DNA Damage Response Inhibitors of DNA-PKcs, PARP, RAD51, ATR and ATM All Sensitize Cancer Cells to X-Rays and Protons

Abstract

<h3>Purpose/Objective(s)</h3> Radiotherapy (RT) controls tumors primarily by inducing DNA damage. However, some tumor cells can repair RT-induced DNA damage and are thus radioresistant. RT could be made more effective by combining it with inhibitors of DNA damage response proteins (DDRi) to selectively render tumor cells more susceptible to RT-induced DNA damage. There are several DDRi currently approved by the FDA or in clinical trials for use as monotherapy, including inhibitors of DNA-PKcs, PARP, Rad 51, ATR and ATM. These proteins function in non-homologous end-joining (classical and alternative), homologous recombination, base excision repair or cell cycle checkpoint and disrupting any of which may enhance RT effectiveness. Because protons produce much more complex DNA lesion compared to x-rays, protons may amplify the effect of DDRis and further enhance potential activation of anti-tumor immune signaling. Our hypothesis is that DDRis sensitize cancer cells to RT and the sensitization effect is amplified by protons more so than x-rays with an enhancement in upstream immune signaling. <h3>Materials/Methods</h3> We used 4 cancer cell lines: H460, H1299, PANC-1 and Panc 10.05 and 5 DDRis targeting DNA-PKcs (NU7441), PARP (AZD2281), Rad 51 (B02), ATR (AZD6738) and ATM (KU55933) at various concentrations (0.1-10 µM). We irradiated cells with 6 MV x-rays or 9.9 keV/µm protons (dose-weighted LET in water) alone or with DDRis at various dose levels to assess cell survival via clonogenic assay, DNA damage via gH2AX and 53BP1 foci, micronuclei (MN) and cGAS-positive micronuclei (NM-cGAS⁺), the latter two being precursors of antitumor immunity. <h3>Results</h3> We observed that DDRis sensitized both x-rays and protons. Protons+ATMi (10 µM) sensitization relative to x-rays alone was increased 3.51-fold (± 0.09). The RBE ranged from 0.79 ± 0.03 to 1.54 ± 0.09 and were negatively correlated with x-rays sensitivity (r = -0.6160), suggesting that the greater the DDRi-induced sensitization, the less benefit there will be from the proton LET effect. Nonetheless, the sensitization relative to x-rays alone from combining DDRi with protons was generally larger (30 ± 3% on average). Generally, the combination of DDRi with RT increased gH2AX and 53BP1 foci at 24 h after radiation, and MN and MN-cGAS⁺ at 24 and 72 h after radiation with protons+DDRi having a higher increase relative to x-rays alone than x-rays+DDRi. <h3>Conclusion</h3> Inhibition of DNA-PKcs, PARP, Rad 51, ATR and ATM substantially sensitized several cancer cell lines to x-rays and protons. Although protons+DDRi reduced proton RBE, the inhibitor-induced sensitization outweighs the RBE loss, resulting in a net sensitization beyond x-rays+DDRi. The increased MN number indicates that pairing RT with DDRis may provide an additional benefit of antitumor immune stimulation. Additional preclinical work is warranted to elucidate the complex interaction of DDRi and RT in vivo.