Myeloid Maturation Research Articles

Abstract 2960: KAT6A and ENL form an epigenetic transcriptional control module to drive critical leukemogenic gene expression programs

Abstract Acute myeloid leukemia (AML) is a poor-prognosis hematological malignancy that is characterized by differentiation blockade and uncontrolled proliferation. Epigenetic programs influence AML cell fate decisions, and their mis-regulation can promote differentiation arrest. Itis therefore of paramount importance to identify the regulators of AML cell differentiation and understand the mechanisms by which they repress myeloid transcriptional programs. Here, we performed a cell-fate selection CRISPR screen for chromatin factors whose inhibition promotes AML cell differentiation. We found that the histone acetyltransferase KAT6A is a critical driver of the differentiation block, and that both genetic and chemical inhibition of KAT6A markedly de-repressed latent myeloid maturation programs, most commonly in MLL-rearranged AML. Inhibition of KAT6A catalytic activity reduced self-renewal, induced cell cycle arrest, and extinguished long-term proliferative capacity of AML cells. Through ChIP-seq, RNA-seq, and extensive analysis of clinical datasets, we found that KAT6A is the likely initiator of a newly-described "writer-reader" module that drives oncogenic gene expression in AML. In this model,KAT6A catalyzes promoter H3K9 acetylation, which is bound by the H3K9ac reader ENL, leading to Super elongation complex (SEC) recruitment and release of paused RNA PolII at oncogenic loci such as MYC. KAT6A is elevated in human AMLs compared to matched normal tissue, and its downregulation correlates with monocytic differentiation transcriptional programs in clinical AML datasets. These findings suggest the potential of targeting both KAT6A and ENL to disrupt this writer-reader module and ablate downstream MYC transcriptional programs inMLL-rearranged AML. Citation Format: Andres Blanco. KAT6A and ENL form an epigenetic transcriptional control module to drive critical leukemogenic gene expression programs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2960.

A Favorable Outcome With CPX-351 Liposome (Vyxeos) in the Management of A Sporadic Monosomy 7 Myelodysplastic Syndrome Related Acute Myeloid Leukemia

Introduction: Although Acute Myeloid Leukemia (AML) has been associated with several environmental factors, in some patients the evolution to AML is preceded by myelodysplastic syndrome (MDS). We report a case of 63-year-old male who was diagnosed with sporadic monosomy 7 MDS/AML who achieved good response to treatment with CPX-351 (Vyxeos) induction chemotherapy. Case information: A 63-year-old male initially presented to the ED for further evaluation of pancytopenia that was discovered incidentally during a routine office visit. Further evaluation revealed his blast count was elevated with presence of Auer rods, myelocytes and basophilia. He was admitted for further work-up of pancytopenia with suspicion being for MDS/AML and bone marrow biopsy for flow cytometric evaluation of the bone marrow was strongly suggestive of acute myeloid leukemia, with morphology suggesting a predisposing MDS. Subsequent molecular cytogenetic [FISH] report confirmed -7/7q abnormality in 80% of nuclei with loss of 1 copy of chromosome 7. The following week, patient was initiated on CPX–351 [Vyxeos; daunorubicin and cytarabine liposome for injection]. Bone marrow biopsy following induction chemotherapy showed 11 to 12% of CD34 positive myeloid blasts with abnormal myeloid maturation, representing notable improvement with greater than 50% reduction in his blast count. Conclusion: Sporadic monosomy 7 MDS/AML that arises without evidence of germline genetic predisposition and in the absence of other predisposing medical conditions or treatments is an uncommon subset of AML. Vyxeos induction chemotherapy may be an excellent option in the management of sporadic monosomy 7 MDS/AML.

Post-operative agranulocytosis caused by intravenous cefazolin: A case report with a discussion of the pathogenesis

A case of post-operative agranulocytosis which occurred in a 66-year-old woman following surgery for endometrial carcinoma is reported. The agranulocytosis had a rapid onset, being detected on the first post-operative day. The causative agent, cefazolin was given to the patient intraoperatively. The agranulocytosis persisted until the 22nd postoperative day. A bone marrow biopsy performed on post-operative day four showed a left-shifted myeloid maturation pattern but not a maturation arrest. The pathogenesis of drug-induced neutropenia/agranulocytosis is discussed. It is postulated that reversible binding of cefazolin to albumin accounts for the prolonged duration of agranulocytosis.

Liver stromal cells restrict macrophage maturation and stromal IL-6 limits the differentiation of cirrhosis-linked macrophages

Myeloid cells are key regulators of cirrhosis, a major cause of mortality worldwide. Because stromal cells can modulate the functionality of myeloid cells invitro, targeting stromal-myeloid interactions has become an attractive potential therapeutic strategy. We aimed to investigate how human liver stromal cells impact myeloid cell properties and to understand the utility of a stromal-myeloid coculture system to study these interactions in the context of cirrhosis. Single-cell RNA-sequencing analyses of non-cirrhotic (n= 7) and cirrhotic (n= 5) human liver tissue were correlated to the bulk RNA-sequencing results of invitro cocultured human CD14+ and primary liver stromal cells. Complimentary mechanistic experiments and flow cytometric analysis were performed on human liver stromal-myeloid coculture systems. We found that stromal-myeloid coculture reduces the frequency CD14+ cell subsets transcriptionally similar to liver macrophages, showing that stromal cells inhibit the maturation of monocytes into macrophages. Stromal cells also influenced invitro macrophage differentiation by skewing away from cirrhosis-linked CD9+ scar-associated macrophage-like cells and towards CD163+ Kupffer cell-like macrophages. We identify IL-6 production as a mechanism by which stromal cells limit CD9+ macrophage differentiation and find that local IL-6 levels are decreased in early-stage human liver disease compared to healthy liver tissue, suggesting a protective role for local IL-6 in the healthy liver. Our work reveals an unanticipated role for liver stromal cells in impeding the maturation and altering the differentiation of macrophages and should prompt investigations into the role of local IL-6 production in the pathogenesis of liver disease. These studies provide a framework for investigating macrophage-stromal interactions during cirrhosis. The impact of human liver stromal cells on myeloid cell maturation and differentiation in liver disease is incompletely understood. In this study, we present a mechanistic analysis using a primary invitro human liver stromal-myeloid coculture system that is translated to liver disease using single-cell RNA sequencing analysis of cirrhotic and non-cirrhotic human liver tissue. Our work supports a role for stromal cell contact in restricting macrophage maturation and for stromal-derived IL-6 in limiting the differentiation of a cirrhotic macrophage subset.

