NG2 is a target gene of MLL-AF4 and underlies glucocorticoid resistance in MLL-r B-ALL by regulating NR3C1 expression.

B-cell acute lymphoblastic leukemia (B-ALL) is the most common pediatric cancer, with long-term overall survival rates of ~85%. However, B-ALL harboring rearrangements of the MLL gene (also known as KMT2A), referred to as MLLr B-ALL, is common in infants and is associated with poor 5-year survival (<30%), frequent relapses, and refractoriness to glucocorticoids (GCs). GCs are an essential part of the treatment backbone for B-ALL and GC resistance is a major clinical predictor of poor outcome. Elucidating the mechanisms of GC resistance in MLLr B-ALL is, therefore, critical to guide therapeutic strategies that deepen the response after induction therapy. Neuron-glial antigen-2 (NG2) expression is a hallmark of MLLr B-ALL and is minimally expressed in healthy hematopoietic cells. We recently reported that NG2 expression is associated with poor prognosis and that anti-NG2 immunotherapy strongly reduces/delays relapse in MLLr B-ALL xenograft models. Despite its contribution to MLLr B-ALL pathogenesis and its diagnostic utility, the role of NG2 in MLLr-mediated leukemogenesis/chemoresistance remains elusive. Here we show that NG2 is an epigenetically regulated direct target gene of the leukemic MLL-AF4 fusion protein. NG2 negatively regulates the expression of the GC receptor NR3C1 and confers GC resistance to MLLr B-ALL cells in vitro and in vivo. Mechanistically, NG2 interacts with FLT3 to render ligand-independent activation of FLT3 signaling (a hallmark of MLLr B-ALL) and downregulation of NR3C1 via AP-1-mediated trans-repression. Collectively, our study elucidates the role of NG2 in GC resistance in MLLr B-ALL through FLT3/AP-1-mediated downregulation of NR3C1, providing novel therapeutic avenues for MLLr B-ALL.