A Role for Mitochondrial Translation in Promotion of Viability in K-Ras Mutant Cells

Abstract

Summary Activating mutations in the KRAS oncogene are highly prevalent in tumors, especially those of the colon, lung, and pancreas. To better understand the genetic dependencies that K-Ras mutant cells rely upon for their growth, we employed whole-genome CRISPR loss of function screens in two isogenic pairs of cell lines. Since loss of essential genes is uniformly toxic in CRISPR-based screens we also developed an shRNA library targeting essential genes. These approaches uncovered a large set of proteins whose loss results in the selective reduction of K-Ras mutant cell growth. Pathway analysis revealed that many of these genes function in the mitochondria. For validation, we generated isogenic pairs of cell lines using CRISPR-based genome engineering, which confirmed the dependency of K-Ras mutant cells on these mitochondrial pathways. Finally, we found that mitochondrial inhibitors reduce the growth of K-Ras mutant tumors in vivo, advancing strategies to target K-Ras-driven malignancy.

Integrated computational and experimental identification of p53, KRAS and VHL mutant selection associated with CRISPR-Cas9 editing

“…As observed for VHL and p53 (Figure 4d and S1, respectively), the effects of KRAS are also independent of gene copy number ( Figure S1). Consistent with these findings, mining the data from genome-wide CRISPR and shRNA screens performed in two KRAS isogenic cell lines (DLD1 and HCT116) 25 revealed that the predicted KRAS CDE+/-genes have marginally significant overlap with the DE+/-genes identified in both the cell lines CRISPR screens (hypergeometric test P<0.1, Supp. Note 2) and not in the corresponding shRNA screens.…”

ACE: A Probabilistic Model for Characterizing Gene-Level Essentiality in CRISPR Screens

“…Three replicates were simulated with an average of 300 reads per sgRNA, with four sgRNA targeting each gene. The noise profile of our simulated counts and relative frequencies of sgRNA in the master library was designed to match the genome-wide CRISPR screens published in Martin et al 2017 (16), in which both the master library and initial infected sgRNA abundances were sequenced along with the depleted samples.…”

“…Differential essentiality can be detected from different levels of sgRNA depletion between samples or panels of sample types. Many experiments evaluate the loss of cells infected with lethal sgRNA by sequencing both the initial infected and the final population of cells (14)(15)(16)(17). Other experiments sequence only the initial pool of sgRNA constructs prior to infection (9,18,19), and use the sgRNA abundances in this master library as a proxy for initial frequencies.…”

CRISPR-based genetic interaction maps inform therapeutic strategies in cancer

“…sgRNAs that show reduced frequency over time indicate genes required for cellular survival or proliferation. Such screens have revealed genotype-specific cancer vulnerabilities such as for the mutant oncogenic Ras cell lines (7–9), which can be interpreted as synthetic lethal genes with oncogenic Ras.…”