SUMMARY It is widely appreciated that T cells increase glycolytic flux during activation, however the role of mitochondrial flux is unclear. Here we have shown that mitochondrial metabolism, in the absence of glucose metabolism, was sufficient to support interleukin-2 (IL-2) induction. Furthermore, we used mice with reduced mitochondrial reactive oxygen species (mROS) production in T cells (T-Uqcrfs−/− mice) to show that mitochondria are required for T cell activation to produce mROS for activation of nuclear factor of activated T cells (NFAT) and subsequent IL-2 induction. These mice could not induce antigen-specific expansion of T cells in vivo, however Uqcrfs1−/− T cells retained the ability to proliferate in vivo under lymphopenic conditions. This suggests that Uqcrfs1−/− T cells were not lacking bioenergetically, but rather lacked specific ROS-dependent signaling events needed for antigen-specific expansion. Thus, mitochondrial metabolism is a critical component of T cell activation through production of complex III ROS.

The EZH2 histone methyltransferase is highly expressed in germinal center (GC) B-cells and targeted by somatic mutations in B-cell lymphomas. Here we find that EZH2 deletion or pharmacologic inhibition suppresses GC formation and functions in mice. EZH2 represses proliferation checkpoint genes and helps establish bivalent chromatin domains at key regulatory loci to transiently suppress GC B-cell differentiation. Somatic mutations reinforce these physiological effects through enhanced silencing of EZH2 targets in B-cells, and in human B-cell lymphomas. Conditional expression of mutant EZH2 in mice induces GC hyperplasia and accelerated lymphomagenesis in cooperation with BCL2. GCB-type DLBCLs are mostly addicted to EZH2, regardless of mutation status, but not the more differentiated ABC-type DLBCLs, thus clarifying the therapeutic scope of EZH2 targeting.

Overexpression of the histone methyltransferase MMSET in t(4;14)+ multiple myeloma patients is believed to be the driving factor in the pathogenesis of this subtype of myeloma. MMSET catalyzes dimethylation of lysine 36 on histone H3 (H3K36me2), and its overexpression causes a global increase in H3K36me2, redistributing this mark in a broad, elevated level across the genome. Here, we demonstrate that an increased level of MMSET also induces a global reduction of lysine 27 trimethylation on histone H3 (H3K27me3). Despite the net decrease in H3K27 methylation, specific genomic loci exhibit enhanced recruitment of the EZH2 histone methyltransferase and become hypermethylated on this residue. These effects likely contribute to the myeloma phenotype since MMSET-overexpressing cells displayed increased sensitivity to EZH2 inhibition. Furthermore, we demonstrate that such MMSET-mediated epigenetic changes require a number of functional domains within the protein, including PHD domains that mediate MMSET recruitment to chromatin. In vivo, targeting of MMSET by an inducible shRNA reversed histone methylation changes and led to regression of established tumors in athymic mice. Together, our work elucidates previously unrecognized interplay between MMSET and EZH2 in myeloma oncogenesis and identifies domains to be considered when designing inhibitors of MMSET function.

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Epigenetic deregulation of gene expression plays a role in the initiation and progression of prostate cancer (PCa). The histone methyltransferase MMSET/WHSC1 (Multiple Myeloma Set Domain) is overexpressed in a number of metastatic tumors, but its mechanism of action has not been defined. In this work, we found that PCa cell lines expressed significantly higher levels of MMSET compared to immortalized, non-transformed prostate cells. Knockdown experiments showed that, in metastatic PCa cell lines, dimethylation of lysine 36 and trimethylation of lysine 27 on histone H3 (H3K36me2 and H3K27me3, respectively) depended on MMSET expression, while depletion of MMSET in benign prostatic cells did not affect chromatin modifications. Knockdown of MMSET in DU145 and PC-3 tumor cells decreased cell proliferation, colony formation in soft agar, and strikingly diminished cell migration and invasion. Conversely, overexpression of MMSET in immortalized, non-transformed RWPE-1 cells promoted cell migration and invasion, accompanied by an epithelial to mesenchymal transition (EMT). Among a panel of EMT-promoting genes analyzed, TWIST1 expression was strongly activated in response to MMSET. Chromatin immunoprecipitation analysis demonstrated that MMSET binds to the TWIST1 locus, leading to an increase in H3K36me2, suggesting a direct role of MMSET in the regulation of this gene. Depletion of TWIST1 in MMSET-overexpressing RWPE-1 cells blocked cell invasion and EMT, indicating that TWIST1 was a critical target of MMSET, responsible for the acquisition of an invasive phenotype. Collectively, these data suggest that MMSET plays a role in PCa pathogenesis and progression through epigenetic regulation of metastasis-related genes.