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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).
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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,...
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... thousand cells were plated and incubated with different concentrations of AZD1775 and AZD6738 for 4 days before measuring surviving cells by crystal violet staining and colorimetry (23). We observed synergistic cell killing by ATR and Wee1 inhibition in all tested cancer cell lines (Table 1 and Supplemental Figure 2C), including the human breast cancer cell lines MDA-MB-231, MCF7, and Zr-75-1 ( Figure 1, B-D), but not in nontumorigenic MCF10A and immortalized mammary epithelial cells (hTERT-HME1) (Figure 1, E and F), as demonstrated in Loewe plots and calculated Bliss combination indices (CIs) (24). A CI below 1 indicates synergy. ...
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... CI below 1 indicates synergy. The synergistic cell killing we observe with Wee1 and ATR inhibitors is unlikely due to off-target effects, because several ATR inhibitors (including ETP-46464 and VE821, Supplemental Figure 3) and knockdown of Wee1 with siRNA (Supplemental Figure 2D) show cooperative lethality as well. Importantly, and in agreement with a conditional synthetic lethality of Wee1 and ATR based on DNA damage, a favorable therapeutic window for the combination treatment is provided by the increased oncogenic stress in cancer cells, as no cooperative lethality is observed in MCF10A and hTERT-HME1. ...
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... latter is more likely, because many cells retain a DNA content below 4n even several hours later. Combined with our observation that cells treated with both ATR and Wee1 inhibitors show frequent centromere fragmentation in mitosis ( Figure 2, E and F), a hallmark of under-replicated cells entering mitosis, the inhibitor-induced shift in DNA-content profile underlines the synergistic contribution of reversible ATR/Wee1 inhibition during S and G2/M phases in causing mitotic catastrophe. ...
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... test synthetic lethality of the combination of Wee1 and ATR inhibitors cancer cell lines stably expressing GFP-tubulin and mCherryhistone H2B enabled us to track the fates of individual cells and their progenies. Our data for MDA-MB-231 show that, unlike Wee1 inhibition (P = 0.0387, one-way ANOVA) (9), ATR inhibition alone does not prolong mitosis ( Figure 2, A and B). However, when ATR and Wee1 inhibition are combined, mitosis is significantly longer (P < 0.0001, one-way ANOVA) ( Figure 2, A and B) and commonly leads to cell death ( Figure 2, C and D). ...
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... data for MDA-MB-231 show that, unlike Wee1 inhibition (P = 0.0387, one-way ANOVA) (9), ATR inhibition alone does not prolong mitosis ( Figure 2, A and B). However, when ATR and Wee1 inhibition are combined, mitosis is significantly longer (P < 0.0001, one-way ANOVA) ( Figure 2, A and B) and commonly leads to cell death ( Figure 2, C and D). The median time between nuclear envelope breakdown and anaphase in control cells or cells treated with AZD6738, AZD1775, or the combination is 35, 45, 160, or 325 minutes, respectively ( Figure 2B). ...
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... data for MDA-MB-231 show that, unlike Wee1 inhibition (P = 0.0387, one-way ANOVA) (9), ATR inhibition alone does not prolong mitosis ( Figure 2, A and B). However, when ATR and Wee1 inhibition are combined, mitosis is significantly longer (P < 0.0001, one-way ANOVA) ( Figure 2, A and B) and commonly leads to cell death ( Figure 2, C and D). The median time between nuclear envelope breakdown and anaphase in control cells or cells treated with AZD6738, AZD1775, or the combination is 35, 45, 160, or 325 minutes, respectively ( Figure 2B). ...
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... when ATR and Wee1 inhibition are combined, mitosis is significantly longer (P < 0.0001, one-way ANOVA) ( Figure 2, A and B) and commonly leads to cell death ( Figure 2, C and D). The median time between nuclear envelope breakdown and anaphase in control cells or cells treated with AZD6738, AZD1775, or the combination is 35, 45, 160, or 325 minutes, respectively ( Figure 2B). Cell death is observed during failed mitosis, after mitotic slippage (when cells have aborted mitosis, as evidenced by the disappearance of the mitotic spindle without cytokinesis), or in interphase after cytokinesis (often with visible micronucleation) ( Figure 2, C and D, and Supplemental Figure 5A). ...
