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Abstract

Lung cancer remains the most common cause of cancer death worldwide. Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, and lung adenocarcinoma (LUAD) is the most frequent subtype of NSCLC. Discovery of the prevalence of distinct driver mutations in LUAD tumors has led to a booming development of targeted therapies blocking the resulting oncogene activation. However, long-term therapeutic outcomes of such treatments are poor, and novel therapeutic targets for mutant LUAD are urgently required. I set out to investigate novel genotype-specific LUAD viability factors, and therefore developed and conducted a pooled CRISPR interference (CRISPRi) dropout screen in eight LUAD cell lines with a custom single guide RNA (sgRNA) library. To compile the screen targets, I analyzed the transcriptional profiles from LUAD patients and selected protein-coding genes and long non-coding RNAs (lncRNAs) differentially overexpressed between LUAD samples with different driver mutational signatures. LncRNAs overexpressed in LUAD compared to normal lung samples were also added to the candidate list to enrich the screen with novel targets, however, neither genotype-specific nor general lncRNA viability factors for LUAD were identified in the screen. The screen revealed that EGFR-mutant LUAD cell lines exhibited resistance to the knockdown of the mitotic regulator CENPE. Conversely, CENPE expression was essential for the viability of EGFR-wildtype cells. Functional validation of the observed divergent responses revealed a direct link between the mutant EGFR activity and the resistance to the CENPE knockdown in LUAD cell lines. Moreover, silencing of CASP8AP2 resulted in the loss of cell viability in all screened LUAD and additional NSCLC cell lines, while, in contrast, non-transformed lung cells tolerated its depletion. I uncovered that expression levels of JUNB, encoding for a subunit of the AP-1 transcription factor, were lower in steady state as well as upon CASP8AP2 silencing in the tolerant cell lines compared to the cancer cells sensitive to the CASP8AP2 loss. Inhibition of AP-1 rescued the loss of viability phenotype in NSCLC cell lines, uncovering the CASP8AP2 – AP-1 functional axis. Overall, my custom CRISPRi screen identified a novel genotype-specific mechanism in EGFR-mutant LUAD cells that were able to overcome disruption of the mitotic regulation and discovered CASP8AP2 as an essential NSCLC viability determinant and a promising candidate for therapeutic targeting acting in cooperation with the AP-1 transcription factor.