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WEE1 inhibition forces S-phase–arrested cancer cells into mitosis. A, assessment of the degree of synergy between gemcitabine and MK-1775 in 25 breast cell lines (23 breast cancer cell lines and 2 breast epithelial cell lines), displayed according to TP53 mutational status. Drug interactions are expressed as a CI. B, SF 50 isobolograms of gemcitabine and MK-1775 combination in MCF7 (wild-type TP53) and CAL120 (mutant TP53) cells. C, CAL120 and MCF7 cells were treated with gemcitabine (Gem), MK-1775, or the combination and subjected to dual pH3 (mitotic cells) and PI fl ow cytometric analysis (FACS). Top, PI alone; bottom, pH3/PI. 2N DNA content indicates cells in G 1 phase. 4N DNA content indicates cells in either G 2 or M phase. D, CAL120 and MCF7 cells were treated with hydroxyurea (HU) for 24 hours or synchronized (synchr) by a double thymidine block (T) after which MK-1775 was added for an additional 8 hours. Mitotic index was determined by pH3/PI FACS analysis for cells with <4N DNA content (green bars) or cells with 4N DNA content (red bars). E, mitotic index after gemcitabine and MK-1775 combination treatment in various breast cancer cell lines. TP53 status (mutant or wild-type, WT) and CI are indicated for each cell line (*, P < 0.01; Student t test compared with <4N DNA mitotic index of MCF7 cells).
Source publication
Inhibition of the protein kinase WEE1 synergizes with chemotherapy in preclinical models and WEE1 inhibitors are being explored as potential cancer therapies. Here, we investigate the mechanism that underlies this synergy. We show that WEE1 inhibition forces S-phase–arrested cells directly into mitosis without completing DNA synthesis, resulting in...
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... investigated whether synergy occurred between gem- citabine and the WEE1 inhibitor MK-1775 in a panel of 25 breast cell lines (23 breast cancer cell lines and 2 breast epithelial cell lines; Fig. 1A; Supplementary Table S1), using the combination index (CI) method of Chou and Talalay (15). Gemcitabine inhibits DNA synthesis by target- ing ribonucleotide reductase, resulting in depletion of the dNTP pool and by competing with endogenous dCTPs for incorporation into DNA. A synergistic interaction between gemcitabine and MK-1775 was ...
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... A synergistic interaction between gemcitabine and MK-1775 was found in 44% (11 of 25) of the cell lines. As shown previously, TP53 wild-type cell lines displayed no evidence of substantial synergy (7), nor did the untransformed breast epithelial cell lines. There was a substantial variation in the degree of synergy in the TP53- mutant cell lines (Fig. ...
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... examine the mechanisms underlying synergy, we ini- tially chose to study 2 model cell lines: MCF7 (nonsyner- gistic, CI = 1.04; TP53 wild-type) and CAL120 (synergistic, CI = 0.61; TP53-mutant; Fig. 1B). MK-1775 treatment alone increased the proportion of cells in mitosis with a full 4N complement of DNA (CAL120 4N mitotic cells: 3.9%-27.8%, MCF7 4N mitotic cells: 1.9%-4.9%; Fig. 1C), most likely refl ecting a subpopulation of G 2 phase cells undergoing appropriately scheduled mitosis. In response to gemcitabine, both cell lines ...
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... synergy, we ini- tially chose to study 2 model cell lines: MCF7 (nonsyner- gistic, CI = 1.04; TP53 wild-type) and CAL120 (synergistic, CI = 0.61; TP53-mutant; Fig. 1B). MK-1775 treatment alone increased the proportion of cells in mitosis with a full 4N complement of DNA (CAL120 4N mitotic cells: 3.9%-27.8%, MCF7 4N mitotic cells: 1.9%-4.9%; Fig. 1C), most likely refl ecting a subpopulation of G 2 phase cells undergoing appropriately scheduled mitosis. In response to gemcitabine, both cell lines arrested in early S-phase (Fig. 1C). Unexpect- edly, the addition of MK-1775 to gemcitabine-arrested cells triggered a large proportion of CAL120 cells to engage mitosis without completing ...
