The Role And Molecular Mechanism Of KMT2D In Acute Myeloid Leukemia Development And Maintenance

Objective:Epigenetic abnormality is an important mechanism in acute myeloid leukemia(AML)development.Lysine-specific methyltransferase 2D(KMT2D),encoding the major mammalian histone-3 lysine-4(H3K4)methyltransferase,is among the most frequently mutated epigenetic genes in human cancers.Recent studies have established the essential tumor suppressor function of KMT2 D in many solid tumors including lung adenocarcinoma and B cell lymphomas.However,the role of KMT2 D in AML has so far not been completely elucidated.There are loss-of-function missense mutations in KMT2 D in persons with AML,suggesting that KMT2 D might be a tumor suppressor in AML.Thus,we sought to examine the role of KMT2 D in AML development and maintenance and explore potential molecular mechanisms underlying the disease.Materials and methods:To delineate the biological function of KMT2 D in AML development,we firstly characterized the mutation and expression profiles of KMT2 D in AML patients in AACR Project GENIE c Bio Portal and Gene Expression Omnibus public database,respectively.Then,we mimicked the expression patterns and genetic alterations of KMT2 D in clinical settings to construct mouse AML models.We used sh RNA interference technology and CRISPR/Cas9 genome editing to down-regulate Kmt2 d expression or disrupt Kmt2 d in mouse hematopoietic stem and progenitor cells(HSPCs),respectively,and constructed mouse AML chimeric transplant models in the context of Nf1 and Trp53 deletion,exploring whether Kmt2 d deficiency promoted AML development.After transplant,we closely monitored recipients and detected the formation of AML cells in the peripheral blood by complete blood counts,blood smear and flow cytometry.When recipients appeared to be dying,we recorded the survival time of recipients post-transplant,performed complete blood counts and blood smear,performed flow cytometry of peripheral blood,bone marrow and spleen cells,constructed mouse secondary transplantation models with bone marrow cells and performed histopathological examination of spleens,livers and sternums to confirm the diagnosis.Besides,we analyzed Kmt2 d expression levels by q RT-PCR,Kmt2 d mutations by T7 EI enzyme digestion and DNA sequencing as well as H3K4 methylation levels by western blotting to evaluate the efficiency and specificity of Kmt2 d sh RNA and sg RNA.These results will clarify the role of Kmt2 d in AML development.In order to study the role of KMT2 D in AML maintenance,we used a doxycycline-induced sh RNA system to construct an inducible Kmt2 d knockdown mice AML model.Then,we extracted bone marrow cells of moribund mouse to generate doxycycline-inducible Kmt2 d knockdown AML cells in vitro.Kmt2 d expression can be repressed in the presence of doxycycline and can be restored in the absence of doxycycline by the regulation of sh Kmt2 d transcription.We analyzed Kmt2 d expression levels by q RT-PCR as well as H3K4 methylation levels by western blotting to evaluate the recovery of Kmt2 d expression after doxycycline withdrawal.To determine the effects of Kmt2 d on AML maintenance,we observed several phenotypic changes after Kmt2 d restoration,including cell growth measured by cell counting,cell proliferation detected and quantified by Ed U staining,cell apoptosis detected and quantified by Annexin V/DAPI staining as well as cell morphology in cytospin.These results will clarify whether Kmt2 d deficiency is required for AML maintenance.We further evaluated the molecular mechanisms underlying KMT2 D in AML development and maintenance.Firstly,we performed RNA-seq to analyze the effects of Kmt2 d expression on transcriptomes of mouse HSPCs and AML cells,discovering pathways involved in Kmt2 d deficiency-induced AML development and maintenance from molecular aspects.Secondly,we conducted a series of relevant experiments in Kmt2 d knockdown and restored AML cells,including q RT-PCR,western blotting and immunofluorescence based on findings from omics data to verify the mechanisms.Thirdly,we further assayed ATAC-seq of Kmt2 d knockdown and restored AML cells to assess the chromatin accessibility and analyzed data of Ch IP-seq for KMT2 D to find genes directly bound by KMT2 D.Combining RNA-seq,ATAC-seq with Ch IP-seq omics data could provide insights into candidate downstream genes of Kmt2 d to comprehensively clarify the molecular mechanisms underlying Kmt2 d in AML development and maintenance.Fourthly,we conducted drug interventions based on molecular mechanisms on Kmt2d-deficient AML mice to explore possible targeted therapies for this disease.Lastly,we constructed a KMT2 