ALBERT

Description: PI3K/Akt pathway is constitutively activated in multiple myeloma (MM). A plethora of studies extensively investigated Akt inhibitors, alone or in combination; however, the outcomes in hematological malignancies were largely unsatisfactory, emphasizing the need for critical preclinical evaluations. Polycomb repressive complex 2 (PRC2) components, EZH2 and its related homolog EZH1, induce H3K27me3 to silence the transcription of target genes. Recent studies ensured that EZH2 inhibition alone is not sufficient to completely disrupt the oncogenic functions of PRC2. With the importance of PRC2 as a therapeutic target in MM, we aimed to investigate the mechanisms by which Akt inhibition may impact PRC2 function, and test whether targeting both EZH2 and EZH1 together with Akt inhibition is a promising treatment strategy for MM. We herein evaluated the cytotoxic effect of TAS-117, a potent and selective non-competitive Akt inhibitor, against different MM cell lines and found that responsive cell lines tended to have significant levels of activated Akt, coupled with low/deleted PTEN. Then, we examined signaling-epigenetic crosstalk on EZH2 level. TAS-117 significantly down-regulated EZH2 mRNA and protein in dose- and time-dependent manners, while H3K27me3 levels were rather maintained or elevated, suggesting compensation by EZH1. As EZH2 is a direct target for E2F1, we focused on Rb-E2F pathway as a regulatory mechanism for EZH2. TAS-117 induced marked down-regulation of E2F1 and E2F2. Moreover, TAS-117 induced the up-regulation of CDKN1B, in addition to the inactivation of cyclins and cyclin dependent kinases, hence, hypo-phosphorylated Rb, thereby stabilizing Rb-E2F1 complex and diminishing free E2F1 available for binding to its target genes, including EZH2 promoter. This prompted us to examine the impact of TAS-117 combination with either dual EZH2/1 inhibitor, UNC1999, or selective EZH2 inhibitor, GSK126. In agreement, UNC1999, but not GSK126, synergistically enhanced TAS-117-induced cytotoxicity, confirmed by combination index calculation, and provoked MM cell apoptosis. As we observed an increase in H3K27me3 levels after TAS-117 treatment, we hypothesized that EZH1 function was augmented. Consistently, we found that EZH1 was markedly up-regulated after TAS-117 treatment in dose- and time-dependent manners. Importantly, EZH1 knockdown significantly enhanced the sensitivity of myeloma cells to TAS-117-induced cytotoxicity. To clarify the molecular mechanisms underlying EZH1 up-regulation, we performed RNA-seq followed by KEGG pathway analysis for up-regulated genes in TAS-117-treated group. We focused on FOXO pathway enrichment as it is a crucial target in MM treatment using Akt inhibitors. We then focused on FOXO3 as it was the main FOXO family gene expressed in MM cells according to our RNA-seq data. We examined the nuclear localization of FOXO3 following TAS-117 treatment. We found that TAS-117 significantly enhanced the nuclear accumulation of FOXO3, as depicted by both the immunostaining images and the digital calculations of the nuclear subset of FOXO3. Murine Ezh1 promoter was shown to be bound by Foxo transcription factors (TFs) in neuronal progenitors, T-regulatory cells, CD8+ cells, and pre-B cells. More than 80% of FOXO3-binding sites share the common binding motif, GTAAACAA, which was found both in human EZH1 (+48 from the TSS) and mouse Ezh1 (+77 from the TSS) promoter regions. So, we hypothesized that FOXO3 may be a regulatory partner for human EZH1 gene in myeloma cells in response to TAS-117 treatment. To this end, we performed ChIP-qPCR analysis for TAS-117-treated and -untreated cells. TAS-117 promoted the binding of FOXO3 to EZH1 promoter, in addition to one of the canonical FOXO3 targets, BIM promoter. To further confirm our results, we expressed shRNA against FOXO3 (shFOXO3) in MM cells which, interestingly, induced the down-regulation of EZH1 mRNA. In conclusion, the present results defined novel signaling-epigenetic crosstalk between PI3K/Akt pathway and PRC2 components, EZH2 and EZH1, and demonstrated that Akt inhibition can differently modulates EZH2 and EZH1 levels via Akt downstream effectors, E2F1 and FOXO3, respectively. Therefore, targeting both EZH2 and EZH1 in addition to Akt inhibition may be a promising rationale to eradicate MM, leading to significant advances in treatment. