We previously demonstrated that cdk1 depletion or inhibition in lung cancer cells reduces BRCA1 focus formation and the activation of DNA damage-induced checkpoint control8
. We have now implicated cdk1 in HR repair in these cells. In response to PARP inhibition, reduced cdk1 activity results in chromosomal aberrations and cell death, in agreement with previous studies demonstrating that HR-deficient cells are hypersensitive to PARP inhibitor therapy10–12
. Furthermore, cdk1 was previously identified in an siRNA library screen designed to identify proteins that when depleted cause sensitivity to PARP inhibitors29
. In contrast to cdk1, cdk2 phosphorylates BRCA2, impairing its interaction with Rad51, thereby limiting HR until cell cycle arrest is accomplished and cdk2 activity extinguished30
. Consistent with these data, depletion of cdk2 did not significantly reduce HR in the cell lines examined, and in several instances, increased the percentage of GFP-positive cells in the gene conversion assay.
In yeast, cdk1 is essential for multiple steps of HR4
. Although cdk1 may directly influence the function of other HR proteins, it is likely that reduced cdk1 activity sensitizes cells to PARP inhibition through disruption of BRCA1 function in lung cancer cells. Cdk1 depletion afforded an increase in sensitivity to PARP inhibition by >100-fold, similar to what is seen in BRCA1-deficient cells11
, and combined depletion of cdk1 and BRCA1 did not sensitize cells to a greater degree than depletion of either alone. Additionally, we previously showed that selective cdk1 inhibition does not affect DNA-end resection in these cells, likely because cdk2 can compensate in this process; such compensation does not occur at the level of BRCA1 focus formation8
Our in vitro
observations were translated in xenograft models, where cdk1 inhibition resulted in a reduction in the PARP inhibitor-mediated increase in BRCA1 but not γ-H2AX foci-containing cells. We also studied mice with lung-specific conditional activating Kras
and inactivating p53
mutations that develop highly aggressive lung adenocarcinomas with short latency compared to those driven by KrasG12D
tumors with concomitant p53 inactivation are also less responsive to cytotoxic therapy than those with wild-type p5332
. The cdk and PARP inhibitor combination induced regression and disease stabilization over 1–3 weeks of treatment in established tumors. Although resistance was documented by 6 weeks, a subset of mice demonstrated sustained response, so that combination therapy significantly increased median survival. Our results suggest a possible approach to lung cancers harboring this genotype that frequently have poor outcomes33
, as well as to other BRCA-proficient tumors.
Importantly, mice treated with both AG024322 and AG014699 had no organ or normal tissue toxicities. In accordance with these observations, cdk1 depletion or inhibition did not sensitize RPE cells or non-transformed HS578TBst breast epithelial cells to PARP inhibition in vitro
. Of note, cdk2 cannot compensate for the loss of cdk1 in cellular proliferation in non-transformed cells to the same degree as in cancer cells34
; consequently, RPE cells were arrested at G2/M when cdk1 was depleted. Following PARP inhibition, single-strand breaks (SSBs) degenerate to DSBs during S phase traversal; non-transformed cells, arrested in G2/M after cdk1 depletion, likely do not accumulate SSBs followed by DSBs, demonstrated by a failure to accumulate γ-H2AX, and are therefore not sensitive to combined cdk1 and PARP inhibition. The data therefore suggest that cdk and PARP inhibitor combinations will have a therapeutic window.
Our data support the clinical development of combined cdk1 and PARP inhibition. Analysis of cdk-mediated BRCA1 phosphorylation suggests that 70–90% reduction in cdk1 activity by small molecule inhibitors results in sensitization to PARP inhibition in vitro, translating to substantial anti-tumor activity in vivo, and serves as a guide for the target degree of inhibition desirable in clinical trials.
In summary, the present study is the first to use targeted kinase inhibition to inactivate BRCA1, handicap the HR DNA repair machinery and selectively sensitize transformd cells to PARP inhibition. This approach avoids the use of toxic DNA damaging chemotherapeutic drugs, thus providing the potential to extend well tolerated PARP inhibition to treatment for BRCA-proficient cancers.