Inhibiting Wee1 and ATR kinases produces tumor-selective synthetic lethality and suppresses metastasis

Abstract

We used the cancer-intrinsic property of oncogene-induced DNA damage as the base for a conditional synthetic lethality approach. To target mechanisms important for cancer cell adaptation to genotoxic stress and thereby to achieve cancer cell-specific killing, we combined inhibition of the kinases ATR and Wee1. Wee1 regulates cell cycle progression, whereas ATR is an apical kinase in the DNA-damage response. In an orthotopic breast cancer model, tumor-selective synthetic lethality of the combination of bioavailable ATR and Wee1 inhibitors led to tumor remission and inhibited metastasis with minimal side effects. ATR and Wee1 inhibition had a higher synergistic effect in cancer stem cells than in bulk cancer cells, compensating for the lower sensitivity of cancer stem cells to the individual drugs. Mechanistically, the combination treatment caused cells with unrepaired or under-replicated DNA to enter mitosis leading to mitotic catastrophe. As these inhibitors of ATR and Wee1 are already in phase I/II clinical trials, this knowledge could soon be translated into the clinic, especially as we showed that the combination treatment targets a wide range of tumor cells. Particularly, the antimetastatic effect of combined Wee1/ATR inhibition and the low toxicity of ATR inhibitors compared with Chk1 inhibitors have great clinical potential.

Keywords: Cancer; Drug therapy; Oncology; Protein kinases.

Figure 1. Wee1 inhibition activates ATR and shows synergistic cancer cell killing with ATR inhibition.

(A) MDA-MB-231 cells were incubated with the indicated inhibitors for Wee1 (AZD1775) or ATR (AZD6738, ETP46464). After 2 hours, cell lysates were harvested and probed for Chk1 and CDK1 phosphorylation by immunoblotting. (B–F) MDA-MB-231, MCF7, Zr-75-1, hTERT-HME1, or MCF10A cells were treated for 4 days with a combination of up to 4 μM AZD6738 and up to 2 μM AZD1775. Survival was assayed by crystal violet staining and each experiment was repeated at least 4 times. Color bars indicate percentage survival normalized to untreated cells. Representative cooperativity screens and Loewe plots for drug cooperativity are shown.

Figure 2. Combined ATR and Wee1 inhibition leads to mitotic defects and cancer cell death.

(A–D) Live cell imaging of MDA-MB-231 expressing mCherry–histone H2B and GFP-tubulin. (A) Cells treated as indicated (ATRi = 1 μM AZD6738, Wee1i = 0.3 μM AZD1775) were monitored by spinning-disk confocal microscopy. Representative images of cells following nuclear envelope breakdown (NEBD) are shown. (B) Quantification of the time from NEBD to anaphase. (C) Representative fates of 5 cells in the 4 treatment groups. (D) Quantification of observed cell fates (n = 56). Of note, when cell death occurred in interphase, the dying cells had previously undergone mitosis following drug addition. (E) Representative images of MDA-MB-231 or T-47D mitotic cells treated as in A. Fixed cells were stained for centromeres (red) and tubulin (green) by immunofluorescence and for DNA with DAPI (blue). Drug-induced clustering of centromeres (white arrows) spatially separated from the main mass of chromosomes (yellow arrow), a feature of centromere fragmentation, is clearly visible. Scale bars: 10 μm. (F) Quantification of cells that are in mitosis (red and blue) and display centromere fragmentation (blue) (n > 1,000), after fixing cells 4 hours after release from a double thymidine block in the presence of the indicated inhibitors. *P < 0.05, ****P < 0.0001 (one-way ANOVA).

Figure 3. Contribution of cell cycle phases, during which ATR and/or Wee1 was inhibited, to overall cell killing.

(A and B) AZD6738 and AZD1775 are reversible inhibitors. Immunoblots of MDA-MB-231 and U-2 OS cells treated as indicated. (A) The ATR inhibitor AZD6738 (1 μM) was added to cells 15 minutes before irradiation with 40 J/m² UV, a strong activator of ATR. One hour after irradiation, AZD6738 was removed and the cells were washed and harvested at indicated times after drug removal. Restoration of ATR activity is observed 1 hour after AZD6738 washout. (B) Cells were incubated for 2 hours with 300 nM Wee1 inhibitor AZD1775, leading to a strong reduction in phospho-CDK1. AZD1775 was then removed and cells washed, leading to restoration of Wee1 activity within 1–2 hours. (C) U-2 OS cells were synchronized by a thymidine-nocodazole block. Six hours after release, cells were treated with 1 μM AZD6738 and/or 300 nM AZD1775. Cell cycle profiles were analyzed by propidium iodide staining and flow cytometry. (D) ATR and/or Wee1 in synchronized cancer cells were transiently inhibited with 1 μM AZD6738 and/or 300 nM AZD1775 during the indicated cell cycle intervals. Survival of drug-treated cells relative to vehicle control was measured after 4 days. Data represent mean ± SD. *P < 0.05, **P < 0.005, and ****P < 0.0001 (one-way ANOVA).

Figure 4. AZD6738 and AZD1775 inhibit ATR and Wee1, respectively, in vivo.

