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Tumors frequently harbor isogenic yet epigenetically distinct subpopulations of multi-potent cells with high tumor-initiating potential-often called Cancer Stem-Like Cells (CSLCs). These can display preferential resistance to standard-of-care chemotherapy. Single-cell analyses can help elucidate Master Regulator (MR) proteins responsible for governing the transcriptional state of these cells, thus revealing complementary dependencies that may be leveraged via combination therapy. Interrogation of single-cell RNA sequencing profiles from seven metastatic breast cancer patients, using perturbational profiles of clinically relevant drugs, identified drugs predicted to invert the activity of MR proteins governing the transcriptional state of chemoresistant CSLCs, which were then validated by CROP-seq assays. The top drug, the anthelmintic albendazole, depleted this subpopulation in vivo without noticeable cytotoxicity . Moreover, sequential cycles of albendazole and paclitaxel-a commonly used chemotherapeutic -displayed significant synergy in a patient-derived xenograft (PDX) from a TNBC patient, suggesting that network-based approaches can help develop mechanism-based combinatorial therapies targeting complementary subpopulations., Competing Interests: Conflict of Interest Disclosure A.C. is founder, equity holder, and consultant of DarwinHealth Inc., a company that has licensed some of the algorithms used in this manuscript from Columbia University. Columbia University is also an equity holder in DarwinHealth Inc. US patent number 10,790,040 has been awarded related to this work, and has been assigned to Columbia University with Dr. Califano as an inventor. P.D. received stock and/or royalties from Oncomed Pharmaceuticals, Quanticel Pharmaceuticals and Forty Seven Inc., as a result of his acknowledgment as a co-inventor on patents licensed from the University of Michigan (US-07723112) and Stanford University (US-09329170, US-09850483, US-10344094, US-11130813), and related to: 1) the discovery of surface markers for the differential purification of cancer stem cell populations from human malignancies; 2) the use of single-cell genomics technologies for the identification of pharmacological targets expressed in cancer stem cell populations; 3) the combination of anti-CD47 and anti-EGFR monoclonal antibodies for the treatment of human colon cancer. P.D. recently owned stock of Eli Lilly and Company. P.Ds spouse is employed by Regeneron Pharmaceuticals Inc., and owns (or recently owned) stock of the following pharmaceutical companies: AbbVie, Amgen, AstraZeneca, Eli Lilly and Company, Gilead Sciences Inc., GlaxoSmithKline (GSK), Johnson & Johnson, Merck & Co., Novartis, Organon & Co., Pfizer, Teva Pharmaceutical Industries Ltd. and Viatris. A.L.K. is on the Scientific Advisory Board of Emendo Biotherapeutics, Karyopharm Therapeutics, Imago BioSciences, and DarwinHealth; is co-Founder and on the Scientific Advisory Board of Isabl; has equity interest in Imago BioSciences, Emendo Biotherapeutics and Isabl; and receives royalty income from Labcorp.

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., Competing Interests: Conflict of Interest The authors declare no potential conflicts of interest. AK is a consultant for Novartis, Rgenta, Blueprint, and Syndax.