Inhibition of S-Adenosylmethionine Synthesis Promotes Erythropoiesis Via Epigenetic Modifications

Abstract Erythroid differentiation involves global gene expression repression, chromatin condensation and enucleation, mitochondria removal and other marked cellular changes. Given the necessity for these dynamic alterations, it is hardly surprising that epigenetic modifications possess important roles for erythropoiesis. S-adenosylmethionine (SAM), a principle methyl donor for DNA and histone methylations, would be involved in this process. Yet little is known about the specific roles for SAM synthesis in erythropoiesis. SAM is synthesized from methionine and ATP via the enzymatic activity of Mat2a and we evaluated the in vivo role of SAM synthesis by treating wild type mice (C57BL/6) with a selective Mat2a inhibitor (cycloleucine). As expected, the Mat2a inhibitor administration (henceforth Mat2ai) reduced SAM in bone marrow (BM) cells (SAM; 3.17±0.43 and 0.93±0.10 area ratio for ctrl and Mat2ai, p < 0.01, n = 4 mice). Interestingly, Mat2ai increased erythropoiesis in BM (Ter119 + cell; 46.3±3.1 and 116.4±14.2×10 6 cells for ctrl and Mat2ai, p < 0.01, n = 8 mice) and in blood (hemoglobin concentrations in peripheral blood; 13.7±0.18 and 16.3±0.26 g/dl for ctrl and Mat2ai, p < 0.01, n = 8 mice). However, serum erythropoietin concentration decreased (erythropoietin; 254.2±34.1 and 42.7±5.70 pg/ml for ctrl and Mat2ai, p < 0.01, n = 10 mice). Therefore, Mat2ai promoted erythropoiesis in vivo without increasing erythropoietin. To reveal the point where the erythroid differentiation was affected, immature and mature erythroblast subsets in BM were assessed. Although immature erythroblasts were not changed by Mat2ai (24.1±2.80 and 23.8±3.86×10 6 cells for ctrl and Mat2ai, p = 0.95, n = 8 mice), mature erythroblasts in BM increased following Mat2ai (18.9±2.48 and 81.2±9.73×10 6 cells for ctrl and Mat2ai, p < 0.01, n = 8 mice). Therefore, Mat2ai promoted erythroid maturation from immature erythroblast in BM. To reveal the mechanistic insight of this promotion of erythroid maturation by Mat2ai, we performed RNA sequencing of immature erythroblast in BM. This analysis revealed that most genes were down-regulated by Mat2ai (differentially expressed genes by Mat2ai; DOWN 2578 genes, UP 72 genes). In line with this notion, transposase-accessible chromatin sequencing (ATAC-seq) of immature erythroblasts revealed that chromatin accessibility was reduced. While DNA methylation analysis (whole genome bisulfite sequence) of immature erythroblasts revealed slightly reduced global DNA methylation (approximately 2%), there were no clear correlations between changes in promotor (or gene-body) DNA methylation and transcription. This result suggests that DNA methylation changes possess limited roles for the erythroid maturation promoted by Mat2ai. On the other hand, we found that an active histone methylation mark (H3K4me3) was selectively reduced by Mat2ai and that the changes of gene expression and H3K4me3 enrichment (revealed by chromatin immunoprecipitation followed by sequencing) correlated (r = 0.66). Therefore, the loss of H3K4me3, but not the DNA methylation, might contribute to the global gene expression repression for erythroid maturation induced by Mat2ai. Finally, in vitro human erythroid differentiation analysis using CD34 + cord blood cells further revealed that therapeutic and genetic inhibition of SAM synthesis induced erythroid maturation, which was cancelled by extracellular administration of SAM. Therefore, SAM synthesis inhibition is a non-erythropoietin trigger for erythroid maturation and this process occurs in human cells. Collectively, we found that SAM synthesis inhibition promoted erythroid maturation in both mouse and human. Histone methylation alteration induced by SAM synthesis inhibition might contribute to this phenomenon. These findings may pave the way to develop a new therapeutic strategy for anemia in erythropoietin independent manner. Disclosures Harigae: Kyowakirin: Other: Subsidies or Donations; Astellas Pharma: Other: Subsidies or Donations; Ono pharma: Honoraria, Other: Subsidies or Donations; Janssen Pharma: Honoraria; Chugai Pharma: Honoraria; Novartis Pharma: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria. Scadden: Magenta Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Clear Creek Bio: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; LifeVaultBio: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Editas Medicines: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics: Current holder of individual stocks in a privately-held company; Clear Creek Bio: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Dainippon Sumitomo Pharma: Other: Sponsored research; FOG Pharma:: Consultancy; Garuda Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; VCanBio: Consultancy; Inzen Therapeutics: Membership on an entity's Board of Directors or advisory committees.

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The BET Inhibitor INCB054329 Primes AML Cells for Venetoclax-Induced Apoptosis

Blood ◽

10.1182/blood-2018-99-111393 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4074-4074

Author(s):

Haley Ramsey ◽

Susu Zhang ◽

Yue Zhao ◽

Melissa Ann Fischer ◽

Agnieszka Ewa Gorska ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Rna Polymerase ◽

