ALBERT

Description: Polycomb repressive complex 2 (PRC2) catalyzes the monomethylation, dimethylation, and trimethylation of histone H3 Lys27 (H3K27) and acts as a central epigenetic regulator that marks the repressive chromatin domain. Embryonic ectoderm development (EED), an essential component of PRC2, interacts with trimethylated H3K27 (H3K27me3) through the aromatic cage structure composed of its three aromatic amino acids, Phe97, Trp364, and Tyr365. This interaction allosterically activates the histone methyltransferase activity of PRC2 and thereby propagates repressive histone marks. In this study, we report the analysis of knock-in mice harboring the myeloid disorder-associated EED Ile363Met (I363M) mutation, analogous to the EED aromatic cage mutants. The I363M homozygotes displayed a remarkable and preferential reduction of H3K27me3 and died at midgestation. The heterozygotes increased the clonogenic capacity and bone marrow repopulating activity of hematopoietic stem/progenitor cells (HSPCs) and were susceptible to leukemia. Lgals3, a PRC2 target gene encoding a multifunctional galactose-binding lectin, was derepressed in I363M heterozygotes, which enhanced the stemness of HSPCs. Thus, our work provides in vivo evidence that the structural integrity of EED to H3K27me3 propagation is critical, especially for embryonic development and hematopoietic homeostasis, and that its perturbation increases the predisposition to hematologic malignancies.

Description: Additional sex combs-like 1 (ASXL1), an epigenetic modulator, is frequently mutated in myeloid neoplasms. Recent analyses of mutant ASXL1 conditional knock-in (ASXL1-MT-KI) mice suggested that ASXL1-MT alone is insufficient for myeloid transformation. In our previous study, we utilized retrovirus-mediated insertional mutagenesis, which exhibited susceptibility of ASXL1-MT-KI hematopoietic cells to transform into myeloid leukemia cells. In this screening, we identified Hematopoietically expressed homeobox (HHEX) gene as one of the common retrovirus integration sites. In this study, we investigated the potential cooperation between ASXL1-MT and HHEX in myeloid leukemogenesis. Expression of HHEX enhanced proliferation of ASXL1-MT expressing HSPCs by inhibiting apoptosis and blocking differentiation, whereas it showed only modest effect in normal HSPCs. Moreover, ASXL1-MT and HHEX accelerated the development of RUNX1-ETO9a and FLT3-ITD leukemia. Conversely, HHEX depletion profoundly attenuated the colony-forming activity and leukemogenicity of ASXL1-MT-expressing leukemia cells. Mechanistically, we identified MYB and ETV5 as downstream targets for ASXL1-MT and HHEX by using transcriptome and ChIP-seq analyses. Moreover, we found that expression of ASXL1-MT enhanced the binding of HHEX to the promoter loci of MYB or ETV5 via reducing H2AK119ub. Depletion of MYB or ETV5 induced apoptosis or differentiation in ASXL1-MT-expressing leukemia cells, respectively. In addition, ectopic expression of MYB or ETV5 reversed the reduced colony-forming activity of HHEX-depleted ASXL1-MT-expressing leukemia cells. These findings indicated that the HHEX-MYB/ETV5 axis promotes myeloid transformation in ASXL1-mutated preleukemia cells.

