ALBERT

Description: Because spermatogonial stem cells (SSCs) are immortal by serial transplantation, SSC aging in intact testes is considered to be caused by a deteriorated microenvironment. Here, we report a cell-intrinsic mode of SSC aging by glycolysis activation. Using cultured SSCs, we found that aged SSCs proliferated more actively than young SSCs and showed enhanced glycolytic activity. Moreover, they remained euploid and exhibited stable androgenetic imprinting patterns with robust SSC activity despite having shortened telomeres. Aged SSCs showed increased Wnt7b expression, which was associated with decreased Polycomb complex 2 activity. Our results suggest that aberrant Wnt7b expression activated c-jun N-terminal kinase (JNK), which down-regulated mitochondria numbers by suppressing Ppargc1a. Down-regulation of Ppargc1a probably decreased reactive oxygen species and enhanced glycolysis. Analyses of the Klotho-deficient aging mouse model and 2-y-old aged rats confirmed JNK hyperactivation and increased glycolysis. Therefore, not only microenvironment but also intrinsic activation of JNK-mediated glycolysis contributes to SSC aging.

Description: Mutations of epigenetic regulators are often found in patients with myelodysplastic syndrome (MDS). Furthermore, DNA methylation inhibitors have a therapeutic impact on MDS. However, it remains unknown how altered DNA methylation promotes the development of MDS. We have shown that concurrent depletion of Tet2 and Ezh2 in hematopoietic cells significantly promotes the development of MDS in vivo by utilizing hypomorphic Tet2 (Tet2KD/KD) mice and Ezh2 conditional knockout mice (Cre-ERT;Ezh2fl/fl)(Muto T, et al. J Exp Med 2014). In order to determine how DNA methylation contributes to the formation of MDS in Tet2KD/KDEzh2Δ/Δ mice, we transplanted wild type (WT), Tet2KD/KD, Cre-ERT;Ezh2fl/fl, and Cre-ERT;Tet2KD/KDEzh2fl/fl fetal liver cells in lethally irradiated CD45.1+ recipient mice, and deleted Ezh2 at 4 weeks post-transplantation. We then performed reduced representation bisulfite sequence (RRBS) in Lin-Sca1+Kit+ (LSK) cells isolated from Tet2KD/KD and Ezh2Δ/Δ mice at 3 and 7 months post-deletion and WT and Tet2KD/KDEzh2Δ/Δ mice at 5 months post-deletion. We defined ≥10% difference of methylation in test cells compared with that in WT cells as hyper- or hypo-differentially methylated regions (DMRs) (p-value

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: Recent reports in RNA biology have suggested that long non-coding RNAs, edited RNAs, and alternatively spliced RNAs (henceforth referred to as non-canonical RNAs) play a relatively unclear but critical role in numerous cells, tissues, and diseases. Of interest to hematology, several recent studies have also shown that these non-canonical RNAs all play various roles in both normal and malignant hematopoiesis, but no study yet has comprehensively examined these non-canonical RNAs in hematopoietic stem cells (HSCs). To this end, we conducted microarray and RNAseq analyses of mouse hematopoietic cells at various stages of differentiation. Microarray analysis detected several thousand possible lincRNAs expressed in hematopoietic stem and progenitor cells (HSPCs), several hundred of which were significantly enriched in CD34-LSKs compared to more differentiated populations. RNAseq analysis provided a more detailed look at these non-coding RNAs, as well as a slightly more conservative estimate of the abundance and structure of the long non-coding RNAs expressed and significantly enriched in HSPCs. In addition to examining lincRNAs, RNAseq data allowed us to profile alternative splicing events and RNA editing events in HSPCs. Interestingly, several hundred potentially significant HSC-specific alternative splicing events and RNA editing events were also discovered. In order to test whether these non-canonical RNAs play a functional role in HSPCs we performed shRNA mediated knockdown of several interesting HSC-specific lincRNAs and alternatively-spliced transcripts. We found that knockdown of several of these candidates was incompatible with hematopoiesis, both in vitro and in vivo. To better understand how these particular non-canonical RNAs function in HSCs, we are currently carrying out RNA FISH studies. In this study, we explored the RNA environment of HSCs and compared it to their downstream progenitors. We discovered several hundred novel non-coding transcripts, hundreds of alternative splicing events and hundreds of RNA editing events unique to HSCs. Functional investigation of some of these events revealed that these non-canonical RNAs do indeed play a critical role in HSC maintenance. The data presented here should eventually be an asset to other HSC researchers interested in the RNA biology of HSCs. Disclosures: No relevant conflicts of interest to declare.

