Myt1 overexpression mediates resistance to cell cycle and DNA damage checkpoint kinase inhibitors.

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.

Automated classification of mitotic catastrophe by use of the centromere fragmentation morphology.

Mitotic catastrophe is a common mode of tumor cell death. Cancer cells with a defective cell-cycle checkpoint often enter mitosis with damaged or under replicated chromosomes following genotoxic treatment. Premature condensation of the under-replicated (or damaged) chromosomes results in double-stranded DNA breaks at the centromere (centromere fragmentation). Centromere fragmentation is a morphological marker of mitotic catastrophe and is distinguished by the clustering of centromeres away from the chromosomes. We present an automated 2-step system for segmentation of cells exhibiting centromere fragmentation. The first step segments individual cells from clumps. We added two new terms, weighted local repelling term (WLRt) and weighted gradient term (WGt), in the energy functional of the traditional Chan-Vese based level set method. WLRt was used to generate a repelling force when contours of adjacent cells merged and then penalized the overlap. WGt enhances gradients between overlapping cells. The second step consists of a new algorithm, SBaN (shape-based analysis of each nucleus), which extracts features like circularity, major-axis length, minor-axis length, area, and eccentricity from each chromosome to identify cells with centromere fragmentation. The performance of SBaN algorithm for centromere fragmentation detection was statistically evaluated and the results were robust.

Upregulation of Myt1 Promotes Acquired Resistance of Cancer Cells to Wee1 Inhibition.

Adavosertib (also known as AZD1775 or MK1775) is a small-molecule inhibitor of the protein kinase Wee1, with single-agent activity in multiple solid tumors, including sarcoma, glioblastoma, and head and neck cancer. Adavosertib also shows promising results in combination with genotoxic agents such as ionizing radiation or chemotherapy. Previous studies have investigated molecular mechanisms of primary resistance to Wee1 inhibition. Here, we investigated mechanisms of acquired resistance to Wee1 inhibition, focusing on the role of the Wee1-related kinase Myt1. Myt1 and Wee1 kinases were both capable of phosphorylating and inhibiting Cdk1/cyclin B, the key enzymatic complex required for mitosis, demonstrating their functional redundancy. Ectopic activation of Cdk1 induced aberrant mitosis and cell death by mitotic catastrophe. Cancer cells with intrinsic adavosertib resistance had higher levels of Myt1 compared with sensitive cells. Furthermore, cancer cells that acquired resistance following short-term adavosertib treatment had higher levels of Myt1 compared with mock-treated cells. Downregulating Myt1 enhanced ectopic Cdk1 activity and restored sensitivity to adavosertib. These data demonstrate that upregulating Myt1 is a mechanism by which cancer cells acquire resistance to adavosertib. SIGNIFICANCE: Myt1 is a candidate predictive biomarker of acquired resistance to the Wee1 kinase inhibitor adavosertib.

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

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.

Prolonged mitotic arrest induced by Wee1 inhibition sensitizes breast cancer cells to paclitaxel.

Wee1 kinase is a crucial negative regulator of Cdk1/cyclin B1 activity and is required for normal entry into and exit from mitosis. Wee1 activity can be chemically inhibited by the small molecule MK-1775, which is currently being tested in phase I/II clinical trials in combination with other anti-cancer drugs. MK-1775 promotes cancer cells to bypass the cell-cycle checkpoints and prematurely enter mitosis. In our study, we show premature mitotic cells that arise from MK-1775 treatment exhibited centromere fragmentation, a morphological feature of mitotic catastrophe that is characterized by centromeres and kinetochore proteins that co-cluster away from the condensed chromosomes. In addition to stimulating early mitotic entry, MK-1775 treatment also delayed mitotic exit. Specifically, cells treated with MK-1775 following release from G1/S or prometaphase arrested in mitosis. MK-1775 induced arrest occurred at metaphase and thus, cells required 12 times longer to transition into anaphase compared to controls. Consistent with an arrest in mitosis, MK-1775 treated prometaphase cells maintained high cyclin B1 and low phospho-tyrosine 15 Cdk1. Importantly, MK-1775 induced mitotic arrest resulted in cell death regardless the of cell-cycle phase prior to treatment suggesting that Wee1 inhibitors are also anti-mitotic agents. We found that paclitaxel enhances MK-1775 mediated cell killing. HeLa and different breast cancer cell lines (T-47D, MCF7, MDA-MB-468 and MDA-MB-231) treated with different concentrations of MK-1775 and low dose paclitaxel exhibited reduced cell survival compared to mono-treatments. Our data highlight a new potential strategy for enhancing MK-1775 mediated cell killing in breast cancer cells.