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... treatment alone increased the proportion of cells in mitosis with a full 4N complement of DNA (CAL120 4N mitotic cells: 3.9%-27.8%, MCF7 4N mitotic cells: 1.9%-4.9%; Fig. 1C), most likely refl ecting a subpopulation of G 2 phase cells undergoing appropriately scheduled mitosis. In response to gemcitabine, both cell lines arrested in early S-phase (Fig. 1C). Unexpect- edly, the addition of MK-1775 to gemcitabine-arrested cells triggered a large proportion of CAL120 cells to engage mitosis without completing DNA synthesis (46.9% mitotic cells with <4N DNA content), an effect that was not seen in MCF7 cells (1.4% mitotic cells with <4N DNA content). Addition of MK-1775 triggered mitosis in ...
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... engage mitosis without completing DNA synthesis (46.9% mitotic cells with <4N DNA content), an effect that was not seen in MCF7 cells (1.4% mitotic cells with <4N DNA content). Addition of MK-1775 triggered mitosis in CAL120 cells, but not in MCF7 cells, that were arrested in early S-phase by hydroxyurea treatment or by a double thymidine block (Fig. 1D), blocking DNA synthesis by depleting the dNTP pool through inhibition of ribonucleotide reductase, sug- gesting that mitotic entry was independent of the mecha- nism of inducing S-phase arrest. Mitosis without completing S-phase was also observed with other chemical inhibitors of WEE1 (PD0166285, WEE1 inhibitor II, and PHCD; Supple- ...
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... 1D), blocking DNA synthesis by depleting the dNTP pool through inhibition of ribonucleotide reductase, sug- gesting that mitotic entry was independent of the mecha- nism of inducing S-phase arrest. Mitosis without completing S-phase was also observed with other chemical inhibitors of WEE1 (PD0166285, WEE1 inhibitor II, and PHCD; Supple- mentary Fig. S1A) and siRNA-mediated silencing of WEE1 ( Supplementary Fig. S1B), confi rming the specifi city of the results obtained with ...
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... of MK-1775 triggered mitosis in CAL120 cells, but not in MCF7 cells, that were arrested in early S-phase by hydroxyurea treatment or by a double thymidine block (Fig. 1D), blocking DNA synthesis by depleting the dNTP pool through inhibition of ribonucleotide reductase, sug- gesting that mitotic entry was independent of the mecha- nism of inducing S-phase arrest. Mitosis without completing S-phase was also observed with other chemical inhibitors of WEE1 (PD0166285, WEE1 inhibitor II, and PHCD; Supple- mentary Fig. S1A) and siRNA-mediated silencing of WEE1 ( Supplementary Fig. S1B), confi rming the specifi city of the results obtained with MK-1775. ...
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... assessed mitotic entry in an expanded set of breast cancer cell lines ( Fig. 1E; Supplementary Fig. S2A and S2B). Unscheduled mitosis, before completion of DNA synthesis, was seen in multiple TP53-mutant cell lines upon gemcitabine and MK-1775 treatment, but not in TP53 wild-type cell lines (Fig. 1E). Time course analysis revealed that unscheduled mitosis occurred earlier (after 6 hours; Supplementary Fig. S3A) and at lower doses of ...
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... assessed mitotic entry in an expanded set of breast cancer cell lines ( Fig. 1E; Supplementary Fig. S2A and S2B). Unscheduled mitosis, before completion of DNA synthesis, was seen in multiple TP53-mutant cell lines upon gemcitabine and MK-1775 treatment, but not in TP53 wild-type cell lines (Fig. 1E). Time course analysis revealed that unscheduled mitosis occurred earlier (after 6 hours; Supplementary Fig. S3A) and at lower doses of MK-1775 (300 nmol/L; Supplementary Fig. S3B) in the synergistic cell lines, whereas nonsynergistic cell lines responded only to 1 μmol/L MK-1775 at later time points (after 12 hours). This suggests ...
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... (Fig. 5D). The expression of mitotic cyclins in normal cells starts at the beginning of S-phase and peaks during G 2 phase in preparation for mitotic entry. In response to DNA damage, expression of the mitotic genes CCNB1 and CDC2 is suppressed in a process mediated by p53 and p21 Waf1/Cip1 (22). After gemcitabine induced early S-phase arrest ( Fig. 1C), TP53 wild-type MCF7 cells expressed relatively low lev- els of mitotic proteins, whereas TP53-mutant CAL120 cells expressed high levels of mitotic proteins and CDK1-Y15 phosphorylation (Fig. 5D). In CAL120 cells, the expression of CDK1 varied with different cell-cycle phases, in a similar manner to cyclin B1 expression ( Supplementary ...
