Epigenetic targeting of PGBD5-dependent DNA damage in SMARCB1-deficient sarcomas.

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 loss of SMARCB1. Through the analysis of tazemetostat-treated patient tumors, 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 combination EZH2 epigenetic therapy. We show that EZH2 inhibition promotes DNA damage in epithelioid and rhabdoid tumor cells, at least in part via its induction of the transposase-derived PGBD5. We leverage this collateral synthetic lethal dependency to target PGBD5-dependent DNA damage by inhibition of ATR but not CHK1 using elimusertib. Consequently, combined EZH2 and ATR inhibition improves 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.

Overcoming clinical resistance to EZH2 inhibition using rational epigenetic combination therapy.

Epigenetic dependencies have become evident in many cancers. Based on antagonism between BAF/SWI/SNF and PRC2 in SMARCB1-deficient sarcomas, we 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 and diverse experimental models, we define molecular mechanisms of tazemetostat resistance in SMARCB1-deficient tumors. We found distinct 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, suggests a general mechanism for effective therapy, and provides prospective biomarkers for therapy stratification, including PRICKLE1. Based on this, we develop a combination strategy to circumvent tazemetostat resistance using bypass targeting of AURKB. This offers a paradigm for rational epigenetic combination therapy suitable for translation to clinical trials for epithelioid sarcomas, rhabdoid tumors, and other epigenetically dysregulated cancers.

Overcoming clinical resistance to EZH2 inhibition using rational epigenetic combination therapy.

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.

MDM2, MDMX, and p73 regulate cell-cycle progression in the absence of wild-type p53.

The p53 tumor suppressor protein, known to be critically important in several processes including cell-cycle arrest and apoptosis, is highly regulated by multiple mechanisms, most certifiably the Murine Double Minute 2-Murine Double Minute X (MDM2-MDMX) heterodimer. The role of MDM2-MDMX in cell-cycle regulation through inhibition of p53 has been well established. Here we report that in cells either lacking p53 or expressing certain tumor-derived mutant forms of p53, loss of endogenous MDM2 or MDMX, or inhibition of E3 ligase activity of the heterocomplex, causes cell-cycle arrest. This arrest is correlated with a reduction in E2F1, E2F3, and p73 levels. Remarkably, direct ablation of endogenous p73 produces a similar effect on the cell cycle and the expression of certain E2F family members at both protein and messenger RNA levels. These data suggest that MDM2 and MDMX, working at least in part as a heterocomplex, may play a p53-independent role in maintaining cell-cycle progression by promoting the activity of E2F family members as well as p73, making them a potential target of interest in cancers lacking wild-type p53.

Acquired resistance to PRMT5 inhibition induces concomitant collateral sensitivity to paclitaxel.

Epigenetic regulators play key roles in cancer and are increasingly being targeted for treatment. However, for many, little is known about mechanisms of resistance to the inhibition of these regulators. We have generated a model of resistance to inhibitors of protein arginine methyltransferase 5 (PRMT5). This study was conducted in KrasG12D;Tp53-null lung adenocarcinoma (LUAD) cell lines. Resistance to PRMT5 inhibitors (PRMT5i) arose rapidly, and barcoding experiments showed that this resulted from a drug-induced transcriptional state switch, not selection of a preexisting population. This resistant state is both stable and conserved across variants arising from distinct LUAD lines. Moreover, it brought with it vulnerabilities to other chemotherapeutics, especially the taxane paclitaxel. This paclitaxel sensitivity depended on the presence of stathmin 2 (STMN2), a microtubule regulator that is specifically expressed in the resistant state. Remarkably, STMN2 was also essential for resistance to PRMT5 inhibition. Thus, a single gene is required for both acquisition of resistance to PRMT5i and collateral sensitivity to paclitaxel in our LUAD cells. Accordingly, the combination of PRMT5i and paclitaxel yielded potent and synergistic killing of the murine LUAD cells. Importantly, the synergy between PRMT5i and paclitaxel also extended to human cancer cell lines. Finally, analysis of The Cancer Genome Atlas patient data showed that high STMN2 levels correlate with complete regression of tumors in response to taxane treatment. Collectively, this study reveals a recurring mechanism of PRMT5i resistance in LUAD and identifies collateral sensitivities that have potential clinical relevance.

Deoxycytidine Release from Pancreatic Stellate Cells Promotes Gemcitabine Resistance.

Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer deaths in the United States. The deoxynucleoside analogue gemcitabine is among the most effective therapies to treat PDAC, however, nearly all patients treated with gemcitabine either fail to respond or rapidly develop resistance. One hallmark of PDAC is a striking accumulation of stromal tissue surrounding the tumor, and this accumulation of stroma can contribute to therapy resistance. To better understand how stroma limits response to therapy, we investigated cell-extrinsic mechanisms of resistance to gemcitabine. Conditioned media from pancreatic stellate cells (PSC), as well as from other fibroblasts, protected PDAC cells from gemcitabine toxicity. The protective effect of PSC-conditioned media was mediated by secretion of deoxycytidine, but not other deoxynucleosides, through equilibrative nucleoside transporters. Deoxycytidine inhibited the processing of gemcitabine in PDAC cells, thus reducing the effect of gemcitabine and other nucleoside analogues on cancer cells. These results suggest that reducing deoxycytidine production in PSCs may increase the efficacy of nucleoside analog therapies. SIGNIFICANCE: This study provides important new insight into mechanisms that contribute to gemcitabine resistance in PDAC and suggests new avenues for improving gemcitabine efficacy.