HDAC Inhibitors Reciprocally Interacts the Wee1 Inhibitor AZD1775 to Abrogate Both the G1/S and G2/M Checkpoints Via Chk1-Related cdc2/Cdk1 Threonine 14 Dephosphorylation in AML Cells

Abstract

Cell cycle checkpoints play essential roles in the DNA damage response (DDR) which halts cell cycle progression to allow cells to complete DNA replication and repair DNA damage in response to genotoxic insults (e.g., by chemotherapy and ionizing radiation). Wee1 is a dual-specificity protein kinase principally involved in regulation of the G2/M checkpoint that delays mitotic onset by phosphorylating Y15 and deactivating cdc2/Cdk1. AZD1775 is a highly selective small molecule ATP-competitive Wee1 inhibitor currently undergoing clinical evaluation combined with genotoxic agents in solid tumor malignancies (particularly in p53-deficient tumors), based on the concept of mitotic lethality. Recently, Wee1 has been identified as a target in AML, in which AZD1775 potentiates ara-C or mTOR inhibitor lethality. Notably, gene aberrations related to the cell cycle (e.g., 66%) or the DDR (e.g., 41%) are frequent in AML and correlate with poor prognosis (NEJM, 2013). These findings provide a rationale for a novel Wee1-targeting strategy in AML combining AZD1775 with HDAC inhibitors (HDACIs) which also interrupt the DDR and have been granted AML Orphan Drug designation. Here we report that AZD1775 synergistically interacts with HDACIs (e.g., Vorinostat; both Merck via NCI/NIH) in both p53 wild type or mutant AML cell lines, including those carrying poor-prognostic mutations (e.g., FLT3-ITD), NGS-defined patient-derived primary AML specimens, and the CD34+/CD123+/CD38- population enriched for leukemia-initiating progenitors, but spares normal CD34+ hematopoietic cells. Significantly, in marked contrast to Y15 dephosphorylation, AZD1775 triggered pronounced phosphorylation of T14, another critical cdc2/Cdk1 inhibitory site, which might compromise checkpoint-abrogation and thus limit anti-leukemic activity of AZD1775. We determined that T14 phosphorylation most likely stems from compensatory Chk1 activation (e.g., marked phosphorylation of all three critical serine sites, including S296, S317, S345), rather than Myt1 activation, a kinase that phosphorylates T14. Significantly, co-administration of HDACIs substantially diminished Chk1 activation as well as T14 phosphorylation in cells exposed to AZD1775. Consequently, combined AZD1775/HDACI exposure led to dephosphorylation of cdc2/Cdk1 on both Y15 and T14 residues, resulting in full (and “inappropriate”) activation. As a consequence, AZD1775/HDACI co-treatment sharply increased S10 phosphorylation of histone H3 (p-H3), a premature mitotic entry indicator. Flow cytometry analysis revealed that whereas both AZD1775 and HDACIs alone modestly increased the p-H3 mitotic index (MI) at 8 h , co-exposure strikingly increased the p-H3 MI (e.g., 3.4- and 3.7- fold increases for AZD1775/Vorinostat or /SBHA, respectively), consistent with premature mitotic entry at this early interval. Interestingly, AZD1775/HDACI co-treatment for 16 h sharply arrested cells in early S-phase and increased newly replicated DNA incorporating EdU, accompanied by persistent increases in premature mitotic entry (e.g., increased p-H3 MI). Fluorescence microscopy demonstrated robust increases in both p-H3- and EdU-positive cells after 16-h co-exposure to AZD1775 and HDACIs. Furthermore, confocal microscopy of AZD1775/HDACI-treated cells (16 h) revealed markedly aberrant mitosis characterized by multiple mitotic abnormalities e.g., anaphase bridging, mono- or multi-polar spindles, centrosome clustering, etc. in p-H3-positive cells. These events were associated with a pronounced increase in DNA damage, manifested by expression of the double-strand break (DSB) indicator γH2A.X, as well as marked caspase activation and PARP cleavage in both p53-wt and -deficient cells. Similar results (e.g., cdc2/Cdk1 dephosphorylation of both Y15 and T14, γH2A.X expression, and PARP cleavage) were observed in a murine AML xenograft model in association with a significant reduction in tumor burden and prolongation of animal survival. In conclusion, these findings suggest that a novel strategy combining AZD1775 with HDACIs may markedly improve the anti-AML effectiveness of Wee1-targeting therapy, due to abrogation of both the G1/S and G2/M checkpoints through disruption of compensatory Chk1-mediated cdc2/Cdk1 T14 phosphorylation, and that this interaction occurs independently of p53 status. DisclosuresNo relevant conflicts of interest to declare.