Clinical And Molecular Characteristics Of MDS Patients Undergoing Transplantation And Exploration On The Targeting Effect Of Chelidonine

Part 1 Prognostic analysis of patients with myelodysplastic syndrome after transplantationBackground Myelodysplastic syndromes(MDS)are a group of clonal hematopoietic stem cell disorders characterized by cytopenias,ineffective hematopoiesis,and increased risk of evolution to acute myeloblastic leukemia.With the growing understanding of the pathophysiology of MDS,epigenetic therapy with hypomethylating agents(HMAs),has become the standard treatment for MDS.However,HMAs are not curative therapies for MDS.Allogeneic hematopoietic stem cell transplantation(HSCT)is the only potentially curative therapeutic option for MDS.For patients who are transplant candidates,selecting the appropriate pre-transplant therapies and determining the optimal time for transplantation with the purpose of reducing transplant-related complications are still facing challenges.Methods We retrospectively analyzed 415 consecutive MDS patients who underwent HSCT between December 2007 and September 2019 at the First Affiliated Hospital of Soochow University and assessed risk factors that affecting the prognosis of MDS after transplantation.Results 1.Among 415 MDS patients who received allogeneic hematopoietic stem cell transplantation,the median age was 40 years,261 males and 154 females.371 patients(89%)received myeloablative conditioning(MAC)regimen and 44 patients(11%)received reduced intensive conditioning(RIC)regimen.According to transplant type,131(32%)received sibling donors transplants,81(20%)received unrelated donors transplants,and 203(49%)received haploid transplants.Grade Ⅱ-Ⅳ acute GVHD occurred in 135 patients(33%)at day 100 after transplantation,and the median onset was 28 days(range:4-98 days).The cumulative incidence of grade Ⅱ-Ⅳ acute GVHD at 30,60 and 90 days after transplantation was 19.6%,31.2%and 32.9%,respectively.Among 374 patients who survived for at least 100 days,chronic GVHD was diagnosed in 142(38%)patients,with the median onset of 5.4 months(range:3.4-34.5 months).Extensive cGVHD occurred in 38 patients.By the last follow-up(2021-06-30),153 cases(36.9%)died,including 115 cases(27.7%)of NRM.The cumulative incidence of NRM at the first,second and third year was 21.7%,25.1%and 26.5%,respectively.42(10.1%)patients had relapsed at the final follow-up,and the cumulative incidence of relapse at the first,third and fifth year was 11%,14%and 16%,respectively.EBV reactivation occurred in 65(15.7%)MDS patients after HSCT,and the median onset time was+55 days(range:4-714 days).The cumulative incidence of EBV viremia in MDS patients was 14.5%at the first year,and 26.0%at the second year,respectively.Among 415 MDS patients after transplantation,116(28%)experienced thrombocytopenia.Among these,48(12%)had primary poor platelet reconstruction and 68(16%)had secondary poor platelet reconstruction.2.Multivariate analysis showed that age≥40 years(P=0.005,HR=1.73),grade Ⅱ-ⅣaGVHD(P<0.001,HR=1.98),extensive cGVHD(P=0.007,HR=1.92),and poor platelet reconstruction(HR=1.82,P=0.003)were independent risk factors affecting OS.AML transformation before transplantation was the most important risk factor affecting RFS in MDS patients(HR=5.94,P=0.001),while receiving MAC conditioning(HR=0.32,P=0.004)and infusion of marrow combined with peripheral blood derived stem cells(HR=0.63,P=0.027)improved RFS after transplantation.EBV reactivation was also identified as an independent risk factor for RFS(HR=3.18,P<0.001).Patients with EBV reactivation after transplantation had significantly shorter RFS,with 1-year and 3-year RFS of 78%and 69%,respectively,compared with those without EBV activation(89%and 88%,respectively)(P=0.006).3.For MDS patients