MDA-MB-231-fluc2-tdTomato xenografts were excised for immunohistochemistry 1 hour after the last administration of the inhibitors to the mice by oral gavage for 5 days (25 mg/kg AZD6738 and/or 60 mg/kg AZD1775 daily). ATR (A) and Wee1 activity (B) was tested by probing for phosphorylation of their respective substrates, ATR Thr1989 and CDK1 Tyr15 (insets show tumor tissue at ×40 magnification). (C and D) DNA damage was tested for with antibodies against γH2AX. (E and F) Ki67 staining was used as a readout for proliferating cells. Scale bars: 100 μm and 25 μm (insets). (G and H) TUNEL assay was used to quantify cell death in excised tumor sections. Scale bars: 20 μm. Data represent mean ± SD. *P < 0.05, ***P < 0.001, and ****P < 0.0001 (one-way ANOVA). DAB = 3,3′-diaminobenzidine.

Figure 5. Combination treatment with ATR and Wee1 inhibitors and normal tissue toxicity.

(A) Mice were treated for 26 days daily with 25 mg/kg AZD6738 and/or 60 mg/kg AZD1775 and tested for adverse effects. (B and C) No significant body weight changes are observed in tumor-bearing immune-deficient NSG or in immunocompetent C57BL/6 mice. (D and E) Although Wee1 inhibition leads to some γH2AX staining in the crypts of NSG mouse ilea (see insets) (D), no significant change in villi length is observed (E). n = 50 refers to 50 measurements in each of 3 mice per group. Scale bars: 100 μm and 25 μm (insets). (F–I) No significant depletion of hematopoietic stem and progenitor cells isolated from treated C57BL/6 mice is observed. Bone marrow cells were isolated from C57BL/6 mice treated as described in A and analyzed with the indicated surface markers by flow cytometry. (F and G) Hematopoietic stem and multipotent progenitor cells stained for CD117 and Sca1. (H and I) The CD117⁺Lin^– population additionally includes myeloid progenitor cells. Data represent mean ± SD.

Figure 6. Evaluation of normal tissue DNA damage.

Tissues from tumor-bearing NSG mice (or immunocompetent C57BL/6 mice without tumors, shown in Supplemental Figure 11) were harvested on the last day (26 d) or 1 week after (33 d) the last day of a 26-day treatment period with AZD6738 and/or AZD1775. While lung, liver, and kidney did not show any signs of DNA damage, some cells in the ileum and spleen were found to stain for γH2AX at the end of the treatment (26 d). However, 1 week later (33 d), ilea and spleens recovered from the drug treatment, as measured by staining for γH2AX. Scale bars: 25 μm and 20 μm (insets).

Figure 7. Combination treatment with ATR and Wee1 inhibitors and tumor control.

(A–E) NSG mice were injected orthotopically with MDA-MB-231-fluc2-tdTomato–labeled breast cancer cells and treated for 26 days (indicated by yellow shades) with 25 mg/kg AZD6738 and/or 60 mg/kg AZD1775 after tumors reached approximately 40 mm³. (A) Tumor progression was monitored weekly by bioluminescence imaging. (B) Tumor growth of mice in the 4 treatment arms (n = 9 per group). (C) Kaplan-Meyer survival curves of treated mice (n = 9 per group). (D and E) Metastasis in regions distal to the primary tumor was assessed 7 weeks after treatment initiation (n = 9 per group). The dotted line indicates background threshold (E). (F and G) To further investigate inhibition of metastasis, a group of MDA-MB-231-fluc2-tdTomato tumors (n = 4 per group) were allowed to grow to approximately 250 mm³ before treatment as in A. Combination treatment leads to tumor shrinkage (F). Unlike control or single-agent-treated mice, those treated with AZD6738 and AZD1775 had no detectable secondary tumors (G). Data represent mean ± SD. *P < 0.05, **P < 0.01, ****P < 0.0001 by 2-way ANOVA (B and F), log-rank Mantel-Cox test (C), or 1-way ANOVA (E and G).

Figure 8. Synergistic killing of breast cancer stem cells by ATR and Wee1 inhibitors.

(A) Isolation of cancer stem cell–enriched subpopulations (side population, SP) from MDA-MB-231 or MCF7 based on their increased dye (DyeCycle Violet, DCV; see supplemental methods) efflux properties. Verapamil, an inhibitor of drug efflux pump proteins, particularly of the ABC transporter family, served as negative control. (B) Isolated SPs show an increased ability to form mammospheres compared with control subpopulations (non–side population, NSP). Representative images of mammospheres are shown. (C and D) Four-day survival assays of cancer stem cell–enriched SPs and control cells (NSPs) isolated from MDA-MB-231 (C) and MCF7 (D). Plated cells were treated with indicated concentrations of AZD1775 and/or AZD6738. Color bars indicate percentage survival normalized to untreated cells. (E) Model of cancer-selective synergistic cell killing by combined ATR and Wee1 inhibition. Cancer cells have higher baseline levels of genotoxic stress than normal cells. Wee1 inhibition increases genotoxic stress, while ATR and Wee1 inhibition together lower cellular DNA-damage response capacity (in the simplified model to the same extent, but potentially higher in cancer cells relying on these 2 kinases for survival). A therapeutic window is created for the selective killing of cancer cells. (F) Cell cycle–dependent effects of ATR and Wee1 inhibition contributing to overall cell death following mitotic catastrophe. HR, homologous recombination.