Target Discovery of Selective Non-Small-Cell Lung Cancer Toxins Reveals Inhibitors of Mitochondrial Complex I.

Selective toxicity among cancer cells of the same lineage is a hallmark of targeted therapies. As such, identifying compounds that impair proliferation of a subset of non-small-cell lung cancer (NSCLC) cell lines represents one strategy to discover new drugs for lung cancer. Previously, phenotypic screens of 202 103 compounds led to the identification of 208 selective NSCLC toxins ( McMillan , E. A. , Cell , 2018 , 173 , 864 ). The mechanism of action for the majority of these compounds remains unknown. Here, we discovered the target for a series of quinazoline diones (QDC) that demonstrate selective toxicity among 96 NSCLC lines. Using photoreactive probes, we found that the QDC binds to both mitochondrial complex I of the electron transport chain and hydroxyacyl CoA dehydrogenase subunit alpha (HADHA), which catalyzes long-chain fatty acid oxidation. Inhibition of complex I is the on-target activity for QDC, while binding to HADHA is off-target. The sensitivity profile of the QDC across NSCLC lines correlated with the sensitivity profiles of six additional structurally distinct compounds. The antiproliferative activity of these compounds is also the consequence of binding to mitochondrial complex I, reflecting significant structural diversity among complex I inhibitors. Small molecules targeting complex I are currently in clinical development for the treatment of cancer. Our results highlight complex I as a target in NSCLC and report structurally diverse scaffolds that inhibit complex I.

Long-term culture and cloning of primary human bronchial basal cells that maintain multipotent differentiation capacity and CFTR channel function.

While primary cystic fibrosis (CF) and non-CF human bronchial epithelial basal cells (HBECs) accurately represent in vivo phenotypes, one barrier to their wider use has been a limited ability to clone and expand cells in sufficient numbers to produce rare genotypes using genome-editing tools. Recently, conditional reprogramming of cells (CRC) with a Rho-associated protein kinase (ROCK) inhibitor and culture on an irradiated fibroblast feeder layer resulted in extension of the life span of HBECs, but differentiation capacity and CF transmembrane conductance regulator (CFTR) function decreased as a function of passage. This report details modifications to the standard HBEC CRC protocol (Mod CRC), including the use of bronchial epithelial cell growth medium, instead of F medium, and 2% O2, instead of 21% O2, that extend HBEC life span while preserving multipotent differentiation capacity and CFTR function. Critically, Mod CRC conditions support clonal growth of primary HBECs from a single cell, and the resulting clonal HBEC population maintains multipotent differentiation capacity, including CFTR function, permitting gene editing of these cells. As a proof-of-concept, CRISPR/Cas9 genome editing and cloning were used to introduce insertions/deletions in CFTR exon 11. Mod CRC conditions overcome many barriers to the expanded use of HBECs for basic research and drug screens. Importantly, Mod CRC conditions support the creation of isogenic cell lines in which CFTR is mutant or wild-type in the same genetic background with no history of CF to enable determination of the primary defects of mutant CFTR.