Description: Polynuclear cells (PNCs) are routinely observed in the bone marrow of MDS patients. They are binuclear, trinuclear or even multinuclear cells with or without micronuclei, the underlying molecular mechanisms for the production of which are largely unknown. Because loss of the long arm of chromosome 7 (7q-) was reported to be associated with the presence of a higher frequency of PNCs, gene(s) preventing bone marrow cells from carrying such nuclear abnormalities may be located at 7q. We previously identified three candidate anti-myeloid tumor suppressor genes, namely Samd9, Samd9L and Miki, from the microdeletion in the 7q21 band frequently detected in JMML patients. SAMD9L-deficient mice develop MDS resembling human diseases associated with 7q-, most likely through enhancement of cytokine signals (Nagamachi et al., Cancer Cell 2013). Miki (mitotic kinetics regulator) translocates from the Golgi apparatus to mitotic centrosomes coincident with the disappearance of the Golgi body after poly-ADP-ribosylation (PARsylation). Miki is indispensable for centrosome maturation [the rapid increase of pericentriolar materials (PCM) during prophase and prometaphase], which is required for the production of robust mitotic spindles to move chromosomes promptly (Ozaki et al., Mol. Cell 2012). Consequently, as observed by time-lapse imaging of HeLa cells expressing histone H2A-GFP, downregulation of Miki by siRNA markedly prolonged the duration of prometaphase to more than several hours (normally around 15 minutes). Chromosomes were scarcely able to align and cells exited from prometaphase either by cell death or by decondensation of each chromosome. In the latter, cells with decondensed chromosomes then fused with one another within 30 minutes to form cells with relatively large nuclei, resulting in PNCs containing various sizes of nuclei including micronuclei. Indeed, reduction of Miki in HeLa cells by siRNA increased the frequency of PNCs from less than 0.5% to 4.5%. To test whether the chaotic chromosome decondensation in prometaphase causes the accumulation of PNCs observed in MDS, we initially used five cell lines derived from MDS associated with 7q-. PARsylated Miki was barely detectable in these cell lines and we found more cells at prometaphase than at metaphase (the ratio of prometa:meta in the lines ranged from 1.7:1 to 5.7:1). In contrast, in seven cell lines expressing PARsylated Miki at high levels, mitotic cells in prometaphase were found less frequently or at roughly the same frequency as those in metaphase (prometa:meta ratio 0.6:1 to 1.3:1). PNCs in five cell lines harboring 7q-were also more frequent (5.9 - 10.2%) than in the seven cell lines expressing high PARsylated Miki (0.8 - 2.4%). In addition, when we reduced Miki expression levels by shRNA in K562 cells, which express PARsylated Miki at high levels, the prometa:meta ratio increased from 1.1:1 to 3.8:1 and PNCs increased from 0.8% to 8.5%. This suggests that, as in HeLa cells, low expression levels of Miki cause prolongation of prometaphase and increase PNCs in blood cells. Fresh bone marrow preparations from 37 patients with MDS were examined to determine whether Miki mRNA-expression levels influence the prometaphase:metaphase ratio and the frequency of PNCs. We found a strong negative correlation (R=-0.59, p

Description: Abstract 1081 α IIbβ3 integrin mutations that result in the complete loss of expression of this molecule on the platelet surface cause Glanzmann's thrombasthenia. This is usually an autosomal recessive, while other mutations are known to cause dominantly inherited macrothrombocytopenia (although such cases are rare). Here, we report a 4-generation pedigree including 10 individuals affected by dominantly inherited macrothrombocytopenia. Six individuals, whose detailed clinical and laboratory data are available, carry a non-synonymous ITGB3gene T2231C alteration resulting in the substitution of leucine at 718 for proline (L718P) in the integrin β3 protein. The patient was 4-year old Japanese girl, who presented with mild bleeding tendency. Her platelet count was 49–72 x109/L with a mean platelet volume of 6.7–10.4 fl. WBC and RBC numbers were normal and there were no morphological abnormalities including inclusions in neutrophils. In the patient, marked platelet anisocytosis and giant platelets were observed in peripheral blood samples. Platelet aggregation induced by ADP and collagen was markedly reduced, but agglutination induced by ristocetin was within the normal range. The α IIbβ3 expression level determined by flow cytometry in the patient was 50–60% of the healthy control. A total of six of her relatives were subsequently found to have low platelet counts and were referred to our institute for further investigation. To isolate a candidate gene alteration responsible for the macrothrombocytopenia, whole exome sequencing analysis was performed using genomic DNA obtained from four affected individuals of the pedigree including the patient. Among the 90 non-synonymous alterations commonly found in the affected individuals, we focused on the heterozygous integrin β3-L718P mutation, because this was recently reported as a candidate mutation responsible for macrothrombocytopenia (Jayo et al, 2010). As far as we could determine, no other non-synonymous gene alterations previously reported