Despite the potential of targeted epigenetic therapies, most cancers do not respond to current epigenetic drugs. The polycomb repressive complex EZH2 inhibitor tazemetostat was recently approved for the treatment of SMARCB1-deficient epithelioid sarcomas, based on the functional antagonism between PRC2 and SMARCB1. Through the analysis of tumors of patients treated with tazemetostat, we recently defined key principles of their response and resistance to EZH2 epigenetic therapy. Here, using transcriptomic inference from SMARCB1-deficient tumor cells, we nominate the DNA damage repair kinase ATR as a target for rational EZH2 combination epigenetic therapy. We showed that EZH2 inhibition promotes DNA damage in epithelioid and rhabdoid tumor cells, at least in part via its induction of piggyBac transposable element derived 5 (PGBD5). We leveraged this collateral synthetic lethal dependency to target PGBD5-dependent DNA damage by inhibition of ATR, but not CHK1, using the ATR inhibitor elimusertib. Consequently, combined EZH2 and ATR inhibition improved therapeutic responses in diverse patient-derived epithelioid and rhabdoid tumors in vivo. This advances a combination epigenetic therapy based on EZH2-PGBD5 synthetic lethal dependency suitable for immediate translation to clinical trials for patients.

Oligonucleotide Table: Complete list of gRNAs and primers used in this study.

Supplementary Table S1: List of patient tumor specimens used for RNA-seq and MSK-IMPACT analysis. *We note that two primary tumors in patients who responded to TAZ harbored deletions of RB1 (patient 2, sample ES_02_T_02) in one tumor and CDKN2A/B in another tumor (patient 5, sample ES_05_T_01). However, these primary tumors were fully resected prior to the initiation of TAZ treatment and did not recur at the primary sites. In the case of patient 2, a later TAZ-responsive metastasis (ES_2_T_03) did not harbor the RB1 loss. In the case of patient 5, a later TAZ-responsive metastasis (ES_05_T_09) did not harbor the CDKN2A/B loss. This suggests that these mutations were subclonal and were not present in tumors exposed to TAZ treatment. Thus, the mutations in these tumors were unlikely to have impacted their response to TAZ. Supplementary Table S2: List of mutations found in all patient tumor specimens in Supplementary table 1 for which MSK-IMPACT data is available.

Uncropped images of all western blots used in this study.

Supplementary Table S6: List of endogenous transposable elements whose expression is up- or downregulated by TAZ treatment. Related to Supplementary Figure S12.

Supplementary Table S3: List of Hallmark gene sets identified by Gene Set Enrichment Analysis (GSEA) up- and down-regulated by TAZ treatment. Related to Supplementary Figure S2C.

Figure S1: Validation of tumor resistance mutations of EZH2. Figure S2: RB1del cells show morphological and transcriptional responses to TAZ. Figure S3: TAZ-treated RB1del cells show evidence of differentiation at the transcript, but not protein level. Figure S4: RB1del cells show increased expression of E2F targets. Figure S5: Characterization of MRT and ES cell lines. Figure S6: Generating and testing CDKN1Adel and CDKN2Adel G401 cells. Figure S7: TAZ-resistant patient tumors show upregulation of cell cycle genes. Figure S8: Transcriptomic analysis of patient tumors nominates putative biomarkers of TAZ sensitivity and resistance. Figure S9: Downstream cell cycle inhibitors overcome resistance to TAZ. Figure S10: TAZ + barasertib increases cell cycle arrest without inducing apoptosis. Figure S11: p16 induction correlates with TAZ response in vivo. Figure S12: Induction of immune-related genes and endogenous transposable elements by TAZ. Figure S13: TAZ may remodel BAF and PRC2 composition by transcriptional regulation of their subunits.

