The Effect Of Host T-cell Methylation Landscapes Of Diffuse Large B-cell Lymphoma On CAR-T Therapy And The Investigation Of The Mechanism Of DNMT3A In Lymphoma

Objectives:Diffuse large B-cell lymphoma(DLBCL),a common lymphoid hematopoietic malignancy,has improved clinical outcomes with widespread rituximab use,but there are still a number of patients at high clinical risk,and existing prognostic scoring models are not good predictors of patient outcomes.New therapies such as chimeric antigen receptor T(CAR-T)cell therapy are promising in relapsed/refractory patients,but little is known about the molecular factors that predict the clinical outcome of CAR-T therapies,and little is known about the impact of host T-cell function on CAR-T therapies.The relevance of epigenetic alterations to immune cell function and the development and progression of lymphoma is of growing concern.In this context,we investigated the following aspects:1)clinical characteristics and risk factors of DLBCL in the rituximab era and constructed new prognostic models to better identify high-risk patients.2)Preliminary investigation of the intrinsic relationship between DNA methylation profiling profiles of DLBCL host T cells and clinical course of CAR-T therapy.3)In vitro studies on the role of DNA methylationcritical enzyme DNA methyltransferase 3A(DNMT3A)in lymphoma cell line.Methods:1.The clinical features,risk factors,and outcomes of 564 patients with newly diagnosed DLBCL at Peking Union Medical College Hospital between January 2009 and May 2017 were retrospectively analyzed.Variables were screened by LASSO regression and nomogram was constructed.2.A total of 13 patients with diffuse large B-cell lymphoma who received CAR-T cell therapy were included in the study.The peripheral blood samples were collected before and after treatment,and CD3+T lymphocytes were sorted by magnetic beads method and tested with a MethylationEPIC(850K)Bead Chip.Differential methylation probe(DMP)and regions of differential methylation were analyzed,and GO,KEGG,and GSEA enrichment analyses were performed.3.In human lymphoma cell line H9,expression of DNMT3A was knocked down by siRNA,proliferation was measured by CCK8,apoptosis was detected by flow cytometry,chemokines and JAK-STAT signaling gene expression was detected by qPCR,and specific mechanisms of DNMT3A regulation in lymphoma were investigated.Results:1.282(50.0%)patients were male.The median age of all patients was 58 years(range,15-90 years).Patients were predominantly of non-GCB subtype(50.7%)and advanced stage(72.0%).Fewer patients had a bulky disease(13.7%),bone marrow(12.6%),or central nervous system involvement(6.4%).The gastrointestinal tract was the most common site of primary extranodal DLBCL involvement,accounting for 15.8%of DLBCL(9.2%in the stomach and 6.6%in the intestine),followed by the thyroid gland(12 cases,2.1%),the testes(11 cases,2.0%),the female genitalia(9 cases,1.6%),and the skeleton(9 cases,1.6%).The 5-year overall survival(OS)of the cohort was 75%.The 5-year OS of patients diferentiated by International Prognostic Index(IPI)score was 90%(score 0-2),73%(score 3),and 51%(score 4-5),respectively.Age>60,Eastern Cooperative Oncology Group(ECOG)>1,Ann Arbor stage Ⅲ-Ⅳ,bone marrow involvement,low level of albumin(ALB),and lymphatic/monocyte ratio(LMR)were independent predictors of OS.The predictive model was developed based on factors including age,bone marrow involvement,LMR,ALB,and ECOG scores.The predictive ability of the model(AUC,0.77)was better than that of IPI(AUC,0.74)and NCCN-IPI(AUC,0.69).The 5-year OS of patients in the low-,intermediate-,and high-risk groups identifed by the new predictive model was 89%,70%,and 33%,respectively.2.The 1-year overall survival rate of 13 patients with R/R DLBCL treated with CAR-T was 78%,with complete remission(CR)of 10 cases at 1 month assessment,and non-CR of 3 patients(2 PR,1 PD).①The mean global methylation level of CD3 T cells in CR group was significantly reduced after infusion compared with the pre-infusion samples(0.578±0.021 vs 0.566 ±0.024,P<0.001).In the 6644 DMPs identified,22.5%were hyper-methylated(1492/6644),77.5%were hypo-methylated(5152/6644),distributed mainly in the genomic regions(48.89%)and open sea regions(79.8%).The KEGG pathway annotation is mainly concerned with pathways in cancer,differentiation of Th17 cells,adhesion junctions,and T-cell receptor signaling,as well as annotated genes enriched for chemokine signaling and differentiation of Thl and Th2 cells.Four DMRs were identified,annotated FOXP1,FGFBP2,SPOCK2,and DIABLO,and enriched for pathways including apoptosis and microRNAs in cancers.The apoptosisassociated gene DIABLO is located in the promoter region and is hypomethylated after CAR-T infusion.② In the non-CR group,a total of 897 DMPs were screened,including 461 hyper-methylation sites and 436 hypo-methylation sites,mainly located in the genomic region(46.54%),whereas for the CpG islands,differences in methylation sites were concentrated in the open sea region(80.3%).The KEGG pathway includes cellular senescence,T-cell receptor signaling,and malaria.Three differentially methylated regions were identified,and their annotated genes were FOXP1,FGFBP2,and SPOCK2.③A total of 1129 DMPs,including 779 hypermethylated and 350 hypomethylated sites,were screened at baseline between the non-CR and CR groups,with no significant difference in mean methylation levels(0.582 ± 0.007 vs 0.578±0.021,P=0.811).Further analysis of the GSEA enrichment revealed that differentially expressed genes were associated with activation of cellular immune functions in T cells,including chemokine production,leukocyte-mediated cytotoxicity,and cytocidal killing.These genes are closely related to T cell receptor signaling pathways and primary immunodeficiency pathways.Three differentially methylated regions were identified,and their annotated genes were FOXP1,FGFBP2,and SPOCK2.3.The meta-oncologic data indicate that DNMT3A is differentially expressed in different tumors and is associated with prognosis.DNMT3A knockdown enhanced lymphoma cell proliferation,reduced apoptosis,activation of the CCL2-CCR2 axis and JAK-STAT signaling,and effective suppression of DNMT3A knockdown-induced activation of JAK-STAT signaling by CCL2 inhibitors.The effect of demethylated drugs on DNMT3 A knockdown was attenuated.Conclusions:1.The new prediction model has better predictive capabilities and can better identify high-risk patients.2.After treatment with CAR-T cells,the mean overall T-cell methylation level decreased significantly in the CR group.Significant differences in the methylation profile were shown at baseline between the CR and the non-CR groups,highexpression of the apoptotic gene DIABLO and immune activation was associated with better therapeutic outcomes.3.DNMT3A affects lymphoma cell line proliferation and apoptosis,regulates JAK-STAT signaling through the CCL2-CCR2 axis,and has an effect on the efficacy of demethylated drugs.