PBX3/LC3B Induced Ensartinib Resistant Mechanism In ALK Positive Non-small Cell Lung Cancer

Purpose: Lung cancer is currently the first malignant tumor in China and worldwide in terms of mortality.Non-small cell lung cancer（NSCLC）is the most common type of lung cancer in clinical practice,accounting for 80-85% of lung cancer.More than half of NSCLC patients are already advanced at the time of diagnosis,and treatment is based on radiotherapy,chemotherapy,molecular targeted therapy and immunotherapy,with a poor prognosis.It has been confirmed that EML4-ALK is one of the driver genes of NSCLC.Ensartinib is a more optimal first-line option for patients with ALK-positive NSCLC and is approved for first-line treatment.And all drugs are inevitably subject to resistance in the clinic.Adequate elucidation of ALK-TKI resistance mechanisms and overcoming drug resistance is an urgent issue that we need to address.Autophagy is a process by which eukaryotic cells use lysosomes to degrade their own cytoplasmic proteins and damaged organelles under the regulation of autophagyassociated genes（ATG）,and several studies in recent years have shown that autophagy is associated with the process of tumor progression after targeted therapy.Some studies have reported that autophagy can enhance the sensitivity of tumor cells to ALK-TKI in vivo and in vitro level,while protective autophagy can promote ALK-TKI resistance,and the specific mechanism of resistance is highly correlated with tumor heterogeneity and drug mechanism of action.Therefore,the mechanism of the role of autophagy in Ensartinib resistance needs to be further investigated.PBX3 is a homologous domain-containing transcription factor of the pro-B-cell leukaemia（PBX）family,and members of the PBX family have a wide range of roles in early developmental and maturational stages.Previous studies have shown that high PBX3 expression is associated with poor tumour prognosis and that PBX3 has a broad role in the maintenance of the malignant phenotype of tumour cells,but there are no reports for PBX3 associated with autophagy or MAP1LC3 B.MAP1LC3B is involved in the formation of autophagic vesicles,and the elevated ratio of LC3II/Ⅰ expression is also a sign of increased autophagic flux,so elucidating the factors regulating the elevated expression of MAP1LC3 B is an important issue to explore the impact of autophagy on the occurrence of drug resistance.In this study,we established Ensartinib-resistant cell lines to assess autophagyinduced drug resistance and investigated the role of PBX3 in regulating autophagic flux and drug sensitivity through transcriptional regulation.In this study,we investigated the novel mechanism by which the PBX3/LC3 B regulates autophagic flux to induce Ensartinib resistance in ALK-positive NSCLC,providing a theoretical basis for the clinical management of Ensartinib resistance in ALK-positive NSCLC patients.Methods: 1.Resistant cell lines with Resistance index（RI）> 10 were established by gradually increasing the concentration of Ensartinib in the cell culture medium,designated H3122 ER and H2228 ER cells,and MTS assays were performed to determine the IC50 of parental and resistant cell lines for comparison.2.Scratch assay to verify the migration ability of drug-resistant cell lines.3.Transwell assay to verify the invasion and migration ability of drug-resistant cell lines.4.Western blot assay was performed to verify the protein expression of EMT related indicators such as Vimentin,N-cadherin and E-cadherin in drug-resistant cell lines.5.Differential gene expression after drug resistance was detected by transcriptome sequencing,GO and KEGG enrichment of genes was performed to analyze pathway activation and identify autophagy-related genes with significant differences.6.Transmission electron microscope observation comparing autophagic fluxes of parental and drug-resistant cell lines.7.The autophagic flux of drugresistant cell lines was verified by CYTO-ID autophagy assay and Western blot assay to detect the protein expression levels of SQSTM1/P62 and MAP1LC3 B.8.Heatmaps were drawn to analyze significantly different transcription factors FOXM1,NKX2-1 and PBX3 and verified by q RT-PCR and Western blot experiments.9.Predicted transcription factor binding sequences,dual luciferase reporter gene assay and gel migration assay（EMSA）were performed to verify the transcriptional regulation of NKX2-1 and PBX3 for MAP1LC3 B.10.The key transcription factor PBX3 was silenced by sh RNA design,autophagy was detected by CYTO-ID,and protein expression levels of SQSTM1/P62 and MAP1LC3B were determined by Western blot assay.11.Establishment of a CDX animal model to validate reversal of drug resistance after silencing PBX3 with autophagic flux.12.The autophagic flux in animal models after silencing PBX3 was verified by immunohistochemistry and Western blot assays.13.Analysis of the relationship between PBX3 expression and patient prognosis using an online database of non-small cell lung cancer patient data.Results: 1.NSCLC cell lines were verified to be