to cause thrombocytopenia or defective platelet function were present in the affected individuals of the pedigree. Resting platelets from affected individuals showed a mild but significant increase of a ligand-mimicking PAC-1 binding relative to healthy individuals. However, in ADP-treated platelets carrying the mutation, only a small increase of affinity to PAC-1 was observed. These findings suggest that α IIbβ3-L718P is partially activated in the absence of inside-out signals such as ADP, but nevertheless cannot be fully activated in the presence of stimulating signals. As previously reported by others, CHO cells expressing α IIbβ3-L718P formed long proplatelet-like protrusions on fibrinogen-coated dishes. This was reported to be mediated by the downregulation of RhoA activity, which is initiated by the binding of c-Src to the C-terminal tail of integrin β3. Indeed, we found that the formation of long cell protrusion was inhibited, when a constitutively-active form of RhoA (Q63L) was introduced into α IIbβ3-L718P-expressing cells. In addition, CHO cells expressing α IIbβ3-L718P (del. 759) mutant, which lacks the C-terminal c-Src binding site of integrin β3, did not form any proplatelet-like protrusions. However, because the enforced expression of a dominant negative form of RhoA (T19N) in α IIbβ3-WT expressing cells did not show typical proplatelet-like protrusions, it is suggested that downregulation of RhoA was required but not sufficient for the formation of proplatelet-like protrusions induced by integrin β3-L718P. In summary, identification of a pedigree showing autosomal dominant inheritance leads to a model whereby the integrin β3-L718P mutation contributes to macrothrombocytopenia most likely through gain-of-function mechanisms. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 645 Background: Acquired aplastic anemia (AA) is a non-clonal hematopoietic disorder caused by the immune system attack on hematopoietic stem cells (HSCs) and has been thought to be rarely associated with intrinsic HSC defects. However, clonal hematopoiesis by HSCs with genetic alterations may not be uncommon in AA since clonal granulocyte populations showing the paroxysmal nocturnal hemoglobinuria (PNH) phenotype or chromosome 6p uniparental disomy (6pUPD) are detected in approximately 50% and 13% of AA patients, respectively (Katagiri T, et al, Blood, 2011). Abnormal HSCs other than those with the PIG-A mutation or 6pUPD may also contribute to hematopoiesis in AA patients due to immune escape or preferential activation of HSCs with genetic alterations in response to immune pressure in the bone marrow (BM). Objectives/methods: To identify novel genetic abnormalities in patients with AA, we carried out whole exome sequencing of peripheral blood leukocytes from 96 AA patients using a next generation sequencer. Among 40 abnormalities found in more than one patient, we selected abnormalities in genes potentially related to HSC regulation and examined the relevance of the genetic alterations to BM failure. Results: Missense mutations of SLIT1, a family of secreted glycoproteins that bind members of the Robo receptor family and regulate axon guidance, were detected in 15 of 96 patients and confirmed by Sanger sequencing in 3 patients who had been in remission for 1–3 years after immunosuppressive therapy at the time of blood sampling. Since previous studies showed that the Slit-Robo pathway negatively regulates proliferation of murine HSCs as well as a human cancer cell line in an autocrine manner (Prasad A et al., J Biol Chem; 283: 26624. 2008) and the break point of chromosome 10 in a patient with immune-mediated AA possessing t(1:10) was located within the SLIT1 gene, we studied SLIT1 and Robo receptor expression in BM mononuclear cells (BMMCs) obtained from healthy volunteers and patients with AA, and in myeloid leukemia cell lines. Although SLIT1 mRNA was scarce in un-stimulated BMMCs from healthy volunteers, SLIT1 gene expression increased 2.5–3.5 fold when incubated in conditioned medium from PHA or LPS stimulated peripheral blood mononuclear cells. The steady state expression level of SLIT1 by BMMCs from AA patients was 6 fold higher than from healthy individuals. SLIT1 mRNA was also detected in several myeloid leukemia cell lines including K562 and MV4-11, and its expression increased 2 fold by culturing the cells for 48 hours with the human stromal cell line HS-5. Examination of Robo receptor expression using antibodies specific to Robo1-4 showed that BM CD34+ cells from healthy individuals and K562 cells expressed Robo1. Recombinant Slit1 inhibited proliferation of K562 in a dose-dependent manner at concentrations of 1 and 5 mg/ml (Figure 1). Conclusions: The presence of clonal leukocytes with SLIT1 mutations in patients with immune-mediated AA suggests that HSCs with these mutations have survival or proliferation advantages over HSCs without SLIT1 mutations. Slit1 secreted from HSCs or hematopoietic progenitor cells in response to inflammatory cytokines may inhibit their own activation by binding to Robo1, but mutant Slit1 may fail to inhibit activation, leading to preferential commitment of the mutant HSCs to hematopoiesis. The Slit-Robo pathway may therefore serve as a novel therapeutic target for immune-mediated BM failure. Disclosures: No relevant conflicts of interest to declare.