Growth Inhibition ◽

Board Of Directors ◽

Research Funding ◽

Advisory Committees ◽

Cycle Arrest ◽

Bet Inhibitors

Abstract Bromodomain and extra-terminal (BET) inhibitors may be efficacious for treatment of acute myeloid leukemia because they attenuate the expression of critical oncogenes including MYC and BCL2. These BET inhibitors (BETi) disrupt the transcriptional elongation process by displacing BET family members BRD2,3, and 4 off of chromatin, and causing RNA polymerase promoter-proximal pausing. We used precision nuclear run-on transcription sequencing (PROseq) to directly measure the effects of INCB054329, a potent BETi, on RNA polymerase II pausing and elongation. We found dramatic reductions on the elongation of key oncogenes such as MYC and BCL2 within 15 min of adding the drug. These effects became more significant over time, eventually affecting nearly two thousand genes. By four hours after drug addition, we found a loss of ribosomal gene expression and a loss of mitochondrial gene expression that is characteristic of genes regulated by MYC, suggesting that these were secondary to turning off MYC expression. When we examined the potential of the BETi INCB054329 for therapeutic efficacy in AML using Alamar Blue assays, which measure cellular redox potential, we noted marked growth inhibition of AML cell lines. However, growth assays and measurements of apoptosis using Annexin V staining found that BETi induced minimal apoptosis and cells were largely cytostatic. BrdU incorporation assays showed that INCB054329 caused the cells to accumulate in the G0/G1 phase of the cell cycle. Metabolic studies indicated that INCB054329 treatment for 48 hours caused disruption of mitochondrial respiration rate and severely reduced glycolytic capacity. Taken together, the growth inhibition, cell cycle arrest and reduced metabolic rate suggests that INCB054329 promoted quiescence in AML cells, but that this is reversible, consistent with the clinical experience of single-agent treatment of hematologic malignancies with BETi. MLL fusion proteins enhance transcription by stimulating RNA polymerase elongation, suggesting INCB054329may provide a therapeutic option to reverse this effect. However, the cell cycle arrest suggested that a second compound may be needed to trigger cell death. We first performed in vivo studies with INCB054329 using a systemic AML xenograft model of MV4-11 cells that express MLL-AF4. Engrafted NSGS mice received INCB054329 in 3 different doses (vehicle vs 10, 30 and 75mg/kg q.d) daily. During treatment, the kinetics of MV-4-11 expansion was monitored via flow cytometry for the detection of human AML in the blood. At approximately 4 weeks after transplant, the vehicle mice became moribund, and all experimental groups were sacrificed for analysis of chimerism. Significant decreases in leukemic expansion were evident in the bone marrow (vehicle vs75mg/kg, p<.001) and spleen (vehicle vs. 75mg/kg, p <.001) of treated mice. As BETi decreases expression of BCL2, we posited that BH3 directed therapy with the BCL-2 inhibitor venetoclax (VEN) could be enhanced by INCB054329. In vitro, we found that the combination of INCB054329 and VEN resulted in significant growth inhibition and apoptosis of treated AML cells. This finding prompted us to test the combination of INCB054329 with VEN in vivo. Mice engrafted with human AML cells received INCB054329 (50mg/kg q.d), VEN (25mg/kg q.d) or the combination. Four weeks after transplant, analyses by flow cytometric measurement of human CD45 of combination treated mice revealed significant decreases of AML cells in the bone marrow (vehicle vs. BRDi/VEN p = 0.004) and spleen (vehicle vs.BRDi/VEN, p = 0.001). Further studies are underway to test this combination in both VEN sensitive and resistant AML primary xenograftmodels. These preliminary data suggest that INCB054329 may serve as a non-cytotoxic priming agent for BH3 directed therapy, and the combination of INCB054329 +VEN may provide a potent therapy in a variety of genetically distinct subtypes of AML. Disclosures Stubbs: Incyte: Employment. Liu:Incyte: Employment. Rathmell:Calithera: Research Funding. Hiebert:Incyte: Research Funding. Savona:Boehringer Ingelheim: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Dasatinib Inhibits FLT3/ITD and PTPN11mutated Acute Myeloid Leukemia Cells Overexpressing SRC Tyrosine Kinases

Blood ◽

10.1182/blood-2019-128016 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1451-1451

Author(s):

Sigal Tavor ◽

Tali Shalit ◽

Noa Chapal Ilani ◽

Yoni Moskovitz ◽

Nir Livnat ◽

...

Keyword(s):

Gene Expression ◽

Acute Myeloid Leukemia ◽

Board Of Directors ◽

Myeloid Leukemia ◽

Drug Sensitivity ◽

Kinase Inhibitors ◽

Advisory Committees ◽

Acute Myeloid

Background: Recent advances in acute myeloid leukemia(AML) targeted therapy improve overall survival. While these targeted therapies can achieve prolonged remissions, most patients will eventually relapseunder therapy. Our recent studies suggest that relapse most often originates from several sub-clones of leukemic stem cells (LSCs), present before therapy initiation, and selected due to several resistance mechanisms. Eradication of these LSCs during treatment induction /remission could thus potentially prevent relapse. The overall goal of the current study was to identify drugs which can be safely administrated to patients at diagnosis and that will target LSCs. Since simultaneously testing multiple drugs in vivo is not feasible, we used an in vitrohigh throughput drug sensitivity assay to identify new targets in primary AML samples. Methods: Drug sensitivity and resistance testing (DSRT) was assessed in vitro (N=46 compounds) on primary AML samples from patients in complete remission (N=29). We performed whole exome sequencing and RNAseq on samples to identify correlations between molecular attributes and in vitro DSRT. Results:Unsupervised hierarchical clustering analysis of in vitro DSRT, measured by IC50, identified a subgroup of primary AML samples sensitive to various tyrosine kinase inhibitors (TKIs). In this subgroup, 52% (9/17) of AML samples displayed sensitivity to dasatinib (defined as a 10-fold decrease in IC50 compared to resistant samples). Dasatinib has broad TKI activity, and is safely administered in the treatment of leukemia. We therefore focused our analysis on predicting AML response to dasatinib, validating our results on the Beat AML cohort. Enrichment analysis of mutational variants in dasatinib-sensitive and resistant primary AML samples identified enrichment of FLT3/ITD (p=0.05) and PTPN11(p=0.05) mutations among dasatinib responders. Samples resistant to dasatinib were enriched with TP53 mutations (p=0.01). No global gene expression changes were observed between dasatinib-sensitive and resistant samples in our cohort, nor in the Beat AML cohort. Following this, we tested the differential expression of specific dasatinib-targeted genes between dasatinib-responding and resistant samples. No significant differences were identified. However, unsupervised hierarchical clustering of dasatinib targeted genes expression in our study and in the Beat AML cohort identified a subgroup of AML samples (enriched in dasatinib responders) that demonstrated overexpression of three SRC family tyrosine kinases:FGR, HCK and LYN as well as PTK6, CSK, GAK and EPHB2. Analysis of the PTPN11 mutant samples revealed that the IC50 for dasatinib in 23 carriers of the mutant PTPN11 was significantly lower compared to the IC50 of PTPN11 wild type samples (p=0.005). LYN was also upregulated (p<0.001) in the mutant samples. We therefore hypothesized that gene expression of dasatinib-targeted genes could be used as a predictive biomarker of dasatinib response among FLT3/ITD carriers. We found that among FLT3/ITD AML carriers in the Beat AML cohort LYN, HCK, CSK and EPHB2 were significantly over-expressed in the dasatinib responding samples (N=27) as compared to the dasatinib resistant samples (N=35). To predict response to dasatinib among FLT3/ITD carriers we used a decision tree classifier based on the expression levels of these four genes. Our prediction model yielded a sensitivity of 74% and specificity of 83% for differentiating dasatinib responders from non-responders with an AUC of 0.84. Based on our findings, we selected FLT3/ITD AML samples and injected them to NSG-SGM3 mice. We found that in a subset of these samples, dasatinib significantly inhibited LSCs engraftment. This subset of FLT3/ITD AML samples expressed higher levels of LYN, HCK,FGR and SRC as compared to the FLT3/ITD samples that were not sensitive to dasatinib therapy in vivo. In summary, we identified a subgroup of AML patients sensitive to dasatinib, based on mutational and expression profiles. Dasatinib has anti-leukemic effects on both blasts and LSCs. Further clinical studies are needed to demonstrate whether selection of tyrosine kinase inhibitors, based on specific biomarkers, could indeed prevent relapse. Disclosures Tavor: Novartis: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; BMS companies: Membership on an entity's Board of Directors or advisory committees.