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... investigated the factors that promote substantial unscheduled mitosis in some TP53-mutant cell lines, but not in others (Fig. 1E). We have previously published whole-genome gene expression profi les for 20 TP53-mutant breast cancer cell lines used in this study (23). The gene expression levels for each individual probe were correlated with gemcitabine/ MK-1775 CI using Pearson correlation coeffi cient (Fig. 5E). We hypothesized that genes correlating positively ...
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... and chemotherapy have focused on disruption of the classical G 2 -M checkpoint as a mechanism by which WEE1 inhibi- tion sensitizes to DNA-damaging agents (6)(7)(8)(9)(10)(11)(12)(13)28). However, we show here that p53-defi cient breast cancer cells engage the intra-S-phase checkpoint and arrest in early S-phase in response to gemcitabine ( Fig. 1C; Supplementary Fig. S2B). Therefore, understanding the role of WEE1 during S-phase rather than the classical G 2 -M checkpoint is key to clarifying the mechanism of sensitivity to WEE1 inhibitors. Multiple p53 and p21 Waf1/Cip1 functions protect against unscheduled mitosis (Fig. 7G). p53 and p21 Waf1/Cip1 inhibit mitotic gene transcription in response to DNA ...
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... before clinical development as the combination modestly sensitizes the breast epithelial cell line MCF10A (Supplementary Fig. S5A). Our data emphasize the potential benefi t of strategies to enhance forced mitotic entry. Only a restricted set of TP53-mutant cell lines showed evidence of synergy between gemcitabine and WEE1 inhibition in vitro (Fig. 1A). Yet, forced mitotic entry could be triggered in the nonsynergistic TP53-mutant cell lines SUM44 and MFM223 ( Supplementary Fig. S3) but only at levels insuffi cient to result in synergy, which suggests that there may be potential to exploit this mechanism of cell death in all TP53-mutant cancer cell ...
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... data emphasize the importance of biomarkers in the clinical development of WEE1 inhibitors, as only a restricted proportion of cancer cell lines show synergy (Fig. 1A). WEE1 inhibitors are in early clinical development and we look for- ward to assessing the expression of CCNB1 and EZH2 in these studies, although further research will be required to ascer- tain whether similar predictors apply to cancer types other than breast cancer. High expression of the mitotic cyclins, and potentially EZH2, may ...
Citations
... Most methods are based on Chromatin ImmunoPrecipitation followed by sequencing (ChIP-seq) to determine genome-wide topoisomerase binding sites or topoisomerase activity as cleavage complexes (TOPccs). Mapping the binding and cleavage sites of TOP1 12 , TOP2A 13 and TOP2B 14,15 in mouse and human cells is critical for understanding the biology and functions of topoisomerases and the sites of actions of the widely used anti-cancer drugs targeting TOP1 and TOP2 [16][17][18] . Yet, the signal to noise ratio (SNR) in topoisomerase ChIP-seq data is generally low, making it difficult to fully elucidate the genomic locations of topoisomerase binding and activity. ...
Both transcription and replication can take place simultaneously on the same DNA template, potentially leading to transcription-replication conflicts (TRCs) and topological problems. Here we asked which topoisomerase(s) is/are the best candidate(s) for sensing TRC. Genome-wide topoisomerase binding sites were mapped in parallel for all the nuclear topoisomerases (TOP1, TOP2A, TOP2B, TOP3A and TOP3B). To increase the signal to noise ratio (SNR), we used ectopic expression of those topoisomerases in H293 cells followed by a modified CUT&Tag method. Although each topoisomerase showed distinct binding patterns, all topoisomerase binding signals positively correlated with gene transcription. TOP3A binding signals were suppressed by DNA replication inhibition. This was also observed but to a lesser extent for TOP2A and TOP2B. Hence, we propose the involvement of TOP3A in sensing both head-on TRCs (HO-TRCs) and codirectional TRCs (CD-TRCs). In which case, the TOP3A signals appear concentrated within the promoters and first 20 kb regions of the 5’ -end of genes, suggesting the prevalence of TRCs and the recruitment of TOP3A in the 5’-regions of transcribed and replicated genes.
