ALBERT

Description: Human T cell leukemia virus 1 (HTLV-1) causes the functionally debilitating disease HTLV-1–associated myelopathy/tropical spastic paraparesis (HAM/TSP) as well as adult T cell leukemia lymphoma (ATLL). Although there were concerns that the mortality of HAM/TSP could be affected by the development of ATLL, prospective evidence was lacking in this area. In this 5-y prospective cohort study, we determined the mortality, prevalence, and incidence of ATLL in 527 HAM/TSP patients. The standard mortality ratio of HAM/TSP patients was 2.25, and ATLL was one of the major causes of death (5/33 deaths). ATLL prevalence and incidence in these patients were 3.0% and 3.81 per 1,000 person-y, respectively. To identify patients at a high risk of developing ATLL, flow cytometry, Southern blotting, and targeted sequencing data were analyzed in a separate cohort of 218 HAM/TSP patients. In 17% of the HAM/TSP patients, we identified an increase in T cells positive for cell adhesion molecule 1 (CADM1), a marker for ATLL and HTLV-1–infected cells. Genomic analysis revealed that somatic mutations of HTLV-1–infected cells were seen in 90% of these cases and 11% of them had dominant clone and developed ATLL in the longitudinal observation. In this study, we were able to demonstrate the increased mortality in patients with HAM/TSP and a significant effect of ATLL on their prognosis. Having dominant clonal expansion of HTLV-1–infected cells with ATLL-associated somatic mutations may be important characteristics of patients with HAM/TSP who are at an increased risk of developing ATLL.

Description: Key PointsATL involves genome-wide reprogramming of the H3K27me3 pattern that is distinct from other cell types. Druggable epigenetic mechanisms are associated with ATL cell development and HTLV-1–mediated transformation.

Description: B-cell receptor (BCR) signaling is a major source of gene expression signature important for B cell survival, functions, and development. The integrated signaling amplitude should be equilibrated; when chronically activated by genetic perturbations or other mechanisms, BCR signaling has been accepted as a stem in the pathogenesis of malignant lymphoma/leukemia. The promiscuous signaling network without appropriate alleviation is an essential property of cancers. Diffuse large B-cell lymphoma (DLBCL) with aggressive phenotypes often associates with BCR signaling activation and epigenetic abnormalities, both of which are well correlated with lymphoma subtypes and disease progression. However, the relationship of the two fundamental characteristics is unknown. It is also still unclear the mechanism by which the expression of functionally important genes is continuously deregulated. microRNAs (miRNAs) are an emerging class of intrinsic buffering molecules that have diverse functions in mainly post-transcriptional regulation through miRNA-RISC (miRISC) formation. Using optimized RISC-capture assay, we revealed that multiple BCR signaling factors were persistently regulated by miRNA in human B cells. Clinical samples from newly diagnosed patients with DLBCL (n=83) showed epigenetic loss of an essential miRNA set (miR-200c, miR-203, miR-31). Conventional screening and RISC profiling identified multiple targets (CD79B, SYK, PKCbII, PLCg1, IKKb, NIK, MYD88, PI3K class I (a/b/d/g), RasGRP3); the miRNA-orchestrated overlapping interactome suggested multiple regulatory windows in signaling pathways, which implied their compensatory roles. The shared components within the interconnected signaling pathways were under constant surveillance by the miRNAs. We demonstrated that simultaneous depletion of the key miRNAs enhanced translation of the multiple targets and caused seamless signaling crosstalk among canonical/noncanonical NF-kB pathways, PI3K-Akt-mTOR and Ras-Erk cascades, and downstream of BCR and BAFF receptors. In addition to the miRNA loss, mTOR activation was also positively correlated with signaling amplitude, offering the possibility of selective therapy targeting the translation machinery. The defenseless signaling crosstalk appeared to be a prerequisite for lymphoma development because the essential miRNA set were functionally lost in all tested lymphoma samples. Surprisingly, a common epigenetic mechanism was identified. Polycomb-mediated H3K27me3 accumulation and reciprocal H3K4me3 loss were frequently observed at the miRNAs loci in primary DLBCL samples. We found that lymphoma-associated deregulation (mutation and/or expression change) of EZH2 and MLL2 cooperated to cause the sustained signaling activities of NF-kB, Ras-Erk, PI3K-Akt and mTOR pathways through miRNA silencing en masse. Of note, the perturbation of histone-modifying polycomb and trithorax groups resulted from variations of pooled miRNAs and signaling integrity. The identified coherent circuit may be a source of pathological robustness and phenotypic convergence. Most malignant B cell clones have some kind of genetic lesions. In particular, recurrent variations have been identified within signaling cascades, where miRNA-formed cooperative gene interference (CGI) could buffer the signaling noise. We re-established some experimental models that mimicked the signaling cascade observed in clinically established malignant clones. We experimentally confirmed the buffering roles of the key miRNAs against genetic mutation/alteration of CARD11, A20, MYD88 and PTEN. Reciprocally, coordinated reduction of the functional miRNAs promoted biological processes caused by genetic perturbations. It is therefore conceivable that systematic redundancy of miRNA is inherently required for robustness against genetic alterations that are acquired in lymphoma evolution. We illustrate an example of a biological masterplan comprised of signaling pathways, compensatory actions of multi-layered miRNAs, translation regulation and epigenetic mechanisms. Malignant cell opts for epigenetic reprogramming to stabilize mutation-driven processes and to surmount the demarcation threshold of transformation. Reversing the plastic characteristics of epigenetic alterations is expected in future clinical settings. Disclosures Yamagishi: Daiichi Sankyo Co., Ltd.: Research Funding. Watanabe:Daiichi Sankyo Co., Ltd.: Research Funding.