Description: Mantle cell lymphoma (MCL) has been mostly incurable, and there is an urgent need to identify targetable molecules for development of a more effective treatment strategy. Bromodomain and extraterminal domain (BET) proteins associate with acetylated histones and facilitate transcription of target genes, and bromodomain-containing 4 (BRD4), a member of BET proteins, recruits the P-TEFb complex to genomic lesions in chromatin and thereby activates RNA Pol II at specific promoter sites of target genes. In addition, super-enhancers have been recognized as regulatory regions with a high level of acetylated histones, mediator complexes and BRD4, and super-enhancers in cancer cells are enriched at oncogenes. Recent studies have shown that BRD4 promotes expression of pivotal molecules in disease development, maintenance and progression in various cancers, including lymphoma. Given, we in this study examined the effect of BRD4 inhibition on human MCL-derived cell lines, Jeko-1, JVM-2, MINO and Z138, and performed broad screening of BRD4-regulated molecules using genome-wide approaches to identify therapeutic targets for MCL. As the results, treatment with a BRD4 inhibitor I-BET151 for 72 h showed a dose-dependent inhibitory effect on cell proliferation in all four cell lines, with half maximal inhibitory concentrations (IC50s) of 15.6 nM, 3.6 nM, 2.6 nM and 3.0 nM in Jeko-1 cells, JVM2 cells, MINO cells and Z138 cells, respectively, which was accompanied by G1/S cell cycle arrest and the induction of apoptosis. Next, we performed comprehensive gene expression profile (GEP) analysis for JVM2 and Z138 cells with or without I-BET151 treatment, and BRD4 chromatin immunoprecipitation sequencing (ChIP-Seq) in JVM2 cells treated with 10 nM I-BET151 or DMSO. Accordingly, GEP analyses revealed that more than 600 genes were commonly upregulated by more than 1.5-fold and downregulated by less than 0.67-fold, respectively, in JVM2 and Z138 cells treated by I-BET151, while ChIP-Seq showed that 7988 BRD4-binding regions were dysregulated by I-BET151, with most of these sites in enhancer regions, and 547 BRD4-binding regions were characterized as super-enhancers. Integrated analysis using the Reactome Pathway Database and the results of GEP and ChIP-Seq showed that a series of genes involved in the B cell receptor (BCR) signaling pathway and IKZF-MYC axis are regulated by BRD4 in MCL cells. To confirm whether each BRD4 target contributes to survival and proliferation of MCL cells, we focused on several candidate targets: the BCR pathway, IKZF and MYB. However, ibrutinib, a Bruton kinase inhibitor, suppressed cell growth in only two of the four cell lines (MINO and JVM2) in a dose-dependent manner, while lenalidomide, an inhibitor of the IKZF family, did not affect cell survival, despite its potency in decreasing IKZF1 and IKZF3 proteins. MYB silencing using shMYB did not decrease cell proliferation in any of the four MCL cell lines. In conclusion, our study disclosed that BRD4 regulates transcription of multiple genes by binding to enhancer region, partly involving super-enhancers and multiple known pathways, such as BCR signaling and the IKZF-MYC axis, which play essential roles in survival of MCL cells. While the efficacy of single targeting of BCR-signaling, IKZF, or MYB was limited, I-BET151 concomitantly inactivated the BCR pathway and IKZF and had a high growth inhibitory efficacy in MCL cells. These results suggest that simultaneous targeting of multiple molecules involved in the BCR pathway and IKZF-MYC axis may overcome resistance to ibrutinib and/or lenalidomide in MCL, and that BRD4 inhibitors are promising candidates for MCL treatment. Disclosures Kuroda: Chugai Pharma: Honoraria, Research Funding. Taniwaki:Bristol-Myers Squibb: Research Funding; Chugai Pharmaceutical Co., Ltd.,: Research Funding; Astellas Pharma Inc,: Research Funding.

Description: High Mobility Group AT-hook 2 (HMGA2) is a chromatin modifier and its overexpression has been found in a subset of patients with myelodysplastic syndrome (MDS). The high level of HMGA2 expression appears to predict poor prognosis in various tumors; however, it remains unclear how HMGA2 dysregulates expression of target genes to facilitate the transformation. To elucidate the mechanisms by which the overexpression of Hmga2 promotes the development of MDS, we generated an Hmga2-expressing Tet2-deficient (Hmga2-Tet2Δ/Δ) mouse model showing the progressive phenotype of MDS. We found that Hmga2-Tet2Δ/Δ mice had neutropenia and anemia, but variable platelet counts, accompanied by elevated frequencies of mutant cells in myeloid cells. Hmga2-Tet2Δ/Δ mice showed a similar median survival to Tet2Δ/Δ mice (274 days vs 290 days), but shorter survival than Hmga2-Tet2wt/wt mice (274 days vs undetermined). Moribund Hmga2-Tet2Δ/Δ mice showed progressive leukopenia and anemia, accompanied by the emergence of dysplastic neutrophils, myeloblasts