Progesterone effect on myeloid cell maturation and cytokine secretion in human placentas following preterm deliveries

Recent studies suggest that immature myeloid cells (IMCs) have a role in parturition and preterm delivery (PTD). In this study, we sought to evaluate the effect of progesterone on IMCs maturation and proinflammatory cytokine secretion in human placentas following PTD( < 37 weeks).

Progesterone effect on myeloid cell maturation and cytokine secretion in human term placentas

Recent studies suggested that immature myeloid cells (IMCs) have a role in parturition. To further investigate this paradigm, we aimed to evaluate the effect of progesterone (P) on IMCs maturation and pro-inflammatory cytokine secretion in human placentas following term deliveries.

Congenital neutropenia: disease models guiding new treatment strategies.

Purpose of reviewMyeloid diseases are often characterized by a disturbed regulation of myeloid cell proliferation, survival, and maturation. This may either result in a severe paucity of functional neutrophils (neutropenia), an excess production of mature cells (myeloproliferative disorders) or in clonal expansions of dysplastic or immature myeloid cells (myelodysplasia and acute myeloid leukemia). Although these conditions can be regarded as separate entities, caused by the accumulation of distinct sets of somatic gene mutations, it becomes increasingly clear that they may also evolve as the prime consequence of a congenital defect resulting in severe neutropenia. Prominent examples of such conditions include the genetically heterogeneous forms of severe congenital neutropenia (SCN) and Shwachman-Diamond Syndrome. CSF3 treatment is a successful therapy to alleviate neutropenia in the majority of these patients but does not cure the disease nor does it prevent malignant transformation. Allogeneic stem cell transplantation is currently the only therapeutic option to cure SCN, but is relatively cumbersome, e.g., hampered by treatment-related mortality and donor availability. Hence, there is a need for new therapeutic approaches.Recent findingsDevelopments in disease modeling, amongst others based on induced pluripotent stem cell and CRISPR/Cas9 based gene-editing technologies, have created new insights in disease biology and possibilities for treatment. In addition, they are fueling expectations for advanced disease monitoring to prevent malignant transformation.SummaryThis review highlights the recent progress made in SCN disease modeling and discusses the challenges that are still ahead of us to gain a better understanding of the biological heterogeneity of the disease and its consequences for patient care.

Integrated analysis of genotype and phenotype reveals clonal evolution and cytogenetically driven disruption of myeloid cell maturation in myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are a heterogenous collection of clonal bone marrow diseases characterized by cytopenias, abnormal karyotypes, molecular abnormalities, and dysplasia by flow cytometry and/or morphology. The progression of MDS to severe cytopenias and/or overt leukemia is associated with the accumulation of additional cytogenetic abnormalities, suggesting clonal evolution. The impact of these accumulated abnormalities on myeloid maturation and the severity of the disease is poorly understood. Bone marrow specimens from 16 patients with cytogenetic abnormalities were flow cytometrically sorted into three myeloid populations: progenitors, immature myeloid cells, and mature myeloid cells. Fluorescence in situ hybridization analysis was performed on each to determine the distribution of chromosomal abnormalities during myeloid maturation. Our findings revealed three distinct distributions of cytogenetic abnormalities across myeloid maturation, each of which corresponded to specific cytogenetic abnormalities. Group 1 had continuous distribution across all maturational stages and contained patients with a single cytogenetic aberration associated with good-to-intermediate prognosis; Group 2 had accumulation of abnormalities in immature cells and contained patients with high-risk monosomy 7; and Group 3 had abnormalities defining the founding clone equally distributed across maturational stages while subclonal abnormalities were enriched in progenitor cells and contained patients with multiple, non-monosomy 7, abnormalities with evidence of clonal evolution. Our findings demonstrate that low-risk abnormalities (e.g., del(20q) and trisomy 8) occurring in the founding clone display a markedly different disease etiology, with respect to myeloid maturation, than monosomy 7 or abnormalities acquired in subclones, which result in a disruption of myeloid cell maturation in MDS.

Acute myeloid leukemia maturation lineage influences residual disease and relapse following differentiation therapy

Acute myeloid leukemia (AML) is a malignancy of immature progenitor cells. AML differentiation therapies trigger leukemia maturation and can induce remission, but relapse is prevalent and its cellular origin is unclear. Here we describe high resolution analysis of differentiation therapy response and relapse in a mouse AML model. Triggering leukemia differentiation in this model invariably produces two phenotypically distinct mature myeloid lineages in vivo. Leukemia-derived neutrophils dominate the initial wave of leukemia differentiation but clear rapidly and do not contribute to residual disease. In contrast, a therapy-induced population of mature AML-derived eosinophil-like cells persists during remission, often in extramedullary organs. Using genetic approaches we show that restricting therapy-induced leukemia maturation to the short-lived neutrophil lineage markedly reduces relapse rates and can yield cure. These results indicate that relapse can originate from therapy-resistant mature AML cells, and suggest differentiation therapy combined with targeted eradication of mature leukemia-derived lineages may improve disease outcome.

Epigenetic Enzyme Mutations in Myeloid Malignancies Are Selected By Chromatin-Remodeling Requirements That Vary By Lineage- and Maturation-Stage