who are candidates for MAC transplantation,receiving nonchemotherapy and proceeding transplantation within 6 months after diagnosis(SC/HMA+<6 months)was regarded as the most appropriate pre-transplant strategy in MDS patients,which could help improve the prognosis of patients.Conclusions 1.Assessment of risk factors affecting the prognosis of MDS after transplantation can help doctors to make more accurate risk assessment and clinical decision.2.The adoption of appropriate pre-transplant treatment strategy can prolong survival and improve the prognosis of patients.Part 2 Prognostic significance of driver gene mutations in MDS patients after transplantationBackground MDS are a heterogenous group of myeloid neoplasms.The IPSS and IPSS-R are the most widely used prognostic models for MDS,yet there are subsets of patients who experience outcomes discordant with their risk score.Some of these differences may be explained by somatic mutations.High-throughput sequencing technologies,especially the development of whole exon sequencing(WES)and next generation sequencing(NGS),provide valuable information for us to clarify the pathogenesis and progression mechanism of MDS,conduct more accurate prognostic risk assessment and develop targeted drugs.Methods We used WES and NGS to detect gene mutation status in bone marrow cells of MDS patients who received HSCT.We also analyzed the correlation between gene mutations with clinical characteristics and evaluated the prognostic impact of gene mutations,especially RAS pathway mutations on MDS patients’ prognosis after HSCT.Results 1.In this study,bone marrow samples from 92 MDS transplant patients were subjected to WES sequencing.Forty-five known driver gene mutations were detected,and 55 patients(59.8%)had at least one mutation.There were 16 genes with a mutation frequency greater than 3%.According to the type of gene mutation,the most common type of gene variation was transcription factor-related genes(BCOR,CEBPA,ETV6,GATA2,NPM1,PHF6,RUNX1,TP53,WT1),with a total of 22 cases.There were 22 cases of RNA splice related genes(U2AF1,SF3B1),12 cases of methylation(IDH1,IDH2,TET2,DNMT3A),11 cases of chromosome modification(ASXL1,SETBP1),and 8 cases of RAS pathway abnormalities,including 3 cases of NRAS mutation,one case of KRAS mutation,2 cases of CBL mutations,2 cases of PTPN11 mutations,one case of KIT mutation and one case of RIT1 mutation.Mutations of U2AF1,TET2 and SETBP1 were associated with poor OS and RFS in MDS patients after transplantation,while mutations of RUNX1 were associated with poor RFS in MDS patients.Patients with mutations in RAS pathway showed poor OS and RFS after transplantation.2.WES identified a total of 22 new tumor driver genes that were associated with OS and/or RFS in MDS patients after HSCT.Twenty-two newly identified tumor driver genes include:LOXHD1 in 8 cases,MYH13 in 8 cases,PIEZO1 in 8 cases,DNAH5 in 7 cases,DPH1 in 7 cases,USH2A in 7 cases,DISP1 in 6 cases,LAMB3 in 6 cases,MYO5B in 6 cases,OASL in 6 cases,SDK2 in 6 cases,ZNF23 in 6 cases,CCDC170 in 5 cases,CHID1 in 5 cases,DIS3L2 in 5 cases,GALNT8 in 5 cases,GCN1 in 5 cases,HTR2B in 5 cases,KLHL32 in 5 cases,MICAL1 in 5 cases,PRDM2 in 5 cases,and SYNE2 in 5 cases.There were 10 genes associated with poor OS after transplantation:DPH1,USH2A,LAMB3,MYO5B,OASL,CHID1,DIS3L2,GALNT8,GCN1 and HTR2B.Nine genes were associated with unfavorable RFS after transplantation:LOXHD1,MYH13,SDK2,ZNF23,CCDC170,KHLH32,MICAL1,PRDM2 and SYNE2.In addition,PIEZO1,DNAH5 and DISP1 mutations are associated with poor OS and RFS in MDS patients after transplantation.3.To further verify the role of RAS pathway mutations in the pathogenesis of MDS,51 driver genes were detected by NGS technique in 211 MDS transplant patients.We combined the results of WES and