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes (POEMS) syndrome is a rare paraneoplastic disease due to an underlying monoclonal plasma cell (PC) dyscrasia. Despite of dynamic symptoms associated with highly elevated VEGF, monoclonal PCs are thought to be quite small, and pathogenic significance of these PCs remains undetermined. In this study, we performed whole exome sequencing (WES), target sequencing, and RNA sequencing of PCs in patients with POEMS syndrome in order to define its genetic profiles. Methods: Patients diagnosed with POEMS syndrome at Chiba University Hospital from July 2014 to June 2016 were enrolled. DNA was extracted from either PCs which were isolated from patients' bone marrow (BM) using CD138 MACS (Miltenyi) or buccal cells as controls. WES and target sequencing were performed using HiSeq2500 (Illumina) and MiSeq (Illumina), respectively. The data of WES and target sequencing were analyzed by Empirical Bayesian mutation Calling (EBCall). Copy number was analyzed using the data of WES. RNA sequencing of PCs isolated by MACS and FACS sorting was conducted using HiSeq 1500 (Illumina). PCs from some patients diagnosed with multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS) were also collected as controls for RNA sequencing. Results: Twenty POEMS patients (M:F 12:8, mean age 42.6, range 16-78; 15 newly diagnosed, 5 refractory or relapsed cases) were included in this study. Regarding the types of M protein, 55% (11/20) were IgA-λ, 25% (5/20) were IgG-λ, and each individual case of the following; IgA-λ+IgG-λ, BJP-λ, IgG-κ, and Castleman's variant with no M protein. The mean serum VEGF was 6,471 pg/ml (range 1,190-13,800), and the mean PCs percentage in the BM was 4.4% (range 0.8-10.5). WES was performed in 15 cases; a total of 359 somatic mutations in 334 genes were revealed in 93.3% of cases (14/15) with a mean number of 23.9 (range 0-119) in each. All these mutated genes were significantly enriched in several pathways related to cell adhesion. Importantly, frequently mutated genes in MM such as NRAS, KRAS, and TP53 were not identified. Among all mutations, 1.7% were frameshift insertions, 2.0% were frameshift deletions, 4.2% were stop gains, 0.8% were non-frameshift deletions, 60.2% were other non-synonymous single nucleotide variants (SNVs), 29.5% were synonymous SNVs, and 1.7% were splicing mutations which were within 2-bp of a splicing junction. Copy-number variations were detected in 33.3% of cases (5/10) including -13 (2 cases), +1q (2 cases), and hyperdiploidy (2 cases). To carry out target sequencing in all 20 cases, we defined 51 target genes which included recurrently mutated genes from our WES data, frequently mutated genes in hematopoietic and lymphoid tissues according to the database (COSMIC), and 15 frequently mutated genes in MM (NRAS, KRAS, TP53, BRAF, CDKN2C, FGFR3, BIRC3, DIS3, CYLD, KDM6A, LRP1B, FAM46C, COL6A3, DNAH5, and KRT6A). A total of 60 somatic mutations were revealed in 65% of cases (13/20), and 9 new somatic mutations were found in the cases in which WES was also performed. Ten recurrently mutated genes were identified; KLHL6 in 20% of cases (4/20), each of LTB, RYR1 in 15% of cases (3/20), and each of EHD1, EML4, HEPHL1, HIPK1, PCDH10, USH2A, and ZNF645 in 10% of cases (2/20). Among frequently mutated genes in MM, only 3 genes (FAM46C, LRP1B, and DNAH5) were mutated, each in a single case. We finally conducted RNA sequencing of the FACS-sorted PCs in 5 POEMS patients compared to 5 MGUS and 4 MM patients. Upregulated genes were significantly enriched in some gene sets, gene ontology terms, and pathways related to immune response and cell adhesion, whereas downregulated genes were related to tumorigenesis. Of note, VEGF was not significantly upregulated in POEMS patients. Principal component analysis distinguished the 3 disease groups of patients with marginal overlaps between POEMS and MGUS, and also MGUS and MM. Conclusions: Our data clearly demonstrate that the genetic profiles of PCs in POEMS syndrome are distinct from those in MM and MGUS. Notably, PCs may not be the main source of extremely elevated VEGF in POMES syndrome. On-going further investigation will help clarify the molecular pathogenesis of POEMS syndrome. Disclosures Ogawa: Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding; Kan research institute: Consultancy, Research Funding.