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... median time between nuclear envelope breakdown and anaphase in control cells or cells treated with AZD6738, AZD1775, or the combination is 35, 45, 160, or 325 minutes, respectively ( Figure 2B). Cell death is observed during failed mitosis, after mitotic slippage (when cells have aborted mitosis, as evidenced by the disappearance of the mitotic spindle without cytokinesis), or in interphase after cytokinesis (often with visible micronucleation) ( Figure 2, C and D, and Supplemental Figure 5A). Mitotic duration seems to correlate with cell death observed during mitosis, with 0, 3.6%, 28.6%, or 64.3% of MDA-MB-231 cells dying in mitosis when treated with vehicle, AZD6738, AZD1775, or combined AZD6738/AZD1775, respectively ( Figure 2D). ...
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... death is observed during failed mitosis, after mitotic slippage (when cells have aborted mitosis, as evidenced by the disappearance of the mitotic spindle without cytokinesis), or in interphase after cytokinesis (often with visible micronucleation) ( Figure 2, C and D, and Supplemental Figure 5A). Mitotic duration seems to correlate with cell death observed during mitosis, with 0, 3.6%, 28.6%, or 64.3% of MDA-MB-231 cells dying in mitosis when treated with vehicle, AZD6738, AZD1775, or combined AZD6738/AZD1775, respectively ( Figure 2D). While ATR inhibition kills 44.6% of the cells, most of the cell death occurs during interphase in daughter cells. ...
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... the majority of cells treated with combined ATR and Wee1 inhibitors died in mitosis, we synchronized cells in S phase by a double thymidine block and inhibited ATR and/or Wee1 after release. Four hours after G1/S release, cells were fixed and stained for tubulin, centromeres, and DNA ( Figure 2E). Wee1 inhibition, but particularly combined ATR/Wee1 inhibition, leads to an increase in mitotic cells ( Figure 2F) in the breast cancer cell lines MDA-MB-231 and T-47D, as well as in HeLa cells (Supplemental Figure 5B). ...
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... hours after G1/S release, cells were fixed and stained for tubulin, centromeres, and DNA ( Figure 2E). Wee1 inhibition, but particularly combined ATR/Wee1 inhibition, leads to an increase in mitotic cells ( Figure 2F) in the breast cancer cell lines MDA-MB-231 and T-47D, as well as in HeLa cells (Supplemental Figure 5B). Furthermore, the majority of the mitotic cells in the combination treatment group show centromere fragmentation, as seen by the clustering of centromeres and kinetochores and their separation form the bulk condensed chromatin (compare mitotic cells treated with combined AZD6738 and AZD1775 to DMSO control in Figure 2E and Supplemental Figure 5B). ...
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... inhibition, but particularly combined ATR/Wee1 inhibition, leads to an increase in mitotic cells ( Figure 2F) in the breast cancer cell lines MDA-MB-231 and T-47D, as well as in HeLa cells (Supplemental Figure 5B). Furthermore, the majority of the mitotic cells in the combination treatment group show centromere fragmentation, as seen by the clustering of centromeres and kinetochores and their separation form the bulk condensed chromatin (compare mitotic cells treated with combined AZD6738 and AZD1775 to DMSO control in Figure 2E and Supplemental Figure 5B). ...
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... washout leads to ATR reactivation within 1 hour, as evidenced by restoration of high Chk1 phospho-S345 levels. We derived from MDA-MB-231, a triple-negative human breast cancer cell line (p53 mutated, BRCA wild type), a cell line that expresses the second-generation, less immunogenic firefly luciferase and the red-fluorescent protein tdTomato (36) (Supplemental Figure 12). In our orthotopic xenograft model, these MDA-MB-231-fluc2-tdTomato cells were injected into the fourth mammary fat pad of 6-to 8-week-old female NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ (NSG) mice according to our approved animal protocol (AC16225). ...
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... a consequence of coordinated effects that Wee1 and ATR have on faithful cell cycle progression, particularly in cells with high baseline DNA damage, a therapeutic window opens to lower the activity of these 2 kinases to levels lethal for cancer cells, but tolerable to normal tissues. This is in stark contrast to Chk1 inhibition, which -particularly when combined with Wee1 inhibition (Supplemental Figure 2) -shows high toxicity in nontransformed cells. As previously pointed out by us Isolated side population cells demonstrate higher mammosphere-forming capabilities as compared with the non-side population cells. ...
