In-Depth Proteomics Reveals Selective EV Protein Sorting and Pathway Perturbation in AML upon Synergistic FLT3 and Hedgehog Pathway Inhibition

Acute myeloid leukemia (AML) is an aggressive cancer mainly affecting bone marrow and blood with a high relapse incidence and mortality rate. Approximately one third of AML patients carry an fms-like tyrosine kinase 3 (FLT3) mutation, which is often associated with GLI expression and Hedgehog signaling, leading to tumor proliferation. AML cells shape their microenvironment into a leukemia-permissive space by releasing extracellular vesicles (EVs). EVs can transfer chemoresistance and thereby play an important role in refractory and relapsing diseases. Here, we discovered a synergistic effect of a combined treatment with the FLT3 inhibitor Crenolanib and the Hedgehog pathway inhibitor HPI-1 in the AML cell lines MOLM-14 and MV4-11. In-depth comparative proteomics revealed alterations in the cellular and the EV proteome upon single or combined inhibition of FLT3 and GLI, highlighting affected pathways. By comparing the cellular and EV proteomes, we report that transport of ribosomal proteins such as RPS26 and RPL27A and ErbB pathway members such as GAB1, GRB2 and SHC1 to EVs is selectively avoided upon treatment with Crenolanib. Ribosomal and ErbB signaling pathway proteins may play an important role in microenvironmental modulation by EVs, and Crenolanib treatment potentially acts by interfering with leukemia niche formation.