Pharmacological Rescue Of Tumor Intrinsic STING Expression And Immune Responsiveness In LKB1 Mutant Lung Cancer Using IAP Inhibitors

Background:Harnessing the power of human immune system to treat patients with cancer has become a core clinical approach for deadly cancers,such as melanoma and lung cancers.However,the diverse primary responsive rate and emerging acquired resistance present a daunting challenge to expand the impact of immunotherapy.Lung cancer is still the leading cause of cancer-related death in the world.And the majority of lung cancer patients are non-small cell lung cancer(NSCLC),which accounts about 85%lung cancer patients.LKB1 inactive mutations(LKB1-Mut)are frequently occurred in NSCLC patients.LKB1-Mut NSCLC is still considered as“undruggable”due to loss-of-function mutations,no targeted therapy and resistance to immunotherapy.However,limited understanding of the gap between LKB1-mut genotype and immune suppression phenotype hampers the therapeutic development.Objective:Explore and discover new approaches or drugs which can enhance immune response for LKB1-mut NSCLC patients.Offer potential therapeutic strategies to reestablish the responsiveness of LKB1-mut NSCLC to immune checkpoint inhibitors or other immunotherapies.Methods:We used immune cell-cancer cell coculture system to investigate whether the expression of LKB1 in NSCLC cells will affect the sensitivity to immune response,indicating that LKB1-Mut NSCLC cells will exhibit different immune response compared to LKB1 wild type(LKB1-WT)NSCLC cells in vitro.And we aimed to discover some small molecules which can enhance immune response towards LKB1-Mut NSCLC cells through high-throughput small molecule screening.We used common molecular biology technologies and approaches to explore potential functions and mechanisms of small molecules or related genes in LKB1-mut NSCLC cells,such as sh RNA knock down,protein inhibitors,q RT-PCR,western blot,and et al.Besides,GST-pull down,Co-immunoprecipitation and TR-FRET were carried out to study protein-protein interaction for molecular mechanism discovery.Moreover,we used syngeneic mouse model with LKB1 mutation to validate whether the small molecules we identified can induce anti-tumor response in vivo.Results:(1)Through immune cell-cancer cell coculture experiments,loss of LKB1 expression in NSCLC cells caused resistance to the immune attack.(2)Through HTi P screening,we found that IAP inhibitors such as birinapant,BV-6,and GDC0152 can induce immune cell-dependent killing towards LKB1-Mut NSCLC cells and had minimal effect on inhibiting LKB1-Mut cancer cell growth without immune cells.(3)IAP inhibitors contribute to anti-cancer immunity towards LKB1-Mut NSCLC cells partially through the modulation of STING expression,which is markedly suppressed in LKB1-Mut NSCLC cells.(4)IAP inhibitors synergize IFNγresponse to restore STING expression via JAK/STAT pathway through degrading c IAP1,which can further activate STING downstream TBK1 and IRF3 phosphorylation.(5)IAP inhibitor birinapant synergizes IFNγto induce STING-mediated apoptosis of LKB1-Mut NSCLC cells in vitro.(6)Using a syngeneic allograft model with LKB1 mutation,we found IAP inhibitor birinapant led to a significant anti-tumor effect and increased the CD8~+T cell proportion in the tumor.However,this anti-tumor effect was totally abolished in the immune-deficient nude mice.(7)c IAP1 can bind with both LKB1 and JAK1.LKB1 can compete with JAK1 binding to c IAP1.Compared with LKB1-WT,LKB1 mutants significantly lose the interaction with c IAP1 due to the loss of LKB1 kinase activity.(8)In LKB1-Mut NSCLC cells,IAP inhibitors treatment can decrease the ubiquitination level of JAK1,which further stabilizes and increases JAK1 protein level and enhances the expression of IFNγtargeting genes,such as IRF1 and TAP1.Conclusions:(1)IAP inhibitors exhibit potent STING-dependent anti-tumor immune repsonse towards LKB1-Mut NSCLC both in vitro and in vivo(2)In LKB1-Mut NSCLC,IAP inhibitor can synergize IFNγto increase STING expression,activate STING downstream TBK1-IRF3signaling pathway,resulting in STING-dependent immune response.(3)LKB1-IAP-JAK trimolecular interactions axis bridges the gap between LKB1-Mut genotype and IFNγresponse or STING expression phenotype.IAP inhibitors can stabilize JAK1 protein,enhance IFNγresponse and increase STING expression through c IAP1 inhibition in LKB1-Mut NSCLC cells.28 figures,27 tables,119 references...