The Significance of IL-3 Receptor beta chain for FLT3-ITD Dependent Oncogeneic Signaling in AML

In AML, activating FLT3 mutations are associated with poor prognosis. FLT3 tyrosine kinase inhibitors (TKIs) have limited clinical activity. The mechanism of this resistance is in most cases unexplained. In the first funding period, we identified mechanisms of FLT3 kinase-independent resistance. In an in vitro model, we identified autocrine secretion of the chemokine CCL5 by leukemia cells as a resistance mechanism and verified it in patient samples (Waldeck et al. 2020). Moreover, we demonstrated that activating JAK1/2/3 mutations can substitute FLT3-dependent CSF2RB activation in vitro and that JAK1/2/3 mutations occur in patients with FLT3 inhibitor resistant AML (Rummelt et al. Leukemia 2020). We demonstrated that his resistance can be overcome by a combination of FLT3 and JAK inhibitors. We found that CSF2RB interacts directly with FLT3-ITD and is phosphorylated in a FLT3-dependent manner. CSF2RB knockdown inhibited cell growth and STAT5 activation, and acted synergistically with FLT3 inhibitors. CSF2RB-deficient bone marrow showed lower transformation potential. In FLT3-ITD positive xenografts, CSF2RB knockdown resulted in reduced STAT5 phosphorylation and prolonged survival. CSF2RB-deficient bone marrow reduced growth of FLT3-ITD-induced AML and prolonged survival in recipient animals. In this follow-up proposal, we aim to elucidate the mechanism of FLT-ITD dependent CSF2RB activation, specifically the role of tyrosines within the CSF3RB binding motif of FLT3-ITD and the significance of bona fide CSF2RB interaction partners (LYN, SRC, SYK) and ER stress signaling as possible mechanisms of FLT3-ITD dependent CSF2RB activation. We will test the highly clinically relevant hypothesis whether the differential transformation capacity and prognostic significance of known FLT3-ITD variants are due to differences in the binding motif for CSF2RB in FLT3-ITD and thus due to CSF2RB binding ability. Based on the elaborated interaction domains, we will validate optimized REPLACE peptides for therapeutic blockade of CSF2RB-dependent transformation and leukemogenesis by FLT3-ITD in vitro and in vivo. Using experimental models that exclude cytokine-dependent effects, we will investigate the cell-intrinsic, FLT3-ITD-dependent transforming effect of CSF2RB in vivo and validate novel combination therapies of FLT3 inhibitors and CSF2RB interface blocking peptidomimetics for clinical use.