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Paroxysmal nocturnal hemoglobinuria (PNH) is a disease of hematopoietic stem cell membrane defects due to acquired PIG-Amutation. Our previous study found some secondary gene mutations in PNH patients by WES. However, it is not clear exactly which mutations are associated with the disease. So, 97 target genes were selected as a target gene panel and tested in 23 PNH patients by DNA sequencing of specific target regions. We found that all PNH patients had other gene mutations except PIG-Amutations, including TTN, NCOR2, CPS1, MUC4, SUZ12, LFNG, CELSR2, JAK2, SETBP1 and KMT2D (Figure1A). Through harmful analysis, KEGG enrichment, GO enrichment analysis and protein interaction analysis, we screened out the secondary mutant gene SUZ12 that may be involved in the cloning proliferation of PNH. We detected the mRNA and protein expression levels of SUZ12 and H3K27me3 methylation in PNH patients and health volunteers, the results showed that the mRNA and protein expression levels of SUZ12 and H3K27me3 methylation in peripheral blood CD59 -cells of PNH patients were higher than those in CD59 + cells of PNH patients and healthy controls (Figure1B). The relative expression level of SUZ12 in peripheral blood CD59 -cells of PNH patients was correlated with (r=0.4162, p=0.0385), CD59 -erythrocyte ratio (r=0.4636, p=0.0196), CD59 -monocyte ratio (r=0.4052, p=0.0495), Flaer -monocyte ratio (r=0.6769, p=0.0004) and Flaer -granulocytic ratio (r=0.6146, p=0.0018), indicating that SUZ12 may be involved in abnormal PNH cloning and proliferation by regulating H3K27me3. To verify the role of SUZ12 in the proliferation of PNH cloning, we used CRISPR/Cas9 to knockdown PIG-A expression in THP-1 cells to construct A PNH cell model, the expression level of PIG-A protein in the cell model was significantly decreased, and the proportion of CD59 - cells accounted was stable at 95%. Then lentivirus transfection was used to knockdown the expression of SUZ12 in PNH cell model. The results showed when the SUZ12 expression was knockdown, the methylation level of histone H3K27me3 was decreased, the cell proliferation activity was decreased, apoptosis was increased, and the cell cycle was arrested at G0/G1 phase. The proportion of CD59 + cells increased gradually from 3 weeks after transfection, and significantly increased at 4 weeks after transfection, while no changes were observed in the empty virus group and control group (Figure1C). Four weeks after lentivirus transfection, the expression of PIG-A protein recovered in SUZ12 knockdown group compared with empty virus group and control group (Figure1D). In conclusion, SUZ12 mutation leads to the overexpression of SUZ12, which can affect cell proliferation, apoptosis and cell cycle by regulating the methylation level of histone H3K27me3, thereby promoting the proliferation of PNH abnormal cloning and participating in the pathogenesis of PNH. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Polycomb repressive complex 2 (PRC2) is a chromatin modifier that regulates stem cells in embryonic and adult tissues. Loss-of-function studies of PRC2 components have been complicated by early embryonic dependence on PRC2 activity and the partial functional redundancy of enhancer of zeste homolog 1 (Ezh1) and enhancer of zeste homolog 2 (Ezh2), which encode the enzymatic component of PRC2. Here, we investigated the role of PRC2 in hematopoiesis by conditional deletion of suppressor of zeste 12 protein homolog (Suz12), a core component of PRC2. Complete loss of Suz12 resulted in failure of hematopoiesis, both in the embryo and the adult, with a loss of maintenance of hematopoietic stem cells (HSCs). In contrast, partial loss of PRC2 enhanced HSC self-renewal. Although Suz12 was required for lymphoid development, deletion in individual blood cell lineages revealed that it was dispensable for the development of granulocytic, monocytic, and megakaryocytic cells. Collectively, these data reveal the multifaceted role of PRC2 in hematopoiesis, with divergent dose-dependent effects in HSC and distinct roles in maturing blood cells. Because PRC2 is a potential target for cancer therapy, the significant consequences of modest changes in PRC2 activity, as well as the cell and developmental stage-specific effects, will need to be carefully considered in any therapeutic context.

