Identification and Characterization Of a Potent FLT3 Inhibitor

Acute myeloid leukemia (AML) is a malignant myeloid disorder for which there is no effective treatment. Gain-of-function mutations of tyrosine kinase FLT3 are frequently found in AML patients. This makes FLT3 an attractive therapeutic target. Currently, several potent FLT3 inhibitors have been developed. However, their clinical efficacy is limited largely due to their poor effectiveness toward the FLT3-D835 mutants which are often present in AML or acquired after treatment of FLT3-ITD-positive AML with tyrosine kinase inhibitors. Needless to say, more potent FLT3 inhibitors targeting both FLT-ITD and FLT3-D835 mutants are needed. In addition, combinations of tyrosine kinase inhibitors with drugs targeting other signaling pathways represent a new trend in anti-cancer drug development. To establish an effective kinase assay for FLT3 inhibitor screening, we generated a protein substrate designated GST-FLT3S which was expressed in E. coli cells as a glutathione S-transferase fusion protein. The protein substrate together with recombinant proteins containing the catalytic domain of wild type and mutant forms of FLT3 expressed in baculovirus was used in biochemical screening of inhibitors. Several potent inhibitors were obtained. Importantly, one of the inhibitors with an oxindole core structure inhibited FLT3 and D835 FLT3 mutants equally well with nanomolar IC50 values. We further analyzed the potency of the inhibitor by performing cell-based assays. The cells used included FLT3-ITD-positive cell line MV-4-11 and an EPO-dependent erythroleukemia cell line transformed by retrovirus mediated expressions of FLT3-ITD and FLT3-D835 mutants. At nanomolar concentrations, the inhibitor blocked growth factor signaling and effectively caused apoptosis and cell cycle arrest. It showed significant advantage over the current available FLT3 inhibitor, sorafenib. Loss-of-function mutations of tumor suppressor p53 are common in solid tumors but relatively rare in AML although its expression is often suppressed. This makes p53 a potential target for anti-AML drug development. We employed MDM2 inhibitor nutlin-3 which blocks the degradation of p53. Importantly, at sub-nanomolar concentrations, FLT3 inhibitors and nutlin-3 synergistically inhibited growth of cells containing FLT3-ITD or FLT3-D835 mutants. Altogether, we developed an effective substrate for screening of FLT3 inhibitors and identified one compound with high potency toward both FLT3-ITD and FLT3-D835. We further demonstrated that targeting FLT3 and p53 simultaneously greatly increases drug potency. Disclosures: No relevant conflicts of interest to declare.