Mechanism Of XPO1 Inhibition Synergizing With PARP1 Inhibition By Targeting Nuclear Export In SCLC

Background: Small cell lung cancer(SCLC)is an extremely aggressive form of lung cancer,with key features including strongest proliferative capacity and the worst prognosis among lung cancers.Currently,platinum-based combination chemotherapy with selective concomitant radiotherapy remains the standard of care for first-line treatment of SCLC.Patient mortality rates have remained stubbornly high in the past decades in SCLC because of having no standard targeted therapies with confirmed advantages.While SCLC almost universally harbors TP53 and RB1 inactivation,making SCLC proliferate rapidly and therefore cause replication pressure.Targeting Poly [ADPribose] polymerase(PARP)activity can supplement the molecular defects of DDR and potentially target the most critical genetic characteristic,especially in SCLC.Present clinical trials have tried to combine PARP inhibitors with chemotherapies in SCLC.But none of strategies obtained positive results in patients’ survival.Therefore,identifying resistance mechanisms and reasonable solutions is critical.This study examined the mechanism and efficacy of nuclear transport inhibition combined with PARP inhibitors in several preclinical models.Purpose: This study focused on compensatory pathways induced by PARP inhibition in SCLC,the subcellular location and mechanism of forkhead box-O3a(FOXO3a)under treatment of PARP inhibition,as well as the in-depth investigation of synergizes efficacy combining PARP and nuclear-export inhibition in vitro and in vivo.Method: 1.MRNA expression levels of PARP1 among over 1000 cancer cell lines were estimated from the Cancer Cell Line Encyclopedia(CCLE).Clinical data and tissue samples of SCLC patients were collected and performed by immunohistochemistry to confirm the PARP1 expression.The correlation between PARP1 expression and overall survival of SCLC patients was analyzed by Kaplan-Meier survival.The RNA-seq and isobaric tags for relative and absolute quantification(ITRAQ)were used to explore the compensatory pathway and protein distribution in the nucleus and cytoplasm under PARP1 inhibition.Western blot and immunofluorescence technique were used to confirm high-throughput sequencing result.2.SCLC cells was infected with FOXO3a-TM plasmid which FOXO3a could not be phosphorylated.Co-immunoprecipitation was performed to prove the binding ability between XPO1 and FOXO3a.By establishing crispr-cas9-mediated XPO1 knockout and performing western blot/immunofluorescence,the specific of FOXO3a and XPO1 binding effect was confirmed.And the combination effect of XPO1 and PARP1 inhibition on FOXO3a subcellular location was validated by western blot and immunofluorescence.The MTT assay and analysis by combination index(CI)using Compu Syn software were performed in order to compare combination regimens with PARP1 inhibition.3.In vitro assays evaluating the efficacy of the combination of PARP1 and XPO1 inhibition used flow cytometry,Chromatin immunoprecipitation quantitative real-time PCR,western blot and immunofluorescence.In vivo efficacy of the combination evaluated used mini-patient-derived xenotransplants(mini-PDXs)and SCLC cells derived xenograft models.The novel dual PARP-XPO1 inhibitor was designed and confirmed its effectiveness by MTT,PARP enzyme assay,immunofluorescence and western blot.Result: 1.Data analysis found that PARP1 was highly expressed in SCLC in m RNA and protein level.Under PARP inhibition treatment,measured changes were found in pathways including PI3K/Akt,AMPK,Wnt and so on.By proteomic quantitative analysis,FOXO3a,the downstream target of PI3K/Akt pathway,exhibited a significant increase in cytoplasm and decrease in nucleus.When shuttling from the nucleus to the cytoplasm,the transcription factor FOXO3a was phosphorylated and lost its function.2.Co-Immunoprecipitation suggested that XPO1 could only bind to phosphorylated FOXO3a.After XPO1 knocked-out,FOXO3a expression in the nucleus was restored regardless of phosphorylation,suggesting that FOXO3a was a XPO1 cargo.FOXO3a was restored in nucleus under PARP1 and XPO1 inhibition.Compared to PARP1+PI3K/Akt inhibition,the combination of PARP1 and XPO1 inhibition exhibited the most drug toxicity on SCLC cells and the least toxicity on normal cells.3.The efficacy of the combination of XPO1 and PARP1 inhibition on apoptosis and DNA damage was significantly improved.The combination inhibited primary SCLC cell proliferation in mini-PDXs models derived from 6 SCLC patients.Mice treated with the combination were significantly prevent the tumor growth and were well tolerated with a weight loss in 10%.Based on the promising results,we developed a novel compound DIR-639 targeting both PARP and XPO1.We preliminarily confirmed the cytotoxicity on SCLC cell lines,as well as the targeting ability on XPO1 and PARP1.Conclusion: 1.PARP1 inhibition leads to FOXO3a phosphorylation and relocalization from nucleus to cytoplasm in SCLC.2.Phosphorylated FOXO3a relocalization depends on nuclear exporter XPO1.3.Combination of PARP1 and XPO1 inhibition prevents SCLC malignant progression in vitro and in vivo.The research not only provides promising strategies for the clinical applications but also opens new avenues for the treatment of drug development.