Abstract 4556: A therapeutic strategy combining the Wee1 inhibitor MK1775 with HDAC inhibitors targets both p53 wild-type or mutant AML cells
Authors: Zhang Yu , Zhou Liang , Chen Shuang , Kmieciak Maciej , Lin Hui , Dai Yun , Grant Steven 
Published: May 2015

Abstract
Wee1 is an essential component of the G2-M cell cycle checkpoint machinery, providing a target for Wee1 inhibitors (e.g., MK1775) designed to potentiate the anti-tumor activity of genotoxic agents via mitotic lethality. In this setting, Wee1 inhibitor actions are primarily p53-dependent. However, recent studies have identified novel S-phase functions of Wee1 that operate independently of p53 status. Such findings present a rationale for combining Wee1 inhibitors with other targeted agents such as HDAC inhibitors (HDACIs) which interrupt DNA replication and repair. To test this hypothesis, interactions between MK1775 and the HDACIs SBHA or vorinostat were investigated in AML cells. Significantly, MK1775 interacted highly synergistically with either HDACI in both p53 wild type (wt) or mutant AML cells, manifested by pronounced apoptosis induction and attenuation of clonogenic survival. Consistent with these findings, shRNA knock-down of Wee1 significantly sensitized cells to HDACIs. Immunobloting revealed that HDACIs markedly enhanced Wee1 inhibition by MK1775, reflected by diminished Wee1 S642 phosphorylation and protein levels, as well as cdc2/Cdk1 Y15 phosphorylation, a direct downstream Wee1 kinase target. In p53-null (U937) or -mutant (MV-4-11) cells, MK1775 triggered S-phase arrest, whereas HDACI co-administration resulted in early S-phase accumulation accompanied by a marked increase in sub-G1 cells. In sharp contrast, in p53 wt MOLM-13 cells, HDACIs clearly induced G2/M arrest, an event abrogated by MK1775, arguing for context-specific cell cycle effects of this strategy. Interestingly, while MK1775/HDACI synergism persisted, shRNA p53 knock-down in p53 wt AML-3 cells partially attenuated lethal effects of combined treatment. Nevertheless, MK1775/HDACI co-treatment strikingly increased DNA damage, manifested by dramatically increases in γH2A.X generation, in both p53 wt and mutant cells. This event was associated with profound caspase activation and PARP cleavage. Notably, the MK1775/HDACI regimen was highly active against patient-derived AML cells harboring either wt or mutant p53, as well as various other mutations in cancer hotspot genes including FLT3, NRAS, KRAS, BRAF, PDGFRA, MET, STK11, SMARCB1, and APC. Furthermore, the CD34+/CD123+/CD38- population, enriched for leukemia-initiating progenitors, but not normal CD34+ hematopoietic cells, was highly susceptible to the Wee1/HDAC inhibition strategy. Finally, co-administration of MK1775 with vorinostat substantially suppressed tumor growth and significantly prolonged animal survival in a murine AML xenograft model. Together, these findings indicate that a strategy combining Wee1 inhibition (e.g., by MK1775) with HDACIs is highly active towards AML cells, including primitive progenitors, through context-specific p53-dependent and -independent mechanisms.
Citation Format: Yu Zhang, Liang Zhou, Shuang Chen, Maciej Kmieciak, Hui Lin, Yun Dai, Steven Grant. A therapeutic strategy combining the Wee1 inhibitor MK1775 with HDAC inhibitors targets both p53 wild-type or mutant AML cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4556. doi:10.1158/1538-7445.AM2014-4556