Study On The Role And Molecular Mechanism Of Albendazole In Enhancing Anti-tumor Immunity Through Down-regulating Tumor PD-L1

Background:Immune checkpoint inhibitors（ICIs）have been widely used in the treatment of various cancers and demonstrated promising outcomes in recent years,especially in non-small cell lung cancer（NSCLC）and melanoma.However,low response rates,susceptibility to recurrence,and high costs have limited the application of clinical immunotherapy.Therefore,screening for drugs that enhance the efficacy of immune checkpoint inhibitors from existing clinical agents and revealing their underlying mechanisms are important research directions to achieve clinical translation and application.It has been reported that albendazole showed effective anti-tumor activity in a variety of tumors,and also increased the counts of CD4⁺and CD8⁺T cells and significantly stimulated the IFN-gamma（a Th1-type cytokine）response in mice and human patients infected with Echinococcus.However,the role of albendazole in regulating antitumor immunity and the underlying mechanism remains unclear.The inhibitory immune checkpoint PD-L1 is highly expressed in most types of tumor cells,also the tumor PD-L1 level is a determinant and a common biomarker for the assessment of the clinical response to anti-PD-L1/PD-1 therapy.Therefore,the regulatory mechanism of PD-L1 expression provides a basis for developing new therapeutic strategies and improving the clinical response rate and efficacy of immunotherapy.UBQLN4 was involved in tumor progression and various pathophysiological processes by regulating the ubiquitination of substrate proteins.However,whether UBQLN4 regulated the PD-L1protein stability has not been reported.Nucleotidase CD73 is an immune checkpoint inhibitory molecule expressed on Treg cells,and our previous results showed that albendazole elevated the Treg cell abundance in both melanoma B16F10 and Lewis Lung Cancer（LLC）tumor-bearing immune-competent C57 mouse models.Therefore,Whether albendazole combined with CD73 inhibitor can synergistically suppress tumors needs to be further elucidated.Objectives:To investigate the novel role and underlying mechanism of albendazole in inhibiting tumor growth and regulating anti-tumor immunity;to study the molecular mechanism of albendazole in regulating tumor PD-L1;to identify the correlation between the expression of UBQLN4-PD-L1 in clinical melanoma samples and tumor progression,clinical prognosis and response to immunotherapy;to evaluate the anti-tumor efficacy of albendazole combined treatment with CD73inhibitors,and to provide molecular targets and new strategies for the diagnosis and cancer immunotherapy.Methods:The melanoma B16F10 and LLC tumor-bearing immune-competent C57 mouse models were constructed to explore the therapeutic effect of albendazole on tumors.Flow cytometry was used to explore the regulating effect on anti-tumor immunity by albendazole.The toxicity of albendazole was confirmed by detecting the mouse body weight,the blood routine examination,the morphology and the organizational structure of the kidney,liver,and spleen of mice.The effect of albendazole on the tumor PD-L1 level was examined by Western blotting,flow cytometry and qRT-PCR.The effect of albendazole on the T cell killing function was investigated by T cells/tumor cells co-cultured.The mechanism of ABZ reducing PD-L1 protein level was identified and validatedthroughimmunoprecipitation-massspectrometry（IP-MS）,Co-immunoprecipitation（co-IP）and extracellular pull-down and the series molecular methods.Using The melanoma B16F10 and LLC tumor-bearing immune-competent WT/CD73^-/-C57 mouse models to explore the combined therapeutic effect of albendazole and CD73knockout in tumors.To explore the synergistic anti-tumor efficacy of albendazole with CD73 inhibitor（APCP）.The level of UBQLN4/PD-L1in the TCGA database and melanoma patients samples with anti-PD-1therapy were detected and assessed by immunofluorescence and bioinformatics,to identify the association of UBQLN4/PD-L1 level with tumor prognosis and anti-PD-1 treatment efficacy.Results:This study found that albendazole inhibited the growth of tumor cells in melanoma B16F10 and LLC tumor-bearing immune-competent C57 mouse models.Albendazole increased the number and activity of CD8⁺T cells,and decreased tumor PD-L1expression with limited toxicity in the mice.Albendazole down-regulated the tumor PD-L1 protein level in a dose-dependent manner,while the mRNA levels of PD-L1 did not significantly change after ABZ treatment.Albendazole induced the antitumor sensitivity of T cells in vitro without affecting PD-1⁺expression on T cells.Mechanistically,we revealed that UBQLN4 bound to PD-L1 through the UBL domain and stabilized PD-L1 protein,albendazole promoted the ubiquitin-mediated degradation of PD-L1 via suppressing UBQLN4.Preclinically,genetic deletion or target inhibition of CD73 showed synergistic therapeutic effects with albendazole treatment in the immune-competent mice models.ABZ treatment significantly increased the GZMB⁺CD8⁺T cell proportion and decreased the Treg level in both melanoma B16F10 and LLC tumor-bearing immune-competent mouse models.UBQLN4 and PD-L1were upregulated in most cancer types,and high expression of UBQLN4and PD-L1 was associated with poor survival in various cancers.Significantly,UBQLN4 and PD-L1 levels were higher in the tumor region of responders compared to nonresponders and correlated with better PFS and OS in anti-PD-1 therapy melanoma patients.Conclusions:Albendazole inhibits tumor growth,enhances CTL activity and down-regulates the expression levels of tumor PD-L1.Albendazole induces ubiquitin-mediated degradation of PD-L1 by suppressing UBQLN4.Albendazole combined with CD73 gene deletion/inhibitor APCP enhances the efficacy of anti-tumor immunity and inhibits tumor growth.UBQLN4/PD-L1 levels were valid predictors for the efficacy of anti-PD-1 therapy.