoiesis resulting in peripheral blood cytopenias. MDS is distinguished from nonneoplastic clonal myeloid proliferations by the presence of morphologic dysplasia and from acute myeloid leukemia by a blast threshold of 20%. The diagnosis of MDS can be challenging because of the myriad other causes of cytopenias: accurate diagnosis requires the integration of clinical features with bone marrow and peripheral blood morphology, immunophenotyping, and genetic testing. MDS has historically been subdivided into several subtypes by classification schemes, the most recent of which are the International Consensus Classification and World Health Organization Classification (fifth edition), both published in 2022. The aim of MDS classification is to identify entities with shared genetic underpinnings and molecular pathogenesis, and the specific subtype can inform clinical decision-making alongside prognostic risk categorization. The current MDS classification schemes incorporate morphologic features (bone marrow and blood blast percentage, degree of dysplasia, ring sideroblasts, bone marrow fibrosis, and bone marrow hypocellularity) and also recognize 3 entities defined by genetics: isolated del(5q) cytogenetic abnormality, SF3B1 mutation, and TP53 mutation. It is anticipated that with advancing understanding of the genetic basis of MDS pathogenesis, future MDS classification will be based increasingly on genetic classes. Nevertheless, morphologic features in MDS reflect the phenotypic expression of the underlying abnormal genetic pathways and will undoubtedly retain importance to inform prognosis and guide treatment., (© 2023 by The American Society of Hematology.)

Acute myeloid leukemia (AML) with TP53 mutation is one of the most lethal cancers and portends an extremely poor prognosis. Based on in silico analyses of druggable genes and differential gene expression in TP53-mutated AML, we identified pololike kinase 4 (PLK4) as a novel therapeutic target and examined its expression, regulation, pathogenetic mechanisms, and therapeutic potential in TP53-mutated AML. PLK4 expression was suppressed by activated p53 signaling in TP53 wild-type AML and was increased in TP53-mutated AML cell lines and primary samples. Short-term PLK4 inhibition induced DNA damage and apoptosis in TP53 wild-type AML. Prolonged PLK4 inhibition suppressed the growth of TP53-mutated AML and was associated with DNA damage, apoptosis, senescence, polyploidy, and defective cytokinesis. A hitherto undescribed PLK4/PRMT5/EZH2/H3K27me3 axis was demonstrated in both TP53 wild-type and mutated AML, resulting in histone modification through PLK4-induced PRMT5 phosphorylation. In TP53-mutated AML, combined effects of histone modification and polyploidy activated the cGAS-STING pathway, leading to secretion of cytokines and chemokines and activation of macrophages and T cells upon coculture with AML cells. In vivo, PLK4 inhibition also induced cytokine and chemokine expression in mouse recipients, and its combination with anti-CD47 antibody, which inhibited the "don't-eat-me" signal in macrophages, synergistically reduced leukemic burden and prolonged animal survival. The study shed important light on the pathogenetic role of PLK4 and might lead to novel therapeutic strategies in TP53-mutated AML., (© 2023 by The American Society of Hematology.)

TP 53-mutant acute myeloid leukemia (AML) remains the ultimate therapeutic challenge. Epichaperomes, formed in malignant cells, consist of heat shock protein 90 (HSP90) and associated proteins that support the maturation, activity, and stability of oncogenic kinases and transcription factors including mutant p53. High-throughput drug screening identified HSP90 inhibitors as top hits in isogenic TP53-wild-type (WT) and -mutant AML cells. We detected epichaperomes in AML cells and stem/progenitor cells with TP53 mutations but not in healthy bone marrow (BM) cells. Hence, we investigated the therapeutic potential of specifically targeting epichaperomes with PU-H71 in TP53-mutant AML based on its preferred binding to HSP90 within epichaperomes. PU-H71 effectively suppressed cell intrinsic stress responses and killed AML cells, primarily by inducing apoptosis; targeted TP53-mutant stem/progenitor cells; and prolonged survival of TP53-mutant AML xenograft and patient-derived xenograft models, but it had minimal effects on healthy human BM CD34+ cells or on murine hematopoiesis. PU-H71 decreased MCL-1 and multiple signal proteins, increased proapoptotic Bcl-2-like protein 11 levels, and synergized with BCL-2 inhibitor venetoclax in TP53-mutant AML. Notably, PU-H71 effectively killed TP53-WT and -mutant cells in isogenic TP53-WT/TP53-R248W Molm13 cell mixtures, whereas MDM2 or BCL-2 inhibition only reduced TP53-WT but favored the outgrowth of TP53-mutant cells. Venetoclax enhanced the killing of both TP53-WT and -mutant cells by PU-H71 in a xenograft model. Our data suggest that epichaperome function is essential for TP53-mutant AML growth and survival and that its inhibition targets mutant AML and stem/progenitor cells, enhances venetoclax activity, and prevents the outgrowth of venetoclax-resistant TP53-mutant AML clones. These concepts warrant clinical evaluation., (© 2023 by The American Society of Hematology.)