Description: Adult T-cell leukemia/lymphoma (ATL) is an aggressive T cell leukemia/lymphoma and is refractory to currently available combination chemotherapy. The unfavorable prognosis results from an inadequate understanding of how diseases are caused and maintained in human T-cell leukemia virus type I (HTLV-1)-infected individuals. To date, direct comprehensive analyses of leukemic cells have identified the intrinsic molecular hallmarks of ATL. Among these, polycomb group (PcG)-mediated epigenetic disruption has been known to be a crucial characteristic of ATL (Yamagishi et al., Cancer Cell, 2012). However, no attempt has been made to determine the global epigenomic status explaining the deregulated gene expression pattern specific to ATL. In this study, we performed integrative analyses of epigenome (n=3) and transcriptome (n=58) of primary ATL patient cells and corresponding normal CD4+ T cells to decipher the ATL-specific 'epigenetic-code' that was critical for cell identity. We found that PcG-mediated tri-methylation at histone H3Lys27 (H3K27me3) was significantly and frequently reprogrammed at over half of genes (53.8%) in ATL cells, the pattern of which appears distinct from other cancer types and PcG-dependent cell lineages such as ES cells and peripheral T lymphocytes. Large proportion of the abnormal gene downregulation occurred at an early stage of disease progression and was explained by the H3K27me3 accumulation. The global H3K27me3 alterations were involved in determination of key genes such as miR-31, CADM1, EVC1/2, CDKN1A, and NDRG2, which are essential for ATL cell survival and other cellular characteristics. In addition, PcG generated diverse outcomes by the remote regulation of a broad spectrum of gene regulators, including various transcription factors, miRNAs, epigenetic modifiers, and developmental genes. Thus, the emerging epigenomic landscape is a fundamental characteristic of ATL. Although EZH2 mutations conferring gain-of-function were undetected in ATL (0/50; 0%), EZH2 level was significantly upregulated and inversely correlated with H3K27me3 targets, indicating that the global alteration of H3K27me3 mark depends on the abundance of EZH2 and other core components of the polycomb repressive complex 2 (PRC2). We found that EZH2 was sensitive to promiscuous signaling network including NF-kB pathway and was functionally affected by HTLV-1 Tax through both NF-kB activation and direct association. The Tax-dependent immortalized cells showed H3K27me3 reprogramming that was significantly similar to that of ATL cells. Of note, the majority of epigenetic silencing has occurred in leukemic cells from indolent type ATL and also in HTLV-1-infected T cells from asymptomatic HTLV-1 carriers. Collectively, our results unveiled that epigenetic reprogramming arises at an early stage of ATL development. Tracing the epigenetic marks and expression patterns in samples obtained from patients of various diagnostic categories, as well as in other biologically relevant models has supported the efficacy and relevance of targeting PRC2. Relief of the cumulative methylation may restore the aberrant transcriptome to ideal expression signature, permitting favorable treatments. According to the expression profiling, EZH2 may represent the first-choice as a druggable target. In addition, peripheral T cells highly express EZH1 that compensates for the EZH2 functions. We found that simultaneous depletion of the two H3K27me3 writers EZH1 and EZH2 significantly diminished cellular H3K27me3 level and dramatically inhibited ATL cell growth compared with single depletion, suggesting that the compensatory actions of EZH1/2 may be critical for ATL. To selectively eliminate the ATL and HTLV-1-infetced populations, we have developed a novel EZH1/2 dual-inhibitor that shows strong inhibitory effects for both of EZH1 and EZH2. Treatment with the new drugs showed significant inhibitory effects on the ATL cell survival derived from patients. Interestingly, the dual-inhibitor reversed the epigenetic disruption and selectively eliminated leukemic and immortalized cells from HTLV-1 infected individuals. Collectively, this approach will achieve potent and selective synthetic lethality by targeting the regulators of H3K27me3 in aggressive and indolent ATL cells, as well as in clonally expanded infected cells, improving medical care and the prevention of disease onset. Disclosures Yamagishi: Daiichi Sankyo Co., Ltd.: Research Funding. Honma:Daiichi Sankyo Co., Ltd: Employment. Adachi:Daiichi Sankyo Co., Ltd: Employment. Araki:Daiichi Sankyo Co., Ltd.: Employment. Watanabe:Daiichi Sankyo Co., Ltd.: Research Funding.