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... thou- sand cells were plated and incubated with different concentrations of AZD1775 and AZD6738 for 4 days before measuring surviving cells by crystal violet staining and colorimetry (23). We observed synergistic cell killing by ATR and Wee1 inhibition in all tested can- cer cell lines (Table 1 and Supplemental Figure 2C), including the human breast cancer cell lines MDA-MB-231, MCF7, and Zr-75-1 ( Figure 1, B-D), but not in nontumorigenic MCF10A and immortal- ized mammary epithelial cells (hTERT-HME1) (Figure 1, E and F), as demonstrated in Loewe plots and calculated Bliss combination indices (CIs) (24). A CI below 1 indicates synergy. ...
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... CI below 1 indicates synergy. The synergistic cell killing we observe with Wee1 and ATR inhibitors is unlikely due to off-target effects, because several ATR inhibitors (including ETP-46464 and VE821, Supplemental Figure 3) and knockdown of Wee1 with siRNA (Supplemental Figure 2D) show cooperative lethality as well. Importantly, and in agreement with a conditional synthetic lethality of Wee1 and ATR based on DNA damage, a favor- able therapeutic window for the combination treatment is provided by the increased oncogenic stress in cancer cells, as no cooperative lethality is observed in MCF10A and hTERT-HME1. ...
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... latter is more likely, because many cells retain a DNA content below 4n even several hours later. Combined with our observation that cells treated with both ATR and Wee1 inhibitors show frequent cen- tromere fragmentation in mitosis ( Figure 2, E and F), a hallmark of under-replicated cells entering mitosis, the inhibitor-induced shift in DNA-content profile underlines the synergistic contribu- tion of reversible ATR/Wee1 inhibition during S and G2/M phases in causing mitotic catastrophe. ...
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... test synthetic lethality of the combination of Wee1 and ATR inhibitors Breast cancer cell lines stably expressing GFP-tubulin and mCher- ry-histone H2B enabled us to track the fates of individual cells and their progenies. Our data for MDA-MB-231 show that, unlike Wee1 inhibition (P = 0.0387, one-way ANOVA) (9), ATR inhibition alone does not prolong mitosis ( Figure 2, A and B). However, when ATR and Wee1 inhibition are combined, mitosis is significantly longer (P < 0.0001, one-way ANOVA) ( Figure 2, A and B) and commonly leads to cell death ( Figure 2, C and D). ...
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... data for MDA-MB-231 show that, unlike Wee1 inhibition (P = 0.0387, one-way ANOVA) (9), ATR inhibition alone does not prolong mitosis ( Figure 2, A and B). However, when ATR and Wee1 inhibition are combined, mitosis is significantly longer (P < 0.0001, one-way ANOVA) ( Figure 2, A and B) and commonly leads to cell death ( Figure 2, C and D). The median time between nuclear envelope breakdown and anaphase in control cells or cells treated with AZD6738, AZD1775, or the combination is 35, 45, 160, or 325 minutes, respectively ( Figure 2B). ...
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... data for MDA-MB-231 show that, unlike Wee1 inhibition (P = 0.0387, one-way ANOVA) (9), ATR inhibition alone does not prolong mitosis ( Figure 2, A and B). However, when ATR and Wee1 inhibition are combined, mitosis is significantly longer (P < 0.0001, one-way ANOVA) ( Figure 2, A and B) and commonly leads to cell death ( Figure 2, C and D). The median time between nuclear envelope breakdown and anaphase in control cells or cells treated with AZD6738, AZD1775, or the combination is 35, 45, 160, or 325 minutes, respectively ( Figure 2B). ...
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... when ATR and Wee1 inhibition are combined, mitosis is significantly longer (P < 0.0001, one-way ANOVA) ( Figure 2, A and B) and commonly leads to cell death ( Figure 2, C and D). The median time between nuclear envelope breakdown and anaphase in control cells or cells treated with AZD6738, AZD1775, or the combination is 35, 45, 160, or 325 minutes, respectively ( Figure 2B). Cell death is observed during failed mitosis, after mitotic slippage (when cells have aborted mitosis, as evidenced by the disappearance of the mitotic spindle without cytokinesis), or in interphase after cyto- kinesis (often with visible micronucleation) ( Figure 2, C and D, and Supplemental Figure 5A). ...
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... median time between nuclear envelope breakdown and anaphase in control cells or cells treated with AZD6738, AZD1775, or the combination is 35, 45, 160, or 325 minutes, respectively ( Figure 2B). Cell death is observed during failed mitosis, after mitotic slippage (when cells have aborted mitosis, as evidenced by the disappearance of the mitotic spindle without cytokinesis), or in interphase after cyto- kinesis (often with visible micronucleation) ( Figure 2, C and D, and Supplemental Figure 5A). Mitotic duration seems to correlate with cell death observed during mitosis, with 0, 3.6%, 28.6%, or 64.3% of MDA-MB-231 cells dying in mitosis when treated with vehicle, AZD6738, AZD1775, or combined AZD6738/AZD1775, respectively ( Figure 2D). ...
