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Hocke, Sandra (2016): A synthetic lethal screen identifies ATR-inhibition as a novel therapeutic approach for POLD1-deficient cancers. Dissertation, LMU München: Faculty of Medicine



ATR (Ataxia Telangiectasia-mutated and Rad3-related) kinase acts as a central regulator and mediator of the replication checkpoint in response to DNA damage and replication stress. To initiate DNA repair, ATR induces a G2/M cell cycle arrest and stabilizes the replication fork during DNA synthesis. Pharmacological inhibition of ATR has recently been demonstrated to eliminate tumor cells in colorectal cancers (CRCs) but the underlying genetic determinants remain unexplained. Identification of these determinants is essential to develop novel tumor therapy strategies. Due to ATRs` essential role in DNA repair, synthetic lethal interactions of DNA repair mechanisms with ATR are suggested to mediate ATR inhibitor specific tumor cell killing. Using the concept of synthetic lethality, a synthetic lethal screen was conducted in a genetically well-defined ATR knock-in model of DLD1 CRC cells to identify potential genetic determinants eliciting ATR inhibitor-specific tumor cell killing. Applying a siRNA library directed against 288 DNA-repair genes, a set of DNA-repair genes was identified whose knockdown caused either the selective killing of DLD1 ATR-deficient cells (n=6) or an ATR genotype-independent cell killing of DLD1 ATR-proficient and DLD1 ATR-deficient cells (n=20). The strongest synthetic lethal effect was observed between ATR and POLD1 confirmed by kinetic and titration analysis upon POLD1 knockdown in ATR-deficient cells. ATR genotype-dependent POLD1 knockdown-induced cell killing was reproducible pharmacologically in POLD1-depleted DLD1 as well as a panel of other CRC cell lines by using chemical inhibitors of ATR or of its major effector kinase CHK1. Mechanistically, POLD1 depletion in DLD1 ATR-deficient cells caused caspase-dependent apoptosis without preceding cell cycle arrest and increased DNA damage along with impaired DNA repair, as demonstrated by elevated and sustained levels of γ H2AX focus formation and pan-nuclear γ H2AX staining. Irradiation-induced spatial co-localization of POLD1 with ATR as well as of POLD1 with γ H2AX at sites of DNA damage was further detected. Notably, inactivating POLD1 mutations have recently been described in four families with multiple colorectal adenomas and CRC. In three of these families endometrial tumors were diagnosed. Considering that whole genome-sequencing might determine the POLD1 mutation rates in different tumor entities, our data could have clinical implications in tumor genotype-based cancer therapy with regard to patients harboring those POLD1-deficient tumors, which might respond to chemical inhibition of the ATR/CHK1-axis. POLD1 deficiency might thus represent a predictive marker for treatment response towards ATR- or CHK1 inhibitors, which are currently tested in clinical trials. Long-term, the development of selective POLD1-targeted drugs might further broaden the applicability of the identified synthetic lethality with ATR-inhibitors.