Normal Hematopoietic Stem/progenitor Cells Research Articles

Cellular taxonomy of the preleukemic bone marrow niche of acute myeloid leukemia.

Leukemias arise from recurrent clonal mutations in hematopoietic stem/progenitor cells (HSPCs) that cause profound changes in the bone marrow microenvironment (BMM) favoring leukemic stem cell (LSC) growth over normal HSPCs. Understanding the cross talk between preleukemic mutated HSPCs and the BMM is critical to develop novel therapeutic strategies to prevent leukemogenesis. We hypothesize that preleukemic-LSCs (pLSCs) induce BMM changes critical for leukemogenesis. Using our AML-murine model, we performed single-cell RNA-sequencing of preleukemic BMM (pBMM) cells. We found normal HSC (nHSC)-regulating LepR+ mesenchymal stem cells, and endothelial cells were decreased, along with increases in CD55+ fibroblasts and pericytes. Preleukemic CD55+ fibroblasts had higher proliferation rates and decreased collagen expression, suggesting extracellular matrix remodeling during leukemogenesis. Importantly, co-culture assays found preleukemic CD55+ fibroblasts expanded pLSCs significantly over nHSCs. In conclusion, we have identified a distinct pBMM and a novel CD55+ fibroblast population that is expanded in pBMM that promote fitness of pLSCs over nHSCs.

Multiomics Profiling Reveals Higher RNA Isoform Diversity and Potential Oncospecific Protein Isoform Targets in HSPCs of MDS and MDS/MPN Patients

Background: Myelodysplastic Syndromes/Neoplasms (MDS) are characterized by somatic driver mutations in hematopoietic stem/progenitor cells (HSPCs) leading to hypermethylation, disruption of normal hematopoiesis and progression to acute myeloid leukemia ( Cazzola M, N Engl J Med 2020). With exception of a minority of fit patients, who are candidates for treatment with the potentially curative allogenic hematopoietic stem cell transplantation (allo HSCT), most elderly patients remain incurable and receive palliative treatment with hypomethylating agents. Due to the high phenotypic overlap between normal and dysplastic HSPCs, eradication of diseased cells remains an unmet therapeutic need for patients not eligible for allo HSCT. Aim: We applied a multiomics profiling strategy to identify novel oncospecific protein isoforms as potential drug targets in HSPCs of MDS and MDS/MPN patients. We hypotesized that isoforms might be more specific biomarkers and targets than consensus RNA and proteins. Methods: We included 91 individuals with MDS and MDS/MPN at diagnosis or follow-up without disease modifying treatment (n=71), unclear cytopenia (n=20) and healthy controls (n=6). All samples were collected through our Swiss MDS Registry/Biobank Platform between 8/2017 and 11/2021. We classified all patients clinically by WHO 2016 and genetically using the Malcovati criteria ( Malcovati L et al., Blood 2017). Targeted sequencing of 65 established myeloid driver genes from bulk DNA was performed using the Illumina sequencing platform. RNA and proteins were extracted from selected CD34/CD117 HSPC cells. RNA profiling was performed using libraries generated with the SMART-Seq mRNA reagent and proteotypic profiling was done by tandem mass-spectrometry. RNA isoforms were identified using StringTie ( Pertea M et al., Nature Biotechnology 2015) and protein isoforms using a reference database constructed from the identified RNA isoforms. RMATS was used to detect differences in alternative splicing events. Differential expression of RNA isoforms was analyzed using Ballgown (Frazee A et al. Nat Biotechnol 2015 ) and conventional differential gene-expression was performed by DESeq2 ( Love MI et al., Genome Biology 2014). Results: Genetic classification identified relevant clonality in 2/3 of patients with clinically unclear cytopenia after morphological and cytogenetic assessment (6 MDS, 7 CCUS, 7 ICUS) ( A). The distribution of somatic driver mutations in our cohort was in line with published literature (data not shown). Compared to controls, patients with MDS and MDS/MPN showed a higher rate of intron retentions (data not shown), higher transcriptional diversity of RNA isoforms ( B), lower numbers of differentially over-expressed RNA isoforms (lower isoform abundancy) ( C) but higher numbers of differentially over-expressed genes (higher gene-expression abundancy)( D). This apparently controversial result reflects the methodological bias where RNA isoforms are merged on the corresponding gene-locus by conventional gene-expression analysis missing the full range of transcriptional (isoform) diversity. This RNA isoform diversity was not clearly associated with clinical (MDS vs MDS/MPN) or genetic subgroups (TP53, spliceosome or cell-signaling mutations). Due to low sample size, we could only observe a tendency of higher RNA isoform diversity in TP53 mutated and lower diversity in cell-signaling mutated subgroups ( C). The intersection of differentially up-regulated RNA isoforms and expressed protein isoforms revealed five novel oncospecific proteins (L-P in E) as potential candidates for specific drug targeting in HSPCs of MDS and MDS/MPN. These included an oncospecific membrane protein (L) as well as a cell-signaling enzyme (M) present in about 25%-30% of the diseased samples. Conclusions: With our multiomics profiling strategy, we identified a higher RNA isoform diversity in HSPCs of MDS and MDS/MPN patients as well as five potential oncospecific protein isoform targets. These potential targets can be missed by differential gene-expression ( D) and are better discernible by the biologically more relevant differential RNA-isoform analysis ( C). L and M have well-established functions in stem-cell biology, leukemic transformation and drug resistance making them promising candidates for further functional validation as disease-specific drug targets.

Cooperative CAR targeting to selectively eliminate AML and minimize escape

Acute myeloid leukemia (AML) poses a singular challenge for chimeric antigen receptor (CAR) therapy owing to its phenotypic heterogeneity and similarity to normal hematopoietic stem/progenitor cells (HSPCs). Here we expound a CAR strategy intended to efficiently target AML while minimizing HSPC toxicity. Quantification of target expression in relapsed/refractory patient samples and normal HSPCs reveals a therapeutic window for gated co-targeting of ADGRE2 and CLEC12A: We combine an attenuated ADGRE2-CAR with a CLEC12A-chimeric costimulatory receptor (ADCLEC.syn1) to preferentially engage ADGRE2posCLEC12Apos leukemic stem cells over ADGRE2lowCLEC12Aneg normal HSPCs. ADCLEC.syn1 prevents antigen escape in AML xenograft models, outperforms the ADGRE2-CAR alone and eradicates AML despite proximate myelopoiesis in humanized mice. Off-target HSPC toxicity is similar to that of a CD19-CAR and can be mitigated by reducing CAR Tcell-derived interferon-γ. Overall, we demonstrate the ability of target density-adapted cooperative CAR targeting to selectively eliminate AML and potentially obviate the need for hematopoietic rescue.

Human acute leukemia uses branched-chain amino acid catabolism to maintain stemness through regulating PRC2 function.

