Modeling DNA trapping of anticancer therapeutic targets using missense mutations identifies dominant synthetic lethal interactions

Abstract

Genetic screens can identify synthetic lethal (SL) interactions and uncover potential anticancer therapeutic targets. However, most SL screens have utilized knockout or knockdown approaches that do not accurately mimic chemical inhibition of a target protein. Here, we test whether missense mutations can be utilized as a model for a type of protein inhibition that creates a dominant gain-of-function cytotoxicity. We expressed missense mutations in the FEN1 endonuclease and the replication-associated helicase, CHL1, that inhibited enzymatic activity but retained substrate binding, and found that these mutations elicited a dominant SL phenotype consistent with the generation of cytotoxic protein–DNA or protein–protein intermediates. Genetic screens with nuclease-defective hFEN1 and helicase-deficient yCHL1 captured dominant SL interactions, in which ectopic expression of the mutant form, in the presence of the wild-type form, caused SL in specific mutant backgrounds. Expression of nuclease-defective hFEN1 in yeast elicited DNA binding-dependent dominant SL with homologous recombination mutants. In contrast, dominant SL interactions with helicase-deficient yCHL1 were observed in spindle-associated, Ctf18-alternative replication factor C (Ctf18-RFC) clamp loader complex, and cohesin mutant backgrounds. These results highlight the different mechanisms underlying SL interactions that occur in the presence of an inhibited form of the target protein and point to the utility of modeling trapping mutations in pursuit of more clinically relevant SL interactions.

Setting molecular traps in yeast for identification of anticancer drug targets

“…Hamza et al (5) provide proof-of-principle for an exciting approach to identify small molecules with therapeutic potential that act through specific mechanisms. Using a putative DNA-protein complex trapping allele of human FEN1, they combine a humanized yeast model with systematic genetic interaction screening to discover genetic interactions that should be recapitulated by a FEN1 inhibitor with trapping properties.…”

“…When connected to phenotypic assays, deep mutational scanning reveals mutant alleles with the desired properties, typically some kind of loss of function. Hamza et al (5) screen FEN1 alleles that were designed to inactivate catalysis but not DNA binding, and so were likely to result in trapping of FEN1-DNA complexes. Deep mutational scanning could be used to make separation of function mutants that lose catalytic activity but that retain protein-DNA or protein-protein interactions (15), exactly the type of trapping mutants that Hamza et al model with their FEN1 alleles.…”

“…The effort of Hamza et al (5) to discover genetic interactions associated with expression of a specific allele of FEN1 complements and leverages major efforts in the yeast community to systematically map genetic interactions. The budding yeast is the only eukaryotic system for which a complete map of synthetic lethal interactions has been generated, through tests of all possible double mutants for synthetic growth defects (16,17).…”

“…In PNAS, Hamza et al (5) start with the premise that small molecules that cause trapping of protein-DNA or protein-protein complexes are desirable, and present a means of identifying trapping inhibitors by screening specific gene alleles in a yeast model (Fig. 1B).…”