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Menin interacts with oncogenic MLL1-fusion proteins, and small molecules that disrupt these associations are in clinical trials for leukemia treatment. By integrating chromatin-focused and genome-wide CRISPR screens with genetic, pharmacologic, and biochemical approaches, we discovered a conserved molecular switch between the MLL1–Menin and MLL3/4–UTX chromatin-modifying complexes that dictates response to Menin–MLL inhibitors. MLL1–Menin safeguards leukemia survival by impeding the binding of the MLL3/4–UTX complex at a subset of target gene promoters. Disrupting the Menin–MLL1 interaction triggers UTX-dependent transcriptional activation of a tumor-suppressive program that dictates therapeutic responses in murine and human leukemia. Therapeutic reactivation of this program using CDK4/6 inhibitors mitigates treatment resistance in leukemia cells that are insensitive to Menin inhibitors. These findings shed light on novel functions of evolutionarily conserved epigenetic mediators like MLL1–Menin and MLL3/4–UTX and are relevant to understand and target molecular pathways determining therapeutic responses in ongoing clinical trials. Significance: Menin–MLL inhibitors silence a canonical HOX- and MEIS1-dependent oncogenic gene expression program in leukemia. We discovered a parallel, noncanonical transcriptional program involving tumor suppressor genes that are repressed in Menin–MLL inhibitor–resistant leukemia cells but that can be reactivated upon combinatorial treatment with CDK4/6 inhibitors to augment therapy responses. This article is highlighted in the In This Issue feature, p. 1

Interplay between metabolism and chromatin signaling have been implicated in cancer initiation and progression. However, whether and how metabolic reprogramming in tumors generates specific epigenetic vulnerabilities remain unclear. Lung adenocarcinoma (LUAD) tumors frequently harbor mutations that cause aberrant activation of the NRF2 antioxidant pathway and drive aggressive and chemo-resistant disease. We performed a chromatin-focused CRISPR screen and report that NRF2 activation sensitized LUAD cells to genetic and chemical inhibition of class I histone deacetylases (HDAC). This association was consistently observed across cultured cells, syngeneic mouse models and patient-derived xenografts. HDAC inhibition causes widespread increases in histone H4 acetylation (H4ac) at intergenic regions, but also drives re-targeting of H4ac reader protein BRD4 away from promoters with high H4ac levels and transcriptional downregulation of corresponding genes. Integrative epigenomic, transcriptomic and metabolomic analysis demonstrates that these chromatin changes are associated with reduced flux into amino acid metabolism andde novonucleotide synthesis pathways that are preferentially required for the survival of NRF2-active cancer cells. Together, our findings suggest that metabolic alterations such as NRF2 activation could serve as biomarkers for effective repurposing of HDAC inhibitors to treat solid tumors.

Supplementary Figures related to Figures 1-6

ChIP-Seq results

Processed data for bulk RNA-seq of primary tumors isolated from KP, KPS-HET and KPS animals

Processed data for bulk ATAC-seq of isogenic SMARCA4-WT and -KO cell lines

Mutations and copy number alterations in PDX models used in this study

Sequences of sgRNAs and primers

Menin interacts with oncogenic MLL1-fusion proteins, and small molecules that disrupt these associations are in clinical trials for leukemia treatment. By integrating chromatin-focused and genome-wide CRISPR screens with genetic, pharmacologic, and biochemical approaches, we discovered a conserved molecular switch between the MLL1–Menin and MLL3/4–UTX chromatin-modifying complexes that dictates response to Menin–MLL inhibitors. MLL1–Menin safeguards leukemia survival by impeding the binding of the MLL3/4–UTX complex at a subset of target gene promoters. Disrupting the Menin–MLL1 interaction triggers UTX-dependent transcriptional activation of a tumor-suppressive program that dictates therapeutic responses in murine and human leukemia. Therapeutic reactivation of this program using CDK4/6 inhibitors mitigates treatment resistance in leukemia cells that are insensitive to Menin inhibitors. These findings shed light on novel functions of evolutionarily conserved epigenetic mediators like MLL1–Menin and MLL3/4–UTX and are relevant to understand and target molecular pathways determining therapeutic responses in ongoing clinical trials.Significance:Menin–MLL inhibitors silence a canonical HOX- and MEIS1-dependent oncogenic gene expression program in leukemia. We discovered a parallel, noncanonical transcriptional program involving tumor suppressor genes that are repressed in Menin–MLL inhibitor–resistant leukemia cells but that can be reactivated upon combinatorial treatment with CDK4/6 inhibitors to augment therapy responses.This article is highlighted in the In This Issue feature, p. 1

