| SECTION IGene mutation patterns of Chinese acute myeloid leukemia patients by targeted next-generation sequencing and bioinformatic analysis.BackgroundAcute myeloid leukemia（AML）is a heterogenous clonal disease originated from myeloid hematopoietic progenitor cells,characterized by uncontrolled accumulation of myeloid blasts in the bone marrow and peripheral blood as well as inhibition of normal hematopoiesis.To date,chemotherapy remains the most important treatment for AML patients,without revolutionary advances showing up for decades.But the disease course and prognosis differ from person to person,and not every patient responses the same way to chemotherapeutics.As many studies revealing the potential role of molecular abnormalities in AML,precision medicine and individualized medicine were put forward.And some variations on genes such as FLT3-internal tandem duplication（FLT3-ITD）have been incorporated into the evaluation system for risk stratification and prognosis.In the era of large-scale application of NGS technique,the study of gene mutations and their roles in disease can better integrate with precision medicine.Previous studies reported many recurrent mutations in patients with AML,from which some were confirmed harmful or beneficial to the patients’ prognosis or therapeutic effect.However,the correlation between most somatic mutations and clinical characteristics and the underlying mechanisms still needs further investigations.What’s more,reports on mutational patterns of Chinese AML patients are limited.PurposesTo provide additional information on the above subjects and evidence to both diagnosis and treatment,we performed targeted NGS of 22 suspected genes on 78 patients.Methods1.PatientsA total of 78 newly diagnosed and follow-up AML patients in Qilu Hospital,Shandong University from February 2016 to October 2017 were enrolled in this study and took NGS assays.The patients included Ml,M2,M3,M4,M5,M6 and not-specified according to French-American-British（FAB）classification system.Complete remission（CR）was defined based on International Working Group Criteria.This study was approved by the Medical Ethical Committee of Qilu Hospital,Shandong University.All the patlients have signed informed consent in accordance with the Declaration of Helsinki.2.Sample preparationsBone marrow aspirates of the 78 AML patients were collected and lysed by red blood cell lysis buffer to obtain mononuclear cells.Genomic DNA for further sequencing was isolated using TIANamp Genomic DNA Kit（Tiangen Biotech,Beijing,China）according to manufacturer’s instructions.3.Targeted next-generation sequencingTargeted NGS of a 22-gene panel and subsequent BLAST analysis were performed by Yuanqi Biomedical Technology（Shanghai,China,Co.,Ltd.）.The panel consists of 84 amplicons captured by pairs of oligonucleotides designed to hybridize flanking targeted regions of interest.The library preparation was performed using at least 200ng of genomic DNA.The sequencing run was conducted on the HiSeq system（Illumina Inc.,San Diego,CA,USA）,with average read depth 2000×.Reads were aligned to human reference genome GRCh37.This gene panel consists of FLT3,NPM1,KIT,CEBPA,DNMT3A,IDH1,IDH2,TET2,EZH2,RUNX1,ASXL1,PHF6,TP53,SF3B1,SRSF2,U2AF1,ZRSR2,NRAS,CBL,SETBP1,ETV6 and JAK2.4.Sanger sequencingA subset of somatic mutations detected by NGS was independently confirmed through Sanger sequencing by BioSune Biotechnology（Shanghai,China,Co.,Ltd.）.Specific primers were designed for targeted mutated regions,followed by Polymerase Chain Reactions（PCR）.Sanger sequencing of the PCR products were performed on an ABI PRISM 3730x1 Genetic Analyzer（Applied Biosystems,Carlsbad,CA,USA）with the BigDye Terminator v3.1 Cycle Sequencing Kit（Life Technologies Corporation,Carlsbad,CA,USA）.Subsequent visualization of the sequence was carried out by SnapGene Viewer 3.2 for windows（GSL Biotech LLC,Chicago,USA）.5.StatisticsThe data were analyzed by SPSS version 24.0 for Windows（SPSS Inc.,Chicago,IL,USA）.Continuous variables with non-normal distribution were present as median（interquartile range）.Mean of two continuous variables with normal distribution were compared by independent samples Student’s test.And non-normal variables were compared by nonparametric test.Fourfold table data were analyzed by chi-square test or Fisher’s exact test.Linear regression and logistic regression analysis were used as univariate and multivariate analysis.Survival analysis were analyzed by Kaplan-Meier Log-rank test and Cox regression analysis.A value of p<0.05 was considered significant.6.Bioinformatic analysisThe impact prediction of a novel point mutation PHF6 H302R to protein or disease was performed through Mutationtaster,PolyPhen2,Mutationassessor and SIFT tools followed by instructions on their websites.The figures of gene mutation profile and landscapes were made by