NGS for re-analysis.Among 303 MDS patients,58 cases(19.4%)had RAS pathway mutations,which was associated with WHO classification(P<0.001),higher marrow blast(P=0.001),higher IPSS risk(P<0.001)and disease progression(P=0.003).These results suggested that RAS pathway mutation was involved in the disease progression of MDS.Conclusions 1.Driver gene mutations affect the prognosis of MDS patients,among which U2AF1,TET2 and SETBP1 mutations are associated with poor OS and RFS,and RUNX1 mutations are associated with poor RFS in MDS patients after transplantation.2.We identified 22 new prognostic driver gene mutations by WES.3.RAS pathway mutation is an adverse factor affecting OS and RFS in MDS patients after transplantation,and participates in the progression of the disease.Part 3 Targeting effect of Chelidonine in myeloid malignancies with RAS pathway mutationsBackground RAS pathway mutations are highly prevalent in hematopoietic malignancies.Apart from FLT3 inhibitors,there are no other drugs targeting RAS pathway.Serine/threonine kinase STK19 is a novel activator of RAS through phosphorylation.Chelidonine was identified as a selective inhibitor of STK19 kinase activity,plays an antitumor role in solid tumors by targeting RAS signaling.Therefore,we explored the role of Chelidonine in hematologic myeloid malignancies with RAS pathway mutations.Methods The effects of Chelidonine on proliferation and apoptosis in cell lines with or without RAS pathway mutations were evaluated in vitro.The changes of downstream pathways and gene expression before and after Chelidonine treatment were detected.The target of Chelidonine was explored by constructing STK19 knockdown plasmid.In addition,we established two CDX models(OCI-AML3 and MV4;11)to observe therapeutic effects of Chelidonine in vivo.Results 1.In vitro,Chelidonine significantly inhibits the proliferation in RAS pathway mutated cell lines,and its effect on wild type of RAS pathway was significantly weakened.Chelidonine significantly increases the percent of cell apoptosis and expression of Caspase 3,Caspase 7 and PARP in cell lines with RAS pathway mutations.Chelidonine inhibits the protein expression of P-MEK,P-ERK and P-AKT in OCI-AML cells(NRAS mut).These results suggest that Chelidonine affects cell proliferation and apoptosis by inhibiting downstream Raf/MEK/ERK pathway and PI3K-AKT pathway.In MV4;11 cells(FLT3-ITD),downstream ERK pathway showed reverse activation,while PI3K-AKT pathway was inhibited after Chelidonine treatment.The gene expression levels of ERK targeted genes HLDA1,DUSP4,ETV4 and SPRY2 were detected by qRT-PCR.qRT-PCR analysis indicated that Chelidonine also decreased the expression of DUSP4,PHLDA1,ETV4 and SPRY2 expression.2.Knockdown of STK19 significantly increases cell apoptosis in OCI-AML3 and MV4;11 cell lines.In shSTK19-OCI-AML3,P-ERK expression was inhibited,suggesting that STK19 affects cell apoptosis by inhibiting downstream Raf/MEK/ERK pathway.However,in shSTK19-MV4;11,P-ERK expression showed reverse activation.Therefore,the downstream pathway warrants further study.3.Chelidonine decreases the proportion of hCD45+cells in bone marrow,reduces the infiltration of bone marrow and spleen,increases the expression of Caspase3,and prolongs the survival time of OCI-AML3 and MV4;11 CDX models,suggesting the anti-tumor effect of Chelidonine in vivo.Conclusions 1.In vitro,Chelidonine inhibits RAS signaling,leading to reduced cell proliferation and induction of apoptosis in a panel of RAS pathway-mutant leukemia cell lines,including OCI-AML3,HL-60,MV4;11 and SKM-1. 2.Chelidonine exerts anti-proliferative and apoptosis-inducing effects by targeting STK19 in OCI-AML3 cells.3.In vivo,Chelidonine induces apoptosis in vivo and has therapeutic effects in OCIAML3 and MV4;11 CDX models.