Block In Myeloid Differentiation Research Articles

Divergent Roles of MLL3 and MLL4 in Hematopoietic Stem Cell Fate Decision

Complex gene regulatory mechanisms, orchestrated by transcription factors (TFs) and epigenetic regulators, govern cell fate decisions in normal and malignant hematopoiesis. While prior works have extensively characterized the TF-networks in blood development, we currently have limited understanding of epigenetic regulation, in particular molecular rules underlying cofactor usage, in hematopoietic cell fate decisions. Kmt2c and Kmt2d are homologous genes that encode transcriptional coactivators, MLL3 and MLL4, respectively. They have been shown to coordinate embryonic stem cell fate transitions and are recurrently mutated in leukemias, offering an ideal paradigm to study epigenetic regulation of hematopoiesis. While MLL3 and MLL4 are often believed to play redundant roles in stem cell fate decisions, few has characterized their molecular distinctions. MLL3 and MLL4 each nucleate a chromatin-bound complex called the Complex of Proteins Associated with Set1 (COMPASS) and both deposit H3K4me1 at enhancers. Despite similar biochemical functions, Kmt2c and Kmt2d loss-of-function mutations have been reported with contrasting phenotypes in hematopoiesis. Kmt2d loss impairs HSC self-renewal and enhances myeloid differentiation at the expense of lymphopoiesis, whereas Kmt2c deletions enhance HSC self-renewal and impair differentiation. We hypothesize that MLL3 and MLL4 are recruited by different transcription factors and bind chromatin at both overlapping and distinct enhancers to convey specific hematopoietic fates. To test whether MLL3 and MLL4 are redundant in licensing myeloid differentiation, we transplanted wildtype, Kmt2c/d single knockout, and Kmt2c/d double knockout (DKO) donor cells into lethally irradiated recipients. Surprisingly, DKO recipients showed significant expansion of phenotypic HSCs, MPPs, and pre-granulocyte-monocyte progenitors (pGMs), typically associated with a myeloid differentiation block. DKO mice progressively developed bone marrow failure 9-months after transplantation, indicating that MLL3/4-COMPASS is necessary to sustain hematopoiesis. To dissect how Kmt2c/d deletions shape lineage specification and whether DKO imposes a differentiation block, we performed Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq) on donor Lin -Kit +Sca1 + (LSK) cells from the transplantation described above. While Kmt2c deletion does not markedly reprogram HSC/MPP identity, Kmt2d deletion results in precocious myeloid differentiation. Most notably, we observed that compound Kmt2c/d deletions fundamentally reprogramed HSCs/MPPs toward a B-lymphoid-like state, instead of imposing a myeloid differentiation block. The data establish a model wherein MLL3 and MLL4 have redundant roles in licensing HSC identity and myeloid potential. However, within the HSC to myeloid differentiation trajectory, MLL3 promotes differentiation while MLL4 promotes self-renewal. To understand how MLL3- and MLL4-COMPASS differentially regulate enhancer accessibility, we performed single-cell transposase-accessible chromatin sequencing (scATAC-seq) on the same donor LSK cells as in the CITE-seq. Compound Kmt2c/d deletions impose a unique epigenomic state on HSCs/MPPs (Figure 1A). Motif enrichment analyses revealed that DKO cells (i) were enriched for PAX5 and EBF1 motifs, consistent with B-lymphoid priming. HSCs (ii) were enriched for AP-1 and NFE2L2 motifs, suggesting that MLL4 may preferentially bind AP-1 and antioxidant response elements associated with HSC identity. Clusters of myeloid progenitors (iii and iv) were populated primarily by Kmt2d-deficient cells and enriched for CEBP and SPI1 motifs, suggesting that MLL3 is preferentially recruited to enhancers bound by myeloid TFs. This study provides several novel insights into epigenetic regulation of hematopoiesis. We have shown that HSC identity and myeloid fate specification are licensed by redundant MLL3/4-COMPASS-dependent TF-cofactor interactions, but once HSCs are established, MLL3- and MLL4-COMPASS engage distinct, antagonistic programs. Furthermore, early B-lymphoid priming is mediated via MLL3/4-COMPASS-independent mechanisms (Figure 1B). Ongoing work will link precise transcriptional codes to divergent cofactor utilization patterns and provide critical insights into how epigenetic regulators are deployed to effect distinct cell fates.

A Multiomic Single-Cell Atlas of Human Myelopoiesis Reveals Cellular and Molecular Drivers of Immunomodulatory Drug-Induced Neutropenia

Neutrophils are crucial innate immune cells which protect the host by killing infectious pathogens. Single cell RNA-sequencing analyses have previously described transcriptional heterogeneity of neutrophils and precursors, however the genome regulatory events underlying the transcriptional changes are less well characterised. Understanding this is important as neutropenia can be caused by dysregulation of the genome regulatory events that orchestrate neutrophil maturation. A good example is the use of immunomodulatory drugs (IMiDs, lenalidomide [LEN] & pomalidomide [POM]) for the treatment of multiple myeloma (MM) which are now standard-of-care, but their use is frequently complicated by neutropenia because of neutrophil maturation impairment. This is due to Cereblon (CRBN)-driven substrate ubiquitination and Ikaros degradation, causing PU.1 downregulation. However, restoration of Ikaros levels cannot fully alleviate the IMiD-induced myeloid differentiation block suggesting other mechanisms might also contribute. Our aim was to build a multiomic atlas of human myelopoiesis and use this to interrogate the molecular drivers of IMiD-induced neutropenia. We developed an ex vivo myeloid cell differentiation assay using human mobilized peripheral blood CD34 + cells from healthy donors and optimised methods for single cell multiomic (combined RNA-seq & ATAC-seq) analysis. This platform was validated orthogonally (immunophenotype, morphology & functional) and employed to construct a multi-omic single-cell landscape of normal human neutrophil differentiation and it's perturbation by IMiDs. Single cell transcriptomes from >110,000 cells from untreated and IMiD-treated samples were analyzed, capturing the full spectrum of human myelopoiesis spanning from early myeloid progenitors through to neutrophil populations (band, segmented, mature) (Fig.1A-left). Ex vivo differentiated neutrophils retained transcriptional signatures reflective of effector functions (granule biogenesis, chemotactic activity, respiratory burst) and mapped to previously described human neutrophil populations in vivo. Ex vivo generated neutrophils showed expected functional properties of phagocytosis and neutrophil extracellular trap formation upon stimulation. Exposure to IMiDs caused a maturation arrest with a 40-50% (p<0.001) decrease in abundance of differentiating metamyelocytes, and neutrophil populations. Crucially, this maturation arrest was due to an accumulation of transcriptionally distinct myeloid precursors that differentiated though an aberrant pathway (population 6, Fig 1A). Furthermore, immature erythroid clusters representing 10% of live cells accumulated specifically in IMiD-treated samples (p<0.001) in absence of erythropoiesis-stimulating cytokines. Simultaneous profiling of gene expression and chromatin accessibility in individual cells confirmed the presence of distinct phenotypes for untreated and IMiD-treated myeloid cells (Fig.1A) enabling the characterisation of the genome landscape of myelopoiesis, which has previously proven challenging. As expected, we observed dysregulation of Ikaros targets including PU.1 upon IMiD treatment (Fig. 1B-frame). The ability to study distinct and aberrant cellular populations at transcriptional and genome regulation level in parallel allowed us to identify several novel regulators of normal and perturbed neutrophil development. For example, we observed perturbed activation patterns of the transcription factors (TFs) zinc finger protein (ZNF467) and interferon regulatory factor 5 (IRF5) and the expression of their downstream targets as illustrated in Fig.1B. ZNF467 orchestrates a transcriptional program supporting terminal neutrophil differentiation in untreated cells (Fig.1B-top right), whilst the regulatory repertoire of IRF5, a hallmark of neutrophil activation is confined primarily to treatment-specific immature populations (Fig.1B-bottom right). We present a multiomic atlas of normal myelopoiesis which we believe will provide an important resource for the study or normal and perturbed neutrophil development. We use this platform to study IMiD-induced neutropenia and identify two aberrant cellular progenitor populations (aberrant myeloid precursors & erythroid progenitors) with distinct molecular properties as the likely cause of myelosuppression.

