SUMMARY Concurrent loss-of-function mutations in STK11 and KEAP1 in lung adenocarcinoma (LUAD) are associated with aggressive tumor growth, resistance to available therapies, and early death. We investigated the effects of coordinate STK11 and KEAP1 loss by comparing co-mutant with single mutant and wild-type isogenic counterparts in multiple LUAD models. STK11/KEAP1 co-mutation results in significantly elevated expression of ferroptosis-protective genes, including SCD and AKR1C1/2/3 , and resistance to pharmacologically induced ferroptosis. CRISPR screening further nominates SCD (SCD1) as selectively essential in STK11/KEAP1 co-mutant LUAD. Genetic and pharmacological inhibition of SCD1 confirms the essentiality of this gene and augments the effects of ferroptosis induction by erastin and RSL3. Together these data identify SCD1 as a selective vulnerability and a promising candidate for targeted drug development in STK11/KEAP1 co-mutant LUAD.

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Summary Activation of mitogenic signaling pathways is a common oncogenic driver of many solid tumors including lung cancer. Although activating mutations in the mitogen-activated protein kinase (MAPK) pathway are prevalent in non-small cell lung cancers, MAPK pathway activity, counterintuitively, is relatively suppressed in the more aggressively proliferative small cell lung cancer (SCLC). Here, we elucidate the role of the MAPK pathway and how it interacts with other signaling pathways in SCLC. We find that the most common SCLC subtype, SCLC-A associated with high expression of ASCL1 , is selectively sensitive to MAPK activation in vitro and in vivo through induction of cell-cycle arrest and senescence. We show strong upregulation of ERK negative feedback regulators and STAT signaling upon MAPK activation in SCLC-A lines. These findings provide insight into the complexity of signaling networks in SCLC and suggest subtype-specific mitogenic vulnerabilities.

2 Patient-derived xenografts (PDXs) constitute a powerful set of preclinical models for in vivo cancer research, reflecting the spectrum of genomic alterations and therapeutic liabilities of human cancers 1-4 . In contrast to either cancer cell lines or genetically engineered mouse models, the utility of PDXs has been limited by the inability to perform targeted genome editing of these tumors. To address this limitation, we have generated a lentiviral platform for CRISPR-Cas9 editing of PDXs using a tightly regulated, inducible Cas9 vector that does not require in vitro culture for selection of transduced cells. We demonstrate the utility of this platform in PDXs (1) to analyze genetic dependencies by targeted gene disruption and (2) to analyze mechanisms of acquired drug resistance by site-specific gene editing using templated homology-directed repair. This flexible system has broad application to other explant models and substantially augments the utility of PDXs as genetically programmable models of human cancer.PDXs recapitulate the complex genotypes and intratumoral heterogeneity of their tumors of origin and are not subject to the selective pressures imposed by in vitro cell culture since they are maintained exclusively in vivo 5-7 . These features have driven the rapid adoption and widespread use of PDXs in preclinical and co-clinical drug development, evaluation of biomarkers and imaging agents, and mechanistic investigation of acquired treatment resistance [8][9][10] . PDXs have also proven to be valuable models of tumor types or variants for which in vitro models are not readily available 11,12 .The ability to genetically manipulate cancer models has played an essential role in defining the functional contributions of individual genes and variants. CRISPR-Cas9 genome editing has greatly expanded our ability to rapidly analyze how specific genes and genotypes contribute to carcinogenesis and tumor maintenance 13,14 . CRISPR-Cas9 can be used to disrupt genes through the introduction of frameshift insertions and 3 deletions (indels) by non-homologous end joining or to make precise genomic alterations through homology-directed repair (HDR) 15 . However, in vivo applications of CRISPR-Cas9 genome editing have been restricted to xenografts of established human cell lines and genetically engineered mouse models (GEMMs) due largely to the technical challenges imposed by the continuous in vivo passaging of PDXs and the need for alternative selection methods. Given the increasing role of PDXs in cancer research, a technological advance is needed to enable direct genome editing of PDXs using CRISPR-Cas9 to further our understanding of cancer biology and facilitate the development of new therapeutic strategies.Tight temporal control of Cas9 activity in the cells that make up established tumors is essential to validate genes required for tumor maintenance and to credential suppressor mutations that may play a role in acquired drug resistance. Several inducible systems have been developed to regulate Cas9 activity at...

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