Description: INTRODUCTION Inappropriate recruitment of functional Polycomb-Group proteins (PcG) may trigger epigenetic unbalance at very specific genomic loci that substantially contribute to the pathogenesis of Acute Myeloid Leukemia (AML). This concept was first described in Acute Promyelocytic Leukemia (APL) in which PcG proteins were abnormally addressed due to the expression of X-RARA fusion proteins and were involved in the treatment response of the disease. For instance, in the context of APL with t(11;17)(q23;q21) translocation, the resulted oncogenic fusion protein PLZF/RARA leads to abnormal recruitment of PcG at the promoters of genes involved in acid-trans-retinoic acid (ATRA) response (Boukarabila et al.). As a consequence of that and compared to other APL subtypes (e-g: PML/RARA), APL with PLZF/RARA are insensitive to ATRA. In the recent years, a repertoire of cis-regulatory enhancer elements has been dissected to reveal important insight about leukemia onset and define new subsets of the disease with different treatment responses (Bhagwat et al). As we previously reported that PLZF displayed epigenetic specificity on enhancers (Poplineau et al.) we questioned the role of PLZF/RARA on these regulatory regions during APL onset. METHODS We performed in vivo comparative epigenomic profiling (H3K27ac, H3K4me1, H3K27me3 and H3K4me3 ChIPseq) between normal myeloid progenitors (granulocyte-monocyte progenitors purified from wild-type mice) and PLZF/RARA transformed mouse progenitors (late promyelocytes purified from mice developing APL). To question the role of PcG in APL onset, we used retroviral overexpression of PLZF/RARA and transduced Lineage negative cells from a conditional KO EZH2 mouse model. Transformation was tested by replating assay and cells were characterized by FACS and morphology analyses. We also performed EZH2 pharmacological inhibition using GSK126 and UNC1999 on a human cell line expressing the fusion protein PLZF/RARA. We analyzed the impact of this inhibition on their transcriptomic signature (RNAseq) and their proliferative capacity. RESULTS Upon PLZF/RARA expression and APL progression, specific cis-regulatory enhancer elements were targeted by the H3K27me3 PcG repressive mark. This gain in poised enhancer regions, upon PLZF/RARA expression reflected a reoriented PcG activity, from enhancers regulating developmental processes to those regulating stress and immune responses. To demonstrate the importance of this H3K27me3 switch for APL progression, we investigated the effect of EZH2 loss during PLZF/RARA transformation. Using a conditional KO EZH2 mouse model, we demonstrated that PLZF/RARA required EZH2 activity to efficiently transform progenitors since EZH2 loss promoted differentiation that altered the replating capacity of the PLZF/RARA expressing cells. In addition, EZH2 inhibition by GSK126 revealed some interesting benefits since it sensitized PLZF/RARA transformed progenitors to ATRA treatment. Moreover, inhibition of EZH2 with GSK126 or UNC1999 induced a decrease in the proliferation advantage of a human PLZF/RARA-inducible cell line. This was linked to a change of its transcriptomic signature towards an expression pattern closer to the one observed in the parent cell line. CONCLUSION Taken together, our data showed that PLZF/RARA modifies H3K27me3 profiles at enhancer regions and requires EZH2 activity for APL onset. Finally, our results suggest that EZH2 inhibition could be a new promising therapeutic approach for retinoic-acid resistant APL. Disclosures No relevant conflicts of interest to declare.