GRAPHICAL ABSTRACT
... 21,22 Inhibition of Wee1 enables cancer cells to enter mitosis prematurely and drive cancer cells into a high level of replicative stress that subsequently leads to mitotic catastrophe and tumor apoptosis. [23][24][25][26] Several Wee1 inhibitors have been investigated in clinical trials, including AstraZeneca's Wee1 inhibitor AZD-1775 (adavosertib, compound 1), for the treatment of solid tumors ( Figure 1). Numerous studies have evaluated the anti-tumor effects of AZD-1775 and have validated the potential for targeting Wee1 kinase. ...
The hit identification stage of a drug discovery program generally involves the design of novel chemical scaffolds with desired biological activity against the target(s) of interest. One common approach is scaffold hopping, which is the manual design of novel scaffolds based on known chemical matter. One major limitation of this approach is narrow chemical space exploration, which can lead to difficulties in maintaining or improving biological activity, selectivity, and favorable property space. Another limitation is the lack of preliminary structure-activity relationship (SAR) data around these designs, which could lead to selecting suboptimal scaffolds to advance lead optimization. To address these limitations, we propose AutoDesigner - Core Design (CoreDesign), a de novo scaffold design algorithm. Our approach is a cloud-integrated, de novo design algorithm for systematically exploring and refining chemical scaffolds against biological targets of interest. The algorithm designs, evaluates, and optimizes a vast range - from millions to billions - of molecules in silico, following defined project parameters encompassing structural novelty, physicochemical attributes, potency, and selectivity. In this manner, CoreDesign can generate novel scaffolds and also explore preliminary SAR around each scaffold using FEP+ potency predictions. CoreDesign requires only a single ligand with quantifiable binding affinity and an initial binding hypothesis, making it especially suited for the hit-identification stage where experimental data is often limited. To validate CoreDesign in a real-world drug discovery setting, we applied it to the design of novel, potent Wee1 inhibitors with improved selectivity over PLK1. Starting from a single known ligand, CoreDesign rapidly explored over 23 billion molecules to identify 1,342 novel chemical series with a mean of 4 compounds per scaffold. Importantly, all chemical series met the predefined property space requirements. To rapidly analyze this large amount of data and prioritize chemical scaffolds for synthesis, we utilize t-Distributed Stochastic Neighbor Embedding (t-SNE) plots of in silico properties. The chemical space projections allowed us to rapidly identify a structurally novel 5-5 fused core meeting all the hit-identification requirements. Several compounds were synthesized and assayed from the scaffold, displaying good potency against Wee1 and excellent PLK1 selectivity. Our results suggest that CoreDesign can significantly speed up the hit-identification process and increase the probability of success of drug discovery campaigns by allowing teams to bring forward high-quality chemical scaffolds de-risked by the availability of preliminary SAR.
... These factors collectively make Wee1 inhibition a potential target in TP53-mutated cancers [53,55]. This mechanism has been supported by the selective efficacy of Wee1 inhibition in multiple TP53-mutated preclinical models, including in breast cancer, non-small cell lung cancer, and glioblastoma [53,56,57]. It should be noted that Wee1 inhibition has also demonstrated efficacy in various cancer cell lines independent of p53 function [58]. ...
Simple Summary
Survival for many pediatric cancers has improved over recent decades. However, for pediatric patients with solid tumors that fail to respond to standard therapies, or relapse after initial response, outcomes generally remain poor, indicating a need for novel and improved treatments. Many cancers have an impaired ability to repair DNA damage, which in excess can become toxic to cells. As such, one potential approach for these challenging cancers is to target the DNA damage repair pathways of cancer cells, with the goal of inducing a lethal amount of DNA damage. This article reviews the current research efforts into targeting DNA damage repair pathways in pediatric extracranial solid tumors. It reviews the biology of DNA damage repair pathways, the biology of several extracranial pediatric cancers, the preclinical research investigating targeting the DNA damage repair in pediatric cancers, and the clinical trials using these agents in patients. This article also reviews the ability to harness a patient’s immune system to kill cancer cells, and the research that has been done investigating ways in which DNA damage can activate the anti-tumor immune response.