The bone marrow microenvironment (BMM) can regulate leukemia stem cells (LSCs) via secreted factors. Increasing evidence suggests that dissecting the mechanisms by which the BMM maintains LSCs may lead to the development of effective therapies for the eradication of leukemia. Inhibitor of DNA binding 1 (ID1), a key transcriptional regulator in LSCs, previously identified by us, controls cytokine production in the BMM, but the role of ID1 in acute myeloid leukemia (AML) BMM remains obscure. Here, we report that ID1 is highly expressed in the BMM of patients with AML, especially in BM mesenchymal stem cells, and that the high expression of ID1 in the AML BMM is induced by BMP6, secreted from AML cells. Knocking out ID1 in mesenchymal cells significantly suppresses the proliferation of cocultured AML cells. Loss of Id1 in the BMM results in impaired AML progression in AML mouse models. Mechanistically, we found that Id1 deficiency significantly reduces SP1 protein levels in mesenchymal cells cocultured with AML cells. Using ID1-interactome analysis, we found that ID1 interacts with RNF4, an E3 ubiquitin ligase, and causes a decrease in SP1 ubiquitination. Disrupting the ID1-RNF4 interaction via truncation in mesenchymal cells significantly reduces SP1 protein levels and delays AML cell proliferation. We identify that the target of Sp1, Angptl7, is the primary differentially expression protein factor in Id1-deficient BM supernatant fluid to regulate AML progression in mice. Our study highlights the critical role of ID1 in the AML BMM and aids the development of therapeutic strategies for AML., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

RNA-binding proteins (RBPs) form a large and diverse class of factors, many members of which are overexpressed in hematologic malignancies. RBPs participate in various processes of messenger RNA (mRNA) metabolism and prevent harmful DNA:RNA hybrids or R-loops. Here, we report that PIWIL4, a germ stem cell-associated RBP belonging to the RNase H-like superfamily, is overexpressed in patients with acute myeloid leukemia (AML) and is essential for leukemic stem cell function and AML growth, but dispensable for healthy human hematopoietic stem cells. In AML cells, PIWIL4 binds to a small number of known piwi-interacting RNA. Instead, it largely interacts with mRNA annotated to protein-coding genic regions and enhancers that are enriched for genes associated with cancer and human myeloid progenitor gene signatures. PIWIL4 depletion in AML cells downregulates the human myeloid progenitor signature and leukemia stem cell (LSC)-associated genes and upregulates DNA damage signaling. We demonstrate that PIWIL4 is an R-loop resolving enzyme that prevents R-loop accumulation on a subset of AML and LSC-associated genes and maintains their expression. It also prevents DNA damage, replication stress, and activation of the ATR pathway in AML cells. PIWIL4 depletion potentiates sensitivity to pharmacological inhibition of the ATR pathway and creates a pharmacologically actionable dependency in AML cells., (© 2023 by The American Society of Hematology.)

Patients treated with cytotoxic therapies, including autologous stem cell transplantation, are at risk for developing therapy-related myeloid neoplasms (tMN). Preleukemic clones (ie, clonal hematopoiesis [CH]) are detectable years before the development of these aggressive malignancies, although the genomic events leading to transformation and expansion are not well defined. Here, by leveraging distinctive chemotherapy-associated mutational signatures from whole-genome sequencing data and targeted sequencing of prechemotherapy samples, we reconstructed the evolutionary life-history of 39 therapy-related myeloid malignancies. A dichotomy was revealed, in which neoplasms with evidence of chemotherapy-induced mutagenesis from platinum and melphalan were hypermutated and enriched for complex structural variants (ie, chromothripsis), whereas neoplasms with nonmutagenic chemotherapy exposures were genomically similar to de novo acute myeloid leukemia. Using chemotherapy-associated mutational signatures as temporal barcodes linked to discrete clinical exposure in each patient's life, we estimated that several complex events and genomic drivers were acquired after chemotherapy was administered. For patients with prior multiple myeloma who were treated with high-dose melphalan and autologous stem cell transplantation, we demonstrate that tMN can develop from either a reinfused CH clone that escapes melphalan exposure and is selected after reinfusion, or from TP53-mutant CH that survives direct myeloablative conditioning and acquires melphalan-induced DNA damage. Overall, we revealed a novel mode of tMN progression that is not reliant on direct mutagenesis or even exposure to chemotherapy. Conversely, for tMN that evolve under the influence of chemotherapy-induced mutagenesis, distinct chemotherapies not only select preexisting CH but also promote the acquisition of recurrent genomic drivers., (© 2023 by The American Society of Hematology.)