Cancer-specific metabolic activities play a crucial role in the pathogenesis of human malignancies. To investigate human acute leukemia-specific metabolic properties, we comprehensively measured the cellular metabolites within the CD34+ fraction of normal hematopoietic stem progenitor cells (HSPCs), primary human acute myelogenous leukemia (AML), and acute lymphoblastic leukemia (ALL) cells. Here, we show that human leukemia cells are addicted to the branched-chain amino acid (BCAA) metabolism to maintain their stemness, irrespective of myeloid or lymphoid types. Human primary acute leukemias had BCAA transporters for BCAA uptake, cellular BCAA, α-ketoglutarate (α-KG), and cytoplasmic BCAA transaminase-1 (BCAT1) at significantly higher levels than control HSPCs. Isotope-tracing experiments showed that in primary leukemia cells, BCAT1 actively catabolizes BCAA using α-KG into branched-chain α-ketoacids, whose metabolic processes provide leukemia cells with critical substrates for the trichloroacetic acid cycle and the synthesis of nonessential amino acids, both of which reproduce α-KG to maintain its cellular level. In xenogeneic transplantation experiments, deprivation of BCAA from daily diet strongly inhibited expansion, engraftment and self-renewal of human acute leukemia cells. Inhibition of BCAA catabolism in primary AML or ALL cells specifically inactivates the function of the polycomb repressive complex 2, an epigenetic regulator for stem cell signatures, by inhibiting the transcription of PRC components, such as zeste homolog 2 and embryonic ectoderm development. Accordingly, BCAA catabolism plays an important role in the maintenance of stemness in primary human AML and ALL, and molecules related to the BCAA metabolism pathway should be critical targets for acute leukemia treatment.

DNA hypermethylation-induced miR-182 silence targets BCL2 and HOXA9 to facilitate the self-renewal of leukemia stem cell, accelerate acute myeloid leukemia progression, and determine the sensitivity of BCL2 inhibitor venetoclax.

Rationale: microRNAs (miRNAs) are frequently deregulated and play important roles in the pathogenesis and progression of acute myeloid leukemia (AML). miR-182 functions as an onco-miRNA or tumor suppressor miRNA in the context of different cancers. However, whether miR-182 affects the self-renewal of leukemia stem cells (LSCs) and normal hematopoietic stem progenitor cells (HSPCs) is unknown. Methods: Bisulfite sequencing was used to analyze the methylation status at pri-miR-182 promoter. Lineage-negative HSPCs were isolated from miR-182 knockout (182KO) and wild-type (182WT) mice to construct MLL-AF9-transformed AML model. The effects of miR-182 depletion on the overall survival and function of LSC were analyzed in this mouse model in vivo. Results: miR-182-5p (miR-182) expression was lower in AML blasts than normal controls (NCs) with hypermethylation observed at putative pri-miR-182 promoter in AML blasts but unmethylation in NCs. Overexpression of miR-182 inhibited proliferation, reduced colony formation, and induced apoptosis in leukemic cells. In addition, depletion of miR-182 accelerated the development and shortened the overall survival (OS) in MLL-AF9-transformed murine AML through increasing LSC frequency and self-renewal ability. Consistently, overexpression of miR-182 attenuated AML development and extended the OS in the murine AML model. Most importantly, miR-182 was likely dispensable for normal hematopoiesis. Mechanistically, we identified BCL2 and HOXA9 as two key targets of miR-182 in this context. Most importantly, AML patients with miR-182 unmethylation had high expression of miR-182 followed by low protein expression of BCL2 and resistance to BCL2 inhibitor venetoclax (Ven) in vitro. Conclusions: Our results suggest that miR-182 is a potential therapeutic target for AML patients through attenuating the self-renewal of LSC but not HSPC. miR-182 promoter methylation could determine the sensitivity of Ven treatment and provide a potential biomarker for it.

Novel Evidence That Intracellular Complement Network (complosome) Is Expressed in Hematopoietic Stem Cells (HSPCs) and Regulates Their Trafficking, Metabolism, and Proliferation in an Intracrine-Dependent Manner By Engaging Pattern Recognition Receptor Nlrp3 Inflammasome

Background. Complement cascade (ComC) proteins, including C3 and C5 components, are synthesized in the liver; however, recent reports indicate their expression in lymphocytes as well (Immunity 2020;52(3):513-527). Accordingly, intracellular ComC proteins have been reported to be cleaved to active C3a and C5a mediators that interact with corresponding receptors in an intracrine-depended manner. This novel regulatory loop operating in lymphocytes has been named ''complosome''. Aim of the study.Because hematopoiesis and lymphopoiesis have a common stem cell origin and similar mechanisms are involved in regulating the biology of both lineages, we asked if complosome is also expressed and functional in normal murine hematopoietic stem progenitor cells (HSPCs). We also address a challenging question if intracellular ComC mediators could activate pattern recognition receptor Nlrp3 inflammasome that, as we reported, regulates trafficking, proliferation and metabolism of HSPCs (Leukemia 2022;36(1):23-32;Stem Cell Rev Rep. 2020 16(5):954-967). Materials and Methods. First, we phenotyped purified murine and human HSPCs for expression of major complement elements, including C3, C5, C3aR, C5aR, and cathepsin L that cleaves intracellular C3 to C3a. Next, we focused on a crucial distal executive part of ComC, protein C5, and evaluated in C5-KO and C5aR-KO mice the number of HSPCs and their clonogeneic potential. We also studied hematopoietic recovery of these animals after sublethal irradiation (650 cGy). Activation of Nlrp3 inflammasome in C5a-KO and C5aR-KO in response to hematopoietic growth factors and cytokines was evaluated by immunofluorescence Glow assay. We also studied homing and engraftment of C5-KO and C5aR-KO mice-derived HSPCs transplanted into normal WT recipients. Finally, we performed a metabolomic analysis of C5-KO, C5aR-KO, and control WT mice in steady-state conditions and after stimulation with selected hematopoietic growth factors and cytokines. Results. We show for the first time that purified murine and human HSPCs express complosome elements, including C3, C5, C3aR, C5aR, and cathepsin L. Next, C5-KO and C5aR-KO mice also have a lower number of HSPCs and clonogenic progenitors in bone marrow (BM), and display defect of hematopoietic recovery from sublethal irradiation. Furthermore, BM cells isolated from these animals had impaired homing and engraftment in normal WT recipients, supported by the observation that HSPCs from complosome deficient mice had a defect in migration to stromal-derived factor-1 (SDF-1), which correlated with decreased activation of Nlrp3 inflammasome. This defect could be alleviated by direct activation of Nlrp3 inflammasome by nigericin. Finally, our metabolic studies revealed a defect in glucose, amino acids, and lipid metabolism in C5-KO and C5aR-KO mice which correlates with complosome deficiency and Nlrp3 inflammasome activation. Conclusions. We provide for the first time evidence that complosome is expressed and functional in normal HSPCs and by engaging Nlrp3 inflammasome regulates metabolism, proliferation, and migration of HSPCs. Thus, our data sheds new intriguing light on innate immunity as a regulator of hematopoiesis that involves intracellular activation of Nlrp3 inflammasome. We currently focus on Nlrp3 inflammasome-released alarmins released from the cells in response to complosome-Nlrp3 inflammasome activation that accounts for observed phenomena. These studies are essential to develop more efficient ex vivo expansion strategies of HSPCs and to understand better intrinsic mechanisms that regulate metabolism of these cells.