Introduction/Methods: Enzymes that modify histone H3 at lysine 27 (H3K27) to thereby regulate gene transcription (epigenetic enzymes) are recurrently inactivated by deletion and/or mutation in myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN) and acute myeloid leukemias (AML). Frustrating understanding of mechanisms is that writers and erasers of the same modification, e.g., methyltransferases that create, and demethylases that remove, H3K27 trimethylation (H3K27me3), a repression ('off') mark, are recurrently inactivated. Moreover, acetyltransferases that write H3K27 acetylation (H3K27ac), an 'on' mark mutually exclusive with H3K27me3, are also recurrently inactivated. One clue to underlying mechanisms is that MDS/MPN/AML present with diverse lineage and maturation phenotypes - perhaps these emerge from, or select for, the diverse epigenetic enzyme mutations. We therefore identified genes upregulated with specific myeloid lineage-commitment, maturation and function fates (~500 genes each) and then examined distributions of H3K27me3 and H3K27ac at these gene-loci in: (i) embryonic stem cells (ESC); (ii) hematopoietic stem and progenitor cells (HSPC); (iii) mature myeloid cells (monocyte [mono], pro-erythroblast, megakaryocyte [MK]); and (iv) AML cells.Results: Terminal-myeloid programs underwent substantial remodeling to gain H3K27ac ‘on’ mark from ESC/HSPC to mature myeloid (Fig.1A). Providing a mechanism for this, the H3K27 acetyltransferases EP300 and CREBBP were recruited into the RUNX1/SPI1 myeloid-lineage master transcription factor (MTF) hub by cooperation between their transcription activating domains. Mutated/translocated RUNX1, or mutated-NPM1 that cytoplasmically dislocated SPI1, disrupted this cooperation, and reverted hub content to default recruitment of histone deacetylases (HDAC) instead. Demonstrating cause-effect, inhibiting these HDAC renewed AML cell maturation to terminal lineage-fates. Meanwhile, MYC-target (proliferation) genes have high baseline H3K27ac in ESC and HSPC and do not require major remodeling during ontogeny (Fig.1A). An H3K27ac remodeling requirement for lineage-maturation but not proliferation/housekeeping explains selection pressure for inactivating mutations in EP300 or CREBBP, that we found in ~1.2% of MDS/MPN/AML in our (n=690) and other series. Consistent with pan-lineage-maturation needs for H3K27ac, the mutations were found in all lineage sub-types. H3K27me3 ‘off’ mark was mostly erased from myeloid programs in HSPC, but was greater at MK vs erythroid, and also at mono vs granulocyte genes (Fig.1B). This implied more need for H327me3 demethylase (KDM6A/UTX) for HSPC commitment into MK vs erythroid, or mono vs granulocyte, lineages. Accordingly, KDM6A was most upregulated in MK and mono-lineage cells (Fig.1C), and myeloid-conditional Kdm6a knockout decreased platelets and increased red cells in the spleen - reported by others: https://doi.org/10.1182/blood.V128.22.1467.1467. RUNX1-ETO has been shown to specifically impede granulocytic but not mono differentiation - https://www.nature.com/articles/2403396 and KDM6A inactivating mutations were significantly more likely to occur secondary to RUNX1-ETO (4-9% BEAT and AMLSG case series) vs other cytogenetics (0.005%). Selection for KDM6A secondary mutations could thus be to channel myeloid precursors toward lineages most efficiently impeded by primary mutations. Although H3K27me3 was substantially erased at all myeloid-commitment and terminal programs (except MK) in HSPC vs ESC, subsequent lineage-commitment and maturation entailed rewriting H3K27me3 at preceding HSPC and alternate lineage-fate programs, including at MTF genes for alternate fates (Fig.1B, D). Primary MDS/MPN/AML and AML cell lines with inactivating mutations/deletions in H3K27 methyltransferase EZH2 thus displayed aberrant co-expression of lineage MTFs and gene expression programs of normally mutually exclusive lineages (Fig.1E-G).Conclusion. Epigenetic remodeling requirements vary by myeloid lineage and maturation stage. Thus, epigenetic enzyme mutations are selected by, and cause, lineage-context of transformation. This knowledge can guide choice of specific epigenetic enzyme inhibitors to remedy the lineage-maturation defects that drive and define myeloid malignancies. [Display omitted] DisclosuresHaferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Maciejewski: Novartis: Consultancy; Bristol Myers Squibb/Celgene: Consultancy; Regeneron: Consultancy; Alexion: Consultancy. Saunthararajah: EpiDestiny: Consultancy, Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Clonotype-Immunophenotype Relationships in TET2 and IDH-Mutant Myeloid Transformation

Large scale molecular profiling studies in AML patients have suggested that stepwise acquisition of somatic mutations is crucial in driving leukemic development. High variant allele frequency (VAF) mutations in epigenetic modifier genes, such as TET2 and IDH1/2, are thought to occur early in AML pathogenesis while oncogenic mutations with typically lower VAF mutations, including FLT3 and NRAS, are suggested to occur late in disease evolution. While bulk DNA sequencing has catalogued co-mutations found in individual AMLs, it cannot unveil the heterogeneity and composition of clones that makes up the disease. Elucidating the architecture and clone-specific molecular profiles at the single cell resolution will be key to understanding how sequential and/or parallel mutation acquisition drives myeloid transformation.To assess the clonal architecture of AML, we previously performed single cell DNA sequencing (scDNA seq) in 146 patients with myeloid malignancies. We have further identified specific mutational combinations driving clonal expansion in TET2- or IDH1/2- mutant AML samples. These studies suggest TET2 and IDH1/2 can cooperate to promote clonal expansion with DNMT3A and NPM1 (Figure 1A). However, TET2 or IDH1/2 mutant clones that acquired KRAS mutations underwent minimal clonal expansion, suggesting mutant-pair specific fitness alterations (Figure 1B). To further identify how co-mutational pairing impacted clonal fitness and differentiation, we integrated the scDNA platform with immunophenotypic profiling of 45 cell surface markers and analyzed new TET2- and IDH1/2- mutant AML samples (Figure 1C). We identified clone-specific differences in lineage markers depending on co-mutational partners. NPM1 co-mutant clones were enriched for more primitive markers (CD33), whereas NRAS co-mutant clones possessed high expression of myeloid differentiation markers (CD14/CD11b), suggestive of clone-specific fitness landscapes across hematopoietic differentiation. We also identified divergent clonotype-immunophenotype patterns in TET2- and IDH2-mutant clones harboring NPM1/RAS mutations, suggesting that initiating mutations may prime mutant clones for very different evolutionary trajectories as they acquire similar mutations in leukemogenesis (Figure 1D).To deterministically delineate the relationship between clonal evolution and myeloid transformation, we generated Cre-inducible single (Tet2 -/-), double (Tet2 -/-/Nras G12D and Tet2 -/-/Npm1 cA/wt), and triple (Tet2 -/-/Npm1 cA/wt/Nras G12D) mutant mice and evaluated differences in chimerism, immunophenotype, and survival. We observed a shortened survival for double and triple mutant mice, compared to Tet2 -/- only mice (Figure 1E). As previously reported, Tet2 -/-/Nras G12D mice developed a CMML-like phenotype. Critically, the addition of Npm1 resulted in a more rapid disease onset and transformation to AML (Figure 1F). Moreover, triple mutant WBM transplanted to form a fully penetrant disease into secondary recipients, while double mutant Tet2 -/-/Nras G12D WBM failed to form disease within 3 months of transplant, suggesting a difference in the cell population responsible for disease propagation. Immunophenotypic alterations were evident with Tet2 -/-/ Nras G12D displaying an increase in Mac1 +Gr1 + cells compared to Tet2 -/-/Npm1 cA/wt/Nras G12D mice which possessed increased Mac1 +Gr1 - cells and expansion of lineage negative cells (Figure 1G). These findings align with the clonotype specific expression patterns observed in clinical specimen and suggest that myeloid transformation and maturation biases are influenced by specific mutational combinations. [Display omitted] DisclosuresMiles: Mission Bio: Honoraria, Speakers Bureau. Bowman: Mission Bio: Honoraria, Speakers Bureau. Carroll: Janssen Pharmaceutical: Consultancy; Incyte Pharmaceuticals: Research Funding. Levine: Astellas: Consultancy; Janssen: Consultancy; Auron: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; QIAGEN: Membership on an entity's Board of Directors or advisory committees; Mission Bio: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Incyte: Consultancy; Imago: Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Research Funding; Prelude: Membership on an entity's Board of Directors or advisory committees; Ajax: Membership on an entity's Board of Directors or advisory committees; Zentalis: Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Lilly: Honoraria; Morphosys: Consultancy.