Description: Recurrent inactivating mutations have been identified in the X-linked plant homeodomain finger protein 6 (PHF6) gene, encoding a chromatin-binding transcriptional regulator protein, in various hematological malignancies. However, the role of PHF6 in normal hematopoiesis and its tumor-suppressor function remain largely unknown. We herein generated mice carrying a floxed Phf6 allele and inactivated Phf6 in hematopoietic cells at various developmental stages. The Phf6 deletion in embryos augmented the capacity of hematopoietic stem cells (HSCs) to proliferate in cultures and reconstitute hematopoiesis in recipient mice. The Phf6 deletion in neonates and adults revealed that cycling HSCs readily acquired an advantage in competitive repopulation upon the Phf6 deletion, whereas dormant HSCs only did so after serial transplantations. Phf6-deficient HSCs maintained an enhanced repopulating capacity during serial transplantations; however, they did not induce any hematological malignancies. Mechanistically, Phf6 directly and indirectly activated downstream effectors in tumor necrosis factor α (TNFα) signaling. The Phf6 deletion repressed the expression of a set of genes associated with TNFα signaling, thereby conferring resistance against the TNFα-mediated growth inhibition on HSCs. Collectively, these results not only define Phf6 as a novel negative regulator of HSC self-renewal, implicating inactivating PHF6 mutations in the pathogenesis of hematological malignancies, but also indicate that a Phf6 deficiency alone is not sufficient to induce hematopoietic transformation.