Novel Evidence That NADPH Oxidase 2 (Nox-2) Enhances Trafficking of Hematopoietic Stem Progenitor Cells (HSPCs) By Involving ROS Mediated Activation of Nlrp3 Inflammasome

Background. NADPH oxidase 2 (Nox-2) is a superoxide-generating enzyme that forms reactive oxygen species (ROS) to regulate the redox state involved in the self-renewal of hematopoietic stem/progenitor cells (HSPCs). Moreover, ROS are released from Nox-2 expressed by cell surface and mitochondria membranes and are potent activators of intracellular pattern recognition receptor Nlrp3 inflammasome, which regulates several aspects of HSPCs biology, including their trafficking, proliferation, and metabolism. Our previous research demonstrated a novel role of Nlrp3 inflammasome in the migration, mobilization, homing, and engraftment of HSPCs (Leukemia 2022;36(1):23-32;Stem Cell Rev Rep. 2020 16(5):954-967). The role of ROS was extensively studied in the proliferation of hematopoietic cells; however, its role in the migration of HSPCs is not very well understood. Aim of the study.Based on the role of Nox-2 in activating Nlrp3 inflammasome, we hypothesized that activation of Nox-2-ROS-Nlrp3 inflammasome axis in both HSPCs and BM microenvironment orchestrates trafficking of HSPCs.Materials and Methods. First, we studied in Transwells chemotaxis of Nox-2 deficient cells to bone marrow (BM) chemoattractants, including SDF-1, S1P, and eATP. Next, to shed more light on the role of Nox2 in the trafficking of HSPCs, we performed in Nox-2-KO animals G-CSF- and AMD3100-induced pharmacological mobilization. We also studied homing and engraftment of Nox-2-KO cells in BM of wild-type (WT) animals and homing and engraftment o WT BM cells in Nox-2-KO recipients. Nox-2-KO mice were also sublethal irradiated, and we followed the recovery of peripheral blood counts in these animals. Activation of Nlrp3 inflammasome in Nox-2-KO murine Sca-1+c-kit+lineage- HSPCs was evaluated by Glow assay. To ''bypass'' Nox-2-ROS activation in control experiments, nigericin has been employed as a direct activator of Nlrp3 inflammasome. Since membrane lipid rafts (MLRs) play a crucial role optimal migration of HSPCs, we evaluated the formation of membrane lipid rafts by employing confocal analysis in Nox-2-KO cells stimulated with SDF-1. We also studied the expression of enzymes (SREBP2, HMGCs, and HMGCR) involved in providing lipid components for MLRs assemble in activated Nox-2-KO cells. Results. We noticed that Nox-2-KO animals have a profound defect in the trafficking of HSPCs as evidenced by impaired chemotaxis to BM chemoattractants, decrease in pharmacological mobilization, and defect in homing and engraftment after transplantation. Nox-2-KO mice have also defect in recovery from sublethal irradiation. At the molecular level, HSPCs from Nox-2-KO mice displayed a defect in activation of Nlrp3 inflammasome and in cholesterol and sphingolipids synthesizing enzymes, which provide lipid components required for optimal assemble of MLRs. These defects were partially ameliorated by using direct activator of Nlrp3 inflammasome that is nigericin. Conclusions. We provide for the first time evidence that Nox-2-ROS-Nlrp3 inflammasome axis operates in normal HSPCs and is crucial for regulating trafficking and post-irradiation recovery of these cells. These effects are mediated by providing components for MLRs required for optimal migration and signaling in HSPCs

Inhibition of O-GlcNAcase Inhibits Hematopoietic and Leukemic Stem Cell Self-Renewal and Drives Dendritic Cell Differentiation via STAT3/5 Signaling.

Myeloid differentiation blockage at immature and self-renewing stages is a common hallmark across all subtypes of acute myeloid leukemia (AML), despite their genetic heterogeneity. Metabolic state is an important regulator of hematopoietic stem cell (HSC) self-renewal and lineage-specific differentiation as well as several aggressive cancers. However, how O-GlcNAcylation, a nutrient-sensitive posttranslational modification of proteins, contributes to both normal myelopoiesis and AML pathogenesis remains largely unknown. Using small molecule inhibitors and the CRISPR/Cas9 system, we reveal for the first time that inhibition of either OGA or OGT, which subsequently caused an increase or decrease in cellular O-GlcNAcylation, inhibits the self-renewal and maintenance of CD34+ hematopoietic stem/progenitor cells (HSPCs) and leukemic stem/progenitor cells and drives normal and malignant myeloid differentiation. We further unveiled the distinct roles of OGA and OGT inhibition in lineage-specific differentiation. While OGT inhibition induces macrophage differentiation, OGA inhibition promotes the differentiation of both CD34+ HSPCs and AML cells into dendritic cells (DCs), in agreement with an upregulation of a multitude of genes involved in DC development and function and their ability to induce T-cell proliferation, via STAT3/5 signaling. Our novel findings provide significant basic knowledge that could be important in understanding AML pathogenesis and overcoming differentiation blockage-agnostic to the genetic background of AML. Additionally, the parallel findings in normal HSPCs may lay the groundwork for future cellular therapy as a means to improve the ex vivo differentiation of normal DCs and macrophages.

Leukotrienes promote stem cell self-renewal and chemoresistance in acute myeloid leukemia.

Acute myeloid leukemia (AML) is characterized by poor clinical outcomes due to high rates of relapse following standard-of-care induction chemotherapy. While many pathogenic drivers have been described in AML, our understanding of the molecular mechanisms mediating chemotherapy resistance remains poor. Therefore, we sought to identify resistance genes to induction therapy in AML and elucidated ALOX5 as a novel mediator of resistance to anthracycline-based therapy. ALOX5 is transcriptionally upregulated in AML patient blasts in comparison to normal hematopoietic stem/progenitor cells (HSPCs) and ALOX5 mRNA, and protein expression is increased in response to induction therapy. In vitro, and in vivo genetic, and pharmacologic perturbation studies confirm that ALOX5 positively regulates the leukemogenic potential of AML LSCs, and its loss does not significantly affect the function of normal HSPCs. ALOX5 mediates resistance to daunorubicin (DNR) and promotes AML cell survival and maintenance through its leukotriene (LT) synthetic capacity, specifically via modulating the synthesis of LTB4 and its binding to LTB receptor (BLTR). Our study reveals a previously unrecognized role of LTs in AML pathogenesis and chemoresistance, whereby inhibition of ALOX5 mediated LTB4 synthesis and function could be combined with standard chemotherapy, to enhance the overall therapeutic efficacy in AML.

Identification of GPAT1-dependent mitochondrial metabolism as a novel therapeutic target for AML

Recent studies have demonstrated that cancer-specific metabolism plays a crucial role in a variety of malignancies, including acute myeloid leukemia (AML). To identify a novel therapeutic target for AML, we conducted a metabolite screen on AML cells and normal hematopoietic stem/progenitor cells (HSPCs) and detected that the metabolism of glycerol-3-phosphate (G3P) is reprogrammed in AML. Glycerol-3-phosphate acyltransferases (GPATs), the first and rate-limiting enzymes in the lipid biosynthesis pathway, convert G3P into lysophosphatidic acid (LPA). Among various GPAT isozymes, GPAT1 was highly expressed in AML cells and silencing it inhibited the cell growth of AML. GPAT1 is located on the outer membrane of the mitochondria and regulates mitochondrial fusion and oxidative phosphorylation (OXPHOS). Silencing GPAT1 promoted mitochondrial fission and reduced OXPHOS. In AML, the GPAT1 inhibitor also suppressed cell proliferation and mitochondrial metabolism. However, this inhibitor had no effect on normal hematopoiesis in vivo. In conclusion, these findings indicate that targeting GPAT1 may be a promising therapeutic strategy for AML, since it suppresses leukemia-specific metabolism without impairing normal HSPCs.