Description: Hematopoietic stem cells (HSCs) reside in hypoxic niche in the bone marrow (BM), where they are maintained in quiescent state. To adapt to this hypoxic microenvironment, HSCs generate ATP mainly from glycolysis, while suppressing oxidative phosphorylation in mitochondria. Through this metabolic regulation, HSCs protect themselves from oxidative stress caused by reactive oxygen species (ROS). ROS levels are controlled by various mechanisms including autophagy, which is one of the critical mechanisms regulating HSC function mainly through maintaining mitochondrial homeostasis. Glycosylation of serine or threonine residues with O-linked N-acetylglucosamine (O-GlcNAcylation) by O-linked N-acetylglucosamine transferase (OGT) is crucial for regulating various protein functions, and dysregulation of O-GlcNAcylation is critically linked to metabolic or degenerative diseases (i.e. diabetes, Alzheimer disease), tumorigenesis and aging. In hematopoietic system, OGT is essential for differentiation and proliferation of T and B cells. However, a role for OGT in the function of hematopoietic stem and progenitor cells (HSPCs) remains elusive. To elucidate a role for OGT in HSPCs, we analyzed the effect of Ogt loss on HSPCs using Ogt-conditional knockout mice. Conditional disruption of Ogt in adult mice led to pancytopenia and significantly reduced numbers of HSPCs in the BM. In addition, Ogt-deficient HSCs exhibited loss of quiescence, increased apoptosis and impaired long-term repopulation as well as self-renewal capacities by serial competitive repopulation assays. Interestingly, ROS levels were significantly increased in Ogt-deficient HSPCs, and the treatment of mice with N-acetyl cysteine (NAC), a scavenger for ROS, at least partially rescued the phenotype of Ogt-deficient hematopoietic cells in in vivo settings. MitoSOX analysis showed that excessive ROS was generated mainly from mitochondria in Ogt-deficient HSCs. In addition, mitochondrial mass was significantly increased in Ogt-deficient HSCs. Assessment of mitochondrial quality by its membrane potential and extracellular flux analysis revealed that mitochondria of Ogt-deficient HSPCs had significantly lower membrane potential and lower spare respiratory capacity. Morphologically, mitochondria in Ogt-deficient HSPCs were enlarged and swollen with disorganized cristae. These findings suggest that inactivation of Ogt leads to loss of HSPCs and impaired HSC homeostasis, which is due, at least in part, to elevated ROS levels with accumulation of defective mitochondria. Since mitophagy (mitochondria-specific autophagy) plays an important role in quality control of mitochondria, we examined gene expressions of mitophagy regulators by RNA sequencing. This revealed that disruption of Ogt led to down-regulation of key mitophagy inducers, Pink1 and Bnip3. These findings together with the accumulation of defective mitochondria indicated that inactivation of Ogt impairs mitophagy probably due to reduced expression of Pink1 and Bnip3. OGT is known to regulate gene expressions by modifying trimethylation of lysine 4 on the histone H3 (H3K4me3) through proteolytic activation of HCF-1, which is a critical component of SET1/COMPASS complex. Chromatin immunoprecipitation (ChIP) assay showed significantly reduced levels of H3K4me3 at the transcriptional start sites of Pink1 and Bnip3 in Ogt-deficient HSPCs. Next we asked whether defective HSPC homeostasis caused by Ogt disruption could be rescued by restoring mitophagy in Ogt-deficient HSPCs through overexpressing Pink1, a critical initiator of mitophagy. As expected, Pink1 overexpression efficiently restored mitophagy in Ogt-deficient HSCs as shown by normalization of mitochondrial mass. Interestingly, a number and apoptosis of Ogt-deficient HSCs were restored to levels similar to those of wild-type HSCs. These data strongly indicate that Pink1 functions as a key downstream effector of OGT in HSC maintenance. In summary, our results revealed that OGT plays an essential role in HSC maintenance by assuring mitochondrial quality through mitophagy. Disclosures No relevant conflicts of interest to declare.

Description: Key Points The level of Sf3b1 expression is critical for the proliferative capacity of hematopoietic stem cells. Haploinsufficiency for Sf3b1 is not sufficient to induce a RARS-like phenotype in mice.

