An In-vitro Study Of Acquired Resistance To Irreversible EGFR-TKIs In Non-small Cell Lung Cancer

Background:Epidermal growth factor receptor(EGFR)T790M mutation is the most frequent mechanism which accounts for about 60% of acquired resistance to first-generation EGFR tyrosine kinase inhibitors(TKIs)in non-small cell lung cancer(NSCLC)patients harboring EGFR activating mutations.The irreversible EGFR-TKIs including the second-generation and third-generation EGFR-TKIs are developed to overcome T790 M mediated resistance.The second-generation EGFR-TKIs inhibit the wide type(WT)EGFR combined with dose-limiting toxicity which limits its application in clinics,while the development of third-generation EGFR-TKIs brings inspiring efficacy either in vitro or in vivo.Unlike the first-and second-generation EGFR-TKIs,the irreversible EGFR-TKIs showed promising kinase inhibitory activity against T790 M mutation,thus they are supposed to prevent or avoid the emergence of T790 M mutation and result in a better outcome as the initial treatment.The rencent evidences showed that the sencond-and third-gneration EGFR-TKIs are highly effective in the first-line treatment of NSCLC with activating EGFR mutations,however,the acquired resistance will inevitably appear and some resistance mechanisms still remain to be uncovered.Understanding the mechanisms of resistance to irreversible EGFR-TKIs plays an important role in the choice of subsequent treatment.In our previous study,we had developed two resistance cell lines by exposing HCC827(exon 19 Del E746-A750)to increasing dose of AZD9291 or afatinib in order to mimic the resistance after initial treatment.Based on the previous study,we identified both of the two cell lines and further evaluated the acquired resistance mechanism and potential therapeutic strategy against AZD9291 and afatinib.Methods: 1.The identification of the resistance cell lines: the sensitivity to indicated agents were evaluated by MTS;The IC50 of AZD9291 or afatinib were evaluated after the cells were frozen in liquid nitrogen for at least 48 h and then re-thawing.The IC50 was also evaluated following AZD9291 withdrawal for at least three months.The ability of cell proliferation from day 1 to day 6 was determined by cell counting and then the doubling time of both resistance cells and parental cells were calculated.The morphology of cells was observed under the light microscope,and EMT-related markers were determined by western-blot assay.2.Screening the potential resistance mechanisms: the next generation sequencing(NGS)including 416 cancer-related genes was used to detected the somatic mutations and copy number variations(CNVs).RNA sequencing was applied to compare the differences in transcriptome of both parental cells and resistance cells.3.Preliminary validation of potential resistance mechanisms: we used western-blot assay to evaluate the the expression and activation of EGFR,MAPK signaling,AKT signaling,and other tyrosine kinase receptors(TKRs).The specific inhibitors were used to block the suspicious signaling associated with drug resistance and MTS assay was to evaluate the growth inhibiton to indicated inhibitors.The combination index(CI)was calculated using Compu Syn software.Results:1.AZD9291 resistance cell lines(HCC827/AZDR):(1)HCC827/AZDR was high-level at 330 fold higher than the parental HCC827 cells and underwent molecule events which were consistent with EMT.(2)HCC827/AZDR lost amplified-EGFR compared with parental HCC827 cells(amplified-EGFR approximately 17×),the result was in line with the loss of EGFR expression.(3)The basal level of AKT activity was down-regulated,but RAS-MAPK pathway maintained in the presence of AZD9291.We found the dual specific phosphatase 6(DUSP6)was down-regulated in HCC827/AZDR.(4)We found that subsequent AKT reactivation after blocking MAPK pathway using trametinib or SCH772984,which was responsible for MAPK inhibitors resistance.We use AKT inhibitor(MK-2206 or GDC-0068)combined with MAPK inhibitor to prevent AKT reactivation.HCC827/AZDR showed moderate sensitivity to this combination.(5)The majority of TKRs expressions were down-regulated,except AXL and IGFBP7.The phospho-AXL(Y779)level was comparable between HCC827 and HCC827/AZDR,while the phospho-IGF1 R in HCC827/AZDR was significantly higher than HCC827 cells.(6)IGF1R signaling inhibitor,NVP-AEW541,had no effect on cell proliferation of HCC827/AZDR cells when administered as a single agent,and 1 ?mol/L NVP-AEW541 cannot reduce ERK1/2 phosphorylation which could effectively block p IGF1 R.The combination of trametinib and NVP-AEW541 effectively inhibited the growth of HCC827/AZDR cells and western-blot analysis showed the ERK phosphorylation and AKT phosphorylation were effectively blocked by this combination.2.Afatinib resistance cell lines(HCC827/AR):(1)HCC827/AR was high-level at 736 fold higher than the parental HCC827 cells.(2)Both HCC827 and HCC827/AR were at high levels(21.6× and 13.2×)of EGFR amplification.HCC827/AR cells exhibited lower m RNA,phosphorylated and total expression levels of EGFR than that in HCC827 cells.The original EGFR pathway was still active and could be effctively blocked by afatinib.(3)AKT phosphorylation could be effectively blocked by 0.01 ?mol/L afatinib in HCC827/AR,while the MAPK pathway maintained in the presence of afatinib.HCC827/AR cells had approximately 8-fold KRAS gene copy number gain,and KRAS was overexpressed compared to HCC827.(4)HCC827/AR cells were resistant to MAPK inhibitors(trametinib or SCH772984)because AKT reactivation after MAPK inhibition,but the activation of AKT pathway can be blocked by afatinib.(5)Both trametinib and SCH772984 with the concentration of 1 ?mol/L could partially restore the sensitivity to afatinib.Conclusions:1.HCC827/AZDR cells:(1)HCC827/AZDR was a high-level laboratory model which was stable overtime.(2)Loss of amplified-EGFR and EGFR expression was considered as an important potential resistance mechanism to AZD9291 in our study.(3)The activation of MAPK pathway which crosstalked with AKT pathway could maintain the proliferation and survival of resistant cells.Blocking MAPK and AKT signaling may be a potential therapeutic strategy following AZD9291 resistance.(4)IGF1R signaling acted as an paralleled alternative signaling responsible for AKT downstream pathways.The combination therapy of IGF1 R inhibitor with MAPK inhibitor may be a potential therapeutic strategy following AZD9291 resistace in our study.2.HCC827/AR cells:(1)HCC827/AR was a high-level laboratory model which was stable overtime.(2)The expression of EGFR was down-regulated in HCC827/AR,but was still responsible for AKT downstream pathways and play an important role in proliferation and survival of resistant cells.(3)KRAS amplification was speculated to activate MAPK downstream pathway,which was considered to be a potential resistance mechanism of afatinib.The combination therapy of MAPK inhibitor with afatinib may be a potential therapeutic strategy following afatinib resistace in our study.