Haploinsufficient Transcription Factors in Myeloid Neoplasms.

Many transcription factors (TFs) function as tumor suppressor genes with heterozygous phenotypes, yet haploinsufficiency generally has an underappreciated role in neoplasia. This is no less true in myeloid cells, which are normally regulated by a delicately balanced and interconnected transcriptional network. Detailed understanding of TF dose in this circuitry sheds light on the leukemic transcriptome. In this review, we discuss the emerging features of haploinsufficient transcription factors (HITFs). We posit that: (a) monoallelic and biallelic losses can have distinct cellular outcomes; (b) the activity of a TF exists in a greater range than the traditional Mendelian genetic doses; and (c) how a TF is deleted or mutated impacts the cellular phenotype. The net effect of a HITF is a myeloid differentiation block and increased intercellular heterogeneity in the course of myeloid neoplasia.

YY1 Knockdown Relieves the Differentiation Block and Restores Apoptosis in AML Cells.

In this study we analyzed the expression of Yin and Yang 1 protein (YY1), a member of the noncanonical PcG complexes, in AML patient samples and AML cell lines and the effect of YY1 downregulation on the AML differentiation block. Our results show that YY1 is significantly overexpressed in AML patient samples and AML cell lines and that YY1 knockdown relieves the differentiation block. YY1 downregulation in two AML cell lines (HL-60 and OCI-AML3) and one AML patient sample restored the expression of members of the CEBP protein family, increased the expression of extrinsic growth factors/receptors and surface antigenic markers, induced morphological cell characteristics typical of myeloid differentiation, and sensitized cells to retinoic acid treatment and to apoptosis. Overall, our data show that YY1 is not a secondary regulator of myeloid differentiation but that, if overexpressed, it can play a predominant role in myeloid differentiation block.

TGFβ Inhibitor A83-01 Enhances Murine HSPC Expansion for Gene Therapy.

Murine hematopoietic stem and progenitor cells (HSPCs) are commonly used as model systems during gene therapeutic retroviral vector development and preclinical biosafety assessment. Here, we developed cell culture conditions to maintain stemness and prevent differentiation during HSPC culture. We used the small compounds A83-01, pomalidomide, and UM171 (APU). Highly purified LSK SLAM cells expanded in medium containing SCF, IL-3, FLT3-L, and IL-11 but rapidly differentiated to myeloid progenitors and mast cells. The supplementation of APU attenuated the differentiation and preserved the stemness of HSPCs. The TGFβ inhibitor A83-01 was identified as the major effector. It significantly inhibited the mast-cell-associated expression of FcεR1α and the transcription of genes regulating the formation of granules and promoted a 3800-fold expansion of LSK cells. As a functional readout, we used expanded HSPCs in state-of-the-art genotoxicity assays. Like fresh cells, APU-expanded HSPCs transduced with a mutagenic retroviral vector developed a myeloid differentiation block with clonal restriction and dysregulated oncogenic transcriptomic signatures due to vector integration near the high-risk locus Mecom. Thus, expanded HSPCs might serve as a novel cell source for retroviral vector testing and genotoxicity studies.

NPM1 mutation reprograms leukemic transcription network via reshaping TAD topology

C-terminal mutation of Nucleophosmin 1 (NPM1C+) was thought to be a primary driving event in acute myeloid leukemia (AML) that reprograms leukemic-associated transcription programs to transform hematopoietic stem and progenitor cells (HSPCs). However, molecular mechanisms underlying NPM1C+-driven leukemogenesis remain elusive. Here, we report that NPM1C+ activates signature HOX genes and reprograms cell cycle regulators by altering CTCF-driven topologically associated domains (TADs). Hematopoietic-specific NPM1C+ knock-in alters TAD topology leading to disrupted regulation of the cell cycle as well as aberrant chromatin accessibility and homeotic gene expression, which results in myeloid differentiation block. Restoration of NPM1 within the nucleus re-establishes differentiation programs by reorganizing TADs critical for myeloid TFs and cell cycle regulators that switch the oncogenic MIZ1/MYC regulatory axis in favor of interacting with coactivator NPM1/p300, and prevents NPM1C+-driven leukemogenesis. In sum, our data reveal that NPM1C+ reshapes CTCF-defined TAD topology to reprogram signature leukemic transcription programs required for cell cycle progression and leukemic transformation.

Abstract A25: A niche directed therapy for the treatment of myelodysplasia and acute myeloid leukemia

Abstract Cells of the surrounding bone marrow microenvironment (niche) have emerged as important regulators of myeloid disease development and progression, leading to myeloproliferative neoplasms, myelodysplasia (MDS) or acute myeloid leukemia (AML). This not only highlights the complexity of the disease but may, at least in part, explain the limitations of current malignant cell targeted therapies to prevent relapse and at the same time opens new avenues for therapeutic intervention. To test this hypothesis, we examined here the therapeutic potential of targeting a potent, niche-driven oncogenic pathway, constitutive activation of b-catenin/Jagged1 signaling in osteoblasts. In humans, this pathway is activated in approximately 40% of MDS and AML patients; and also following hypermethylation of its regulators in MDS patients. Its activation levels increase with disease severity, correlate with MDS to AML transformation and with del(5q)-associated myeloid malignancies. In mice, it leads to MDS rapidly progressing to AML. To test its therapeutic potential, we inhibited Jagged1. We generated a chimeric human-mouse neutralizing antibody that efficiently and specifically binds JAG1 (anti-JAG1) and inhibits Notch1-induced signaling. Administration of anti-JAG1 in leukemic mice with activated b-catenin/Jag1 in their osteoblasts rescued anemia, thrombocytopenia, neutrophilia and lymphocytopenia, relieved myeloid differentiation block and eliminated blasts. Body weight increased with time and lethality was abrogated in treated mice. Blood chemistry profiling indicated lack of any toxicity following treatment as indicated by normal liver and kidney function and absence of inflammation, dyslipidemia or pancreatitis. In contrast, chemotherapy at a dose simulating the induction regimen used in patients, dramatically exacerbated anemia, thrombocytopenia and lymphocytopenia without decreasing blasts leading to increased lethality due to bone marrow failure. Emphasizing relevance to human disease, anti-JAG1 treatment of patient-derived samples with activated b-catenin/JAG1 in their osteoblasts, inhibited MDS and AML cell growth and survival and promoted myeloid and erythroid differentiation through its actions on osteoblasts. Responsiveness was observed across patients belonging to diverse disease subtypes and categories including patients with adverse cytogenetics and high-risk groups. Confirming the specificity of anti-JAG1 action, no effect was observed in cells from patients without activated b-catenin/JAG1 in their osteoblasts or healthy subjects and the magnitude of the response correlated with the levels of b-catenin/JAG1 activation in osteoblasts. These results suggest the therapeutic efficacy of blocking JAG1 and its superiority to chemotherapy in osteoblastic, b-catenin-driven MDS/AML that could impact 1/3 of MDS and AML patients. In addition, they suggest that targeting the niche may be an approach to avoid toxicity and overcome MDS/AML cell mutation dependence and clonal resistance that follows standard of care, and therefore, prevent relapse. Citation Format: Ioanna Mosialou, Abdullah M Ali, Rachel Adams, Adam Corper, Catherine M Woods, Xiaomin Fan, Azra Raza, Stavroula Kousteni. A niche directed therapy for the treatment of myelodysplasia and acute myeloid leukemia [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A25.