Activation of apoptosis in malignant cells is an established strategy for controlling cancer and is potentially curative. To assess the impact of concurrently inducing the extrinsic and intrinsic apoptosis-signaling pathways in acute myeloid leukemia (AML), we evaluated activity of the TRAIL receptor agonistic fusion protein eftozanermin alfa (eftoza; ABBV-621) in combination with the B-cell lymphoma protein-2 selective inhibitor venetoclax in preclinical models and human patients. Simultaneously stimulating intrinsic and extrinsic apoptosis-signaling pathways with venetoclax and eftoza, respectively, enhanced their activities in AML cell lines and patient-derived ex vivo/in vivo models. Eftoza activity alone or plus venetoclax required death receptor 4/5 (DR4/DR5) expression on the plasma membrane but was independent of TP53 or FLT3-ITD status. The safety/tolerability of eftoza as monotherapy and in combination with venetoclax was demonstrated in patients with relapsed/refractory AML in a phase 1 clinical trial. Treatment-related adverse events were reported in 2 of 4 (50%) patients treated with eftoza monotherapy and 18 of 23 (78%) treated with eftoza plus venetoclax. An overall response rate of 30% (7/23; 4 complete responses [CRs], 2 CRs with incomplete hematologic recovery, and 1 morphologic leukemia-free state) was reported in patients who received treatment with eftoza plus venetoclax and 67% (4/6) in patients with myoblasts positive for DR4/DR5 expression; no tumor responses were observed with eftoza monotherapy. These data indicate that combination therapy with eftoza plus venetoclax to simultaneously activate the extrinsic and intrinsic apoptosis-signaling pathways may improve clinical benefit compared with venetoclax monotherapy in relapsed/refractory AML with an acceptable toxicity profile. This trial was registered at www.clinicaltrials.gov as #NCT03082209., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

The challenge of eradicating leukemia in patients with acute myelogenous leukemia (AML) after initial cytoreduction has motivated modern efforts to combine synergistic active modalities including immunotherapy. Recently, the ETCTN/CTEP 10026 study tested the combination of the DNA methyltransferase inhibitor decitabine together with the immune checkpoint inhibitor ipilimumab for AML/myelodysplastic syndrome (MDS) either after allogeneic hematopoietic stem cell transplantation (HSCT) or in the HSCT-naïve setting. Integrative transcriptome-based analysis of 304 961 individual marrow-infiltrating cells for 18 of 48 subjects treated on study revealed the strong association of response with a high baseline ratio of T to AML cells. Clinical responses were predominantly driven by decitabine-induced cytoreduction. Evidence of immune activation was only apparent after ipilimumab exposure, which altered CD4+ T-cell gene expression, in line with ongoing T-cell differentiation and increased frequency of marrow-infiltrating regulatory T cells. For post-HSCT samples, relapse could be attributed to insufficient clearing of malignant clones in progenitor cell populations. In contrast to AML/MDS bone marrow, the transcriptomes of leukemia cutis samples from patients with durable remission after ipilimumab monotherapy showed evidence of increased infiltration with antigen-experienced resident memory T cells and higher expression of CTLA-4 and FOXP3. Altogether, activity of combined decitabine and ipilimumab is impacted by cellular expression states within the microenvironmental niche of leukemic cells. The inadequate elimination of leukemic progenitors mandates urgent development of novel approaches for targeting these cell populations to generate long-lasting responses. This trial was registered at www.clinicaltrials.gov as #NCT02890329., (© 2023 by The American Society of Hematology.)