Description: Proteasome inhibitors (PIs) such as bortezomib and carfilzomib play a central role in the treatment of multiple myeloma (MM). However, the almost inevitable resistance to PIs necessitates the search for novel strategies to improve patient outcome. The methyltransferase EZH2 and its homolog EZH1 are components of polycomb repressive complex 2 (PRC2), inducing H3K27me3 and repressing the transcription of target genes. Recent studies have linked EZH2 to tumorigenesis including MM. In this study, we investigated the molecular mechanism of PRC2 inhibition as a partner of PIs for the treatment of MM. We first examined the impact of proteasome inhibition on EZH2. Bortezomib as well as carfilzomib remarkably decreased EZH2 protein, and downregulated its mRNA in dose- and time-dependent manners. As EZH2 is a downstream target of E2F1, the effects of bortezomib on RB-E2F pathway were investigated. Bortezomib downregulated E2F1 protein and mRNA with notable decrease of phosphorylated RB protein, due to accumulation of cyclin-dependent kinase inhibitors such as p21 and p27. ChIP assay revealed that bortezomib significantly inhibited the binding of E2F1 to EZH2 promoter, and E2F1 overexpression resulted in upregulation of EZH2 in MM cells. These data suggest that bortezomib transcriptionally downregulates EZH2 via modulating RB-E2F pathway. Next we used lentiviral vectors to overexpress EZH2 in RPMI8226 cells and observed diminished sensitivity to bortezomib in EZH2-overexpressing cells compared to cells transduced with an empty vector. Remarkably, the combined treatment of bortezomib and UNC1999, a dual inhibitor of EZH2 and EZH1, restored the sensitivity of MM cells to bortezomib. Notably, UNC1999 enhanced the cytotoxicity induced by bortezomib in vitro and in vivo partly through enhanced apoptosis. Carfilzomib also demonstrated strong synergy with UNC1999 in vitro, suggesting broad application of this strategy. To characterize the mechanism of action of PRC2 inhibition alone and in combination with proteasome inhibition, we performed RNA sequencing (RNA-seq) of MM.1S cells treated with UNC1999, bortezomib or the combination of both agents versus DMSO-treated cells and chromatin immunoprecipitation sequencing (ChIP-seq) for H3K27me3 of UNC1999 versus DMSO-treated MM.1S cells. Importantly, we identified the direct targets of UNC1999 as those with significantly enhanced expression (〉1.5 fold UNC1999/Control) and remarkable reduction of H3K27me3 (≥ 2-fold). These genes included NR4A1, EGR1 and LTB. EGR1 and LTB are known tumor suppressor candidates in MM, while NR4A1 is implicated in myeloid and lymphoid malignancies. Upregulation of NR4A1 and reduction of H3K27me3 at the NR4A1promotor were confirmed using manual RT-PCR and ChIP, respectively. Notably, overexpression of NR4A1 significantly inhibited the growth of MM cells, suggesting a tumor suppressive role for NR4A1 in MM. Notably, MYC (c-Myc), a major contributor to the pathogenesis of MM, was greatly downregulated in NR4A1-overexpressing cells. MYC is reportedly a direct target of NR4A1 that suppresses its expression. We found that UNC1999 downregulated MYC mRNA and protein. Moreover, the combination of UNC1999 and bortezomib remarkably suppressed MYC-related gene sets. Gene set enrichment analysis (GSEA) showed that while PRC2 genes were positively enriched in UNC1999- and combination-treated cells, they were not significantly enriched in bortezomib-treated cells. In addition, although bortezomib downregulated EZH2, EZH1 and H3K27me3 mark were not affected in bortezomib-treated cells. This suggested that inhibition of EZH2 alone is not enough to completely suppress PRC2 function. Therefore, we compared the combination of bortezomib and UNC1999 with that of bortezomib and a specific EZH2 inhibitor, GSK126. UNC1999 induced much better synergistic activity with bortezomib than GSK126 as evidenced by the combination index, associated with further reduction of the levels of H3K27me3. This underlines the importance of dual inhibition of EZH2 and EZH1 to fully block PRC2 activity. In conclusion, our findings demonstrate that the combination of dual inhibition of EZH2 and EZH1 together with proteasome inhibition cooperatively blocks PRC2 function, resulting in derepression of tumor suppressors such as NR4A1 and inhibition of MYC. Thus, this combination is a promising new therapeutic option for the treatment of MM. Disclosures No relevant conflicts of interest to declare.

