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Background: Genetic perturbation screens with single-cell readouts have enabled rich phenotyping of gene function and regulatory networks. These approaches have been challenging in vivo, especially in adult disease models such as cancer, which include mixtures of malignant and microenvironment cells. Glioblastoma (GBM) is a fatal cancer, and methods of systematically interrogating gene function and therapeutic targets in vivo, especially in combination with standard of care treatment such as radiotherapy, are lacking., Results: Here, we iteratively develop a multiplex in vivo perturb-seq CRISPRi platform for single-cell genetic screens in cancer and tumor microenvironment cells that leverages intracranial convection enhanced delivery of sgRNA libraries into mouse models of GBM. Our platform enables potent silencing of drivers of in vivo growth and tumor maintenance as well as genes that sensitize GBM to radiotherapy. We find radiotherapy rewires transcriptional responses to genetic perturbations in an in vivo-dependent manner, revealing heterogenous patterns of treatment sensitization or resistance in GBM. Furthermore, we demonstrate targeting of genes that function in the tumor microenvironment, enabling alterations of ligand-receptor interactions between immune and stromal cells following in vivo CRISPRi perturbations that can affect tumor cell phagocytosis., Conclusion: In sum, we demonstrate the utility of multiplexed perturb-seq for in vivo single-cell dissection of adult cancer and normal tissue biology across multiple cell types in the context of therapeutic intervention, a platform with potential for broad application., (© 2024. The Author(s).)

Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase and emerging therapeutic target that is overexpressed in most castration-resistant prostate cancers and implicated as a driver of disease progression and resistance to hormonal therapies. Here we define the lineage-specific action and differential activity of EZH2 in both prostate adenocarcinoma and neuroendocrine prostate cancer (NEPC) subtypes of advanced prostate cancer to better understand the role of EZH2 in modulating differentiation, lineage plasticity, and to identify mediators of response and resistance to EZH2 inhibitor therapy. Mechanistically, EZH2 modulates bivalent genes that results in upregulation of NEPC-associated transcriptional drivers (e.g., ASCL1) and neuronal gene programs in NEPC, and leads to forward differentiation after targeting EZH2 in NEPC. Subtype-specific downstream effects of EZH2 inhibition on cell cycle genes support the potential rationale for co-targeting cyclin/CDK to overcome resistance to EZH2 inhibition., (© 2024. The Author(s).)

Background: Hundreds of functional genomic screens have been performed across a diverse set of cancer contexts, as part of efforts such as the Cancer Dependency Map, to identify gene dependencies-genes whose loss of function reduces cell viability or fitness. Recently, large-scale screening efforts have shifted from RNAi to CRISPR-Cas9, due to superior efficacy and specificity. However, many effective oncology drugs only partially inhibit their protein targets, leading us to question whether partial suppression of genes using RNAi could reveal cancer vulnerabilities that are missed by complete knockout using CRISPR-Cas9. Here, we compare CRISPR-Cas9 and RNAi dependency profiles of genes across approximately 400 matched cancer cell lines., Results: We find that CRISPR screens accurately identify more gene dependencies per cell line, but the majority of each cell line's dependencies are part of a set of 1867 genes that are shared dependencies across the entire collection (pan-lethals). While RNAi knockdown of about 30% of these genes is also pan-lethal, approximately 50% have selective dependency patterns across cell lines, suggesting they could still be cancer vulnerabilities. The accuracy of the unique RNAi selectivity is supported by associations to multi-omics profiles, drug sensitivity, and other expected co-dependencies., Conclusions: Incorporating RNAi data for genes that are pan-lethal knockouts facilitates the discovery of a wider range of gene targets than could be detected using the CRISPR dataset alone. This can aid in the interpretation of contrasting results obtained from CRISPR and RNAi screens and reinforce the importance of partial gene suppression methods in building a cancer dependency map., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Systematic identification of signaling pathways required for the fitness of cancer cells will facilitate the development of new cancer therapies. We used gene essentiality measurements in 1,086 cancer cell lines to identify selective coessentiality modules and found that a ubiquitin ligase complex composed of UBA6, BIRC6, KCMF1, and UBR4 is required for the survival of a subset of epithelial tumors that exhibit a high degree of aneuploidy. Suppressing BIRC6 in cell lines that are dependent on this complex led to a substantial reduction in cell fitness in vitro and potent tumor regression in vivo. Mechanistically, BIRC6 suppression resulted in selective activation of the integrated stress response (ISR) by stabilization of the heme-regulated inhibitor, a direct ubiquitination target of the UBA6/BIRC6/KCMF1/UBR4 complex. These observations uncover a novel ubiquitination cascade that regulates ISR and highlight the potential of ISR activation as a new therapeutic strategy., Significance: We describe the identification of a heretofore unrecognized ubiquitin ligase complex that prevents the aberrant activation of the ISR in a subset of cancer cells. This provides a novel insight on the regulation of ISR and exposes a therapeutic opportunity to selectively eliminate these cancer cells. See related commentary Leli and Koumenis, p. 535. This article is highlighted in the In This Issue feature, p. 517., (©2022 The Authors; Published by the American Association for Cancer Research.)

