Polycomb-Dependent Epigenetic Landscape in Adult T Cell Leukemia (ATL); Providing Proof of Concept for Targeting EZH1/2 to Selectively Eliminate the HTLV-1 Infected Population

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.