Abstract
DNA damage is fundamental to tumorigenesis, and the inability to repair DNA damage is a hallmark of many human cancers. DNA is repaired via the DNA damage repair (DDR) apparatus, which includes five major pathways. DDR deficiencies in cancers give rise to potential therapeutic targets, as cancers harboring DDR deficiencies become increasingly dependent on alternative DDR pathways for survival. In this review, we summarize the DDR apparatus, and examine the current state of research efforts focused on identifying vulnerabilities in DDR pathways that can be therapeutically exploited in pediatric extracranial solid tumors. We assess the potential for synergistic combinations of different DDR inhibitors as well as combinations of DDR inhibitors with chemotherapy. Lastly, we discuss the immunomodulatory implications of targeting DDR pathways and the potential for using DDR inhibitors to enhance tumor immunogenicity, with the goal of improving the response to immune checkpoint blockade in pediatric solid tumors. We review the ongoing and future research into DDR in pediatric tumors and the subsequent pediatric clinical trials that will be critical to further elucidate the efficacy of the approaches targeting DDR.
... It has been shown previously that WEE1 inhibition not only abrogates the G2/M checkpoint but can also drive S-phase cells under replication stress into premature mitoses (32). Such a scenario would be in line with the aberrant mitoses with pannuclear DNA damage described above. ...
... inhibitor combinations. For instance, Aarts and coworkers showed that adavosertib forces S-phase cells into premature mitoses if DNA replication is stalled by gemcitabine (32). PARP (50) or ATR(51) inhibitors also result in replication stress and lead to similar aberrant mitoses in combination with WEE1 inhibition. ...
Cancer homeostasis depends on a balance between activated oncogenic pathways driving tumorigenesis and engagement of stress response programs that counteract the inherent toxicity of such aberrant signaling. Although inhibition of oncogenic signaling pathways has been explored extensively, there is increasing evidence that overactivation of the same pathways can also disrupt cancer homeostasis and cause lethality. We show here that inhibition of protein phosphatase 2A (PP2A) hyperactivates multiple oncogenic pathways and engages stress responses in colon cancer cells. Genetic and compound screens identify combined inhibition of PP2A and WEE1 as synergistic in multiple cancer models by collapsing DNA replication and triggering premature mitosis followed by cell death. This combination also suppressed the growth of patient-derived tumors in vivo. Remarkably, acquired resistance to this drug combination suppressed the ability of colon cancer cells to form tumors in vivo. Our data suggest that paradoxical activation of oncogenic signaling can result in tumor-suppressive resistance.
Significance
A therapy consisting of deliberate hyperactivation of oncogenic signaling combined with perturbation of the stress responses that result from this is very effective in animal models of colon cancer. Resistance to this therapy is associated with loss of oncogenic signaling and reduced oncogenic capacity, indicative of tumor-suppressive drug resistance.
... However, more recent work showed that protein synthesis in G2 phase is not strictly required for mitotic entry (Lockhead et al., 2020). Moreover, inhibition of Wee1 can force mitotic entry even before a cell has entered G2 phase, raising the question why a long G2 phase exists (Aarts et al., 2012). ...
At completion of DNA replication, the mitotic kinases CDK1 and PLK1 are activated. Their activities increase slowly through early G2 phase, but the reason for this low-level activity before mitotic entry is not clear. Using a combination of experiments and mathematical modelling, we find that gradual CDK1 activation through G2 phase stimulates production of mitotic factors and coordinates activation of CDK1 and PLK1. We find that inhibition of CDK1 during G2 phase limits transcription of mitotic factors.
Conversely, the duration of premature mitosis by forced activation of CDK1 is inversely related to the time-point in G2 when mitosis is triggered. Forced CDK1 activation not only leads to a lack of mitotic factors, but also decouples CDK1 and PLK1 activation. Accordingly, we find that duration of forced mitosis by WEE1 inhibition can be partially rescued by expression of constitutively active PLK1. Our results show a function for slow mitotic kinase activation through G2 phase and suggests a mechanism for how the timing of mitotic entry is linked to preparation for mitosis.
Highlights
Slow Cdk1 activation through G2 phase coordinates expression of mitotic proteins and activation of mitotic kinases
The duration of forced mitosis by WEE1 inhibition depends on when in G2 mitosis is triggered
Forced CDK1 activation decouples CDK1 and PLK1 activation
The duration of forced mitosis can be partially rescued by expression of constitutively active PLK1
... The intracellular DNA damage response (DDR) pathway represents a sophisticated protein network system that plays a crucial role in maintaining genome integrity. Dysfunctions in the intracellular DDR have been implicated in numerous diseases including tumors, neurodegenerative disorders, and immune deficiencies [4]. The hallmark of tumor cells lies in their genomic instability, primarily characterized by alterations in chromosome number and structure, as well as MSI [5]. ...