Chromatin Accessibility Analysis Reveals Epigenetic Evolution Is a Common Mechanism of Relapse in Acute Myeloid Leukemia

Introduction: Acute myeloid leukemia (AML) is associated with a poor prognosis and high rates of relapse despite aggressive treatments including high dose chemotherapy. To understand the clonal dynamics and genetic evolution of relapsed AML, we analyzed a cohort of 142 previously published genotyped and paired diagnosis-relapse AML samples. 40% of queried cases exhibited no major changes in somatic mutations upon relapse, and genetically stable clonal structure correlated with increased relapse probability. Thus, we hypothesized epigenetic reprogramming plays a role in these cases and AML relapse in general. Here, we examine the epigenetic landscape of relapsed AML and characterize the cis and trans regulatory elements that correlate with AML relapse in the presence or absence of genetic evolution.Methods: We identified 27 viable cryopreserved paired diagnosis and relapse samples from the same patient treated at Stanford with high-dose chemotherapy. Leukemic blasts and (when possible) leukemia stem cell (LSC) enriched populations were FACS purified and prepared for genotyping with a myeloid malignancy targeted sequencing panel as well as ATAC-seq for chromatin accessibility profiling. Single-cell ATAC-seq was further performed on select samples to investigate regulatory reprogramming in cell subpopulations. We then performed integrative analysis to uncover the interplay between genetic lesions, epigenetic regulatory programs, and gene accessibility in relapsed AML.Results: Genotyping analysis of these AML specimens revealed that 40% of samples exhibited no changes in AML-related genetic alterations upon relapse (hereby referred to as “stable” samples). Chromatin accessibility analysis revealed these stable samples had a distinct epigenetic signature, modulating similar gene accessibility programs and sharing enhancer loci that become accessible across all stable relapse samples. These sequences included genes involved in chromatin organization and compaction, as well as those involved in transcriptional control of hematopoietic differentiation and myeloid cell maturation. We also observed several regulatory signatures present at relapse specific for AML subtypes, including NPM1/FLT3 double mutant AML and those with mutations in transcription factors such as CEBPA or RUNX1.We then performed single-cell ATAC-seq on genetically stable samples to further characterize the sub-clonal epigenetic dynamics between the diagnosis and relapse cells. Several samples exhibited regulatory heterogeneity in multiple cell subpopulations that changed significantly at relapse. One subpopulation of interest was characterized by increased GATA and RUNX family transcription factor motif accessibility at relapse, indicating a shift toward a less differentiated progenitor cell phenotype. In addition, we identified a subpopulation of cells at diagnosis that were epigenetically similar to the major epigenetic states present at relapse, indicating that selection for specific epigenetic subclones may occur in AML patients during therapy in the absence of additional genetic lesions.Finally, given the critical role of LSCs in AML pathogenesis and their possible role as a reservoir for AML relapse, we analyzed LSC-enriched subpopulations in a subset of our cohort. ATAC-seq analysis indicated these cells shared several LSC-specific epigenetic features between samples, are distinguished from leukemia blasts by distinct regulatory programs, and undergo epigenetic remodeling between initial diagnosis and relapse. Gene accessibility analysis also revealed a shared LSC gene expression signature that also shifted at relapse. These data indicate a specific, distinct epigenomic signature for LSC enriched cell populations, and that epigenetic evolution at relapse occurs intracellularly, rather than reflecting heterogeneity in cellular subpopulations upon AML relapse.Conclusion: This study reveals that epigenetic remodeling in the absence of genetic evolution is a mechanism through which AML relapse occurs. We show that chromatin reorganization of genes and regulatory sequences occurs in these AML cells, leading to a permissive cell state that might be resistant to conventional treatment. Ongoing work includes dissecting the subclonal structure of these AML cells and identifying the relationship between gene regulatory networks that contribute to relapse. DisclosuresEdiriwickrema: Nanosive SAS: Patents & Royalties. Majeti: BeyondSpring Inc.: Membership on an entity's Board of Directors or advisory committees; CircBio Inc.: Membership on an entity's Board of Directors or advisory committees; Kodikaz Therapeutic Solutions Inc.: Membership on an entity's Board of Directors or advisory committees; Coherus Biosciences: Membership on an entity's Board of Directors or advisory committees; Acuta Capital Partners: Consultancy; Gilead: Patents & Royalties: inventor on a number of patents related to CD47 cancer immunotherapy licensed to Gilead Sciences, Inc..

