Phenotypic enhancement of thymidylate synthetase pathway inhibitors following ablation of Neil1 DNA glycosylase/lyase.
Inhibition of thymidine biosynthesis is a clinically-validated therapeutic approach for multiple cancers. Inhibition of thymidylate synthetase (TS) leads to a decrease in cellular TTP levels, replication stress and increased genomic incorporation of uridine (dUMP). Thus, inhibitors of this pathway (such as methotrexate) can drive a multitude of downstream cell cycle checkpoint and DNA repair processes. Genomic dUMP is recognized by the base excision repair (BER) pathway. Using a synthetic lethal siRNA-screening approach, we systematically screened for components of BER that, when ablated, enhanced methotrexate response in a high content γ-H2A.X bioassay. We observed specific ablation of the mixed function DNA glycosylase/lyase Neil1 phenotypically enhanced several inhibitors of thymidine biosynthesis, as well as cellular phenotypes downstream of gemcitabine, cytarabine and clofarabine exposure. These synthetic lethal interactions were associated with significantly enhanced accumulation of γ-H2A.X and improved growth inhibition. Significantly, following TS pathway inhibition, addition of exogenous dTTP complemented the primary Neil1 γ-H2A.X phenotypes. Similarly, co-depletion of Neil1 with Cdc45, Cdc6, Cdc7 or DNA polymerase β (PolB) suppressed Neil1 phenotypes. Conversely, co-depletion of Neil1 with the Rad17, Rad9 ATR, ATM and DNA-PK checkpoint/sensor proteins appears primarily epistatic to Neil1. These data suggest Neil1 may be a critical mediator of BER of incorporated dUMP following TS pathway inhibition.
Merck Research Laboratory, Palo Alto, CA, USA.
Lorena
L
Taricani
Frances
F
Shanahan
Rob H
RH
Pierce
Timothy J
TJ
Guzi
David
D
Parry
D015415:Biomarkers / Q000378:metabolism; D002460:Cell Line; D000071877:Checkpoint Kinase 1; D045647:DNA Glycosylases / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair; D004791:Enzyme Inhibitors / Q000378:metabolism; D005493:Folic Acid Antagonists / Q000378:metabolism; D006657:Histones / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008727:Methotrexate / Q000378:metabolism; D019384:Nucleic Acid Synthesis Inhibitors / Q000378:metabolism; D010641:Phenotype; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D011799:Quinazolines / Q000378:metabolism; D013876:Thiophenes / Q000378:metabolism; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
D015415:Biomarkers; D004791:Enzyme Inhibitors; D005493:Folic Acid Antagonists; C468783:H2AX protein, human; D006657:Histones; D019384:Nucleic Acid Synthesis Inhibitors; D011799:Quinazolines; D013876:Thiophenes; D013940:Thymidylate Synthase; D011494:Protein Kinases; D000071877:Checkpoint Kinase 1; D045647:DNA Glycosylases; C473634:NEIL1 protein, human; C068874:raltitrexed; D008727:Methotrexate