Increased c-Jun Expression and Reduced GATA2 Expression Promote Aberrant Monocytic Differentiation Induced by Activating PTPN11 Mutants

Abstract

ABSTRACT Juvenile myelomonocytic leukemia (JMML) is characterized by myelomonocytic cell overproduction and commonly bears activating mutations in PTPN11 . Murine hematopoietic progenitors expressing activating Shp2 undergo myelomonocytic differentiation, despite being subjected to conditions that normally support only mast cells. Evaluation of hematopoietic-specific transcription factor expression indicates reduced GATA2 and elevated c-Jun in mutant Shp2-expressing progenitors. We hypothesized that mutant Shp2-induced Ras hyperactivation promotes c-Jun phosphorylation and constitutive c-Jun expression, permitting, as a coactivator of PU.1, excessive monocytic differentiation and reduced GATA2. Hematopoietic progenitors expressing activating Shp2 demonstrate enhanced macrophage CFU (CFU-M) compared to that of wild-type Shp2-expressing cells. Treatment with the JNK inhibitor SP600125 or cotransduction with GATA2 normalizes activating Shp2-generated CFU-M. However, cotransduction of ΔGATA2 (lacking the C-terminal zinc finger, needed to bind PU.1) fails to normalize CFU-M. NIH 3T3 cells expressing Shp2E76K produce higher levels of luciferase expression directed by the macrophage colony-stimulating factor receptor ( MCSFR ) promoter, which utilizes c-Jun as a coactivator of PU.1. Coimmunoprecipitation demonstrates increased c-Jun-PU.1 complexes in mutant Shp2-expressing hematopoietic progenitors, while chromatin immunoprecipitation demonstrates increased c-Jun binding to the c- Jun promoter and an increased c-Jun-PU.1 complex at the Mcsfr promoter. Furthermore, JMML progenitors express higher levels of c- JUN than healthy controls, substantiating the disease relevance of these mechanistic findings.