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... death is observed during failed mitosis, after mitotic slippage (when cells have aborted mitosis, as evidenced by the disappearance of the mitotic spindle without cytokinesis), or in interphase after cyto- kinesis (often with visible micronucleation) ( Figure 2, C and D, and Supplemental Figure 5A). Mitotic duration seems to correlate with cell death observed during mitosis, with 0, 3.6%, 28.6%, or 64.3% of MDA-MB-231 cells dying in mitosis when treated with vehicle, AZD6738, AZD1775, or combined AZD6738/AZD1775, respectively ( Figure 2D). While ATR inhibition kills 44.6% of the cells, most of the cell death occurs during interphase in daughter cells. ...
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... the majority of cells treated with combined ATR and Wee1 inhibitors died in mitosis, we synchronized cells in S phase by a double thymidine block and inhibited ATR and/or Wee1 after release. Four hours after G1/S release, cells were fixed and stained for tubulin, centromeres, and DNA ( Figure 2E). Wee1 inhibition, but particularly combined ATR/Wee1 inhibition, leads to an increase in mitotic cells ( Figure 2F) in the breast cancer cell lines MDA-MB-231 and T-47D, as well as in HeLa cells (Supplemental Figure 5B). ...
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... hours after G1/S release, cells were fixed and stained for tubulin, centromeres, and DNA ( Figure 2E). Wee1 inhibition, but particularly combined ATR/Wee1 inhibition, leads to an increase in mitotic cells ( Figure 2F) in the breast cancer cell lines MDA-MB-231 and T-47D, as well as in HeLa cells (Supplemental Figure 5B). Furthermore, the majority of the mitotic cells in the combination treatment group show centromere fragmentation, as seen by the clustering of cen- tromeres and kinetochores and their separation form the bulk con- densed chromatin (compare mitotic cells treated with combined AZD6738 and AZD1775 to DMSO control in Figure 2E and Sup- plemental Figure 5B). ...
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... inhibition, but particularly combined ATR/Wee1 inhibition, leads to an increase in mitotic cells ( Figure 2F) in the breast cancer cell lines MDA-MB-231 and T-47D, as well as in HeLa cells (Supplemental Figure 5B). Furthermore, the majority of the mitotic cells in the combination treatment group show centromere fragmentation, as seen by the clustering of cen- tromeres and kinetochores and their separation form the bulk con- densed chromatin (compare mitotic cells treated with combined AZD6738 and AZD1775 to DMSO control in Figure 2E and Sup- plemental Figure 5B). ...
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... a consequence of coordinated effects that Wee1 and ATR have on faithful cell cycle progression, particularly in cells with high baseline DNA damage, a therapeu- tic window opens to lower the activity of these 2 kinases to levels lethal for cancer cells, but tolerable to normal tissues. This is in stark contrast to Chk1 inhibition, which -particularly when com- bined with Wee1 inhibition (Supplemental Figure 2) -shows high toxicity in nontransformed cells. As previously pointed out by us Isolated side population cells demonstrate higher mammosphere-forming capabilities as compared with the non-side population cells. ...
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Citations
... 11,12 Protein kinases catalyze protein phosphorylation and are involved in various cellular processes, 13,14 and their mutation or abnormal expression has been associated with carcinogenesis, metastasis, and chemoresistance. [15][16][17] Several kinases have become important molecular targets and biomarkers for cancer. 18,19 CDC2-like kinase (CLK) family, which consists of four isoforms: CLK1, CLK2, CLK3, and CLK4, is known for its bidirectional phosphorylation specificity for regions rich in serine-arginine residues, and modulation of pre-mRNA splicing. ...