Description: Epigenetic program is a molecular basis of cellular identity and functions. We have shown the transcriptome (Cancer Cell, 2012) and the underlying histone methylation patterns (Blood, 2016) of adult T-cell leukemia-lymphoma (ATL). The epigenetic landscape of ATL is defined as "genome-wide H3K27me3 accumulation". Similar accumulation has been detected in multiple cancers, particularly in hematological malignancies. Relief of the cumulative methylation may restore the aberrant transcriptome to ideal expression signature, leading to a promising treatment. Due to variation characteristics, EZH2 is recognized as a druggable target. However, extrinsic regulation of methylation pattern is still challenging without knowledge of molecule(s) and their combination that needs to be targeted to reprogram the epigenome in anticipation of synthetic lethality. To redefine the conventional concept, we first examined expression and global occupancy patterns of H3K27 methyltransferases EZH1 and EZH2. EZH2 expression was low in CD4+ T cells and significantly high in ATL. In contrast, basal EZH1 was very high in mature lymphocytes compared with undifferentiated lineages and also high in ATL. ChIP-on-chip analysis revealed significant changes in distributions of both EZH1 and EZH2 in primary ATL cells, which caused abnormal H3K27me3 accumulation. Importantly, over 80% of H3K27me3 are specifically induced by either EZH1 (28.9%) or EZH2 (14.4%), or by both (39.3%). Moreover, they have differential functions by regulating specific targets; EZH2 suppresses various transcription regulators, establishing complex gene regulatory network. EZH1 would rather directly induce H3K27me3 at genes involving functional processes such as lymphocyte activation. We detected EZH1 and EZH2 in functional polycomb repressive complex 2 (PRC2) in ATL and DLBCL cells. Unexpectedly, EZH1/2 were co-localized with H3K4me3 at a small population of the lymphoma genome. The "EZH1/2 + H3K4me3" non-canonical targets such as cell cycle activators were highly expressed in ATL. We next compared functions of EZH1/2. Single depletion of EZH1 or EZH2 suppressed T- and B-lymphoma cell proliferation, which supports their unique functions. The broad reduction of H3K27me3 was achieved by EZH2 inhibition; however, genome-wide ChIP demonstrated that EZH2 inhibition or depletion triggered strong compensatory action of EZH1, followed by rebound accumulation of H3K27me3 at many functional genes in EZH1+ lymphoma cells. We found that a transcription factor YY1 affects PRC2 recruitment and EZH1 compensation after EZH2 inhibition. Double depletion of EZH1+EZH2 or YY1+EZH2 surmounted the epigenetic homeostasis. In addition, depletion of EZH1 significantly enhanced effects of EZH2 inhibitors in ATL and DLBCL cells (5.47~205-fold). These data indicate requirements for the aberrant epigenome, namely, fluctuating EZH2 (upregulation or mutation) and underlying stable EZH1. We have succeeded in developing an unprecedented EZH1/2 dual-inhibitor (IC50 9.0 nM (EZH1); 10 nM (EZH2)). The new drug successfully inhibited genome-wide EZH1/2 occupancies without compensation, resulting in effective H3K27me3 reduction and gene reactivation compared with the single inhibitor. Of note, the dual-inhibitor suppressed expression of the non-canonical EZH1/2 + H3K4me3 target genes. Besides H3K27me3 reprogramming, EZH1/2 inhibition could modulate genes through H3K4me3 regulation and indirect influence on the secondary regulatory network and signaling pathways such as NF-kB. The EZH1/2 dual-inhibitor significantly blocked primary ATL cell survival and in vivo tumor growth of ATL and DLBCL models. Furthermore, we evaluated drug efficacy in various models of hematological malignancies including ATL, DLBCL (EZH2 WT and mutant), Burkitt lymphoma, and PTCL, which are all originated from EZH1+ differentiated lymphocytes. All of them showed higher sensitivity against the EZH1/2 inhibitor compared with the EZH2 solo inhibitor (5.2~200-fold). We did not detect strong adverse effects in normal T cells. These integrated molecular and phenotypic analyses provide a novel concept "EZH1+EZH2 dual-targeting" and demonstrate validity of the new drug in the treatment of malignant lymphomas. Taking these results, a Phase I clinical trial against T- and B-cell non-Hodgkin lymphomas