The Roles And Mechanisms Of SAMHD1-mediated STING In Diffuse Large B-cell Lymphoma

Lymphoma is a malignant disease which generates from the lymphatic system and is one of the most common kinds of tumor in China.Although modern biomedicine has progressed in diagnosing and treating,there are still many problems in clinical diagnosis and treatment.Exploration of the molecular characteristics and pathogenic evolution of lymphoma is essential for providing precise and individualized treatment for patients.As the most common subtype of adult B-cell lymphoma,diffuse large B-cell lymphoma(DLBCL)has different clinical manifestations,immunophenotypes,and genetic features.Although new immunotherapy and targeted therapy significantly improve the long-term survival rate of DLBCL patients,about 40%of patients face drug resistance or relapse after remission,leading to a huge social and economic burden.Therefore,increasing studies mainly focus on investigating pathogenic molecules,biological functions,and mechanisms.These results are expected to provide precise therapeutic targets for DLBCL patients.Genomic sequencing illustrated that B cells underwent a clonal expansion and somatic hypermutation during the malignant transformation,indicating the genomic instability of DLBCL.As is well known,homeostasis of the intracellular deoxyribonucleoside triphosphate(dNTP)pool is critical for DNA replication and damage repair.SAM domain and HD domain-containing protein-1(SAMHD1)is a common nucleoside triphosphate hydrolase in eukaryotic cells.As a vital regulator of dNTP homeostasis,SAMHD1 is involved in gene mutation,cell cycle regulation,DNA damage repair,and DNA damage signaling activation.Cyclic GMP-AMP syntheses(cGAS)-stimulator of interferon genes(STING)pathway is a classical DNA damage sensing pathway involved in anti-microbial infection,immune regulation,and cell death regulation.Several types of cell death can be induced by this signaling pathway,including necroptosis,apoptosis,and pyroptosis,where the interaction of multiple cell death pathways is known as PANoptosis.Targeted activation of STING was found to suppress tumor growth and enhance the efficacy of immunotherapy.As a result,several STING agonists have achieved impressive effects on solid tumors,such as lung and ovarian cancers.However,the roles and regulatory mechanisms of the STING pathway in DLBCL remain unclear.Our study explores the functions and mechanisms of SAMHD1 and SAMHD1-induced STING in DLBCL.The results highlight the mechanisms of SAMHD1 deficiency-induced STING activation and STING-mediated PANoptosis.Our findings are expected to provide novel therapeutic strategies for DLBCL patients.Part Ⅰ.Expression level and biological function of SAMHD1 in DLBCLObjective:DLBCL is the most common subtype of B-cell lymphoma characterized by clinical heterogeneity.About 30%of DLBCL patients face refractory,relapsed,or primary resistance.Besides,some patients cannot achieve complete remission by using rescue treatments.Molecular characteristics,biological function,and mechanisms of pathogenic molecules are investigated to provide novel therapeutic targets,which contribute to improving treatment responses and the survival of patients.Since DLBCL features genomic instability,targeted DNA damage response(DDR)is expected to enhance the efficacy of anti-tumor therapy.SAMHD1 is a nucleoside triphosphate hydrolase involved in DDR,cell cycle regulation,and cell death regulation.However,molecular expression,function,and clinical significance of SAMHD1 in DLBCL are unveiled.Thus,we attempted to explore the expression and biological role of SAMHD1 in DLBCL.Our findings clarified the expression levels of SAMHD1 in the lymph node tissue of patients and further established the correlation between SAMHD1 expression and patients’ clinical characteristics.Meanwhile,human DLBCL cell lines were used to investigate the roles of SAMHD1 on cell viability.These results provide a new marker for stratification and targeted therapy in DLBCL.Materials and methods:1.Bioinformatics analysis of the GEO and TCGA database.2.Collection of paraffin lymph node tissues from primary DLBCL and reactive hyperplasia(RHL),followed by statistics of patients’ clinical