Imetelstat, a Telomerase Inhibitor, Is Capable of Depleting Myelofibrosis Hematopoietic Stem Cells and Progenitor Cells

Treatment of myelofibrosis (MF) patients with imetelstat (Imet), a telomerase inhibitor, has been reported to lead to clinical, morphologic and molecular remissions in a subset of patients (Tefferi A, et al. N Engl J Med. 2015; 373:908), suggesting that Imet has disease-modifying activity. The precise mechanism by which Imet induces such responses has however not been reported. In this study, we investigated the effects of Imet on MF hematopoietic stem/progenitor cells (HSC)/(HPC) to address this question.Cord blood (CB) CD34+ cells were cultured with cytokines alone or cytokines plus increasing doses of Imet (1.8uM, 3.75uM, 7.5μM) or mismatched oligonucleotide control (Mis), for 1-2 weeks. Similar numbers of Lin-CD34+, Lin-CD34+CD38- cells, CD34+ cells that expressed aldehyde dehydrogenase (ALDH) and assayable HPCs (CFU-GM+BFU-E+CFU-GEMM) were generated under each of these culture conditions (P all>0.05). By contrast, culture of MF CD34+ cells (n=14; 9 spleens and 5 peripheral blood) with 7.5uM Imet led to reduction in the numbers of Lin-CD34+ (P<0.05), Lin-CD34+CD38- (P=0.07), CD34+ALDH+ cells (P=0.05), and HPCs (CFU-GM+BFU-E+CFU-GEMM) (P<0.05) as compared with cells treated with Mis. Furthermore, Imet treatment (7.5uM) for 1 week reduced the absolute numbers of JAK2V617F+ CFU-GM by 45.8±22.5% (P=0.09, n=4) and the absolute number of JAK2V617F homozygous CFU-GM by 53.6±19.3% (P=0.03, n=4) as compared to treatment with Mis. These findings suggest Imet can preferentially deplete malignant MF HPCs, resulting in the reduction of malignant cell burden.We further investigated the effects of Imet on normal and MF HSCs in vivo by treating NOD/SCID/IL2R null (NSG) mice transplanted with CB or MF splenic CD34+ cells directly with vehicle alone, Mis or Imet. The mice receiving CB CD34+ cells (5-10 ×105/mouse, n=3) were treated with 5mg/kg, 15mg/kg or 30mg/kg Imet using IP injection 3 times a week for 4 weeks. Four months after the transplantation (3 months after the treatment), the number of hCD45+ cells and the number of hCD34+ HSCs and HPCs in the marrows of mice receiving 5 mg/kg Imet were not reduced as compared to mice receiving vehicle alone or equal doses of Mis. By contrast mice receiving 15mg/kg Imet had modest reduction in the numbers of hCD45+ cells in the marrow (reduced by 20.9% as compared to vehicle alone and by 33.9% as compared to equal dose of Mis). In addition, the numbers of hCD45+ cells and hCD34+ cells were reduced by 25.4% and 38.0%, respectively in the marrow of the mice treated with 30mg/kg Imet as compared to the mice treated with vehicle alone. These findings indicate that the ability of Imet treatment to deplete normal HSCs is dose-dependent. In order to limit the effects of Imet on the normal HSC, 15mg/kg Imet treatment for 4 weeks was selected to treat mice transplanted with MF CD34+ cells. Four month after the transplantation, the number of hCD45+ cells in the marrow was dramatically depleted by Imet treatment in mice receiving CD34+ cells from 2 MF patients (% of hCD45+ cells relative to vehicle alone treatment: Pt 1: Mis: 54.2%, Imet: 15.7%; Pt 2: Mis: 4.1%, Imet: 0.3%). The various lineage cells belonging to both myeloid and lymphoid lineages generated by the grafts of these 2 patients were also correspondingly depleted. Moreover, depletion of hCD34+ cells in the same mice was also achieved by Imet treatment (% of hCD34+ cells relative to vehicle alone treatment: Pt 1: Mis: 44.5%, Imet: 21.9%; Pt 2: Mis: 7.0%, Imet: 4.1%). These data suggest that Imet is capable of selectively eliminating MF HSCs.To explore the mechanisms by which Imet might affect MF CD34+ cells, the effect of the drug on apoptosis was studied. Two days after the treatment of MF CD34+ cells (n=4) with 7.5uM Imet, the percentage of MF CD34+ Annexin V+ PI− cells was significantly greater than the percentage of cells treated with Mis (P=0.002) or cytokines alone (P=0.003). The absolute number of CD34+ Annexin V+ PI− cells present in Imet containing cultures was 3.4±1.0- and 5±1.4-fold greater than in cultures treated with Mis or cytokines alone, respectively. Furthermore, Imet treatment did not induce apoptosis of CB CD34+ cells (n=3). These findings suggest that Imet is capable of selectively promoting apoptosis and inhibiting the proliferation of phenotypically and functionally defined MF but not normal HSCs/HPCs. Imet, therefore, represents a potentially promising drug for the treatment of MF which appears to affect primitive MF HSCs. DisclosuresHuang:Janssen Research & Development, LLC: Employment. Wang:Janssen Research & Development, LLC: Research Funding.

Recreating the Hematopoietic Microenvironment in Vitro Using Microfluidic “on-a-Chip” Technology Enables the Study of Interactions of Normal and Malignant HSPC with Specific Bone Marrow Niches

