Study On The New Mechanism Of Acquired Resistance To The Third-generation EGFR-TKI Osimertinib And Its Treatment Strateg

Introduction:Lung cancer is the leading cause of death in our country.Patients with driver-positive advanced non-small cell lung cancer(NSCLC)have entered the era of targeted therapy.The third representative of epidermal growth factor receptor-tyrosine kinase inhibitor,represented by osimeritinib etc.,EGFR-TKI has been widely used in the treatment of patients with advanced NSCLC with EGFR-sensitive mutations.It can not only overcome the first and second generation of EGFR-TKI drug resistance caused by T790 M,but also be applied in the first-line treatment of patients with advanced non-small cell lung cancer with driver positive genes,effectively improving the near and long-term efficacy and quality of life of these patients.However,acquired resistance occurs in almost all patients with delayed treatment of EGFR-TKI.In addition,the mechanism of third-generation resistance to EGFR-TKI,such as osimeritinib,is complex,and the current research is not very clear.For the research on the mechanism of third-generation EGFR-TKI resistance,osimeritinib is the earliest on the market and the most widely used.Therefore,current studies mainly focus on the mechanism of osimeritinib resistance.However,the mechanism of osimeritinib resistance is complex,and the mechanism of osimeritinib resistance is different between first-line and second-line use.Recent studies have found that the mechanism of osimeritinib resistance mainly includes the following aspects: 1.Drug resistance mutations at EGFR sites,such as C797 S,L718Q and G796 D mutations;2.2.Activation of downstream signaling pathways,such as BRAF V600 E and PIK3 CA mutations;3.Bypass signal activation,such as c-Met amplification.Among the above resistance mechanisms,EGFR C797 S mutation and c-Met amplification were the most common resistance mechanisms,accounting for only about 20%.At the same time,there is a lack of effective treatment strategies after third-generation EGFR-TKI resistance,and the therapeutic effect of patients after drug resistance is poor,which seriously limits the clinical efficacy of patients with advanced NSCLC with EGFR-sensitive mutations.In summary,ofthe known osimeritinib resistance mechanisms,there is no one class of resistance mechanisms with a prominent incidence,let alone effective clinical strategies to overcome osimeritinib resistance.At the same time,osimeritinib combined with other pathway inhibitors is commonly used in clinical treatment for osimeritinib resistant patients,but the effect is not satisfactory and also needs to be improved.Therefore,acquired drug resistance has become a bottleneck limiting the clinical efficacy of third-generation EGFR-TKI such as osimeritinib.It is an urgent problem to further study the mechanism of drug resistance of third-generation EGFR-TKI such as osimeritinib and explore the treatment strategies after drug resistance.Methods:In this study,whole exome sequencing,whole transcriptome sequencing and proteomic analysis of multi-series polyclonal subtype osimertinib resistant cell lines were conducted to discover the possible important mechanism of osimertinib resistance.si RNA transfection,lentivirus transfection,CCK8,ELISA,flow cytometry,immunofluorescence,immunohistochemistry,invasion assay,Western The important role of sialtransferase ST3GAL4 in mediating osimertinib resistance was verified by blot and human tumor cell line xenograft model(CDX).Subsequently,glycosylation site analysis was performed to identify the glycosylation modification sites of c-Met,the key regulatory protein downstream of ST3GAL4,and HPLC-MS/MS was used to detect N-glycosylation sites,deglycosylation mutants,and IP-mediated endogenous ubiquitination,in order to clarify how ST3GAL4 regulates the stability and degradation of c-Met.And activate the detailed mechanism of the downstream signaling pathway.Finally,we searched for compounds that effectively inhibit ST3GAL4 through high-throughput small-molecule compound library screening,and observed the therapeutic effects of the selected drugs on ST3GAL4-mediated