Fructose Treatment Targets Myeloid Leukemogenesis in IDH2 Mutants By Exhausting Alpha-Ketoglutarate Pools

Acute Myeloid Leukemia (AML) is a heterogeneous group of hematological malignancies and represents the most frequent cause of leukemia-related deaths. The uncontrolled growth of myeloblasts in the Bone Marrow (BM) leads to accelerated metabolic processes and high consumption of nutrients, especially glucose. Isocitrate dehydrogenase (IDH) 1 and 2 are frequently mutated in human cancer, affecting up to 15% of AML patients. The neo-morphic activity conferred by IDH 1 or 2 mutations (mIDH1/2) converts α-ketoglutarate (α-KG) to the R-enantiomer of 2-hydroxyglutarate (2-HG), which competitively inhibits α-KG-dependent dioxygenases (Figueroa et al., 2010, Su et al.,2018, Li et al.,2017) and contributes to a block in myeloid differentiation and leukemic transformation. mIDH1/2 are clinically validated therapeutic targets as specific inhibitors have been approved by the FDA for the treatment of AML(DiNardo et al., 2018; Rohle et al., 2013; Stein et al., 2019; Wang et al., 2013) However, only 40% or less of mIDH AML patients respond to IDH inhibitor therapy (DiNardo et al., 2018; Stein et al., 2017) and little is known of how IDH mutations can be exploited for new metabolic vulnerabilities. We recently reported a novel metabolic dependency in AML cells that express low levels of the fructose transporter GLUT5. In fructose conditions these AMLs rewired their metabolism away from glycolysis and toward the serine synthesis pathway (SSP), (Jeong and Savino, Cell Metabolism 2021). Inhibiting the SSP by blocking PHGDH under these metabolic conditions resulted in reduced tumor burden. Additionally, patients with FLT-ITD alterations were sensitive to SSP blockade (Bjelosevic et. al. 2021). Although we previously found that myeloid leukemia cells grow normally in fructose, AML cell lines engineered to express IDH2 R140Q (TF1 knock in, and transduced MOLM13, U937 and OCIAML2) had a 50% reduction in cellular growth and 1.6-fold increase in differentiation (CD15 marker). Remarkably, a similar effect was observed in mIDH1 or 2 chondrosarcoma cell lines, suggesting that this metabolic program is more generally applicable. Excitingly, this new fructose induced therapeutic vulnerability could be recapitulated in vivo. We found that the fructose treated mice could delay myeloid leukemogenesis by 9-10 days (MOLM13 IDH2 R140Q) or in a primary patient sample successfully engrafted with IDH2 R140Q (n=6 mice/group p=0.0012; 3 weeks, I.P. 4 g/kg). To understand the basic mechanism for this new metabolic vulnerability, we first focused on the flux though the SSP. We found an increase in the end product of the SSP, glycine, as measured by LC-MS in the fructose condition compared to glucose. This SSP addiction under fructose was further demonstrated by reduced growth upon PHGDH inhibition (mIDH2: 75% reduction vs 50% WT IDH2 p=.0004; NCT503 5uM). Utilization of the SSP results in the generation of cytosolic α-KG, the substrate of IDH mutant enzymes. Thus, we hypothesized that mIDH AML cells might display enhanced dependency due to the requirement of α-KG for SSP, mIDH2 activity and to sustain the otherwise defective TCA cycle. Consistent with α-KG's central role in fructose driven metabolic addiction, we rescued the cell growth reduction by treating with cell permeable a-KG. Furthermore, in fructose conditions, we observed significant accumulation of the TCA cycle intermediate malate as well as aspartate, suggesting an engagement of the malate-aspartate shuttle in maintaining the redox balance. Previously, we found that AML cell lines (i.e THP1 cells) with moderate to high abundance of surface GLUT5, were not dependent on SSP in fructose. Surprisingly, by introducing IDH2 R140Q or by treating the cells with exogenous D-2HG, we found a 2-fold reduction of surface GLUT5 protein. This reduction of GLUT5 rewired the metabolism toward the SSP-addiction and resulted in a 50% reduction in cell growth in fructose. Interestingly, GLUT5 was controlled through transcriptional regulation of the locus as early as 8hrs post D-2HG treatment. Importantly, this relationship was found to be associated in patients, where PHGDH expression was significantly increased in mIDH2 AML patients with low vs high GLUT5 (n=15, n=16 p<.038) compared to other mutational subtypes (n=155, n=155 ns). In summary, we find a surprising new metabolic dependency based on sugar conditions in mIDH2 AML that could pave the way for a new therapeutic strategy.

Therapeutic Anti-Jagged1 Antibody Targeting Osteoblast-Related Myeloid Dysplasia to Overcome Standard of Care Resistance

Acute myeloid leukemia (AML) and myelodysplasia (MDS) are increasingly recognized as not only genetic but niche-dependent malignancies prompting the question of whether targeting signals from supporting stromal cells may provide a more stable target for therapy than emerging leukemic clones. Whereas standard of care and malignant cell targeted therapies fail to eliminate resistant clones or prevent the expansion of new ones, inhibition of oncogenic or tumor promoting signals from the stroma could overcome mutation dependance and clonal resistance and be an efficient approach to manage disease and prevent relapse. Here, we inhibited a potent, niche-driven tumorigenic pathway, constitutive activation of b-catenin/Jagged1 signaling in osteoblasts. This pathway is activated following hypermethylation of its regulators in MDS patients; is activated in approximately 40% of MDS and AML patients; its activation levels increase with disease severity and progression; correlate with MDS to AML transformation and del(5q)-associated myeloid malignancies and leads to MDS rapidly progressing to AML in mice. To examine the therapeutic potential of targeting this pathway we generated a human-mouse chimeric antibody that efficiently and specifically binds JAG1 (anti-JAG1) and inhibits Notch1 signaling. Administration of anti-JAG1 in leukemic mice with osteoblastic active b-catenin rescued anemia, thrombocytopenia, neutrophilia and lymphocytopenia, restored LT-HSCs (the leukemia initiating population), relieved myeloid differentiation block and eliminated blasts. Body weight increased and lethality was abrogated in treated mice. In contrast, chemotherapy at a dose simulating induction regimen used in humans, dramatically exacerbated anemia, thrombocytopenia and lymphocytopenia without decreasing blasts leading to increased lethality due to bone marrow failure. A lower chemotherapy dose induced a physiologically relevant response in wild type mice, but failed to improve hematopoiesis and myeloid differentiation block in leukemic mice and as a result AML progression and lethality was not affected. Emphasizing relevance to human disease, anti-JAG1 treatment of patient-derived samples with activated b-catenin in their osteoblasts, inhibited MDS and AML cell growth and survival and promoted myeloid and erythroid differentiation irrespective of disease category through its actions on osteoblasts. These findings suggest anti-JAG1 may provide a broad therapeutic approach that is effective in 1/3 of MDS and AML patients with cytogenetically distinct types of disease. The therapeutic potential of targeting the bone marrow stromal niche in hematological malignancies can be a treatment strategy that may evade relapse and overcome standard of care resistance and toxicity.