In high-throughput functional genomic screens, each gene product is commonly assumed to exhibit a singular biological function within a defined protein complex or pathway. In practice, a single gene perturbation may induce multiple cascading functional outcomes, a genetic principle known as pleiotropy. Here, we model pleiotropy in fitness screen collections by representing each gene perturbation as the sum of multiple perturbations of biological functions, each harboring independent fitness effects inferred empirically from the data. Our approach (Webster) recovered pleiotropic functions for DNA damage proteins from genotoxic fitness screens, untangled distinct signaling pathways upstream of shared effector proteins from cancer cell fitness screens, and predicted the stoichiometry of an unknown protein complex subunit from fitness data alone. Modeling compound sensitivity profiles in terms of genetic functions recovered compound mechanisms of action. Our approach establishes a sparse approximation mechanism for unraveling complex genetic architectures underlying high-dimensional gene perturbation readouts., Competing Interests: Declaration of interests F.V. receives research support from Novo Ventures. A.T. is a consultant for Cedilla Therapeutics, Foghorn Therapeutics, and The Center for Protein Degradation (Deerfield) and is an SAB member and holds equity in Turbine Simulated Cell Technologies. W.C.H. is a consultant for Thermo Fisher, Solasta Ventures, MPM Capital, KSQ Therapeutics, iTeos, Tyra Biosciences, Jubilant Therapeutics, RAPPTA Therapeutics, Frontier Medicine, Calyx, and Function Oncology., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Prognostically relevant RNA expression states exist in pancreatic ductal adenocarcinoma (PDAC), but our understanding of their drivers, stability, and relationship to therapeutic response is limited. To examine these attributes systematically, we profiled metastatic biopsies and matched organoid models at single-cell resolution. In vivo, we identify a new intermediate PDAC transcriptional cell state and uncover distinct site- and state-specific tumor microenvironments (TMEs). Benchmarking models against this reference map, we reveal strong culture-specific biases in cancer cell transcriptional state representation driven by altered TME signals. We restore expression state heterogeneity by adding back in vivo-relevant factors and show plasticity in culture models. Further, we prove that non-genetic modulation of cell state can strongly influence drug responses, uncovering state-specific vulnerabilities. This work provides a broadly applicable framework for aligning cell states across in vivo and ex vivo settings, identifying drivers of transcriptional plasticity and manipulating cell state to target associated vulnerabilities., Competing Interests: Declaration of interests B.M.W. reports research support from Celgene and Eli Lilly and consulting for BioLineRx, Celgene, G1 Therapeutics, and GRAIL. W.C.H. is a consultant for Thermo Fisher, Solasta Ventures, MPM Capital, Tyra Biosciences, iTeos, Frontier Medicines, Function Oncology, KSQ Therapeutics, Jubilant Therapeutics, RAPPTA Therapeutics, and Paraxel. A.K.S. reports compensation for consulting and/or SAB membership from Merck, Honeycomb Biotechnologies, Cellarity, Repertoire Immune Medicines, Ochre Bio, Third Rock Ventures, Hovione, Relation Therapeutics, FL82, and Dahlia Biosciences. A.J.A. has consulted for Oncorus, Arrakis Therapeutics, and Merck & Co. and has research funding