One potential cause of cancer is genomic instability that arises in normal cells due to years of DNA damage in the body. The clinical application of radiotherapy and cytotoxic drugs to treat cancer is based on the principle of damaging the DNA of cancer cells. However, the benefits of these treatments also have negative effects on normal tissue. While there have been notable advancements in molecular-driven therapy and immunotherapy for colorectal cancer (CRC), a considerable portion of patients with advanced CRC do not experience any benefits from these treatments, leading to a poor prognosis. In recent years, targeted therapy aimed at suppressing the DNA damage response (DDR) in cancer cells has emerged as a potential treatment option for CRC patients, offering them more choices for treatment. Currently, the integration of DDR and clinical intervention remains in the exploratory phase. This review primarily elucidates the fundamental principles of DDR inhibitors, provides an overview of their current clinical application status in CRC, and discusses the advancements as well as limitations observed in relevant studies.
... This inherent genomic instability of tumor cells provides a vulnerability that can be exploited. Particularly inhibition of kinases regulating the G2/M checkpoint can force cells to enter mitosis with damaged DNA leading to cancer cell death (Aarts et al., 2012;Do et al., 2013;Schmidt et al., 2017;Lewis et al., 2019;Bukhari et al., 2022). The G2/M (DNA damage) checkpoint kinases are potential therapeutic targets because cancer cells have a defective G1/S checkpoint making them more reliant on the G2/M checkpoint. ...
... Wee1 inhibition by Adavosertib disrupts the cell cycle by three independent mechanisms. First, Adavosertib treatment in G1 or early S-phase leads to unscheduled replication origin firing (De Witt Hamer et al., 2011;Aarts et al., 2012). Secondly, and importantly for cancer therapy, the upregulation of Cdk1 activity by Wee1 inhibition can force cancer cells to enter mitosis with under-replicated or unrepaired chromosomes leading to mitotic catastrophe and chromosome fragmentation (Figures 6A, B) (Aarts et al., 2012;Duda et al., 2016;Lewis et al., 2017). ...
... First, Adavosertib treatment in G1 or early S-phase leads to unscheduled replication origin firing (De Witt Hamer et al., 2011;Aarts et al., 2012). Secondly, and importantly for cancer therapy, the upregulation of Cdk1 activity by Wee1 inhibition can force cancer cells to enter mitosis with under-replicated or unrepaired chromosomes leading to mitotic catastrophe and chromosome fragmentation (Figures 6A, B) (Aarts et al., 2012;Duda et al., 2016;Lewis et al., 2017). Third, Cdk1 re-phosphorylation and cyclin B degradation are key steps required for mitotic exit (Jin et al., 1998;Chow et al., 2011;Visconti et al., 2012;2015;Lewis et al., 2017). ...
Cell cycle checkpoint kinases serve as important therapeutic targets for various cancers. When they are inhibited by small molecules, checkpoint abrogation can induce cell death or further sensitize cancer cells to other genotoxic therapies. Particularly aberrant Cdk1 activation at the G2/M checkpoint by kinase inhibitors causing unscheduled mitotic entry and mitotic arrest was found to lead to DNA damage and cell death selectively in cancer cells. Promising drugs inhibiting kinases like Wee1 (Adavosertib), Wee1+Myt1 (PD166285), ATR (AZD6738) and Chk1 (UCN-01) have been developed, but clinical data has shown variable efficacy for them with poorly understood mechanisms of resistance. Our lab recently identified Myt1 as a predictive biomarker of acquired resistance to the Wee1 kinase inhibitor, Adavosertib. Here, we investigate the role of Myt1 overexpression in promoting resistance to inhibitors (PD166285, UCN-01 and AZD6738) of other kinases regulating cell cycle progression. We demonstrate that Myt1 confers resistance by compensating Cdk1 inhibition in the presence of these different kinase inhibitors. Myt1 overexpression leads to reduced premature mitotic entry and decreased length of mitosis eventually leading to increased survival rates in Adavosertib treated cells. Elevated Myt1 levels also conferred resistance to inhibitors of ATR or Chk1 inhibitor. Our data supports that Myt1 overexpression is a common mechanism by which cancer cells can acquire resistance to a variety of drugs entering the clinic that aim to induce mitotic catastrophe by abrogating the G2/M checkpoint.