Isocitrate Dehydrogenase 2 Mutation Drives Bone Marrow Macrophage Dysfunction without a Complete Block in Hematopoietic Differentiation

Myelodysplastic syndromes (MDS) represent a broad category of myeloid malignancies characterized by myeloid skewed hematopoiesis, blood cytopenias and increased risk of transformation to leukemia. While not common, isocitrate dehydrogenase 2 (IDH2) mutations are represented in clonal hematopoiesis and in MDS, are found in patients with higher International Prognosis Scoring System (IPSS) disease scores and confer a higher risk of transformation to leukemia. IDH2 mutations lead to metabolic dysfunction and DNA hypermethylation due to the accumulation of D-2-hydroxyglutarate (2HG), an oncometabolite which disrupts hematopoiesis. However, the mechanisms that induces clonal progression and transformation remains incompletely understood. Furthermore, the intrinsic and extrinsic impact of IDH mutation on mature myeloid cell functions and the bone marrow microenvironment (BMME) remains to be elucidated. Using RAW264.7 cells, a monocyte/macrophage-like murine cell line, we retrovirally infected cells with human wt IDH2 or the R172K IDH2 mutant, a common IDH2 mutation. After confirming a 30-fold increase of 2HG (p<.005) and observing evidence of hypermethylation in the IDH2 mutant RAW cells, we tested several key functions of bone marrow macrophages: ability to engulf apoptotic cells (efferocytosis), erythropoietic support, and cytokine production. After labeled apoptotic neutrophils were fed as efferocytic targets to RAW cells at a 10:1 ratio for 6 hours, we observed a 10% decrease of efferocytosis in IDH2 mutants (p < 0.05). Next, we cocultured RAW cells with primary murine late-stage erythroid progenitors and quantified their capacity to form colonies (CFU-E) in the presence of erythropoietin. Compared to wt, IDH2 mutant RAW cells inhibited CFU-E formation by 25% (p < 0.0075). Cytokine array on cell culture supernatant from mutant RAW cells had elevated proinflammatory chemokines/cytokines (CCL5, CXCL10, TNF-a) and decreased cytokines related to inflammation resolution and efferocytosis (GDF-15, Gas6, Endostatin, CCL2). To test these findings further in vivo we generated a murine bone marrow transplant model with retrovirally infected hematopoietic stem cells (HSC: lineage-, c-Kit+, Sca-1+) containing the human wt or R172K IDH2 mutation with an mCherry reporter and transplanted into CD45.1 congenic mice (10 mutant, 10 wt, 1:1 male:female). Mutant IDH2 HSCs demonstrated better engraftment (85-90%) compared to wt (70-80%) and also had higher levels of mCherry positivity within CD45.2+ cells (80-90% mutant vs 40-60% in wt). Longitudinal blood draws showed macrocytic anemia, thrombocytopenia, and elevated reticulocytes in IDH2 mutants by 16 weeks post-transplant. In contrast to current literature, IDH2 mutant cells did not exhibit a complete block in myeloid cell differentiation, as the mutant clone gave rise to mature monocytes, macrophages, and neutrophils. We also harvested bone marrow at 16 weeks post transplantation. Compared to wt IDH2, the mutant IDH2 bone marrow cells exhibited myeloid skewing hematopoiesis and a 3-fold increase in bone marrow monocytes (CD45+, Ly6G-, Ly6C++, F4/80+, p < 0.00001). In the IDH2 mutant bone marrow cells, myeloid progenitors were also expanded: common myeloid progenitors (CMPs: 4-fold increase p < 0.001, CD45+, lineage-, c-Kit+, Sca-1-, CD34+, CD16/32-), and granulocytic monocytic precursors (GMPs: 3-fold increase p < 0.0001, CD45+, c-Kit+, Sca-1-, CD34+, CD16/32+). We also observed a previously unreported loss of long-term hematopoietic stem cells (LT-HSC: 13.5-fold reduction, p < 0.00001, CD45+, lineage-, c-Kit+, Sca-1+, Flt3-, CD48-, CD150+). When cultured ex vivo, there was loss of efferocytic capacity in IDH2 mutant bone marrow derived macrophages fed normal apoptotic neutrophils at a 10:1 ratio for 3 or 24 hours. In summary, we identify defects in efferocytic function and cytokine production in monocytes and macrophages derived from IDH2 mutant hematopoietic stem and progenitor cells. We have previously shown that defects in the efferocytic function of bone marrow macrophages induce increases in inflammatory signals and contribute to skewing of hematopoiesis. Since these macrophage dysfunctions have not previously been attributed to IDH2 single mutation, they represent a potential novel mechanism that could alter the bone marrow microenvironment and impact progression of MDS and transformation to leukemia.

NPM1 Mutation Reprograms Leukemic Transcription Network Via Reshaping CTCF-Defined TAD Topology

