Abstract
Chemical inhibitors of the checkpoint kinases have shown promise in the treatment of cancer, yet their clinical utility may be limited by a lack of molecular biomarkers to identify specific patients most likely to respond to therapy. To this end, we screened 112 known tumor suppressor genes for synthetic lethal interactions with inhibitors of the CHEK1 and CHEK2 checkpoint kinases. We identified eight interactions, including the Replication Factor C (RFC)-related protein RAD17. Clonogenic assays in RAD17 knockdown cell lines identified a substantial shift in sensitivity to checkpoint kinase inhibition (3.5-fold) as compared to RAD17 wild-type. Additional evidence for this interaction was found in a large-scale functional shRNA screen of over 100 genotyped cancer cell lines, in which CHEK1/2 mutant cell lines were unexpectedly sensitive to RAD17 knockdown. This interaction was widely conserved, as we found that RAD17 interacts strongly with checkpoint kinases in the budding yeast Saccharomyces cerevisiae. In the setting of RAD17 knockdown, CHEK1/2 inhibition was found to be synergistic with inhibition of WEE1, another pharmacologically relevant checkpoint kinase. Accumulation of the DNA damage marker γH2AX following chemical inhibition or transient knockdown of CHEK1, CHEK2 or WEE1 was magnified by knockdown of RAD17. Taken together, our data suggest that CHEK1 or WEE1 inhibitors are likely to have greater clinical efficacy in tumors with RAD17 loss-of-function.
Highlights
Loss-of-function of cell cycle checkpoints is frequent in tumors [1, 2]
We found that chemical inhibition of CHEK1/2 with AZD7762 resulted in a dose dependent increase in γH2AX, with significantly greater induction of γH2AX seen in RAD17 knockdown samples relative to nonsilencing controls in both HeLa (p < 0.05, Figure 4A) and LN428 cell lines (p < 0.05, Supplementary Figure 6a)
Assuming these cell lines are a reasonable surrogate for human tumors, it suggests that for each patient with a tumor sensitive to checkpoint kinase inhibition, as many as nine patients with resistant tumors will be treated with ineffective therapy
Summary
Loss-of-function of cell cycle checkpoints is frequent in tumors [1, 2]. Because such tumors have increased reliance on the remaining elements of cell cycle control, targeting the kinases that regulate cell cycle checkpoints has been proposed as an anti-cancer therapeutic strategy [2,3,4,5]. The recent report of a remarkable and possibly curative response to the CHEK1 and CHEK2 (CHEK1/2) inhibitor AZD7762 in a small-cell tumor with RAD50 mutation illustrates what is possible when a targeted therapy is given to a susceptible tumor [19]. This case highlights the importance of using molecular markers to prospectively identify patients with susceptible tumors so that they can be put on effective therapy
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