The Effect Of Myeloproliferative Neoplasm Driver Mutation Genes On Neutrophil NAP Expression

Background and Objective:Classic myeloproliferative neoplasms(MPN)are a group of clonal hematopoietic stem cell disorders characterized by the proliferation of one or more lineages of myeloid cells,including the red,granulocytic,and megakaryocytic lineages,including PV,ET,and PMF.Mutations in JAK2,MPL and CALR genes mainly affect red,granulocyte-monocyte and megakaryocyte myeloid cells of MPN patients.At the cellular level,researchers have demonstrated by cloning experiments that JAK2,MPL,and CALR mutations occur mainly in the myeloid hematopoietic stem cell stage,resulting in the development of red,granulocyte-monocyte,and megakaryocyte myeloid cells that also carry JAK2,MPL,and CALR mutations.Neutrophils play a key role in host defense,by phagocytosis and digestion of microorganisms.Available clinical data indicate that elevated granulocyte counts are the main clinical phenotype of MPN myelofibrosis;elevated granulocyte counts are a major risk factor for the development of thrombotic complications and are included as the main basis for clinical assessment of thrombotic risk.These clinical phenomena suggest a role for the driver mutation in the development of MPN,and we hypothesize that the mutants within the affected granulocytes interact with specific network signals to generate functions that are different from those of granulocytes in physiological conditions and thus participate in the development of MPN disease.Neutrophil alkaline phosphatase(NAP),the product of tissue non-specific alkaline phosphatase or liver/bone/kidney alkaline phosphatase genes,is stored in cytoplasmic secretory vesicles,located on their membrane surface,and appears with secretory vesicles in the lobulated nucleus of neutrophils at the most advanced stage of maturation,constituting a reservoir of membrane proteins required for neutrophil-mediated inflammatory responses.The activity of this enzyme reflects the degree of maturation and function of mature granulocytes,and increases gradually as the cells mature,providing an important indicator of neutrophil maturation.Under physiological conditions,neutrophils have increased expression of NAP in host defense and are involved in phagocytosis and digestion of microorganisms;NAP is also abnormally expressed in MPN patients,and we speculate that pathological changes in this abnormal expression may be involved in the development of MPN disease,and its specific pathological significance needs to be further investigated.Earlier,after the discovery of JAK2V617F mutation,some investigators confirmed that JAK2V617F mutation could increase NAP expression in patients with PV,ET,and PMF,elucidating the molecular basis of increased NAP in neutrophils from MPN patients at the molecular level.In recent years,MPL and CALR mutations have been identified in JAK2V617F mutation-negative patients,but the effects of these mutations on neutrophil NAP in MPN patients are not clear.In view of this,the effect of each MPN driver mutation gene on neutrophil NAP expression was elucidated in this study.Methods:1.Clinical study of the effect of myeloproliferative neoplasm driver mutation genes on NAP expression(1)Patients with initial MPN consultation at our hospital were collected,and the study inclusion criteria were:not using any MPN disease-related cytoreduced or aspirin therapy at the initial consultation;having a record of NAP test results at the initial consultation at our hospital;accompanied by JAK2,MPL,CALR gene mutations and functionally positive.(2)The patient’s JAK2V617F mutation was detected by allele-specific PCR;JAK2exon12,MPL exon10 and CALR exon9 mutations were detected by PCR combined with Sanger generation sequencing method.(3)The patient’s NAP was detected by neutrophil alkaline phosphatase chemical staining method.(4)Patients with JAK2V617F mutation load detection were quantified by digital PCR.2.Cellular model study of the effect of myeloproliferative tumor driver mutation genes on NAP expression(1)Cell models were constructed by applying a lentiviral overexpression vector system to transduce JAK2 wild type(JAK2WT),JAK2 mutant(JAK2V617F),MPL wild type(MPLWT),MPL mutant(MPLW515L,MPLA497-L498LVIAins4),CALR wild type(CALR WT),CALR mutant(CALR WT),and CALR mutant(CALRType1,CALR Type2),and empty vector(MSCV)in NB4 cell line.