Preclinical Development Hsp90 Inhibitor–Mediated Disruption of Chaperone Association of ATR with Hsp90 Sensitizes Cancer Cells to DNA Damage

Following DNA damage that results in stalled replication fork, activation of ATR-CHK1 signaling induces the DNA damage response (DDR) in transformed cells. In the present studies on human cervical and breast cancer cells, we determined the effects of hsp90 inhibition on the levels and accumulation of DNA damage/ repair–associated proteins following exposure to g-ionizing radiation (IR; 4 Gy). We show that hsp90 inhibition with 17-allylamino-demehoxygeldanamycin or the novel, nongeldanamycin analogue AUY922 (resorcinylic isoxazole amide; Novartis Pharma) dose-dependently reduced the levels of ATR and CHK1 without affecting ATM levels. AUY922-mediated depletion of ATR and CHK1 was associated with an increase in their polyubiquitylation and decreased binding to hsp90. Cotreatment with bortezomib partially restored AUY922-mediated depletion of ATR and CHK1 levels. Additionally, treatment with AUY922 reduced the accumulation of ATR, p53BP1, and CHK1 but not g-H2AX to the sites of DNAdamage. Following exposure to IR, AUY922 treatment abrogated IR-induced phospho (p)-ATR and p-CHK1 levels, but significantly enhanced g-H2AX levels. AUY922 treatment also increased IR-induced accumulation of the cells in G2–M phase of the cell cycle, inhibited the repair of IR-induced DNA damage, and augmented IR-mediated loss of clonogenic survival. Short hairpin RNA–mediated depletion of ATR also inhibited IR-induced p-ATR and p-CHK1, but increased g-H2AX levels, sensitizing cancer cells to IR-induced apoptosis and loss of clonogenic survival. These findings indicate that ATR is a bona fide hsp90 client protein and post-IR administration of AUY922, by inhibiting ATR-CHK1–mediated DDR, sensitizes cancer cells to IR.Mol Cancer Ther; 10(7); 1194–206. 2011 AACR.