Description: MPN harbors altered hematopoietic stem cell (HSC) function, resulting in skewed hematopoiesis and extramedullary hematopoiesis with splenomegaly. Mutations such as JAK2V617F and insertion/deletion of CALR exon9 have been established as phenotypic drivers of MPN. In addition, mutations in epigenetic modifiers and aberrant expressions of microRNAs play a crucial role in disease progression and clonal expansion. We have shown that almost all patients with myelofibrosis (MF) highly express HMGA2 (Harada-Shirado et al, BJH, 2015) and that transgenic mice expressing HMGA2 without 3'UTR including let7 binding sites (∆Hmga2 : H) (Ikeda et al, Blood, 2011) develop mild MPN. Moreover, we (Sashida et al, JEM, 2016) and other groups (Shimizu et al, JEM, 2016; Yang et al, Blood, 2016) showed that loss of EZH2 induces endogenous expression of HMGA2 and provokes severe MF in mice carrying JAK2V617F. Thus, we hypothesized that HMGA2 plays a central role in the disease progression of MPN. To clarify this, we generated ∆Hmga2/JAK2V617F transgenic mice (HJ), which developed severe leukocytosis, thrombocytosis, anemia, giant splenomegaly and shorter survival period, but did not progress fibrosis compared with transgenic mice carrying JAK2V617F alone (J) (Ueda et al, ASH, 2015). Compared with J, HJ also showed an increased bone marrow (BM) lineage-Sca1+Kit+(LSK) cells and growth advantage in competitive serial BM transplants. Encouraged by these findings, we further investigated the mechanism that HMGA2 exacerbates disease phenotype, and elucidated up-stream and down-stream factors of HMGA2. First, we sought cause of aggressive phenotypes. In BM cells of HJ mice, STAT3 and STAT5 were drastically upregulated in both expression and phosphorylation. Despite severer anemia, formations of EPO-independent erythroid colonies and proportions of CD71+Ter119+ erythroblasts in BM were not different between HJ and J. To explain this discrepancy, we measured spontaneous apoptosis. Erythroblasts were more susceptible to apoptosis in HJ as well as aged H, compared with J, suggesting that HMGA2 contributes to apoptosis under stressed condition. Next, we compared gene expression profiles of BM LSK cells between HJ and J by RNA sequence to seek genes altered by addition of HMGA2 expression to JAK2V617F. To clarify the role of endogenous expression of HMGA2 induced by EZH2 deletion in hematopoiesis with JAK2V617F in addition to direct effects of EZH2 deletion, we also studied RNA sequence in BM LSK cells of Ezh2-/-JAK2V617F-Tg mice (EJ). Upregulated genes were highly overlapped in between HJ and EJ. Relative to J, 200 genes including oncogenic Lmo1 were commonly upregulated, while 314 and 739 were exclusively upregulated in HJ and EJ, respectively. Thus, some of the EZH2 targets may be also targets of HMGA2. Pathway analysis revealed that chaperon-related pathways involving SCAP1 and SREBF1/2 were commonly upregulated. On the other hand, TGFB pathway was upregulated in EJ but not in HJ, and it may explain the discrepancy that addition of HMGA2 in JAK2V617F hematopoiesis did not progress fibrosis despite severe phenotype of MPN, but deletion of EZH2 exacerbated fibrosis as well as MF patients with high expression of HMGA2. To verify this finding, we measured expression of HMGA2 and its main suppressor let7, and performed target sequencing in blood samples from 16 MF patients. Because all of them highly expressed HMGA2, we also evaluated selected essential thrombocythemia (ET) patients (N=10 for HMGA2 low, 10 for HMGA2 high). While high HMGA2 expression was consistently correlated with low let-7 expression in ET, only half of MF showed low expression of let7. The other half of MF harbored mutations in components or modifiers of polycomb, including EZH2, ASXL1 and spliceosomes. These findings are compatible with the finding that loss of polycomb function upregulates HMGA2. In fact, the genetic depletion of HMGA2 or administration of HMGA2 