Synthetic lethal interactions between ATR and DNA polymerases as a novel concept for individualized cancer therapy
The chemical inhibition of the kinase ATR, a central regulator of the DNA damage response, eliminates subsets of cancer cells in different tumors. This effect is at least partly attributable to the synthetic lethal relationship between ATR and certain DNA repair genes. In a previous study of our lab...
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|The chemical inhibition of the kinase ATR, a central regulator of the DNA damage response, eliminates subsets of cancer cells in different tumors. This effect is at least partly attributable to the synthetic lethal relationship between ATR and certain DNA repair genes. In a previous study of our laboratory, POLD1 and PRIM1 were identified to act synthetically lethal with ATR. Thus, this dissertation was divided into two subprojects to characterize the synthetic lethal relationship between ATR and each of the two identified proteins individually.
The first subproject addressed the characterization of the synthetic lethal relationship between ATR and PRIM1, the catalytic subunit of primase of the polymerase α-primase complex. Applying a genetic ATR knock-in model of colorectal cancer cells, we confirmed that siRNA-mediated PRIM1 depletion inhibits proliferation of ATR-deficient cells, and excluded both off-target effects of the applied siRNA targeting PRIM1, and artifacts due to clonal variation by using an ATR re-expressing cell clone. We expanded these data by demonstrating in a panel of different cancer cell lines that not only genetically induced ATR deficiency but also chemical inhibition of ATR or its main effector kinase CHK1 inhibits proliferation upon PRIM1 depletion. Mechanistically, PRIM1 depletion in ATR-deficient cells caused S-phase stasis with no evidence for increased DNA damage followed by Wee1-mediated activation of caspase 8 and consequently of apoptosis. As PRIM1 inactivation sensitizes cancer cells to ATR and CHK1 inhibitors, alterations in PRIM1 or other components of the polymerase α-primase complex could represent a novel concept for the individualized cancer therapy using ATR pathway inhibitors.
The second subproject addressed the further characterization of the synthetic lethal relationship between ATR and POLD1, the catalytic subunit of polymerase δ. In the previous study of our laboratory, we demonstrated that not only genetically induced ATR deficiency but also chemical inhibition of the ATR pathway inhibits
proliferation upon POLD1 depletion. To extend these data, we now characterized the impact of defined POLD1 variants on the sensitivity to ATR pathway inhibitors.
Therefore, we used the CRISPR/Cas9 technique in the colorectal cancer cell line DLD-1, which harbors four heterozygous POLD1 variants, to establish heterozygous POLD1-knockout clones with exclusive expression of distinct POLD1 variants. These knockout clones served as a model to determine the functional significance of individual POLD1 variants. We demonstrated that of the four variants analyzed onlyPOLD1R689W impairs POLD1 function, as shown by compensatory ATR pathway activation and impaired DNA replication. Moreover, the POLD1-R689W variant in cancer cells led to a strong decrease of cell survival in vitro and decelerated growth of murine xenograft tumors in vivo upon treatment with ATR pathway inhibitors, which further corroborates a potential clinical relevance of our data. Our here established and characterized functional model could thus be used to complement algorithm-based models to predict the pathogenicity of tumor-specific variants of uncertain significance, and improve their accuracy. Furthermore, our data enable a novel and potentially clinically relevant concept for the individualized and genotype-based therapy of POLD1-deficient cancers with ATR pathway inhibitors.
Taken together with the in the literature identified variants of uncertain significance of POLD1 and POLE as well as defects in other polymerases in cancer the data
of this dissertation illustrate the emerging role of tumor-specific alterations in DNA polymerases as novel therapeutic targets. The comprehensive identification and
functional characterization of all polymerases and their possible synthetic lethal partners is thus essential in future studies.