Activation Of IGF-1R Pathway And ALK G1269A Mutation Confer Resistance To Crizotinib Treatment In ALK Positive Lymphoma

Objective:ALK-positive anaplastic large cell lymphoma?ALCL?represents a subset of non-Hodgkin's lymphoma that is treated with crizotinib,a dual ALK/MET inhibitor.Despite the remarkable initial response,ALCLs eventually develop resistance to crizotinib.ALK inhibitor resistance in tumors is a complex and heterogeneous process with multiple underlying mechanisms,including ALK gene copy number amplification,ALK kinase domain secondary mutation,and the activation of various bypass signaling pathways.To overcome resistance,multiple promising next-generation ALK kinase inhibitors and rational combinatorial strategies are being developed.Previous studies have reported that IGF-1R?type 1 insulin-like growth factor receptor?plays important roles in the occurrence of several malignant tumors and is widely expressed in ALK positive anaplastic large cell lymphoma.Our previous study demonstrated that IGF-1R was highly expressed in crizotinib-resistant ALK positive ALCL cell lines.In this study,we explored the mechanisms of activation of IGF-1R pathway and ALK mutation induced resistance to crizotinib treatment in ALK positive ALCL.The results of our study would provide new strategy for crizotinib-resistant patients.Methods:1.Karpas299 cells were cultured by increasing concerntration of crizotnib to generate crizotinib-resistant cell lines.2.The cell proliferation activity was detected by MTS assay.3.The secretion of IGF-1 in cell supernatant was measured by ELISA.4.Flow cytometry?AnnexinV/PI double staining?to detect apoptosis.5.The protein expression were detected by Western Blot.6.Electroporation transfection to knockdown gene ecpression.7.Cell migration ability was measured by Transwell assay.8.Statistical analysis.Each experiment was repeated 3 times and data analyzed using SPSS version 21.0?IBM,Armonk,NY,USA?.All values are expressed as mean±standard deviation????±s?.The difference between two groups was evaluated by t-test.P<0.05 was considered to be statistically significant.Results:1.Generation and characterization of crizotinib-resistant cells.The ALCL cell line Karpas299 expresses NPM-ALK and is highly sensitive to crizotinib treatment.To explore the mechanisms of crizotinib resistance,we generated a crizotinib-resistant Karpas299 cell line by culturing the cells in the presence of increasing concentrations of crizotinib for 4 months.We maintained the crizotinib-resistant Karpas299?Karpas299CR?cells in 400n?of crizotinib.2.Activation of the IGF-1R pathway and ALK G1269A mutation coexist in crizotinib-resistant cells.We first examined whether resistant lines might harbor an activating mutation within the ALK kinase domain by analyzing the mutation status of ALK in parental and resistant cells by ultra-deep next generation sequencing.In Karpas299CR cells,we observed a C?G substitution at position 1269 with a mutation rate of 49.79%.Previous studies reported that this 1269 C?G substitution results in decreased affinity of ALK for crizotinib.We next generated Karpas299CR single-cell subclones by limiting dilution method and selected a G1269A-mutated subline for further study,which we named Karpas299CR^G1269A.To examine the resistance of Karpas299CR^G1269A to crizotinib,we detected the effect of crizotinib on the cell growth rate and phosphorylation status of ALK and its downstream effectors.Cell viability assays confirmed that the Karpas299CR^G1269A cell line was resistant to crizotinib.Consistent with the cell viability results,crizotinib treatment had limited impact on the proliferation of resistant cell lines.To determine whether the acquired resistance of crizotinib is due to alternative signaling pathways,we analyzed several pathways by western blot.Both resistant cell lines showed that crizotinib treatment resulted in a significant increase in ALK phosphorylation compared with parental cells,and a corresponding increase in the phosphorylation of IGF-1R and its downstream proteins,such as STAT3 or ERK was also observed in resistant cell lines.3.Crizotinib-resistant cells maintain survival and apoptosis resistance compared to parental cells.We performed western blot analysis on lysates from parental and crizotinib-resistant cells treated with either DMSO or increasing concentrations of crizotinib.In Karpas299WT cells,crizotinib reduced IGF-1R,STAT3,AKT and ERK phosphorylation in a dose-dependent manner,but these effects were not observed in Karpas299CR and Karpas299CR^G1269A cells.The phosphorylation of ALK and its crucial downstream signaling intermediates ERK1/2 and STAT3 was maintained in the resistant cell lines even at substantially high doses of crizotinib compared to parental cells.We also examined changes in the DNA damage-associated protein PARP,pro-apoptotic protein PUMA,and anti-apoptotic protein survivin as well as caspase-3 and caspase-9cleavage in response to crizotinib treatment.In Karpas299WT cells,survivin was significantly downregulated after 24 h of treatment with crizotinib,while significant increases in PARP,PUMA,caspase-3 and caspase-9 cleavage in response to crizotinib were observed.These changes were not seen in Karpas299CR and Karpas299CR^G1269A1269A cells.To further study the biological effects of the inhibition of NPM-ALK on the growth and survival of ALCL cells,we examined the level of apoptosis in cells treated with either crizotinib or DMSO by flow cytometry.In Karpas299WT cells treated