information.3.Immunohistochemical staining(IHC)of lymph node tissues.4.Collection of peripheral venous blood samples from primary DLBCL and peripheral blood mononuclear cells(PBMCs)from healthy donors.5.Purification of CD19+B cells from peripheral blood by immunomagnetic bead sorting.6.Cultivation of human-derived DLBCL cell lines.7.Western blotting(WB)analysis determined protein expression levels.8.Lentivirus transfection constructed SAMHD1 overexpression(LU-SAMHD1)and knockdown(SAMHD1-KD)modes.9.CCK-8 assay detected cell viability.10.Construction of xenograft model in SCID Beige mice.11.Statistical analysis.Results:1.Public database analysis showed high mRNA levels of SAMHD1 in DLBCL cohorts.IHC confirmed that SAMHD1 protein levels in lymph nodes of patients were higher than that of RHL.Statistically,primary DLBCL was marked by a 60%(60/100)positive rate,while RHL featured a 35%(7/20)positive rate.2.Clinical information analysis revealed that SAMHD1-positive patients were featured with reduced serum LDH and B-symptom deficiency.Survival analysis showed that the overall survival(OS)of SAMHD1-positive patients was poor than that of SAMHD1-negative patients.Additionally,SAMHD1 expression was not related to treatment responses.3.LV-SAMHD1 and SAMHD1-KD sequences were transfected into SAMHD1-positive LY1 and LY3 cells.CCK-8 assay further detected cell viability of stably transfected cells.The results showed that DLBCL cell viability was increased by SAMHD1 overexpression and decreased by SAMHD1 deficiency.The xenograft mice model revealed that silencing SAMHD1 in LY1 cells significantly suppressed in vivo tumor growth.Conclusions:SAMHD1 is highly expressed in DLBCL tissues and cells.High SAMHD1 level is associated with poor prognosis,implying that SAMHD1 may be a biomarker for prognostic assessment in DLBCL.SAMHD1-positive expression promotes DLBCL tumor growth,while SAMHD1 deficiency induces the cell viability.Therefore,SAMHD1 can be used as a therapeutic target for DLBCL.Part Ⅱ.SAMHD1 deficiency-induced STING mediates PANoptosis in DLBCLObjective:Previous studies demonstrated that SAMHD1 might inhibit the aberrant activation of innate immune signalings by maintaining genetic stability.The cGAS-STING pathway is an intracellular DNA damage-sensing pathway for initiating innate immune responses,which functions to induce immune responses and remodel the tumor microenvironment.Targeted activation of STING can induce cell death to inhibit tumor growth and further reduce tumor burden.Given that the cGAS-STING pathway exhibits dramatic anti-tumor effects,biological functions and mechanisms of STING in DLBCL are unclear.This study investigated the function of STING and the mechanisms of STING-mediated cell death.Our results elucidated that SAMHD1 deficiency induced DNA damage to activate the STING pathway.Moreover,STING induced PANoptosis by activating MLKL,CASP3,and GSDME.Our findings provided a theoretical basis for the application of STING agonists in DLBCL.Materials and methods:1.CRISPR-Cas9 gene editing technology-mediated STING knockout(KO).2.Transcriptomic sequencing(RNA-sequencing,RNA-seq)and bioinformatics analysis were used to examine the mechanisms of SANWD1.3.Annexin V-PE/7AAD and Annexin V-FITC/PI double-staining assays for cell apoptosis detection.4.Immunofluorescence(IF)staining detected protein localization and expression levels.5.Comet assay evaluated intracellular DNA fragment abundance.6.Lactate dehydrogenase(LDH)release assay determined supernatant LDH levels.7.Electron microscopic observed cell morphology.8.Lentivirus-mediated overexpression of STING and SAMHD1-KD.9.CCK-8 assay detected cell viability.10.WB analysis examined the expression of cell death pathways.11.Statistical analysis.Results:1.Flow cytometry showed a significant increase in the apoptosis rate of SAMHD1-KD DLBCL cells.In addition,we found that SAMHD1-deficient cells featured multiple modes of cell death morphology and increased LDH levels.2.RNA-seq verified that DNA damage repair and double-strand breaks were enriched in SAMHD1-KD LY1 cells.Consistently,SAMHD1 silencing increased the expression of nuclear DNA damage marker H2AX and the abundance of double-stranded DNA in DLBCL cells.3.GSEA