RNA-Seq results

CRISPR screen results

Processed data for bulk RNA-seq of isogenic SMARCA4-WT and -KO cell lines

SMARCA4/BRG1 encodes for one of two mutually exclusive ATPases present in mammalian SWI/SNF chromatin remodeling complexes and is frequently mutated in human lung adenocarcinoma. However, the functional consequences of SMARCA4 mutation on tumor initiation, progression, and chromatin regulation in lung cancer remain poorly understood. Here, we demonstrate that loss of Smarca4 sensitizes club cell secretory protein–positive cells within the lung in a cell type–dependent fashion to malignant transformation and tumor progression, resulting in highly advanced dedifferentiated tumors and increased metastatic incidence. Consistent with these phenotypes, Smarca4-deficient primary tumors lack lung lineage transcription factor activities and resemble a metastatic cell state. Mechanistically, we show that Smarca4 loss impairs the function of all three classes of SWI/SNF complexes, resulting in decreased chromatin accessibility at lung lineage motifs and ultimately accelerating tumor progression. Thus, we propose that the SWI/SNF complex via Smarca4 acts as a gatekeeper for lineage-specific cellular transformation and metastasis during lung cancer evolution.Significance:We demonstrate cell-type specificity in the tumor-suppressive functions of SMARCA4 in the lung, pointing toward a critical role of the cell-of-origin in driving SWI/SNF-mutant lung adenocarcinoma. We further show the direct effects of SMARCA4 loss on SWI/SNF function and chromatin regulation that cause aggressive malignancy during lung cancer evolution.This article is highlighted in the In This Issue feature, p. 275

Processed data for CUT&RUN of isogenic SMARCA4-WT and -KO cell lines

Supplementary Figures and Legends

Supplementary Figures S1-S8, with legends

To study genetic factors influencing the progression and therapeutic responses of advanced prostate cancer, we developed a fast and flexible system that introduces genetic alterations relevant to human disease directly into the prostate glands of mice using tissue electroporation. These electroporation-based genetically engineered mouse models (EPO-GEMM) recapitulate features of traditional germline models and, by modeling genetic factors linked to late-stage human disease, can produce tumors that are metastatic and castration-resistant. A subset of tumors with Trp53 alterations acquired spontaneous WNT pathway alterations, which are also associated with metastatic prostate cancer in humans. Using the EPO-GEMM approach and an orthogonal organoid-based model, we show that WNT pathway activation drives metastatic disease that is sensitive to pharmacologic WNT pathway inhibition. Thus, by leveraging EPO-GEMMs, we reveal a functional role for WNT signaling in driving prostate cancer metastasis and validate the WNT pathway as therapeutic target in metastatic prostate cancer.Significance:Our understanding of the factors driving metastatic prostate cancer is limited by the paucity of models of late-stage disease. Here, we develop EPO-GEMMs of prostate cancer and use them to identify and validate the WNT pathway as an actionable driver of aggressive metastatic disease.This article is highlighted in the In This Issue feature, p. 890

Supplementary Table 1

Figure S1. Expression of 14q32 miRNAs in tumor samples from lung adenocarcinoma patients according to EGFR, KRAS and TP53 mutation status. Figure S2. Methylation analysis of imprinting control regions of the 14q32 cluster in lung adenocarcinoma patients. Figure S3. Association of disease-free survival and N stage in never-smoker lung adenocarcinoma patients. Figure S4. Cell viability of lung adenocarcinoma cells after increasing 14q32 miRNA levels by CRISPR activation technology. Figure S5. Differential expression of genes adjacent to the 14q32 miRNA cluster activated by CRISPR activation technology. Figure S6. Association of genes modulated by 14q32 miRNAs with disease-free survival in lung adenocarcinoma patients. Figure S7. Cell viability of lung adenocarcinoma cells after increasing levels of 14q32 miRNAs by transfection of specific miRNA mimics. Figure S8. Validation of the migratory and invasive effects of miR-323b, miR-487a, and miR-539 on lung adenocarcinoma cells with different genetic background.

Mutations in the TP53 tumor suppressor gene are common in many cancer types, including the acute myeloid leukemia (AML) subtype known as complex karyotype AML (CK-AML). Here, we identify a gain-of-function (GOF) Trp53 mutation that accelerates CK-AML initiation beyond p53 loss and, surprisingly, is required for disease maintenance. The Trp53R172H mutation (TP53R175H in humans) exhibits a neomorphic function by promoting aberrant self-renewal in leukemic cells, a phenotype that is present in hematopoietic stem and progenitor cells (HSPC) even prior to their transformation. We identify FOXH1 as a critical mediator of mutant p53 function that binds to and regulates stem cell–associated genes and transcriptional programs. Our results identify a context where mutant p53 acts as a bona fide oncogene that contributes to the pathogenesis of CK-AML and suggests a common biological theme for TP53 GOF in cancer.Significance:Our study demonstrates how a GOF p53 mutant can hijack an embryonic transcription factor to promote aberrant self-renewal. In this context, mutant Trp53 functions as an oncogene to both initiate and sustain myeloid leukemia and suggests a potential convergent activity of mutant Trp53 across cancer types.This article is highlighted in the In This Issue feature, p. 813