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Modeling Diamond Blackfan Anemia in Vivo Using Human Induced Pluripotent Stem Cells

Blood ◽

10.1182/blood.v124.21.359.359 ◽

2014 ◽

Vol 124 (21) ◽

pp. 359-359

Author(s):

Sergei Doulatov ◽

Linda T Vo ◽

Elizabeth R Macari ◽

Stephanie S Chou ◽

Manav Gupta ◽

...

Keyword(s):

Board Of Directors ◽

Pluripotent Stem Cells ◽

Disease Modeling ◽

Erythroid Differentiation ◽

Advisory Committees ◽

Diamond Blackfan Anemia ◽

Equity Ownership ◽

Induced Pluripotent

Abstract Human induced pluripotent stem cells (iPSCs) represent a promising source of patient-specific cells for disease modeling, drug screens and cellular therapies. However, the inability to derive engraftable human hematopoietic stem and progenitor cells has limited their use for modeling of hematological diseases. We previously reported a strategy to respecify lineage-restricted CD34+hematopoietic precursors derived from iPSCs into multilineage progenitors that can be expanded in vitro and transplanted in vivo. Five transcription factors, HOXA9, ERG, RORA, SOX4 and MYB, enable expansion and maintenance of primitive CD34+CD38- cells, and allow their differentiation upon transgene silencing. Respecified iPSC-derived hematopoietic progenitors give rise to robust short-term engraftment with myeloid and erythroid lineages. Notably, the erythrocytes undergo definitive maturation and hemoglobin switching to express β-globin in vivo. This system presents a useful platform for modeling hematological disorders due to its capacity to generate large numbers of engraftable disease cells for in vitro and in vivo screens. Congenital anemias, such as Diamond Blackfan anemia (DBA) represent a defined system for understanding red blood cell development and more common idiopathic anemias. To model this disease in vitro, we combined factor-induced respecification with stepwise erythroid maturation. We show that respecified iPSCs from DBA patients recapitulate the defect in erythroid differentiation. Consistent with clinical observations, early erythroblasts show impaired proliferation marked by increased apoptosis and p21 expression, while terminal maturation is unaffected. Interestingly, while early passage iPSC lines display a profound erythroid defect, continuous passage restores a near-normal capacity for erythroid differentiation. Furthermore, transplanted DBA iPSC-derived progenitors give rise to normal myeloid, but fail to generate erythroid, engraftment. This validates this system as a powerful tool for disease modeling and drug discovery. Mice transplanted with DBA iPSC-derived progenitors treated with conventional anemia drugs, such as dexamethasone, show a modest improvement in human erythroid output suggesting the need for novel treatments. Using high-throughput chemical screens, we have identified several signaling pathways which may be attractive therapeutic targets in DBA and other anemias. Further characterization is currently underway to better target defective erythropoiesis and dissect underlying disease mechanisms. Disclosures Zon: FATE Therapeutics, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other; Scholar Rock: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other; Stemgent: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

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Spatial Transcriptomics Reveals DPP4 As Novel Marker of a More Proliferative Phenotype in Early AML Progression

Blood ◽

10.1182/blood-2021-151917 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3310-3310

Author(s):

Christa Haase ◽

Karin Gustafsson ◽

Shenglin Mei ◽

Jelena Milosevic ◽

Shu-Chi Yeh ◽

...

Keyword(s):

Bone Marrow ◽

Disease Progression ◽

Board Of Directors ◽

Hematologic Malignancy ◽

New Technology ◽

Publicly Traded ◽

Advisory Committees ◽

Privately Held

Abstract Acute myeloid leukemia (AML) is a hematologic malignancy with poor prognosis for which the standard-of-care chemotherapy treatment regimen has remained virtually unchanged over the past 40 years. We have employed "Image-Seq", a new technology that was developed in our laboratory, to study spatial variations in early leukemia progression in a mouse model of HoxA9-Meis1 AML. We visualized leukemia cells with differing proliferative phenotype using intravital microscopy, captured these cells under image guidance from individual bone marrow microenvironments and studied their differential expression by single-cell RNA sequencing. This analysis identified DPP4 as a key upregulated gene in AML cells from more proliferative bone marrow compartments and associated DPP4 expression with a cell cluster enriched in progenitor cell markers for HoxA9-Meis1 AML, including Flt3, Itgb7 and Ddx4. Strikingly, DPP4 is not expressed in vitro, and its expression in vivo (as quantitated by FACS analysis) correlated with disease progression and marked a more proliferative phenotype both at the 1-week and 2-week time-points during disease progression. Disclosures Sykes: Clear Creek Bio: Current equity holder in publicly-traded company; SAFI Biosolutions: Consultancy, Current equity holder in publicly-traded company; Keros Therapeutics: Consultancy. Scadden: Magenta Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; VCanBio: Consultancy; LifeVaultBio: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Inzen Therapeutics: Membership on an entity's Board of Directors or advisory committees; Garuda Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; FOG Pharma: Consultancy; Fate Therapeutics: Current holder of individual stocks in a privately-held company; Editas Medicines: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Dainippon Sumitomo Pharma: Other: sponsored research; Clear Creek Bio: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees.