A Novel CSF3R Activating Mutation Identified in a Patient with Chronic Neutrophilic Leukemia

Mutations in CSF3R, the growth factor receptor that signals to drive neutrophil production, are commonly found in Chronic Neutrophilic Leukemia (CNL). There are two main types of CSF3R mutations in CNL: membrane-proximal point mutations and cytoplasmic truncating mutations. The membrane proximal point mutations-notably T618I-are the most common in CNL. These point mutations lead to the loss of glycosylation sites, causing ligand-independent dimerization of the receptor and enhanced activation of downstream signaling pathways. Previously, these glycan-altering mutations were thought to reside in a relatively restricted region near the membrane spanning 610-618 that contains an N-linked glycosylation site and three putative O-linked glycosylation sites.A 55-year old man presented with a WBC of 50,000 and a predominant neutrophilia with 84% neutrophils and bands and no immature cells. A bone marrow examination showed a markedly hypercellular bone marrow exhibiting myeloid-predominant maturing trilineage hematopoiesis with left-shifted myeloid maturation, mild megakaryocytic atypia (small, hypolobated forms), mild reticulin fibrosis (grade 1 of 3). He had multi-station small volume lymphadenopathy and a spleen that measured 16.6 cm craniocaudally. The findings were most consistent with chronic neutrophilic leukemia.Targeted NGS analysis revealed three mutations in CSF3R: Q739* (VAF 26%), Q741* (VAF 24%) and N579Y (VAF 52%). ASXL1 G646fs (38% VAF) and ETNK1 H243Q (VAF 12%) mutations were also present. The truncating mutations in CSF3R were reported as pathogenic, but the N579Y mutation was listed as a variant of uncertain significance. When truncating mutations occur in CNL, they most frequently occur alongside an activating point mutation. This, combined with the knowledge that N579 was previously mapped as a site of receptor glycosylation prompted us to assess the oncogenicity of this variant.We undertook several parallel approaches to characterize this variant. First, a cytokine independent growth assay was performed in Ba/F3 cells. The CSF3R N579Y variant allowed these cells to proliferate in the absence of cytokine support, similarly to the well characterized CSF3R T618I variant. Furthermore, it enabled growth independent from the ligand for CSF3R (GCSF), indicating that this mutation likely also confers ligand-independent dimerization. In an orthogonal approach, we found that CSF3R N579Y enables primary murine hematopoietic cells to grow in cytokine-free methylcellulose to a similar extent as CSF3R T618I.To understand the downstream consequences of receptor activation we undertook immunoblot analysis and discovered that CSF3R N579Y robustly enhances JAK/STAT and MAPK signaling downstream of the receptor. Because of the know activation of JAK by CSF3R mutations, a JAK1/2 inhibitor (ruxolitinib) has been under investigation for treatment of CSF3R-mutant CNL. We find that CSF3R N579Y confers sensitivity to ruxolitinib in both Ba/F3 cells and murine colony forming unit assays. Furthermore, ruxolitinib inhibited colony formation of CD34 positive cells isolated from a bone marrow aspirate from the patient whose leukemia harbored CSF3R N579Y, CSF3R Q739* and CSF3R Q741* mutations. Collectively, our data suggests that N579Y is a pathogenic and potentially actionable variant and that N-linked glycosylation outside of the membrane-proximal region is necessary to maintain the receptor in an inactive state. [Display omitted] DisclosuresSanz-Altamira: Kura Oncology: Consultancy. DeAngelo: Autolus: Consultancy; Forty-Seven: Consultancy; Incyte: Consultancy; Jazz: Consultancy; Novartis: Consultancy, Research Funding; Pfizer: Consultancy; Servier: Consultancy; Takeda: Consultancy; Abbvie: Research Funding; Blueprint: Research Funding; Amgen: Consultancy; Agios: Consultancy; Glycomimetrics: Research Funding. Maxson: Ionis Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Clinical Significance of Mitochondrial DNA Content in Acute Promyelocytic Leukemia

Used in the clinical practice for more than three decades, the all-trans retinoic acid (ATRA) rendered acute promyelocytic leukemia (APL) the most curable subtype of acute myeloid leukemia, and currently, its combination with arsenic trioxide (ATO) exceeded all expectations for a chemotherapy-free protocol. In terms of metabolic importance, ATRA can also modulate the mitochondria-mediated cellular metabolism and promote a shift from a glycolytic-driven metabolism to an oxidative phosphorylation profile, although this effect has never been demonstrated in APL. As part of the cellular metabolic machinery, mitochondrial DNA (mtDNA) content has been reported to be altered in different types of solid tumors with clinical implication on patient treatment outcomes, although its clinical significance in acute leukemias has not been investigated to the same extent. Particularly in acute promyelocytic leukemia (APL), the role of mtDNA content on prognostication is completely unknown. Considering that mostly APL samples display a glycolytic-driven metabolism, it is conceivable that APL patients harboring high mtDNA content may present a better response to ATRA-based therapies. To test this hypothesis, we determined the mtDNA content in samples from patients with APL enrolled in the International Consortium on Acute Promyelocytic Leukemia study (Rego et al. Blood. 2013 Mar 14;121(11):1935-43) and analyzed its relationship to treatment outcomes. Diagnostic bone marrow (BM) mononuclear cells from 156 consecutive patients with APL (median age: 35 years, range: 18-82 years; 45% male) were obtained at diagnosis. For comparison purposes, we also included peripheral blood (PB) from 293 age- and sex-adjusted healthy volunteers. First, we determined whether mtDNA content could be compared between PB mononuclear cells and BM. To do so, we measured the mtDNA content of 22 APL patients, for whom paired samples were available at the time of diagnosis and detected a strong correlation between PB and BM samples (Pearson correlation coefficient, r=0.78, 95% confidence interval, CI: 0.54 to 0.9). Next, we used the values of mtDNA higher than the 95 th percentile of healthy subjects (≥1.63. Note: this value represents a fold change relative to healthy control) to define APL patients with high mtDNA content. Patients that presented values within the range of normal control samples (<1.63) were classified as normal mtDNA content. The median follow-up among survivals was 40 months (95%CI: 34-47 months). Of the 131/156 patients who achieved complete remission, 18 patients (14%) relapsed. mtDNA content had no impact on complete remission achievement (84% for normal mtDNA versus 83% for high mtDNA; P=0.924) or overall survival (78% for normal mtDNA versus 80% for high mtDNA; P=0.69). In contrast, patients with high mtDNA content had a significantly high 5-year disease-free survival rate (86%, 95%CI: 78-95%) than patients with normal mtDNA content (61%, 95%CI: 46-82%). Considering non-relapse death as a competing cause of failure, the 5-year cumulative incidence of relapse (CIR) for patients with high and normal mtDNA content were 35% (95%CI: 16-49%) and 10% (95%CI: 2-17%), respectively. The multivariate Cox proportional hazards model showed that mtDNA content was independently associated with CIR (hazard ratio, HR: 0.31, 95%CI: 0.12-0.8) considering PETHEMA/GIMEMA risk of relapse subgroups and age as confounders. To functionally evaluate the metabolic alterations in APL cells upon ATRA treatment, NB4 cell line was treated with ATRA (1 µM) for 48 and 72 hours. In vitro analyses demonstrated (as expected) that the treatment with resulted in increased levels of myeloid maturation markers (CD11b/CD11c/CD15), with morphological changes being only observed at 72 hours. Metabolically, we observed an increase in mitochondrial mass and potential upon ATRA-treatment after 48 hours, which was also reflected by increase in the mtDNA content (2-fold increase in comparison with the vehicle). Together, these findings demonstrate an important, but not completely understood role for mtDNA content in APL. DisclosuresSilveira: BMS/Celgene: Research Funding; Servier/Agios: Research Funding; Abbvie: Speakers Bureau; Astellas: Speakers Bureau. Pagnano: EMS: Other: Lecture; Jansenn: Other: Lecture; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pintpharma: Other: Lecture.

