Combination of RUNX1 inhibitor and gemcitabine to mitigate chemo‐resistance in pancreatic ductal adenocarcinoma by modulating BiP/PERK/eIF2a-axis-mediated endoplasmic reticulum stress

Abstract

Abstract Background: Gemcitabine (GEM)-based chemotherapy is the standard treatment for pancreatic ductal adenocarcinoma (PDAC). However, the development of drug resistance limits its efficacy, and the specific mechanisms remain largely unknown. RUNX1, a key transcription factor in hematopoiesis, also involved in the malignant progression of PDAC, but was unclear in the chemoresistance of PDAC. Methods: We detected the expression of RUNX1 in the PDAC tissues by RT-PCR, immunohistochemistry (IHC) and western blot. The clinical significance of RUNX1 in PDAC was confirmed by single-or multivariate analysis and survival analysis. We established the stable expression cell lines with shRUNX1 and RUNX1 construct, and GEM-resistant cell lines was succeeded yet. The role of RUNX1 in GEM resistance was determined by CCK8 assay, plate colony formation assay and apoptosis analysis in vitro and in vivo. To explore the mechanism, we performed bioinformatic analysis using single-cell RNA sequencing(scRNA-seq) data from PDAC to screen for the endoplasm reticulum (ER) stress that was indispensable for RUNX1 in GEM resistance. We observed the cell morphology in ER stress by transmission electron microscopy and validated RUNX1 in gemcitabine resistance depended on the BiP/PERK/eIF2a pathway by in vitro and in vivo oncogenic experiments, using ER stress inhibitor(4-PBA) and PERK inhibitor (GSK2606414). The correlation between RUNX1 and BiP expression was assessed using TCGA RNA-seq dataset and validated by RT-PCR, immunostaining and western blot. The mechanism of RUNX1 regulating BiP was confirmed by ChIP-PCR and dual luciferase assay. Finally, the effect of RUNX1 inhibitor on PDAC was conducted in vivo mouse models, including subcutaneous xenograft and patient-derived xenograft (PDX) mouse models. Results: RUNX1 was aberrantly expressed in PDAC and was closely associated with responses to GEM-based chemotherapy. Silencing of RUNX1 could reverse GEM resistance in drug-resistant cell lines, and its inhibitor Ro5-3335 displayed a synergistic effect in inhibiting tumor growth in GEM-resistant xenograft and PDX mouse models, enhancing the anti-tumor activity of GEM. In detail, forced expression of RUNX1 in PDAC cells suppressed apoptosis induced by GEM exposure, which was reversed by the ER stress inhibitor 4-PBA and GSK2606414. RUNX1 modulating ER stress mediated gemcitabine resistance was supported by the analysis of our single cell RNA sequencing data. Consistently, silencing of RUNX1 strongly inhibited the GEM-induced activation of BiP and PERK/eIF2a signaling, one of the major pathways involved in ER stress. It was identified that RUNX1 directly binds to the BiP promoter, a key direct ER stress initiator, and stimulates BiP expression to enhance the capacity for cell adaptation, which in turn facilitates GEM resistance in PDAC cells. Conclusions: This study identified RUNX1 as a predictive biomarker for response to GEM-based chemotherapy. RUNX1 inhibition may represent an effective strategy for overcoming GEM resistance in PDAC cells.