Flt3/ITD Confers Resistance to FLT3 Inhibitor AC220 By up-Regulating Runx1
Authors: Hirade Tomohiro , Abe Mariko , Onishi Chie , Yamaguchi Seiji , Fukuda Seiji 
Published: October 2019

Abstract
FMS-like tyrosine kinase 3 (FLT3) is a membrane type tyrosine kinase and has important roles for the proliferation and differentiation of the hematopoietic cells. The Internal Tandem Duplications of FLT3 (FLT3/ITD) is detected in approximately 30 % of patients with acute myeloid leukemia (AML) and the prognoses of FLT3/ITD+ AML are very poor. While a number of inhibitors targeting FLT3 tyrosine kinase have been developed, few drugs are effective for the FLT3/ITD+ AML because of emergence of resistant cells against the drugs. Recently, AC220 (Quizartinib), a second generation class III tyrosine kinase inhibitor (TKI) for FLT3/ITD+ AML was developed and used in clinical trials. Although AC220 is a more potent and specific inhibitor for FLT3/ITD compared to the other TKIs, report demonstrates that prolonged exposure to AC220 can generate resistant clones to AC220 in FLT3/ITD+ cells (Smith et al. Nature 2012). These findings underscore the need to develop additional therapeutic strategies to overcome the resistance of FLT3/ITD+ AML to TKIs. However, the mechanism responsible for drug resistance of FLT3/ITD+ AML cells remains to be investigated.
We previously reported that mRNA expression of RUNX1, a core-binding transcription factor that regulates the differentiation and proliferation of hematopoietic stem cells, is significantly higher in FLT3/ITD+AML cells compared to FLT3/ITD-AML cells (Hirade et al. ASH 2013). Although loss of RUNX1 function (i.e. RUNX1/ETO fusion gene) contributes to the development of AML, RUNX1 also promotes survival of AML cells (Goyama et al. JCI 2013) and can function as an oncogene in cancer cells (Kilbey et al. Cancer Research 2010). These findings lead us hypothesize that RUNX1 may confer resistance of AML cells to TKIs. In the present study, we investigated if Runx1 is involved in the refractory phenotype of Flt3/ITD+cells to AC220.
Transduction of Flt3/ITD into IL3-dependent mouse 32D cells allowed the cells proliferate in a growth factor independent fashion, concomitant with up-regulation of Runx1 mRNA level, similar to the patients’ samples with FLT3/ITD+AML. Silencing Runx1 expression using shRNA resulted in 70% reduction of Flt3/ITD+32D cells that proliferated in the absence of growth factors. Similarly, incubating the Flt3/ITD+32D cells with 0.5nM AC220 inhibited their factor independent proliferation by 95%, which was further accentuated up to 99% by the combination with shRNA mediated silencing of Runx1. Although the number of factor independent Flt3/ITD+32D cells cultured in the presence of 2nM AC220 rapidly declined within 96 hours, the residual cells subsequently re-proliferate within 14 days and became no longer sensitive to AC220. Surprisingly, the expression of Runx1 mRNA in the resistant cells to AC220 was 5.0±0.2 fold higher (P<0.05) compared to control Flt3/ITD+32D cells sensitive to AC220. Silencing Runx1 using shRNA abrogated the proliferation of AC220-resistant Flt3/ITD+32D cells cultured in the presence of 2nM AC220, leading to 99.5% reduction in the viable cells.
Our data indicates that knocking down Runx1 expression enhances the cytotoxic effect of AC220 on Flt3/ITD+32D cells and that Runx1 expression is significantly up-regulated by the AC220 resistance cells. Moreover, Runx1 knockdown recovered the cytotoxicity of AC220 in the refractory Flt3/ITD+32D cells, demonstrating that Flt3/ITD confers resistance to AC220 by up-regulating the expression of Runx1. These findings demonstrate that antagonizing RUNX1 may represent potential therapeutic strategy in the patients with FLT3/ITD+ AML that become refractory to AC220.

Disclosures
No relevant conflicts of interest to declare.