Effect And Mechanism Study Of Palmitoyl-transferase DHHC21 On Regulating Differentiation Of Acute Myeloid Leukemia

ObjectiveIntracellular expression of genes and regulation of signaling networks often induce malignant phenotypes of tumors,such as sustained proliferation,apoptotic resistance,chemoresistance,metastasis and recurrence.There are hundreds of different post-translational modifications in cells that regulate the localization,stability,interaction,and transcription of key proteins of signaling networks to affect tumor progression.Post-translational lipid modification can enhance the liposolubility of substrates,endowing it with a better affinity for the plasma membrane,endoplasmic reticulum,Golgi apparatus or other membrane structures.The S-palmitoylation catalyzes the attachment of palmitic acid to the cysteine residue of the substrates,and its linkage is reversible.To date,studies have found that numerous tumor-key-regulated proteins are S-palmitoylated.Therefore,it is important for the tumor-theraptic study to find a new strategy to intervene the S-palmitoylation of tumor proteins.Currently,the palmitoyltransferase DHHC protein family,which mediates the protein S-palmitoylation,have received extensive attention.In view of the close relation between DHHCs and various diseases such as tumor or nervous system diseases,DHHCs are considered to possess important biological functions.Many studies have found the frequent deletions and abnormal amplification of DHHCs,which played different roles in tumors.Acute myeloid leukemia(Acute Myeloid Leukemia,AML)is a blood system tumor with clinical and biological heterogeneity.In recent years,researches related to AML treatment have gradually entered a bottleneck from rapid development.In clinical,major chemotherapy drugs for AML are cytarabine,anthracycline antibiotics and all-trans retinoic acid.After the standard chemotherapy,the long-term disease-free survival rate of patients is only 30-40%and the prognosis is poor for the chemotherapy resistance and recurrence.Therefore,there is a need to find new potential targets and intervention strategies for AML treatment.However,studies have found that DHHC9 affects T cell acute lymphoblastic leukemia mediated by N-Ras and DHHC14 promotes the development of acute biphenotype leukemia,suggesting that DHHCs may have important biological functions in leukemia.On previous studies,we found that the inhibition of DHHC activity induces the differentiation of AML.Thus,our study is dedicated to further clarifying the key DHHC that regulates AML differentiation and investigating the molecular mechanism involved in the regulation of AML differentiation,in order to find new molecular targets and potential intervention strategies for clinical AML treatment.MethodsOur research subjects were HL60 and NB4(AML cell lines).The differentiation induced by the palmitoyltransferase inhibitor 2BP in AML cells was detected by NBT reduction assay and RG staining assay.The expression of different DHHCs in various tumors were investigated on the Oncomine database.DHHC3 and DHHC21 expression in normal bone marrow cells and AML cells were compared on the Oncomine database.The silencing efficiency of shDHHCs were confirmed by Quantitative-Real-time PCR.The expression of differentiation marker CD11b was detected by flow-cytometry when DHHCs were knockout.The differentiation of AML cells were detected by NBT reduction assay and RG staining assay when DHHCs were knockout.Cell growth curves were performed with cell-counting assay when DHHC21 was knockout.Time-dependent changes in expression of differentiation markers CDllb and CD14 in AML cells was detected by flow-cytometry when DHHC21 were knockout.The differentiation of AML cells were detected by NBT reduction assay and RG staining assay when DHHC21 were knockout.The mRNA level of STAT1,PU.1 and CEBPβwere detected by Quantitative-Real-time PCR in AML cells when DHHC21 were knockout.With the overexpression of m-DHHC21 or m-DHHC21-C120S,the AML differentiation induced by shDHHC21 were detected by NBT reduction assay and RG staining assay.The differentiation induced by shDHHC21 were detected by NBT reduction assay when treated with LY-294002,PD-98059,SB-203580,SP-600125 or R041-5253.The gene expression profile of shDHHC21 in AML cells was established by genechip.The functional clustering analysis of altered genes were established successfully by the Metabase.The mRNA level of altered genes were detected by Quantitative-Real-time PCR when DHHC21 were knockout.The differentiation induced by shDHHC21 when MAFB,shIRF9 or shPLSCR1 was knockout were detected by NBT reduction assay.Results①The effect of the inhibition of different DHHC isoform on AML differentiation.After the treated with different concentrations of palmitoyltransferase activity inhibitor 2BP,the NBT-reducing ability of AML cells was enhanced and its nuclear morphology exhibited differentiation-related transformation.The expression of DHHC3 and DHHC21 were the highest,followed by DHHC8,DHHC9 and DHHC14,while the expression of other DHHCs were relatively low on the Oncomine database.The expression of DHHC21 in AML cells was higher than that of normal bone marrow cells which showed significant difference,while DHHC3 did not.After knockout of DHHC3,DHHC7,DHHC21 or DHHC23,only shDHHC21 specifically enhanced the expression of CD11b,NBT-reducing ability and differentiation-related transformation of nuclear morphology.② Confirmation of AML differentiation induced by shDHHC21.shDHHC21 enhanced the expression of the differentiation markers CD11b and CD 14 with certain time-effect relationship.The slience of DHHC21 enhanced the NBT-reducing ability and differentiation-related transformation of nuclear morphology in AML cells.The mRNA levels of STATI,PU.1 or CEBPP were enhanced by shDHHC21.Overpression of m-DHHC21 could inhibit the NBT-reducing ability and nuclear morphology transforms of AML cells,but m-DHHC21-C120S could not③Role of classical differentiation signal in shDHHC21-induced AML differentiation.The AML cell differentiation induced by shDHHC21 was blocked through the inhibition of PI3K activity,MEK activity or JNK activity.The AML cell differentiation induced by shDHHC21 was not blocked through the inhibition of P38-MAPK activity or RARa activity.④The exploration of the molecular mechanism of DHHC-regulating AML differentiation.The gene expression profile of shDHHC21 in AML cells was successfully established by gene-chip.It was found that many genes showed significant changes on the 24,48 or 72 h(57,137 or 169 genes were up-regulated,118,44,50 genes were down-regulated correspondingly).There were simultaneously 31 genes that are up-regulated and 5 genes that are down-regulated.The mRNA levels of the altered genes(AGPAT9，CLEC7A,MAFB,PARP9,IFIT2,IRF9,IFIT5,FPR1,IF16 and PLSCR1)by shDHHC21 were indeed significantly up-regulated in vitro The NBT reduction ability enhanced by shDHHC21 was not significant affected by silencing of MAFB,IRF9 or PLSCR1.ConclusionOur study found that the palmitoyltransferase inhibitor 2BP could induce differentiation of AML cells.When searching for the key DHHC of the differentiation of AML cells,DHHC21 was found to specifically induce AML cell differentiation compared to other DHHC isoforms,which reasonably explained the abnormal expression of DHHC21 in clinical AML patients.And we have defined the direction of differentiation of shDHHC21-induced AML cells-monocyte/macrophage differentiation.The differentiation that was regulated by DHHC21 was dependent of its palmitoyltransferase activity.Further studies have found that shDHHC21 induces AML cells differentiation through regulating monocyte/macrophage differentiation-related transcription factors and signaling networks such as PI3K,MEK,and JNK.In addition,we also found that the AML differentiation induced by shDHHC21 is independent of RARa.Our study not only found a new biological function of DHHC21 and the mechanism that regulates AML cell differentiation,but also provided a potential differentiation therapeutic target for clinical AML treatment.