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.

Myt1 inhibition of Cyclin A/Cdk1 is essential for fusome integrity and premeiotic centriole engagement in Drosophila spermatocytes.

Regulation of cell cycle arrest in premeiotic G2 phase coordinates germ cell maturation and meiotic cell division with hormonal and developmental signals by mechanisms that control Cyclin B synthesis and inhibitory phosphorylation of the M-phase kinase, Cdk1. In this study, we investigated how inhibitory phosphorylation of Cdk1 by Myt1 kinase regulates premeiotic G2 phase of Drosophila male meiosis. Immature spermatocytes lacking Myt1 activity exhibit two distinct defects: disrupted intercellular bridges (fusomes) and premature centriole disengagement. As a result, the myt1 mutant spermatocytes enter meiosis with multipolar spindles. These myt1 defects can be suppressed by depletion of Cyclin A activity or ectopic expression of Wee1 (a partially redundant Cdk1 inhibitory kinase) and phenocopied by expression of a Cdk1F mutant defective for inhibitory phosphorylation. We therefore conclude that Myt1 inhibition of Cyclin A/Cdk1 is essential for normal fusome behavior and centriole engagement during premeiotic G2 arrest of Drosophila male meiosis. The novel meiotic functions we discovered for Myt1 kinase are spatially and temporally distinct from previously described functions of Myt1 as an inhibitor of Cyclin B/Cdk1 to regulate G2/MI timing.

Chk1 and Wee1 kinases coordinate DNA replication, chromosome condensation, and anaphase entry.

Defects in DNA replication and chromosome condensation are common phenotypes in cancer cells. A link between replication and condensation has been established, but little is known about the role of checkpoints in monitoring chromosome condensation. We investigate this function by live analysis, using the rapid division cycles in the early Drosophila embryo. We find that S-phase and topoisomerase inhibitors delay both the initiation and the rate of chromosome condensation. These cell cycle delays are mediated by the cell cycle kinases chk1 and wee1. Inhibitors that cause severe defects in chromosome condensation and congression on the metaphase plate result in delayed anaphase entry. These delays are mediated by wee1 and are not the result of spindle assembly checkpoint activation. In addition, we provide the first detailed live analysis of the direct effect of widely used anticancer agents (aclarubicin, ICRF-193, VM26, doxorubicin, camptothecin, aphidicolin, hydroxyurea, cisplatin, mechlorethamine and x-rays) on key nuclear and cytoplasmic cell cycle events.

Drosophila myt1 is the major cdk1 inhibitory kinase for wing imaginal disc development.

Mitosis is triggered by activation of Cdk1, a cyclin-dependent kinase. Conserved checkpoint mechanisms normally inhibit Cdk1 by inhibitory phosphorylation during interphase, ensuring that DNA replication and repair is completed before cells begin mitosis. In metazoans, this regulatory mechanism is also used to coordinate cell division with critical developmental processes, such as cell invagination. Two types of Cdk1 inhibitory kinases have been found in metazoans. They differ in subcellular localization and Cdk1 target-site specificity: one (Wee1) being nuclear and the other (Myt1), membrane-associated and cytoplasmic. Drosophila has one representative of each: dMyt1 and dWee1. Although dWee1 and dMyt1 are not essential for zygotic viability, loss of both resulted in synthetic lethality, indicating that they are partially functionally redundant. Bristle defects in myt1 mutant adult flies prompted a phenotypic analysis that revealed cell-cycle defects, ectopic apoptosis, and abnormal responses to ionizing radiation in the myt1 mutant imaginal wing discs that give rise to these mechanosensory organs. Cdk1 inhibitory phosphorylation was also aberrant in these myt1 mutant imaginal wing discs, indicating that dMyt1 serves Cdk1 regulatory functions that are important both for normal cell-cycle progression and for coordinating mitosis with critical developmental processes.

Drosophila Myt1 is a Cdk1 inhibitory kinase that regulates multiple aspects of cell cycle behavior during gametogenesis.

The metazoan Wee1-like kinases Wee1 and Myt1 regulate the essential mitotic regulator Cdk1 by inhibitory phosphorylation. This regulatory mechanism, which prevents Cdk1 from triggering premature mitotic events, is also induced during the DNA damage response and used to coordinate cell proliferation with crucial developmental events. Despite the previously demonstrated role for Myt1 regulation of Cdk1 during meiosis, relatively little is known of how Myt1 functions at other developmental stages. To address this issue, we have undertaken a functional analysis of Drosophila Myt1 that has revealed novel developmental roles for this conserved cell cycle regulator during gametogenesis. Notably, more proliferating cells were observed in myt1 mutant testes and ovaries than controls. This can partly be attributed to ectopic division of germline-associated somatic cells in myt1 mutants, suggesting that Myt1 serves a role in regulating exit from the cell cycle. Moreover, mitotic index measurements suggested that germline stem cells proliferate more rapidly, in myt1 mutant females. In addition, male myt1 germline cells occasionally undergo an extra mitotic division, resulting in meiotic cysts with twice the normal numbers of cells. Based on these observations, we propose that Myt1 serves unique Cdk1 regulatory functions required for efficient coupling of cell differentiation with cell cycle progression.

Drosophila Wee1 kinase regulates Cdk1 and mitotic entry during embryogenesis.

Cyclin-dependent kinases (Cdks) are the central regulators of the cell division cycle. Inhibitors of Cdks ensure proper coordination of cell cycle events and help regulate cell proliferation in the context of tissues and organs. Wee1 homologs phosphorylate a conserved tyrosine to inhibit the mitotic cyclin-dependent kinase Cdk1. Loss of Wee1 function in fission or budding yeast causes premature entry into mitosis. The importance of metazoan Wee1 homologs for timing mitosis, however, has been demonstrated only in Xenopus egg extracts and via ectopic Cdk1 activation . Here, we report that Drosophila Wee1 (dWee1) regulates Cdk1 via phosphorylation of tyrosine 15 and times mitotic entry during the cortical nuclear cycles of syncytial blastoderm embryos, which lack gap phases. Loss of maternal dwee1 leads to premature entry into mitosis, mitotic spindle defects, chromosome condensation problems, and a Chk2-dependent block of subsequent development, and then embryonic lethality. These findings modify previous models about cell cycle regulation in syncytial embryos and demonstrate that Wee1 kinases can regulate mitotic entry in vivo during metazoan development even in cycles that lack a G2 phase.

ATM is required for telomere maintenance and chromosome stability during Drosophila development.

ATM is a large, multifunctional protein kinase that regulates responses required for surviving DNA damage: including DNA repair, apoptosis, and cell cycle checkpoints. Here, we show that Drosophila ATM function is essential for normal adult development. Extensive, inappropriate apoptosis occurs in proliferating atm mutant tissues, and in clonally derived atm mutant embryos, frequent mitotic defects were seen. At a cellular level, spontaneous telomere fusions and other chromosomal abnormalities are common in atm larval neuroblasts, suggesting a conserved and essential role for dATM in the maintenance of normal telomeres and chromosome stability. Evidence from other systems supports the idea that DNA double-strand break (DSB) repair functions of ATM kinases promote telomere maintenance by inhibition of illegitimate recombination or fusion events between the legitimate ends of chromosomes and spontaneous DSBs. Drosophila will be an excellent model system for investigating how these ATM-dependent chromosome structural maintenance functions are deployed during development. Because neurons appear to be particularly sensitive to loss of ATM in both flies and humans, this system should be particularly useful for identifying cell-specific factors that influence sensitivity to loss of dATM and are relevant for understanding the human disease, ataxia-telangiectasia.