including ATL is now under way in Japan. Disclosures Yamagishi: Daiichi Sankyo Co., Ltd.: Research Funding. Honma:Daiichi Sankyo Co., Ltd.: Employment. Adachi:Daiichi Sankyo Co., Ltd.: Employment. Ohsugi:Daiichi Sankyo Co., Ltd.: Research Funding. Utsunomiya:Daiichi Sankyo Co., Ltd.: Speakers Bureau. Tsukasaki:Daiichi Sankyo Co., Ltd.: Consultancy; Takeda: Research Funding. Tobinai:Zenyaku Kogyo: Honoraria; Takeda: Honoraria, Research Funding; Mundipharma KK: Honoraria, Research Funding; Kyowa Hakko Kirin: Research Funding; Celgene: Research Funding; Daiichi Sankyo Co., Ltd.: Consultancy; SERVIER: Research Funding; Ono Pharmaceutical: Research Funding; Eisai: Honoraria, Research Funding; Chugai Pharma: Research Funding; Abbvie: Research Funding; Janssen Pharmaceuticals: Honoraria, Research Funding; GlaxoSmithKline: Research Funding; HUYA Bioscience: Honoraria. Araki:Daiichi Sankyo Co., Ltd.: Employment.

Description: The retrovirus human T-cell leukemia virus type 1 (HTLV-1) integrates into the host genome and persists for the lifetime of the host. There are tens of thousands of different infected clones in a HTLV-1 carrier and each clone can be identified by its unique viral integration site. Only about 5% of infected people develop the hematological malignancy, adult T-cell leukemia-lymphoma (ATL). However, it is unclear how a certain infected clone, among various different ones, is selected as a malignant clone. It has been reported that viral integration alters transcripts of the cellular host genes adjacent to the integration site, even generating truncated or virus-host chimeric transcripts. Because each infected clone has a unique viral integration site, each clone possibly has unique virus-host chimeric transcripts, which were not present in the host before infection. Therefore, we hypothesized that the integrated provirus generates virus-host chimeric transcripts that may play a role in the clonal selection of the HTLV-1-infected cell. We previously reported HTLV-1 DNA-capture-seq using biotinylated DNA-probes for the viral genome, to increase the sensitivity and efficiency of viral-sequences detection. In this study, we used HTLV-1 RNA-capture-seq for PBMCs samples from ATL patients to test the hypothesis in a highly sensitive manner. The results showed the presence of chimeric transcripts in 19 out of 30 ATL patients. We next quantified the abundance of chimeric transcripts by droplet digital PCR, and found that the expression levels of chimeric transcripts were similar to those of viral RNAs containing splice junction of HBZ, in 5 of 19 chimeric transcripts positive ATL cases, although the levels varied among different ATL cases. To identify the whole sequences of the chimeric transcripts, we performed Oxford Nanopore sequencing. This approach revealed that the HTLV-1 provirus generates various splicing chimeric transcripts with the host genes in both viral sense and antisense orientations. The transcriptional start site of most of the sense chimeric transcripts was the R region of the 5'- or 3'-long terminal repeats (LTRs) in the proviral sequences, indicating that the chimeric transcripts were generated using the viral promoters because the LTRs work as a promoter for the viral transcripts. Given the structure of the chimeric transcripts with the viral promotors, the expression of the fused host genes could be enhanced by generating the chimeric transcripts. We evaluated the mRNA expression of the fused host genes of the chimeric transcripts by RNA-seq, and the results correlated with those obtained by ddPCR. To clarify the impact of viral integration on the clonal expansion, we analyzed HTLV-1-infected Jurkat cells. The clonality analysis of infected cells by HTLV-1 DNA-capture-seq showed that some infected clones were remarkably expanded for 4-6 months culture. We also confirmed that some of them harbored virus-host chimeric transcripts by HTLV-1 RNA-capture-seq. This study revealed the expression levels and the structures of virus-host chimeric transcripts in ATL patients. We are currently investigating the functional role of chimeric transcripts in the clonal proliferation of infected cells in vitro. Disclosures Uchimaru: Daiichi Sankyo Co., Ltd..: Research Funding. Kimura:Novartis: Honoraria, Research Funding; Ohara Pharmaceutical Co.: Research Funding.