... WEE1 inhibition has been shown to induce premature mitotic entry of S-phase-arrested cells after induction of DNA damage 25 . To evaluate mitotic entry, we used live cell microscopy on control or ATIP3-depleted cells ( Figure S3A) that were synchronized at the G1/S boundary and released in the presence or absence of AZD1775. ...
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.
... PARPi and WEE-like kinase 1 inhibitors WEE1 modulates cell cycle progression through the S and G2/M checkpoints via regulation of CDK1 and 2, and inhibition has been shown to promote premature mitotic entry and mitotic catastrophe [106,107]. Inhibitors of WEE1 have therefore been evaluated as a candidate for combination with PARP inhibition. The combination was shown to be synergistic in PARPi-resistant models across several histologies [108][109][110]. ...
Poly (ADP-ribose) polymerase inhibitors (PARPi) have significantly changed the treatment landscape for tumours harbouring defects in genes involved in homologous repair (HR) such as BRCA1 and BRCA2 . Despite initial responsiveness to PARPi, tumours eventually develop resistance through a variety of mechanisms. Rational combination strategies involving PARPi have been explored and are in various stages of clinical development. PARPi combinations have the potential to enhance efficacy through synergistic activity, and also potentially sensitise innately PARPi-resistant tumours to PARPi. Initial combinations involving PARPi with chemotherapy were hindered by significant overlapping haematologic toxicity, but newer combinations with fewer toxicities and more targeted approaches are undergoing evaluation. In this review, we discuss the mechanisms of PARPi resistance and review the rationale and clinical evidence for various PARPi combinations including combinations with chemotherapy, immunotherapy, and targeted therapies. We also highlight emerging PARPi combinations with promising preclinical evidence.
... Adavosertib (AZD1775) is a highly selective ATP-competitive small-molecule inhibitor of Wee1, with a halfmaximal inhibitory concentration (IC 50 ) of 5.2 nmol/L in in vitro kinase assays [7,8]. Inhibition of Wee1 releases tumor cells from DNA-damage-induced arrest at the G2/M boundary, so that unrepaired DNA damage may be taken into mitosis (M phase); as cancer cells show higher levels of endogenous damage than normal cells, as well as exhibiting loss of one or more DDR capabilities, this is predicted to preferentially enhance cancer cell death through mitotic catastrophe compared with normal cells [2,9]. ...
Purpose
Adavosertib may alter exposure to substrates of the cytochrome P450 (CYP) family of enzymes. This study assessed its effect on the pharmacokinetics of a cocktail of probe substrates for CYP3A (midazolam), CYP2C19 (omeprazole), and CYP1A2 (caffeine).
Methods
Period 1: patients with locally advanced or metastatic solid tumors received ‘cocktail’: caffeine 200 mg, omeprazole 20 mg, and midazolam 2 mg (single dose); period 2: after 7- to 14-day washout, patients received adavosertib 225 mg twice daily on days 1–3 (five doses), with cocktail on day 3. After cocktail alone or in combination with adavosertib administration, 24-h pharmacokinetic sampling occurred for probe substrates and their respective metabolites paraxanthine, 5-hydroxyomeprazole (5-HO), and 1′-hydroxymidazolam (1′-HM). Safety was assessed throughout.
Results
Of 33 patients (median age 60.0 years, range 41–83) receiving cocktail, 30 received adavosertib. Adavosertib co-administration increased caffeine, omeprazole, and midazolam exposure by 49%, 80%, and 55% (AUC0–12), respectively; AUC0–t increased by 61%, 98%, and 55%. Maximum plasma drug concentration (Cmax) increased by 4%, 46%, and 39%. Adavosertib co-administration increased 5-HO and 1′-HM exposure by 43% and 54% (AUC0–12) and 49% and 58% (AUC0–t), respectively; paraxanthine exposure was unchanged. Adavosertib co-administration decreased Cmax for paraxanthine and 5–HO by 19% and 7%; Cmax increased by 33% for 1′-HM. After receiving adavosertib, 19 (63%) patients had treatment-related adverse events (six [20%] grade ≥ 3).
Conclusion
Adavosertib (225 mg bid) is a weak inhibitor of CYP1A2, CYP2C19, and CYP3A.
ClinicalTrials.gov
NCT03333824
