NPM1 Mutant Mediated PML Delocalization And Stabilization Enhances Autophagy And Cell Proliferation In Leukemic Cells

Objective: Acute myeloid leukemia(AML)is a clinically,cytogenetically,and molecularly heterogeneous disease.Mutation in nucleophosmin(NPM1)gene is one of the most frequently genetic lesions in AML.Previous researches have defined NPM1 mutation as a driver genetic event in AML,whereas the precise molecular mechanism for the involvement of NPM1-mA in AML malignant transformation remains to be fully elucidated.Autophagy is a catabolic process involving the degradation of intracellular misfolded proteins,and damaged organelles through lysosomal pathway to maintain homeostasis.Increasing researches have demonstrated the critical role of autophagy in tumor development and treatment outcome.More recently,analysis of primary AML samples have revealed the aberrant intracellular distribution of the tumor suppressor promyelocytic leukemia(PML)protein in NPM1-mutated AML blasts.The aim of the study is to investigate the effect of NPM1-mA on autophagy,further undertake whether autophagy is beneficial to NPM1 mutation mediated cell proliferation,and preliminarily explore the involvement of aberrant PML in NPM1 mutation mediated autophagy in leukemic cells.Methods: Firstly,we monitored autophagic activity upon NPM1 mutant expression by gain-and loss-of NPM1 mutation type A(NPM1-mA)function experiments,autophagy inducer(rapamycin)and inhibitor(3-methyladenine,3-MA)were used to examine the involvement of autophagy in NPM1-mA mediated cell proliferation.The expression and intracellular distribution of PML in primary AML blasts and cultured cells were analysed by qRT-PCR,western blot,immunocytochemical(ICC)and immunofluorescence(IF)assays,respectively.Co-immunoprecipitation(Co-IP)was performed to determine the interaction between mutant NPM1 and PML.The levels of PML protein were checked following NPM1-mA overexpression and knockdown.PML protein stability was examined by using cycloheximide(CHX)and MG132 treatment.In vitro experiments were conducted to observe the effect of PML knockdown on autophagy and cell proliferation and its potential mechanisms.Finally,a rescue assay was performed to confirm the critical role of PML in NPM1-mA mediated autophagy and cell proliferation.Results: Our data showed that NPM1-mA overexpression enhanced autophagic activity,and treatment with 3-MA could alleviate the enhancement of NPM1-mA mediated growth advantage in vitro.Conversely,the depletion of NPM1-mA impaired autophagy,and the addition of rapamycin could rescue the inhibiting effect of NPM1-mA depletion on cell proliferation.Meanwhile,the expression and altered intracellular distribution of PML were detected in NPM1-mutated AML primary blasts and cultured cells.Co-IP assays confirmed the interaction between mutant NPM1 and PML.In addition,overexpression of NPM1-mA upregulated,whereas depletion of NPM1-mA downregulated PML protein levels.Further experiments revealed that NPM1-mA could mediate PML stabilization through inhibiting proteolysis.Next,in vitro experiments demonstrated that PML knockdown inhibited cell proliferation and colony formation ability in OCI-AML3 cells,which possibly due to the impaired autophagy caused by AKT downregulation.Finally,we found that rescuing PML expression could bypass the inhibiting effect of NPM1-mA depletion on autophagy and cell proliferation in OCI-AML3 cells.Conclusions: Our data suggested that NPM1 mutant could promote autophagy and cell proliferation.Mechanically,NPM1-mA interacted with PML,aiding to PML cytoplasmic dislocation was well as stabilization;PML could mediate autophagy partially due to AKT activation,consequently contributed to cell proliferation in leukemic cells.These findings indicate an attractive therapeutic avenue for autophagy inhibition and PML targeting in the treatment of NPM1-mutated AML.