C-terminal mutation of Nucleophosmin 1 (NPM1C+) was thought to be a primary driving event in acute myeloid leukemia (AML) that reprograms leukemic-associated transcription programs to transform hematopoietic stem and progenitor cells (HSPCs). However, it remains poorly understood how NPM1C+ mutation results in altered myeloid master transcription program and HOX expression profile leading to AML disease and whether NPM1 mutant associated transcription signature can be targeted to prevent pathogenesis of acute myeloid leukemia. Here, we report that NPM1C+ reprograms MIZ-1/MYC regulatory axis by altering NPM1-associated CTCF-driven topologically associated domains (TADs) that switches the balance of MIZ1 interaction with coactivator NPM1/p300 and corepressors MYC/G9A complexes to control cell cycle progression and myeloid lineage-specific PU.1/CEBPa transcription networks leading to impairment of myeloid differentiation and leukemic transformation. To test the hypothesis that mislocalization of the NPM1C+ mutant to cytoplasm may alter CTCF mediated three-dimensional (3D) TADs, we carried out Hi-C to examine whether NPM1C+ mutation altered hematopoietic TAD integrity by comparing mouse bone marrow (BM) Lin-cKit+ (LK) stem- and progenitor-enriched cells purified from WT and NPM1C+ knock-in animals, which develop a myeloproliferative neoplasm/myelodsplasia-like condition with a long latency characterized by enlarged spleens, leukocytosis, and thrombocytopenia. Hematopoietic-specific NPM1C+ knock-in alters TAD topology leading to altered chromatin accessibility and NPM1 mutant associated gene expression signature, which results in disrupted regulation of the cell cycle and myeloid master transcription factors, and eventually myeloid differentiation block. In contract, Retention of NPM1 in OCI-AML3 nucleus, a NPM1C+ mutant driven AML cells, reverses NPM1C+-driven signature TAD formation and gene transcription leading to proper cell cycle control and myeloid differentiation. Mechanistically, we demonstrated that cytoplasmic mislocalization of NPM1C+ mutant exerts two coordinated effects: (a) alteration of CTCF dependent TAD structure, and (b) MYC-MIZ-1 transcription axis. Both of these regulate target genes critical for cell cycle control and myeloid master transcription factors, Pu.1 and CEBPA, mediated myeloid differentiation. Re-activation of Pu.1 or CEBPa within the nucleus by CRISPR-dCas9 mediated endogenous promoter activation restores myeloid differentiation programs by reorganizing TADs critical for myeloid TFs and cell cycle regulators, and prevents NPM1C+-driven gene expression. FinalIy, when we transplanted NPM1OE, activated Pu.1, or activated CEBPA OCI-AML3 cell line or primary NPM1C+ AML patient cells into NSG mice along with control cells, NPM1 overexpression or re-activation of PU.1 or CEBPA in OCI-AML3 cells or NPM1C+ primary AML patient cells prolonged transplanted or PDX mice survival and mitigate AML leukemic blast in transplant or PDX mouse models. In sum, our data reveal that NPM1C+ reshapes CTCF-defined TAD topology to reprogram signature leukemic transcription programs required for cell cycle progression and leukemic transformation. Restoration of myeloid transcription program in nucleus reversed NPM1C+-driven transcription signature and promotes myeloid differentiation leading to mitigation of AML.

PB1781: THERAPEUTIC POTENTIAL OF DNA DAMAGE RESPONSE IN ACUTE MYELOBLASTIC LEUKEMIA

Background: Genome integrity is of utmost importance for cell survival and homeostasis maintenance. However, DNA integrity is threatened by endogenous and exogenous sources. DNA lesions can block genome replication and transcription, and if unrepaired or repaired incorrectly can lead to mutations and genomic instability. Hence, cells have developed a variety of mechanisms to protect genome integrity and repair damaged DNA, collectively termed the DNA damage response (DDR). These mechanisms comprise DNA damage recognition, damage tolerance, signal transduction, transcriptional regulation, cell cycle control, and DNA repair. Key DDR elements include sensor proteins that detect damage (e.g. PARP1 and 2), mediators and signaling transducers (e.g. DNA-PK) that regulate downstream targets, and effector molecules (e.g. CHK1 and 2). Increased DNA damage and altered DDR are critical features of genetic instability presumably implicated in the pathogenesis of acute myeloblastic leukemia (AML). AML is a clonal malignant disease of hematopoietic stem and/or progenitor cells characterized by a block in myeloid differentiation and increased proliferation. This neoplasia is considered the most common acute leukemia in adults and is characterized by recurrent genetic abnormalities. Recently, specific recurrent chromosomal translocations and genetic mutations typical of AML have been associated with DNA repair defects. Aims: Our goal is to assess the potential of DDR as a therapeutic target in AML, using in vitro models, in order to identify new therapeutic approaches. Methods: Five AML cell lines of different molecular and cellular subtypes were used (HEL, HL-60, K-562, LAMA-84, and NB-4). Cells were incubated in the absence and presence of increasing concentrations of two DDR inhibitors, CCT245737 (CHK1 inhibitor) and Niraparib (PARP1/2 inhibitor). Cell density and viability were assessed, for 72 hours, by trypan blue assay. Afterward, resorting to flow cytometry, the cell cycle was assessed using propidium iodide/RNAse, and the cell death was determined using Annexin V/7-AAD double staining as well as by optic microscopy (May-Grünwald-Giemsa staining). Results were statistical analyzed, considering a significance level of 95%. Results: CCT245737 reduced cell proliferation and viability in a dose-, time- and cell line-dependent manner. NB-4 was the most sensitive cell line, with an IC50 at 48 hours of 54 µM. In contrast, LAMA-84 was the most resistant cell line with an IC50 at 48 hours of 203 µM, 3.7-fold higher than NB-4. PARP1/2 inhibition also reduced cell proliferation and viability in a dose-, time- and cell line-dependent manner. Once again NB-4 was the most sensitive cell line, with an IC50 at 48 hours of 25 µM and LAMA-84 was the most resistant with an IC50 at 48 hours of 66 µM, 2.7-fold higher than NB-4. Both inhibitors induced cell death by apoptosis since an increase in the percentage of cells in late apoptosis was observed. This result is in agreement with the observed by optic microscopy. We also detected a cytostatic effect since a cell cycle arrest in the S phase was observed after treatment with both inhibitors. Summary/Conclusion: AML cell lines have different sensibilities to DDR inhibitors, particularly CCT245737 and niraparib, with the NB-4 cell line appearing to be the most sensitive to DDR inhibition (namely CHK1 and PARP1/2 inhibition), whereas LAMA-84 appears to be the most resistant to this inhibition. In conclusion, this work may help to identify new therapeutic approaches that could eventually improve AML patients’ outcomes.

The BCAT1 CXXC Motif Provides Protection against ROS in Acute Myeloid Leukaemia Cells.

The cytosolic branched-chain aminotransferase (BCAT1) has received attention for its role in myeloid leukaemia development, where studies indicate metabolic adaptations due to BCAT1 up-regulation. BCAT1, like the mitochondria isoform (BCAT2), shares a conserved CXXC motif ~10 Å from the active site. This CXXC motif has been shown to act as a ‘redox-switch’ in the enzymatic regulation of the BCAT proteins, however the response to reactive oxygen species (ROS) differs between BCAT isoforms. Studies indicate that the BCAT1 CXXC motif is several orders of magnitude less sensitive to the effects of ROS compared with BCAT2. Moreover, estimation of the reduction mid-point potential of BCAT1, indicates that BCAT1 is more reductive in nature and may possess antioxidant properties. Therefore, the aim of this study was to further characterise the BCAT1 CXXC motif and evaluate its role in acute myeloid leukaemia. Our biochemical analyses show that purified wild-type (WT) BCAT1 protein could metabolise H2O2 in vitro, whereas CXXC motif mutant or WT BCAT2 could not, demonstrating for the first time a novel antioxidant role for the BCAT1 CXXC motif. Transformed U937 AML cells over-expressing WT BCAT1, showed lower levels of intracellular ROS compared with cells over-expressing the CXXC motif mutant (CXXS) or Vector Controls, indicating that the BCAT1 CXXC motif may buffer intracellular ROS, impacting on cell proliferation. U937 AML cells over-expressing WT BCAT1 displayed less cellular differentiation, as observed by a reduction of the myeloid markers; CD11b, CD14, CD68, and CD36. This finding suggests a role for the BCAT1 CXXC motif in cell development, which is an important pathological feature of myeloid leukaemia, a disease characterised by a block in myeloid differentiation. Furthermore, WT BCAT1 cells were more resistant to apoptosis compared with CXXS BCAT1 cells, an important observation given the role of ROS in apoptotic signalling and myeloid leukaemia development. Since CD36 has been shown to be Nrf2 regulated, we investigated the expression of the Nrf2 regulated gene, TrxRD1. Our data show that the expression of TrxRD1 was downregulated in transformed U937 AML cells overexpressing WT BCAT1, which taken with the reduction in CD36 implicates less Nrf2 activation. Therefore, this finding may implicate the BCAT1 CXXC motif in wider cellular redox-mediated processes. Altogether, this study provides the first evidence to suggest that the BCAT1 CXXC motif may contribute to the buffering of ROS levels inside AML cells, which may impact ROS-mediated processes in the development of myeloid leukaemia.