analysis showed that the STING-mediated DNA damage signaling pathway was increased by SAMHD1 deficiency.In addition,STING,RIPK1/3,and ASC transcription were increased.4.Lentiviral transfection was used to construct STING overexpression cells,while the CRISPR-Cas9 gene editing technique established STING-KO cells.DLBCL cells with overexpressed-STING showed cell membrane swelling,which was not found in STING-KO cells.Flow cytometry showed that STING overexpression significantly promoted cell apoptosis.Consistently,the release level of LDH was positively correlated with STING expression.CCK-8 assay further verified that the cell viability was inhibited by STING overexpression and increased by STING-KO.5.WB revealed that LV-STING induced PANoptosis by activating MLKL,CASP3,and GSDME proteins.In contrast,STING-KO reduced the protein expression of p-MLKL,cleaved-CASP3,and GSDME-N in DLBCL cells.In addition,STING-KO decreased the activation of effector molecules mediated by SAMHD1 deficiency.Conclusions:Our results show that SAMHD1 deficiency induces DNA damage and dsDNA accumulation to activate the STING pathway in DLBCL cells.STING overexpression induces PANoptosis by activating MLKL,CASP3,and GSDME,which indicates the importance of STING in cell death induction.These findings lay the foundation for applying STING agonists in lymphoma.Part Ⅲ.STING agonist DMXAA enhances the efficacy of a PD-L1 inhibitor in DLBCLObjective:The STING pathway is a well-known DNA damage-sensing pathway that plays a vital role in auto-immune diseases,anti-tumor immune responses,and cell death regulation.So far,STING agonists have become a hot spot for drug investigations and have been approved for clinical trials.Research data showed that STING agonists exhibited significant anti-tumor efficacy alone or combined with immune checkpoint inhibitors.In recent studies,programmed death receptor-1/programmed death ligand-1(PD-1/PD-L1)is regarded as the potential therapeutic target for refractory/relapsed DLBCL;however,the response to anti-PD-1/PD-L1 therapy is differential in DLBCL patients.Optimizing the therapeutic strategies and exploring effective therapeutic strategies are urgent clinical issues.Therefore,we attempted to investigate the application value of STING agonists in DLBCL.We found that targeted activation of STING promoted PD-L1 overexpression,further enhancing the efficacy of PD-L1 inhibitors in DLBCL.These findings may provide a new therapeutic strategy for DLBCL patients.Materials and methods:1.Drug combination assay followed by the combination index(CI)calculation.2.CCK-8 assay evaluated cell viability.3.Immunoblot analysis examined protein expression levels.4.Construction of xenograft model in BALB/c mice.5.Subcutaneous tumor formation and intraperitoneal injection of drugs.6.Statistical analysis.Results:1.CCK-8 assay showed that the stimulant DMXAA could inhibit the cell viability in vitro.Further investigation revealed that DMXAA could induce cell death,represented by increased apoptosis rates,LDH release,and cell death effector expression.Of note,genetic deletion of STING decreased the inhibitory effects of DMXAA.In addition,cell viability of SAMHD1-positive LY1 cells could be suppressed by DMXAA treatment.2.WB further revealed the increased expression of PD-L1 in LV-STING cells and decreased PD-L1 proteins in STING-KO cells.We further examined the functions of BMS1166 in DLBCL cells with differentially expressed STING.The results showed that STING overexpression enhanced the inhibitory effects of BMS1166,while STING deletion attenuated drug effects.3.Drug combination experiments of DMXAA and BMS1166 were designed.In vitro assay revealed that the combination regimen suppressed the proliferation of LY1 cells by a moderate synergistic effect.Besides,drug combination significantly suppressed tumor growth in vivo compared with BMS1166 monotherapy.Conclusions:STING agonist DMXAA induces cell death and reduces cell viability in DLBCL.Targeted activation of STING promotes PD-L1 overexpression,further enhancing the inhibitory effects of PD-L1 inhibitor BMS 1166 in DLBCL.STING agonist combined with PD-L1 blockade is expected to enhance the efficacy of PD-L1 immunotherapy in DLBCL.