On the other hand, regardless of inhibition of downstream signaling pathways, H3122 CR3 cells remained significantly less sensitive to the blend of crizotinib and gefitinib than parental H3122 cells handled with crizotinib alone . To determine if the mitigated response may possibly indicate that the H3122 CR3 cells fail to undergo apoptosis in response to blend treatment method with crizotinib and gefitinib, we performed annexin V staining of parental and resistant cells. Whereas remedy of parental H3122 cells with crizotinib induced marked apoptosis right after 72 hours, remedy of H3122 CR3 cells with crizotinib, gefitinib, or the combination failed to induce apoptosis . To investigate the molecular basis for this acquiring, we examined both protein and mRNA ranges of BIM, a crucial mediator of apoptosis in cancers addicted to kinases .
Whereas BIM protein appeared to become dephosphorylated and upregulated in H3122 CR3 cells taken care of with combined crizotinib and gefitinib, the upregulation of BIM was substantially much less than that observed in parental H3122 cells taken care of Raf Inhibitors with crizotinib alone . Accordingly, BIM mRNA was decrease within the H3122 CR3 cells . That is consistent with our latest findings that BIM mRNA might possibly account for different BIM protein ranges plus the differing potential of oncogeneaddicted cancers for undergoing apoptosis . Collectively, these results recommend that, whereas EGFR activation may possibly mediate acquired crizotinib resistance, EGFR activation won’t fully describe the acquired resistance phenotype, and combined ALK and EGFR kinase inhibition in crizotinibresistant disease could not be as effective as crizotinib in treating crizotinibsensitive disorder. These in vitro findings spurred us to find out regardless if there may be proof for EGFR activation as being a resistance mechanism in patient specimens.
We thus examined the resistant tumors from your 18 ALKpositive patients who had relapsed on crizotinib . About the basis of immunohistochemical staining for phosphoEGFR, we detected EGFR activation in all but among the BAF312 resistant specimens with ample tissue for IHC examination . In 9 situations, we were capable of evaluate the resistant tumor specimen with the unique diagnostic specimen obtained ahead of crizotinib therapy. In 4 of your nine cases, we detected enhanced EGFR activation during the resistant compared using the corresponding delicate sample , supporting a possible function for EGFR in mediating crizotinib resistance. On top of that, certainly one of the 4 instances with proof of EGFR activation also had a secondary ALK mutation.
Therefore, a lot more than one particular mechanism of resistance could possibly contribute to the growth of crizotinib resistance inside a single patient, recapitulating the heterogeneity of resistance mechanisms observed during the H3122 cell line models. Unexpectedly, we detected EGFR activation in all but certainly one of the pretreatment specimens .