Supplementary Data - Tables

Supplementary Data - Materials and Methods

Suppl. Figure Legends

Prime editing leverages Cas proteins fused to reverse transcription enzymes and prime editing guide RNAs (pegRNAs) to programmably engineer diverse genetic alterations without DNA double strand breaks or exogenous donor templates. Prime editing is powerful, but the process of pegRNA design remains a challenge that limits its widespread adoption by the community. Although a number of computational pegRNA design tools have been developed, their capability for rapid and systematic design of pegRNAs for variant engineering and high-throughput genetic screens is limited. Here, we describePrimeEditingGuideGenerator (PEGG): a computational pipeline for rapid design of pegRNAs and paired ‘sensor’ pegRNA libraries. PEGG is a user-friendly Python package that generates and visualizes pegRNA designs for installing desired mutations while integrating user-defined properties like variable length of primer binding site and reverse transcription template regions, and outputs ready-to-order pegRNA oligos and libraries containing optimized sequence adapters for pooled cloning. PEGG (including documentation) can be accessed athttps://pegg.readthedocs.io.

Single nucleotide variants (SNVs) comprise the majority of cancer-associated genetic changes and can have diverse effects on protein function. Despite a comprehensive catalogue of SNVs across human cancers, little is known about their impact on tumor initiation and progression. To enable the functional interrogation of cancer-associated SNVs, we developed a murine system for temporal and regulatable in vivo cytosine base editing (iBE). The iBE mice show robust, doxycycline-dependent expression across a broad range of tissues with no evidence of DNA or RNA off-target effects. Transient iBE induction drives efficient creation of individual or multiple SNVs in intestinal, lung, and pancreatic organoids, while temporal iBE regulation allows controlled sequential genome editing. Moreover, in situ delivery of plasmid-based or synthetic sgRNAs to target tissues facilitates the simple and rapid generation of pre-clinical cancer models. Overall, iBE is a powerful in vivo platform to define and interrogate the genetic drivers of cancer.

Background and aims Hepatocellular carcinoma (HCC) is among the most common cancer types worldwide, yet patients with HCC have limited treatment options. There is an urgent need to identify drug targets that specifically inhibit the growth of HCC cells. Approach and results We used a CRISPR library targeting ~2,000 druggable genes to perform a high-throughput screen and identified adenylosuccinate lyase (ADSL), a key enzyme involved in the de novo purine synthesis pathway, as a potential drug target for HCC. ADSL has been implicated as a potential oncogenic driver in some cancers, but its role in liver cancer progression remains unknown. CRISPR-mediated knockout of ADSL impaired colony formation of liver cancer cells by affecting AMP production. In the absence of ADSL, the growth of liver tumors is retarded in vivo. Mechanistically, we found that ADSL knockout caused S-phase cell cycle arrest not by inducing DNA damage but by impairing mitochondrial function. Using data from patients with HCC, we also revealed that high ADSL expression occurs during tumorigenesis and is linked to poor survival rate. Conclusions Our findings uncover the role of ADSL-mediated de novo purine synthesis in fueling mitochondrial ATP production to promote liver cancer cell growth. Targeting ADSL may be a therapeutic approach for patients with HCC.

The SS18-SSX fusion drives oncogenic transformation in synovial sarcoma by bridging SS18, a member of mSWI/SNF complex, to Polycomb repressive complex 1 (PRC1) target genes. Here we show that the SSX C-terminus, via its SSXRD domain, directs SS18-SSX chromatin binding independently of SS18. SSXRD specific targeting is mediated by interaction with mono ubiquitinated H2A (H2AK119ub1) and histone MacroH2A with which the fusion overlaps genome wide. Variant Polycomb Repressive Complex 1.1 (PRC1.1) acts as the main depositor of H2AK119ub1 and is therefore required for SS18-SSX occupancy. Importantly, the SSX C-terminus not only depends on H2AK119ub1 for localization but also further increases it by promoting PRC1.1 complex stability. Consequently, high H2AK119ub1 levels are a feature of murine and human synovial sarcomas. These results reveal an SSX/PRC1 autoregulatory feedback loop that reinforces fusion chromatin binding and therefore its oncogenic activity, and could play a role in a wider range of cancers and physiological settings where SSX proteins are overexpressed.