T-cell acute lymphoblastic leukemia (T-ALL) is a T-cell malignancy characterized by cell subsets and enriched with leukemia-initiating cells (LICs). β-Catenin modulates LIC activity in T-ALL. However, its role in maintaining established leukemia stem cells remains largely unknown. To identify functionally relevant protein interactions of β-catenin in T-ALL, we performed coimmunoprecipitation followed by liquid chromatography-mass spectrometry. Here, we report that a noncanonical functional interaction of β-catenin with the Forkhead box O3 (FOXO3) transcription factor positively regulates LIC-related genes, including the cyclin-dependent kinase 4, which is a crucial modulator of cell cycle and tumor maintenance. We also confirm the relevance of these findings using stably integrated fluorescent reporters of β-catenin and FOXO3 activity in patient-derived xenografts, which identify minor subpopulations with enriched LIC activity. In addition, gene expression data at the single-cell level of leukemic cells of primary patients at the time of diagnosis and minimal residual disease (MRD) up to 30 days after the standard treatments reveal that the expression of β-catenin- and FOXO3-dependent genes is present in the CD82+CD117+ cell fraction, which is substantially enriched with LICs in MRD as well as in early T-cell precursor ALL. These findings highlight key functional roles for β-catenin and FOXO3 and suggest novel therapeutic strategies to eradicate aggressive cell subsets in T-ALL., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Fms-like tyrosine kinase 3 (FLT3) is often overexpressed or constitutively activated by internal tandem duplication (ITD) and tyrosine kinase domain (TKD) mutations in acute myeloid leukemia (AML). Despite the use of receptor tyrosine kinase inhibitors (TKI) in FLT3-ITD-positive AML, the prognosis of patients is still poor, and further improvement of therapy is required. Targeting FLT3 independent of mutations by antibody-drug conjugates (ADCs) is a promising strategy for AML therapy. Here, we report the development and preclinical characterization of a novel FLT3-targeting ADC, 20D9-ADC, which was generated by applying the innovative P5 conjugation technology. In vitro, 20D9-ADC mediated potent cytotoxicity to Ba/F3 cells expressing transgenic FLT3 or FLT3-ITD, to AML cell lines, and to FLT3-ITD-positive patient-derived xenograft AML cells. In vivo, 20D9-ADC treatment led to a significant tumor reduction and even durable complete remission in AML xenograft models. Furthermore, 20D9-ADC demonstrated no severe hematotoxicity in in vitro colony formation assays using concentrations that were cytotoxic in AML cell line treatment. The combination of 20D9-ADC with the TKI midostaurin showed strong synergy in vitro and in vivo, leading to reduction of aggressive AML cells below the detection limit. Our data indicate that targeting FLT3 with an advanced new-generation ADC is a promising and potent antileukemic strategy, especially when combined with FLT3-TKI in FLT3-ITD-positive AML., (© 2023 by The American Society of Hematology.)

Germ line loss-of-function heterozygous mutations in the RUNX1 gene cause familial platelet disorder with associated myeloid malignancies (FPDMM) characterized by thrombocytopenia and a life-long risk of hematological malignancies. Although gene therapies are being considered as promising therapeutic options, current preclinical models do not recapitulate the human phenotype and are unable to elucidate the relative fitness of mutation-corrected and RUNX1-heterozygous mutant hematopoietic stem and progenitor cells (HSPCs) in vivo long term. We generated a rhesus macaque with an FPDMM competitive repopulation model using CRISPR/Cas9 nonhomologous end joining editing in the RUNX1 gene and the AAVS1 safe-harbor control locus. We transplanted mixed populations of edited autologous HSPCs and tracked mutated allele frequencies in blood cells. In both animals, RUNX1-edited cells expanded over time compared with AAVS1-edited cells. Platelet counts remained below the normal range in the long term. Bone marrows developed megakaryocytic dysplasia similar to human FPDMM, and CD34+ HSPCs showed impaired in vitro megakaryocytic differentiation, with a striking defect in polyploidization. In conclusion, the lack of a competitive advantage for wildtype or control-edited HSPCs over RUNX1 heterozygous-mutated HSPCs long term in our preclinical model suggests that gene correction approaches for FPDMM will be challenging, particularly to reverse myelodysplastic syndrome/ acute myeloid leukemia predisposition and thrombopoietic defects.