Under normal conditions, hematopoietic stem/progenitor cells (HSPC) reside within specific bone marrow (BM) niches. These are comprised of an array of different cell types located strategically to provide a myriad of chemical signals and physical interactions that maintain HSPC and orchestrate the process of hematopoiesis. In human myeloid malignancies, the BM niche is remodeled by malignant cells, which displace resident HSPC, and create self-reinforcing malignant niches that drive disease progression, drug-resistance, and relapse. Attaining a mechanistic understanding of the major determinants of HSPC function in the human BM has been difficult, due to challenges associated with visualizing and modeling the complex niche environment in humans. The creation of accurate models of normal and malignant human BM niches would fill a critical gap, and enable the development of therapies targeting deviant cells and/or signaling pathways, to disrupt self-reinforcing malignant niches and reestablish normal hematopoiesis. We showed that microfluidic “on-a-chip” technologies using bioengineered 3D human tissue constructs, in the presence of physiological flow, can model normal physiology and disease processes and enable drug screening. Herein, we sought to exploit the high physiological relevance and single cell resolution offered by “on-a-chip” technology to recreate the BM microenvironment in vitro and study, in real-time, the interactions of normal and malignant HSPC with each specific niche cell type. To accomplish this objective, we constructed a microfluidic platform using polydimethylsiloxane (PDMS) and photolithography, in which microchannels allow continuous flow of media and HSPC through each of the specific niches, reproducing, in effect, a primitive circulatory system. To recreate the various BM niches, mesenchymal cells (Stro-1+; MSC), arterial endothelium (CD146+NG2+; AEC), and sinusoidal endothelium (CD146+NG2-; SEC) were magnetically-sorted from adult human BM. An aliquot of Stro-1+ cells was also induced to undergo osteogenic differentiation, to generate differentiated osteoblasts (OB). To create the in situ patterned 3D niche-on-a-chip (NOC), the chambers of the microfluidic device were filled with AEC, SEC, MSC, or OB embedded in a hyaluronic acid (HA)-based hydrogel precursor, and the cellularized constructs were formed by UV photopolymerization through the apertures of a photomask. Following flushing of uncrosslinked hydrogel precursor solution, the niche constructs were allowed to equilibrate for 3 days, under constant media flow using a four-channel precision microperistaltic pump. U937, MOLM13, and normal CD34+ cells were each labeled with Qtracker 605, and independently perfused into the main channel of a separate NOC, from which theyenter and evenly disperse into four equidistant chambers, each of which housed a distinct BM niche (AEC, SEC, MSC, or OB). LIVE/DEAD staining confirmed viability of the HSPC and niche cells for > 5 days following HSPC infusion, and immunocytochemistry demonstrated continued expression of appropriate phenotypic markers by each specific niche cell. At 24h post-infusion, U937 cells already exhibited a marked predilection for AEC, and this persisted throughout the 5-day observation period, with roughly 4.5-times more U937 cells engrafting within the AEC niche than the SEC or MSC niches, and 3-times more engrafting within the AEC niche than the OB niche. In contrast, MOLM13 cells exhibited a marked preference for the MSC and SEC niches, a moderate affinity for the OB niche, and engrafted only minimally within the AEC niche. In contrast to malignant cells, normal CD34+ HSPC engrafted preferentially within the SEC and OB niches, exhibited moderate engraftment within the MSC niche, and did not engraft the AEC niche. In conclusion, our studies establish the feasibility of using microfluidic “on-a-chip” technology to recreate the various niches within the BM microenvironment, and provide proof that this novel system can be used to study the interactions of normal and malignant HSPC with distinct cells of the niche. We are currently using this system to delineate the signaling pathways responsible for the observed preferential HSPC/niche cell interactions, with the ultimate goal of using this knowledge to develop more effective treatments for hematological malignancies and enhance engraftment following HSPC transplant. DisclosuresNo relevant conflicts of interest to declare.

Inhibition of the C5a-C5aR Axis By the L-Aptamer Aon-D21 As a New Strategy to Ameliorate Uncontrolled Migration/Motility of Hematopoietic Stem Cells - Potential Therapeutic Implications for Paroxysmal Nocturnal Hemoglobinuria

Background. Several mechanisms have been postulated for orchestrating the mobilization of hematopoietic stem/progenitor cells (HSPCs), and it has been proposed that activation of the complement cascade plays a crucial role in the initiation and execution of the egress of HSPCs from bone marrow (BM) into peripheral blood (PB). In support of this notion, we demonstrated that mice deficient in the mannan-binding lectin (MBL) pathway, which activates the proximal part of the complement cascade (Leukemia. 2017, 31, 262-265), as well as mice deficient in the fifth component of the complement cascade (C5), which is the central molecule of the distal part of the complement cascade, are poor mobilizers (Leukemia. 2014, 28, 2148-2154). While enhancing mobilization is of clinical importance for poor mobilizers, inhibition of increased motility of HSPCs could be of therapeutic importance in patients suffering from paroxysmal nocturnal hemoglobinuria (PNH) (J Cell Mol Med. 2015, 19, 2193-201) . It is well known that in PNH patients pathological activation of the complement cascade leads to accelerated lysis of erythrocytes due to formation of C5b-C9 (also known as the membrane attack complex, MAC). As a consequence of this unwanted activation, the bioactive lipid sphingosine-1-phosphate (S1P) is released from lysed erythrocytes, and this major chemoattractant of HSPCs preferentially increases motility of PNH-affected HSPCs in the bone marrow (BM) microenvironment (Leukemia 2010;24:976-85). This increased motility promotes their expansion by enabling them to outcompete normal HSPCs for BM niches. To inhibit the complement cascade activation that initiates this sequence of events, monoclonal antibodies are currently employed against C5 (eculizumab). Nevertheless, recent clinical data prompt for developing other more potent inhibitors of complement cascade activation (Clin Immunol 2017, 180, 25-32). Aim of the study. To better address the involvement of the distal part of complement cascade activation in regulating the motility of HSPCs, we performed experiments in mice that do not express the receptor C5aR, which binds the C5 cleavage fragments, C5a and desArgC5a. We also tested in an in vivo model the plasma-stable, mixed L-RNA/L-DNA-based aptamer, AON-D21, that is directed against C5a to inhibit the in vivo activity of the C5aR-C5a axis. Hypothesis. We hypothesized that inhibition of the C5aR -C5a/desArgC5a axis could be an efficient strategy to inhibit the uncontrolled motility of HSPCs during hemolytic episodes . Results. We present evidence that C5aR-deficient mice are poor mobilizers of HSPCs, which, together with the fact that motility of these cells is impaired after administration of the anti-C5a aptamer AON-D21, confirms the critical role of the C5a/desArgC5a-C5aR axis in enhancing the motility of HSPCs. Conclusions. We propose that novel drugs that inhibit the C5a-C5aR axis, such as the newly developed L-aptamer AON-D21 or C5aR antagonists, can find practical application in preventing the enhanced motility of HSPCs that would otherwise lead to the expansion of PNH clones in the BM of PNH-affected patients. Since eculizumab as recently reported may not be able to efficiently block C5 cleavage by serine proteases such as thrombin (Clin Immunol 2017, 180, 25-32) and close bidirectional interactions between the coagulation cascade and the complement system are well described, AON-D21 could play a supportive role. Moreover, inhibitors of the C5a-C5aR axis could also find application in other situations when complement cascade is hyperactivated such as for example in Atypical Hemolytic Uremic Syndrome. DisclosuresHoehlig:Aptarion Biotech AG: Employment, Patents & Royalties. Vater:Aptarion Biotech AG: Employment, Patents & Royalties.

TLR7/8 agonist treatment induces an increase in bone marrow resident dendritic cells and hematopoietic progenitor expansion and mobilization

There is accumulating evidence suggesting that toll-like receptor (TLR) signals play an important role in the regulation of hematopoietic stem/progenitor cells (HSPCs). TLR7/8 stimulation induces the myeloid differentiation of normal HSPCs and acute myeloid leukemia cells. However, the in vivo effect of TLR7/8 agonists on hematopoiesis is largely unknown. Here, we show that, similar to TLR4 and TLR2, treatment with the TLR7/8 agonist R848 induces an expansion of phenotypic hematopoietic stem cells (HSCs) with reduced repopulating potential and HSPC mobilization. In contrast to chronic TLR4 stimulation, treatment with R848 for 5 days did not induce a significant increase in myeloid-biased HSCs. Treatment with R848 results in a significant increase in classic dendritic cells (DCs) in the bone marrow, but a decrease in common dendritic cell progenitors and pre-DCs. Phenotypic analysis of DCs revealed that R848 treatment is associated with altered expression of certain chemokines, activation markers, and migratory receptors. Together, these data indicate that systemic administration of a TLR7/8 agonist has unique effects on hematopoiesis, including the expansion of DCs in the bone marrow, that might have clinical relevance to augment responses to certain immunotherapies, such as cancer vaccines and immune checkpoint blockade.