Metastatic gastric carcinoma is a highly lethal cancer that responds poorly to conventional and molecularly targeted therapies. Despite its clinical relevance, the mechanisms underlying the behavior and therapeutic response of this disease are poorly understood owing, in part, to a paucity of tractable models that faithfully recapitulate different subtypes of the human disease. To close this gap, we developed methods to somatically introduce different oncogenic lesions directly into the stomach epithelium and show that genotypic configurations observed in patients produce metastatic gastric cancers that recapitulate the histological, molecular, and clinical features of all non-viral molecular subtypes of the human disease. Applying this platform to both wild-type and immune-deficient mice revealed previously unappreciated links between the genotype, organotropism and immune surveillance of metastatic cells that produced distinct patterns of metastasis that were mirrored in patients. Our results establish and credential a highly portable platform for producing autochthonous cancer models with flexible genotypes and host backgrounds, which can unravel mechanisms of gastric tumorigenesis or test new therapeutic concepts aimed at improving outcomes in gastric cancer patients.

We present GuideScan2 for memory-efficient, parallelizable construction of high-specificity CRISPR guide RNA (gRNA) databases and user-friendly gRNA/library design in custom genomes. GuideScan2 analysis identified widespread confounding effects of low-specificity gRNAs in published CRISPR knockout, interference and activation screens and enabled construction of a ready-to-use gRNA library that reduced off-target effects in a novel gene essentiality screen. GuideScan2 also enabled the design and experimental validation of allele-specific gRNAs in a hybrid mouse genome.

Anticancer drug development campaigns often fail due to an incomplete understanding of the therapeutic index differentiating the efficacy of the agent against the cancer and its on-target toxicities to the host. To address this issue, we established a versatile preclinical platform in which genetically defined cancers are produced using somatic tissue engineering in transgenic mice harboring a doxycycline-inducible short hairpin RNA against the target of interest. In this system, target inhibition is achieved by the addition of doxycycline, enabling simultaneous assessment of efficacy and toxicity in the same animal. As proof of concept, we focused on CDK9—a cancer target whose clinical development has been hampered by compounds with poorly understood target specificity and unacceptable toxicities. We systematically compared phenotypes produced by genetic Cdk9 inhibition to those achieved using a recently developed highly specific small molecule CDK9 inhibitor and found that both perturbations led to robust antitumor responses. Remarkably, nontoxic levels of CDK9 inhibition could achieve significant treatment efficacy, and dose-dependent toxicities produced by prolonged CDK9 suppression were largely reversible upon Cdk9 restoration or drug withdrawal. Overall, these results establish a versatile in vivo target validation platform that can be employed for rapid triaging of therapeutic targets and lend support to efforts aimed at advancing CDK9 inhibitors for cancer therapy.

Inactivation of SMARCA4/BRG1, the core ATPase subunit of mammalian SWI/SNF complexes, occurs at very high frequencies in non–small cell lung cancers (NSCLC). There are no targeted therapies for this subset of lung cancers, nor is it known how mutations in BRG1 contribute to lung cancer progression. Using a combination of gain- and loss-of-function approaches, we demonstrate that deletion of BRG1 in lung cancer leads to activation of replication stress responses. Single-molecule assessment of replication fork dynamics in BRG1-deficient cells revealed increased origin firing mediated by the prelicensing protein, CDC6. Quantitative mass spectrometry and coimmunoprecipitation assays showed that BRG1-containing SWI/SNF complexes interact with RPA complexes. Finally, BRG1-deficient lung cancers were sensitive to pharmacologic inhibition of ATR. These findings provide novel mechanistic insight into BRG1-mutant lung cancers and suggest that their dependency on ATR can be leveraged therapeutically and potentially expanded to BRG1-mutant cancers in other tissues. Significance: These findings indicate that inhibition of ATR is a promising therapy for the 10% of non-small cell lung cancer patients harboring mutations in SMARCA4/BRG1.