(2)Cell model NAP expression was induced at different time points by applying ATRA and G-CSF co-induction culture.(3)Cell model NAP assay was quantified using the phosphate p-nitrophenyl ester(p NPP)method.3.Proteomics and phosphorylation-modified proteomics to study the mechanism of NAP expression regulation by myeloproliferative tumor driver mutation genes(1)NB4-JAK2V617F,NB4-MPLW515L,NB4-CALR Type1 and NB4-MSCV cell models were collected after 96h co-induction culture with ATRA and G-CSF,and liquid chromatography-mass spectrometry was applied to detect the proteomics and phosphorylation modification proteomics of each group of cells.(2)GO protein annotations were obtained from Uni Prot-GOA database;protein structural domain annotations were performed using the software Inter Pro Scan and the corresponding Inter Pro structural domain database;KEGG pathway annotations were performed using the KEGG online service tool KAAS;subcellular localization annotations were performed using the software wolfpsort for predicting subcellular localization.modification site The software Mo Mo,motif-x algorithm was used for motif characterization.(4)Protein functional enrichment using Fisher’s exact test.cluster analysis based on protein functional enrichment using heatmap.2,a function in the R language package gplots,to draw heat maps.Protein interaction network analysis was performed using STRING(v.10.5)protein network interaction database comparison.Expression pattern clustering analysis was performed using the Mfuzz clustering method.Phosphorylation site upstream kinase prediction was performed using i GPS 1.0software.Phosphokinase activity prediction was performed using the GSEA method.Results:1.Clinical study of the effect of myeloproliferative neoplasm driver mutation genes on NAP expression(1)Comparison of NAP score values and positivity rates in patients with each subtype of MPNAll 111 patients with PV had elevated NAP score values;135 of 180 patients with ET had elevated values,16 were within the normal range,and 29 were decreased;72of 97 patients with PMF had elevated values,6 were within the normal range,and 19were decreased.there was a statistical difference between patients in the PV group compared with those in the ET group(P<0.0001),patients in the PV group compared with those in the PMF group(P<0.0001),and patients in the ET group compared with those in the PMF group(P<0.0001).(P<0.0001),and there was no statistical difference between patients in the ET group and those in the PMF group(P=0.0508).There was a statistical difference between the patients in the PV group and the ET group(P<0.0001),and between the patients in the PV group and the PMF group(P<0.0001).There was a statistical difference between the patients in the PV group and the ET group(P<0.0001),and there was no statistical difference between the patients in the ET group and the PMF group(P=0.5755).(2)Comparison of NAP score values,positive rate and mean score values among patients with each mutation of MPNThe NAP score values were increased in all 313 patients with JAK2V617F mutation and 3 patients with JAK2 exon12 mutation;increased in 2 of 14 patients with MPL mutation,within the normal range in 2 patients,and decreased in 10patients;increased in 1 of 58 patients with CALR mutation,within the normal range in 20 patients,and decreased in 37 patients.All JAK2 mutation groups(including JAK2V617F and JAK2 exon12)were statistically different compared with MPL mutation group and CALR mutation group(P<0.0001),MPL mutation group was not statistically different compared with CALR mutation group(P=0.4634),JAK2V617F mutation group was not statistically different compared with JAK2 exon12 mutation group compared to the JAK2 exon12 mutation group(P=0.9040).The NAP positivity rate was increased in all 313 patients with JAK2V617F mutation and 3 patients with JAK2 exon12 mutation;increased in 2 of 14 patients with MPL mutation,within the normal range in 2 cases,and