Comparative Activity and Off-Target Effects in Cells of the CHK1 Inhibitors MK-8776, SRA737, and LY2606368.

DNA damage activates the checkpoint protein CHK1 to arrest cell cycle progression, providing time for repair and recovery. Consequently, inhibitors of CHK1 (CHK1i) enhance damage-induced cell death. Additionally, CHK1i elicits single agent cytotoxicity in some cell lines. We compared three CHK1i that have undergone clinical trials and exhibited different toxicities. Each CHK1i inhibits other targets at higher concentrations, and whether these contribute to the toxicity is unknown. We compared their sensitivity in a panel of cell lines, their efficacy at inhibiting CHK1 and CHK2, and their ability to induce DNA damage and abrogate damage-induced S phase arrest. Published in vitro kinase analyses were a poor predictor of selectivity and potency in cells. LY2606368 was far more potent at inhibiting CHK1 and inducing growth arrest, while all three CHK1i inhibited CHK2 at concentrations 10- (MK-8776 and SRA737) to 100- (LY2606368) fold higher. MK-8776 and SRA737 exhibited similar off-target effects: higher concentrations demonstrated transient protection from growth inhibition, circumvented DNA damage, and prevented checkpoint abrogation, possibly due to inhibition of CDK2. Acquired resistance to LY2606368 resulted in limited cross-resistance to other CHK1i. LY2606368-resistant cells still abrogated DNA damage-induced S phase arrest, which requires low CDK2 activity, whereas inappropriately high CDK2 activity is responsible for sensitivity to CHK1i alone. All three CHK1i inhibited protein synthesis in a sensitive cell line correlating with cell death, whereas resistant cells failed to inhibit protein synthesis and underwent transient cytostasis. LY2606368 appears to be the most selective CHK1i, suggesting that further clinical development of this drug is warranted.

Activation of CDC25A phosphatase is limited by CDK2/cyclin A-mediated feedback inhibition.

Cyclin-dependent kinase (CDK) 1 complexed with cyclin B is a driver of mitosis, while CDK2 drives S phase entry and replicon initiation. CDK2 activity increases as cells progress through S phase, and its cyclin partner switches from cyclin E to cyclin A. Activation of CDK2 requires dephosphorylation of tyrosine-15 by CDC25A. DNA damage activates the checkpoint protein CHK1, which phosphorylates and degrades CDC25A to prevent activation of CDK2 and protect from cell cycle progression before damage is repaired. CHK1 inhibitors were developed to circumvent this arrest and enhance the efficacy of many cancer chemotherapeutic agents. CHK1 inhibition results in the accumulation of CDC25A and activation of CDK2. We demonstrate that inhibition of CDK2 or suppression of cyclin A also results in accumulation of CDC25A suggesting a feedback loop that prevents over activation of this pathway. The feedback inhibition of CDC25A targets phosphorylation of S88-CDC25A, which resides within a CDK consensus sequence. In contrast, it appears that CDK complexes with cyclin B (and possibly cyclin E) stabilize CDC25A in a feed-forward activation loop. While CDK2/cyclin A would normally be active at late S/G2, we propose that this feedback inhibitory loop prevents over activation of CDK2 in early S phase, while still leaving CDK2/cyclin E to catalyze replicon initiation. One importance of this observation is that a subset of cancer cell lines are very sensitive to CHK1 inhibition, which is mediated by CDK2/cyclin A activity in S phase cells. Hence, dysregulation of this feedback loop might facilitate sensitivity of the cells.

Inhibition of Protein Synthesis Induced by CHK1 Inhibitors Discriminates Sensitive from Resistant Cancer Cells.

The DNA-damage-activated checkpoint protein CHK1 is required to prevent replication or mitosis in the presence of unrepaired DNA damage. Inhibitors of CHK1 (CHK1i) circumvent this checkpoint and enhance cell killing by DNA-damaging drugs. CHK1i also elicit single-agent cytotoxicity in a small subset of cell lines. Resolving the mechanisms underlying the single-agent activity may permit patient stratification and targeted therapy against sensitive tumors. Our recent comparison of three CHK1i demonstrated that they all inhibited protein synthesis only in sensitive cells. LY2606368, the most selective of these CHK1i, was used in the current study. Comparison across a panel of cell lines demonstrated that sensitive cells died upon incubation with LY2606368, whereas resistant cells underwent growth inhibition and/or cytostasis but failed to die. Sensitive cells exhibited inhibition of protein synthesis, elevated DNA damage, impaired DNA repair, and subsequently death. The consequence of CHK1 inhibition involved activation of cyclin A/CDK2 and MUS81, resulting in DNA damage. This damage led to activation of AMPK, dephosphorylation of 4E-BP1, and inhibition of protein synthesis. Inhibition of MUS81 prevented activation of AMPK, while inhibition of AMPK enhanced DNA repair and cell survival. The activation of AMPK may involve a combination of LKB1 and CaMKKß. This study raises questions concerning the potential importance of the inhibition of protein synthesis in response to other drugs, alone or in combination with CHK1i. It also highlights the importance of clearly discriminating among growth inhibition, cytostasis, and cell death, as only the latter is likely to result in tumor regression.

Sensitivity of cells to ATR and CHK1 inhibitors requires hyperactivation of CDK2 rather than endogenous replication stress or ATM dysfunction.

DNA damage activates cell cycle checkpoint proteins ATR and CHK1 to arrest cell cycle progression, providing time for repair and recovery. Consequently, inhibitors of ATR (ATRi) and CHK1 (CHK1i) enhance damage-induced cell death. Intriguingly, both CHK1i and ATRi alone elicit cytotoxicity in some cell lines. Sensitivity has been attributed to endogenous replications stress, but many more cell lines are sensitive to ATRi than CHK1i. Endogenous activation of the DNA damage response also did not correlate with drug sensitivity. Sensitivity correlated with the appearance of γH2AX, a marker of DNA damage, but without phosphorylation of mitotic markers, contradicting suggestions that the damage is due to premature mitosis. Sensitivity to ATRi has been associated with ATM mutations, but dysfunction in ATM signaling did not correlate with sensitivity. CHK1i and ATRi circumvent replication stress by reactivating stalled replicons, a process requiring a low threshold activity of CDK2. In contrast, γH2AX induced by single agent ATRi and CHK1i requires a high threshold activity CDK2. Hence, phosphorylation of different CDK2 substrates is required for cytotoxicity induced by replication stress plus ATRi/CHK1i as compared to their single agent activity. In summary, sensitivity to ATRi and CHK1i as single agents is elicited by premature hyper-activation of CDK2.