Platinum resistance represents a major barrier to the survival of patients with ovarian cancer (OC). Cdc2‐like kinase 2 (CLK2) is a major protein kinase associated with oncogenic phenotype and development in some solid tumors. However, the exact role and underlying mechanism of CLK2 in the progression of OC is currently unknown. Using microarray gene expression profiling and immunostaining on OC tissues, we found that CLK2 was upregulated in OC tissues and was associated with a short platinum‐free interval in patients. Functional assays showed that CLK2 protected OC cells from platinum‐induced apoptosis and allowed tumor xenografts to be more resistant to platinum. Mechanistically, CLK2 phosphorylated breast cancer gene 1 (BRCA1) at serine 1423 (Ser1423) to enhance DNA damage repair, resulting in platinum resistance in OC cells. Meanwhile, in OC cells treated with platinum, p38 stabilized CLK2 protein through phosphorylating at threonine 343 of CLK2. Consequently, the combination of CLK2 and poly ADP‐ribose polymerase inhibitors achieved synergistic lethal effect to overcome platinum resistance in patient‐derived xenografts, especially those with wild‐type BRCA1. These findings provide evidence for a potential strategy to overcome platinum resistance in OC patients by targeting CLK2.
... CHK1 inhibitors are known to induce p-S345-CHK1 through a CHK1 activity-dependent phosphatase cycle, where inhibition of CHK1 causes the accumulation of p-S345-CHK1 [24]. Inhibition of WEE1 by AZD1775 has also been shown to induce p-S345-CHK1 by activating ATR in other cancer cells [25], which may circumvent the effectiveness of WEE1 inhibition. AZD1775induced p-S345-CHK1 was also observed in other prostate cancer cells, such as H660 and C4-2B, where increased p-S345-CHK1 was accompanied by increased pHH3 and γH2AX (Fig. 4B). ...
WEE1 and CHEK1 (CHK1) kinases are critical regulators of the G2/M cell cycle checkpoint and DNA damage response pathways. The WEE1 inhibitor AZD1775 and the CHK1 inhibitor SRA737 are in clinical trials for various cancers, but have not been thoroughly examined in prostate cancer, particularly castration-resistant (CRPC) and neuroendocrine prostate cancers (NEPC). Our data demonstrated elevated WEE1 and CHK1 expressions in CRPC and NEPC cell lines and patient samples. AZD1775 resulted in rapid and potent cell killing with comparable IC50s across different prostate cancer cell lines, while SRA737 displayed time-dependent progressive cell killing with 10- to 20-fold differences in IC50s. Notably, their combination synergistically reduced the viability of all CRPC cell lines and tumor spheroids in a concentration- and time-dependent manner. Importantly, in a transgenic mouse model of NEPC, both agents alone or in combination suppressed tumor growth, improved overall survival, and reduced the incidence of distant metastases, with SRA737 exhibiting remarkable single agent anticancer activity. Mechanistically, SRA737 synergized with AZD1775 by blocking AZD1775-induced feedback activation of CHK1 in prostate cancer cells, resulting in increased mitotic entry and accumulation of DNA damage. In summary, this preclinical study shows that CHK1 inhibitor SRA737 alone and its combination with AZD1775 offer potential effective treatments for CRPC and NEPC.
... No reuse allowed without permission. [41][42][43] . Our data provide further molecular insights into this phenotype and support the model in which centromere detachment from DNA is orchestrated by the action of nucleases. ...
Aneuploidy, a hallmark of cancer, is a prominent feature associated with poor prognosis in breast cancer. Here, we screened a panel of cell cycle kinase inhibitors to identify novel targets for highly aneuploid breast cancers. We show that increasing aneuploidy in breast cancer cells sensitizes to the inhibition of WEE1 kinase. Upon exposure to WEE1 inhibitor, aneuploid cells exhibit aberrant mitosis characterized by the detachment of centromere proteins from centromeric DNA and pulverization of chromosomes. The occurrence of such phenotype is driven by excessive levels of replication stress and DNA damage during S-phase, that in turn trigger major defects in the subsequent mitosis. We show that DNA2 helicase/nuclease, that regulates replication of centromeric DNA, is the key player responsible for severe chromosome pulverization in mitosis. The heightened vulnerability of aneuploid cells to WEE1 inhibition, coupled with underlying molecular mechanisms, provides a rationale for clinical exploration of WEE1-targeted therapies against aneuploid breast cancers.
Impact Statement
Increased vulnerability of aneuploid cells to WEE1 inhibition is orchestrated by the DNA2 nuclease/helicase. These findings open new therapeutic strategies in the context of personalized medicine in breast cancer.
... Additional DNA damage repair pathways under evaluation include ATR, a kinase involved in DNA damage response, and Wee1, which regulates the cell-cycle. In combination, inhibition of these targets in vivo shows a synergistic effect in disrupting mitosis [77]. Multiple phase II studies are underway targeting ATR and Wee1 (as monotherapy or in combination with PARP inhibitors) in cholangiocarcinoma (NCT03878095, NCT04298021) [78]. ...
