CRISPR-mediated interrogation of small cell lung cancer

Abstract

Small cell lung cancer (SCLC) is a highly aggressive neuroendocrine lung carcinoma that remains among the most lethal of solid tumor malignancies. Despite decades of research, treatment outcomes for SCLC remain very poor, highlighting the need for novel approaches to target the disease. Recent genomic sequencing studies have identified multiple recurrently altered genes in human SCLC tumors, many of which remain to be functionally validated. Genetically engineered mouse models (GEMMs) of SCLC have been developed that recapitulate many key features of human SCLC. These models have been used extensively to investigate various aspects of SCLC biology, including tumor initiation, progression and metastasis. The development of the CRISPR-Cas9 system has greatly facilitated genome editing in mammalian cells, leading to its widespread adoption for various applications in cancer biology. We have utilized this system in two complementary ways to investigate the molecular mechanisms involved in SCLC initiation, progression and maintenance. Firstly, we have adapted the CRISPR-Cas9 system for use in GEMMs of SCLC, to enable rapid modeling and functional validation of candidate tumor suppressor genes in vivo. Using this system, we have demonstrated that p107, a member of the retinoblastoma family that is mutated in a significant fraction of human SCLC tumors, is a functional tumor suppressor in SCLC. Notably, loss of p107 in SCLC tumors resulted in significant phenotypic differences compared with loss of its close relative, p130. We also demonstrated that CRISPR-induced mutations can be used to infer lineage relationships between primary and metastatic tumors in the same animal. Secondly, we have performed a CRISPR-based genetic screen, utilizing a custom sgRNA library targeting the druggable genome, to identify novel SCLC-specific genetic vulnerabilities. We found that SCLC cells displayed enhanced sensitivity towards disruption of several key metabolic pathways, including the de novo pyrimidine biosynthesis pathway. Pharmacological inhibition of Dhodh, a key enzyme in this pathway, reduced the viability of SCLC cells in vitro and strongly suppressed SCLC tumor growth in vivo, validating this pathway as a promising therapeutic target in SCLC. Taken together, the work presented here demonstrates the utility of the CRISPR-Cas9 system for performing functional interrogation of SCLC.