D mutated human AML cell line MOLM-13 with CRISPR/Cas9 gene editing technology and analyzed transcriptomes of AML patients with low KMT2 D expression in TARGET-AML cohort,evaluating whether the mechanisms of KMT2 D in human AML were similar to these in mouse AML models.Results:In the study of the biological function of KMT2 D in AML development,we found that there are often loss-of-function missense mutations of KMT2 D and generally reduced expression of KMT2 D in leukemia cells of AML patients compared with healthy control samples.We showed Kmt2 d loss using sh RNA and CRISPR/Cas9 technologies,by cooperating with Trp53 and Nf1 loss,promoted AML in mice.Specifically,compared with controls(sh Ren or sg Scramble),recipients of Kmt2d-deficent HSPCs(Kmt2d sh RNA or sg RNA)had shorter lifespans,displayed an increasing amount of white blood cells,developed AML as evidenced by phenotypic analyses of flow cytometry(CD117+;Gr-1/CD11b+;CD3?/B220-)and morphologic analyses of peripheral blood,displayed significantly enlarged spleen and liver with histological analyses revealing the prominent infiltration of leukemia cells and the destruction of organ structures.Kmt2 d was repressed or mutated and the levels of H3K4me1 and H3K4me2 were significantly reduced in Kmt2d-deficient AML cells,consistent with its H3K4 methyltransferase activity.Therefore,both knockdown and mutation of Kmt2 d can promote the development of AML.In the study of the biological function of KMT2 D in AML maintenance,using doxycycline-induced sh RNA system we successfully constructed an inducible Kmt2 d knockdown mice AML model.Then,we extracted bone marrow cells of moribund mouse and successfully generated inducible Kmt2 d knockdown AML cells in vitro.As expected,doxycycline withdrawal from the medium led to the enhancement of Kmt2 d expression and the corresponding enhancement of genome-wide H3K4me1 and H3K4me2 concentrations in AML cells.We explored characteristic alterations following Kmt2 d restoration and found that the growth rate of AML cells slowed down,the percentages of Edu-positive proliferating cells were reduced,while the percentages of Annexin V-positive apoptotic cells were increased.Therefore,the aberrant AML cell growth cannot be maintained without the repression of Kmt2 d.In the study of the molecular mechanisms of KMT2 D in AML development and maintenance,we conducted multi-omics analyses and biofunctional experiments.Firstly,RNA-seq data showed that knocking down Kmt2 d expression in HSPCs up-regulated genes in functional categories linked to ribosome biogenesis,whereas,restoring Kmt2 d expression in AML cells down-regulated aforementioned genes.Indeed,we found that compared with Kmt2d-deficent AML cells,Kmt2d-restored AML cells exhibited down-regulation of genes regulating ribosome biogenesis and genes coding ribosomal proteins,decreased r RNA content,decreased nucleolar size and content and inactive protein synthesizing process with reduced nascent peptides.Therefore,Kmt2 d deficiency may promote AML through regulating ribosome biogenesis.In addition,further analyses showed that mTOR signaling pathway that controls ribosome biogenesis was activated in Kmt2d-deficienct HSPCs and AML cells,whereas,its activity decreased with the restoration of Kmt2 d expression.Next,we combined RNA-seq,ATAC-seq with Ch IP-seq omics data and found that KMT2 D could directly bind enhancer regions of DDIT4 and LMNA,negative regulators of mTOR signaling pathway,and via histone methyltransferase activity and chromatin remodeling thereby regulate the expression of these genes.Together,Kmt2 d deficiency promotes AML development and maintenance through regulating mTOR signaling pathway and ribosome biogenesis.Remarkably,pharmacological inhibition of ribosome biogenesis with a novel RNA polymerase I inhibitor CX-5461 alleviated Kmt2d deficiency-induced AML development and maintenance.Last but not least,we found that MOLM-13 cells with KMT2 D mutation and leukemia cells of AML patients with low KMT2 D expression also had activated mTOR signaling pathway and ribosome biogenesis.Conclusions:Our study establishes the tumor suppressor function of KMT2 D in AML,whose deficiency promotes AML development in mice.Besides,KMT2 D deficiency is required for AML maintenance.KMT2 D can directly bind enhancer regions of DDIT4 and LMNA,negative regulators of mTOR signaling pathway,and via histone methyltransferase activity and chromatin remodeling regulate gene expression,the activity of mTOR signaling pathway and ribosome biogenesis,and ultimately suppress AML development and maintenance.Our genetic modifications of Kmt2 d in mice can mimic real-world clinical settings well.