Purpose of ReviewCholangiocarcinoma remains difficult to treat with a poor prognosis. Nevertheless, the treatment landscape is rapidly evolving to include chemotherapy, immunotherapy, and targeted therapies. This paper will summarize recent developments in targeted therapies.Recent FindingsGemcitabine/cisplatin plus durvalumab or pembrolizumab is the standard first-line treatment for patients with advanced cholangiocarcinoma. Multiple alterations have been identified with corresponding approved targeted agents, including fibroblast growth factor receptor inhibitors pemigatinib, infigratinib, and futibatinib; the isocitrate dehydrogenase 1 inhibitor ivosidenib; and the inhibitors of encoded kinases of NTRK1-3 entrectinib and larotrectinib. In tumors with microsatellite instability or deficient mismatch repair, pembrolizumab is approved. Dabrafenib and trametinib also received tumor agnostic approval for BRAF V600E mutations. Additional emerging targets include HER2 and DNA repair pathways.SummaryMultiple targeted therapies are currently approved in the treatment of advanced cholangiocarcinoma, and many more molecular alterations or pathways have been identified as promising therapeutic targets.
... Earlier research has demonstrated that in small-cell lung cancer, the activation of Chk1 through the upregulation of AXL and MET pathways is a significant mechanism of resistance to AZD-1775 [47]. Thus, combining ATR with Chk1 inhibition can be a rational approach to overcoming resistance to Wee1 inhibition, and it has demonstrated a synergistic anti-tumor effect in multiple types of cancer cells [48][49][50]. While the application of ATR inhibitors in urothelial carcinoma is still in its early stages of research, a potential new therapeutic approach involves combining Wee1 and ATR inhibition with conventional platinum-based chemotherapy. ...
Urothelial carcinoma (UC) is characterized by a high incidence of TP53 mutation, and overcoming resistance to cisplatin-based chemotherapy in UC is a major concern. Wee1 is a G2/M phase regulator that controls the DNA damage response to chemotherapy in TP53-mutant cancers. The combination of Wee1 blockade with cisplatin has shown synergistic efficacy in several types of cancers, but little is known regarding UC. The antitumor efficacy of the Wee1 inhibitor (AZD-1775) alone or in combination with cisplatin was evaluated in UC cell lines and a xenograft mouse model. AZD-1775 enhanced the anticancer activity of cisplatin by increasing cellular apoptosis. AZD-1775 inhibited the G2/M checkpoint, improving the sensitivity of mutant TP53 UC cells to cisplatin by enhancing the DNA damage process. We confirmed that AZD-1775 combined with cisplatin reduced tumor volume and proliferation activity and increased the markers of cell apoptosis and DNA damage in the mouse xenograft model. In summary, the Wee1 inhibitor AZD-1775 combined with cisplatin elicited a promising anticancer efficacy in UC, and constitutes an innovative and promising therapeutic strategy.
... WEE1 inhibition results in premature mitotic entry resulting in mitotic catastrophe in cells with unrepaired DNA damage [9,13]. To determine if the increase in mitotic catastrophe observed in FBH1 KO cells was due to an increase in unscheduled mitotic entry, we treated cells with 400 nM WEE1i and examined phosphorylated histone H3 positive Ser 10 (pH3+) after 8 hrs by flow cytometry (Fig 4A, S3). ...
WEE1 kinase phosphorylates CDK1 and CDK2 to regulate origin firing and mitotic entry. Inhibition of WEE1 has become an attractive target for cancer therapy due to the simultaneous induction of replication stress and inhibition of the G2/M checkpoint. WEE1 inhibition in cancer cells with high levels of replication stress results in induction of replication catastrophe and mitotic catastrophe. To increase potential as a single agent chemotherapeutic, a better understanding of genetic alterations that impact cellular responses to WEE1 inhibition is warranted. Here, we investigate the impact of loss of the helicase, FBH1, on the cellular response to WEE1 inhibition. FBH1-deficient cells have a reduction in ssDNA and double strand break signaling indicating FBH1 is required for induction of replication stress response in cells treated with WEE1 inhibitors. Despite the defect in the replication stress response, FBH1-deficiency sensitizes cells to WEE1 inhibition by increasing mitotic catastrophe. We propose loss of FBH1 is resulting in replication-associated damage that requires the WEE1-dependent G2 checkpoint for repair.