MECOM gene overexpression in pediatric patients with acute myeloid leukemia

Background Acute myeloid leukemia (AML) is characterized by blocked or aberrant differentiation of hematopoietic stem cells. The MECOM gene overexpression in hematopoietic progenitors induces myeloid differentiation block, resulting in increased self-renewal and survival of these transformed progenitors. However, its exact role in AML remains unclear. We aimed to estimate the prevalence of MECOM overexpression among pediatric AML patients, and assess its impact on clinical outcome. Patients and Methods Real-time quantitative polymerase chain reaction and Livak method (2ΔΔCt) were used to determine relative MECOM expression level among 243 pediatric patients with AML. MECOM overexpression was considered if the cumulative relative expression was above 1 (2-ΔΔCt) and was designated as MECOMpos . Results Of 243 AML patients tested 57(23.5%) demonstrated MECOMpos . Patients with MECOMpos had significantly lower median age. The frequency of MECOMpos was significantly higher among AML patients with 11q23 abnormalities, complex karyotypes and among high- and intermediate-risk groups compared to low-risk group (p = .014). MECOMpos patients had significantly lower overall survival (OS) (38.7 vs. 78.9%, p < .001), event-free survival (EFS) (37.3% vs. 68.4%, p < .001), and had higher cumulative incidence of relapse (49.5% vs. 23.5%, p = .002) at 36 months compared to MECOMneg patients. Multivariate analysis revealed that MECOMpos was an adverse prognostic factor for OS (hazards ratio (HR) = 2.11, 95% confidence interval (CI) 1.24–3.60, p = .006) and EFS (HR= 1.71, 95% CI 1.07–2.75, p = .025). The logistic regression model showed that MECOMpos was an independent prognostic factor regardless of minimal residual disease status post first induction therapy in the intermediate-risk group (odds ratio 2.89; 95% CI 1.19–6.57, p = .018). Conclusion The aberrant MECOM gene expression is an adverse prognostic factor, especially in patients without previously known cytogenetic risk factors. Our results suggest the potential benefit from pretreatment screening for MECOM gene overexpression in newly diagnosed AML patients for better risk stratification and treatment adjustment.

Modeling Clonal Progression in SF3B1-Mutant Myelodysplastic Syndrome

SF3B1-mutant myelodysplastic syndrome (MDS) has recently been proposed as a distinct disorder characterized by ring sideroblasts, ineffective erythropoiesis and good prognosis. Selected co-occurring genetic abnormalities were reported associated with significantly worse outcome and suggested as exclusion criteria for the proposed entity. However, it remains unclear how a limited spectrum of co-occurring drivers affects SF3B1-mutant MDS biology to determine evolution from a relatively indolent condition to high risk malignancy.To gain a better insight into the clonal progression of SF3B1-mutant MDS, we analyzed SF3B1 co-mutations in a cohort of 176 SF3B1-mutated patients diagnosed with a myeloid neoplasm. RUNX1 and STAG2 were the only co-mutated genes found significantly associated with advanced disease phenotype (i.e. MDS with excess blasts and secondary acute myeloid leukemia) (OR=18.36 (2.18-862.91), P=0.001, and OR= Inf (3.57-Inf), P<.001, respectively), and reduced overall survival (P<.001). Based on these data, we hypothesized that acquisition of RUNX1 or STAG2 co-mutations in patients with SF3B1-mutant MDS drives progression to high-risk neoplasms.RUNX1 and STAG2 control hematopoietic stem and progenitor cell self-renewal and differentiation by regulation of gene expression. To explore the biological impact of RUNX1 or STAG2 loss in the context of SF3B1-mutant MDS, we disrupted RUNX1 or STAG2 using CRISPR/Cas9 in an induced pluripotent stem cell (iPSC) model of MDS-RS that we previously established by reprogramming of bone marrow cells from SF3B1-mutant individuals. This patient-derived system allows hematopoietic progenitor expansion through doxycycline-mediated expression of 5 transcription factors (5F-HPC) and multilineage differentiation upon doxycycline withdrawal. We asked how RUNX1 or STAG2 disruption affected SF3B1-mutant HPCs self-renewal and differentiation in our model of MDS-RS, and sought to define the underlying transcriptional changes.RUNX1-edited cells maintained significantly higher proportion of CD34 + HPCs, suggesting that RUNX1 mutation increases self-renewal capacity of SF3B1-mutant progenitors. Consistently, SF3B1/RUNX1 double-mutant 5F-HPCs showed positive enrichment of HSC-specific gene signatures. This was accompanied by broad upregulation of inflammatory programs, recapitulating the activated gene signatures we identified in SF3B1/RUNX1 co-mutated patients in our cohort. RUNX1 disruption promoted myeloid skewing at the expense of erythroid differentiation in SF3B1-mutant cells. Consistent with this, granulocyte-monocyte progenitors (GMP) and myelo-erythroid transcriptional programs were positively and negatively enriched, respectively. By contrast, in SF3B1/STAG2 double-mutant 5F-HPCs, both erythroid and myeloid populations were reduced compared to SF3B1 single mutant controls, suggesting a block in both myeloid and erythroid differentiation, despite the presence of pro-differentiation signals. This was supported by a profound down-regulation of genes involved in the response to external stimulus and suppression of GMP specific transcriptional signature.In summary, we identified RUNX1 and STAG2 mutations as main drivers of disease progression in SF3B1-mutant patients, and generated extensive cell line panels to interrogate their functional interaction with mutant SF3B1. By applying CRISPR/Cas9 editing to our MDS-RS model, we could overcome limited availability of primary MDS samples to show that RUNX1 or STAG2 co-mutations drive progression through distinct biological mechanisms in SF3B1-mutant HPCs. DisclosuresNo relevant conflicts of interest to declare.

IDH Mutated AML: Beyond Enasidenib and Ivosidenib Monotherapy

Introduction Mutations in the isocitrate dehydrogenase (IDH) genes, IDH1 or IDH2, occur in approximately 20% of patients with acute myeloid leukemia (AML) and promote leukemogenesis through a block in normal myeloid differentiation and through the production of the oncometabolite 2-hydroxyglutarate (2HG).1 Selective oral inhibitors of mutant IDH1 and IDH2, leading to restoration of normal myeloid differentiation and reduction of 2HG have been developed, and are now approved as single-agent therapy, based on clinical efficacy observed within the original first-in-human trials.

