The Therapy Efficacy And Underlying Mechanism Of Sirolimus In Acquired Pure Red Cell Aplasia

ObjectivesAcquired pure red cell aplasia(aPRCA)is a rare syndrome with a variety of etiologies.It is unclear whether sirolimus is effective in newly diagnosed patients with aPRCA even though it has been demonstrated that sirolimus is an effective treatment for patients with refractory/relapsed/intolerant aPRCA after cyclosporine A(CsA)therapy.The present study aims to:1)compare the safety and efficacy of sirolimus versus CsA as the frontline therapy for patients with newly diagnosed aPRCA;2)determine the changes in gene expression profile of peripheral blood mononuclear cells from primary aPRCA patients before and after sirolimus therapy;and 3)assess the role of sirolimus in inducing K562 cells to differentiate into erythroid cells and the underlying molecular mechanisms.Methods1)Patients diagnosed with primary aPRCA at Peking Union Medical College Hospital from July 2020 through April 2021 were prospectively assigned to sirolimus or CsA treatment based on their clinical status.The enrolled patients were given sirolimus(1 mg/d,oral,qd)or CsA(3-5 mg/kg/d,oral,qd)for more than 6 months and followed up for more than 12 months unless disease progression,death,or treatment discontinuation due to various reasons,such as side effects or withdrawing consents.The primary endpoint of this study was treatment response including complete response(CR),partial response(PR),no response(NR),overall response(OR)and relapse.The secondary endpoint of this study was safety of sirolimus versus CsA in patients with newly diagnosed aPRCA.2)We prospectively collected the clinicopathological features of patients with primary aPRCA at Peking Union Medical College Hospital from August 2021 to December 2021.After obtaining the agreement of each patient for blood sample collection,we obtained a total of 9ml peripheral blood sample of each enrolled patients before and after 12-month of sirolimus treatment.The harvested peripheral blood sample was then used for peripheral blood mononuclear cells(PBMC)isolation by Ficoll,and RNA in PBMC was extracted by TRIZOL method for subsequent experiments.The gene-expression profile of PBMC from patients before and after sirolimus treatment was identified by RNA-seq.3)Human chronic myeloid leukemia cells K562 were treated with 10/100nM sirolimus,respectively.Benzidine staining was used to detect the benzidine positive cells in each group.The expression level of globin mRNA of K562 cells in each group was detected by qRT-PCR.The expression of CD71,a marker on the surface of erythroid cells,was detected and analyzed by flow cytometry in each group.The proliferation activity of K562 cells in each group was assessed by CCK-8 assay.The expression level of SHP1 and mTOR was detected in K562 cells post treament with 10/100nM sirolimus for 48h.Furthermore,K562 cell with or without SHP1 silence via transfection of siRNA were treated with 100nM sirolimus for 48h,the percentage of benzidine staining positive cells,the expression level of globin at mRNA level and CD71 at protein level were detected by benzidine staining,qRT-PCR and flow cytometry,respectively.Results1)Totally,56 patients with newly diagnosed aPRCA were enrolled:26 patients received sirolimus and 30 patients received CsA.Although the two groups had similar overall response rate(ORR)and complete response rate(CRR)at 3 and 6-months and the end of follow-up,the sirolimus group had significantly higher ORR(80.8%vs 60.0%,P=0.039)and CRR(73.1%vs.43.3%,P=0.035)at 12-month,and a lower relapse rate(15.3%vs.30.0%,P=0.044)at a median of 18 months follow-up(range,12-22).Furthermore,the serum creatinine level was significantly improved in the sirolimus group(P=0.0038)whereas it was significantly deteriorated in the CsA group(P=0.0018)at the end of follow-up.The level of TNF-α and erythropoietin were significantly decreased 6 months post-sirolimus treatment(P=0.0073 and 0.010,respectively),but not after CsA treatment.2)By comparing the gene expression profile of peripheral blood mononuclear cells from patients with primary aPRCA before and after sirolimus treatment,we identified 1639 significantly upregulated genes at mRNA expression level(the top five upregulated-genes were SYTL5,VSNL1,LINC00707,BTBD17 and LINC01954)and 2421 significantly downregulated genes at mRNA expression level(the top five downregulated-genes were KLHDC8A,IGKV1-8,SNORD3B-1,PRAME and LINC00520),which indicated that sirolimus could play a therapeutic role in primary aPRCA by regulating the expression level of these dysregulated genes.According to GO enrichment analysis results,we found that the immune-related biological processes significantly enriched by the differentially expressed genes in peripheral blood mononuclear cells of aPRCA patients after sirolimus treatment include B-cell-mediated immunity and circulating immunoglobulin-mediated humoral immune response.By analyzing the functional interaction of differentially expressed genes in peripheral blood mononuclear cells of patients with aPRCA,we found that TNF,CD40,HLADRB1 and SERPING1 are the core regulatory genes in the B-cell-mediated immune molecular network;Also,TNF,SERPENG1,C1QA,IGLL5 and IGHV3-11 are the hub genes in the molecular network of humoral immune response.Differential genes in peripheral blood mononuclear cell of patients with aPRCA after sirolimus treatment showed that PGBD4,MIR553,FIBCD1,BSCL2 and GTPBP1 genes(expression profiles)in peripheral blood mononuclear cell might be used as new molecular markers to predict the clinical efficacy of sirolimus in aPRCA patients according to WGCNA.3)The proportion of benzidine-staining positive cells were significantly increased in K562 cells after treatment with 10/100nM sirolimus for 48/72/96h,respectively.The proliferation activity was decreased in K562 cells post treatment with 10/100nM sirolimus for 24/48/72h,respectively.After treating K562 cells with 10/100nM sirolimus for 72h,the expression level of α、γ globin mRNA and erythroid surface marker CD71 were significantly upregulated in each group,respectively.Furthermore,SHP1 was significantly increased while mTOR was markedly decreased in K562 cells post treatment with 10/100nM sirolimus for 48h.Additionally,we treated K562 cells with or without SHP1 knockdown by 100nM sirolimus for 48h,showing that proportion of benzidine staining positive cells,the expression level of CD71 at protein level or the expression level of y globin at mRNA level were significantly decreased in K562 cells with SHP1 knockdown when compared with the cells without SHP1 knockdown.Conclusions1)Our results support that sirolimus could be used to treat patients with newly diagnosed primary aPRCA.2)RNA-seq analysis results revealed the changes in gene expression profile of peripheral blood mononuclear cells from primary aPRCA patients before and after sirolimus treatment.Sirolimus can play a therapeutic role by regulating the expression level of many dysregulated genes in aPRCA patients.After sirolimus treatment,the significantly changed immune processes in peripheral blood mononuclear cells induce B-cell-mediated immunity and circulating immunoglobulin-mediated humoral immunity.PGBD4,MIR553,FIBCD1,BSCL2 and GTPBP1 genes(expression profiles)in peripheral blood mononuclear cell may be used as new molecular markers to predict the clinical efficacy of sirolimus in aPRCA patients.3)Sirolimus treatment can induce the erythroid differentiation of K562 cells by upregulating SHP1.