Polycomb repressive complex 2 (PRC2) plays a key role in hematopoietic stem and progenitor cell (HSPC) function. Analyses of mouse mutants harboring deletions of core components have implicated PRC2 in fine-tuning multiple pathways that instruct HSPC behavior, yet how PRC2 is targeted to specific genomic loci within HSPCs remains unknown. Here we use short hairpin RNA-mediated knockdown to survey the function of PRC2 accessory factors that were defined in embryonic stem cells (ESCs) by testing the competitive reconstitution capacity of transduced murine HSPCs. We find that, similar to the phenotype observed upon depletion of core subunit Suz12, depleting Jarid2 enhances the competitive transplantation capacity of both fetal and adult mouse HSPCs. Furthermore, we demonstrate that depletion of JARID2 enhances the in vitro expansion and in vivo reconstitution capacity of human HSPCs. Gene expression profiling revealed common Suz12 and Jarid2 target genes that are enriched for the H3K27me3 mark established by PRC2. These data implicate Jarid2 as an important component of PRC2 that has a central role in coordinating HSPC function.

Deregulation of polycomb group complexes polycomb repressive complex 1 (PRC1) and 2 (PRC2) is associated with human cancers. Although inactivating mutations in PRC2-encoding genes EZH2, EED, and SUZ12 are present in T-cell acute lymphoblastic leukemia and in myeloid malignancies, gain-of-function mutations in EZH2 are frequently observed in B-cell lymphoma, implying disease-dependent effects of individual mutations. We show that, in contrast to PRC1, PRC2 is a tumor suppressor in Eµ-myc lymphomagenesis, because disease onset was accelerated by heterozygosity for Suz12 or by short hairpin RNA-mediated knockdown of Suz12 or Ezh2. Accelerated lymphomagenesis was associated with increased accumulation of B-lymphoid cells in the absence of effects on apoptosis or cell cycling. However, Suz12-deficient B-lymphoid progenitors exhibit enhanced serial clonogenicity. Thus, PRC2 normally restricts the self-renewal of B-lymphoid progenitors, the disruption of which contributes to lymphomagenesis. This finding provides new insight regarding the functional contribution of mutations in PRC2 in a range of leukemias.

EZH2 is the catalytic component of the polycomb repressive complex 2 (PRC2), which also contains the non-catalytic subunits suppressor of zeste 12 (SUZ12) and embryonic ectoderm development (EED). This complex methylates histone H3 at lysine 27 (H3K27) which, together with H3K4 methylation, generates a bivalent “code” that primes genes for either expression or silencing. EZH2 is highly expressed in stem cells and many proliferating cells, downregulated in differentiated cells and frequently altered in cancer in ways that point to a context dependent role for this gene. For instance, overexpression of EZH2 has been described in prostate and breast cancer, where this overexpression is associated with invasive growth metastatic potential and poor clinical outcome. More recently, both gain and loss of function mutations of EZH2 were identified in human cancer. In particular, activating mutations of EZH2 affecting a tyrosine at position 641 located within the SET domain (Y641) were observed in lymphoma. In striking contrast to wild type EZH2 which catalyzes the monomethylation of H3K27 very efficiently and shows less efficient catalytic activity in the subsequent di- and trimethylation reactions, the mutation generates a neomorphic protein with enhanced catalytic activity and efficiency for di- and tri-methylation, hinting toward a “cooperation” of both wild type and mutant EZH2 to increase total H3K27me3 levels and representing a functional equivalent of EZH2 overexpression in human lymphoma. Gain of function mutations in EZH2 are frequent in Diffuse Large B-cell Lymphoma (DLBCL; 22%) and in Follicular Lymphoma (FL; 7%) and recent data implicate EZH2 as an oncogene in DLBCL and FL lymphomas. In contrast to DLBCL and FL, EZH2 mutations have so far never been identified in MYC-driven B-cell Non-Hodgkin Lymphoma (B-NHL), and EZH2 expression is suppressed in Burkitt’s Lymphoma (BL), a lymphoma for which a MYC translocation is pathognomonic, suggesting that in this context EZH2 could have a role different from its role in DLBCL and FL. We probed the role of EZH2 in MYC-driven lymphomagenesis by mimicking the loss of function mutations by RNAi mediated suppression of EZH2 and the gain of function mutations by over-expression of the Y641 mutant. Our results show that suppression of EZH2, but not overexpression of the EZH2 Y641 mutant, accelerates MYC-driven lymphomagenesis by attenuating apoptosis. Our model recapitulates the transcriptional signature of a subset of B-NHLs driven by MYC overactivation and EZH2 suppression. Taken together, our data imply EZH2 as a tumor suppressor in the context of MYC activation and thus raise a warning for the use of EZH2-targeted therapies in some B-NHLs subtypes. Disclosures No relevant conflicts of interest to declare.