osimertinib resistance through human tumor cell line xenograft tumor model(CDX),human tumor tissue derived graft tumor model(PDX)and human cases.Results:In this study,through a series of experiments,we found that high expression of sialtransferase ST3GAL4 was in osimertinib series drug-resistant cell lines,and further detection of blood and tissue samples of drug-resistant patients also found high expression of ST3GAL4.By endogenous overexpression of ST3GAL4 in osimertinib-sensitive non-small cell lung cancer cell lines,we further demonstrated that overexpression of ST3GAL4 induces acquired osimertinib resistance.Aiming at how ST3GAL4 regulates osimertinib resistance,we conducted proteomic and glycoprotein modification analysis of ST3GAL4 overexpressingcell lines,and identified c-Met as a key downstream target of osimertinib resistance induced by ST3GAL4.Moreover,based on the analysis of glycoprotein site data,we found that ST3GAL4 catalyzed the glycosylation site of N785 on c-Met protein and antagonized K48-linked ubiquitin-dependent c-Met degradation,then over-activating c-Met/AKT and C-Met /ERK signaling pathways.Finally,we found through small-molecule compound library screening that the second generation of ALK-TKI brigatinib could effectively inhibit osimertinib resistance caused by ST3GAL4 overexpression.The results were confirmed in cell derived xenografts(CDX),patient derived xenografts(PDX),and clinical cases.Conclusions:In summary,this study for the first time proposed that sialtransferase ST3GAL4 can mediate the development of osimertinib resistance of the third generation EGFR-TKI,and clarified that ST3GAL4 improves the stability of the c-Met by glycosylating the N785 site.Decreased K-48-associated ubiquitination levels of c-Met protein lead to activation of the bypass c-Met/AKT and c-Met/ERK signaling pathways leading to acquired osimertinib resistance.In addition to the discovery of the above new regulatory mechanism of osimertinib resistance,we found that the second generation of ALK-TKI brigatinib can effectively overcome the ST3GAL4-mediated osimertinib resistance through small-molecule drug library screening,and brigatinib is a new choice and a new direction for the future effective post-line treatment of patients with osimertinib resistance caused by the increase of ST3GAL4.It provides a solid theoretical basis for the follow-up clinical research.Background: The mechanisms of osimertinib resistance are complicated and have not been well characterized.Moreover,the proportion of known osimertinib resistance mechanisms is very low.Thus,it is necessary to explore treatment strategies for various possible or even rare drug resistance mechanisms.Methods: The resistance mechanism of osimertinib-resistant patients was detected through next-generation sequencing.Two murine pro-B cells,BA/F3-EGFRDel19 and BA/F3-EGFRDel19-T790 M,and four EGFR mutant NSCLC cells were stably transfected with human PIK3CG(L468M)or PIK3CA(H1047R)c DNAs.We conducted cell proliferation assays and used cell line–derived xenograft(CDX)and patient-derived xenograft(PDX)models to evaluate the anti-proliferative effects of aspirin in combination with osimertinib in vivo and in vitro.Results: We first observed that PIK3 CG mutations led to acquired resistance to osimertinib in a patient and further confirmed that both PIK3 CG and PIK3 CA mutations caused osimertinib resistance in vitro.Mechanistically,the expression of PI3Kγ or PI3Kα was up-regulated after PIK3 CG or PIK3 CA lentivirus transfection,respectively.Furthermore,we also observed activation of the downstream protein kinase B(AKT)/mammalian target of rapamycin(m TOR)and AKT/Bcl-2-like protein 11(BIM)pathways,which can be effectively suppressed by aspirin in a PI3K-dependent manner.Lastly,our results from in vivo studies indicate that aspirin can synergize with osimertinib to reverse osimertinib resistance caused by PIK3 CG or PIK3 CA mutations in CDX and PDX models.Conclusions: Herein,we first confirmed that mutations in PIK3 CG or PIK3 CA can lead to resistance to osimertinib,and aspirin combined therapy may be a method to reverse this osimertinib resistance.