Acquired Point Mutations of TKD Are Responsible for Sorafenib Resistance in FLT3-ITD Mutant AML,
Authors: Zhang Weiguo , Konopleva Marina , Jacamo Rodrigo O , Borthakur Gautam , Chen Wenjing , Cortes Jorge E. , Ravandi Farhad , Andreeff Michael 
Published: October 2019

Abstract
Abstract 3505
Activating mutations of fms-like tyrosine kinase 3 (FLT3), including Internal tandem duplications (ITD) mutations and point mutations of tyrosine kinase domain (TKD), can be detected in 1/3 of acute myeloid leukemia (AML) patients, and are associated with higher risk of relapse. (1) We reported that sorafenib, by directly targeting mutant FLT3-ITD, exerted anti-leukemia effects in AML with FLT3-ITD mutations in Phase I/II clinical trial and increased the complete remission rate to 100% (CR and CRp) in combination with Idarubicin/cytarabine.(2, 3) However, resistance to sorafenib develops in most AML patients with ITD mutations during prolonged therapy and leads to relapse.(3) Therefore, understanding the mechanisms of sorafenib resistance remains a challenge that may aid approaches to overcoming drug resistance in this dismal prognostic group of AML patients. The present study found that acquired point mutations in the TKD1 and TKD2 domains of the FLT3 gene play a crucial role by elevating levels of phosphorylated FLT3 and its downstream signaling proteins.
cDNA-based mutation analysis of the FLT3 gene was performed in blood/bone marrow samples from AML patients who showed sensitivity to sorafenib during the first cycles of therapy in Phase I/II trials and then became resistant. Results showed that 50% (3 of 6 cases) developed single or multiple acquired point mutations of the FLT3 gene, which included mutations of D651G(H), G619C and I687F in TKD1 and/or E858K in TKD2. Further, a murine sorafenib-resistant AML cell line (Ba/F3-ITD-Res) was developed by long-term/low-dose exposure to sorafenib in vitro. Sequence analysis identified acquired point mutations of N676D and Y842C. To further investigate the correlation of these point mutations with drug resistance, selected single point mutations were individually introduced into Ba/F3-ITD cells by lentiviral infection. Apoptosis induction was measured after exposure to sorafenib. Results showed varying degrees of resistance in sublines with different point mutations and their EC50s for apoptosis induction were 0.69, 0.61 and 19 μM in D651G, N676D and Y842C mutation-containing cells, respectively. By comparison, EC50 for Ba/F3-ITD was 0.16 μM. However, cells with multiple concomitant point mutations (such as N676D plus Y842C) displayed impressive resistance (EC50 = 32μM). These results suggest that single mutations in either TKD1 or TKD2 are associated with resistance of sorafenib-induced apoptosis, and that structural alterations of TKD2 are more critical than those of TKD1. Further, concomitant mutations in both TKDs strongly impair response to sorafenib, suggesting a pivotal role for the structural integrity of both TKDs in maintaining sensitivity of FLT3-ITD AML cells to sorafenib.
To better understand mechanisms of resistance, relevant biomarkers were investigated by Western blot in these cells with point mutations. The results showed that TKD1 mutations (D651G and/or N676D) increased basal levels of phospho–FLT 3 and its downstream targets phospho–ERK, –Stat5, –AKT and –S6K, suggesting that mutations of TKD1 result in increased FLT3 activation. However, one TKD2 mutation (Y842C) which does not result in increased basal levels of phospho-FLT3, was nevertheless resistant to sorafenib-induced suppression of FLT3 targets, implying that Y842C may interfere with the binding of sorafenib to FLT3 by a conformational shift of the adenosine triphosphate (ATP)–binding pocket, which reduces the accessibility for sorafenib and mediates resistance. Notably, cells with concomitant N676D and Y842C mutations in both TKDs displayed higher basal phosphorylation levels of FLT3 and its downstream targets, resulting in increased resistance to sorafenib-induced suppression of phosphorylation, suggesting that synergistic resistance to sorafenib may result from multiple mutations in by both, TKD1 and TKD2.
Our findings provide an improved understanding of acquired FLT3-ITD/TDK point mutations associated with sorafenib resistance in FLT3-ITD-mutant AML, and might be useful in developing agents that have the potential of overcoming resistance of FLT3-ITD inhibitor in AML.

Disclosures:
Ravandi: Bayer: Research Funding; Onyx: Research Funding.