Approximately 20-30% of human lung adenocarcinomas (LUAD) harbor loss-of-function (LOF) mutations in Kelch-like ECH Associated-Protein 1 (KEAP1), which lead to hyperactivation of the nuclear factor, erythroid 2-like 2 (NRF2) antioxidant pathway and correlate with poor prognosis(1-3). We previously showed that Keap1 mutation accelerates KRAS-driven LUAD and produces a marked dependency on glutaminolysis(4). To extend the investigation of genetic dependencies in the context of Keap1 mutation, we performed a druggable genome CRISPR-Cas9 screen in Keap1-mutant cells. This analysis uncovered a profound Keap1 mutant-specific dependency on solute carrier family 33 member 1 (Slc33a1), an endomembrane-associated protein with roles in autophagy regulation(5), as well as a series of functionally-related genes implicated in the unfolded protein response. Targeted genetic and biochemical experiments using mouse and human Keap1-mutant tumor lines, as well as preclinical genetically-engineered mouse models (GEMMs) of LUAD, validate Slc33a1 as a robust Keap1-mutant-specific dependency. Furthermore, unbiased genome-wide CRISPR screening identified additional genes related to Slc33a1 dependency. Overall, our study provides a strong rationale for stratification of patients harboring KEAP1-mutant or NRF2-hyperactivated tumors as likely responders to targeted SLC33A1 inhibition and underscores the value of integrating functional genetic approaches with GEMMs to identify and validate genotype-specific therapeutic targets.

Epithelial plasticity, reversible modulation of a cell's epithelial and mesenchymal features, is associated with tumor metastasis and chemoresistance, leading causes of cancer mortality. Although different master transcription factors and epigenetic modifiers have been implicated in this process in various contexts, the extent to which a unifying, generalized mechanism of transcriptional regulation underlies epithelial plasticity remains largely unknown. Here, through targeted CRISPR/Cas9 screening, we discovered two histone-modifying enzymes involved in the writing and erasing of H3K36me2 that act reciprocally to regulate epithelial-to-mesenchymal identity, tumor differentiation, and metastasis. Using a lysine-to-methionine histone mutant to directly inhibit H3K36me2, we found that global modulation of the mark is a conserved mechanism underlying the mesenchymal state in various contexts. Mechanistically, regulation of H3K36me2 reprograms enhancers associated with master regulators of epithelial-to-mesenchymal state. Our results thus outline a unifying epigenome-scale mechanism by which a specific histone modification regulates cellular plasticity and metastasis in cancer. Significance: Although epithelial plasticity contributes to cancer metastasis and chemoresistance, no strategies exist for pharmacologically inhibiting the process. Here, we show that global regulation of a specific histone mark, H3K36me2, is a universal epigenome-wide mechanism that underlies epithelial-to-mesenchymal transition and mesenchymal-to-epithelial transition in carcinoma cells. These results offer a new strategy for targeting epithelial plasticity in cancer. This article is highlighted in the In This Issue feature, p. 747

Cancers develop as a result of driver mutations1,2 that lead to clonal outgrowth and the evolution of disease3,4. The discovery and functional characterization of individual driver mutations are central aims of cancer research, and have elucidated myriad phenotypes5 and therapeutic vulnerabilities6. However, the serial genetic evolution of mutant cancer genes7,8 and the allelic context in which they arise is poorly understood in both common and rare cancer genes and tumour types. Here we find that nearly one in four human tumours contains a composite mutation of a cancer-associated gene, defined as two or more nonsynonymous somatic mutations in the same gene and tumour. Composite mutations are enriched in specific genes, have an elevated rate of use of less-common hotspot mutations acquired in a chronology driven in part by oncogenic fitness, and arise in an allelic configuration that reflects context-specific selective pressures. cis-acting composite mutations are hypermorphic in some genes in which dosage effects predominate (such as TERT), whereas they lead to selection of function in other genes (such as TP53). Collectively, composite mutations are driver alterations that arise from context- and allele-specific selective pressures that are dependent in part on gene and mutation function, and which lead to complex-often neomorphic-functions of biological and therapeutic importance.

The chromatin adaptor Menin interacts with oncogenic fusion proteins encoded byMLL1-rearrangements (MLL1-r), and small molecules that disrupt these associations are currently in clinical trials for the treatment of leukemia. By integrating chromatin-focused and genome-wide CRISPR screens with genetic, pharmacological, and biochemical approaches in mouse and human systems, we discovered a molecular switch between the MLL1-Menin and MLL3/4-UTX chromatin modifying complexes that dictates response to Menin-MLL inhibitors. We show that MLL1-Menin safeguards leukemia survival by impeding binding of the MLL3/4-UTX complex at a subset of target gene promoters. Disrupting the interaction between Menin and MLL1 leads to UTX-dependent transcriptional activation of a tumor suppressor gene-program that is crucial for a therapeutic response in murine and human leukemia. We establish the therapeutic relevance of this mechanism by showing that CDK4/6 inhibitors allow re-activation of this tumor-suppressor program in Menin-inhibitor insensitive leukemia cells, mitigating treatment resistance. The discovery of a molecular switch between MLL1-Menin and MLL3/4-UTX complexes on chromatin sheds light on novel functions of these evolutionary conserved epigenetic mediators and is particularly relevant to understand and target molecular pathways determining response and resistance in ongoing phase 1/2 clinical trials.