cBioPortal.The comprehensive analysis of TCGA public AML dataset were calculated and plotted by cBioPortal and UALCAN.The correlation between gene mutations and drug sensitivity was integrated by the Genomics of Drug Sensitivity in Cancer（GDSC）database.Results1.Mutation profile78 newly diagnosed and follow-up AML patients with a median age of 53 years（range,14-77）,56%males,were included in this study.Among the 78 cases,62（80%）harbored gene mutations,range 1 to 6,and nearly half of them（29/62）carried only one mutation.Mutational frequencies were 26%for FLT3,17%for NRAS,15%for NPM1,14%for DNMT3A and IDH2,12%for ASXL1,9%for KIT,IDH1,TET2 and U2AF1,6%for CEBPA and SRSF2,4%for EZH2 and SF3B1,3%for TP53,SETBP1 and ETV6,and 1%for RUNX1,PHF6,CBL and JAK2.No mutation was found in ZRSR2 coding regions.Most of the mutations occurred at the critical domains.2.Correlation of gene mutations with clinical and laboratory characteristicsPatients were divided into two groups by age:the old age AML（≥60 years old）and young age AML（<60 years old）.The Fisher’s exact test revealed that mutations of SRSF2 were correlated with old age（p=0.034）.In a univariate analysis,compared to the wild type,patients with FLT3 mutations showed higher bone marrow blasts,peripheral blood blasts,abnormal cell proportion detected by flow cytometry and WBC counts.Patients with TET2 mutations showed higher peripheral blood blasts.In the multivariate analysis of peripheral blood blasts and WBC counts,FLT3 and TET2 were both risk factors.As to therapeutic effect,TET2 mutations were related with non-remission（NR）after the first cycle of standard induction chemotherapy.Although patients with SRSF2 mutations were all NR after the first cycle of standard induction chemotherapy,there wasn’t any difference with CR group in significance.3.Survival analysisWith regard to overall survival（OS）,the patients with more than three mutations（NPM1 and CEBPA not included）had shorter OS than patients with less than three mutations.Patients with mutations on epigenetic regulator genes—DNMT3A,TET2,ASXL1,IDH1/2 and EZH2 had shorter OS than patients with wild type.And patients with IDH1/2 mutations had shorter OS than those with wild type.For the whole cohort,a multivariate cox regression analysis revealed FLT3-ITD,DNMT3 A,IDH1,TET2 and SRSF2 as independent prognostic factors of OS.For non-M3 patients,FLT3-ITD,DNMT3A,IDH1 and SRSF2 were found to be independent prognostic factors of OS in the multivariate analysis.For old age AML patients,FLT3-ITD,DNMT3A and IDH1 were independent prognostic factors of OS in the multivariate analysis.For patients with cytogenetically intermediate risk,FLT3-ITD,DNMT3A,and IDH1 were independent prognostic factors of OS in the multivariate analysis.4.Novel mutations and Sanger sequencing validationWe identified several novel forms of mutations that were not ever recorded by the Cosmic database（http://cancer.sanger.ac.uk/cosmic）,including one point mutation and fourteen indel mutations.Six of the indel mutated samples were tested by Sanger sequencing for validation,and five were also detected frameshift.Based on the result,only one FLT3 c.17951796ins30 mutation cannot be identified by Sanger sequencing probably due to its low variant allele frequency（VAF）（5.47%）.Next,Mutationtaster,PolyPhen2,Mutationassessor and SIFT tools were used to predict the impact of PHF6 H302R mutation on protein and disease.It was expected consistently to be disease causing/probably damaging/high impact/affecting protein function by all the four prediction software.5.Comparison with TCGA public AML datasetIn 2013,one study conducted by the Cancer Genome Atlas Research Network analyzed the whole genome sequencing of 200 AML patients,of whom 181 were Caucasians,15 were black or African Americans,2 not reported and only 2 were Asian.Our work,focused on only Chinese population,yield the similar results with Caucasians AML except for frequencies of 6 genes-NRAS（17%vs 8%,p=0.034）,NPM1（15%vs 27%,p=0.041）,DNMT3A（14%vs 25%,p=0.049）,ASXL1（12%vs 2.5%,p=0.004）,SRSF2（6%vs 0.5%,p=0.007）and RUNX1（1%vs 13%,p=0.003）.6.Gene mutations and drug sensitivities in AMLWe searched Genomics of Drug Sensitivity in Cancer（GDSC）database,the largest public resource for information on drug sensitivity in cancer cells and molecular markers of drug response,and got some exciting findings between mutations in AML and drug sensitivities.AML cell lines with mutations in U2AF1 have significant higher sensitivity to Quizartinib,Sorafenib and Cabozantinib,and those with mutations in TET2 have higher sensitivity to VNLG/124 and Bexarotene.While AML cell lines with mutations in TP53 have lower sensitivity to Bleomycin and Nutlin-3a（-）,and those with mutations in NRAS have lower sensitivity to TL-1-85 and NG-25.Conclusions1.80%percent of patients harbored at least one mutation.2.The mutations of FLT3 and