Convergence of oncogenic cooperation at single-cell and single-gene levels drives leukemic transformation

Cancers develop from the accumulation of somatic mutations, yet it remains unclear how oncogenic lesions cooperate to drive cancer progression. Using a mouse model harboring NRasG12D and EZH2 mutations that recapitulates leukemic progression, we employ single-cell transcriptomic profiling to map cellular composition and gene expression alterations in healthy or diseased bone marrows during leukemogenesis. At cellular level, NRasG12D induces myeloid lineage-biased differentiation and EZH2-deficiency impairs myeloid cell maturation, whereas they cooperate to promote myeloid neoplasms with dysregulated transcriptional programs. At gene level, NRasG12D and EZH2-deficiency independently and synergistically deregulate gene expression. We integrate results from histopathology, leukemia repopulation, and leukemia-initiating cell assays to validate transcriptome-based cellular profiles. We use this resource to relate developmental hierarchies to leukemia phenotypes, evaluate oncogenic cooperation at single-cell and single-gene levels, and identify GEM as a regulator of leukemia-initiating cells. Our studies establish an integrative approach to deconvolute cancer evolution at single-cell resolution in vivo.

MiR-378-3p Knockdown Recapitulates Many of the Features of Myelodysplastic Syndromes

Myelodysplastic syndromes (MDS) are clonal neoplasms of the hematopoietic stem cell that result in aberrant differentiation of hematopoietic lineages caused by a wide range of underlying genetic, epigenetic, and other causes. Despite the myriad origins, a recognizable MDS phenotype has been associated with miRNA aberrant expression. A model of aberrant myeloid maturation that mimics MDS was generated using a stable knockdown of miR-378-3p. This model exhibited a transcriptional profile indicating aberrant maturation and function, immunophenotypic and morphologic dysplasia, and aberrant growth that characterizes MDS. Moreover, aberrant signal transduction in response to stimulation specific to the stage of myeloid maturation as indicated by CyTOF mass cytometry was similar to that found in samples from patients with MDS. The aberrant signaling, immunophenotypic changes, cellular growth, and colony formation ability seen in this myeloid model could be reversed with azacytidine, albeit without significant improvement of neutrophil function.

The Effect of the MK0339 Neutrophil Elastase Inhibitor on Proliferation and Maturation of iPSC Derived from Patients with ELANE, TCIRG1 and Whim Syndrome Associated Neutropenia

Genetic neutropenias are heterogeneous group of hematological diseases characterized by impaired survival and maturation of myeloid precursors in bone marrow. G-CSF and HSCT are currently the only effective treatment options. MK-0339 is a potent, cell permeable, orally absorbed inhibitor of neutrophil elastase (NE), previously investigated in preclinical and clinical studies by Merck/DuPont as a potential anti-inflammatory drug. We have previously reported that MK-0339 increases neutrophil proliferation in cellular models of ELANE associated neutropenia (2016 [ASH Annual Meeting Abstracts] Blood. 2016;128:406; 2017 J Leuk Biol [in press]). To examine the specificity of these responses, we investigated the effects of MK-0339 on iPSCs from healthy volunteers and compared its effects on differentiation of iPSC derived from patients with ELANE, TCIRG-1 and CXCR4 associated neutropenia.We generated induced pluripotent stem cells (iPSC) from dermal fibroblasts from healthy volunteers and neutropenic patients harboring ELANE, TCIRG1 and CXCR4 mutations and developed a feeder free, reproducible and efficient protocol for myeloid differentiation.In our initial experiments, we observed that iPSC lines derived from patients with ELANE mutations (P139L and G214R) consistently grew much slower and formed much smaller colonies than the controls using both mTeSR1/matrigel and DEF-CS feeder free culture medium systems. We observed more than 3-fold growth reduction of patient derived lines compared to healthy volunteers.We next tested the effects of MK-0339 under the same culture conditions. Growth of control and ELANE patient cell lines was enhanced, and the cells appeared more robust, after addition of 1uM MK-0339. We were surprised to see a similar proliferative effect (~40% increase) with Ramos (human Burkitt's lymphoma cell line) cells cultured in the presence of 1uM MK-0339, although neither the Ramos cells nor the ELANE iPSCs had detectable levels of ELANE expression. The enhanced growth of control and patient iPSCs and the results with Ramos cells suggested that the effects of MK-0339 are not solely mediated via inhibition of NE.We next compared myeloid differentiation and CD34+ cell proliferation of iPSC from patients with ELANE, TCIRG1 and CXCR4 associated neutropenia in HPC expansion medium, using granulocytic differentiation markers and flow cytometry. Myeloid maturation was impaired and inefficient for both ELANE (P139L and G214R) patient derived cell lines. Myeloid differentiation of the TCIRG1 patient derived was also impaired. Introduction of MK-0339 restored the impaired myeloid differentiation capacity of both ELANE and TCIRG1 positive patient derived cell lines. An iPSC line derived from a WHIM (CXCR4 mutation) patient appeared to have normal maturation and proliferation. Western blot analysis revealed elevated levels of endoplasmic reticulum chaperone GRP78/BiP in ELANE and TCIRG1 mutant cell lines which was corrected by MK-0339 treatment.These studies demonstrate heretofore unreported proliferative effects of MK-0339 on cell lines not expressing neutrophil elastase. They suggest important mechanisms beyond enzyme inhibition to account for these findings. Although preliminary, we believe that this work suggests that MK-0339 may be a novel, oral therapy for both ELANE and non-ELANE associated neutropenia. DisclosuresDale:University of Washington: Employment, Research Funding; National Institutes of Health: Research Funding; WedMD/Medscape: Membership on an entity's Board of Directors or advisory committees; Wolter Kluwer: Other: Editor, Current Opinions in Hematology; Philips: Research Funding; X4Pharma: Research Funding; Shire: Other: Independent Review Board; Omeros: Other: DSMB; Coherus: Consultancy; Boheringer-Ingelheim: Consultancy; Prolong: Consultancy; Hospira: Consultancy; Genkyotex: Other: DSMB (work completed 6/2015); Genzyme (now owned by Sanofi-Aventis): Consultancy, Patents & Royalties, Research Funding; Cellerant: Membership on an entity's Board of Directors or advisory committees; Sanofi Aventis: Consultancy, Editor, Current Opinions in Hematology, Honoraria; Amgen: Consultancy, Research Funding; American College of Physicians: Other: Editor and author; GlaxoSmithKline: Equity Ownership; Johnson&Johnson: Equity Ownership.