... Several clinical trials have shown that WEE1 inhibitors can be safely used in combination with different chemotherapeutic agents as well as concurrent chemotherapy with radiation therapy. Ongoing clinical trials testing novel agents of WEE1 inhibitors, such as ATR and PAPR inhibitors and anti-PDL1 immunotherapies, are underway and could better define the role of WEE1 inhibitors in the future, in terms of efficacy in terms of good safety profile compared to monotherapy and/or standard of care, if any of the novel therapeutic combinations would show superior antitumor efficacy [53,54]. In our study, EZH2 was highly expressed in lung cancers with positive KRAS expression, and the correlation was significant in lung adenocarcinoma (r = 0.3129 and p < 0.001) (Fig. 4H). ...
Backgrounds
It has been observed that high levels of enhancer of zeste homolog 2 (EZH2) expression are associated with unsatisfactory prognoses and can be found in a wide range of malignancies. However, the effects of EZH2 on Lung Adenocarcinoma (LUAD) remain elusive. Through the integration of bioinformatic analyses, the present paper sought to ascertain the effects of EZH2 in LUAD.
Methods
The TIMER and UALCAN databases were applied to analyze mRNA and protein expression data for EZH2 in LUAD. The result of immunohistochemistry was obtained from the HPA database, and the survival curve was drawn according to the library provided by the HPA database. The LinkedOmics database was utilized to investigate the co-expressed genes and signal transduction pathways with EZH2. Up- and down-regulated genes from The Linked Omics database were introduced to the CMap database to predict potential drug targets for LUAD using the CMap database. The association between EZH2 and cancer-infiltrating immunocytes was studied through TIMER and TISIDB. In addition, this paper explores the relationship between EZH2 mRNA expression and NSCLC OS using the Kaplan–Meier plotter database to further validate and complement the research. Furthermore, the correlation between EZH2 expression and EGFR genes, KRAS genes, BRAF genes, and smoking from the Cancer Genome Atlas (TCGA) database is analyzed.
Results
In contrast to paracancer specimens, the mRNA and protein levels of EZH2 were higher in LUAD tissues. Significantly, high levels of EZH2 were associated with unsatisfactory prognoses in LUAD patients. Additionally, the coexpressed genes of EZH2 were predominantly associated with numerous cell growth-associated pathways, including the cell cycle, DNA replication, RNA transport, and the p53 signaling pathway, according to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways. The results of TCGA database revealed that the expression of EZH2 was lower in normal tissues than in lung cancer tissues (p < 0.05). Smoking was associated with elevated EZH2 expression (p < 0.001). EZH2 was highly expressed in lung cancers with positive KRAS expression, and the correlation was significant in lung adenocarcinoma (r = 0.3129, p < 0.001). CMap was applied to determine the top 15 positively correlated drugs/molecules and the top 15 negatively correlated drugs/molecules. MK-1775, MK-5108, fenbendazole, albendazole, BAY-K8644, evodiamine, purvalanol-a, mycophenolic-acid, PHA-793887, and cyclopamine are potential drugs for patients with lung adenocarcinoma and high EZH2 expression.
Conclusions
Highly expressed EZH2 is a predictor of a suboptimal prognosis in LUAD and may serve as a prognostic marker and target gene for LUAD. The underlying cause may be associated with the synergistic effect of KRAS, immune cell infiltration, and metabolic processes.
... U251 and U87 cells were stably transfected with luciferase. The growth and progression of the intracranial tumors were monitored weekly using IVIS bioluminescence imaging (BLI) as previously described [40]. The body weight of the mice was monitored weekly, and the mice were euthanized when weight loss reached 20% or when mice showed development of neurological symptoms. ...
Glioblastoma (GBM) is the most common and aggressive malignant primary brain tumor in adults. The standard treatment achieves a median overall survival for GBM patients of only 15 months. Hence, novel therapies based on an increased understanding of the mechanistic underpinnings of GBM are desperately needed. In this study, we show that elevated expression of 28S rRNA (cytosine-C(5))-methyltransferase NSUN5, which methylates cytosine 3782 of 28S rRNA in GBM cells, is strongly associated with the poor survival of GBM patients. Moreover, we demonstrate that overexpression of NSUN5 increases protein synthesis in GBM cells. NSUN5 knockdown decreased protein synthesis, cell proliferation, sphere formation, migration, and resistance to temozolomide in GBM cell lines. NSUN5 knockdown also decreased the number and size of GBM neurospheres in vitro. As a corollary, mice harboring U251 tumors wherein NSUN5 was knocked down survived longer than mice harboring control tumors. Taken together, our results suggest that NSUN5 plays a pro-tumorigenic role in GBM by enabling the enhanced protein synthesis requisite for tumor progression. Accordingly, NSUN5 may be a hitherto unappreciated target for the treatment of GBM.
