SUMMARY Small cell lung cancer (SCLC) is an aggressive and lethal neuroendocrine lung cancer type. Most patients initially respond to chemotherapy but relapse occurs within months and genetic alterations that drive chemoresistance are poorly understood. Beyond amplification of MYC family members and epigenetic silencing of SLFN11, the field has an extremely poor understanding of genes that promote SCLC chemoresistance. We developed a novel system in which we genetically alter highly chemosensitive patient derived xenograft (PDX) models of SCLC to identify perturbations that confer resistance to cisplatin/etoposide (CIS-ETO) in vivo. Lentiviral overexpression of either MYCN or MYCL caused complete switch to chemoresistance (Grunblatt et al, 2020). To systematically identify SCLC chemoresistance drivers, we expanded use of this PDX lentiviral transduction system to perform in vivo CRISPR inactivation screens. We identified sgRNAs targeting multiple components of the SAGA (Spt- Ada-Gcn5 acetyltransferase) chromatin modifying complex as screen hits and confirmed that deleting the SAGA member USP22, a deubiquitylase, indeed confers chemoresistance in two SCLC PDX models, while return of USP22 to a USP22-null SCLC PDX model re-sensitizes to chemotherapy. Our overarching hypothesis is that suppressing the expression of USP22 and SAGA complex members drives chemoresistance in SCLC, and that transcriptional changes caused by SAGA suppression are critical. Aim 1, we will interrogate how genetically perturbing multiple SAGA complex members, including USP22 and TADA1, in PDX models alters the in vivo response to chemotherapy. Aim 2 employs genomic and proteomic approaches to develop a deep molecular understanding of the USP22-regulated genes and pathways that contribute to chemotherapy response in SCLC and uses human patient data to prioritize key SAGA targets for functional study. Decades of studying chemotherapy response in SCLC cell lines grown in vitro have provided little insight into how chemoresistance emerges, suggesting that key aspects of this process are not recapitulated under tissue culture conditions. Our novel system prioritizes the study of chemoresistance using in vivo approaches with potential to provide foundational knowledge to help prevent chemoresistance or re-sensitize chemoresistant SCLC to chemotherapy.

Project Narrative Small cell lung cancer (SCLC) patients tend to respond remarkably well to chemotherapy, but, through largely unknown mechanisms, chemoresistance rapidly emerges. We found that single gene perturbation of chemosensitive patient derived xenograft models of SCLC can confer switch to chemoresistance and we took advantage of this finding to perform in vivo functional screens. This proposal is focused on understanding a pathway that emerged from our screens that, when suppressed, potently drive SCLC chemoresistance in vivo.