Targeting of the Alox12-12-HETE in Blast Crisis Chronic Myeloid Leukemia Inhibits Leukemia Stem/Progenitor Cell Function.

IntroductionChronic myeloid leukemia (CML) is a myeloid malignancy characterized by the oncogene BCR-ABL. CML responds well to therapy targeting BCR-ABL in the chronic phase but is resistant to treatment when it progresses to the blast phase (BP). This study attempted to address whether arachidonate 12-lipoxygenase (Alox12) confers to CML drug resistance.Materials and MethodsWe analyzed the expression of Alox12 using Western blotting, ELISA, and RT-PCR methods. Loss of functional analysis was performed using cellular activity assays on CML and normal hematopoietic stem/progenitor cells (HSPCs).ResultsAlox12 and 12-Hydroxyeicosatetraenoic acid (12-HETE) are overexpressed in BP-CML but not HSPCs, and that Alox12-12-HETE axis is regulated by BCR-ABL. The Alox12-12-HETE axis is required for CML. Specific Alox12 inhibitor inhibits colony formation, survival, and self-renewal capacity in BP-CML HSPCs, and to a significantly greater extent than in normal HSPCs. Of note, the Alox12 inhibitor significantly augments dasatinib’s efficacy in BP-CML HSPCs. Mechanism studies show that Alox12 inhibition does not affect activities of essential signaling pathways involved in maintaining stem cell function, such as Wnt, p53, and bone morphogenetic protein (BMP). In contrast, we show that Alox12 inhibition disrupts nicotinamide adenine dinucleotide phosphate (NADPH) homeostasis and induces oxidative stress and damage in CML HSPCs and committed cells.ConclusionAlox12-12-HETE axis is a specific and critical regulator of BP-CML HSPCs functions. Pharmacological inhibition of Alox12 may be useful in BP-CML.

Unexpected Novel Findings That Caspase-1-KO Mice Are Poor Mobilizers and Engraft Poorly with Wild Type Bone Marrow Cells - Indicating a Presence of an Autocrine Feedback Loop Involving Interleukin 1b and Interleukin 18 Signaling That Potentiates Nlrp3 Inflammasome Activity, Both in HSPCs and in the BM Microenvironment for Optimal Stem Cell Trafficking

Background . Our recent results indicate that Nlrp3 inflammasome expressed in hematopoietic stem/progenitor cells (HSPCs) and in the bone marrow (BM) microenvironment are required for optimal mobilization of murine cells (Leukemia 2020 Jun;34(6):1512-1523, Stem Cell Rev Rep. 2019 Jun;15(3):391-403). Moreover, Nlrp3 inflammasome expressed in these cellular contexts are also required for normal homing and engraftment after hematopoietic transplantation (Stem Cell Rev and Rep (2020) in press. https://doi.org/10.1007/s12015-020-10005-w). Consistent with these results, Nlrp3-KO mice are poor mobilizers, cells from Nlrp3-KO mice engraft poorly, and, as transplant recipients, Nlrp3-KO mice show a delay in hematopoietic recovery after transplantation of normal BM cells. Activation of Nlrp3 inflammasomes leads to the release in caspase-1-dependent manner from HSPCs and cells in the BM microenvironment of two potent pro-inflammatory cytokines, interleukin 1b (IL-1b) and interleukin 18 (IL-18). Hypothesis. Based on the aforementioned results, we envisioned that the activated Nlrp3 inflammasome caspase-1 autocrine feedback loop involving IL-1bor IL-18 signaling potentiates HSPCs trafficking by amplifying Nlrp3 inflammasome activation in HSPCs and the BM microenvironment.Materials and Methods. We employed normal control and caspase-1-KO mice for mobilization and homing/engraftment experiments. Mice were mobilized with G-CSF or AMD3100 in the absence or presence of administered IL-1b or IL-18, and we measured i) the total number of white blood cells (WBCs) and ii) the number of clonogenic colony-forming unit granulocyte/macrophage (CFU-GM) progenitors and Sca-1+c-kit+lineage- (SKL) cells circulating in PB. Next, caspase-1-KO cells were transplanted into lethally irradiated wild type animals, while caspase-1-KO mice were transplanted with wild type BMMNCs. We then evaluated homing and engraftment by measuring the number of PKH27-labeled cells, the number of clonogenic progenitors 24 hours after transplantation, and the numbers of day-12 CFU-S colonies and day-12 CFU-GM progenitors in the BM of recipient mice. In control experiments, to provide proof of our hypothesis, we also mobilized Nlrp3-KO mice with AMD3100 or G-CSF in the presence of administered IL-1b and IL-18. Results. We found that IL-1b or IL-18 alone mobilizes HSPCs and, significantly, that caspase-1-KO mice were poor mobilizers. Radiation chimera experiments revealed that this pro-mobilizing defect is dependent on the lack of caspase-1 in hematopoietic cells. To our surprise, administration of IL-1b and IL-18 did not improve G-CSF- or AMD3100-induced mobilization in Nlrp3-KO animals. We also found that both of these pro-inflammatory cytokines activate Nlrp3 inflammasomes in an Myd88-dependent manner. Moreover, HSPCs from caspase-1-KO mice show defective migration in response to a BM-released SDF-1 gradient and other supportive homing chemoattractants (sphingosine-1 phosphate; S1P and extracellular adenosine triphosphate; eATP). Finally, caspase-1-KO mice recipients were also engrafted poorly with normal HSPCs. To explain this phenomenon, we observed that the Nlrp3 inflammasome-caspase-1-IL-1b axis is important in upregulating SDF-1 and other key HSPC chemoattractants in the BM microenvironment conditioned for transplantation. Conclusions. We demonstrate for the first time that an Nlrp3 inflammasome-caspase-1-IL-1b/IL18 autocrine feedback mechanism operating in HSPCs and the BM microenvironment is involved in the normal trafficking of HSPCs during mobilization and homing/engraftment. This research demonstrates the unexpected role of caspase-1, which is situated downstream of the Nlrp3 inflammasome and directs the release of the potent pro-inflammatory cytokines IL-1b and IL18 that by feedback mechanism, in turn, activate Nlrp3 inflammasomes in HSPCs and the BM microenvironment, which is required for optimal trafficking of HSPCs. These results are important for understanding the novel casapase-1-mediated autocrine mechanisms involved not only in pharmacological mobilization but also in the inflammatory response of the organism to external challenges, such as infection and tissue/organ damage. Disclosures No relevant conflicts of interest to declare.