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Btm-3566, a Novel Activator of the Mitochondrial Stress Response Promotes Robust Therapeutic Responses in Vitro and In Vivo in Diffuse Large B-Cell Lymphoma

Blood ◽

10.1182/blood-2021-145637 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 684-684

Author(s):

Adrian Schwarzer ◽

Matheus Oliveira ◽

Marc-Jens Kleppa ◽

Andy Anantha ◽

Alan Cooper ◽

...

Keyword(s):

Stress Response ◽

B Cell ◽

Board Of Directors ◽

Research Funding ◽

Advisory Committees ◽

Double Hit ◽

Large B Cell ◽

Privately Held

Abstract Relapsed/refractory diffuse large B-cell lymphomas (r/r-DLBCL) are a therapeutic challenge, especially in patients not suitable for high dose chemotherapy, stem cell transplantation or patients who fail CAR-T-cell therapy. r/r-DLBCLs are highly heterogeneous both clinically and molecularly, which imposes a pressing need to develop novel therapies to improve outcomes in patients independently of the molecular subtype. We describe here BTM-3566, a first-in-class compound with activity against a variety of B-cell malignancies but with greatest effect in DLBCL. BTM-3566 activates the mitochondrial integrated stress response (ISR) through a novel mechanism regulated by the mitochondrial protein FAM210B. BTM-3566 induces apoptosis in DLBCL lines in vitro and complete tumor regression in vivo in DLBCL PDX mouse models harboring genetic alterations associated with poor prognosis. BTM-3566 is an oral small molecule based on a pyrazolothiazol-backbone, developed for treatment of diffuse large B-cell lymphoma (DLBCL). BTM-3566 induces apoptosis and complete cell killing in DLBCL lines a with an IC 50 of ~200 - 500 nM. Responsive DLBCL cell lines include ABC, GCB, and double-hit and triple-hit lymphoma lines. Pharmacokinetic studies in mice showed suitability for once daily dosing, with > 50% of oral bioavailability and close to 6 hours of serum half-life. 14-day dosing in mice and dogs demonstrated excellent tolerability at therapeutic doses. BTM-3566 showed stability in human hepatocytes (IC < 5 ml/min*kg) as well and a favorable in vitro safety profile. In xenograft models using the double-hit DLBCL line SU-DHL-10, BTM-3566 treatment resulted in complete regression in all tumor-bearing animals. Most importantly, no subsequent tumor growth occurred for 2 weeks after cessation of therapy, indicating that treatment with BTM-3566 resulted in a durable complete remission in this model of double-hit DLBCL. Expansion studies into human DLBCL PDX models harboring a range of high-risk genomic alterations, including Myd88 mutations and MYC and BCL2 rearrangements, demonstrated response in 100% of the lines with complete tumor regression in 6 of 8 PDX models tested (Table 1). Transcriptome and proteome analyses revealed that BTM-3566 strongly activated the ATF4-integrated stress response (ISR), indicated by phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) and subsequent upregulation of the transcription factor ATF4. Of the four eIF2a-kinases in the human genome we determined, using CRISPR-Cas 9 gene depletion, that HRI was uniquely required for BTM-3566 eIF2a phosphorylation, induction of ATF4 ISR and apoptosis. HRI is described as being activated by mitochondrial-related stress, including heme depletion, increased ROS generation or blockage of mitochondrial ATP synthesis which result in an increase in mitochondrial proteostasis including activation of mitochondrial protease OMA1. We determined that BTM-3566 activates OMA1 without acting as a classical mitochondrial toxin. Treatment with BTM-3566 induced OMA1-dependent OPA1 processing and mitochondrial fragmentation in as little as 30 minutes of treatment, in the absence of any reduction in mitochondrial oxygen consumption or membrane depolarization. This data indicates that BTM-3566 represents a new class of compounds that activate the mitochondrial protease OMA1. Gene expression-based profiling of BTM-3566 sensitivity in over 400 cancer cell lines showed that FAM210B, a mitochondrial membrane protein, negatively correlated with response to BTM-3566. Notably, overexpression of FAM210B completely prevents OMA1 activation and causes complete resistance to BTM-3566-induced apoptosis in DLBCL cell line BJAB and the Burkitt lymphoma cell line Ramos. Thus, FAM210B serves as a strong predictor of BTM-3566 sensitivity, as well as revealing a novel mechanism of regulation of OMA1 activation. In summary, we describe here a novel, highly potent activator of the mitochondrial ISR, which is well tolerated in mice and dogs, has favorable pharmacokinetics and induces robust DLBCL regression in-vivo. An IND application in B-cell malignancies will be completed by early Q1 2022 with initiation of first in human clinical trials the first half of 2022. Figure 1 Figure 1. Disclosures Schwarzer: Bantam Pharmaceutical: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Anantha: Bantam Pharmaceutical: Consultancy, Current Employment, Current holder of stock options in a privately-held company. Cooper: Bantam Pharmaceutical: Consultancy. Hannink: Bantam Pharmaceutical: Research Funding. Hembrough: Bantam Pharmaceutical: Consultancy. Levine: Bantam Pharmaceutical: Consultancy, Current holder of individual stocks in a privately-held company. Luther: Bantam Pharmaceutical: Current holder of individual stocks in a privately-held company, Ended employment in the past 24 months, Membership on an entity's Board of Directors or advisory committees. Stocum: Bantam Pharmaceutical: Current Employment, Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees. Liesa-Roig: Bantam Pharmaceutical: Research Funding. Kostura: Bantam Pharmaceutical: Consultancy, Current holder of stock options in a privately-held company, Patents & Royalties: Named Inventor on patents with assignment to Bantam Pharmaceutical.

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MCT1 As Molecularly Validated Predictive Marker for Lenalidomide-Maintenance Therapy in Multiple Myeloma

Blood ◽

10.1182/blood-2019-130734 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3187-3187

Author(s):

Jacob Stroh ◽

Anja Seckinger ◽

Michael Heider ◽

Ruth Eichner ◽

Martina Emde ◽

...