from Mirati Therapeutics, Syros, Deerfield, and Novo Ventures that are unrelated to this project. S.R.M. is a founder of Travera. S.R. has equity in Amgen. J.M.C. has received research funding from Merck, Tesaro, AstraZeneca, Bayer, and Esperas Pharma; has served as a consultant to Bristol Myers Squibb; and has received travel funding from Bristol Myers Squibb. A.R. is an employee of AstraZeneca and an equity holder in Celsius Therapeutics and NucleAI. Y.Y.L. reports equity from g.Root Biomedical Services. A.D. Cherniack reports research support from Bayer. R.J.S reports research support from Merck and consulting and/or SAB membership for Bristol Myers Squibb, Merck, Novartis, and Pfizer. G.I.S. reports sponsored research support from Eli Lilly, Merck KGaA/EMD-Serono, Merck, and Sierra Oncology and has served on advisory boards for Pfizer, Eli Lilly, G1 Therapeutics, Roche, Merck KGaA/EMD-Serono, Sierra Oncology, Bicycle Therapeutics, Fusion Pharmaceuticals, Cybrexa Therapeutics, Astex, Almac, Ipsen, Bayer, Angiex, Daiichi Sankyo, Seattle Genetics, Boehringer Ingelheim, ImmunoMet, Asana, Artios, Atrin, Concarlo Holdings, Syros, Zentalis, CytomX Therapeutics, Blueprint Medicines, and Kymera Therapeutics. T.J. is a member of the Board of Directors of Amgen and Thermo Fisher Scientific; a co-Founder of Dragonfly Therapeutics and T2 Biosystems; serves on the Scientific Advisory Board of Dragonfly Therapeutics, SQZ Biotech, and Skyhawk Therapeutics; and is the President of Break Through Cancer. None of these affiliations represent a conflict of interest with respect to the design or execution of this study or interpretation of data presented in this manuscript. T.J.’s laboratory currently also receives funding from the Johnson & Johnson Lung Cancer Initiative and The Lustgarten Foundation for Pancreatic Cancer Research, but this funding did not support the research described in this manuscript. K.N. reports research support from Revolution Medicines, Evergrande Group, Pharmavite, and Janssen; is a member of SABs for SeaGen, BiomX, and Bicara Therapeutics; and has consulted for X-Biotix Therapeutics and Redesign Health. A.K.S., P.S.W., A.W.N., S.R., W.C.H., A.J.A., B.W.M., and J.G.R. have filed a patent (Publication number WO/2021/081491; International application number PCT/US2020/057342) related to this work., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

The RecQ DNA helicase WRN is a synthetic lethal target for cancer cells with microsatellite instability (MSI), a form of genetic hypermutability that arises from impaired mismatch repair 1-4 . Depletion of WRN induces widespread DNA double-strand breaks in MSI cells, leading to cell cycle arrest and/or apoptosis. However, the mechanism by which WRN protects MSI-associated cancers from double-strand breaks remains unclear. Here we show that TA-dinucleotide repeats are highly unstable in MSI cells and undergo large-scale expansions, distinct from previously described insertion or deletion mutations of a few nucleotides 5 . Expanded TA repeats form non-B DNA secondary structures that stall replication forks, activate the ATR checkpoint kinase, and require unwinding by the WRN helicase. In the absence of WRN, the expanded TA-dinucleotide repeats are susceptible to cleavage by the MUS81 nuclease, leading to massive chromosome shattering. These findings identify a distinct biomarker that underlies the synthetic lethal dependence on WRN, and support the development of therapeutic agents that target WRN for MSI-associated cancers.