decreased in 10 cases;increased in 14 of 58 patients with CALR mutation,within the normal range in 17cases,and decreased in 27 cases.The NAP positivity rate was statistically different in all JAK2 mutation groups compared with the MPL mutation group and CALR mutation group(P<0.0001),no statistical difference was found in the MPL mutation group compared with the CALR mutation group(P=0.4678),and no statistical difference was found in the JAK2V617F mutation group compared with the JAK2exon12 mutation group(P=0.1046).The median mean integral value of NAP was 2.000(1.070-3.870)in patients with JAK2V617F mutation,2.580(1.920-2.970)in patients with JAK2 exon12mutation,1.085(0-1.750)in patients with MPL mutation,and 1.065(0-1.970)in patients with CALR mutation.All JAK2 mutation groups(including JAK2V617F,JAK2 exon12)were statistically different compared with MPL mutation and CALR mutation groups(P<0.0001),MPL mutation group was not statistically different compared with CALR mutation group(P=0.8281),and JAK2V617F mutation group was compared with JAK2 exon12 mutation group There was no statistical difference(P=0.2145).(3)Comparison of NAP score values,positivity rates and mean score values for each mutant MPN subtypeAll 108 patients with JAK2V617F-PV,133 patients with JAK2V617F-ET,72patients with JAK2V617F-PMF,and JAK2 exon12-PV had increased NAP score values;2 of 10 patients with MPL-ET had increased values,2 had normal values and6 had decreased values;all 4 patients with MPL-PMF had decreased values;37patients with CALR-ET mutation had increased values,2 had normal values and 6had decreased values.There was a statistically significant difference between the NAP score values in the JAK2V617F-PV group compared to the JAK2V617F-ET group(P<0.0001),but the NAP score values in the JAK2V617F-PV group were different from those in the JAK2V617F-ET group.JAK2V617F-PMF and JAK2 exon12-PV groups were not statistically significantly different(P>0.05);the JAK2V617F-PV,JAK2V617F-ET,JAK2V617F-PMF,and JAK2exon12-PV groups were all higher than the MPL-ET,MPL-PMF,CALR-ET,and CALR-PMF groups,all with statistically significant differences(P<0.05);however,there were no statistically significant differences between the MPL-ET,MPL-PMF,CALR-ET,and CALR-PMF groups(P>0.05).All patients with JAK2V617F-PV,JAK2V617F-ET,JAK2V617F-PMF,and JAK2 exon12-PV had increased NAP positivity;4 of 10 patients with MPL-ET had increased,1 was within the normal range,and 5 had decreased;all 4 patients with MPL-PMF had decreased;37 patients with CALR-ET mutation The rate of NAP positivity in the JAK2V617F-PV group was statistically significantly different from that in the JAK2V617F-ET group(P<0.0001),and the rate of NAP positivity in the JAK2V617F-ET,JAK2V617F-ET,and JAK2V617F-PV groups was statistically significantly different from that in the JAK2V617F-ET group(P<0.0001).JAK2V617F-PMF,and JAK2 exon12-PV were not statistically significantly different between the two comparisons(P>0.05),and the JAK2V617F-PV,JAK2V617F-ET,JAK2V617F-PMF,and JAK2exon12-PV groups were all higher than the MPL-ET,MPL-PMF,and CALR-ET,and CALR-PMF,with statistically significant differences(P<0.05),but no statistically significant differences(P>0.05)were found between the two comparisons of MPL-ET,MPL-PMF,CALR-ET,and CALR-PMF.The median mean integral value of NAP was 2.265(1.220-3.520)in JAK2V617F-PV patients,1.820(1.070-3.470)in JAK2 V617F-ET patients,2.100(1.190-3.780)in JAK2V617F-PMF patients,and 2.580 in JAK2 exon12 patients(1.920-2.970),MPL-ET patients 1.200(0-1.750),MPL-PMF patients NAP 1.000(1.000-1.330),CALR-ET patients 1.110(0-1.930),CALR-PMF patients 1.000(0-1.970).jak2v617f-PV,JAK2V617F-PMF,and JAK2exon12-PV patients all had higher mean NAP score values than patients in the JAK2V617F-ET group,with statistically significant differences(P<0.05);JAK2V617F-PV,JAK2V617F-ET,JAK2V617F-PMF,and JAK2exon12-PV groups were all higher than those of patients in the MPL-ET,MPL-PMF,CALR-ET,and CALR-PMF groups,with statistically significant differences(P<0.05);however,there was no statistically significant difference between the mean point values of patients in the MPL-ET,MPL-PMF,CALR-ET,and CALR-PMF groups(P>0.05).