Essential epigenetic dependencies have become evident in many cancers. Based on the functional antagonism between BAF/SWI/SNF and PRC2 in SMARCB1-deficient sarcomas, we and colleagues recently completed the clinical trial of the EZH2 inhibitor tazemetostat. However, the principles of tumor response to epigenetic therapy in general, and tazemetostat in particular, remain unknown. Using functional genomics of patient tumors and diverse experimental models, we sought to define molecular mechanisms of tazemetostat resistance in SMARCB1-deficient sarcomas and rhabdoid tumors. We found distinct classes of acquired mutations that converge on the RB1/E2F axis and decouple EZH2-dependent differentiation and cell cycle control. This allows tumor cells to escape tazemetostat-induced G1 arrest despite EZH2 inhibition, and suggests a general mechanism for effective EZH2 therapy. This also enables us to develop combination strategies to circumvent tazemetostat resistance using cell cycle bypass targeting via AURKB, and synthetic lethal targeting of PGBD5-dependent DNA damage repair via ATR. This reveals prospective biomarkers for therapy stratification, including PRICKLE1 associated with tazemetostat resistance. In all, this work offers a paradigm for rational epigenetic combination therapy suitable for immediate translation to clinical trials for epithelioid sarcomas, rhabdoid tumors, and other epigenetically dysregulated cancers.

Supplementary Table S4: List of mutations found in all MRT and ES cell lines used in this study as determined by targeted MSK-IMPACT sequencing. Related to Figure 3A. Supplementary Table S5: List of PDX models used in this study, with clinical characteristics of the original tumor specimens, followed by a list of mutations found in all PDX models, as determined by targeted MSK-IMPACT sequencing.

NUT carcinoma (NC) is one of the most common types of undifferentiated carcinomas affecting young adults with a dismal prognosis. NUT carcinomas often involve chromosomal translocations, leading to the production of BRD4-NUT fusion protein that generates large domains of hyperactive chromatin and activates oncogenic gene expression. Bromodomain and extraterminal domain (BET) bromodomain inhibitors offer a direct means to block BRD4-mediated gene activation but have shown limited clinical efficacy in patients. In this issue of Cancer Research, Huang and colleagues report an unexpected discovery of a synthetic lethal NC dependency on Polycomb repressive complex 2 (PRC2)-mediated gene repression, including EZH2, the catalytic subunit of PRC2. EZH2 is highly expressed in NC patient tumors and a specific inhibitor of its methyltransferase activity, tazemetostat, exhibits potent antitumor cell activity. While the repressed and activated chromatin domains in NC cells are distinct, the resultant gene expression changes exhibit convergent features, including dysregulation of CDKN2A and the E2F-RB1 axis. As a result, combined treatment of NC tumors with tazemetostat and the BET inhibitor mivebresib produces marked antitumor therapeutic synergy in vitro and in vivo, associated with enhanced suppression of RB1 function through convergent remodeling of NC gene expression. This study advances epigenetic cooperativity as a distinct mode of gene expression dysregulation in NC and nominates a compelling combination epigenetic strategy for investigation in clinical trials for patients. See related article by Huang et al., p. 3956.

Translocations involving the NUP98 gene produce NUP98-fusion proteins and are associated with a poor prognosis in acute myeloid leukemia (AML). MLL1 is a molecular dependency in NUP98-fusion leukemia, and therefore we investigated the efficacy of therapeutic blockade of the menin-MLL1 interaction in NUP98-fusion leukemia models. Using mouse leukemia cell lines driven by NUP98-HOXA9 and NUP98-JARID1A fusion oncoproteins, we demonstrate that NUP98-fusion-driven leukemia is sensitive to the menin-MLL1 inhibitor VTP50469, with an IC50 similar to what we have previously reported for MLL-rearranged and NPM1c leukemia cells. Menin-MLL1 inhibition upregulates markers of differentiation such as CD11b and downregulates expression of proleukemogenic transcription factors such as Meis1 in NUP98-fusion-transformed leukemia cells. We demonstrate that MLL1 and the NUP98 fusion protein itself are evicted from chromatin at a critical set of genes that are essential for the maintenance of the malignant phenotype. In addition to these in vitro studies, we established patient-derived xenograft (PDX) models of NUP98-fusion-driven AML to test the in vivo efficacy of menin-MLL1 inhibition. Treatment with VTP50469 significantly prolongs survival of mice engrafted with NUP98-NSD1 and NUP98-JARID1A leukemias. Gene expression analysis revealed that menin-MLL1 inhibition simultaneously suppresses a proleukemogenic gene expression program, including downregulation of the HOXa cluster, and upregulates tissue-specific markers of differentiation. These preclinical results suggest that menin-MLL1 inhibition may represent a rational, targeted therapy for patients with NUP98-rearranged leukemias.