Feeder-free and serum-free in vitro assay for measuring the effect of drugs on acute and chronic myeloid leukemia stem/progenitor cells

Research on chronic and acute myeloid leukemia (CML/AML) is focused on the development of novel therapeutic strategies to eliminate leukemic stem/progenitor cells that are responsible for drug resistance and disease relapse. Methods to culture hematopoietic stem/progenitor cells (HSPCs) from blood or bone marrow samples are indispensable for investigating disease pathogenesis and delineating drug responses in individual patients. A key challenge in this area is that primary leukemic cells grow poorly in culture or rapidly differentiate and lose their hematopoietic potential. Access to patient samples can also be limiting or cell numbers too low to enable large-scale assays and/or to obtain reproducible quantitative data. Here we describe a feeder cell-free and serum-free liquid culture system for the expansion of CD34+ HSPCs from CML/AML samples and healthy control tissues. Following 7 or 14 days of culture, CD34+ cells are expanded 30- to 65-fold or 400- to 800-fold, yielding a purity of ∼80% and ∼60% CD34+ cells, respectively. This system was adapted to a 96-well format to measure the sensitivity of leukemic and normal HSPCs to cytotoxic drugs after only 7 days. The assay requires only 103 cells per well to determine drug IC50 values and can be performed with uncultured and culture-expanded cells. Importantly, resulting IC50 values strongly correlate with those obtained in the classic colony-forming unit (CFU) assay. Compared with the CFU assay, this novel 96-well liquid-based assay designed specifically for leukemic and normal HSPCs is faster and simpler, with more flexible readout methods for selecting candidates for further drug development.

Human Acute Leukemia Is Addicted to Branched-Chain Amino Acid Metabolism to Maintain Leukemia Stemness

Hematopoietic stem cells (HSCs) have capabilities to self-renew, maintaining an undifferentiated status, as well as to proliferate and mature into blood cells. Similarly, cancer stem cells (CSCs) self-renew and propagate to form cancer tissues. In human acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), CSC-like populations that can reconstitute human leukemia in immuno-deficient mice have been found, and are called leukemic stem cells or leukemia-initiating cells (LICs). In order to obtain "cure" by eradicating LICs, it should be critical to understand molecular machineries as to how LICs self-renew and expand to exert their cancer stemness properties. Recent studies revealed that several specific metabolism pathways actively contribute to the maintenance of stemness in several types of stem cells including ES/iPS cells. However, little is known about the LICs-specific metabolic activity. To clarify metabolic features of human LICs, we comprehensively analyzed 116 cellular metabolites of human CD34+ normal hematopoietic stem progenitor cells (HSPCs) (n=10), CD34+ AML (n=30) and CD34+ ALL cells (n=18). The metabolome analysis revealed that CD34+ AML and ALL cells commonly contain extremely high levels of branched chain amino acids (BCAA) as compared to normal CD34+ HSPCs. AML and ALL cells, but not normal HSCsexpressed BCAA-metabolism related enzymes as well as BCAA-transporters at high levels, and actively transport BCAAs into cytoplasm. Enzymatic inhibition of BCAA metabolism induced apoptosis in LICs but not in normal HSCs. Furthermore, deprivation of BCAA from daily diet in mice xeno-transplanted with human LICs caused significant inhibition of their reconstitution activities in vivo. Serial transplantation experiments revealed that hCD34+AML/ALL cells from BCAA-free-dieted mice exhibited extremely impaired reconstitution of human AML/ALL in secondary recipients fed with conventional diet, but those from mice with control diet developed AML/ALL with a high leukemia burden. To clarify how BCAA metabolism pathway regulates the leukemia-initiating activity, the global gene expression of primary CD34+AML /ALL cells and cell lines including THP-1(AML), Kasumi-9(B-ALL), Jurkat cells (T-ALL) were compared before and after pharmacological BCAA-metabolism inhibition and BCAA-deprivation. Gene set enrichment analysis (GSEA) revealed BCAA metabolism inhibition induced the expression of ES-related PRC2 target genes, which should be suppressed in ES cells via H3K27me3 histone modification, and ChIP-Seq analysis confirmed the significantly decreased H3K27me3 level around thetranscription start site ofPRC2 target genes both in CD34+ AML and ALL cells. Furthermore, BCAA metabolism inhibition or BCAA-deprivation resulted in the down regulation of EZH2 and EED, critical components of PRC2, at mRNA and protein level in human AML/ALL cells. Thus, BCAA-metabolism activity is a common metabolic machinery to maintain PRC2 function through potentiating EZH2 and EED expression in AML/ALL. We also found that human AML/ALL cells, but not normal HSPCs are dependent on BCAA metabolism for maintaining energy production through TCA cycle activity, leading to the adequate supply of alpha ketoglutarate (α-KG), an intermediate metabolite of TCA cycle. Since BCAT1, a catalytic enzyme for BCAA, requires α-KGas a substrate, the active energy production driven by BCAA metabolism contributes to prevent α-KG exhaustion to maintain the high BCAA catabolism activity in human AML/ALL. Consistent with persistent α-KG supply through enhanced BCAA metabolism, primary CD34+AML/ALL cells, which highly express BCAT1 did not exhibit decreased cellular α-KG level as compared to normal HSPCs in our metabolome analysis. Thus, the energy production and regulation of PRC2 function driven by enhanced BCAA metabolism constitute a vicious molecular cycle to maintain the leukemia stemness through the maintaining α-KG level in human acute leukemia. In summary, the current study demonstrates that human LICs are addicted to the activated BCAA metabolism to maintain their leukemia stemness irrespective of their lineage origin, and that the BCAA metabolic pathway is a generic therapeutic target in human acute leukemias. Disclosures Akashi: Sumitomo Dainippon, Kyowa Kirin: Consultancy; Celgene, Kyowa Kirin, Astellas, Shionogi, Asahi Kasei, Chugai, Bristol-Myers Squibb: Research Funding.

Whole Genome Bisulfite Sequencing of 63 Primary AML Samples Identifies a Unique DNA Hypermethylation Signature for Mutant IDH1/2 Cases That Is Different from That of TET2 Mutant AML