Relapse is a leading cause of death after allogeneic hematopoietic stem cell transplantation (allo-HSCT) for acute myeloid leukemia (AML). However, the underlying mechanisms remain poorly understood. Natural killer (NK) cells play a crucial role in tumor surveillance and cancer immunotherapy, and NK cell dysfunction has been observed in various tumors. Here, we performed ex vivo experiments to systematically characterize the mechanisms underlying the dysfunction of bone marrow-derived NK (BMNK) cells isolated from AML patients experiencing early relapse after allo-HSCT. We demonstrated that higher levels of active transforming growth factor β1 (TGF-β1) were associated with impaired effector function of BMNK cells in these AML patients. TGF-β1 activation was induced by the overexpression of glycoprotein A repetitions predominant on the surface of CD4+ T cells. Active TGF-β1 significantly suppressed mTORC1 activity, mitochondrial oxidative phosphorylation, the proliferation, and cytotoxicity of BMNK cells. Furthermore, pretreatment with the clinical stage TGF-β1 pathway inhibitor, galunisertib, significantly restored mTORC1 activity, mitochondrial homeostasis, and cytotoxicity. Importantly, the blockade of the TGF-β1 signaling improved the antitumor activity of NK cells in a leukemia xenograft mouse model. Thus, our findings reveal a mechanism explaining BMNK cell dysfunction and suggest that targeted inhibition of TGF-β1 signaling may represent a potential therapeutic intervention to improve outcomes in AML patients undergoing allo-HSCT or NK cell-based immunotherapy., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Targeting altered tumor cell metabolism might provide an attractive opportunity for patients with acute myeloid leukemia (AML). An amino acid dropout screen on primary leukemic stem cells and progenitor populations revealed a number of amino acid dependencies, of which methionine was one of the strongest. By using various metabolite rescue experiments, nuclear magnetic resonance-based metabolite quantifications and 13C-tracing, polysomal profiling, and chromatin immunoprecipitation sequencing, we identified that methionine is used predominantly for protein translation and to provide methyl groups to histones via S-adenosylmethionine for epigenetic marking. H3K36me3 was consistently the most heavily impacted mark following loss of methionine. Methionine depletion also reduced total RNA levels, enhanced apoptosis, and induced a cell cycle block. Reactive oxygen species levels were not increased following methionine depletion, and replacement of methionine with glutathione or N-acetylcysteine could not rescue phenotypes, excluding a role for methionine in controlling redox balance control in AML. Although considered to be an essential amino acid, methionine can be recycled from homocysteine. We uncovered that this is primarily performed by the enzyme methionine synthase and only when methionine availability becomes limiting. In vivo, dietary methionine starvation was not only tolerated by mice, but also significantly delayed both cell line and patient-derived AML progression. Finally, we show that inhibition of the H3K36-specific methyltransferase SETD2 phenocopies much of the cytotoxic effects of methionine depletion, providing a more targeted therapeutic approach. In conclusion, we show that methionine depletion is a vulnerability in AML that can be exploited therapeutically, and we provide mechanistic insight into how cells metabolize and recycle methionine., (© 2022 by The American Society of Hematology.)

We have developed a deep-scale proteome and phosphoproteome database from 44 representative acute myeloid leukemia (AML) patients from the LAML TCGA dataset and 6 healthy bone marrow-derived controls. After confirming data quality, we orthogonally validated several previously undescribed features of AML revealed by the proteomic data. We identified examples of posttranscriptionally regulated proteins both globally (ie, in all AML samples) and also in patients with recurrent AML driver mutations. For example, samples with IDH1/2 mutations displayed elevated levels of the 2-oxoglutarate-dependent histone demethylases KDM4A/B/C, despite no changes in messenger RNA levels for these genes; we confirmed this finding in vitro. In samples with NPMc mutations, we identified several nuclear importins with posttranscriptionally increased protein abundance and showed that they interact with NPMc but not wild-type NPM1. We identified 2 cell surface proteins (CD180 and MRC1/CD206) expressed on AML blasts of many patients (but not healthy CD34+ stem/progenitor cells) that could represent novel targets for immunologic therapies and confirmed these targets via flow cytometry. Finally, we detected nearly 30 000 phosphosites in these samples; globally, AML samples were associated with the abnormal phosphorylation of specific residues in PTPN11, STAT3, AKT1, and PRKCD. FLT3-TKD samples were associated with increased phosphorylation of activating tyrosines on the cytoplasmic Src-family tyrosine kinases FGR and HCK and related signaling proteins. PML-RARA-initiated AML samples displayed a unique phosphorylation signature, and TP53-mutant samples showed abundant phosphorylation of serine-183 on TP53 itself. This publicly available database will serve as a foundation for further investigations of protein dysregulation in AML pathogenesis., (© 2022 by The American Society of Hematology.)

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the most effective treatment for selected patients with acute myeloid leukemia (AML) and relies on a "graft-versus-leukemia" effect (GVL) where donor T lymphocytes mediate control of malignant cell growth. However, relapse remains the major cause of death after allo-HSCT. In various malignancies, several immunoregulatory mechanisms have been shown to restrain antitumor immunity, including ligand-mediated engagement of inhibitory receptors (IRs) on effector cells, and induction of immunosuppressive cell subsets, such as regulatory T cells (Tregs) or myeloid-derived suppressor cells (MDSCs). Relapse after HSCT remains a major therapeutic challenge, but immunoregulatory mechanisms involved in restraining the GVL effect must be better deciphered in humans. We used mass cytometry to comprehensively characterize circulating leukocytes in 2 cohorts of patients after allo-HSCT. We first longitudinally assessed various immunoregulatory parameters highlighting specific trends, such as opposite dynamics between MDSCs and Tregs. More generally, the immune landscape was stable from months 3 to 6, whereas many variations occurred from months 6 to 12 after HSCT. Comparison with healthy individuals revealed that profound alterations in the immune equilibrium persisted 1 year after HSCT. Importantly, we found that high levels of TIGIT and CD161 expression on CD4 T cells at month 3 after HSCT were distinct features significantly associated with subsequent AML relapse in a second cross-sectional cohort. Altogether, these data provide global insights into the reconstitution of the immunoregulatory landscape after HSCT and highlight non-canonical IRs associated with relapse, which could open the path to new prognostic tools or therapeutic targets to restore subverted anti-AML immunity., (© 2022 by The American Society of Hematology.)