sensitive to three ALK-TKIs.The results of the MTS assay showed that H3122 and H2228 cell lines were the most sensitive to Ensartinib and had the lowest IC50.2.Construction of NSCLC cell lines resistant to Ensartinib.Successful construction of resistant cell lines can be determined by the calculated RI（RI > 10）.3.Drug-resistant cell lines exhibited a mesenchymal transformation phenotype.Microscopic observation and Western blot assay showed that the drug-resistant cell lines exhibited epithelial-mesenchymal transformation.4.Drug-resistant cell lines showed increased invasive migration.Scratch assay and Transwell assay demonstrated enhanced invasive migration of drug-resistant cell lines.5.Transcriptome sequencing to analyze the altered gene expression in drug-resistant cell lines.We obtained 4275 differential genes between H3122 parental and drugresistant cells and 3111 differential genes between H2228 parental and drug-resistant cells by transcriptome sequencing,and 1144 differential genes were common to the four groups of cells;further analysis of differential gene expression and volcano plotting were performed,and 1856 genes were up-regulated and 1255 genes were down-regulated in H2228 ER cells compared with the parental cells.H3122 ER cells had 2148 genes upregulated and 2127 genes down-regulated compared with the parents.Analysis of differential genes for GO and KEGG enrichment analysis of pathway activation showed that differential genes in drug-resistant cell lines were mainly enriched in lysosomes,autophagic vesicle maturation,molecular chaperone-mediated autophagy,autophagic cell death and positive regulation of DNA-binding transcription factor activity pathways.Thus autophagy may be the main cause of drug resistance phenomenon.6.Autophagic fluxes were increased in drug-resistant cell lines.The increased autophagic lysosomes in the resistant cell lines were observed by transmission electron microscopy of the parental and resistant cell lines;the autophagic flux of the resistant cell lines was found to be increased by selective labelling of accumulated autophagic vesicles with CYTOID^? autophagy detection reagent;the protein expression levels of the autophagy-related genes SQSTM1/P62 and MAP1LC3 B were determined in the resistant cell lines.7.Screening for transcription factors that regulate autophagic flux.A heat map of differential gene clustering was first drawn based on the expression of transcription factors enriched to differential genes from transcriptome sequencing.Three transcription factors,FOXM1,NKX2-1 and PBX3,were screened,and PBX3 was verified to bind to the promoter region of MAP1LC3 B and positively regulate MAP1LC3 B transcription by dual luciferase reporter gene assay and EMSA assay.8.Silencing PBX3 affects autophagic flux in drug-resistant cell lines.CYTO-ID^? autophagy detection reagent was used to selectively label cumulative autophagic vesicles,and the autophagic flux of drug-resistant cell lines was found to be reduced after silencing;the reduced autophagic flux of drug-resistant cell lines was determined by detecting the protein expression levels of autophagy-related genes SQSTM1/P62 and MAP1LC3 B in drug-resistant cell lines.9.Silencing of PBX3 affects the sensitivity of drug-resistant cell lines to Ensartinib sensitivity.The IC50 changes after PBX3 silencing were examined by MTS assay,and it was found that silencing of PBX3 partially reversed the resistance of resistant cell lines to Ensartinib.10.Animal models validated that silencing of PBX3 could reverse resistance by reducing autophagic flux.In vivo observations in the animal model showed no significant difference in the final tumor volume and tumor weight formed in the sh PBX3 group compared to the shvector group,while the sh PBX3+Ensartinib group showed a significant decrease in tumor volume and weight compared to the sh PBX3 group,demonstrating that silencing PBX3 can partially reverse drug resistance at the in vivo level.Immunohistochemical staining and Western blot experiments on the removed tumors to detect changes in the expression profile of MAP1LC3 B,SQSTM1/P62 demonstrated that silencing PBX3 at the in vivo level could reverse drug resistance by reducing autophagic flux.11.Databases to validate the relationship between high PBX3 expression and prognosis of NSCLC patients.High PBX3 expression was found to be positively correlated with poor prognosis in NSCLC patients in TCGA and GEO databases.Conclusions: 1.H3122 and H2228 parental cells are the most sensitive to Ensartinib among the three ALK inhibitors;2.Ensartinib-resistant cell lines were established with phenotypic changes of enhanced migration and invasion;3.PBX3 regulates MAP1LC3B transcription to increase autophagic flux in Ensartinib-resistant cell lines;4.In vivo and in vitro experiments demonstrated that silencing PBX3 partially reverses Ensartinib resistance;5.High PBX3 expression was associated with poor prognosis in NSCLC patients;6.PBX3/LC3 B regulates autophagic flux to induce nonsmall cell lung cancer resistance to Ensartinib.