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Summary This standardized protocol describes the preparation of PCR amplified and purified samples from human cell lines passaged and collected from CRISPR screening. High-quality samples can be used to perform next-generation sequencing (NGS) to uncover changes in sgRNA abundance from the timepoint at which library-transduced cells are selected to the timepoint when the screen is ended. Here, we describe proper calculation methods for library representation and show how to overcome potential issues often encountered by researchers. For complete information on the use and execution of this protocol, please refer to Wohlhieter et al. (2020) .

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., Graphical Abstract, In Brief Wohlhieter et al. explore the global changes in gene expression and oncogenic signaling pathways driven by concurrent loss of function in two tumor suppressor genes, STK11 and KEAP1. They identify a molecular vulnerability, in which co-mutant cells depend on ferroptosis protective mechanisms for survival, and highlight SCD1 as an essential gene and promising drug target.

[Objectives] Lung adenocarcinoma accounts for approximately half of lung cancer cases. Twenty to 50% of tumors of this type harbor mutations affecting epidermal growth factor receptor (EGFR) expression or activity, which can be therapeutically targeted. EGFR inhibitors in this context exhibit high efficacy and are currently used in the clinical setting. However, not all adenocarcinomas harboring EGFR mutations respond to therapy, so predictive biomarkers of therapeutic outcomes, as well as novel therapies sensitizing these tumors to EGFR inhibition, are needed., [Materials and methods] We performed in vitro gene overexpression/silencing and tumorigenic surrogate assays, as well as in vitro and in vivo combination treatments with Fibroblast Growth Factor Receptor (FGFR)/EGFR inhibitors. At the clinical level, we determined FGFR4 expression levels in tumors from patients treated with EGFR inhibitors and correlated these with treatment response., [Results] We describe a cooperative interaction between EGFR and FGFR4, which results in their reciprocal activation with pro-oncogenic consequences in vitro and in vivo. This cooperation is independent of EGFR activating mutations and increases resistance to different EGFR inhibitors. At the therapeutic level, we provide evidence of the synergistic effects of the combination of EGFR and FGFR inhibitors in high FGFR4-expressing, EGFR-activated tumors in vitro and in vivo. Correlated with these results, we found that patients treated with EGFR inhibitors relapse earlier when their tumors exhibit high FGFR4 expression., [Conclusions] We propose a novel predictive biomarker for EGFR-targeted therapy, and a highly efficacious combinatory therapeutic strategy to treat EGFR-dependent; this may may extend the use of appropriate inhibitors beyond EGFR-mutated adenocarcinoma patients., L.P.A. was funded by the Comunidad de Madrid, CAM, (B2017/BMD3884), ISCIII (PI14/01964, PIE15/00076, PI17/00778 and DTS17/00089) and CIBERONC (CD16/12/00442), and co-funded by FEDER from Regional Development European Funds (European Union). I.F. is funded by the AECC (AIO2015) and Consejería de Igualdad, Salud y Políticas Sociales de la Junta de Andalucía (PI-0029-2013) and ISCIII (PI16/01311), and co-funded by FEDER from Regional Development European Funds (European Union). AC was supported by grants from the Spanish Ministry of Economy and Competitiveness Plan Estatal de I + D+I 2013–2016, ISCIII (PI15/00045) and CIBERONC (CD16/12/00275), and co-funded by FEDER from Regional Development European Funds (European Union). S.M.P. is funded by the Consejería de Salud y Bienestar Social (PI-0046-2012), the Fundación Mutua Madrileña (2014) and ISCIII (PI17/00033). A.Q is funded by the ISCIII (FI12/00429). L.O. is funded by the Ministerio de Educación, Cultura y Deporte (FPU13/02595).

Introduction: There is substantial evidence for the onco- genic effects of fi broblast growth factor receptor 1 (FGFR1) in many types of cancer, including lung cancer, but the role of this receptor has not been addressed speci fi cally in lung adenocarcinoma. Methods: We performed FGFR1 and EGFR overexpression and co-overexpression assays in adenocarcinoma and in inmortalized lung cell lines, and we also carried out surrogateandinteractionassays.Weperformedmono- therapy and combination EGFR /FGFR inhibitor sensitivity assays in vitro and in vivo in cell line – and patient- derived xenografts. We determined FGFR1 mRNA expression in a cohort of patients with anti – EGFR ther- apy – treated adenocarcinoma. Results: We have reported a cooperative interaction between FGFR1 and EGFR in this context, resulting in increased EGFR activation and oncogenic signaling. We have provided in vitro and in vivo evidence indicating that FGFR1 expression in- creases tumorigenicity in cells with high EGFR activation in EGFR-mutated and EGFR wild-type models. At the clinical level, we have shown that high FGFR1 expression levels pre- dict higher resistance to erlotinib or ge fi tinib in a cohort of patients with tyrosine kinase inhibitor – treated EGFR-mutated and EGFR wild-type lung adenocarcinoma. Dual EGFR and FGFR inhibition in FGFR1-over expressing, EGFR-activated models shows synergistic effects on tumor growth in vitro and in cell line – and patient-derived xenografts, suggesting that patients with tumors bearing these characteristics may bene fi t from combined EGFR/FGFR inhibition. Conclusion: These results support the extended the use of EGFR inhibitors beyond monotherapy in the EGFR-mutated adenocarcinoma setting in combination with FGFR in- hibitors for selected patients with increased FGFR1 over- expression and EGFR activation. ISCIII PI14/01964 PIE15/00076 PI17/00778 DTS17/00089 PI15/00045 PI17/00033 PI16/01311 FI12/00429 CIBERONC CD16/12/00442 FEDER CD16/12/00442 PI16/01311 Spanish Ministry of Economy and Competitiveness PI15/00045 Ministry of Health and Social Welfare of Junta de Andalucía PI-0046-2012 C-0040-2016 Ministry of Equality, Health and Social Policies of the Junta de Andalucía PI- 0029-2013 Comunidad de Madrid B2017/BMD3884 Ministry of Education, Culture and Sports FPU13/02595