Abstract 3420: The novel Chk1 inhibitor MK8776 sensitizes AML cells to HDAC inhibitors by targeting the intra-S checkpoint and DNA replication and repair.
Authors: Chen Shuang , Kmieciak Maciej , Zhou Liang , Lin Hui , Pei Xin-Yan , Dai Yun , Grant Steven 
Published: August 2014

While histone deacetylase inhibitors (HDACIs, e.g., vorinostat) are approved for CTCL, they have limited single-agent activity in AML. Consequently, new mechanisms and targeted agents capable of improving HDACI anti-leukemic efficacy are urgently needed. Recently, new insights into HDACI mechanisms of actions have implicated interruption of DNA replication and DNA damage checkpoints, raising the possibility that agents targeting Chk1, a key modulator that monitors the DNA replication process and mediates activation of DNA damage checkpoints, may improve HDACI activity. To test this hypothesis, interactions between the novel Chk1 inhibitor MK8776 and the HDAC inhibitor (HDACI) vorinostat were examined in human AML cells harboring wild-type (wt) or deficient p53 and/or FLT3-ITD. MK-8776 synergistically potentiated vorinostat-mediated apoptosis in various AML cell lines, although lethality was significantly more pronounced in p53-deficient cells. Notably, AML cells carrying FLT3-ITD were particularly sensitive to the MK8776/vorinostat regimen, regardless of p53 status. Synergistic interactions were associated with inhibition of Chk1 activity (e.g., diminished Ser296 phosphorylation), interference with the intra-S phase checkpoint (cell cycle analysis), disruption of DNA replication (EdU incorporation), and down-regulation of proteins involved in DNA replication (e.g., CDT1) and repair (e.g., CtIP and BRCA1), resulting in sharp increases in DNA damage, reflected by enhanced γH2A.X formation and increased Chk2 Thr68 phosphorylation, and apoptosis. Moreover, AML cells expressing kinase-dead Chk1 (D130A) or Chk1 shRNA were significantly more sensitive to HDACIs compared to their wt-counterparts, and displayed down-regulation of CtIP and BRCA1 phosphorylation following HDACI exposure. Interestingly, MK8776 induced Ser15, but not Ser20, phosphorylation and accumulation of p53 in p53-wt cells, events that were largely blocked by HDACIs. Moreover, p53 knock down by shRNA significantly sensitized these cells to MK8776 or HDACIs, particularly in combination. Finally, the MK8776/vorinostat regimen was active in primary AML blasts, particularly against CD34+/CD38-/CD123+ populations enriched for leukemia-initiating cells. In contrast, identical regimens were relatively sparing toward normal cord blood CD34+ cells. Together, these findings indicate that the novel Chk1 inhibitor MK8776 markedly potentiates HDACI lethality in AML cells displaying various genetic backgrounds through mechanisms involving disruption of DNA replication, the intra-S checkpoint, and DNA repair. They also argue that leukemic cells, and particularly those bearing oncogenic mutations associated with poor prognosis e.g., p53 deletion/mutation or FLT3-ITD, may be more susceptible than their wt-counterparts to this strategy.

Citation Format: Shuang Chen, Maciej Kmieciak, Liang Zhou, Hui Lin, Xin-Yan Pei, Yun Dai, Steven Grant. The novel Chk1 inhibitor MK8776 sensitizes AML cells to HDAC inhibitors by targeting the intra-S checkpoint and DNA replication and repair. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3420. doi:10.1158/1538-7445.AM2013-3420