Mechanistic Distinctions between CHK1 and WEE1 Inhibition Guide the Scheduling of Triple Therapy with Gemcitabine

Abstract

Combination of cytotoxic therapy with emerging DNA damage response inhibitors (DDRi) has been limited by tolerability issues. However, the goal of most combination trials has been to administer DDRi with standard-of-care doses of chemotherapy. We hypothesized that mechanism-guided treatment scheduling could reduce the incidence of dose-limiting toxicities and enable tolerable multitherapeutic regimens. Integrative analyses of mathematical modeling and single-cell assays distinguished the synergy kinetics of WEE1 inhibitor (WEE1i) from CHEK1 inhibitor (CHK1i) by potency, spatiotemporal perturbation, and mitotic effects when combined with gemcitabine. These divergent properties collectively supported a triple-agent strategy, whereby a pulse of gemcitabine and CHK1i followed by WEE1i durably suppressed tumor cell growth. In xenografts, CHK1i exaggerated replication stress without mitotic CDK hyperactivation, enriching a geminin-positive subpopulation and intratumoral gemcitabine metabolite. Without overt toxicity, addition of WEE1i to low-dose gemcitabine and CHK1i was most effective in tumor control compared with single and double agents. Overall, our work provides quantitative insights into the mechanisms of DDRi chemosensitization, leading to the rational development of a tolerable multitherapeutic regimen.Significance: Multiple lines of mechanistic insight regarding DNA damage response inhibitors rationally guide the preclinical development of a tolerable multitherapeutic regimen.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/11/3054/F1.large.jpg Cancer Res; 78(11); 3054-66. ©2018 AACR.

Figure 1. WEE1i and CHK1i synergise with gemcitabine with different potency.

(A) Combination assay. MIA PaCa-2 cells were treated for 72 hours. Data were analysed with two synergy mathematical models. Combinations of 10-30 nM GEM with a pair of non-inhibitory (10) equivalent concentrations of MK1775 and MK8776 are boxed in white. Bar graph shows the mean synergy score within the boxed surface. Data are represented as mean ± SEM, n=3. A two-tailed t-test was performed, *p≤0.05. (B) Clonogenic assay. Panc-1 cells were treated for 72 hours (30 nM GEM, 300 nM MK1775, 1 µM MK8776), and were left to grow after washout for 10 days. Data are represented as mean ± SEM, n=3. A one-way ANOVA analysis was performed, *p≤0.05, ****p≤0.0001. (C) Quantitative immunofluorescence of MIA PaCa-2 cells treated for 24 hours (10 nM GEM, 300 nM MK1775, 1 µM MK8776). Each blue, green or red dot marks a cell positive for γH2AX, RPA32 S4/8 or both, respectively. (D) Quantification of overlap between γH2AX (red) and RPA32 S4/8 (green) in MIA PaCa-2 cells treated for 24 hours (10 nM GEM, 300 nM MK1775, 1 µM MK8776). Percentage of γH2AX-positive cells in RPA32 S4/8-positive population is in green; percentage of RPA32 S4/8-positive cells in γH2AX-positive population is in red. Red arrowhead denotes γH2AX-positive cell; yellow arrowhead denotes γH2AX/RPA32 S4/8 double-positive cell. Data are represented as mean ± SEM, n=5. At least 2000 cells per condition per replicate were analysed. A two-tailed t-test was performed, *p≤0.05. Scale bar, 25 µm.

Figure 2. Mitotic stress underlies WEE1i cytotoxicity.

(A-B) S/G2 duration of MIA PaCa-2 FastFUCCI cells treated as indicated. At least 100 cells per condition were analysed. Data are represented as mean ± SEM. A one-way ANOVA analysis was performed, ****p≤0.0001. (C) Cell cycle duration of MIA PaCa-2 FastFUCCI cells treated with DMSO or 10 nM GEM+300 nM MK1775. A total of 243 cells were analysed. Data are represented as mean ± SEM. A two-tailed t-test was performed, *p≤0.05, **p≤0.01, ****p≤0.0001. (D) Percentage of MIA PaCa-2 FastFUCCI cells in the first and second cycles that underwent division or not following 10 nM GEM+300 nM MK1775. Fraction of non-dividing cells was further categorised according to cell fate. A total of 122 cells were analysed.

