Investigating the predictors of sensitivity to the Chk1 inhibitor PF-00477736 in melanoma

© 2013 Britanto Dani WicaksonoChk1 and Chk2 are two of the most important and highly studied checkpoint kinases that help protect cells from endogenous or exogenous insults which could damage DNA. When activated they arrest cell cycle at specific checkpoints in order for cells to repair damage before resuming the cell cycle and mitosis. Recently, several inhibitors of Checkpoint kinases have been developed which were designed to sensitise TP53 mutant cancers with defective G1 checkpoints to DNA damaging chemotherapy. However, a recent study has demonstrated that TP53 mutation alone does not predict sensitivity to Chk1 inhibitors. Moreover, inhibition of Chk1 by PF-00477736 (PF-736) as a single agent was also recently shown to be effective in mediating p53-dependent apoptosis in lymphoma cells by increasing the level of MYC oncogene-induced DNA damage. Other reports have also shown that mutant BRAF and RAS (NRAS) which are the predominant oncogenes found in melanoma could induce DNA damage in epithelial cells and thyroid cancers, respectively. This led to the hypothesis that melanomas which are predominantly TP53 wild-type and BRAF or NRAS mutant would be responsive to Chk1 inhibitors as single agents. Screening for drug sensitivity across a panel of 30 melanoma cell lines showed a wide range of IC50 values following 3 days treatment with PF-736. BRAF and TP53 mutation status were not found to affect sensitivity to PF-736. Due to unavailability of NRAS mutation data at the time of analysis, it is still unconfirmed whether BRAF and NRAS as the source of oncogene DNA damage in melanoma influence the sensitivity of melanoma to PF-736 treatment. Several cell lines categorised as sensitive was revealed to have high Total Growth Inhibition (TGI) values. This suggests that there may be genetic variations in the cells that have high TGI particularly in cell death activation pathways that affect the sensitivity of these cells to Chk1 inhibition. Previously, it has been reported that up-regulation of p53 negative-regulators such as Mdm4 and Bcl-2 family anti-apoptotic proteins were responsible for the defective p53-mediated apoptosis responses in p53 wild-type melanomas. Overexpression of BCL2 or BCLxl was able to prevent apoptosis in C002-M1, a cell line that underwent apoptosis following PF-736 treatment. However, MCL1 overexpression did not result in similar effect. Assessment of Mdm4 basal protein levels did not reveal any significant correlation with PF-736 IC50. Further assessment utilising MDM4 knockdown by shRNA in the A375 cell line expressing high Mdm4 protein expression did not result in sensitisation to PF-736. These data suggested that the increased levels of Bcl-2 or Bcl-XL proteins were able to decrease the level of apoptosis but did not significantly alter sensitivity to PF-736 in C002-M1 cell line. Additionally knockdown of MDM4 levels did not alter PF-736 IC50 in a cell line with high Mdm4 protein expression. Hence it is possible that other cellular characteristics such as the level of inherent DNA damage recently reported to be important in sensitivity of melanoma to Checkpoint kinase inhibition, could provide more insight in revealing factors that determine the sensitivity of PF-736 in melanoma. γH2AX is a marker of double strand break which can be utilised as a measure of DNA damage. In an attempt to assess γH2AX as a biomarker of sensitivity, the levels of inherent γH2AX and fold change following PF-736 treatment was determined. The result showed that high level of inherent γH2AX showed a significant correlation with low PF-736 IC50, suggesting that high levels of endogenous DNA damage is associated with sensitivity to PF-736. Furthermore, the average fold increase in levels of γH2AX after PF-736 treatment across the entire cell line panel also significantly correlated with PF-736 IC50. Additionally, we discovered that increase in pChk2 T68 levels following PF-736 treatment also correlated with response to PF-736 in the 30 melanoma cell line panel. Increase in pChk1 S345 following Chk1 inhibition, previously reported as a potential pharmacodynamic biomarker of Chk1 inhibition was confirmed across the panel following the same PF-736 treatment demonstrating the biomarkers identified were associated with Chk1 inhibition. A significant component of this project was to identify predictors of sensitivity using a pharmacogenomic whole genome-wide approach and a candidate approach. For the candidate approach, gene expression of selected genes from pathways related to the DNA damage response pathway for the 30 melanoma cell line panel were analysed with IC50 from SRB assay utilising linear regression analysis to search for significant correlation between sensitivity to PF-736 and gene expression. Gene expression of NBN, CHK2, RAD21 and RAD54B, which are components of the DSB repair particularly HR repair, significantly associated with sensitivity to PF-736. An unbiased Pearson correlation analysis between whole-genome gene expression microarray data and IC50 data from SRB assay of the 30 melanoma cell line panel also showed good correlation between DSB repair genes and sensitivity to PF-736. From this analysis, RAD21 and RAD54B were showed to have a good correlation with sensitivity to PF-736. An SVM prediction model built using the gene expression data pre-processed using the Pearson correlation analysis to include only genes that are differentially expressed across the panel and their sensitivity to PF-736 based on IC50, to discover a gene set that can classify melanoma cell lines between sensitive and resistant to PF-736. The result showed that the SVM model was able to predict at 100% accuracy with a cut-off of 2048 nM. Some of the genes included in the gene set for the SVM prediction model were genes that are important for DSB repair particularly HR such as RAD21, RAD54B and NSMCE2 confirming the importance of this specific DNA repair pathway in association with sensitivity to PF-736 particularly in melanoma. In conclusion, this study has revealed the importance of the DSB Response pathway in predicting the sensitivity to the Chk1 specific inhibitor PF-736 in melanoma. As there are also several other cancers which exhibit oncogene-induced DNA damage and aberrant DSB repair pathways, this study may help in predicting the sensitivity of these types of cancers in with single agent Chk1 therapy. Furthermore, this project has verified that melanoma cell lines are sensitive to Chk1 inhibition therapy as a single agent and that the level of inherent DNA damage may provide insight into the sensitivity of melanoma to Chk1 inhibition. Moreover, candidate and genome-wide pharmacogenomic analyses have revealed the importance of DSB repair pathway and Bcl-2 family anti-apoptotic genes in predicting sensitivity to Chk1 inhibition. Although functional study on C002-M1 cell line did not show evidence that the level of Bcl-2 family anti-apoptotic proteins can affect sensitivity, the western blot data, candidate gene and genome wide predictor analysis across the 30 melanoma cell line panel showed that there is a significant correlation between Bcl-2 family anti-apoptotic proteins particularly Bcl-XL to sensitivity to PF-736, which warrants further investigation. As a future direction, the current predictor model will need to be tested with a validation set of samples, to test its robustness in predicting sensitivity to single agent Chk1 inhibition therapy. To progress further the assessment of the usefulness of Chk1 inhibition therapy in melanoma, an investigation on Chk1 inhibition in combination with DNA damaging chemotherapy which was shown to be promising in the preliminary study, will need to be carried out