The covalent attachment of ubiquitin (Ub) or Ub chains to cellular proteins is a versatile post-translational modification involved in a variety of eukaryotic cellular events. Recently, the post-translational modification of Ub itself by phosphorylation has emerged as an important component of the Ub-signaling system. Specifically, Ub phosphorylation at serine-65 was shown to activate parkin-mediated mitochondrial quality control. However, the impact of phosphorylation on Ub structure and interactions is poorly understood. Here we investigate the recently reported structural changes in Ub upon serine-65 phosphorylation, namely, the equilibrium between a native-like and a novel, alternate conformer of phosphorylated Ub (pUb). We show that this equilibrium is pH-dependent, and the two pUb conformers are linked to the different charge states of the phosphate group. We examined pUb binding to a known Ub-receptor and found that the alternate conformer is binding incompetent. Furthermore, serine-65 phosphorylation affects the conformational equilibrium of K48-linked Ub dimers. Lastly, our crystal structure of S65D Ub and NMR data indicate that phosphomimetic mutations do not adequately reproduce the salient features of pUb. Our results suggest that the pH-dependence of the conformations and binding properties of phosphorylated Ub and polyUb could provide an additional level of modulation in Ub-mediated signaling.

Protein ubiquitination plays a role in essentially every process in eukaryotic cells. The attachment of ubiquitin (Ub) or Ub-like (UBL) proteins to target proteins is achieved by parallel but distinct cascades of enzymatic reactions involving three enzymes: E1, E2, and E3. The E1 enzyme functions at the apex of this pathway and plays a critical role in activating the C-terminus of ubiquitin or UBL, which is an essential step that triggers subsequent downstream transfer to their cognate E2s resulting in the fidelity of the Ub/UBL conjugation machinery. Despite the central role of the E1 enzyme in protein modification, a quantitative method to measure Ub/UBL activation by E1 is lacking. Here, we present a mass spectrometry-based assay to accurately measure the activation of Ub/UBL by E1 independent of the E2/E3 enzymes. Our method does not require radiolabeling of any components and therefore can be used in any biochemical laboratory having access to a mass spectrometer. This method allowed us to dissect the concerted process of E1-E2-catalyzed Ub conjugation in order to separately characterize the process of Ub activation and how it is affected by select mutations and other factors. We found that the hydrophobic patch of Ub is important for the optimal activation of Ub by E1. We further show that the blockers of the Ub-proteasome system such as ubistatin and fullerenol inhibit Ub activation by E1. Interestingly, our data indicate that the phosphorylation of Ub at the S65 position augments its activation by the E1 enzyme.

Malaria and other vector borne diseases claim lives and cause illness, especially in less developed countries. Although well understood methods, such as spraying and insecticidal nets, are identified as effective deterrents to malaria transmission by mosquitoes, the nations that have the greatest burden from the disease also struggle to deploy such measures sufficiently. More targeted and up to date information is needed to identify which regions of malaria-endemic countries are most likely to be at risk of malaria in the near future. This will allow national governments, local officials and public health workers to deploy protective equipment and personnel where they are most needed. This paper explores the role of environmental data generated via satellite remote sensing as an ingredient to a Malaria Early Warning System. Data from remote sensing satellites can cover broad geographical areas frequently and consistently. Much of the relevant data may be accessed by malaria-endemic countries at minimal cost via international data sharing polices. While previous research studies have demonstrated the potential to assign malaria risk to a geographic region based on indicators from satellites and other sources, there is still a need to deploy such tools in a broader and more operational manner to inform decision making on malaria management. This paper describes current research on the use of satellite-based environmental data to predict malaria risk and examines the barriers and opportunities for implementing Malaria Early Warning Systems enabled by satellite remote sensing. A Systems Architecture Framework analyses the components of a Malaria Early Warning System and highlights the need for effective coordination across public and private sector organizations.