Mutations in IDH1, IDH2, and TET2 occur in more than 30% of AML patients, and are thought to contribute to leukemogenesis by disrupting TET2-mediated DNA demethylation, either directly or indirectly. While IDH1/2 and TET2 mutations are both predicted to result in hypermethylation, the specific methylation changes associated with these mutations are poorly understood. Additionally, we recently showed that CpG island (CGI) hypermethylation is common across virtually all AML subtypes, and it has been yet unclear whether IDH or TET2 hypermethylation is distinct from these 'generic', AML-associated changes. To investigate genome-wide methylation in IDH and TET2 mutated AML samples, we performed whole genome bisulfite sequencing (WBGS) on 15 primary AML samples with canonical IDH mutations (IDH1R132C, N=6; IDH1R132G, N=1; IDH2R140Q, N=7; and IDH2R172K, N=1) and 9 samples with biallelic loss-of-function mutations in TET2. These data were analyzed with WGBS data from primary CD34+ hematopoietic stem/progenitor cells (HSPCs) from 6 normal adult bone marrow donors, and 39 additional primary AML samples (representing 9 AML mutational categories) to define the methylation phenotypes specifically associated with IDH and/or TET2 mutations. Differential methylation analysis of each AML subtype vs. HSPCs identified a mean of 8,622 (53,73-13,773) AML-associated Differentially Methylated Regions (DMRs) in each mutational category (mean size: 986 bp), of which 6% to 97% were hypermethylated in the AML samples vs. HSPCs. IDH-mutant AML samples were the most hypermethylated subtype, with 97% of the 6,392 identified DMRs having increased methylation compared to HSPCs. The TET2-mutant samples were hypermethylated at fewer loci, representing 71% of the 5,892 identified DMRs vs HSPCs. Analysis of all AML subtypes demonstrated that 55% (16,275/29,534) of identified DMRs were shared across multiple subtypes, with more pronounced overlap among hypermethylated DMRs (&gt;70%). To distinguish mutation-specific changes in IDH and TET2 mutated AMLs from commonly hypermethylated loci across AML subtypes, we conducted a joint analysis of either IDH or TET2 mutant AMLs with data from all other AML samples and normal HSPCs. We performed hierarchical clustering of the methylation values at each DMR, and used the clustering topology to identify DMRs where the single outlier branch represented either the IDH or TET2 AML samples. We identified 2,024 DMRs (mean size: 998 bp) specific to IDH mutant AML samples, of which 100% were hypermethylated with respect to normal HSPCs (mean DMR b value of 0.60 vs 0.35 in HSPCs). IDH outlier DMRs were enriched for functional genomic elements relative to the set of commonly hypermethylated loci, including CGIs (65% of DMRs; 2.4-fold enrichment) and potential enhancers marked by H3K27ac in HSPCs (82% of DMRs; 1.4-fold enrichment); overlap with promoter regions was reduced (20% of DMRs; 0.77-fold decrease). Outlier analysis of TET2 mutated samples demonstrated that most changes were also present in other AML types, with only 93 unique DMRs (mean size 552 bp), and a subtler hypermethylation phenotype (83% of DMRs were hypermethylated, with a mean b value of 0.51 vs. 0.32 in HSPCs). TET2 DMRs were also enriched for overlap with CGIs with respect to background, but to a lesser extent than IDH outliers (52% of DMRs; 1.9-fold). Intersection of IDH and TET2-associated DMRs showed that TET2-specific DMRs represented a subset of IDH hypermethylated regions, with 60% of TET2 DMRs overlapping a DMR independently identified in the IDH samples. Conversely, the vast majority of IDH outliers (97%) were identified only in IDH mutated AMLs. This comprehensive, genome-wide analysis demonstrates that IDH mutations result in a clear hypermethylation phenotype in primary AML samples that is far more expansive than the observed TET2-associated methylation changes. IDH-specific methylation changes were enriched for CGIs located outside of promoters and were distinct from the AML-associated CGI hypermethylation that occurs in other AML subtypes. TET2 mutations were associated with far fewer hypermethylated DMRs, and only a small fraction of mutation-specific changes occurred in bothIDH and TET2 samples. Our results demonstrate that the methylation phenotypes of IDH and TET2 mutant AML samples do not phenocopy, and suggest that IDH-associated hypermethylation must have a TET2-independent component. Disclosures No relevant conflicts of interest to declare.

Overexpression or Deficiency of RUNX3 Results in Defective Normal Human Hematopoietic Development Similar to RUNX1-ETO Expression in AML

A common abnormality in AML (observed at a frequency of ~12%) is the t(8;21)(q22;q22) encoding the RUNX1-ETO fusion protein. Previously we showed that RUNX1-ETO can block the differentiation of normal human primary CD34+ hematopoietic stem progenitor cells (HSPC) and promote their self-renewal (consistent with its putative initiating role; Tonks et al 2003, 2004). To identify the genes that may mediate this phenotype, transcriptome analysis was performed (Tonks et al 2007). We now report that RUNX1-ETO significantly downregulates the expression of RUNX3 (2.1±0.6 fold in CD34+ HSPC; p&lt;0.05). Furthermore, we also show that RUNX3 mRNA is significantly downregulated in t(8;21) patients compared to normal human bone marrow by 4 fold (p&lt;0.001). Interestingly, in non-t(8;21) AML, RUNX3 expression is overexpressed compared with normal HSC, suggesting that both down and upregulation of RUNX3 may contribute to the pathogenesis of AML. To investigate this further, we examined the impact of RUNX3 expression on normal human hematopoiesis. Initially, we investigated the role of RUNX3 in erythropoiesis (which is severely disrupted by RUNX1-ETO). We mimicked the effects of RUNX1-ETO through shRNA-mediated knockdown (KD) of RUNX3 in CD34+ HSPC which co-expressed GFP. Following infection, GFP+ CD13low cells were isolated by FACS to yield a population of cells enriched for erythroid progenitors. Under clonal conditions, RUNX3-KD cells formed significantly less erythroid colonies than control (1.9±0.7 fold, p&lt;0.05), matching the impairment seen in erythroid progenitors expressing RUNX1-ETO. To examine this phenotype in more detail, we studied the effect of RUNX3-KD on the EPO-independent and EPO-dependent stages of erythroid development. RUNX3-KD significantly inhibited growth during the EPO-independent development (suppressed by 1.7±0.1 fold; p&lt;0.001) and showed impaired differentiation with reduced CD36 expression (1.5±0.3 fold, p&lt;0.01), retention of HLA-DR (1.2±0.1 fold, p&lt;0.01) and CD34 (1.2±0.1 fold, p&lt;0.05). These cells showed partial response to EPO but again growth response was suppressed (2±0.8 fold compared to control). Developmentally, RUNX3-KD cells failed to upregulate GlyA to the same levels as controls (suppressed by 1.3±0.1 fold p&lt;0.05), suggesting that the inhibition of differentiation persisted in the EPO-dependent phase of development. In summary, KD of RUNX3 in HSPC leads to an impairment in erythropoiesis similar to the phenotype induced by RUNX1-ETO, which suggests that suppression of RUNX3 by this fusion protein contributes to the RUNX1-ETO phenotype. We next assessed the effect of RUNX3 overexpression on erythroid development as above, this time using retrovirus co-expressing RUNX3 and DsRed. Interestingly, RUNX3 overexpression also suppressed erythroid colony formation by 1.7±0.7 fold (p&lt;0.01) compared to control, however, in a serial replating strategy, RUNX3 cells were able to form 2.7±0.6 fold more erythroid colonies than control, which suggests that RUNX3 promotes self-renewal. In the EPO-independent phase of development RUNX3 overexpression significantly inhibited the growth of erythroid progenitors by 2.7±1.3 fold (p&lt;0.05), possibly arising from reduced erythroid commitment (CD36+CD13neg reduced by 1.5±0.2 fold, p&lt;0.01). Erythroid committed cells showed a greater proliferative response to EPO (4.6±2.8 fold greater than controls) and showed phenotypic evidence of inhibition of development: decreased expression of GlyA (suppressed by 1.3±0.1 fold, p&lt;0.01), retention of CD36 (1.4±0.2 fold, p&lt;0.05) and 20% increased cell size (p&lt;0.05) compared to controls. Morphologically, these cells appeared to be blocked in an early/intermediate stage of development. These data imply that RUNX3 overexpression impairs erythroid differentiation and blocks terminal differentiation. Overall, these results show that both the overexpression and KD of RUNX3 in normal HSPC leads to a block in erythroid differentiation. Further, our preliminary data suggests that ectopic expression of RUNX3 in CD34+ HSPC selectively inhibits granulocytic development. These experimental models indicate that the level of RUNX3 expression is an important regulator of normal human hematopoietic development. We are currently determining the underlying mechanisms by which RUNX3 perturbs normal hematopoiesis, which ultimately may lead to new therapies for AML patients. Disclosures Knapper: Daiichi Sankyo: Honoraria; Jazz: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; Tolero: Membership on an entity's Board of Directors or advisory committees.