CD34 is a target for covalent EGFR inhibitors to eliminate stem/progenitor cells in acute and chronic myeloid leukemia

Abstract The effect of epidermal growth factor receptor (EGFR) inhibitors on acute myeloid leukemia (AML) was discovered over one decade ago. However, clinical trials of EGFR inhibitors in AML have yielded controversial outcomes. Leukemia cells lack EGFR expression, and the mechanism by which EGFR inhibitors affect leukemia cell growth is unknown, obscuring the precise subset of AML patients that might be targeted by these compounds. Since myeloid leukemia arises from malignant stem/progenitors, here we evaluated the effect of EGFR inhibitors on primary leukemia stem/progenitors that expressed the stem cell marker CD34 which were sorted from leukemia patients. EGFR inhibitors induced significant apoptosis of primary CD34+ but not CD34− cells derived from AML and chronic myeloid leukemia (CML) patients both in vitro and in patient-derived xenotransplantation model. Using two EGFR inhibitors osimertinib and afatinib, we demonstrated binding and covalent adducts of the inhibitors with the cysteine(C) 199 residue of the CD34 protein, which downregulated phosphorylation of tyrosine 329(Y329) of CD34, leading to the dissociation of CD34 from tyrosine kinase Src and thereafter the inhibition of STAT3 phosphorylation. Most importantly, administration of osimertinib yielded clinical responses in two CD34-high AML patients identified by quantitative proteomics with reduced levels of Y329 phosphorylation of CD34 after treatment. Collectively, these findings delineate a novel molecular pathway whereby EGFR inhibitors kill leukemia and reveal that the CD34 antigen is a targetable signaling molecule that mediates cell survival signals via connecting to Src-STAT3 pathway.