Genetic determinants of cellular addiction to DNA polymerase theta

Abstract

Polymerase theta (Pol θ, gene name Polq) is a widely conserved DNA polymerase that mediates a microhomology-mediated, error-prone, double strand break (DSB) repair pathway, referred to as Theta Mediated End Joining (TMEJ). Cells with homologous recombination deficiency are reliant on TMEJ for DSB repair. It is unknown whether deficiencies in other components of the DNA damage response (DDR) also result in Pol θ addiction. Here we use a CRISPR genetic screen to uncover 140 Polq synthetic lethal (PolqSL) genes, the majority of which were previously unknown. Functional analyses indicate that Pol θ/TMEJ addiction is associated with increased levels of replication-associated DSBs, regardless of the initial source of damage. We further demonstrate that approximately 30% of TCGA breast cancers have genetic alterations in PolqSL genes and exhibit genomic scars of Pol θ/TMEJ hyperactivity, thereby substantially expanding the subset of human cancers for which Pol θ inhibition represents a promising therapeutic strategy.

Mechanistic Basis for Microhomology Identification and Genome Scarring by Polymerase Theta

“…Fig. 4A) – the most frequent Pol θ-dependent repair products for a given chromosome break, and serve as a biomarker for TMEJ activity (Feng et al ., 2019). However, some MHD are favored during NHEJ (Fig.…”

“…Past work from our group and others has determined that in some contexts, Pol θ-dependent repair can include microhomologies more distal than 15 bp from 3’ termini. These contexts include cells deficient in regulation of end resection (cells defective in KU or 53BP1 ) or wild type cells after a much longer recovery period than is used here (24 hours) (Wyatt et al ., 2016; Schimmel et al ., 2017; Feng et al ., 2019). Given our results with pre-resected substrates, we suggest this second class of Pol θ-dependent products reflects loss of 3’ terminal ssDNA before Pol θ could be engaged, rather than a fundamental change in the preference of Pol θ for break proximal microhomologies.…”

“…6B), consistent with a requirement for TMEJ for viability of several BRCA1 / 2 defective cancer cell lines (Ceccaldi et al ., 2015; Mateos-Gomez et al ., 2015). Indeed, Pol θ is required for viability in the context of a wide variety of DNA damage response defects, and thus is as an attractive target for therapy in as many as 30% of all breast tumors (Higgins and Boulton, 2018; Feng et al ., 2019). Assessment of TINS in tumor genomes thus holds promise as a biomarker for deciding when TMEJ should be targeted for cancer therapy.…”

“…TMEJ overlaps with previously defined Alternative Non-Homologous End Joining (Alt-NHEJ) and Microhomology Mediated End Joining (MMEJ) pathways (Boulton and Jackson, 1996; Kabotyanski et al ., 1998; Ma et al ., 2003), though the extent these definitions overlap is not clear. In mammals, TMEJ is both more frequent and essential for viability in cells deficient in NHEJ (Wyatt et al ., 2016; Saito, Maeda and Adachi, 2017; Zelensky et al ., 2017) or HR (Ceccaldi et al ., 2015; Mateos-Gomez et al ., 2015; Feng et al ., 2019). A specific requirement in BRCA -deficient contexts for Pol θ has identified this protein as a therapeutic target in BRCA -deficient breast cancers (Higgins and Boulton, 2018).…”

Hyperactive end joining repair mediates resistance to DNA damaging therapy in p53-deficient cells

“…Prior studies have demonstrated that cells with NHEJ deficiency exhibit a compensatory increase in alternative end-joining repair mediated by DNA polymerase theta (Pol θ, gene POLQ ) 4,33,34 . Polymerase theta dependent end joining (TMEJ) of DNA DSBs is characterized by deletions and templated insertions that are flanked by short tracts of sequence identity, or microhomology (MH) 4 .…”