Altered Enhancer Activity Represses CEBPE Expression in MLL -Rearranged Leukemia

Mixed lineage leukemia (MLL)-rearranged leukemia is characterized by the presence of MLL fusion proteins resulting from chromosomal translocation between MLL and its partner genes. The resultant fusion proteins can confer aberrant epigenetic patterns at gene regulatory elements to disrupt transcriptional program, and ultimately leads to differentiation blockage at early hematopoietic progenitor stage. CCAAT enhancer binding proteins (C/EBPs) are key transcription factors involved in myeloid lineage commitment and maturation, among which CEBPE is crucial in terminal differentiation of granulocytes/macrophages. We found that Cebpe is markedly downregulated in murine hematopoietic stem/progenitor cells (HSPCs) expressing MLL-EEN, which could be associated with differentiation blockage in MLL -rearranged leukemia. However, how CEBPE is epigenetically dysregulated by MLL fusion proteins remains unclear. In murine granulocytes, we demonstrated long-range chromatin interaction between Cebpe promoter and enhancer by chromatin conformation capture (3C) assay, conferring the strong expression of Cebpe . Strikingly, we noticed gain of DNA methylation at Cebpe promoter in MLL-EEN expressing HSPCs, suggesting the disruption of chromatin interaction through aberrant epigenetic alterations resulted in Cebpe repression. Interestingly, CEBPE expression was also found significantly lower in human MLL acute myeloid leukemia (AML) cell lines (HL60, NB4, U937 and Kasumi-1), when compared to non-MLL AML cell lines (ML-2, MV4:11, Monomac-6 and THP-1). DNA hypermethylation was observed at both promoter and predicated enhancer regions in MLL AML cells, which were absent in non-MLL AML cells. In addition, reduction of H3K27ac enrichment at the predicted enhancer was found in MLL AML cells. Analysis of the public available RNA Polymerase II (RNAPII) ChIP-seq data demonstrated RNAPII binding at both CEBPE promoter and enhancers in non-MLL AML cell lines, but it was absent at the enhancer region in MLL AML cells. These results suggest a common epigenetic mechanism which disrupts the CEBPE enhancer-promoter interaction in MLL -rearranged leukemia. Besides, treatment of MLL AML cell lines with DNA methylation inhibitor 5'-aza-cytidine (5-aza-C) demonstrated induction of CEBPE and myeloid marker CD11b . While all -trans retinoid acid failed to trigger cell differentiation in ML-2 AML cells, co-treatment with 5-aza-C resulted in further induction of CEBPE and CD11b, compared to 5-aza-C treatment per se . Collectively, our work shows that aberrant DNA methylation mediated by MLL fusion proteins represses CEBPE expression through altering its enhancer activity, which subsequently leads to cell differentiation blockage in MLL AML cells. This study provides novel insights on the aberrant epigenetic regulation in MLL -rearranged leukemia and facilitates the development of differentiation therapy for leukemia. DisclosuresNo relevant conflicts of interest to declare.

Expression of the Tumor Suppressor WT1 Is Induced By PML-Rara in Acute Promyelocytic Leukemia. Â

