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Aug 21, 2013

Literature Review: New Combination of Targets Shows Promise for Leukemia

Researchers looked at the combination of a CHK1 inhibitor and an HDAC inhibitor for their effect on AML cells.

Literature Review: New Combination of Targets Shows Promise for Leukemia

The authors of this study show that the Chk1 inhibitor MK-8776 can sensitize human leukemia cells to HDAC inhibitors. [© Guido Vrola - Fotolia.com]

  • Click Image To Enlarge +
    Figures A–B. MK-8776 synergistically interacts with HDAC inhibitors (HDACIs) to induce apoptosis in leukemia cell lines with various genetic backgrounds. (A) U937, MV-4-11, OCI-AML-3, and MOLM-13 cells were characterized by immunoblotting analysis (upper panels). Dose responses to MK-8776 were then examined in these lines by Annexin-V-FITC/PI staining and flow-cytometry (lower panel). (B) Cells were exposed (48 h for OCI-AML-3; 24 h for others) to MK-8776 ± 1.5 µM vorinostat, apoptosis was measured.

    Acute myeloid leukemia (AML) has been the subject of intense research efforts in recent years with clinical trials ongoing. A recent focus for this disease has been in combination therapies. The authors* here looked at the combination of a CHK1 (checkpoint kinase) inhibitor and an HDAC inhibitor (histone deacetylase) for their effect on AML cells with various genetic backgrounds (wild-type or mutant p53 and wild-type FLT3 or FLT3-ITD).

    Defects in p53 are much more common in relapsed/refractory AML than at initial AML presentation, and FLT3-ITD AML carries a worse prognosis than many other types of AML. CHK1 acts at various checkpoints including the intra-S phase checkpoint as shown with flow cytometry. Checkpoints are important components of the DNA-damage response in cells, and causing DNA-damage while targeting the cells' ability to cope with the insult is one strategy for the development of cancer therapeutics.

    The CHK1 inhibitor studied here, MK-8776, is currently in clinical trials in combination with DNA-damaging agents, such as cytarabine, for the treatment of AML. CHK1 inhibitors have also been previously shown to target cancer stem cells in non–small-cell lung cancer and pancreatic cancer. Cells with p53 mutations had a greater susceptibility to MK-8776.

  • Click Image To Enlarge +
    Figures C–D. (C) Alternatively, immunoblotting was performed to detect cleavage of caspase-9 and PARP. (D) After 24-h exposure to 500 nM MK-8776 ± HDACIs, a soft-agar assay was performed to assess the colony-forming capacity of U937 cells. CF, cleaved fragment.

    The HDAC inhibitor investigated here was vorinostat, and HDAC inhibitors are important for modifying chromatin structure, which impacts gene expression. HDAC inhibitors are also known to lead to DNA damage. The synergy between MK-8776 and vorinostat in a variety of leukemia cell lines was tested by looking at apoptosis and colony-forming assays (see Figures A–D).

    Co-administration of the two compounds led to large increases in caspase-3 and caspase-9 cleavage as well as PARP degradation. Synergy was observed in the AML cells with these two inhibitors regardless of the genetic background of the cells, and there was a sharp increase in the DNA damage (enhanced γH2A.X). The synergy observed could also be replicated with kinase-dead CHK1 or CHK1 shRNA and the HDAC inhibitor.

    The combination was also active in primary AML blasts but was relatively less toxic to normal CD34+ hematopoietic cells. Interestingly, the combination also had an effect on CHK1 mRNA levels. This particular combination is intriguing, and it will be interesting to see whether the synergy occurs in vivo as well.

  • *Abstract from Molecular Cancer Therapeutics 2013, Vol. 12: 878–889

    Interactions between the novel Chk1 inhibitor MK-8776 and the 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-wild type (wt) or -deficient leukemia cell lines, while p53 knock-down by 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 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 apoptosis.

    Moreover, leukemia cells expressing kinase-dead Chk1 (D130A) or Chk1 shRNA were significantly more sensitive to HDACIs compared to their wild-type counterparts, and displayed down-regulation of CtIP and BRCA1 phosphorylation following HDACI exposure. Finally, the MK-8776/vorinostat regimen was active in primary 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 (e.g., p53 deletion/mutation or FLT3-ITD) may also be susceptible to this strategy.


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