TET2 were correlated with peripheral blasts and WBC.3.The mutation of TET2 was correlated with the therapeutic effect of first cycle chemotherapy.4.Mutations of FLT3-ITD,DNMT3A,IDH1,TET2 and SRSF2 were risk factors of 05.5.15 novel mutations were found.6.A summary of the mutational patterns of Caucasian AML and comparison with Chinese AML.7.A summary of the correlation of gene mutations and drug sensitives.SECTION ⅡThe role and mechanism of Irf8 in T cell acute lymphoblastic leukemiaBackgroundAcute T cell lymphoblastic leukemia（T-ALL）is a malignant hematopoietic disease caused by the malignant transformation of T lineage precursors when gene mutations happen.In recent years,with the improvement of T-ALL treatment,most patients can achieve complete remission,but the recurrence rate is still high,with long-term survival rate only 25-50%.Therefore,in-depth study of the pathogenesis of T-ALL,and to find a new targeted therapy strategy,not only has important scientific significance,but also has potential clinical value.Notchl is a member of the evolutionarily conserved endothelial growth factor-like transmembrane receptor protein（Notch）family.Its signal transduction is closely related to cell proliferation,differentiation and apoptosis.In 2004,WENG et al first proposed that more than half of children and adults with T-ALL had activated Notchl mutations,and Notchl became a recognized T-ALL oncogene.Notchl can promote the differentiation of T cells and inhibit the differentiation of B cells in the process of lymphocyte formation,and plays a role in multiple stages of T cell formation and development.Studies have shown that the selective inactivation of Notchl signaling pathway can stop the proliferation and differentiation of T cells,while the mutation of Notchl can lead to the sustained activation of downstream signals,and then promote the occurrence of T-ALL.On leukemia mechanism of "second strike" doctrine that only Notchl activation mutation is not enough to cause leukemia,the synergistic effect of other molecules is needed to lead to T-ALL together.Interferon regulatory factor 8（Irf8）is a transcript factor in the family of interferon regulatory factors that play an important role in regulating the pedigree of the hematopoietic lineage.Studies have shown that Irf8 promotes the differentiation of granulocytic and B lineage cells and inhibits monocyte-macrophage and T lineage cell differentiation.It has been reported that Irf8 expression is decreased in myeloid leukemia,with splicing mutations occurring in acute myeloid leukemia（AML）,and increased expression of wild-type and mutant Irf8 transcripts correlates with a reduction in recurrence-free survival.There are also reports that expression of Irf8 exists in different subtypes of B lymphoma,which can be used as a new diagnostic indicator.In addition,a number of studies have shown that IrfB was found methylation alteration in many cancers such as nasopharyngeal carcinoma,esophageal cancer,gastric cancer,breast cancer,non-small cell lung cancer,kidney cancer and other diseases,proving itself as a tumor suppressor gene.Abnormal methylation led to the downregulation of its expression and played a carcinogenic effect.It was also reported that in regulative dendritic cells,Notch-RBP-J signaling pathway mediated histone methylation modification of Irf8 in the chromatin fold state through the MLL and PRC2,inhibiting its expression level.In macrophages,RBP-J can promote Irf8 protein synthesis.Studying the methylation,gene expression and molecular function of Irf8 in T-ALL will help reveal the interaction and mechanism of Irf8 and Notchl in the development of T-ALL,and provide a new idea for the diagnosis and treatment of T-ALL.Previous studies found that Yes-associated protein（YAP）was overexpressed in bone marrow mononuclear cells of Irf8 knockout mice than wide type.YAP is an important member of the HIPPO pathway,which is involved in important physiological processes such as organ development,tissue homeostasis and regeneration.Recent studies showed that YAP,an oncoprotein,was crucial for the development or growth of most solid tumors.Its activation induceed the maintenance,proliferation,chemoresistance and metastasis of cancer stem cells.In leukemia,it has been reported that YAP is overexpressed in chronic myeloid leukemia（CML）cells and inhibition of YAP function can inhibit CML cell proliferation and promote apoptosis.In addition,verteporfin,a YAP inhibitor,potentiates imatinib in vitro and inhibits leukemia in vivo.However,the expression level and function of YAP in T-ALL are seldom reported.Therefore,a scientific hypothesis is proposed that:expression of Irf8 is downregulated at the lymphoid progenitor cells due to the abnormal methylation of Irf8,thereby inhibiting the differentiation of the B lineage,promoting the production of T lineage cells,and