PDF file - 1275K, Supplemental Table 1. Synergism between MK-8776 and SBHA or vorinostat in various human leukemia cell lines. Supplemental Figure S1. SBHA or vorinostat significantly potentiates lethality of MK-8776 in leukemia cell lines. Supplemental Figure S2. SBHA or vorinostat enhances Chk1 inhibition by MK-8776. Supplemental Figure S3. Disruption of Chk1 function increases S phase population and sensitizes cells to SBHA or vorinostat, in association with Cdt1 downregulation. Supplemental Figure S4. MK-8776/HDACIs abrogate intra-S-phase checkpoint and induce DNA damage in association with disruption of the CtIP/BRCA1 pathway that mediates DNA repair via HR. Supplemental Figure S5. Knock-down of p53 by shRNA sensitizes in p53-wt leukemia cells to the MK-8776/SBHA regimen, and this regimen is active against leukemia CD34+/CD38-/CD123+ primitive population. Supplemental Figure S6. The MK-8776/HDACI regimen is active against primary AML cells, while spare normal CD34+ cells.
ARTICLE ABSTRACTInteractions between the novel Chk1 inhibitor MK-8776 and the histone deacetylase (HDAC) inhibitor (HDACI) vorinostat were examined in human leukemia cells harboring wild-type (wt) or deficient p53. MK-8776 synergistically potentiated vorinostat-mediated apoptosis in various p53-wt or -deficient leukemia cell lines, whereas p53 knockdown by short hairpin RNA (shRNA) sensitized p53-wt cells to lethality of this regimen. Leukemia cell lines carrying FLT3-ITD were also sensitive to the MK-8776/vorinostat regimen. Synergistic interactions were associated with inhibition of Chk1 activity, interference with the intra-S-phase checkpoint, disruption of DNA replication, and downregulation 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 γ-H2A.X formation, and apoptosis. Moreover, leukemia cells expressing kinase-dead Chk1 (D130A) or Chk1 shRNA were significantly more sensitive to HDACIs compared with their wt counterparts and displayed downregulation of CtIP and BRCA1 phosphorylation following HDACI exposure. Finally, the MK-8776/vorinostat regimen was active in primary acute myelogenous leukemia (AML) blasts, particularly against the CD34+/CD38−/CD123+ population 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 MK-8776 markedly potentiates HDACI lethality in leukemia cells displaying various genetic backgrounds through mechanisms involving disruption of the intra-S checkpoint, DNA replication, and DNA repair. They also argue that leukemic cells, including those bearing oncogenic mutations associated with poor prognosis, for example, p53 deletion/mutation or FLT3-ITD, may also be susceptible to this strategy. Mol Cancer Ther; 12(6); 878–89. ©2013 AACR.