In addition, phosphorylation of CHK1 on serine 317 was observed following G?6976 treatment, indicating increased ATR activity. This observation selleck is consistent with other studies indicating an increase in ATR mediated phosphorylation of CHK1 fol lowing inhibition of CHK1 kinase activity. Also, as predicted, siRNA knockdown Inhibitors,Modulators,Libraries of FANCA or BRCA2 in HeLa cells resulted in an increase in the G2 M percentage of cells, consistent with a compensatory increase in CHK1 activation of the G2 M checkpoint in these cells. FANCD2 knockdown sensitizes zebrafish embryos to G?6976 To ensure that the hypersensitivity of FA deficient cells to CHK1 inhibition was not an artifact of our cell model sys tems, we used an in vivo, whole organism approach. We have previously described a FANCD2 knockdown model in zebrafish using a morpholino approach.
We treated zebrafish embryos with an increasing concentration of the FANCD2 morpholino after 1 uM G?6976 treatment. The specificity of G?6976 for CHK1 inhibition in the in vivo zebrafish model Inhibitors,Modulators,Libraries was previously demonstrated by our group. In the absence of FANCD2 morpholino, treatment with 1 uM of G?6976 yielded no detectable phenotype. However, a combined loss of the FA pathway and CHK1 function resulted in enhanced lethality of zebrafish embryos. This result confirms the synthetic lethality between FA pathway and CHK1 inactivation in an in vivo model. FA specific tumoricidal effect of CHK1 inhibition and cisplatin was synergistic The selectivity of CHK1 inhibition for FA defective tumor is modest. However, CHK1 inhibition is under clinical trial in combination with cis platin and other DNA damaging agents.
Many of these DNA damaging agents, including Inhibitors,Modulators,Libraries cisplatin are also shown to have FA specific tumoricidal activities. We, there fore, tested the effect of combining CHK1 inhibition with cisplatin treatment. As shown in Figure 6A, the FA defi cient 2008 line was hypersensitive to cisplatin treatment and CHK1 inhibition by G?6976. In response to either G?6976 or cisplatin, the 2008 cells exhibited a two fold increase in sensitivity relative to the 2008F. When sub jected to a combined CHK1 inhibition and cisplatin treat ment, this differential sensitivity was magnified to approximately ten fold. When fit into the Chou Talalay mutually nonexclusive modal, the Combination Index was 0. 7, supporting a synergistic effect.
Inhibitors,Modulators,Libraries Combination of ATM and CHK1 inhibition induces synergistic killing of FA deficient tumor cells We previously demonstrated that Fanconi Anemia pathway deficient tumor Inhibitors,Modulators,Libraries cells are hypersensitive this article to inhibi tion of the Ataxia Telangiectasia Mutated kinase. Having observed the synergistic FA specific effect of CHK1 inhibition and cisplatin treatment, we wished to determine whether such synergism could be achieved by combining ATM and CHK1 inhibition.