A tool to study SARS-CoV-2 Work with infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires high-level biocontainment facilities, making it important to develop safer molecular tools that can potentially be used under less stringent conditions. Self-replicating RNAs known as replicons have long been used to study pathogenic RNA viruses; however, developing replicons to study SARS-SoV-2 has been challenging because of its large genome. Ricardo-Lax et al . used a yeast-based system to construct SARS-CoV-2 replicons that cannot assemble infectious virus because they lack the spike protein required for host cell entry. Transfecting cells with a spike-expressing plasmid and separately with the replicon generates replicon delivery particles (RDPs) that are only capable of one cycle of infection. The replicons and the RDPs can be used in different contexts for drug screening, and viral assays. —VV

SMARCA4/BRG1 encodes for one of two mutually exclusive ATPases present in mammalian SWI/SNF chromatin remodeling complexes and is frequently mutated in human lung adenocarcinoma. However, the functional consequences of SMARCA4 mutation on tumor initiation, progression, and chromatin regulation in lung cancer remain poorly understood. Here, we demonstrate that loss of Smarca4 sensitizes club cell secretory protein–positive cells within the lung in a cell type–dependent fashion to malignant transformation and tumor progression, resulting in highly advanced dedifferentiated tumors and increased metastatic incidence. Consistent with these phenotypes, Smarca4-deficient primary tumors lack lung lineage transcription factor activities and resemble a metastatic cell state. Mechanistically, we show that Smarca4 loss impairs the function of all three classes of SWI/SNF complexes, resulting in decreased chromatin accessibility at lung lineage motifs and ultimately accelerating tumor progression. Thus, we propose that the SWI/SNF complex via Smarca4 acts as a gatekeeper for lineage-specific cellular transformation and metastasis during lung cancer evolution. Significance: We demonstrate cell-type specificity in the tumor-suppressive functions of SMARCA4 in the lung, pointing toward a critical role of the cell-of-origin in driving SWI/SNF-mutant lung adenocarcinoma. We further show the direct effects of SMARCA4 loss on SWI/SNF function and chromatin regulation that cause aggressive malignancy during lung cancer evolution. This article is highlighted in the In This Issue feature, p. 275

Significance It has long been observed that activation of p53 can yield two main cell fates: either apoptotic cell death or cell cycle arrest. However, what determines this cell fate remains to be fully elucidated. Here we show that the state of mitochondrial priming prior to activation of p53 can determine cell fate. Highly primed cells commit to apoptosis, while less-primed cells commit to cell cycle arrest. We show that modulation of priming in either direction can alter cell fate; increasing priming in poorly primed cells yields apoptosis, while decreasing priming in highly primed cells yields arrest. Drugs that increase priming enforce an apoptotic fate, suggesting a p53-independent strategy for augmenting efficacy of cancer chemotherapy drugs that kill via p53., Reactivation of p53 in established tumors typically results in one of two cell fates, cell cycle arrest or apoptosis, but it remains unclear how this cell fate is determined. We hypothesized that high mitochondrial priming prior to p53 reactivation would lead to apoptosis, while low priming would lead to survival and cell cycle arrest. Using a panel of Kras-driven, p53 restorable cell lines derived from genetically engineered mouse models of lung adenocarcinoma and sarcoma (both of which undergo cell cycle arrest upon p53 restoration), as well as lymphoma (which instead undergo apoptosis), we show that the level of mitochondrial apoptotic priming is a critical determinant of p53 reactivation outcome. Cells with high initial priming (e.g., lymphomas) lacked sufficient reserve antiapoptotic capacity and underwent apoptosis after p53 restoration. Forced BCL-2 or BCL-XL expression reduced priming and resulted in survival and cell cycle arrest. Cells with low initial priming (e.g., lung adenocarcinoma and sarcoma) survived and proceeded to arrest in the cell cycle. When primed by inhibition of their antiapoptotic proteins using genetic (BCL-2 or BCL-XL deletion or BAD overexpression) or pharmacologic (navitoclax) means, apoptosis resulted upon p53 restoration in vitro and in vivo. These data demonstrate that mitochondrial apoptotic priming is a key determining factor of cell fate upon p53 activation. Moreover, it is possible to enforce apoptotic cell fate following p53 activation in less primed cells using p53-independent drugs that increase apoptotic priming, including BH3 mimetic drugs.

