CRISPR-SID: Identifying EZH2 as a druggable target for desmoid tumors via in vivo dependency mapping.

Cancer precision medicine implies identification of tumor-specific vulnerabilities associated with defined oncogenic pathways. Desmoid tumors are soft-tissue neoplasms strictly driven by Wnt signaling network hyperactivation. Despite this clearly defined genetic etiology and the strict and unique implication of the Wnt/ß-catenin pathway, no specific molecular targets for these tumors have been identified. To address this caveat, we developed fast, efficient, and penetrant genetic Xenopus tropicalis desmoid tumor models to identify and characterize drug targets. We used multiplexed CRISPR/Cas9 genome editing in these models to simultaneously target a tumor suppressor gene (apc) and candidate dependency genes. Our methodology CRISPR/Cas9 selection-mediated identification of dependencies (CRISPR-SID) uses calculated deviations between experimentally observed gene editing outcomes and deep-learning-predicted double-strand break repair patterns to identify genes under negative selection during tumorigenesis. This revealed EZH2 and SUZ12, both encoding polycomb repressive complex 2 components, and the transcription factor CREB3L1 as genetic dependencies for desmoid tumors. In vivo EZH2 inhibition by Tazemetostat induced partial regression of established autochthonous tumors. In vitro models of patient desmoid tumor cells revealed a direct effect of Tazemetostat on Wnt pathway activity. CRISPR-SID represents a potent approach for in vivo mapping of tumor vulnerabilities and drug target identification.

B2 and ALU retrotransposons are self-cleaving ribozymes whose activity is enhanced by EZH2.

Transposable elements make up half of the mammalian genome. One of the most abundant is the short interspersed nuclear element (SINE). Among their million copies, B2 accounts for ∼350,000 in the mouse genome and has garnered special interest because of emerging roles in epigenetic regulation. Our recent work demonstrated that B2 RNA binds stress genes to retard transcription elongation. Although epigenetically silenced, B2s become massively up-regulated during thermal and other types of stress. Specifically, an interaction between B2 RNA and the Polycomb protein, EZH2, results in cleavage of B2 RNA, release of B2 RNA from chromatin, and activation of thermal stress genes. Although an established RNA-binding protein and histone methyltransferase, EZH2 is not known to be a nuclease. Here, we provide evidence for the surprising conclusion that B2 is a self-cleaving ribozyme. Ribozyme activity depends on Mg+2 and monovalent cations but is resistant to protease treatment. However, contact with EZH2 accelerates cleavage rate by >100-fold, suggesting that EZH2 promotes a cleavage-competent RNA conformation. B2 modification-interference analysis demonstrates that phosphorothioate changes at A and C nucleotides can substitute for EZH2. B2 nucleotides 45 to 55 and 100 to 101 are essential for activity. Finally, another family of SINEs, the human ALU element, also produces a self-cleaving RNA and is cleaved during T-cell activation as well as thermal and endoplasmic reticulum (ER) stress. Thus, B2/ALU SINEs may be classified as "epigenetic ribozymes" that function as transcriptional switches during stress. Given their high copy numbers, B2 and ALU may represent the predominant ribozyme activity in mammalian cells.

EZH2 promotes neoplastic transformation through VAV interaction-dependent extranuclear mechanisms.

Recently, we reported that the histone methyltransferase, EZH2, controls leukocyte migration through interaction with the cytoskeleton remodeling effector, VAV, and direct methylation of the cytoskeletal regulatory protein, Talin. However, it is unclear whether this extranuclear, epigenetic-independent function of EZH2 has a profound impact on the initiation of cellular transformation and metastasis. Here, we show that EZH2 increases Talin1 methylation and cleavage, thereby enhancing adhesion turnover and promoting accelerated tumorigenesis. This transforming capacity is abolished by targeted disruption of EZH2 interaction with VAV. Furthermore, our studies demonstrate that EZH2 in the cytoplasm is closely associated with cancer stem cell properties, and that overexpression of EZH2, a mutant EZH2 lacking its nuclear localization signal (EZH2ΔNLS), or a methyl-mimicking Talin1 mutant substantially promotes JAK2-dependent STAT3 activation and cellular transformation. Taken together, our results suggest a critical role for the VAV interaction-dependent, extranuclear action of EZH2 in neoplastic transformation.

Semi-Quantitative Mass Spectrometry in AML Cells Identifies New Non-Genomic Targets of the EZH2 Methyltransferase.

Alterations to the gene encoding the EZH2 (KMT6A) methyltransferase, including both gain-of-function and loss-of-function, have been linked to a variety of haematological malignancies and solid tumours, suggesting a complex, context-dependent role of this methyltransferase. The successful implementation of molecularly targeted therapies against EZH2 requires a greater understanding of the potential mechanisms by which EZH2 contributes to cancer. One aspect of this effort is the mapping of EZH2 partner proteins and cellular targets. To this end we performed affinity-purification mass spectrometry in the FAB-M2 HL-60 acute myeloid leukaemia (AML) cell line before and after all-trans retinoic acid-induced differentiation. These studies identified new EZH2 interaction partners and potential non-histone substrates for EZH2-mediated methylation. Our results suggest that EZH2 is involved in the regulation of translation through interactions with a number of RNA binding proteins and by methylating key components of protein synthesis such as eEF1A1. Given that deregulated mRNA translation is a frequent feature of cancer and that eEF1A1 is highly expressed in many human tumours, these findings present new possibilities for the therapeutic targeting of EZH2 in AML.