Using Single Cell Technology To Analyze The Pathogenesis And Treatment Mechanism Of Essential Thrombocythemi

Objectives:Philadelphia chromosome-negative myeloproliferative neoplasms(PhMPNs)are a group of clonal heterogeneous diseases of the hematopoietic system characterized by the proliferation of mature blood cells of one or multiple myeloid lineages.Essential thrombocythemia(ET),polycythemia vera(PV),and primary myelofibrosis(PMF)are three major subtypes of Ph-MPNs.JAK2V617F is the most frequent driver mutation in Ph-MPNs.However,the mechanism by which the same JAK2(Janus kinase 2)V617F mutation in hematopoietic stem cells(HSCs)triggers different MPN subtypes,especially the link between disease subtypes and HSC heterogeneity,is incompletely understood.Furthermore,the therapeutic effects of medicines such as Interferon-α(IFNα)on HSCs of MPN patients as well as the underlying mechanism remain to be elucidated.Therefore,this study aims to reveal the characteristics of HSCs from patients with distinct JAK2V617F+ MPN subtypes(ET and PV),exploring the association between these characteristics and the pathogenesis of specific MPN subtype.More importantly,this study is aimed at investigating the HSC heterogeneity in JAK2V617F+ ET patients with or without therapy by single cell technology,resolving the association between HSC heterogeneity and the pathogenesis and therapeutic responses of ET.Methods:(1)HSCs from patients with newly diagnosed JAK2V617F+ ET and PV,as well as normal controls(NC)were sorted to perform rare-cells RNA-seq,followed by bioinformatics analysis and experimental validation by flow cytometry.(2)3’-Target-seq(single-cell transcriptome sequencing with parallel single-cell mutation detection)was developed by modifying the single-cell RNA-seq(Smart-seq2)to precisely identify JAK2 homozygous mutant(HOMO),heterozygous mutant(HET)and wild-type(WT)cells in a single individual with ET.(3)Single HSCs from patients with newly diagnosed JAK2V617F+ ET and NCs were sorted to perform 3’-Target-seq,followed by bioinformatics analysis and experimental validation by single-cell quantitative real-time PCR,ex vivo megakaryocyte(Mk)differentiation,and colony-forming unit(CFU)assays to resolve the pathogenesis of ET.(4)Single HSCs from JAK2V617F+ ET patients treated with IFNα alone or in combination with hydroxyurea(HU)were sorted to perform 3’-Target-seq,followed by comprehensive bioinformatics analysis of integrated data from patients with non-treated ET(NT-ET),treated ET(T-ET)and NC to elucidate the transcriptional signatures of HOMO HSCs and HET HSCs upon treatment.Results:(1)Significant Mk lineage priming was exhibited in HSCs from newly diagnosed JAK2V617F+ ET patients,whereas preferential lineage skewing was not observed in HSCs from PV patients.(2)The elevated IFNα response signaling was observed in HSCs from newly diagnosed JAK2V617F+ ET patients.(3)transient,lowdose IFN stimulation promoted megakaryopoiesis of HSPCs or HSCs.(4)JAK2V617F expanded the proportion of Mk-primed HSCs with enhanced Mk lineage priming potential in newly diagnosed ET patients.(5)JAK2V617F+ HSCs in ET exhibited hyperresponsiveness to transient,low-dose IFN stimulation toward megakaryopoiesis.(6)Upon treatment,HOMO HSCs showed a quiescent signature,whereas HET HSCs underwent enhanced apoptosis in patients with ET.Conclusions:3’-Target-seq(single-cell transcriptome sequencing with parallel singlecell Genotyping)was successfully developed.HSC heterogeneity is linked to the pathogenesis and therapeutic responses of JAK2V617F+ ET.The expanded JAK2V617F+Mk-primed HSCs,elevated IFN response signaling,and the hyperresponsiveness of JAK2V617F+ HSCs to IFN signaling toward megakaryopoiesis are involved in the pathogenesis of JAK2V617F+ET.Upon treatment,JAK2V617FF+ Mk-primed HSCs were reduced.Interestingly,JAK2V617F+ HSCs in ET exhibited distinct signatures:homozygous mutant HSCs had a quiescent signature,whereas their heterozygous counterparts underwent enhanced apoptosis,which might be one of the potential therapeutic mechanisms of medicines such as IFNα.Objectives:Philadelphia chromosome-negative myeloproliferative neoplasms(PhMPNs)are a group of clonal heterogeneous diseases of the hematopoietic system characterized by the proliferation of mature blood cells of one or multiple myeloid lineages.Essential thrombocythemia(ET)is one of the major subtypes of Ph-MPNs.The JAK-STAT hyperactivation caused by JAK2(Janus kinase 2)、CALR(Calreticulin)and MPL(Myeloproliferative leukemia virus oncogene)mutations has been considered to participate in the pathogenesis of ET.However,the similarities and differences in the pathogenesis of these distinct types of ET(JAK2/CALR/MPL-mutated ET)are not fully understood.The comprehensive and precise analysis for single HSCs from these distinct types of ET is lacked and urgently necessary.Therefore,based on the research from part 1 of the thesis,this study aims to depict the single-cell transcriptional profiles of HSCs from patients with JAK2/CALRIMPL-mutated ET and unravel the gene expression signatures of mutant HSCs and wild-type HSCs from these patients,elucidating the potential similarities and differences in the pathogenesis of JAK2/CALR/MPL-mutated ET.Methods:(1)3’-Target-seq(single-cell transcriptome sequencing with parallel singlecell mutation detection)was modified and applied further,adding the detecting methods for mutations of insertions,deletion and base substitution,to identify precisely homozygous mutant(HOMO),heterozygous mutant(HET)and non-mutant wild-type(WT)cells in patients with CALR/MPL-mutated ET.(2)Single HSCs from patients with newly diagnosed CALR-mutated ET,MPL-mutated ET were sorted to perform 3’-Targetseq,followed by integrated bioinformatics analysis of the data from JAK2/CALR/MPLmutated ET and normal controls(NC)based on the research from part 1 of the thesis.(3)The gene expression profiles of mutant HSCs and wild-type HSCs were analyzed to identify the gene expression signatures of JAK2/CALR/MPL mutant HSCs,revealing potential the similarities and differences in the pathogenesis of JAK2/CALR/MPLmutated ET.Results:(1)In patients with CALR-mutated ET,CALR mutant HSCs exhibited high-level expression of genes related to cell cycle and cell proliferation.(2)CALR-type2 mutant HSCs showed higher-level expression of gene sets related to cell cycle,IFN response,and Mk lineage priming than CALR-typel mutant HSCs.(3)In patients with MPLmutated ET,MPL mutant HSCs exhibited dysregulation of metabolism-related genes.(4)In patients with JAK/CALR/MPL-mutated ET,JAK2/CALR/MPL mutant HSCs had shared enriched gene sets(cell cycle,lipid metabolism,interferon response,Mk lineage priming),but the degrees of the enrichment were different.CALR mutant HSCs were mainly characterized by the proliferative signature,MPL mutant HSCs were primarily characterized by the signature of metabolic dysregulation,while JAK2 mutant HSCs were mostly characterized by the signatures of strong interferon response and Mk lineage priming.Conclusions:In this study,we preliminarily depicted single-cell transcriptional profiles of HSCs from patients with JAK2-mutated ET,CALR-mutated ET and MPL-mutated ET and unraveled the gene expression signatures of JAK2/CALR/MPL mutant HSCs,suggesting the potential similarities and differences in the pathogenesis of different types of ET,but further investigations are still necessary in the future.