with crizotinib for24 h,approximately 15–30%of the cells were Annexin V-positive.In contrast,no significant increase in the number of Annexin V-positive cells was seen for Karpas299CR and Karpas299CR^G1269A cells treated with crizotinib compared to the controls.4.Additive effects of crizotinib and IGF-1R inhibitor in Karpas299CR cells but not Karpas299CR^G1269A cells.To assess if IGF-1R was activated by IGF-1 in the Karpas299cell line,we examined the secretion of IGF-1 in Karpas299 parental and crizotinib-resistant cells.The secretion level of IGF-1 in Karpas299CR cells was increased by more than three-fold.IGF-1 cotreatment resulted in protection against the growth inhibitory effects of crizotinib in Karpas299WT cells.Furthermore,the stimulation of crizotinib-treated Karpas299WT cells with IGF-1 resulted in sustained downstream signaling activation of IGF-1R,as evidenced by the phosphorylation of STAT3,AKT and ERK.IGF-1R knockdown was able to inhibit this response.We next tested the ability of an IGF-1R inhibitor,alone or combined with crizotinib,to impede the growth of crizotinib-resistant cells.The IGF-1R inhibitor,picropodophyllotoxin,shows moderate single agent activity in ALCL cells.The combination of crizotinib and picropodophyllotoxin partially restored crizotinib sensitivity in Karpas299CR cells.Furthermore,crizotinib and picropodophyllotoxin combination treatment inhibited the phosphotylation of ALK,IGF-1R,STAT3,AKT,and ERK in Karpas299CR cells to a greater extent than either inhibitor alone.Together,these results indicate that the addition of picropodophyllotoxin partially restored the sensitivity of Karpas299CR cells to the growth inhibitory effects of crizotinib.In addition,we examined whether the NPM-ALK G1269A mutation or IGF-1R pathway activation was the main cause of crizotinib resistance in Karpas299CR^G1269A cell lines.We found no significant difference between the proliferation of Karpas299CR^G1269A1269A cells treated with crizotinib alone compared to the combination of crizotinib with picropodophyllotoxin.Western blot analysis showed that the combination treatment with crizotinib and picropodophyllotoxin did not have the same impact as observed in Karpas299CR cells.Furthermore,the combination of IGF-1R inhibitor plus crizotinib resulted in more significant inhibition of growth and apoptosis in Karpas299CR cells.These results demonstrated that resistant Karpas299CR^G1269A cells remain addicted to ALK signaling and picropodophyllotoxin could not restore crizotinib sensitivity in these cells.This observation suggests that IGF-1R signaling operates as a resistance mechanism in Karpas299CR cells but not Karpas299CR^G1269A cells.5.Second-generation ALK inhibitors overcome crizotinib resistance.Although both Karpas299CR and Karpas299CR^G1269A cells showed resistance to crizotinib,the two cell lines may be sensitive to structurally distinct ALK kinase inhibitors.Thus,we examined the effects of five second-generation ALK inhibitors,alectinib,ceritinib,TAE684,ASP3026 and AP26113,on cell proliferation.As expected,all five second-generation ALK inhibitors were effective in inhibiting crizotinib-resistant cell proliferation and with lower IC₅₀ values compared to crizotinib.However,alectinib and ASP3026 had a weaker inhibition on resistant cells compared to the other three inhibitors,especially in Karpas299CR^G1269A cells.To confirm these data,we investigated the ALK,STAT3,AKT and ERK phosphorylation status in crizotinib-resistant cell lines treated with each of the five inhibitors.ALK phosphorylation and downstream signaling were completely abrogated or strongly inhibited in Karpas299CR cells treated with each of the five inhibitors.However,alectinib and ASP3026 did not display the same degree of activity in Karpas299CR and Karpas299CR^G1269A cells.We suspect that this phenomenon is caused by the presence of ALK G1269A mutation.6.Dual ALK/IGF-1R inhibition represser cell proliferation and cell signaling pathways in Karpas299CR cells but not Karpas299CR^G1269A cells.AP26113 and certinib are second-generation TKIs against ALK and show selectivity against IGF-1R.Since Karpas299CR^G1269A cells were more susceptible to the activation of ALK signaling than IGF-1R signaling,AP26113 showed more significant inhibition of viability in Karpas299CR cells than in Karpas299CR^G1269A cells when combined with the IGF-1R inhibitor picropodophyllotoxin.The same phenomenon was observed when combining ceritinib with picropodophyllotoxin.Western blot analysis in Karpas299CR cells showed that combination treatment with the ALK inhibitors and picropodophyllotoxin inhibited IGF-1R,AKT,and ERK phosphorylation to a greater extent than either inhibitor alone.However,in Karpas299CR^G1269A cells,the combination treatment did not have the same effect as in Karpas299CR cells.Conclusion:1.IGF-1 induced ALK+ALCL cells resistance to crizotinib via activating IGF-1R expression and upregulating downstream STAT3?AKT and ERK.2.The combinatioan of IGF-1R inhibitor with crizotinib overcomed the resistance of crizotinib in ALK+ALCL crizotinib-resistant cell lines.3.Crizotinib resistance was also mediated by the ALK G1269A mutation in ALK+ALCL crizotinib-resistant cell lines.4.Second-generation ALK inhibitors effectively overcomed the resistance of crizotinib in ALK+ALCL crizotinib-resistant cell lines.5.The combination of second-generation ALK inhibitors ceritinib and AP26113 and IGF-1R inhibitor had an additive effect on on ALK+ALCL crizotinib-resistant cells.