Inactivating mutations in Wilms Tumor 1 (WT1) are found in 5-10% of cases of acute myeloid leukemia (AML). Paradoxically, wild type WT1 is expressed highly in most cases of AML compared to normal hematopoietic progenitors, and high levels of WT1 have been associated with poor outcomes. Therefore, it is not yet clear whether loss-of-function WT1 mutations-or paradoxically, wild type WT1 overexpression-both promote disease progression in AML. We performed a genome-wide screen for mutations in acute promyelocytic leukemia (APL), a morphologic subtype of AML characterized by the PML-RARA fusion. We identified WT1 mutations in 11/42 patients, suggesting that WT1 mutations may cooperate specifically with PML-RARA in APL. Further, expression analysis demonstrated a high level of wild type WT1 expression in non-mutated cases. To test the effect of Wt1 inactivation in a mouse model of APL, we bred mice carrying a floxed Wt1 allele (give Baylor reference) to transgenic mice expressing tamoxifen-inducible Cre recombinase (ERT2-Cre). Tamoxifen treatment leads to deletion of the C-terminal zinc finger domains of Wt1, mimicking some of the mutations that are commonly found in AML/APL cases. These mice were crossed with mice expressing the PML-RARA fusion cDNA in early myeloid progenitors with Cathepsin G (Ctsg) regulatory sequences (Ctsg-PML-RARA). Bone marrow cells from Ctsg-PML-RARA x Wt1fl/fl x ERT2-Cre mice were transplanted into irradiated recipients. Tamoxifen-treated mice (i.e. Wt1 deficient) developed APL with a significantly shortened latency compared to untreated controls (198 days vs. endpoint not reached, N=8-10 each group, p=.0001), suggesting that Wt1 loss-of-function mutations can cooperate with PML-RARA to drive APL. Next, to test the effect of wild type Wt1 overexpression on hematopoietic cells, bone marrow cells from wild type or Ctsg-PML-RARA mice was transduced with retroviruses expressing full length Wt1 cDNA or an empty vector control, and subjected to serial replating. As previously reported, untransduced bone marrow cells from Ctsg-PML-RARA mice were able to form colonies in methylcellulose week after week, while cells overexpressing wild type Wt1 were selected against during serial replating, as demonstrated by a gradual reduction in GFP percentage and Wt1 expression, as measured by western blotting. Interestingly, in contrast to human AML/APL, Wt1 expression could not be detected in a panel of murine tumors with initiating mutations in DNMT3A, PML-RARA, or MLL-AF9 . Taken together, these findings suggest that Wt1 expression suppresses APL development in mice.To reconcile these findings with the high level of WT1 expression in human AML/APL, we speculated that in human progenitor cells, WT1 expression may be induced by leukemia-associated oncogenes (like PML-RARA) as a physiologic, inhibitory response to oncogenic stress. In this case, subsequent inactivating mutations in WT1 would provide an additional growth advantage. Given the observed interspecies difference between WT1/Wt1 expression levels in mouse and human leukemia cells, we used human umbilical cord blood-derived CD34+ cells transduced with retroviruses expressing PML-RARA to model early events in human APL development. CD34+ cells expressing PML-RARA share some characteristics of fully transformed APL cells, including enhanced proliferation in liquid culture, and reduced expression of the myeloid maturation markers CD11b and CD14. WT1 mRNA expression is low in freshly isolated CD34+ cells, but rapidly increases in cells expressing PML-RARA over two weeks in culture (10.5-fold increase compared to empty-vector transduced controls, N=3 separate experiments, p<0.05). Western blotting confirmed robust induction of WT1 protein in transformed cells compared to empty-vector transduced controls, suggesting that WT1 expression is a normal consequence of PML-RARA activation in human hematopoietic progenitor cells. In summary, these findings support a cooperating role for WT1 mutations in APL development, and a potential mechanism to explain the high level of wild type WT1 expression observed in APL cells. Further experiments will be required to determine the downstream mechanisms by which WT1 antagonizes APL development, and whether these observations are generalizable to other AML subtypes. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Combinatorial Use of Bexarotene Plus ATRA for the Treatment of Acute Myeloid Leukemia

Aim: All-trans retinoic acid (ATRA) results in high response rates in acute promyelocytic leukemia (APL), but has not had a significant impact on non-APL forms of acute myeloid leukemia (AML), even though retinoic acid receptors have been implicated in hematopoietic maturation and self-renewal. RXRs bind as heterodimers with RARs, and are expressed at higher levels in AML cells than are RARs. We sought to determine whether natural RXR ligands exist in primary leukemia cells, whether RXR ligands might augment or inhibit leukemic cell growth, and whether RXRs might be rational targets for AML therapeutics.Background: Retinoid receptors are nuclear hormone receptors, induced during terminal myeloid maturation with highest expressed in M4 and M5 AML. Retinoids serve as intracellular messengers and are activating ligands for the retinoic acid receptors (RAR) and retinoid X receptors (RXR).Methods: We developed an in vivo reporter assay that measures intracellular, activating nuclear receptor ligands in mouse bone marrow cells. We generated transgenic mice with GFP regulated by a UAS promoter. The yeast Gal4 transcription factor recognizes UAS promoter sequences. When a fusion of the Gal4-DNA binding domain (DBD) and a retinoid receptor ligand-binding domain (LBD) is expressed in UAS-GFP bone marrow cells, and an activating ligand is present, the cells express GFP. Leukemia was generated in bone marrow cells from these mice and from Mx1-Cre x Rxrαfl/fl x Rxrβfl/fl mice using MSCV-MLL-AF9 retrovirus.Results: We found that intermediate levels of RXRA ligands are present in mouse MLL-AF9 leukemia cells in vivo. To determine if activation of RXRA is required for MLL-AF9 cell growth, we transduced MSCV-MLL-AF9 retrovirus into bone marrow cells from Mx1-Cre+ x Rxrαfl/fl x Rxrβfl/fl mice and transplanted these into recipient mice. Mx1-Cre is “leaky.” Without external activation of Mx1-Cre (i.e. without pIpC treatment) we found that all subsequent leukemias were associated with deleted (“floxed out”) Rxra and Rxrb alleles, suggesting that the deleted allele was positively selected for. In secondary transplants, we found that MLL-AF9 leukemias with deleted Rxrα and Rxrβ were associated with shorter survival when compared with MLL-AF9 leukemias with wild type Rxrα and Rxrβ, suggesting that naturally occurring RXR ligands suppressed leukemic growth. To determine if pharmacologic activation of retinoid receptors could inhibit MLL-AF9 cell growth, we treated MLL-AF9 cells ex vivo with ATRA and bexarotene. We found that ATRA modestly inhibited growth, and this effect reached a plateau at 62 nM (this may partially explain the limited clinical efficacy of ATRA in non-APL AML) (see Figure). In contrast, the EC50 of bexarotene was 125 nM and the EC50 decreased to 54 nM when bexarotene was combined with 62 nM ATRA. Higher doses of ATRA decreased the bexarotene EC50 further to 43 nM and resulted in near complete culture collapse. Finally, to determine whether combination bexarotene and ATRA inhibited cell growth by inducing differentiation or apoptosis, we examined cytomorphology, cell division by Celltrace Violet staining (similar to CFSE), and Annexin V. Unexpectedly, we observed no evidence of differentiation, but rather increased Annexin V associated with ATRA and bexarotene-induced growth arrest.Conclusion: RXRs act as tumor suppressors in MLL-AF9 murine leukemia cells in vivo, and the activation of RXR/RAR heterodimers with bexarotene and all-trans retinoic acid leads to synergistic apoptosis and growth arrest. Additional experiments are now being performed to test the efficacy of this dug combination in vivo and assess their potential use in elderly relapsed/refractory AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.