Figure 3. Inimical effects of WEE1i are spatiotemporally defined.

(A) Immunoblotting for MIA PaCa-2 cells treated for 24 hours. The graph shows densitometric analysis of CDK1 Y15/CDK1 or H3 S10/H3, relative to DMSO. (B) Quantification of DNA content of mitotic MIA PaCa-2 cells treated for 24 hours (10 nM GEM, 300 nM MK1775, 1 µM MK8776). Data are represented as mean, normalised to DMSO. A one-way ANOVA analysis was performed, ****p≤0.0001. (C) Quantification of mitotic Panc-1 cells in S phase. Cells were treated with 10 µM EdU for 45 minutes followed by 3 µM MK1775 for 1 hour. S and non-S phase cells were identified based on EdU and DNA contents. Percentage of H3 S10-positive cells is shown. (D) Quantification of damaged Panc-1 cells in S phase, treated as in (C). Percentage of γH2AX-positive cells is shown. (E-F) Quantification of mitotic Panc-1 cells harbouring ssDNA. Cells were grown with 10 µM BrdU for 48 hours, treated with 3 µM MK1775 and immunostained for native BrdU. In (E), the first column shows positive control, where sample was acid-denatured to confirm BrdU incorporation. Scale bar, 10 µm. In (F), percentage of mitotic cells is in black and percentage of native BrdU-positive mitotic cells (out of the respective mitotic fractions) in red. At least 2000 cells were analysed per time-point. Inset shows the total native BrdU intensity per mitotic cell. A one-way ANOVA analysis was performed, ****p≤0.0001.

Figure 4. A gemcitabine/CHK1i/WEE1i regimen enhances tumour cell suppression.

(A) Schematics of the spatiotemporal effects of WEE1i and CHK1i. (B) Correlative analysis between WEE1 and CHK1 mRNA expression in 967 tumour cell lines from the Cancer Cell Line Encyclopedia project. Pearson correlation coefficient r and p values are indicated. (C) Kaplan-Meier analysis of RNASeq V2 data on WEE1 or CHK1 expression and patient survival in indicated primary tumour samples. Tumours with mRNA expression Z-score +1.5 were considered as tumours with high expression. Data were sourced from the TCGA Research Network. (D-E) Real-time growth kinetics of MIA PaCa-2 cells treated as indicated (10 nM GEM, 1 µM MK8776, 20 nM CHIR124, 300 nM MK1775). Data are represented as mean ± SEM, n=3.

Figure 5. In vivo studies show antitumour potential of the triple regimen.

(A) Quantification of immunoblotting of tumour samples from MIA PaCa-2 xenografts treated and harvested as indicated. Data are represented as mean ± SEM, n=3. (B) Quantification of immunohistochemistry of tumour samples from (A). γH2AX and H3 S10 were used as marker of DNA damage and mitosis, respectively. Middle line marks the mean. A two-tailed t-test was performed, *p≤0.05. (C) Quantification of geminin-positive cells in tumour samples from (A). Data are represented as mean ± SEM, n=3. A two-tailed t-test was performed, *p≤0.05. Scale bar, 50 µm. (D) Pharmacokinetic profile of GEM. Tumour samples from MIA PaCa-2 xenografts treated with either 25 mg/kg GEM or 25 mg/kg GEM+MK8776 were analysed for the active metabolite of GEM (dFdCTP) at specified time-points. Area under the curve (AUC) and p values are indicated. (E) Change in tumour volume of MIA PaCa-2 xenografts. Mice were treated as indicated for four consecutive weekly cycles. Black triangle on the x-axis denotes start of each dosing cycle. Data are represented as mean ± SEM, n=3.

Figure 6. Combination of GEM, CHK1i and WEE1i maximises synergy space.

Rationale for GEM/CHK1i/WEE1i triple combination. GEM and CHK1i at optimal non-cytotoxic concentrations enforces synergy primarily via S-phase deregulation. Complementing this combination with WEE1i expands the synergy space of GEM+CHK1i by more robust induction of G2 bypass and mitotic stress.