XL880 at the MET inhibitor PHA665752 effectively

Compromises the IR induced DNA damage XL880 activation by destabilizing the ATR CHK1 CDC25B pathway. This is in accordance with previous studies that showed reduction of gemcitabine or irinotecan induced CHK1 phosphorylation using the CHK1 inhibitors XL844 or CHIR 124.35,36 Regarding downstream CHK1 signaling, the literature considers CDC25C, and to a lesser extent CDC25A, as the major tyrosine phosphatase substrates of CHK1.26 Here, we surveyed the impact of PHA665752 on CDC25B, whose biological role is not fully clear yet. Interestingly, our observations that show a consequent reduction of CDC25B phosphorylation in response to CHK1 inhibition by PHA665752 support few previous studies that already suggested CDC25B as a potential CHK1 substrate38,39 and reinforce the newly described MET DDR signaling axis.
Another important difference between the aforementioned reports that used CHK1 inhibitors and this work is that PHA665752 affects the signaling cascade upstream of CHK1 by blocking already ATR, the major kinase that phosphorylates CHK1. This observation GW3965 supports our assumption that PHA665752 activity is not elicited through an off target inhibition of CHK1. This premise was however best validated by the observation that PHA665752 was capable of reducing DNA damage dependent activation of ATR and CHK1 only in cells expressing the PHA665752 sensitive MET variant, while no parallel inhibitory effects on pATR and pCHK1 were seen in the PHA665752 resistant cells. Targeting DDR checkpoint effectors such as CHK1 and CHK2 is expected to function through abrogation of their DNA damage induced cell cycle arrests.
25 For example, depletion of CHK1 by a short hairpin RNA withdraws MDA MB 231 cells from an S phase arrest induced by the topoisomerase I inhibitor SN38,40 and similar results were seen when combining gemcitabine with the CHK1 small molecule inhibitor PF477736 in HT29 cells.33 To validate whether a comparable outcome is seen due to MET inhibition, we examined cell cycle distribution of tumor cells following irradiation. As already mentioned, the data demonstrated a reduction by approximately 70 of S phase IR arrested GTL 16 cells that were treated by the MET inhibitor in parallel to IR. These findings support the idea that inhibition of a MET ATR CHK1 CDC25B axis by PHA665752 leads to abrogation of an S phase arrest, similarly to CHK1 inhibitors.
In summary, the findings presented in this study establish a direct role for an RTK system in the maintenance of the genome integrity via interaction with the cellular DDR machinery. On the therapeutic level, the findings reinforce the potential benefit for the integration of MET inhibitors in cancer therapeutics not only for suppressing tumor growth dependent MET activity but also for improving the response of tumors with aberrant MET signaling to DNA damaging modalities, widely applied in clinical oncology. Materials and Methods Cell lines. GTL 16 cells were provided by the laboratory of Dr. Paolo Comoglio. NIH3T3 cells expressing the MET mutated variants M1268T and Y1248H were from Dr. Laura Schmidt. Chemicals. PHA665752, 5 3 carbonyl} 1H pyrrol 2 yl methylene 1,3 dihydro 2H indol 2 one was dissolved in DMSO, and adriamycin 7 oxy 6,9,11 trihydroxy 9 4 methoxy