Small cell lung cancer (SCLC) has limited therapeutic options and an exceptionally poor prognosis. Understanding the oncogenic drivers of SCLC may help define novel therapeutic targets. Recurrent genomic rearrangements have been identified in SCLC, most notably an in-frame gene fusion between RLF and MYCL found in up to 7% of the predominant ASCL1-expressing subtype. To explore the role of this fusion in oncogenesis and tumor progression, we used CRISPR/Cas9 somatic editing to generate a Rlf–Mycl-driven mouse model of SCLC. RLF–MYCL fusion accelerated transformation and proliferation of murine SCLC and increased metastatic dissemination and the diversity of metastatic sites. Tumors from the RLF–MYCL genetically engineered mouse model displayed gene expression similarities with human RLF–MYCL SCLC. Together, our studies support RLF–MYCL as the first demonstrated fusion oncogenic driver in SCLC and provide a new preclinical mouse model for the study of this subtype of SCLC. Significance: The biological and therapeutic implications of gene fusions in SCLC, an aggressive metastatic lung cancer, are unknown. Our study investigates the functional significance of the in-frame RLF–MYCL gene fusion by developing a Rlf–Mycl-driven genetically engineered mouse model and defining the impact on tumor growth and metastasis. This article is highlighted in the In This Issue feature, p. 2945

The coronavirus disease 2019 (COVID-19) pandemic has claimed the lives of over one million people worldwide. The causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a member of the Coronaviridae family of viruses that can cause respiratory infections of varying severity. The cellular host factors and pathways co-opted during SARS-CoV-2 and related coronavirus life cycles remain ill defined. To address this gap, we performed genome-scale CRISPR knockout screens during infection by SARS-CoV-2 and three seasonal coronaviruses (HCoV-OC43, HCoV-NL63, and HCoV-229E). These screens uncovered host factors and pathways with pan-coronavirus and virus-specific functional roles, including major dependency on glycosaminoglycan biosynthesis, sterol regulatory element-binding protein (SREBP) signaling, bone morphogenetic protein (BMP) signaling, and glycosylphosphatidylinositol biosynthesis, as well as a requirement for several poorly characterized proteins. We identified an absolute requirement for the VMP1, TMEM41, and TMEM64 (VTT) domain-containing protein transmembrane protein 41B (TMEM41B) for infection by SARS-CoV-2 and three seasonal coronaviruses. This human coronavirus host factor compendium represents a rich resource to develop new therapeutic strategies for acute COVID-19 and potential future coronavirus pandemics., Graphical Abstract, Highlights • Genome-wide CRISPR screens for SARS-CoV-2 and seasonal coronavirus host factors • Identification of host factors and pathways with pan-coronavirus and discrete roles • Coronaviruses co-opt multiple biological pathways • TMEM41B is a critical pan-coronavirus host factor, Schneider et al. conducted parallel genome-wide CRISPR knockout screens with SARS-CoV-2 and three seasonal coronaviruses to identify pan-coronavirus and virus-specific host factor requirements. They identified an interconnected network of host factors required by these four viruses and validated TMEM41B as a pan-coronavirus host factor required for a post-entry step in the coronavirus life cycle.

The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has devastated the global economy and claimed more than 1.7 million lives, presenting an urgent global health crisis. To identify host factors required for infection by SARS-CoV-2 and seasonal coronaviruses, we designed a focused high-coverage CRISPR-Cas9 library targeting 332 members of a recently published SARS-CoV-2 protein interactome. We leveraged the compact nature of this library to systematically screen SARS-CoV-2 at two physiologically relevant temperatures along with three related coronaviruses (human coronavirus 229E [HCoV-229E], HCoV-NL63, and HCoV-OC43), allowing us to probe this interactome at a much higher resolution than genome-scale studies. This approach yielded several insights, including potential virus-specific differences in Rab GTPase requirements and glycosylphosphatidylinositol (GPI) anchor biosynthesis, as well as identification of multiple pan-coronavirus factors involved in cholesterol homeostasis. This coronavirus essentiality catalog could inform ongoing drug development efforts aimed at intercepting and treating coronavirus disease 2019 (COVID-19) and help prepare for future coronavirus outbreaks., Graphical Abstract, Building upon a published SARS-CoV-2 protein interactome, Hoffmann et al. use a custom CRISPR library to determine which of these interacting host proteins are essential for infection by SARS-CoV-2 virus as well as three seasonal coronaviruses. These factors represent potential targets to combat COVID-19 and perhaps future coronavirus outbreaks.

