mTORC1 and DNA-PKcs as novel molecular determinants of sensitivity to Chk1 inhibition

Abstract

Background: Chk1 inhibitors are currently under clinical evaluation as single agents and in combination with cytotoxic chemotherapy. Understanding determinants of sensitivity and novel combinations is critical for further clinical development.

Methods: Potentiation of mTOR inhibitor cytotoxicity by the Chk1 inhibitor V158411 was determined in p53 mutant colon cancer cells. DNA damage response, expression levels of repair proteins, cell cycle effects and the contribution of alternative DSB repair pathways were further evaluated by western blotting and high content analysis.

Results: mTOR inhibitors AZD8055, RAD-001, rapamycin and BEZ235 induced synergistic cytotoxicity with the Chk1 inhibitor V158411 in p53 mutant colon cancer cells. Reduced FANCD2, RAD51 and RPA70, core proteins in homologous recombination repair (HRR) and interstrand crosslink repair (ICLR), following inhibition of mTOR was associated with increased V158411 induced DSBs and caspase 3-independent cell death. Dual mTOR and Chk1 inhibition activated DNA-PKcs. Cells defective in DNA-PKcs exhibited increased resistance to V158411 with Chk1 expression closely correlated to DNA-PKcs expression in various types of cancer.

Conclusions: Down regulation of proteins involved in HRR or ICLR by mTOR inhibitors is associated with increased sensitivity of human tumours to Chk1 inhibitors such as V158411. High levels of DNA-PKcs may be a potential biomarker to stratify patients to Chk1 inhibitor therapy alone or in combination with mTOR inhibitors.

Keywords: Chk1; DNA-PKcs; Double strand break repair; V158411; mTOR.

Figure 1

The Chk1 inhibitor V158411 and the mTOR inhibitors AZD8055, RAD‐001, rapamycin and BEZ235 demonstrate synergistic combinatorial activity in colon cancer cell lines. (A) GI50 and combination GI50 (cGI50) values in HT29, SW620 and Colo205 cancer cells grown anchorage dependently (2D), anchorage independently (3D) or as multicellular tumour spheroids (Sph) following 72 or 168 h treatment with rapamycin and 400 nM V158411. Pf values are indicated above the bars and were calculated from the average GI50 and cGI50 where Pf equals average GI50/average cGI50. *, P < 0.05; **, P < 0.01. (B) Growth inhibition values for 0.04 μM AZD8055, RAD‐001, rapamycin or BEZ235, or 0.01 μM gemcitabine in combination with 0 or 0.4 μM V158411 for 72 or 168 h. Values are the average of 3 determinations ± SD. (C) HT29 cells were treated with 0.1 μM AZD8055, RAD‐001, rapamycin, BEZ235 or gemcitabine in combination with 0 or 0.4 μM V158411. Loss of membrane integrity was assessed using NucGreen DEAD stain by high content analysis following 72 h compound treatment.

Figure 2

mTOR/DDR signalling pathway changes. HT29 cells were treated with 0.1 μM of the mTOR inhibitors AZD8055, RAD‐001, rapamycin or BEZ235, or gemcitabine in combination with 0 or 0.4 μM of the Chk1 inhibitor V158411 for 48 h. Expression levels of proteins involved in mTOR signalling (A) or DNA damage signalling (B) were determined by immunoblotting.

Figure 3

Inhibition of mTOR increases V158411 induced DNA DSB. HT29 cells were treated with 0.1 μM of the mTOR inhibitors AZD8055, RAD‐001, rapamycin or BEZ235, or gemcitabine in combination with 0 or 0.4 μM of the Chk1 inhibitor V158411 and pH2AX (S139) expression determined by high content analysis. (A) The fraction of γH2AX positive cells was determined by high content image analysis with Harmony software. The fold increase in γH2AX positive nuclei compared to V158411 + DMSO treatment alone is indicated above the bars. ***, P < 0.001. (B) Cell cycle phase of γH2AX positive nuclei was determined by high content image analysis using Harmony software following counterstaining for EdU and pHH3 (S10). Values are the average of 4 determinations ± SD.

Figure 4

Inhibition of Chk1 in combination with mTOR inhibition modulates key proteins involved in homologous recombination and intra‐strand crosslink repair. HT29 cells were treated with 0.1 μM of the mTOR inhibitors AZD8055, RAD‐001, rapamycin or BEZ235, or gemcitabine in combination with 0 or 0.4 μM of the Chk1 inhibitor V158411 for 48 h. (A) Expression levels of proteins involved in DNA damage signalling and repair were determined by immunoblotting. (B) Densitometric analysis of FANCD2, FANCF, RAD51 or RPA70 expression levels were determined. Expression was normalised to actin protein levels. (C) Expression levels of pDNA‐PKcs and DNA‐PKcs were determined by immunoblotting and (D) quantified by densitometric analysis. Values are the average of 3 independent determinations ± SD.

Figure 5

DNA‐PKcs expression but not non‐homologous end joining confers sensitivity to Chk1 inhibition. (A) Parental CHO‐AA8 or the DNA‐PKcs defective V3 or Ku80 defective xrs‐6 variants were treated with the Chk1 inhibitor V158411 for 24 h and clonogenic survival determined. Values are the average of ≥2 replicates in 3 independent experiments ± SD. (B) DNA‐PKcs defective M059J cells and the DNA‐PKcs corrected variant M059J‐Fus1 were treated with V158411 for 24 h and clonogenic survival determined. Values are the average of duplicate measurements in 3 independent experiments ± SD. (C) The correlation between PRKDC (DNA‐PKcs) and CHEK1 (Chk1) mRNA expression levels was determined from publicly available datasets in HCC, lung, ovarian, breast, and colon tumours.

Figure 6

Model of the involvement of mTOR and DNA‐PKcs in DNA damage response signalling.