Cancer cells that survive checkpoint adaptation contain micronuclei that harbor damaged DNA.

We have examined the relationship between checkpoint adaptation (mitosis with damaged DNA) and micronuclei. Micronuclei in cancer cells are linked to genomic change, and may induce chromothripsis (chromosome shattering). We measured the cytotoxicity of the cancer drug cisplatin in M059K (glioma fibroblasts, IC50 15 µM). Nearly 100% of M059K cells were positive for histone γH2AX staining after 48 h treatment with a cytotoxic concentration of cisplatin. The proportion of micronucleated cells, as confirmed by microscopy using DAPI and lamin A/C staining, increased from 24% to 48%, and the total micronuclei in surviving cells accumulated over time. Promoting entry into mitosis with a checkpoint inhibitor increased the number of micronuclei in cells whereas blocking checkpoint adaptation with a Cdk inhibitor reduced the number of micronuclei. Interestingly, some micronuclei underwent asynchronous DNA replication, relative to the main nuclei, as measured by deoxy-bromo-uracil (BrdU) staining. These micronuclei stained positive for histone γH2AX, which was linked to DNA replication, suggesting that micronuclei arise from checkpoint adaptation and that micronuclei may continue to damage DNA. By contrast the normal cell line WI-38 did not undergo checkpoint adaptation when treated with cisplatin and did not show changes in micronuclei number. These data reveal that the production of micronuclei by checkpoint adaptation is part of a process that contributes to genomic change.

Measurement of Cdk1/Cyclin B Kinase Activity by Specific Antibodies and Western Blotting.

Quantitative measurement of enzyme activity is a valuable approach to study how cells function. We present a method to measure the activity of the enzyme Cdk1/cyclin B. This enzyme is required by all eukaryotic cells to enter mitosis. Therefore, a biochemical assay to measure Cdk1/cyclin B activity can be used to identify cell populations that are in mitosis or to detect inhibitors of Cdk1/Cyclin B in vitro. A key distinction of the method presented here, compared to others, is that it uses a recombinant protein, a specific antibody, and a western blot apparatus, which makes the technique available to cell and molecular biology laboratories who do not wish to use radioisotopes, which are commonly required for other protein kinase assays.

Natural product extracts of the Canadian prairie plant, Thermopsis rhombifolia, have anti-cancer activity in phenotypic cell-based assays.

Many plant species within the terrestrial ecological zones of Canada have not yet been investigated for anti-cancer activity. We examined the scientific literature describing the endemic flora from the prairie ecological zone and selected the species, Thermopsis rhombifolia, locally known as the buffalo bean, for investigation of its anti-cancer potential. We tested it in cell-based assays using phenotypic screens that feature some of the hallmarks of cancer. An ethanolic extract prepared from T. rhombifolia was cytotoxic to HT-29 (colon) and SH-SY5Y (brain) cancer cell lines, and showed little cytotoxicity to a normal human cell line (WI-38). In phenotypic assays, we identified activities in the extracts that target cell death, cell cycle and cell adhesion. These data highlight the anti-cancer potential of previously untested plants found in northern ecological zones and the feasibility of using pertinent phenotypic assays to examine the anti-cancer potential of natural product extracts.

A western blot assay to measure cyclin dependent kinase activity in cells or in vitro without the use of radioisotopes.

We developed a quantitative method to measure the activity of cyclin-dependent kinases (Cdks) by western blotting, without radioisotopes. We prepared a recombinant protein substrate based upon the natural Cdk1 substrate, PP1Cα. By combining this substrate in a western blot method using fluorochrome based antibodies and phospho-imager analysis, we measured the Km of ATP binding to Cdk1 to be 3.5 µM. We then measured Cdk1 activity in cell extracts from interphase or mitotic cells, and demonstrated that previously identified Cdk inhibitors could be detected by this assay. Our data show that we have a safe, reliable assay to identify Cdk1 inhibitors and measure Cdk1 activity.