Mechanisms and markers of CHK1 inhibitors sensitivity in melanoma

Abstract

Landscape for the treatment of cancer patients has recently been changed with the arrival of targeted therapies in different malignancies with the drugs targeting specific mutations and genetic alterations such as, EGF-receptor blockers and BRAF inhibitors, and the drugs such as PARP inhibitors which exploit defects in the cancers and cause synthetic lethality. In this thesis, I investigated to understand the mechanism of action of a drug that targets the cell cycle checkpoint regulator, checkpoint kinase 1 (CHK1) and explored its potential therapeutic usage in specific cancer types. Checkpoint kinase 1 inhibitor (CHK1i) as a single-agent treatment is effective in some of the cancer types with high levels of replication stress, including melanoma. However, the mechanism and manner of cell-killings induced by CHK1i single-agent treatment is still poorly understood. To identify the patient population who can benefit from CHK1i single-agent treatment, it is important to understand how single-agent CHK1i induced the cell killing. CHK1i has been investigated in pre-clinical studies and clinical trials, and shown to enhance the efficacy of chemotherapeutic drugs, particularly those that promote replication stress such as gemcitabine.Most of those investigations were based on the previous notion that CHK1i abrogates the G2/M phase checkpoint to cause mitotic catastrophe and results in cancer cell death. However, considering numerous roles of CHK1 playing in cell cycle and DNA damage response pathway, this mechanism alone may not represent the entirety of sensitivity to CHK1i. CHK1 has different roles in S and G2/M phases of cell cycle such as controlling the G2/M checkpoint, stabilising the stalled replication fork, and inhibiting the apoptosis together with other DNA damage repair and cell cycle checkpoint regulators. When one of these other regulators/pathways has defects, inhibiting CHK1 can be synthetically lethal to the cancer cells. Another CHK1 function in S phase is regulating CDC25A; CHK1 triggers the destabilisation of CDC25A upon DNA damage and replication stress.The normal role of CDC25A in S phase progression is activation of CDK2 which is required for progression into and through S phase. CDC25A dysregulation and overexpression has been reported in various cancers and shown to create increased replication stress in the cells. Tumours with high level of replication stress have been suggested to be selectively susceptible to the inhibition of CHK1 and its upstream regulator, ATR.In this thesis, it was firstly shown that S phase cell cycle checkpoint defect is a common feature of CHK1i-hypersensitive melanoma cell lines, and this defect is often associated with failure to degrade CDC25A in response to replication stress. Furthermore, CDC25A over-expression and/or dysregulation contributes to CHK1i sensitivity in hypersensitive melanoma cell lines. Secondly, it was demonstrated that CHK1i induced high level of replication stress via RPA hyperphosphorylation and subsequently RPA depletion occurred in h...