The online version of this article (doi:10.1186/1475-2867-12-45) contains supplementary material, which is available to authorized users.
Amy D Guertin, Melissa M Martin contributed equally to this work.
AG, XQ, NM, YL, JL, IF, YB, AB, and SS are employees of Merck Research Laboratories. The author(s) declare that they have no competing interests.
AG and MM both participated in proliferation assays, carried out flow cytometry for γH2AX, and performed Western blot analyses of cell line studies. YL and JL performed the combination screen of MK-1775 and MK-8776 on solid tumor cell lines. BR, YB, and IF performed synergy analyses of the screen. MH performed xenograft efficacy studies and xenograft sample collection. XQ and NM conducted and quantitated immunohistochemistry assays on xenograft tumor samples. AB contributed to study design and concept. CT contributed to study design, concept, and manuscript preparation. SS participated in proliferation assays, study design and coordination, figure construction, and drafting of the manuscript. All authors read and approved the final manuscript.
Inhibition of kinases involved in the DNA damage response sensitizes cells to genotoxic agents by abrogating checkpoint-induced cell cycle arrest. CHK1 and WEE1 act in a pathway upstream of CDK1 to inhibit cell cycle progression in response to damaged DNA. Therapeutic targeting of either CHK1 or WEE1, in combination with chemotherapy, is under clinical evaluation. These studies examine the overlap and potential for synergy when CHK1 and WEE1 are inhibited in cancer cell models.
Small molecules MK-8776 and MK-1775 were used to selectively and potently inhibit CHK1 and WEE1, respectively.
In vitro, the combination of MK-8776 and MK-1775 induces up to 50-fold more DNA damage than either MK-8776 or MK-1775 alone at a fixed concentration. This requires aberrant cyclin-dependent kinase activity but does not appear to be dependent on p53 status alone. Furthermore, DNA damage takes place primarily in S-phase cells, implying disrupted DNA replication. When dosed together, the combination of MK-8776 and MK-1775 induced more intense and more durable DNA damage as well as anti-tumor efficacy than either MK-8776 or MK-1775 dosed alone. DNA damage induced by the combination was detected in up to 40% of cells in a treated xenograft tumor model.
These results highlight the roles of WEE1 and CHK1 in maintaining genomic integrity. Importantly, the strong synergy observed upon inhibition of both kinases suggests unique yet complimentary anti-tumor effects of WEE1 and CHK1 inhibition. This demonstration of DNA double strand breaks in the absence of a DNA damaging chemotherapeutic provides preclinical rationale for combining WEE1 and CHK1 inhibitors as a cancer treatment regimen.
Additional file 1: Figure S1. Synergistic interaction of MK-1775 and MK-8776 in 39 solid tumor cell lines. A, Cell lines are grouped according to cancer type. Observed synergy is reported for each line as vBliss, which is the volumetric difference between the surface of predicted combination effect and the surface of observed combination effect as illustrated in parts B and C, (see Methods for explanation of Bliss synergy predictions). B, The A2058 melanoma cell line is an example of synergy. Four concentrations each of MK-1775 and MK-8776 were titrated and proliferation at 96 hours was plotted as a fraction of DMSO treated control A2058 cells. The predicted effect on proliferation (using Bliss synergy model) is represented as the upper surface on the plot whereas the observed effect on proliferation is represented by black dots. Observed effects are connected by vertical lines to the corresponding Bliss predicted effect for those concentrations. C, As in part B but showing the KPL1 cell line as an example of lack of synergy between MK-1775 and MK-8776. (PPT 134 KB)12935_2012_363_MOESM1_ESM.ppt
Additional file 2: Figure S2. Synergistic interaction of MK-1775 and MK-8776 in primary human renal epithelial (HRE) cells. A, Proliferation assay results (72 hours) in HRE cells showing the WEE1 inhibitor MK-1775 titrated in addition to either vehicle (DMSO), or the indicated fixed concentration of the CHK1 inhibitor, MK-8776 (compare to Figure 1). B, Proliferation assay results (72 hours) in HRE cells exposed to 8-point titrations of both MK-1775 (starting 4 μM then 1-to-3 dilutions) and MK-8776 (starting 10 μM then 1-to-3 dilutions) are expressed as surface plots for Bliss predicted additivity and actual observed response (compare to Additional file 1: Figures S1B and S1C). The observed vBliss was 0.06 (compare to Additional file 1: Figure S1A). (PPT 422 KB)
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- Unique functions of CHK1 and WEE1 underlie synergistic anti-tumor activity upon pharmacologic inhibition
Amy D Guertin
Melissa M Martin
Nathan R Miselis
Stuart D Shumway
- BioMed Central
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