Detailed genomic and epigenomic analyses of MECOM (the MDS1 and EVI1 complex locus) have revealed that inversion or translocation of chromosome 3 drives inv(3)/t(3;3) myeloid leukemias via structural rearrangement of an enhancer that upregulates transcription of EVI1. Here, we identify a novel, previously unannotated oncogenic RNA-splicing derived isoform of EVI1 that is frequently present in inv(3)/t(3;3) acute myeloid leukemia (AML) and directly contributes to leukemic transformation. This EVI1 isoform is generated by oncogenic mutations in the core RNA splicing factor SF3B1, which is mutated in >30% of inv(3)/t(3;3) myeloid neoplasm patients and thereby represents the single most commonly cooccurring genomic alteration in inv(3)/t(3;3) patients. SF3B1 mutations are statistically uniquely enriched in inv(3)/t(3;3) myeloid neoplasm patients and patient-derived cell lines compared with other forms of AML and promote mis-splicing of EVI1 generating an in-frame insertion of 6 amino acids at the 3' end of the second zinc finger domain of EVI1. Expression of this EVI1 splice variant enhanced the self-renewal of hematopoietic stem cells, and introduction of mutant SF3B1 in mice bearing the humanized inv(3)(q21q26) allele resulted in generation of this novel EVI1 isoform in mice and hastened leukemogenesis in vivo. The mutant SF3B1 spliceosome depends upon an exonic splicing enhancer within EVI1 exon 13 to promote usage of a cryptic branch point and aberrant 3' splice site within intron 12 resulting in the generation of this isoform. These data provide a mechanistic basis for the frequent cooccurrence of SF3B1 mutations as well as new insights into the pathogenesis of myeloid leukemias harboring inv(3)/t(3;3)., (© 2022 by The American Society of Hematology.)

Mutations in chromatin regulator ASXL1 are frequently identified in myeloid malignancies, in particular ∼40% of patients with chronic myelomonocytic leukemia (CMML). ASXL1 mutations are associated with poor prognosis in CMML and significantly co-occur with NRAS mutations. Here, we show that concurrent ASXL1 and NRAS mutations defined a population of CMML patients who had shorter leukemia-free survival than those with ASXL1 mutation only. Corroborating this human data, Asxl1-/- accelerated CMML progression and promoted CMML transformation to acute myeloid leukemia (AML) in NrasG12D/+ mice. NrasG12D/+;Asxl1-/- (NA) leukemia cells displayed hyperactivation of MEK/ERK signaling, increased global levels of H3K27ac, upregulation of Flt3. Moreover, we find that NA-AML cells overexpressed all the major inhibitory immune checkpoint ligands: programmed death-ligand 1 (PD-L1)/PD-L2, CD155, and CD80/CD86. Among them, overexpression of PD-L1 and CD86 correlated with upregulation of AP-1 transcription factors (TFs) in NA-AML cells. An AP-1 inhibitor or short hairpin RNAs against AP-1 TF Jun decreased PD-L1 and CD86 expression in NA-AML cells. Once NA-AML cells were transplanted into syngeneic recipients, NA-derived T cells were not detectable. Host-derived wild-type T cells overexpressed programmed cell death protein 1 (PD-1) and T-cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT) receptors, leading to a predominant exhausted T-cell phenotype. Combined inhibition of MEK and BET resulted in downregulation of Flt3 and AP-1 expression, partial restoration of the immune microenvironment, enhancement of CD8 T-cell cytotoxicity, and prolonged survival in NA-AML mice. Our study suggests that combined targeted therapy and immunotherapy may be beneficial for treating secondary AML with concurrent ASXL1 and NRAS mutations., (© 2022 by The American Society of Hematology.)