and anisocytosis in the PB and BM and dysplastic megakaryocytes in the BM. Hmga2-Tet2Δ/Δ mice had mildly increased spleen weights, and expanded myeloid cells and HSPCs in the spleen without the deposition of fibrosis. During a 12-month observation, we found that Hmga2-Tet2Δ/Δ mice developed lethal MDS/MPN overlap disease (47%), MDS (33%), MPN (13%), and AML (7%), while 6 out of 11 Tet2Δ/Δ mice developed MPN (55%). Hmga2-Tet2wt/wt mice subsequently showed similar blood counts in PB and died without the expansion of leukemic or dysplastic blood cells. Therefore, Hmga2 overexpression did not transform wild-type HSCs but promoted the development of MDS in the absence of Tet2 in vivo. In order to elucidate the molecular mechanisms underlying the transformation of Hmga2-Tet2Δ/Δ cells, we initially performed gene expression profiling by a RNA sequencing analysis in LSK HSPCs isolated from WT, Hmga2-Tet2wt/wt, Tet2Δ/Δ, and Hmga2-Tet2Δ/Δ mice at a pre-disease stage and those isolated from two Hmga2-Tet2Δ/Δ MDS/MPN and AML mice. Hmga2-Tet2Δ/Δ leukemic cells were placed closer to one out of two Hmga2-Tet2Δ/Δ cells at the pre-disease stage, but clearly apart from the other genotype cells, indicating that Hmga2 overexpression and Tet2 loss result in the accumulation of alterations in the transcriptional program during the development of MDS.In order to clarify the mechanisms by which the overexpression of Hmga2 alters the transcriptional program in Tet2-deficient cells, we performed the ChIP-sequencing of FLAG-tagged Hmga2 in bone marrow progenitor cells isolated from WT, Hmga2-Tet2wt/wt, and Hmga2-Tet2Δ/Δ mice. The numbers of Hmga2-binding peaks were markedly lower in Tet2-deficient cells than in Hmga2-Tet2wt/wt cells (2227 peaks versus 11500 peaks). Furthermore, annotated genes adjacent to Hmga2-binding sites partially overlapped in both genotype cells, whereas 2965 out of 3843 genes identified in Tet2 wild-type cells lost the binding peaks of Hmga2 upon the deletion of Tet2. Based on the DNA-binding capacity of Hmga2, the loss of Tet2 remodeled the binding sites of Hmga2 via change in DNA methylation in Hmga2-binding flanking regions, which were not observed in the presence of Tet2, leading to significant enrichments in genes involved in cell-to-cell adhesion and cell morphogenesis in Hmga2-Tet2Δ/Δ cells. Furthermore, we found that the overexpression of Hmga2 and loss of Tet2 resulted in the activation of oncogenic pathways (e.g. TGF-b, TNF-a), but suppressed the expression of genes in the unfolded protein response. Notably, the inhibition of bile acid metabolism to reactivate the unfolded protein response markedly attenuated the proliferation of Hmga2-Tet2Δ/Δ cells. These combinatory effects on the transcriptional program and cellular functions were not redundant to those in either single mutant cell, supporting Hmga2 being a proto-oncogene because its overexpression alone was not sufficient to develop MDS in vivo. Thus, Hmga2 overexpression exerts synergistic, but also gain-of-function effects with the loss of Tet2 to target these key biological pathways and promotes the transformation of Tet2-deficient stem cells. This study also provides a new rationale for targeting the unfolded protein response in MDS cells expressing HMGA2. Disclosures No relevant conflicts of interest to declare.

Description: The establishment of cancer cell lines, which have different metastatic abilities compared with the parental cell, is considered as an effective approach to investigate mechanisms of metastasis. A highly metastatic potential mouse colon cancer cell subline, Colon-26MGS, was derived from the parental cell line Colon-26 by in vivo selection using continuous subcutaneous implanting to immunocompetent mice. To clarify the mechanisms involved in the enhancement of metastasis, morphological characteristics, cell proliferation, and gene expression profiles were compared between Colon-26MGS and the parental cell. Colon-26MGS showed over 10 times higher metastatic ability compared with the parental cell, but there were no differences in morphological characteristics and in vitro proliferation rates. In addition, the Colon-26MGS-bearing mice exhibited no marked change of splenocyte population and lung pre-metastatic niche with tumor-free mice, but there were significant differences compared to Colon-26-bearing mice. RNA-seq analyses indicated that immune costimulatory molecules were significantly up-regulated in Colon-26MGS. These results suggest that Colon-26MGS showed not only higher metastatic activity, but also less induction property of host immune response compared to parental Colon-26. Colon-26MGS has proven to be a novel useful tool for studying multiple mechanisms involving metastasis enhancement.

Description: Key Points Fbxl10 is a bona fide oncogene in vivo. Fbxl10 overexpression in HSCs induces mitochondrial metabolic activation and enhanced expression of Nsg2.