cooperating with Notchl to convert the CLP into leukemia initiating cells,Downstream molecules YAP promote the maintenance of leukemia cells or promote drug resistance,accelerate the occurrence and development of T-ALL.PurposesTo study the role and molecular mechanism of Irf8 cooperating with Notchl activation in promoting the development of T-ALL.Methods1.Bioinformatics analysisThe published T-ALL chip data on Oncomine database were analyzed and calculated,and the differences in the Irf8 gene expression between T-ALL patients and healthy controls were compared.The Methylmer online site was used to analyze the Irf8 promoter region to predict the density of CpG islands.2.Cell culture293T cells are routinely cultured in DMEM medium containing 10%fetal bovine serum.293T cells in good condition were seeded 8 × 106-1 × 107 per dish on the night before packaging virus.Mice lin-cells were cultured in IMDM medium containing 15%fetal bovine serum.CCRF-CEM and Jurkat cells were routinely cultured in RPMI 1640 medium containing 10%fetal bovine serum.3.MSCV-Notchl-IRES-GFP virus packagingThree kinds of plasmids were extracted from the MSCV-Notchl-IRES-GFP,pKat and VSVG bacterial cultures and transfected 293T cells at a ratio of 7:5:3.After 8 hours,the medium was replaced with fresh medium.Collect the supernatant,which contains MSCV-Notchl-IRES-GFP virus particles.4.In vivo experiment to verify that knockdown of Irf8 synergizes with Notchl activating mutations to promote the development of T-ALLIrf8-/-and WT mice are routinely housed.Bone marrow cells of Irf8-/-and WT mouse were isolated,and sorted with lineage beads to acquire hematopoietic stem/progenitor cells（lin-cells）.TPO,FLT3 and SCF cytokines were added for overnight pre-stimulation.After 8 hours,replace with fresh medium.Observe fluorescence under a fluorescence microscope 48 hours later.The proportion of GFP cells was detected by flow cytometry.Injecte C57BL/6J mice which have been lethally irradiated by caudal vein.After 8 weeks,mice gradually developed T-ALL.5.Construction of Irf8-GFP eukaryotic expression vector and virus packagingIrf8 CDS region specific cloning primers containing restriction sites were designed to clone the Irf8 CDS region from highly expressed Irf8 cell line and inserted into the MSCV-IRES-GFP vector to construct the eukaryotic expression vector MSCV-Irf8-IRES-GFP.Virus packaging process was the same with MSCV-Notchl-ICN-IRES-GFP.6.To construct a T-ALL cell line stably expressing Irf8CCRF-CEM and Jurkat cells were infected with Irf8-GFP virus and sorted GFP-positive cells by flow cytometry to acquire cell lines stably expressing Irf8.7.Cell proliferation,cell cycle and drug sensitivity testingAdjust the cell density and seed into 96-well plates,test the proliferation with CCK8 assay.PI staining was used to detect the cell cycle.Annexin V-APC/7-AAD double staining was used to detect drug-induced apoptosis rate.8.YAP expression detectionThe bone marrow mononuclear cells of Irf8-/-and WT mice were collected and extracted the total RNA and total protein.The differences of YAP mRNA and protein expression levels were detected by RT-PCR and western blot.Results1.Expression of Irf8 in T-ALL patientsBy searching the T-ALL chip data published on the oncomine database,it was found that Irf8 showed lower expression in four different research chips compared to the healthy control.2.Knockout of Irf8 synergistic Notchl activation to promote the development of T-ALLBone marrow mononuclear cells of Irf8-/-and WT mice were extracted,sorted hematopoietic stem/progenitor cells（lin-cells）by lineage beads,infected with Notchl-ICN-GFP retrovirus and injected C57BL/6J mice that were radiated under lethal dose.gradually developed disease after 8 weeks.The survival of Irf8-/--Notchl mice was shortened compared with that of WT-Notchl mice.The bone marrow smears showed that leukemia cells were in a more primitive stage.However,there is no significant difference between the appearance,weight and spleen size.3.Overexpression of Irf8 in T-ALL cell line arrests cell cycle and promotes apoptosisIrf8-overexpressed CCRF-CEM cell line was detected cell cycle arrest by flow cytometry,increased apoptosis,which indicated the tumor suppressor effect of Irf8.4.Irf8 negatively regulates YAPPotential downstream target genes were screened by searching the GEO database for published chips of Irf8overexpression or knockdown.RT-PCR and western blot analysis of Irf8-/-and WT mouse bone marrow mononuclear cells showed that YAP was highly expressed in Irf8-/-mice.Conclusions1.The expression of Irf8 was down-regulated in T-ALL.2.T-ALL cell lines overexpressing Irf8 decreased proliferation,blocked cell cycle and increased drug-induced apoptosis.3.Loss of Irf8 synergized with Notchl activation to accelerate T-ALL.4.Irf8 negatively regulates oncoprotein YAP. |
PDF Full Text Request