RUNX1 Mutations Lead to a Myeloid Differentiation Block in Association with Enhanced Proliferation By Altering the RUNX1 Transcriptional Program

RUNX1 (AML1) is a transcription factor critically involved in normal haematopoiesis. Inactivating RUNX1 mutations have been frequently described in a variety of myeloid neoplasms, including high-risk myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Here, we aimed to functionally and molecularly define the actions of a dominant negative mutant.Overexpression of the RUNX1 mutant S291fs300X (RUNX1 mt) in cord blood (CB) CD34+ cells caused a decline in erythroid colony formation (p= 0.01) which was associated with an immature morphology and an increased number of Annexin V positive erythroid cells. The negative phenotype coincided with a strong reduction in GATA-1 and GATA-2 expression in RUNX1mt CD34+ CB cells after 24-36 hours transduction. On the contrary, the CFU-GM colonies showed a greater than 3-fold enhanced replating capacity compared to control. It appeared that the replating potential was predominantly present in the CD34+CD38- and CD14-CD15- cell population.Upon long-term MS5 stromal co-cultures of transduced RUNX1 mt CB CD34+ cells a rather homogenous cell population was obtained after 8-10 weeks that localized underneath the stromal layer. This cell population was phenotypically defined by CD34+/CD38-. The interactions with the stroma appeared to be important to limit proliferation and retain quiescence. Suspension cultures with myeloid growth factors (IL-3, SCF) of RUNX1 mt CB CD34+ cells provided a similar cell population after 10 weeks of culture. These cells were growth factor-dependent and were phenotypically defined by CD34+/CD38+/CD33+/CD45RA+/CD123+, resembling a GMP phenotype which could be propagated for approximately 20 weeks in suspension. Karyotype analyses of this cell population demonstrated no abnormalities while integration site analysis showed multiple different integration sites that differed between individual experiments, suggesting that the myeloid differentiation block was related to the RUNX1 mutation and not to aspecific integration site effects. Comparable results were obtained with adult normal bone marrow CD34+ cells or peripheral blood CD34+ cells transduced with the RUNX1 mt.Morphological analysis after 8-10 weeks of culture showed monoblastic cells with limited macrophage differentiation but without signs of granulocytic differentiation, even in the presence of G-CSF. This might might be due to downregulation of C/EBPα, one of the key targets of RUNX1. Therefore, week 10 RUNX1 mt CB CD34+ cells were transduced with a retroviral C/EBPα-ER overexpression vector. Re-expression of C/EBPα by tamoxifen induction resulted in a reduction in cell proliferation, decline of undifferentiated blasts and an increase in CD15 expression.In our goal to asses RUNX1 mt target binding and gene expression changes at early time points we used induced pluripotent stem cells (iPS cells) with conditional expression of RUNX1 mt. Expression of RUNX1 mt during early differentiation leads to a differentiation block with the maintenance of CD34+ cells. Genome-wide binding analysis of these RUNX1 mt cells revealed RUNX1 occupancy at similar regions as observed in primary AMLs with a RUNX1 mutation. These binding sites were enriched for RUNX and ETS motifs and associated with genes important for differentiation and chromatin regulation. Moreover, expression analysis revealed a severely altered RUNX1 transcription program in the presence of the RUNX1 mt.Together these results reveal that the RUNX1 mutant S291fs300X, as a single oncogenic hit, induces cell proliferation and a myeloid differentiation block by altering the RUNX1 gene program. DisclosuresNo relevant conflicts of interest to declare.

A Metabolic Interplay Coordinated By Hlx Balances Hematopoietic Stem Cell Maintenance and Differentiation

Upregulation of the non-clustered homeobox transcription factor HLX (H2.0-like Homeobox) is frequently observed in patients with acute myeloid leukemia, but its mechanism of action remains obscure. Since developmental pathways are often reactivated in cancer, we asked whether hlx1 plays a role during hematopoietic development in zebrafish. Endothelial specific overexpression of human HLX led to aberrant production of hematopoietic stem and progenitor cells (HSPCs) and increased numbers of immature myeloid cells at 48 hpf (hours post fertilization), thereby mimicking a preleukemic phenotype. On the other hand, hlx1 morphants had diminished numbers of HSPCs, a defect that could be rescued by overexpressing human HLX under control of either an endothelial or HSPC specific promoter. To delve into the molecular mechanism of HLX function, we performed expression analysis of sorted endothelial cells from zebrafish that overexpress HLX and hlx1 morphants. Overexpression of HLX led to downregulation of mitochondrial electron chain enzymes (NDUFS4, SDHD, CYTB, COX7A2) and upregulation of the peroxisome proliferator activated receptor family member δ (PPARδ). Downregulation of hlx1 led to diminished expression of PPARδ. Analysis of open chromatin by ATAC-seq revealed altered chromatin accesibility around the vicinity of these genes in hlx1 morphants. To validate functionally these results we measured the mitochondrial membrane potential by Tetramethylrhodamine, Methyl Ester, Percholate (TMRM). TMRM was decreased upon HLX overexpression and increased in hlx1 morphants. These results suggested that HLX regulates the fate of hematopoietic cells by fine tuning mitochondrial membrane potential and PPARδ. Indeed PPARδ agonists could rescue HSPC generation in zebrafish morphants, while PPARδ antagonists relieved the differentiation block of myeloid cells. Direct regulation of mitochondrial genes and PPARδ by HLX was established by chromatin immunoprecipitation in human cell lines. To test whether there is a conserved, intrinsic role of HLX in primary hematopoietic progenitor cells, we used human progenitor CD34+ cells. In accordance with our results in zebrafish, overexpression of HLX led to increased numbers of colonies in colony forming unit assays and a myeloid differentiation block, whereas downregulation greatly compromised the ability of CD34+ cells to form colonies. HLX overexpression resulted in diminished oxidative phosphorylation and mitochondrial membrane potential whereas downregulation of HLX coincided with increased mitochondrial membrane potential. PPARδ agonists and antagonists could rescue TMRM staining and the colony forming capacity in HLX knockdown CD34+ cells and alleviated the myeloid differentiation block upon HLX overexpression, respectively. In summary, our findings show that HLX balances hematopoietic stem and progenitor cell maintenance and differentiation by modulating their metabolic status. DisclosuresNo relevant conflicts of interest to declare.