Keyword(s):

Gene Expression ◽

Board Of Directors ◽

Research Funding ◽

Maintenance Treatment ◽

Xenograft Model ◽

Predictive Marker ◽

High Dose ◽

Advisory Committees

Introduction: Lenalidomide-based maintenance therapy is the currently approved standard of care for multiple myeloma (MM) patients after high-dose melphalan and autologous stem cell transplantation (HD-Mel), which significantly prolongs progression-free (PFS) and overall survival (OS). For patients with del17p bortezomib based maintenance treatment is considered overcoming adverse prognosis of this aberration. Predictive markers of response to lenalidomide maintenance have remained elusive. We have previously shown that IMiDs exert their anti-MM activity via destabilization of MCT1 and CD147 and combined overexpression reduces response to lenalidomide-treatment in vitro and in an in vivo MM xenograft model (Eichner et al. Nature Medicine 2016). Methods: CD138-purified myeloma cell samples of 654 patients receiving high-dose melphalan therapy and autologous stem cell transplantation and either bortezomib (n=101), thalidomide (n=98) or lenalidomide (n=455) maintenance treatment were assessed by gene expression profiling (GEP) using U133 2.0 plus DNA microarrays, 316 by RNA-sequencing (RNA-seq). Expression of CD147 and MCT1 were assessed and correlated with PFS and OS data. Gene expression based risk scores, including UAMS70-gene, Rs-score and gene expression based proliferation index were assessed alongside routine iFISH-analysis. Survival curves and median time to progression were computed with nonparametric survival estimates for censored data using the Kaplan-Meier method. Difference between the curves were tested using the G-rho Log-rank test. Landmark analysis was performed by defining an alternative start point (landmark) at 12 months. In vitro, CD147 and MCT1 were lentivirally overexpressed in MM1S cells, which were subjected to lenalidomide or bortezomib treatment and proliferation analysis. Xenografted MM-tumors were followed by 18FDG-PET and analyzed by immunohistochemistry. Results: Patients with high gene expression levels of MCT1 showed significantly reduced PFS (31.9 vs. 48.2months in MCT1high vs. MCT1low,P=.03) and OS (75.9 months vs. not reached (NR) months in MCT1high vs. MCT1low; P=.001) in case of lenalidomide maintenance. Likewise, patients with thalidomide maintenance showed reduced PFS (34.8 vs. 43.7 months in MCT1high vs. MCT1low, P=.23) and significantly shorter OS (83.6 months vs. not reached (NR) months in MCT1high vs. MCT1low;P=.03). For bortezomib based maintenance, MCT1 expression had no significant impact on PFS (39.8 months vs. 32.6 months in MCT1high vs. MCT1low) and OS (125.8 months vs. 129.8 months in MCT1high vs. MCT1low). No association with other prognostic factors was found. As still differences between MCT1high vs. MCT1lowexpression myeloma cells might be attributed to undiscerned molecular factors and for functional validation, we lentivirally overexpressed CD147 and MCT1 in human myeloma cell lines. Overexpression of MCT1 significantly reduced cytotoxicity of lenalidomide, while no change was observed in MM cells treated with bortezomib. We subsequently validated our results in vivo. Functional investigations in the mechanism of MCT1 impact on cellular survival are ongoing. Conclusion: Taken together MCT1 expression as potential predictive marker for response to IMiD-based maintenance treatment. Both PFS and OS were significantly reduced in patients with high gene expression levels of MCT1. In vitro and in vivo (xenograft model), MCT1 overexpression reduced sensitivity to lenalidomide unlike bortezomib treatment. Disclosures Salwender: Bristol-Myers Squibb: Honoraria, Other: Travel or accommodations; Janssen Cilag: Honoraria, Other: Travel or accommodations; AbbVie: Honoraria; Celgene: Honoraria, Other: Travel or accommodations; Sanofi: Honoraria, Other: Travel or accommodations; Takeda: Honoraria, Other: Travel or accommodations; Amgen: Honoraria, Other: Travel or accommodations. Bertsch:Sanofi: Other: travel support; Celgene: Other: travel support. Goldschmidt:Chugai: Honoraria, Research Funding; Amgen: Consultancy, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive Biotechnology: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Molecular Partners: Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Research Funding; Dietmar-Hopp-Stiftung: Research Funding; John-Hopkins University: Research Funding; John-Hopkins University: Research Funding; MSD: Research Funding; Mundipharma: Research Funding; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Weisel:Takeda: Consultancy, Honoraria; GSK: Honoraria; Sanofi: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Juno: Consultancy; Bristol-Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Adaptive Biotech: Consultancy, Honoraria. Scheid:Celgene: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria; Takeda: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria. Bassermann:Celgene: Consultancy, Research Funding.

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The Genomic and Epigenomic Landscapes of Blast Crisis Transformation in Chronic Myeloid Leukemia

Blood ◽

10.1182/blood.v126.23.3737.3737 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3737-3737 ◽

Cited By ~ 3

Author(s):

S. Tiong Ong ◽

Thushangi Pathiraja ◽

Asif Javed ◽

Xin Xuan Sheila Soh ◽

Simeen Malik ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Chronic Myeloid Leukemia ◽