In an effort to identify novel drugs targeting fusion-oncogene-induced acute myeloid leukemia (AML), we performed high-resolution proteomic analysis. In AML1-ETO (AE)-driven AML, we uncovered a deregulation of phospholipase C (PLC) signaling. We identified PLCgamma 1 (PLCG1) as a specific target of the AE fusion protein that is induced after AE binding to intergenic regulatory DNA elements. Genetic inactivation of PLCG1 in murine and human AML inhibited AML1-ETO dependent self-renewal programs, leukemic proliferation, and leukemia maintenance in vivo. In contrast, PLCG1 was dispensable for normal hematopoietic stem and progenitor cell function. These findings are extended to and confirmed by pharmacologic perturbation of Ca++-signaling in AML1-ETO AML cells, indicating that the PLCG1 pathway poses an important therapeutic target for AML1-ETO+ leukemic stem cells., (© 2022 by The American Society of Hematology.)

Acute myeloid leukemia (AML) is a clonal hematopoietic stem and progenitor cell malignancy characterized by poor clinical outcomes. Major histocompatibility complex class I polypeptide-related sequence A and B (MICA/B) are stress proteins expressed by cancer cells, and antibody-mediated inhibition of MICA/B shedding represents a novel approach to stimulate immunity against cancers. We found that the MICA/B antibody 7C6 potently inhibits the outgrowth of AML in 2 models in immunocompetent mice. Macrophages were essential for therapeutic efficacy, and 7C6 triggered antibody-dependent phagocytosis of AML cells. Furthermore, we found that romidepsin, a selective histone deacetylase inhibitor, increased MICB messenger RNA in AML cells and enabled subsequent stabilization of the translated protein by 7C6. This drug combination substantially increased surface MICA/B expression in a human AML line, pluripotent stem cell-derived AML blasts and leukemia stem cells, as well as primary cells from 3 untreated patients with AML. Human macrophages phagocytosed AML cells following treatment with 7C6 and romidepsin, and the combination therapy lowered leukemia burden in a humanized model of AML. Therefore, inhibition of MICA/B shedding promotes macrophage-driven immunity against AML via Fc receptor signaling and synergizes with an epigenetic regulator. These results provide the rationale for the clinical testing of this innovative immunotherapeutic approach for the treatment of AML., (© 2022 by The American Society of Hematology.)

Changes in gene regulation and expression govern orderly transitions from hematopoietic stem cells to terminally differentiated blood cell types. These transitions are disrupted during leukemic transformation, but knowledge of the gene regulatory changes underpinning this process is elusive. We hypothesized that identifying core gene regulatory networks in healthy hematopoietic and leukemic cells could provide insights into network alterations that perturb cell state transitions. A heptad of transcription factors (LYL1, TAL1, LMO2, FLI1, ERG, GATA2, and RUNX1) bind key hematopoietic genes in human CD34+ hematopoietic stem and progenitor cells (HSPCs) and have prognostic significance in acute myeloid leukemia (AML). These factors also form a densely interconnected circuit by binding combinatorially at their own, and each other's, regulatory elements. However, their mutual regulation during normal hematopoiesis and in AML cells, and how perturbation of their expression levels influences cell fate decisions remains unclear. In this study, we integrated bulk and single-cell data and found that the fully connected heptad circuit identified in healthy HSPCs persists, with only minor alterations in AML, and that chromatin accessibility at key heptad regulatory elements was predictive of cell identity in both healthy progenitors and leukemic cells. The heptad factors GATA2, TAL1, and ERG formed an integrated subcircuit that regulates stem cell-to-erythroid transition in both healthy and leukemic cells. Components of this triad could be manipulated to facilitate erythroid transition providing a proof of concept that such regulatory circuits can be harnessed to promote specific cell-type transitions and overcome dysregulated hematopoiesis., (© 2021 by The American Society of Hematology.)

Acute leukemias (ALs) of ambiguous lineage are a heterogeneous group of high-risk leukemias characterized by coexpression of myeloid and lymphoid markers. In this study, we identified a distinct subgroup of immature acute leukemias characterized by a broadly variable phenotype, covering acute myeloid leukemia (AML, M0 or M1), T/myeloid mixed-phenotype acute leukemia (T/M MPAL), and early T-cell precursor acute lymphoblastic leukemia (ETP-ALL). Rearrangements at 14q32/BCL11B are the cytogenetic hallmark of this entity. In our screening of 915 hematological malignancies, there were 202 AML and 333 T-cell acute lymphoblastic leukemias (T-ALL: 58, ETP; 178, non-ETP; 8, T/M MPAL; 89, not otherwise specified). We identified 20 cases of immature leukemias (4% of AML and 3.6% of T-ALL), harboring 4 types of 14q32/BCL11B translocations: t(2,14)(q22.3;q32) (n = 7), t(6;14)(q25.3;q32) (n = 9), t(7;14)(q21.2;q32) (n = 2), and t(8;14)(q24.2;q32) (n = 2). The t(2;14) produced a ZEB2-BCL11B fusion transcript, whereas the other 3 rearrangements displaced transcriptionally active enhancer sequences close to BCL11B without producing fusion genes. All translocations resulted in the activation of BCL11B, a regulator of T-cell differentiation associated with transcriptional corepressor complexes in mammalian cells. The expression of BCL11B behaved as a disease biomarker that was present at diagnosis, but not in remission. Deregulation of BCL11B co-occurred with variants at FLT3 and at epigenetic modulators, most frequently the DNMT3A, TET2, and/or WT1 genes. Transcriptome analysis identified a specific expression signature, with significant downregulation of BCL11B targets, and clearly separating BCL11B AL from AML, T-ALL, and ETP-ALL. Remarkably, an ex vivo drug-sensitivity profile identified a panel of compounds with effective antileukemic activity., (© 2021 by The American Society of Hematology.)