AML1/ETO and FLT3-ITD Cooperate to Induce Acute Myeloid Leukemia in Mice

Acute myeloid leukemia (AML), the most common form of acute leukemia in adults, accounts for more than 40% of all leukemia deaths. The AML1-ETO (A) fusion gene resulting from t(8;21) is a recurring genetic alteration in human AML, typically associated with a favorable outcome. Internal tandem duplications (ITD) in the juxtamembrane domain of Fms-like tyrosine kinase 3 (Flt3) result in constitutive ligand-independent signaling. Flt3ITD is one of the most frequently identified molecular abnormalities in AML, and when present, portends unfavorable clinical outcomes. AML1/ETO and FLT3ITD can co-exist in human AML, indicating that these lesions cooperate to promote disease development. Neither AML1/ETO nor Flt3ITD alone are sufficient to induce AML in mice. To determine whether coordinated expression of AML1/ETO and Flt3ITD would cause AML, Mx1-Cre x LSL AML1-ETO/+ x FLT3ITD/ITD (MAFF) mice were generated. In this system, an Mx1 promoter-driven Cre recombinase removes a lox-stop-lox (LSL) cassette upstream of a knock-in human AML1/ETO fusion gene in hematopoietic progenitor cells, while FLT3ITD is constitutively expressed. MAFF mice developed AML at 4-5 weeks of age with 100% penetrance. The AML was associated with severe leukocytosis, splenomegaly and dense bone marrow infiltration by immature-appearing blasts, and was universally lethal. Flow cytometry of bone marrow from MAFF mice revealed a striking expansion of cells harboring a hematopoietic stem cell (HSC) and multipotent progenitor (MP) cell phenotype, indicative of a block in normal myeloid differentiation. No lymphoid leukemias were observed. Interestingly, two copies of the FLT3ITD allele were required for AML to develop in this model. Transplantation of as few as 105 bone marrow cells from primary MAFF mice into lethally-irradiated secondary recipients resulted in 100% engraftment and lethality, with a leukemia identical to that which developed in primary MAFF mice, albeit with delayed latency. Mixed bone marrow chimeras were generated using MAFF marrow along with marrow from syngeneic, wild-type C57BL/6 or Rag2-/-γC-/- mice (lacking mature B, T and NK cells). Chimeras generated with MAFF and Rag2-/-γC-/- marrow succumbed more rapidly than those with MAFF and wild-type marrow, indicating that adaptive immunity and/or NK cells provided some level of active immune surveillance against MAFF AML. MAFF mice were crossed with Rosa26-LSLSIY/+ mice (referred to as MAFFS mice) in order to incorporate inducible expression of a tractable peptide antigen (SIYRYYGL; SIY) in leukemia cells. Through the use of mixed bone marrow chimeric approaches, we are now capable of monitoring SIY antigen-specific CD8+ T cell responses in MAFFS AML-bearing animals, including contexts in which the SIY antigen is shared (leukemia-associated antigen) or expressed only by leukemia cells (leukemia-specific antigen). In conclusion, we have generated the first autochthonous murine AML model in which the role of host immune surveillance can be interrogated. We expect that the MAFF AML model will also be useful for studies regarding leukemia biology and treatment. DisclosuresNo relevant conflicts of interest to declare.

OSU-2S, a Novel PKC Activator, Mediates PKC Dependent Cell Death, Differentiation and Cell Cycle Arrest in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a near incurable disease with 5-year survival rates of 10% in older patients. Poor tolerance to chemotherapy, chemo resistance and high rate of relapse warrants less toxic and more effective regimens in AML. AML leads to a block in normal myeloid differentiation, leading to an accumulation of undifferentiated blasts. Thus, therapies directed towards promoting these cells to differentiate re-establishes the ability of AML cells to undergo cell death, reducing the leukemic population. Such differentiation therapy with all-trans retinoic acid (ATRA) has been successful in acute promyelocytic leukemia (APL), but not in other AML subtypes resistant to ATRA, thus presenting a promising and unexploited avenue for targeted therapies. There is accumulating evidence that Protein Kinase C (PKC) activation induces myeloid differentiation. However, since most PKC activators are either tumor promoters or have inconclusive activity, their potential for use in AML has yet to be realized. OSU-2S is a non-immunosuppressive derivative of FTY720 (a sphingosine analogue with anti-tumorigenic properties) and has previously been reported to directly activate PKC to mediate cytotoxicity, showing promising in vitro and in vivo activity against a number of leukemias and lymphomas, and other malignancies such as hepatocellular carcinoma. This impelled us to evaluate the activity of OSU-2S in causing cell death and differentiation in AML. We found that OSU-2S resulted in increased activation of PKC in HL-60 cells. In addition to effecting significant dose-dependent cytotoxicity against AML cell lines HL-60, MV4-11 and MOLM-13 [mean decrease in viability with OSU-2S in HL-60: 85±7% (p=0.08), MV4-11: 74±20% (p=0.02), MOLM-13: 60±25% (p=0.04)], and in primary samples from leukemic patients, including high risk FLT3-ITD mutated AMLs (65±8% decrease in viability with OSU-2S, p<0.0001, n=13, dose trend p<0.0001), OSU-2S caused a significant upregulation of CD11b, a cell-surface marker associated with myeloid maturation, in HL-60 and primary AML samples [(25±5% and 14±7% increase in CD11b+ cells with OSU-2S vs vehicle control in HL-60 (p =0.0126) and primary AML (p=0.043) cells respectively]. In addition, OSU-2S treated HL-60 and primary AML cells exhibited morphological features indicative of differentiation, as evidenced by lower nuclear to cytoplasmic ratio, and highly vacuolated and granulated cytoplasm. The observed differentiation activity prompted us to test if OSU-2S affected the leukemia-initiating cell population. Colony forming assays showed OSU-2S significantly reduced the number of colony forming units-leukemic (CFU-L) in primary AML blasts (52±13% decrease in CFU-L in response to OSU-2S vs vehicle control; p=0.018, n=4). OSU-2S mediated cytotoxicity was significantly abrogated in the presence of PKC inhibitor Bisindolylmaleimide (BIS) in HL-60 cell line [39±8% decrease in OSU-2S induced cytotoxicity in the presence of BIS vs control (p=0.008), n=3] as well as in primary AML cells [41±16% decrease in OSU-2S induced cytotoxicity (p=0.02), n=6], indicating that OSU-2S mediates its cytotoxic effects in AML via PKC activation.To further gain insight into the mechanism of cell death and differentiation effected by OSU-2S, we performed RNA-Sequencing analysis of total RNA isolated from MV4-11 cells treated with OSU-2S. Ingenuity Pathway Analysis on these transcripts showed significant changes in gene expression affecting cell cycle regulation and proliferation (p=10-13). Cell cycle analysis of MV4-11, HL-60 and U-937 cells by PI staining followed by flow cytometry confirmed a decrease in cells in G2/M phase of the cell cycle associated with increased cells in S but not Go/G1 phase (69.63% decrease in G2/M phase cells with OSU-2S vs vehicle control, p=0.04). Current ongoing studies in cell line-derived xenografted mouse models of AML indicate favorable therapeutic effect of OSU-2S in providing survival advantage in AML. Tumor burden monitoring by bioluminescent imaging shows reduction in tumor burden following OSU-2S treatment. Our results show promising pre-clinical efficacy of OSU-2S against AML, and warrants further studies to establish clinical efficacy as a potential therapeutic agent. Ongoing studies are also directed towards studying the involvement and role of PKC and other target molecules in the cell death and differentiation effected by OSU-2S. DisclosuresWalker:Gilead: Research Funding. Mims:Novartis: Honoraria. Chen:The Ohio State University: Patents & Royalties: OSU-2S. Byrd:Pharmacyclics: Research Funding; Acerta Pharma: Research Funding; Janssen: Research Funding; Genentech: Research Funding; The Ohio State University: Patents & Royalties: OSU-2S. Vasu:Boehringer-Ingelheim: Research Funding; Stemline Therapeutics: Research Funding.