Flt3 Mutations in Proximity to an Ubiquitin Dependent Endocytosis Motif Suspend Its Hdm2 Modulation.

Acute myeloid leukemia (AML) frequently features mutations in the receptor tyrosine kinase Flt3 and elevated expression of the oncogenic E3 ubiquitin ligase Hdm2. Additional to the p53 inhibitory effect of Hdm2, Hdm2 appears involved in endocytosis of cell surface receptors. In this study we explore the possibility of Flt3 modulation by Hdm2 in primary AML cells and cell lines (NB4 and MV4–11) with wild type Flt3 (Flt3-wt) or mutated Flt3 (Flt3-ITD). Flt3 ligand (FL), small molecular inhibitors and small interfering RNA (siRNA) were used to elucidate the relation between Flt3 and Hdm2 on protein level, mRNA expression and modulation of apoptosis. The basal level of Flt3 is higher in AML patients with Flt3-ITD than in patients with Flt3-wt. Flt3-ITD affects a ubiquitin endocytosis motif that in some patients are duplicated, possibly resulting in enhanced receptor cycling. Down-regulation of Flt3-wt by FL, small interfering RNA or PKC412 resulted in elevated level of Hdm2. Similarly, Hdm2 attenuation resulted in increased Flt3 protein expression. Flt3-ITD responded less to Flt3 down-regulation, and was only weakly responding to Hdm2 modulation. We demonstrate that modulation of Flt3 or Hdm2 results in reciprocal regulation, and that Flt3 with internal tandem duplications may suspend its Hdm2 modulation. Together, Flt3-ITD results in dysregulated receptor turnover and elevated Hdm2 thus interconnecting the two pathways of Flt3 and p53, both related to chemoresistance in AML.