Protein-coding and noncoding RNAs can be decorated with a wealth of chemical modifications, and such modifications coordinately orchestrate gene expression during normal hematopoietic differentiation and development. Aberrant expression and/or dysfunction of the relevant RNA modification modulators/regulators ("writers," "erasers," and "readers") drive the initiation and progression of hematopoietic malignancies; targeting these dysregulated modulators holds potent therapeutic potential for the treatment of hematopoietic malignancies. In this review, we summarize current progress in the understanding of the biological functions and underlying mechanisms of RNA modifications in normal and malignant hematopoiesis, with a focus on the N6-methyladenosine modification, as well as discuss the therapeutic potential of targeting RNA modifications for the treatment of hematopoietic malignancies, especially acute myeloid leukemia., (© 2021 by The American Society of Hematology.)

Bone marrow (BM) microenvironment contributes to the regulation of normal hematopoiesis through a finely tuned balance of self-renewal and differentiation processes, cell-cell interaction, and secretion of cytokines that during leukemogenesis are altered and favor tumor cell growth. In pediatric acute myeloid leukemia (AML), chemotherapy is the standard of care, but >30% of patients still relapse. The need to accelerate the evaluation of innovative medicines prompted us to investigate the role of mesenchymal stromal cells (MSCs) in the leukemic niche to define its contribution to the mechanism of leukemia drug escape. We generated a humanized 3-dimensional (3D) niche with AML cells and MSCs derived from either patients (AML-MSCs) or healthy donors. We observed that AML cells establish physical connections with MSCs, mediating a reprogrammed transcriptome inducing aberrant cell proliferation and differentiation and severely compromising their immunomodulatory capability. We confirmed that AML cells modulate h-MSCs transcriptional profile promoting functions similar to the AML-MSCs when cocultured in vitro, thus facilitating leukemia progression. Conversely, MSCs derived from BM of patients at time of disease remission showed recovered healthy features at transcriptional and functional levels, including the secretome. We proved that AML blasts alter MSCs activities in the BM niche, favoring disease development and progression. We discovered that a novel AML-MSC selective CaV1.2 channel blocker drug, lercanidipine, is able to impair leukemia progression in 3D both in vitro and when implanted in vivo if used in combination with chemotherapy, supporting the hypothesis that synergistic effects can be obtained by dual targeting approaches., (© 2021 by The American Society of Hematology.)

RNA-binding proteins (RBPs) are critical regulators of transcription and translation that are often dysregulated in cancer. Although RBPs are increasingly recognized as being important for normal hematopoiesis and for hematologic malignancies as oncogenes or tumor suppressors, RBPs that are essential for the maintenance and survival of leukemia remain elusive. Here we show that YBX1 is specifically required for maintaining myeloid leukemia cell survival in an N6-methyladenosine (m6A)-dependent manner. We found that expression of YBX1 is significantly upregulated in myeloid leukemia cells, and deletion of YBX1 dramatically induces apoptosis and promotes differentiation coupled with reduced proliferation and impaired leukemic capacity of primary human and mouse acute myeloid leukemia cells in vitro and in vivo. Loss of YBX1 has no obvious effect on normal hematopoiesis. Mechanistically, YBX1 interacts with insulin-like growth factor 2 messenger RNA (mRNA)-binding proteins (IGF2BPs) and stabilizes m6A-tagged RNA. Moreover, YBX1 deficiency dysregulates the expression of apoptosis-related genes and promotes mRNA decay of MYC and BCL2 in an m6A-dependent manner, which contributes to the defective survival that results from deletion of YBX1. Thus, our findings have uncovered a selective and critical role of YBX1 in maintaining myeloid leukemia survival, which might provide a rationale for the therapeutic targeting of YBX1 in myeloid leukemia., (© 2021 by The American Society of Hematology.)