Determining Synthetic Lethal Targets in DNA Polymerase Epsilon P286R Mutants Using Genome-wide CRISPR-Cas9 Knockout Screens

Abstract

Genomic instability is an important hallmark of cancer development and is often a product of impaired or abnormal DNA replication. For instance, DNA Polymerase Epsilon (POLE) is responsible for the bulk of DNA replication on the leading strand, and somatic mutations mapping to the ‘proof-reading’ exonuclease domain, such as POLEP286R/+, are commonly detected in tumors of the endometrium and gastrointestinal tract. POLE exonuclease domain mutations are frequently associated with increased mutational burden and can be further sub-classified as either ‘microsatellite stable’ (MSS) or ‘instable’ (MSI), depending on the functionality of compensatory mismatch repair (MMR) pathways. Genetic dependencies in MSS cancers bearing POLE mutations are poorly understood, however, previous studies modeling analogous mutations in yeast demonstrated increased levels of replication stress irrespective of MMR status. We therefore predicted that human MSS POLE mutant cancer cells would show enhanced sensitivity to pharmacologic inhibition of ATR, the master-regulator of replication fork stability. Furthermore, our in-silico predictions indicate that DNA damage repair (DDR) genes downstream of ATR, including FANCM, MSH2, and MSH6, are more heavily relied upon in patient-derived MSS POLE mutant cancer cell lines relative to those without POLE mutations. Towards this end, we used CAS9 to create human U2OS-derived POLEP286R/+ knock-in cell lines. Indeed, we observed distinct replication fork abnormalities and reduced viability in the presence of ATRi in POLEP286R/+ cells relative to isogenic controls. To validate our in-silico predictions and to identify additional synthetic-lethal targets in-vitro, we designed a custom CRISPR knockout (KO) library with particular emphasis on genes known to be involved in DNA replication and repair, including ample representation of targets previously implicated in ATR-dependent signaling pathways. We further engineered and optimized our POLE isogenic cell line pairs to enable doxycycline-inducible expression of CAS9 following introduction of the KO library. The results of our pilot screen are currently pending next-generation sequencing and analysis. Future works will be focused on putative target validation and additional mechanistic inquiry, the summation of which will provide important insight towards the development of novel therapies capable of selectively killing MSS POLE mutant cancers.