Abstract 1336: Bortezomib induces the degradation of FLT3-ITD tyrosine kinase in acute myeloid leukemia through an autophagy-dependent mechanism
Authors: Larrue Clément , Saland Estelle , David Marion , Sarry Jean-Emmanuel , Manenti Stéphane , Récher Christian 
Published: May 2015

Abstract
Internal tandem duplication mutations of the Fms-like tyrosine kinase 3 (FLT3-ITD) are found in 30% of Acute Myeloid Leukemias and are associated with a very poor outcome. Although the activity of FLT3 inhibitors such as AC-220 is currently evaluated in phase II and III trials with promising results, some studies suggest that leukemic cells can develop various mechanisms of resistance including mutations of the tyrosine kinase domain (TKD).
In the present work, we show that AML cell lines harboring FLT3-ITD mutation, MV4-11 and MOLM-14, are exquisitely sensitive to the proteasome inhibitor bortezomib. Importantly, although bortezomib induced apoptosis in all primary AML samples (n=24), FLT3-ITD mutated cells (n=13) are much more sensitive than non-mutated samples (n=11). Moreover we observed a positive correlation between FLT3-ITD mutation load (measured by the FLT3-ITD/FLT3wt ratio) and bortezomib activity among FLT3-ITD samples (p=0.0084, r=0.719). At the molecular level, bortezomib induced autophagy, as shown by an increase of LC3-II protein expression and by electron microscopy analysis. This autophagy was mainly cytotoxic as cell death was inhibited by the autophagy pharmacological inhibitor, 3-methyladenine. Before cell death occurs, we observed that bortezomib induced a downregulation of FLT3-ITD both in cell lines and primary samples. Accordingly, cell signalling dependent on FLT3-ITD (ERK/Akt/STAT5) was inhibited upon bortezomib treatment. Others proteasome inhibitors such as MG-132 or carfilzomib were also able to downregulate FLT3-ITD protein level. Interestingly, 3-methyladenine restored FLT3-ITD expression and signaling after bortezomib exposure, suggesting a role for autophagy in the downregulation of the mutated receptor. Using confocal microscopy, we showed a co-localization between FLT3-ITD and LC3 structures after bortezomib exposure. Electron microscopy analyses confirmed the presence of the receptor in autophagy vesicles. Furthermore, the membrane trafficking inhibitor dynasore inhibited autophagy, cell death and degradation of FLT3-ITD induced by bortezomib. We also showed that bortezomib overcomes the resistance to AC-220 in a MOLM-14 ITD/TKD model, by triggering the degradation of the receptor. Finally, in vivo studies using a mouse xenograft model, confirmed that bortezomib was able to decrease FLT3-ITD protein level and inhibit leukemic cell growth.
Altogether, our results show that proteasome inhibitors induce a downregulation of the oncogenic tyrosine kinase FLT3-ITD in an autophagic-dependent manner, suggesting that they could be used as an alternative strategy in AML with FLT3-ITD mutations.
Citation Format: Clément Larrue, Estelle Saland, Marion David, Jean-Emmanuel Sarry, Stéphane Manenti, Christian Récher. Bortezomib induces the degradation of FLT3-ITD tyrosine kinase in acute myeloid leukemia through an autophagy-dependent mechanism. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1336. doi:10.1158/1538-7445.AM2014-1336