An unexpected revelation of cancer genome studies has been frequent abnormality in genes for factors that modify chromatin, underscored in this issue of Blood by reports from Score et al and Kroeze et al of inactivating mutations and chromosome loss in SUZ12, EED and JARID2 in myelodysplastic syndrome (MDS) and myeloproliferative disease (MPD).

Abstract 2456 Leukemia carrying a chromosomal translocation involving the mixed-lineage leukemia (MLL) gene is particular resistant to current therapy, and is associated with frequent relapse. Current evidence points towards self-renewing leukemic stem cells (LSC) as the driver of relapse. The eradication of LSC is thought to be critical for successful anti-leukemia therapy. Recent studies suggest that the multi-protein complex polycomb repressive complex 2 (PRC2) is required for MLL-rearranged AML. PRC2 contains the core proteins EZH2, EED and SUZ12, which have intrinsic histone methyltransferase activity specific to trimethylation of histone H3 on lysine 27. It is involved in the epigenetic silencing of tumor suppressor genes (e.g., p16 and p19). In this study we determined the effect of a PRC2-associated inhibitor (hereafter named PAI) on the PRC2 components in LSC isolated from bone marrow cells of mice with established leukemia induced by the MLL-AF9 oncogene. Treatment with PAI (0.1 to 0.5 μM) for 24 hours in culture media depleted protein levels of EZH2 and EED in LSC. Following treatment, quantitative real-time PCR and immunoblotting analysis demonstrated increased mRNA and protein levels of p16 and p19, as well as inhibition of cell cycle progression. PAI treatment also induced a 10-fold increase of apoptosis in LSC. As a consequence, colony formation assays showed that LSC treated for 24 hours with 0.1 μM PAI completely lost their ability to form compact colonies in methylcellulose. We are currently assessing the effects of PAI in in vivo models of MLL-rearranged leukemia where preliminary data indicate that we are able to achieve pharmacologic inhibition of the PRC2 activity. Collectively, these findings indicate that PAI depletes the levels of PRC2 components, and eliminates LSC, thus representing a potentially promising novel treatment for AML. Disclosures: No relevant conflicts of interest to declare.

Abstract 65 Epigenetic pathways act to control gene expression in a heritable fashion without altering DNA sequence, typically involving the control of chromatin structure. Several lines of evidence implicate the involvement of epigenetics in the pathogenesis of human cancer, however it remains uncertain to what extent manipulating epigenetic pathways can fully rectify malignant cellular states for therapeutic benefit. To systematically explore this issue, we have developed a novel RNAi screening pipeline that can distinguish the epigenetic requirements for normal and malignant hematopoiesis. This was achieved by first constructing a custom shRNA library targeting all known enzymatic complexes that regulate chromatin structure (1100 shRNAs in total). Next, each shRNA was delivered systematically to cells derived from a mouse model of chemotherapy-resistant acute myeloid leukemia driven by the oncogenes MLL-AF9 and NRAS, or to several non-transformed hematopoietic cell lines of different lineages. shRNAs were scored for their capacity to differentially inhibit growth of leukemic cells without influencing growth of non-leukemic cells. Each of the identified genes was then evaluated in vivo for its influence on normal reconstitution of the hematopoietic system following transplantation of shRNA-infected hematopoietic stem and progenitor cells into lethally irradiated recipient mice. Each gene was likewise suppressed in leukemia cells in vivo using both constitutive and conditional RNAi vectors. The net result of the in vivo testing was identification of 6 genes encoding different regulators of chromatin structure whose suppression provides therapeutic benefit in a mouse model of therapy-resistant AML, without significantly influencing the production of normal blood lineages. In support of the accuracy of our screening protocol, one of the identified genes from the screen encodes the protein Menin, a known MLL-AF9 cofactor essential for disease initiation and shown to be dispensable for steady-state hematopoiesis in knockout mice. Two of the identified hits in our screen are the genes Eed and Suz12, which encode two subunits of the PRC2 Polycomb complex (Eed and Suz12), which catalyzes histone H3K27 methylation to suppress gene expression. Inhibiting PRC2 function in MLL-AF9 leukemia cells leads to monocytic differentiation, as revealed by FACS, RT-qPCR, and cell morphology analysis. Microarray experiments coupled with chromatin immunoprecipitation identified a core program of myeloid fate determinants that are suppressed by PRC2 via H3K27 methylation. In addition, we observe that MLL-AF9 directly occupies several Polycomb gene promoters to upregulate their expression in leukemic cells. Our findings highlight an unexpected alliance between the MLL-AF9 oncogene (a Trithorax protein) and PRC2 (a Polycomb complex), which act together to block myeloid differentiation in AML. Our findings also highlight the utility of employing RNAi in vivo to identify novel therapeutic targets in otherwise chemotherapy-resistant disease models. Disclosures: No relevant conflicts of interest to declare.

