The Impact Of LKB1 On PD-1 Blockade Therapy In Non-small Cell Lung Cancer

Significant improvement in non-small cell lung cancer(NSCLC)therapy has been achieved over the past few years by the utilization of PD-1/PD-L1 blockade.It brings a revolution in the treatment of advanced NSCLC,which become an indispensable part for NSCLC treatment.Yet,a majority of patients receive no benefit from these therapeutic drugs,especially a subset of the patients harboring driver gene mutations.In NSCLC,patients with a high level of intratumoral PD-L1 often associated with superior clinical outcomes in PD-1/PD-L1 blockade.Therefore,it is currently believed that intratumoral expression of PD-L1 could serve as a predictor to screen patients,who are more likely respond to PD-1/PD-L1 blockade.Encoded by the gene serine and threonine kinase 11(STK11),liver kinase B1(LKB1)is a tumor suppressor,which phosphorylates and activates 13 members of the AMP-activated protein kinase(AMPK)superfamily kinases.Loss of LKB1,which often co-occurs with activated KRAS mutation,is found in 15%-35% of NSCLCs.Previous studies demonstrated that LKB1 mutations are correlated with low PD-L1 expression in KRAS-mutated NSCLC.Remarkably,this subtype of NSCLC was shown to be resistant to PD-1/PD-L1 blockade.However,direct links between LKB1 and PD-L1 are lacking,and also,the mechanisms by which LKB1 regulates PD-L1 and its implications on PD-1/PD-L1 blockade therapy remain largely unknown.In the current study,we analyzed the relationship between LKB1 and PD-L1 in NSCLC patients through The Cancer Genome Atlas(TCGA)data analysis and immunohistochemical staining(IHC).The impact of LKB1 on PD-L1 expression was further validated by genetical manipulations of LKB1 in NSCLC cells and the downstream signals involved in this process were also assessed.To determine the combinational effect of LKB1-AMPK activators and PD-1 blockade,metformin was applied to immunocompetent C57BL/6 mice treated with PD-1 blockade in LKB1-intact and LKB1-compromised tumors.A remarkable positive correlation between PD-L1 and LKB1 expression in NSCLC tissues was identified based on TCGA analysis and the IHC study.Knockdown of LKB1 decreased PD-L1 mRNA and protein expression in TC-1 cells,whereas overexpression of LKB1 increased the levels of PD-L1 mRNA and protein in A549 cells.AMPK activator metformin reversed the reduction of PD-L1 expression in LKB1-knockdown TC-1 cells.AMPK inhibitor compound C markedly inhibited PD-L1 expression in TC-1 cells and precluded LKB1 overexpression-sponsored PD-L1 upregulation in A549 cells.These results demonstrate a role of AMPK in LKB1-mediated upregulation of PD-L1 in NSCLC.Previous studies showed that LKB1 inactivation increases serine utilization,synthesis of S-adenylmethionine(SAM),expression of DNA methyltransferases(DNMTs),as well as a significant elevation of DNA methylation.In the current study,we found that methylation of PD-L1 promoter was only marginally changed in cells with or without LKB1 although knockdown of LKB1 decreased the expression of DNMT3 A,a methyltransferase promoting DNA methylation.Nuclear factor E2-related transcription factor 2(NRF2)was reported to be a transcriptional activator of PD-L1.Abnormal alterations in Kelch-like ECH-associated protein 1(KEAP1),a negative regulator of NRF2,have also been reported in LKB1-deficient KRAS-mutated tumors.We previously reported that activation of AMPK is involved in the regulation of KEAP1-NRF2.We,therefore,determined the role of KEAP1/NRF2 signaling in LKB1-induced PD-L1 gene transcription.Our results revealed that LKB1 depletion significantly elevated the levels of KEAP1 and reduced the expression of NRF2 in TC-1 cells,whereas overexpression of LKB1 bore an opposite effect on the expression of KEAP1 and NRF2 in A549 cells.More importantly,NRF2 activator dimethyl fumarate(DMF)markedly increased the levels of PD-L1 mRNA in LKB1-knockdown TC-1 cells.In contrast,NRF2 inhibitor ML385 notably impaired the increase of PD-L1 mRNA induced by LKB1 overexpression in A549 cells.Taken together,we conclude that LKB1 upregulates PD-L1 in NSCLC cells via AMPK and KEAP1/NRF2 signaling.Application of single anti-PD-1 antibody markedly suppressed the growth of LKB1-intact tumors,with no regression of LKB1-compromised tumors,suggesting that expression of LKB1 is pivotal in response to PD-1 blockde therapy.The expression of PD-L1 was notably elevated in both LKB1-intact and LKB1-compromised tumors of mice that treated with metformin compare to that in mice without metformin administration,however,combined usage of metformin and anti-PD-1 antibody was only efficient in LKB1-intact tumors,but not in LKB1-compromised tumors.We further demonstrated that CCL5 and CXCL12,two chemokines aiding in recruitment of lymphocytes and dendritic cells in the tumor microenvironment,were reduced in LKB1-compromised tumors compared to those in LKB1-intact tumors,whereas chemokines CXCL5 and CXCL7,which promote the recruitment of neutrophils,displayed a contrary alteration.Furthermore,alterations of these chemokines in LKB1-compromised tumors of mice treated with metformin and/or anti-PD-1 antibody were similar as that in mice treated with saline and/or isotype IgG,suggesting that these treatments do not change the immunosuppressive phenotype of LKB1-compromised tumors.In summary,in the current study,we demonstrated a positive regulation of LKB1 on PD-L1 expression in NSCLC via AMPK and KEAP1/NRF2 signaling.Activation of LKB1-AMPK with metformin improves the therapeutic effect of PD-1 blockade in preclinical model of NSCLC with wild-type LKB1,but not in LKB1-compromised tumors,which possesses an immunosuppressive tumor microenvironment.Metformin comprises a promising combined therapeutic strategy of PD-1 blockade for NSCLC with wild-type LKB1.