NPM1 Mutant Maintains ULK1 Protein Stability To Promote Autophagic Cell Survival In Leukemia

Objective: Nucleophosmin(NPM1)mutations are the most frequent genetic alteration in acute myeloid leukemia(AML).Because of unique clinical features and gene expression profile,NPM1-mutated AML has been defined as an entity in the 2016 updated WHO classification of myeloid neoplasms,and aberrant cytoplasm-dislocated NPM1 mutant is a distinct biological characterization of this disease.Our group previously revealed that NPM1 mutant elevated autophagy activity and autophagy activation contributed to leukemic cell survival.However,the molecular mechanisms by which cytoplasmic NPM1 mutant involving in the autophagy pathway has not been fully elucidated.Methods: Firstly,the expression levels of autophagy-related genes in AML with NPM1 mutations were analyzed by using TCGA and GEO databases.The expression of ULK1,ATG3 and ATG7 further were verified in human myeloid leukemia cell lines and primary AML cases by western blot and qRT-PCR.Next,the interaction of NPM1-mA and ULK1 was detected by Co-immunoprecipitation(Co-IP)and immunofluorescence(IF)assays.Furthermore,the protein and mRNA levels were checked following NPM1-mA depletion or overexpression.ULK1 protein stability was observed following cycloheximide treatment.The phosphorylation levels of ATG13 as a ULK1 substrate were examined upon NPM1-mA knockdown or overexpression.Subsequently,the effects of NPM1-mA on TRAF6-mediated ubiquitination of ULK1 were detected upon NPM1-mA overexpression.In addition,following ULK1 knockdown or overexpression,the levels of autophagy markers(LC3 II,p62)were detected by western blot,and cell proliferation in vitro was assessed by colony formation and CCK-8 assay.Finally,rescue experiments were performed to observed the change in autophagy activity and cell proliferation ability in NPM1-mA-silenced OCI-AML3 cells following ULK1 overexpression or in NPM1-mA-enforced K562 and NB4 cells following ULK1 inhibitor SBI-0206965 treatment.Results: Firstly,ULK1 expression levels were relatively high expressed in NPM1-mutated AML.Secondly,Co-IP and IF assays showed that NPM1-mA physically interacted with ULK1 protein.In addition,NPM1-mA enhanced the ULK1 protein levels,whereas ULK1 mRNA levels remained unchanged.Mechanically,NPM1-mA positively regulates ULK1 protein stability and kinase activity.It is noteworthy that ectopically expressed NPM1-mA led to a significant increase of TRAF6-dependent ubiquitination of ULK1.Moreover,ULK1 up-regulatedLC3Ⅱ levels,down-regulated p62 levels,and promoted cell proliferation in vitro.Finally,the rescue experiment confirmed that restoring ULK1 expression or ULK1 inhibitor SBI-0206965 treatment partially rescued the effect of NPM1-mA on autophagy activation and cell survival.Conclusions: cytoplasmic NPM1-mA could interact with ULK1 protein and positively regulated ULK1 protein levels.Mechanically,NPM1-mA promoted TRAF6-dependent K63 ubiquitination and further maintained ULK1 stability and kinase activity,which contributed to autophagy activation and leukemic cell survival.These findings provide a new understanding of NPM1 mutant in the regulation of autophagy activation in NPM1-mutated leukemia.