inhibitor neuropsin diminished phenotype of EJ mice and prolonged survival. Our findings suggest importance of HMGA2 as a therapeutic target of MPN. Disclosures Komatsu: Shire: Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Description: Ezh1 and Ezh2, the catalytic components of polycomb-repressive complex 2 (PRC2), negatively control gene expression by catalyzing mono, di, and tri-methylation of histone H3 at lysine 27 (H3K27me1/me2/me3). Loss-of-function mutations of EZH2, but not those of EZH1, have been found in patients with hematologic malignancies such as myelodysplastic syndrome (MDS), myeloproliferative neoplasms (MPNs), and MDS/MPN overlap disorders. We previously demonstrated that hematopoietic cell-specific Ezh2 knockout mice (Ezh2Δ/Δ) developed hematologic malignancies including MDS and MDS/MPN. Although deletion of Ezh1, another enzymatic component of PRC2, (Ezh1-/-) did not significantly affect global H3K27me3 levels or hematopoiesis, deletion of both Ezh1 and Ezh2 in mice (Ezh1-/-Ezh2Δ/Δ) caused rapid exhaustion of hematopoietic stem cells (HSCs). Given that only Ezh1 and Ezh2 are known as enzymatic components of PRC2, we concluded that residual PRC2 enzymatic activity is required for HSC maintenance and development of hematologic malignancies in the setting of EZH2 insufficiency frequently observed in MDS. However, the role of Ezh1 in Ezh2-insufficient hematologic malignancies is still not fully understood since hematopoiesis could not be maintained in Ezh1-/-Ezh2Δ/Δ mice. Here we analyzed the impact of Ezh1 heterozygosity on Ezh2-null hematopoiesis (Ezh1+/-Ezh2Δ/Δ), in which PRC2 activity is mediated by a single allele of Ezh1, for better understanding of Ezh2-deficient hematologic malignancies. We first transplanted BM cells from Ezh1+/-Ezh2flox/flox CD45.2 mice with CD45.1 wild-type competitor cells into lethally irradiated CD45.1 recipient mice and deleted Ezh2 by intraperitoneal injection of tamoxifen. Ezh1+/-Ezh2Δ/Δ cells exhibited a lower repopulation capacity than Ezh2Δ/Δ but established persistent repopulation for at least 6 months after the deletion of Ezh2 while double knockout cells (Ezh1-/-Ezh2Δ/Δ) were outcompeted by competitor cells immediately. We next transplanted BM cells from Ezh1+/-Ezh2flox/flox CD45.2 mice without CD45.1 wild-type competitor cells into lethally irradiated CD45.1 recipient mice and deleted Ezh2 by intraperitoneal injection of tamoxifen. Importantly, recipient mice reconstituted with Ezh1+/-Ezh2Δ/Δ cells exhibited MDS-like phenotypes including anemia and morphological myelodysplasia, which were more pronounced than those of Ezh2Δ/Δ mice. Ezh1+/-Ezh2Δ/Δ mice also showed more advanced hematological abnormalities such as erythroid differentiation block, increased apoptosis of erythroid cells, and extramedullary hematopoiesis in the spleen than Ezh2Δ/Δ mice did. These results suggest that Ezh1 heterozygosity promotes the development of myelodysplasia in the setting of Ezh2insufficiency. Next we examined the molecular mechanism by which the loss of Ezh1 promotes myelodysplasia. Western blot and ChIP-sequence analyses revealed that global levels of H3K27me3 were not significantly changed but H3K27me3 levels at promoter regions of the PRC2 target genes were obviously reduced by Ezh1 heterozygosity in Ezh2Δ/Δ HSPCs. As a consequence, PRC2 target genes were highly de-repressed in Ezh1+/-Ezh2Δ/Δ LSK HSPCs compared with Ezh2Δ/Δ HSPCs. Among these, several genes appeared to be associated with MDS such as S100A9, encoding an inflammatory protein implicated in dyserythropoiesis in MDS. Furthermore, gene set enrichment analysis showed that the genes highly expressed in myeloid cells were positively enriched by Ezh1 heterozygosity in Ezh2Δ/ΔHSPCs. These findings indicate that dosage of Ezh1 is critical in the maintenance of Ezh2-insufficient hematopoiesis as well as the progression of MDS with Ezh2 insufficiency. Disclosures No relevant conflicts of interest to declare.