... An alternative approach is drug targeting WEE1. Inhibition of WEE1 was shown to sensitise trastuzumab-resistant BCSCs to chemotherapy-induced apoptosis, and co-administration of an ATR and WEE1 inhibitor showed an elevated synergistic cytotoxic effect in BCSCs isolated from an orthotopic breast cancer xenograft mouse model [158,159]. Mechanistically, this was due to a lack of intra-S and G 2 /M checkpoint, thereby allowing cells with under-replicated and damaged DNA to progress prematurely through the cell cycle leading to mitotic catastrophe. Moreover, since CHK1 signalling is controlled by ATR activation, co-administration of RS agents should potentiate cytotoxicity. ...
Simple Summary
A major theory of cancer development is that cancer originates from a specialised type of tumour cell called the cancer stem cell (CSC). Although CSCs comprise a relative subpopulation to the overall heterogeneous tumour mass, they are responsible for cancer establishment, progression, metastasis, and relapse. The eradication of CSCs is therefore vital for long-term patient remission and survival; however, achieving this is challenging as these cells are highly drug resistant compared to the bulk of cancer cells. CSC drug resistance is multifaceted, occurring through multiple extrinsic and intrinsic mechanisms, including an improved ability to repair chemo/radiotherapy-induced DNA lesions. This review summarises the evidence supporting the notion that CSCs display enhanced DNA repair efficiency relative to the bulk tumour population, the possible mechanisms by which this occurs, and discusses strategies of targeting the DNA damage response within CSCs to improve the efficacy of cancer treatment.
Abstract
First-line cancer treatments successfully eradicate the differentiated tumour mass but are comparatively ineffective against cancer stem cells (CSCs), a self-renewing subpopulation thought to be responsible for tumour initiation, metastasis, heterogeneity, and recurrence. CSCs are thus presented as the principal target for elimination during cancer treatment. However, CSCs are challenging to drug target because of numerous intrinsic and extrinsic mechanisms of drug resistance. One such mechanism that remains relatively understudied is the DNA damage response (DDR). CSCs are presumed to possess properties that enable enhanced DNA repair efficiency relative to their highly proliferative bulk progeny, facilitating improved repair of double-strand breaks induced by radiotherapy and most chemotherapeutics. This can occur through multiple mechanisms, including increased expression and splicing fidelity of DNA repair genes, robust activation of cell cycle checkpoints, and elevated homologous recombination-mediated DNA repair. Herein, we summarise the current knowledge concerning improved genome integrity in non-transformed stem cells and CSCs, discuss therapeutic opportunities within the DDR for re-sensitising CSCs to genotoxic stressors, and consider the challenges posed regarding unbiased identification of novel DDR-directed strategies in CSCs. A better understanding of the DDR mediating chemo/radioresistance mechanisms in CSCs could lead to novel therapeutic approaches, thereby enhancing treatment efficacy in cancer patients.
... In addition, the combination down-regulates PD-L1 immune ligand and other immune receptors that are involved in tumor immune evasion [87]. Adavoserib synergizes with ATR inhibitor ceralasertib (AZD6738) in killing cells of various cancer origins [88]. Mechanistically, ATR inhibition prevents feedback activation of ATR produced by WEE1 inhibition. ...
Genes participating in the cellular response to damaged DNA have an important function to protect genetic information from alterations due to extrinsic and intrinsic cellular insults. In cancer cells, alterations in these genes are a source of genetic instability, which is advantageous for cancer progression by providing background for adaptation to adverse environments and attack by the immune system. Mutations in BRCA1 and BRCA2 genes have been known for decades to predispose to familial breast and ovarian cancers, and, more recently, prostate and pancreatic cancers have been added to the constellation of cancers that show increased prevalence in these families. Cancers associated with these genetic syndromes are currently treated with PARP inhibitors based on the exquisite sensitivity of cells lacking BRCA1 or BRCA2 function to inhibition of the PARP enzyme. In contrast, the sensitivity of pancreatic cancers with somatic BRCA1 and BRCA2 mutations and with mutations in other homologous recombination (HR) repair genes to PARP inhibitors is less established and the subject of ongoing investigations. This paper reviews the prevalence of pancreatic cancers with HR gene defects and treatment of pancreatic cancer patients with defects in HR with PARP inhibitors and other drugs in development that target these molecular defects.