Polycomb Group (PcG) proteins are key regulators of normal and leukemic hematopoiesis (Lessard and Sauvageau, Nature 2003; Lessard and al., Genes Dev 1999). The eed gene is part of a PcG complex together with Ezh2 and Suz12 . Evidence from our laboratory indicates that eed plays a crucial role in the negative regulation of the proliferative capacity of lymphoid and myeloid progenitors, suggesting that this PcG gene is a tumor suppressor. Interestingly, the human eed locus is in a region of chromosome 11 (11q14.2-22.3) which is associated with recurrent deletions in several reported cases of B-CLL, mantle cell lymphoma (MCL) and T-PLL. We now show that when exposed to ionizing radiation at 5 weeks of age, both eed hypo/hypo and eed null/+ mutant mice developed mono- or oligoclonal B and T cell lymphomas with much shorter latencies than observed in littermate controls (50% lethality at 16 wks; 27 wks and 34 wks post-irradiation for eed hypo/hypo , eed null/+ and controls, respectively). We reasoned that new pathways involved in tumorigenesis could be at play in this eed sensitized background, potentially allowing for the identification of new oncogenic collaborators. Therefore, a proviral insertional mutagenesis screen was conducted. Neonatal MMLV infection of eed mutant mice dramatically accelerated the occurrence of B and T cell lymphomas (50% lethality at 16 wks; 22 wks and 26 wks post-infection for eed hypo/hypo, eed hypo/+ and controls, respectively). To characterize retroviral insertions inverse-PCR (IPCR) was performed using genomic DNA isolated from 32 eed hypo/hypo and 27 wild-type tumors. More than 780 IPCR products were recovered and purified. To date, at least 140 retroviral insertion sites (RIS) have been cloned, sequenced and mapped against the mouse genome. Of these, 7 insertion targeting gene loci known to be cancer-related, like Pim1 (n= 7 tumors) , Notch1 (n= 14 tumors) and c-Myc (n= 3 tumors) have been identified. In addition, 8 new common insertion sites (CIS), such as Socs1 (n= 6 tumors) , Gadd45g (n= 5 tumors) and Fgfr3 (n= 6 tumors) have also been identified. Rearrangements of the Pim1 locus were found in more than 22% of the eed 1989/1989 mutant mice whereas none could be found in wild-type littermates. Moreover, RT-PCR analysis indicates that the Pim1 gene is clearly overexpressed in 6 out of 16 eed 1989/1989 tumors analyzed compared to wild-type, further supporting a key role for the Pim1 pathway in eed sensitized tumor cells. Thus, the data presented in this study provide evidence that eed has tumor suppressing function. It also points to a novel mechanism of tumor suppression by a PcG protein. Based on these results, it will be interesting to study the status of eed and of the newly identified CISs in human diseases associated with deletions of 11q14.2-22.3.