(4)Correlation analysis of JAK2V617F mutation burdern and NAP expressionAmong the 61 patients,26 were PV,18 were ET,and 17 were PMF.the JAK2V617F mutational load was significantly higher in PV patients and PMF patients than in ET patients,with statistically significant differences(P=0.0023,P=0.0018),respectively,while there was no significant difference between PV and PMF.NAP score values were significantly higher in PV patients and PMF patients than in ET patients,with statistically significant differences(P<0.0001,P=0.0020),respectively,while there were no significant differences between PV and PMF.Linear correlation analysis showed a significant positive correlation between mutation load and NAP score values in 61 patients,i.e.,NAP score values increased with higher mutation load and were statistically significant(r~2=0.1919,P=0.0005).The rate of NAP positivity was significantly higher in PV patients and PMF patients than in ET patients,with statistically significant differences(P<0.0001,P=0.0247),respectively,while there was no significant difference between PV and PMF.Linear correlation analysis showed a positive correlation between mutational load and NAP positivity rate in 61 patients,i.e.,NAP positivity rate increased with higher mutational load(r~2=0.09692,P=0.0184).The mean NAP score values were significantly higher in PV patients and PMF patients than in ET patients,with statistically significant differences(P<0.0062,P=0.0115),respectively,while there was no significant difference between PV and PMF.Linear correlation analysis showed a significant positive correlation between mutational load and mean NAP score value in 61 patients,i.e.,mean NAP score value increased with higher mutational load and was statistically significant(r~2=0.2428,P<0.0001).2.Cellular model studies of the effect of myeloproliferative tumor driver mutation genes on NAP expression(1)Comparison of NAP expression after induction in JAK2 mutant gene and control gene NB4 cell modelsThe NAP expression in NB4,NB4-MSCV,NB4-JAK2WT and NB4-JAK2V617F groups increased gradually with the extension of induction differentiation time;the NB4-JAK2V617F group started to be higher than the control groups at 48h;at 96h of induction,the NAP expression in NB4-JAK2V617F group was significantly higher than the control groups.(2)Comparison of NAP expression in MPL mutant gene and control gene NB4 cell model after inductionWith the extension of induction differentiation time,the NAP expression in NB4group,NB4-MSCV group and NB4-MPLWT group showed a gradual increase,and the expression changes in NB4-MPLW515L group and NB4-MPLA497-L498LVIA ins4 group were not obvious;at the beginning of induction 96h,NB4-MPLW515L,NB4-MPLA497-L498 LVIAins4 mutant groups were statistically different from NB4,NB4-MSCV,and NB4-MPLWT control groups,respectively.(3)Comparison of NAP expression in CALR mutant and control gene NB4 cell models after inductionWith the extension of induction differentiation time,the NAP expression in NB4group,NB4-MSCV group and NB4-CALRWT group showed a gradual increase,and the expression changes in NB4-CALRtype1 group and NB4-CALRtype2 group were not obvious;at the beginning of induction 96h,the NB4-CALRtype1 and NB4-CALRtype2 mutation groups were different from NB4,NB4-MSCV,and NB4-CALRWT control groups were statistically different compared to the NB4,NB4-MSCV,and NB4-CALRWT control groups.3.Proteomics and phosphorylation-modified proteomics to investigate the mechanism of NAP expression regulation by myeloproliferative tumor driver mutant genes(1)Analysis of proteomic results in each mutant group cell modelThe differential proteins of NB4-JAK2V617F,NB4-MPLW515L and NB4-CALR Type1 mutation models were functionally enriched,and the clustering analysis was performed according to the enrichment results.The results showed that there were no significantly enriched differential proteins that met the screening criteria.