Board Of Directors ◽

Candidate Genes ◽

Myeloid Leukemia ◽

Research Funding ◽

Blast Crisis ◽

Advisory Committees ◽

Cpg Sites

Abstract The transition from chronic phase (CP) to blast crisis (BC) chronic myeloid leukemia (CML) is characterized by reprogramming of the CML transcriptome (Radich et al. PNAS 2006), and shortened survival. Current models propose genomic instability as causal in BC transformation with enhanced DNA damage and impaired DNA repair inducing genetic mutations (ranging from large chromosomal aberrations to point mutations), altered gene function, and eventually BC transformation (Perrotti et al. JCI 2010). Consistent with this model are the phenomena of BC clonal evolution, and the increased frequency of ABL kinase domain mutations found in BC. Because different mutational processes are associated with distinct cancer-specific mutation signatures (Alexandrov et al. Nature 2013), this model also predicts the existence of a CML-specific mutation signature. In addition, recent work has highlighted the importance of epigenetic alterations in hematologic malignancies (Shih et al., Nat. Rev. Cancer, 2012). However, we lack a complete understanding of the type or frequency of genetic alterations in BC, and the relative contribution of genetic vs. epigenetic events in reprogramming the BC transcriptome. To address these knowledge gaps, we analyzed the CML progression genome, epigenome, and transcriptome in 12 CP/BC sample pairs. Whole-genome sequencing revealed the CML genome to be relatively stable with respect to structural variations, indels, and somatic single nucleotide variants. The average number of nonsynonymous coding mutations per BC genome was 5, placing the BC coding genome in the same mutation frequency range as AML and ALL genomes (Alexandrov et al. Nature 2013). In addition, we identified a novel mutation signature in all CML samples suggesting a CML-specific mutational process. 1175 genes were 'hit' by genomic, mostly copy number, alterations in >1 sample, and included TCR genes and Ikaros (IKZF1) among lymphoid BC pairs. Only 21 recurrently altered genes were affected by somatic SNVs or indels, with resistance-associated ABL1 mutations being commonest. We next used DNA methylation arrays to assess the BC epigenome, and found 20,651 CpG sites (out of 455,187) to be hyper-methylated, and 3225 to be hypo-methylated in BC compared to CP. Combined methylome and transcriptome analysis demonstrated an inverse relationship between methylation and expression changes at a subset of CpG sites enriched at promoters. Genes with increased methylation/decreased expression or decreased methylation/increased expression included those involved in cell cycle control/heme biosynthesis, and molecular mechanisms of cancer/G-protein coupled receptor signaling/MAPK signaling respectively. Unsupervised methylation-based clustering segregated samples into CP, lymphoid BC and myeloid BC groups, recapitulating expression-based clustering, and further supporting a functional role for DNA methylation in BC transcriptional reprogramming. We next performed an integrative analysis by combining the genome, methylome, and transcriptome datasets, and included data from 34 additional CML samples. Top ranking candidate genes included epigenetic modifiers, and hematopoetic differentiation- and stem cell-related genes. Functional analysis of candidate genes and epigenetic processes using genetic and epigenetic drug-based approaches are ongoing. In summary, we conclude that: 1. The genomic and epigenomic landscapes in BC are characterized by a modest number of recurring events in the former, but consistent and striking differences in the latter, 2. The BC methylome is functionally associated with the robust gene expression changes found in BC, and 3. Epigenetic modifier drugs may be of use in reversing the gene expression changes characteristic of BC. Disclosures Chuah: Children International: Honoraria; Novartis: Honoraria; Bristol Meyers Squibb: Honoraria. Takahashi:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Sysmex: Research Funding, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Speakers Bureau; Masis: Consultancy; Otsuka: Membership on an entity's Board of Directors or advisory committees; Astellas: Speakers Bureau; BMS: Honoraria, Research Funding, Speakers Bureau.

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Transcriptional Signaling Centers Govern Human Erythropoiesis and Harbor Genetic Variations of Red Blood Cell Traits

Blood ◽

10.1182/blood-2018-99-118637 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1277-1277

Author(s):

Avik Choudhuri ◽

Eirini Trompouki ◽

Brian J. Abraham ◽

Leandro Colli ◽

William Mallard ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Transcription Factors ◽

Blood Cell ◽

Board Of Directors ◽

Red Blood Cell ◽

Erythroid Differentiation ◽

Regulatory Elements ◽

Advisory Committees ◽

Equity Ownership

Abstract Single Nucleotide Polymorphisms (SNPs) identified through genome-wide association studies (GWAS) provide insight into the mechanism of human genetic diseases, and majority of functional GWAS mutations target genomic regulatory elements. During erythroid differentiation of human CD34+ cells, we mapped regulatory DNA elements (enhancers and open chromatin regions) by H3K27Ac ChIP-seq and ATAC-seq, and studied the SNPs that reside within these DNA regulatory elements. We followed genomic binding of lineage restricted GATA transcription factors and also chose to examine the binding of the BMP signal responsive transcription factor SMAD1 in CD34+ cells during erythropoiesis. By overlapping their genomic occupancy with stage-matched RNA-seq, we found that SMAD1, in association with GATA-factors, serves as marker of genes responsible for differentiation at every step of erythropoiesis. ChIP-seq for other crucial signaling transcription factors, such as WNT-responsive and TGFb-responsive factors (TCF7L2 and SMAD2, respectively) demonstrated a remarkable co-existence of such factors at GATA+SMAD1 co-bound regions nearby stage-specific genes. We defined such regions as "Transcriptional Signaling Centers (TSC)" where multiple signaling transcription factors converge with master transcription factors to determine optimum stage-specific gene expression in response to growth factors. Our bioinformatics-algorithms demonstrated that PU1 and FLI1 binding sites were present in progenitor-specific TSCs whereas KLF1 and NFE2 sites were enriched in TSCs of red blood cells. We performed CRISPR-CAS9 mediated perturbations of each of the PU1, GATA and SMAD1 motifs separately in a representative progenitor TSC in K562 and HUDEP2 cells. Similar to loss of PU1 and GATA motifs, loss of SMAD1 motif selectively inhibited expression of the associated gene and showed defects in erythroid differentiation, demonstrating that TSCs are important to provide optimum gene expression and proper erythroid differentiation. To determine if such TSCs are targeted by GWAS mutations, we have studied 1270 lead and additional 27,799 SNPs in linkage disequilibrium with lead SNPs that are associated with six critical red blood cell traits - hemoglobin concentration (Hb), hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) and red blood cell count (RBC). Surprisingly, we observed that, out of the 3831 "functional" SNPs that fall within non-exonic H3K27Ac enhancers, while only 5% (188) of RBC-SNPs target only blood-master-transcription-factor motifs, at least 48% (1821) of them reside on various signaling pathway associated transcription factor motifs including SMADs (BMP/TGFb signaling), RXR/ROR (nuclear receptor signaling), FOXO/FOXA (FOX signaling), CREBs (cAMP signaling) and TCF7L2 (WNT signaling). Additionally, these RBC-trait-SNPs are specifically enriched in GATA+SMAD1 co-bound TSCs and fall within signaling factor binding sites. We validated such SNPs that target SMAD-motifs. The SNP rs9467664 is associated with the MCV-trait near HIST1H4A, a gene that increases in expression during differentiation. Using gel-shift assay, we found that SMAD1 binding is compromised when the major allele T changes to minor allele A under MCV-trait. Remarkably, eQTL analysis using microarray gene expression profiles of peripheral blood obtained from the Framingham Heart Studies revealed that expression of HIST1H4A is significantly more in a population with T-allele than that with A-allele. This demonstrates that inhibition of SMAD1 binding by the SNP causes a loss of allele-specific HIST1H4A expression. Another MCV-associated SNP rs737092 targets a SMAD motif within an erythroid-specific TSC near RBM38 gene. T-allele, in comparison with C-allele, that retains SMAD1 binding showed more expression in luciferase-based reporter assays specifically under BMP stimulation suggesting that rs737092 compromise BMP-responsiveness. Taken together, our study provides the first evidence that naturally occurring GWAS variations directly impact gene expression from signaling centers by modulating binding of signaling transcription factors under stimulation. Such aberrant signaling events over time could lead to "signalopathies", ultimately resulting in phenotypic variations of RBC traits. Disclosures Abraham: Syros Pharmaceuticals: Equity Ownership. Young:Omega Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Syros Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Camp4 Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