Description: Early T cell precursor (ETP) acute lymphoblastic leukemia (ALL) has been identified as a new pathologic entity with poor outcome in patients with T-ALL. In contrast to cortical T-ALL, ETP-ALL has been characterized by the activating mutations in genes regulating cytokine signaling and the inactivating mutations in the polycomb repressor complex 2 (PRC2) component genes including EZH2. EZH2 catalyzes trimethylation of histone H3 at lysine 27 (H3K27me3) that mediates silencing of critical genes for cell proliferation and differentiation. Given that loss-of-function mutations in EZH2 are often found in ETP-ALL, EZH2 may play a tumor suppressor role in the pathogenesis of ETP-ALL. To determine how EZH2 dysfunction promotes the development of ETP-ALL in vivo, we generated a novel mouse model of ETP-ALL by utilizing Ezh2 and p53 conditional knockout mice. We harvested bone marrow cells from Cre-ERT (WT), Cre-ERT;Ezh2flox/flox (Ezh2 KO), Cre-ERT;Trp53 flox/flox (p53 KO) and Cre-ERT;Trp53 flox/flox;Ezh2flox/flox (DKO) mice, and transplanted them into lethally-irradiated Ly5.1+ recipient mice. We deleted p53 and Ezh2 via administration of tamoxifen 4 weeks post-transplantation. We observed that p53 KO mice died dominantly due to CD3+ CD8+ cortical T-ALL (median survival; 137 days), as previously reported. While 2 out of 8 DKO mice developed CD8+ cortical T-ALL, we found that 6 out of 8 DKO mice developed CD4- CD8- (DN) T-ALL with a longer latency (median survival; 189 days). At the time of sacrifice, DKO mice showed significantly enlarged thymus due to the expansion of c-Kit+ CD44+ CD25- DN1 and c-Kit+ CD44+ CD25+ DN2 cells, which were markedly compromised in differentiation into DN3 and further down to CD4+ CD8+ (DP) cells. Histological analysis demonstrated the expansion of cytoplasmic CD3+ (cyCD3) tumor cells in the thymus of DKO mice with DN T-ALL. Thus, DKO mice faithfully recapitulated the phenotypic features of human ETP-ALL, indicating that the loss of Ezh2 is prerequisite to the development of c-Kit+ CD44+ CD25+ cyCD3+ DN ALL in mice lacking p53. To understand the underlying molecular mechanism in the pathogenesis of Ezh2 loss-induced ETP-ALL, we performed gene expression analysis in DN1, DN2 and DN3 cells isolated from WT and DKO mice at pre-disease and ETP-ALL stages. Since NOTCH1 activating mutations are not frequently found in patients with ETP-ALL, we observed no significant difference in activation of Notch1-target genes between WT and DKO ETP-ALL cells. In contrast, Ras-target genes were significantly activated in DKO ETP-ALL DN2 cells relative to WT DN2 cells. Thus, our ETP-ALL mouse model shows active Ras signaling but lacks Notch1 activation, consistent with the molecular features of human ETP-ALL. Furthermore, we found that the genes critical for T-cell commitment, including Tcf7 or Bcl11b, were transcriptionally repressed in DKO ETP-ALL DN2 cells compared to WT DN2 cells, while the signature genes of both HSCs and myeloid cells were retained in DKO ETP-ALL DN2 cells. Taken together, Ezh2 and p53 deletions cooperate to activate the function of HSCs and impede the transcriptional program of T-cell differentiation at the DN2 stage with sustaining myeloid potential. To determine how H3K27me3 modification contributed to induce ETP-ALL in the absence of Ezh2, we performed H3K27me3-chromatin immunoprecipitation (ChIP) sequencing in WT and DKO ETP-ALL DN1/2 cells. We found that H3K27me3 marks were lost or kept at low levels at the promoter regions of T-cell differentiation regulators in ETP-ALL cells. Thus, we sought to determine whether altered DNA hypermethylation contributed to silencing the expression of T-cell differentiation regulators. The transduction of either Tcf7 or Bcl11b expression alone was not sufficient to induce differentiation of DKO DN1/2 cells in vitro, however, we found that treatment of decitabine, a demethylating agent, clearly induced the differentiation of DKO DN1/2 cells beyond the DN3 stage in vitro, implying that Ezh2 loss and p53 loss cooperatively induced aberrant DNA hypermethylation, thereby impeding the differentiation of DN1/2 cells. In conclusion, we demonstrated that combined deletion of Ezh2 and p53 altered the epigenetic regulation to an extent not seen in either deletion alone, and induced highly penetrant ETP-ALL characterized by the molecular profile similar to that in patients with ETP-ALL harboring mutations in the PRC2 components. 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: Abstract 2416 TET2 mutations are frequently observed in myeloid malignancies including myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN) and MDS/MPN. Several groups have already reported that deletion of Tet2 in mice leads to dysregulated hematopoietic stem cell self-renewal and subsequent development of myeloid malignancies. Of note, mice hypomorphic or heterozygous for the Tet2 allele have been reported to show similar phenotypes as those of Tet2-null mice, suggesting that haploinsufficiency of Tet2 plays a role in the development of myeloid malignancies. However, little is known about long-term cell autonomous effects of Tet2 loss in hematopoietic cells: most of the reports were based on relatively short-term observations or did not exclude the influence of Tet2 loss in the niche cells. To study long-term cell autonomous effect of Tet2 loss in hematopoietic cells, we analyzed the hematopoiesis of wild-type recipient mice reconstituted with fetal liver cells from Tet2 hypomorphic mice for a longer period up to 1 year. Tet2 gene trap mice (Tet2trap/trap), in which the gene trap vector was inserted into the exon 2 of Tet2 just before the first coding exon, express Tet2 mRNA at the level approximately 20% of that of the wild-type (WT) mice (Shide et al. Leukemia 2012). We transplanted fetal liver cells from E14.5 WT or Tet2trap/trap mice into lethally irradiated recipient mice. At 4 months after transplantation, the recipient mice reconstituted with Tet2trap/trap cells showed a significantly increased proportion of monocytes in peripheral blood (PB) compared with those with WT cells (WT=5.59±2.57%, Tet2trap/trap=12.67±7.45%, p=0.01). While there were no significant differences between the two groups in the bone marrow (BM) compartments including the numbers of Lineage−Sca-1+c-Kit+ (LSK) hematopoietic stem/progenitor cells, extramedullary hematopoiesis in the spleen was markedly enhanced in the recipients with Tet2trap/trap cells. The proportion of LSK, granulocyte/macrophage progenitors (GMPs) and megakaryocyte/erythroid progenitors (MEPs) in the spleen of recipient mice reconstituted with WT and Tet2trap/trap cells were 0.002±0.001% vs 0.006±0.001% (p