(2)Analysis of the phosphorylation-modified proteomic results in mutant cell modelThe proteins corresponding to the differential phosphosites of NB4-JAK2V617F,NB4-MPLW515L and NB4-CALR Type1 mutant models were enriched for function,and clustering analysis was performed according to the enrichment results.The results showed that there were no significant enrichment proteins corresponding to differential phosphosites that met the screening criteria.The results of expression pattern clustering analysis showed that there were three significantly differentially expressed phosphosites MESD-S221(Protein ID:Q14696),RAB11FIP1-S156(Protein ID:Q6WKZ4),NDRG1-T346(Protein ID:Q92597),in which MESD-S221 was up-regulated in the JAK2V617F group and down-regulated in the MPL and CALR mutant groups,while RAB11FIP1-S156 and NDRG1-T346were down-regulated in the JAK2V617F group and up-regulated in the MPL and CALR mutant groups.The predicted upstream phosphokinases at the phosphorylation sites showed 41NDRG1-T346-related upstream kinases,and we analyzed the 41 kinases by Pubmed database among which PKN1 kinase was expressed in neutrophils and the rest were not expressed in neutrophils.The kinase activity of the predicted NDRG1-T346upstream kinase PKN1 was continued to be screened,and this kinase activity was among the 23 kinases mentioned above,which tended to be in a suppressed state in the JAK2V617F group and unchanged in the MPL and CALR mutant groups.neither MESD-S221 nor RAB11FIP1-S156 predicted its associated upstream kinase.Conclusion:1.Through comparative analysis of NAP indexes in patients with each subtype of MPN,we found that NAP was increased in almost all PV patients,but about 80%or more of ET and PMF patients showed increased NAP,and most of the remaining patients showed significantly reduced NAP.This suggests that NAP expression is suppressed in MPN patients,which is different from the previous traditional view that NAP is increased in MPN patients.This study has important guiding value for clinical MPN diagnosis.2.Through the comparative analysis of each index of NAP in patients with each driver mutation of JAK2,MPL,and CALR,we confirmed that the heterogeneity of differential NAP expression in MPN patients was significantly correlated with the driver mutations of JAK2,MPL,and CALR,i.e.,JAK2 mutations showed increased NAP,and MPL and CALR mutations showed decreased or suppressed NAP expression.It is suggested that JAK2,MPL,and CALR driver mutations may be the intrinsic molecular basis of NAP expression changes in MPN patients.3.By comparing the NAP indicators of JAK2 mutations in different subtypes of MPN,we found that the higher the load of JAK2V617F mutation in neutrophils,the higher the NAP expression;the heterozygous mutation of JAK2exon12 affected NAP to the same extent as the pure mutation load of AK2V617F.It is suggested that the mutation load of JAK2 and the mutation of different sites of JAK2 also have different degree of effect on NAP expression.4.In this part of the study,we constructed NB4 models of JAK2,MPL,CALR mutations and wild-type overexpression by lentiviral transduction technique,and the results showed that JAK2 mutation promoted NAP significantly,while MPL and CALR mutations suppressed NAP expression significantly.This model confirmed the causal regulation of JAK2,MPL,and CALR driver mutations with NAP and was consistent with the findings of previous clinical data.5.By resolution at the proteomic level,we did not find significant enrichment of differentially expressed protein molecules in each mutation group cell model of NB4-JAK2V617F,NB4-MPLW515L,and NB4-CALR Type1.It is suggested that the intrinsic mechanism of differential regulation of NAP by JAK2 mutation with MPL and CALR may not be signaling by causing increased expression levels of downstream signaling molecules,but mainly regulating the level of phosphosites.6.By resolving the phosphorylation modification at the proteomic level,we identified three significantly enriched differentially expressed phosphosites,namely MESD-S221,RAB11FIP1-S156,and NDRG1-T346,in each mutant group cell model of NB4-JAK2V617F,NB4-MPLW515L,and NB4-CALRType1.and their upstream phosphokinase prediction as well as functional analysis were performed.The data suggest that all three phosphosites may be involved in the mechanism underlying the differential regulation of NAP by JAK2 mutations with MPL and CALR.