DHX15 Is Required For T-cell Development And Leukemia

Part Ⅰ.DHX15 is required for T-cell development and leukemiaPurpose:T cells are continually generated in the thymus in a highly dynamic process comprising discrete steps of differentiation.Deregulation of these processes is often associated with leukemogenesis.This study focuses on the functional significance and regulatory mechanisms of DHX15,an outstanding member of the DEAD/H-box helicase family,in T-cell development and leukemia.Methods:The specifically elevated expression of DHX15 in T-ALL was confirmed via bioinformatics analysis using a series of public datasets.The role of DHX15 in T-ALL cell growth and viability was evaluated by a series of loss-of-function assays with shorthairpin RNA(shRNA)in vitro.T-ALL xenograft was performed to validate the requirement of DHX15 in T-ALL progression in vivo.NOTCH1-induced T-ALL model in conditional Dhx15 knockout(cKO)mice(Lck-Cre;Dhx15fl/fl)was established to demonstrate the DHX15 dependency in T-ALL initiation.Single-cell transcriptomics combined with flow cytometry analysis were carried out to observe T-cell development upon DHX15 withdrawal.RNA sequencing(RNA-seq)using DHX15-depleted human and mouse T-ALL cells was performed to investigate downstream effectors of DHX15 in T-ALL.Biochemistry experiments including qPCR,immunoblot,coimmunoprecipitation,CHX chasing assay and ubiquitination assay were used to study regulatory mechanism of DHX15 on MYC.The biological significance of DHX15MYC axis in T-cell development and leukemia was validated by MYC rescue assays in both physiological and pathological contexts.Results:Gene expression studies based on multiple human leukemia datasets showed that DHX15 was overexpressed in T-ALL patient samples when compared with normal T or bone marrow(BM)cells.DHX15 depletion in T-ALL cells resulted in substantial growth inhibition and robust apoptosis.Consistently,DHX15 loss in T-ALL xenograft dampened tumor progession in vivo and prolonged animal survival.More importantly,Dhx15 knockout inhibited NOTCH1-induced T-ALL initiation in mice.scRNA-seq and flow cytometry analysis revealed that DHX15 depletion in T-cell progenitors impaired normal T-cell development and,more specifically,hindered burst proliferation during CD4"CD8"(DN)-to-CD4+CD8+(DP)transition.Mechanistically,abrogation of DHX15 repressed MYC transcriptional program in both normal and transformed Tlymphoblasts.In-depth molecular investigation demonstrated that DHX15 physically bond to MYC and enhanced MYC protein stability by antagonizing FBXW7-mediated polyubiquitylation and degradation.Enforced MYC expression significantly rescued the DHX15-deficient phenotypes,supporting MYC as a prominent downstream effector mediating the pro-proliferative role of DHX15.Conclusion:This study identifies DHX15 as an essential regulator for T-cell development and leukemogenesis.DHX15 enhances MYC oncoprotein stability at post-translational level to magnify MYC transcriptional output.We thus provide a unique mechanism that the DHX15-MYC axis exploited by normal thymocytes for development,is hijacked by transformed T cells to augment proliferation and survival.These findings exemplify that DEAD/H-box helicases may orchestrate diverse cellular processes,beyond RNA metabolism,to promote development and drive tumorigenesis.Part Ⅱ.Recombination activating gene 1 and 2 are novel dimeric Notchl targets in T-cell developmentPurpose:Recombination activating gene 1 and 2(RAG1 and RAG2)play central roles in assembling functional antigen receptors on T and B cells.Although RAG1/2 expression is highly restricted to lymphoid cells,it remains unclear how RAG gene expression is precisely regulated in T cells.This study aimed to clarify the molecular mechanisms underlying the regulation of RAG1/2 by dimeric NOTCH1 transcription complex(NTC).Methods:Public gene expression data were reanalyzed to identify novel dimeric NOTCH1 targets in T cells.RAG1/2 mRNA from T cells with or without NOTCH1 inhibition were measured by qPCR.ChIP-seq analysis was performed to search the binding sites of NOTCH1 on RAG locus.ChIP-qPCR,duel-luciferase assay and EMSA were used to validate the binding and activation of sequence-paired sites(SPS)within RAG2-2.8 Kb region by dimeric NTC.In vitro T-cell development expriments by OP9 co-culture system were carried out to measure the activation of RAG1/2 by dimeric NTC in the physiological scenario.Results:Gene expression studies based on public datasets show that RAG1/2 were dimerically activated by NOTCH1 in T cells.NOTCH1 signaling withdrwal in thymocytes resulted in substantial inactivation of RAG1/2.While wild-type intracellular NOTCH1(ICN1)could strongly upregulate the RAG1/2 in thymocytes,dimeric deficiency in ICN1(R1984A)abrogated the trans-activating capacity.ChIPseq analysis identified a sequence-paired sites(SPS)for NTC dimer within RAG2-2.8 Kb region.ChIP-qPCR,duel-luciferase assay and EMSA showed that NTC directly bind to RAG2 SPS and activate RAG1/2 in a dimeric dependent manner.OP9 co-culture system showed NTC dimerization is indispensable for the transcariptional induction of RAG1/2 during early T-cell development.Conclusion:These results reveal NOTCH1 dimerization-mediated transcription as one of the mechanisms for optimally activating RAG1/2 expression in thymocytes.Our data suggest a new role of dimeric NOTCH1 in compelling efficient TCRβ rearrangements during T-cell development.