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In Vivo Assessment of Intracellular Dynamics Comparing Injection Versus Oral Azacitidine in a Phase IIb Investigator Initiated Clinical Trial

Blood ◽

10.1182/blood-2019-123921 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4247-4247

Author(s):

Ashwin Unnikrishnan ◽

Xin Ying Lim ◽

Swapna Joshi ◽

Andrea C. Nunez ◽

Lachlin Vaughan ◽

...

Keyword(s):

Cell Cycle ◽

Clinical Trial ◽

Dna Methylation ◽

Bone Marrow ◽

High Risk ◽

Board Of Directors ◽

Research Funding ◽

Dna Demethylation ◽

Advisory Committees

Introduction: 5'-Azacitidine (AZA), a DNA demethylating agent, is the primary drug for the treatment of high-risk Myelodysplastic Syndrome (MDS) and Chronic Myelomonocytic Leukaemia (CMML). Response is associated with improved survival. However, only half of patients respond, and these responses are rarely durable. We recently reported that primary AZA resistance is associated with a molecular signature of cell cycle quiescence within bone marrow (BM) hematopoietic progenitor cells (Unnikrishnan et al, Cell Reports, 20:572-585 (2017)). As DNA incorporation of the deoxyribonucleic form of AZA (5-aza-2′-deoxycytidine, DAC) occurs during DNA replication, cell cycle quiescence is predicted to lead to less DAC in DNA and concomitantly less DNA demethylation. We recently developed a quantitative multi-parameter assay, AZA-MS (Unnikrishnan, Vo et al, Leukemia 32:900-910 (2018)), to measure the intracellular dynamics of AZA in patients. Using AZA-MS, we reported data supporting the predicted resistance model. CC486 is an oral formulation of AZA. A 28-day cycle of CC486 involves 21 continuous days (21/28) versus the standard 7/28 subcutaneous (SC) injection AZA scheme. Whether levels of in vivo DAC incorporation into DNA during a cycle of CC486 are comparable with that of SC AZA is unknown. AZA-MS provides us with a unique opportunity to empirically assess the in vivo intracellular dynamics of SC versus oral AZA. Study Design and Methods: To directly assess in vivo DAC incorporation and concomitant DNA demethylation with SC AZA and CC486 in the same patient, we initiated a phase II clinical trial (NCT03493646; Fig A). MDS (IPSS; intermediate-2 or high-risk), CMML (bone marrow [BM] blasts 10-29%) and AML (20-30%) patients were recruited for six cycles of SC AZA (75mg/m^2/day for 7/28 days) followed by six cycles of CC486 (100mg bid for 21/28 days in C7-C8 and 150mg bid for 21/28 in C9-C12). Clinical response was assessed at the end of C6 and C12 using International Working Group criteria. Clinical responders and non-responders to SC AZA at C6 received CC486 from C7 onwards. From each patient, 36 peripheral blood (PB) samples and five BM samples were collected over the study period. DNA, RNA and intracellular fractions were isolated from the PB MNCs, for intracellular DAC/AZA measurements by AZA-MS (primary endpoint; Fig A). BM MNCs were utilised for AZA-MS as well as flow cytometry-based cell cycle measurements (secondary endpoint). Results: 31 of 42 consented patients have commenced treatment since trial opening (Fig B-C). We applied the AZA-MS assay on the longitudinal PB and BM samples collected from the seven patients who had completed six months AZA and commenced CC486 as at 26th June 2019 (Fig D). DAC incorporation into DNA and DNA methylation levels were quantified within the same cells, in addition to measuring other parameters (Fig E). As represented by patient 61213-005 (Fig F) who had a complete response (CR) at cycle 6, after 7 days of injection AZA we observed robust incorporation of DAC within PB MNCs (left panel, Fig F) together with concomitant DNA demethylation (right panel, Fig F). DAC levels diminished upon cessation of AZA within a cycle, with corresponding increases in DNA methylation. There were quantitatively higher levels of DAC incorporated in DNA during SC AZA cycles versus CC486. The trend observed is also appreciated from 2.3x higher area under the curve (AUC) measurements in 61213-005 during the SC AZA cycle. DAC incorporation was higher at C9/10 (CC486 150mg bid 21/28) than at C7/8 (CC486 100mg bid 21/28) without appreciable changes in DNA demethylation. During SC AZA cycles, higher DAC levels (top panel, Fig G) and greater DNA methylation (lower panel, Fig G) were seen in the BM MNCs. In a non-responding patient at cycle 6 (61290-002, SD), we saw less DAC incorporation and DNA demethylation (Fig H). We also observed a positive correlation between baseline proportions of cycling BM cells (LIN-CD34+CD38+) and the amount of DAC incorporated in BM MNCs at C1 day 8 (Fig I). Conclusion: AZA-MS can be used to reliably measure in vivo DAC incorporation and concomitant DNA demethylation in PB MNCs and inform appropriate CC486 dosing. Figure Disclosures Unnikrishnan: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Fong:Astellas: Consultancy; Novartis: Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Amgen: Consultancy, Research Funding, Speakers Bureau. Roncolato:St. George Hospital: Employment. Enjeti:Roche: Honoraria, Speakers Bureau; Bayer and Sanofi: Honoraria, Speakers Bureau; Astellas: Consultancy; Novartis: Consultancy; Abbvie: Consultancy. Hertzberg:BMS: Membership on an entity's Board of Directors or advisory committees; F. Hoffmann-La Roche Ltd: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees. Polizzotto:Janssen: Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding; ViiV: Research Funding. Pimanda:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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CD62L Expression Level Dictates the Cell Fate of Myeloid Progenitors in Mice and Humans

