In vivo multiplexed screen reveals a critical role of Keap1/Nrf2 pathway in small cell lung cancer [TUBAseq]
Approximately 15% of all lung cancer cases are small cell lung cancer (SCLC), which originates from neuroendocrine cells lining the airways (bronchi) of the lung. SCLC is known for its aggressive nature and swift growth, resulting in a five-year survival rate of around 6%. Understanding the genetic pathways that drive tumor development remains an ongoing challenge. In our study, we employed an in vivo multiplexed approach, known as Tuba-seq, to investigate the impact of the functional loss of 18 putative tumor suppressors. Our screening unveiled Pten and Keap1 as top candidates; knocking out either one promoted both tumor initiation and progression. We identified the Keap1/Cul3/Nrf2 pathway as a pivotal regulator for SCLC development, particularly in regulating the susceptibility of SCLC to ferroptosis. Our work not only established an in vivo multiplexed approach for assessing the role of tumor suppressors in SCLC but also uncovers a previously unappreciated role of Keap1 in SCLC. Overall design: In this study a multiplex approach was used to examine the consequences of loss of putative 18 tumor suppressors (TS) in vivo. The 18 TSs were selected based on their mutation prevalence in human SCLC patients, in addition to the genes of personal interest. To stimulate SCLC development  Rb1;p53;p130;p107 quadruple knockout mice (hereafter referred to as QKO) was used. To study the roles of putative tumor suppressors, the QKO mice was bred with H11loxP-STOP-loxP-Cas9 mice (QKO;Cas9). The sgRNA for each putative tumor suppressor gene was cloned into the Lenti-U6-sgRNA/PGK-Cre vector. To facilitate the multiplexed study, the Tuba-seq approach was utilized. A unique barcode was assigned to each Lenti-sgRNA/Cre vector: an sgRNA ID (a specific eight-nucleotide sequence) and a tumor ID (a string of hemi-random nucleotides). All the barcoded vectors (including 18 putative tumor suppressors and one non-targeting control) were pooled at an equal ratio, encapsulated them into a lentivirus pool using a PEG8000-mediated precipitation method. The lentivirus titer was determined by FACS with 3T3 cells expressing Cre-reporter. Next, small cell lung cancer formation was induced. The barcoded lentivirus pool was injected into the QKO;Cas9 mice, with each mice receiving 9.7×10^5 ifu (infectious units) of virus. The entire lung was harvested from each mouse at the survival end point (median survival time ~13 weeks).