Study On The Molecular Pathogenesis And Targeted Therapy In Acute Myeloid Leukemia

This thesis consists of three parts.I.Conditional knock-in of Dnmt3 a R878H initiates acute myeloid leukemia in mice.II.The RNA helicase RIG-I regulates the differentiation of granulopoiesis by binding the m RNA of RNF138.III.Treatment of acute myeloid leukemia M2 b with homoharringtonine in combination with nilotinib.This project focused on the study of acute myeloid leukemia.Through a number of advanced technologies such as conditional knock-in mouse model,transcriptome sequencing,methylated DNA immunoprecipitation sequencing?Me DIP-seq?,as well as RNA-binding protein immunoprecipitation-sequencing?RIP-Seq?,we revealed the molecular mechanism underlying Dnmt3 a mutation-induced acute myeloid leukemia and a novel potential therapeutic target,and we found that RIG-I as a RNA helicase could bind the endogenous RNA of RNF138 to regulate granulocyte differentiation,we also proved that the combination of HHT and Nilotinib produced a synergistic effect on killing AML-M2 b leukemia cells.Our study has thus provided a theoretical basis for further elucidating the molecular mechanism of leukemogenesis in acute myeloid leukemia and exploring new targeted therapeutic strategies.Part I Conditional knock-in of Dnmt3 a R878H initiates acute myeloid leukemia in miceDNMT3A is frequently mutated in acute myeloid leukemia?AML?,with DNMT3 A R882H as the hotspot.To further explore the role of DNMT3 A R882H mutation in the pathogenesis of AML,we generated Dnmt3 a R878H?corresponding to human DNMT3 A R882H?conditional knock-in mouse model,so that Dnmt3 a mutant could be driven by the endogenous promoter/enhancer in hematopoietic system.We discovered that Dnmt3a_R878H/WT mice developed AML characterized by segmental expansion of immature cells in bone marrow,along with extramedullary infiltration including splenomegaly and lymphadenectasis,approximately 4–6 months after interferon induction.The transcriptome and DNA methylation profiling of leukemic cells revealed significant changes in expression of genes related to proliferation and differentiation in hematopoietic cells.Further molecular mechanisticstudy showed that DNMT3 A mutations could increase CDK1 protein level through m TOR activation associated with DNA hypomethylation,leading to abnormal proliferation.Interestingly,the m TOR inhibitor rapamycin prolonged the lifespan of Dnmt3a_R878H/WT mice.In addition,rapamycin also exerted significant inhibitory effects on both human AML cell lines and fresh leukemic cells of AML patients containing DNMT3 A mutations.Our study suggest the m TOR pathway as a potential drug target in DNMT3 A mutation-related AML.Part II RNA helicase RIG-I regulates granulocytic differentiation through binding to RNF138Retinoic acid inducible gene-I?RIG-I?is highly expressed during the process of differentiation induced by the treatment of acute promyelocytic leukemia?APL?with all-trans retinoic acid?ATRA?.Previous work of our laboratory showed that knockout of Rig-I in mice could lead to abnormal hematopoietic phenotype such as myeloproliferative neoplasms,granulocyte hyperplasia.RIG-I,also known as DEAD box protein 58?DDX58?,is a DEx D/H box containing RNA helicase which can identify a variety of viral RNA to activate intracellular signaling pathways.However,the molecular mechanisms underlying RNA helicase RIG-I-mediated regulation of granulocytic differentiation through binding to endogenous RNA without virus infection,are poorly understood.In this study,we performed RIP-Seq analysis to screen the endogenous RNAs that interact with RIG-I,We found that RIG-I could interact with a series of endogenous RNA in the process of inducing granulocyte differentiation.Among them,verification experiments showed that RNF138 m RNA could be significantly enriched by RIG-I,therefore we focused on investigation of the interaction between RIG-I and RNF138.RIG-I could negatively regulate RNF138 by combining with its m RNA to reduce its RNA stability,and then cause the upregulation of LEF1 which is a target protein of RNF138.LEF1 is kown as a key transcription factor of Wnt signaling pathway and plays an important role in the proliferation and differentiation of hematopoietic cells.Further investigation showed that overexpression of RNF138 could reverse the cell differentiation induced by RIG-I to a certain extent,suggesting that RIG-I might regulate granulocytic differentiation through binding to the m RNA of RNF138.Our results thus revealed a novel molecular mechanism underlying RIG-I as a RNA helicase in regulating granulocytic differentiation via binding to endogenous RNA.Part III Treatment of acute myeloid leukemia M2 b with homoharringtonine in combination with nilotinibM2b subtype acute myeloid leukemia?AML-M2b?occurs as a multi-step process,in addition to AML1-ETO fusion gene,other events such as C-KIT mutations are also important in leukemogenesis.Therefore,drugs targeting C-KIT mutations are hopeful to improve the prognosis of AML-M2 b.The homoharringtonine?HHT?is a natural active alkaloid with anti-leukemia feature and has a good clinical effect on AML patients. Many studies have shown that HHT can down-regulate the expression of C-KIT D816 V in tumor cells by inhibiting translation.Nilotinib is a novel tyrosine kinase inhibitor which could inhibit C-KIT receptor tyrosine kinase activity.In this study,a dose-dependent growth inhibition was observed following the treatment with HHT or Nilotinib in Kasumi-1 cells,while the combination of these two drugs produced a synergistic effect.In vivo,HHT combined with Nilotinib could significantly inhibit the proliferation of leukemic cells,and prolong the lifespan of leukemic mice.The results of this study may provide a theoretical basis for the clinical application of HHT and Nilotinib in the treatment of AML-M2b.