Blood ◽

10.1182/blood-2020-134753 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 26-27

Author(s):

Yusuke Ito ◽

Fumio Nakahara ◽

Yuki Kagoya ◽

Mineo Kurokawa

Keyword(s):

Gene Expression ◽

Transcription Factors ◽

Board Of Directors ◽

Transcriptome Analysis ◽

Research Funding ◽

Advisory Committees ◽

Differentiation Potential ◽

Cell Assay ◽

Colony Forming Cell

Hematopoietic cells are hierarchically differentiated from hematopoietic stem cells through several progenitors. Recent studies showed that each progenitor population has significant heterogeneity and some of the subsets have skewed differentiation potential. However, it has not been elucidated how and when common myeloid progenitors (CMPs) and granulocyte-monocyte progenitors (GMPs) acquire different fates. We hypothesized that progenitor cells with skewed differentiation potential had acquired a part of gene expression profiles of more differentiated cells. By analyzing publicly available single-cell RNA sequencing data of human and murine CMPs and GMPs, we thoroughly explored surface markers with heterogeneous expression patterns and identified CD62L as a candidate to refine the differentiation potential of CMPs and GMPs. First, at CMP level, we clarified that CD62L-low CMPs had genuine CMP potential, whereas CD62L-high CMPs were mostly restricted to GMP potentials in both mice and humans. CD62L expression on CMPs was widely distributed and when divided into low, middle, and high fraction, colony forming-cell assay showed that murine CD62L-high CMPs mostly differentiated into CD11b-positive granulocytes and macrophages (97.4 ± 1.7%), whereas CD62L-low CMPs produced many erythroid and megakaryocytic colonies (38.7 ± 3.3%), and human CMPs had similar tendency. Also, liquid culture assay showed that murine CD62L-low CMPs differentiated into both GMPs (43.3 ± 4.1%) and megakaryocyte-erythrocyte progenitors (MEPs) (20.6 ± 3.7%), while CD62L-high CMPs mainly produced GMPs (82.8 ± 1.7%) and the proportion of MEPs was only 1.3 ± 0.1%. As for in vivo kinetics, we transplanted CD62L-low and high CMPs from GFP-expressing mice into irradiated wild-type mice, thus donor-derived platelets can be detected as GFP-positive. On day 7 after transplantation of each 15,000 cells, GFP-positive platelets from CD62L-low CMPs accounted for 9.8 ± 2.7% of all platelets, meanwhile CD62L-high CMPs accounted for only 0.20 ± 0.14%, which reinforced our hypothesis. Moreover, we performed transcriptome analysis using data of murine and human CMPs and focused on several transcription factors. Gata1, Klf1, Tal1 and Gfi1b are important transcription factors for erythroid and megakaryocytic differentiation, and these expressions were exclusively high in CD62L-low CMPs. On the other hand, Spi1 and Irf8 are important for differentiation into granulocytes and monocytes, and these expressions were higher in CD62L-high CMPs, which further corroborated the role of CD62L as a marker for refining the differentiation fate of CMPs. Second, at GMP level, we found that part of CD62L-neg GMPs possess remaining CMP potential and CD62L-low GMPs were skewed to granulocyte differentiation. CD62L expression on GMPs was mostly positive, but when we defined lowest 10% of GMPs as CD62L-neg GMPs, colony-forming cell assay in mice showed that part of CD62L-neg GMPs produced erythroid and megakaryocytic colonies (3.9 ± 2.6%), which suggested that this subset still possesses CMP potential. In vivo transplantation experiment using GFP-positive mice showed that CD62L-neg GMPs produced GFP-positive platelets slightly (0.02 ± 0.01%), but no platelets from CD62L-positive GMPs. Also, single-cell RNA-seq data revealed that part of CD62L-neg GMPs had CMP-specific gene expression pattern, suggesting that the bona fide GMPs were restricted to CD62L-positive GMPs. Next, we divided GMPs into CD62L-low and high GMPs, and performed colony-forming cell assay. CD62L-low GMPs produced granulocyte colony (CFU-G) 73.2 ± 6.1% and macrophage colony (CFU-M) 17.4 ± 3.3%, whereas CD62L-high GMPs produced CFU-G 46.6 ± 1.7% and CFU-M 42.9 ± 2.7%, which suggested that CD62L-low GMPs were granulocyte-skewed population. Also, we performed in vivo transplantation assay using Ly5.1 and Ly5.2 mice. On day 5 after transplantation, murine CD62L-low GMPs produced more neutrophils (87.6 ± 3.1%) in spleen than bulk GMPs (78.9 ± 1.9 %), which further confirmed this hypothesis. In summary, our in vitro and in vivo experiment and transcriptome analysis revealed that CMPs and GMPs had high heterogeneity. CD62L expression level refines the definition of CMPs and GMPs in both mice and humans, and elucidates the differentiation mechanism of myeloid cells in more detail. Disclosures Nakahara: Bristol-Myers Squibb Company: Honoraria; Eisai Co., Ltd.: Honoraria; Astellas Pharma Inc.: Honoraria. Kagoya:NIPPON SHINYAKU CO.,LTD.: Research Funding; Bristol-Myers Squibb Company: Research Funding; Kyowa Kirin Co., Ltd.: Research Funding. Kurokawa:Eisai: Research Funding, Speakers Bureau; Teijin: Research Funding; Bioverativ Japan: Consultancy; Celgene: Consultancy, Speakers Bureau; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Nippon Shinyaku: Research Funding, Speakers Bureau; Sumitomo Dainippon Pharma: Research Funding, Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Boehringer Ingelheim: Speakers Bureau; Ono: Research Funding, Speakers Bureau; Jansen Pharmaceutical: Speakers Bureau; Shire Plc: Speakers Bureau; MSD: Consultancy, Research Funding, Speakers Bureau; Chugai: Consultancy, Research Funding, Speakers Bureau; Sanwa-Kagaku: Consultancy; Pfizer: Research Funding; Otsuka: Research Funding, Speakers Bureau; Astellas: Research Funding, Speakers Bureau; Kyowa Kirin: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Research Funding, Speakers Bureau.

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