ABSTRACT
Low success rates during drug development are due, in part, to the difficulty of defining drug mechanism-of-action and molecular markers of therapeutic activity. Here, we integrated 199,219 drug sensitivity measurements for 397 unique anti-cancer drugs with genome-wide CRISPR loss-of-function screens in 484 cell lines to systematically investigate cellular drug mechanism-of-action. We observed an enrichment for positive associations between the profile of drug sensitivity and knockout of a drug's nominal target, and by leveraging protein-protein networks, we identified pathways underpinning drug sensitivity. This revealed an unappreciated positive association between mitochondrial E3 ubiquitin-protein ligase MARCH5 dependency and sensitivity to MCL1 inhibitors in breast cancer cell lines. We also estimated drug on-target and off-target activity, informing on specificity, potency and toxicity. Linking drug and gene dependency together with genomic data sets uncovered contexts in which molecular networks when perturbed mediate cancer cell loss-of-fitness and thereby provide independent and orthogonal evidence of biomarkers for drug development. This study illustrates how integrating cell line drug sensitivity with CRISPR loss-of-function screens can elucidate mechanism-of-action to advance drug development.
Subject(s)
Antineoplastic Agents/pharmacology , CRISPR-Cas Systems , Drug Development/methods , Drug Screening Assays, Antitumor/methods , Gene Regulatory Networks/drug effects , Genetic Fitness/drug effects , Protein Interaction Maps/drug effects , Antineoplastic Agents/toxicity , Biomarkers/metabolism , Cell Line, Tumor , Gene Knockout Techniques , Gene Regulatory Networks/genetics , Genetic Fitness/genetics , Genomics , Humans , Linear Models , Membrane Proteins/genetics , Membrane Proteins/metabolism , Myeloid Cell Leukemia Sequence 1 Protein/antagonists & inhibitors , Pharmaceutical Preparations/metabolism , Software , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolismABSTRACT
Micro-RNAs (miRNAs) can be specifically expressed in forensically relevant body fluids such as blood or saliva. The aim of the study was to develop a simultaneous extraction and analysis protocol that allows for the acquisition of a DNA profile and the identity of the body fluid using a single process. DNA and micro-RNA were extracted from blood and saliva before undergoing a cDNA synthesis step by using stem-loop reverse transcription PCR. The resulting extracts containing DNA and cDNA synthesized from body fluid-specific miRNA markers then underwent standard STR analysis using a modified ABI AmpFâSTR(®) NGM SElect™ kit. In all samples, a full DNA profile was obtained along with additional peaks corresponding to the miRNA marker targeted. In all cases, blood samples profiled exhibited a peak indicating the presence of the blood-specific miRNA marker and the saliva sample profiled exhibited a peak indicating the presence of the saliva-specific miRNA marker.