SMARCA4 (BRG1) encodes for one of two mutually-exclusive ATPases present in SWI/SNF chromatin remodeling complexes, and is among the most frequently mutated genes in human lung adenocarcinoma. Despite its prevalence, the functional consequences of SMARCA4 alterations on lung cancer initiation and progression remain poorly understood. Using genetically engineered mouse models, patient-derived xenografts, and single-cell epigenomic profiling, we demonstrate that loss of Smarca4 sensitizes CCSP+ cells within the lung to malignant transformation and tumor progression, resulting in highly advanced dedifferentiated tumors and increased metastatic incidence. Consistent with these phenotypes, Smarca4-deficient tumors lack lung lineage transcription factor activities and instead show activation of embryonic stem cell-like programs, resembling a metastatic cell state. Thus, the SWI/SNF complex - via Smarca4 - acts as a gatekeeper for lineage-specific cellular transformation and metastasis during lung cancer evolution. Citation Format: Carla P. Concepcion, Sai Ma, Lindsay M. LaFave, Arjun Bhutkar, Manyuan Liu, Lydia P. DeAngelo, Jonathan Y. Kim, Isabella Del Priore, Adam J. Schoenfeld, Vinay K. Kartha, Peter M. Westcott, Francisco J. Sánchez-Rivera, Kevin Meli, Manav Gupta, Gregory J. Riely, Natasha Rekhtman, Charles M. Rudin, Carla F. Kim, Aviv Regev, Jason D. Buenrostro, Tyler Jacks. Smarca4 inactivation promotes lineage-specific transformation and early metastatic features in the lung [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2506.

Treating KRAS-mutant lung adenocarcinoma (LUAD) remains a major challenge in cancer treatment given the difficulties associated with directly inhibiting the KRAS oncoprotein. One approach to addressing this challenge is to define mutations that frequently co-occur with those in KRAS, which themselves may lead to therapeutic vulnerabilities in tumors. Approximately 20% of KRAS-mutant LUAD tumors carry loss-of-function mutations in the KEAP1 gene encoding Kelch-like ECH-associated protein 1 (refs. 2, 3, 4), a negative regulator of nuclear factor erythroid 2-like 2 (NFE2L2; hereafter NRF2), which is the master transcriptional regulator of the endogenous antioxidant response. The high frequency of mutations in KEAP1 suggests an important role for the oxidative stress response in lung tumorigenesis. Using a CRISPR-Cas9-based approach in a mouse model of KRAS-driven LUAD, we examined the effects of Keap1 loss in lung cancer progression. We show that loss of Keap1 hyperactivates NRF2 and promotes KRAS-driven LUAD in mice. Through a combination of CRISPR-Cas9-based genetic screening and metabolomic analyses, we show that Keap1- or Nrf2-mutant cancers are dependent on increased glutaminolysis, and this property can be therapeutically exploited through the pharmacological inhibition of glutaminase. Finally, we provide a rationale for stratification of human patients with lung cancer harboring KRAS/KEAP1- or KRAS/NRF2-mutant lung tumors as likely to respond to glutaminase inhibition.

Developmental and lineage plasticity have been observed in numerous malignancies and have been correlated with tumor progression and drug resistance. However, little is known about the molecular mechanisms that enable such plasticity to occur. Here, we describe the function of the plant homeodomain finger protein 6 (PHF6) in leukemia and define its role in regulating chromatin accessibility to lineage-specific transcription factors. We show that loss of Phf6 in B-cell leukemia results in systematic changes in gene expression via alteration of the chromatin landscape at the transcriptional start sites of B-cell- and T-cell-specific factors. Additionally, Phf6KO cells show significant down-regulation of genes involved in the development and function of normal B cells, show up-regulation of genes involved in T-cell signaling, and give rise to mixed-lineage lymphoma in vivo. Engagement of divergent transcriptional programs results in phenotypic plasticity that leads to altered disease presentation in vivo, tolerance of aberrant oncogenic signaling, and differential sensitivity to frontline and targeted therapies. These findings suggest that active maintenance of a precise chromatin landscape is essential for sustaining proper leukemia cell identity and that loss of a single factor (PHF6) can cause focal changes in chromatin accessibility and nucleosome positioning that render cells susceptible to lineage transition.

The heterogeneity of cellular states in cancer has been linked to drug resistance, cancer progression and presence of cancer cells with properties of normal tissue stem cells1,2. Secreted Wnt signals maintain stem cells in various epithelial tissues, including in lung development and regeneration3–5. Here we report that murine and human lung adenocarcinomas display hierarchical features with two distinct subpopulations, one with high Wnt signaling activity and another forming a niche that provides the Wnt ligand. The Wnt responder cells showed increased tumour propagation ability, suggesting that they have features of normal tissue stem cells. Genetic perturbation of Wnt production or signaling suppressed tumour progression. Small molecule inhibitors targeting essential post-translational modification of Wnt reduced tumour growth and dramatically decreased proliferative potential of the lung cancer cells, leading to improved survival of tumour-bearing mice. These results indicate that strategies for disrupting pathways that maintain stem-like and niche cell phenotypes can translate into effective anti-cancer therapies.