Role of Enhanced Autophagy in Resistance of FLT3-ITD AML Stem Cells to FLT3 TKI Treatment
Authors: Qiu Shaowei , Paterson Andrew J , Abraham Ajay , He Jianbo , Shah Mansi , Agarwal Puneet , Mullen Amanda K , Li Hui , Harris Mason W , Anderson Nicholas R , Bhatia Ravi 
Published: October 2019

Abstract
The FLT3-ITD mutation occurs in 25-30% of AML patients, and is associated with increased relapse rate and poor survival. FLT3 tyrosine kinase inhibitors (TKI) have shown encouraging results, but responses are not sustained in most patients. Therefore there is considerable interest in understanding mechanism contributing to resistance and preservation of FLT3-ITD AML stem cells after TKI treatment. Autophagy is an important cellular response that maintains cell survival under stress. Here we investigated the regulation of autophagy in FLT3-ITD+ AML cells, and its role in resistance to FLT3 TKI treatment.
Expression of a FLT3-ITD construct in FLT3-ITD negative OCI-AML3 cells using lentiviral vectors resulted in increased autophagic flux compared with wild-type FLT3 or non-transduced controls. However, treatment of FLT3-ITD+ MV4-11 and Molm13 AML cells, or primary FLT3-ITD+ CD34+ cells, with the potent FLT3 TKI AC220 (Quizartinib) did not significantly alter autophagic flux. These results suggest that FLT3-ITD expression increases autophagy but in a kinase-independent manner.
MV4-11 and Molm13 cells were transduced with the GFP-LC3-RFP-LC3 ΔG construct (Kaizuka T, et al,2016) to measure autophagic flux. Autophagyhigh AML cells showed lower growth rate compared with Autophagylow cells (0.65, p=0.025), and reduced sensitivity to inhibition by AC220 (0.49 vs 0.71, p=0.003). Treatment of MV4-11 and Molm13 cells with the potent autophagy inhibitor Lys05 in combination with AC220 resulted in significantly enhanced inhibition of AML cell growth (MV4-11: 0.83 vs 0.54, p<0.001; Molm13: 0.58 vs 0.43, p=0.02) and increased induction of apoptosis (MV4-11: 0.81 vs 0.54, p<0.001; Molm13: 0.73 vs 0.49, p=0.024) compared to AC220 alone. We further show that inhibition of autophagy by shRNA-mediated knockdown of the critical autophagy gene ATG5 enhanced sensitivity of AML cells to AC220 treatment (MV4-11: 0.91 vs 0.65, p=0.005; Molm13: 0.88 vs 0.64, p=0.004). Treatment with Lys05 also inhibited autophagic flux in primary FLT3-ITD+ AML CD34+ stem/progenitor cells and resulted in significantly increased inhibition of cell cycle arrest (G0 stage: 40.3% vs 61.8%), increased myeloid differentiation (CD11b: 1 vs 1.24, p=0.009; CD14: 1 vs 1.21, p=0.0024), and increased cell death when combined with AC220 (0.58% vs 0.78%, p=0.0245). These results indicate that increased autophagy in FLT3-ITD+ AML cells is associated with TKI resistance, and that autophagy inhibition enhances sensitivity of FLT3-ITD+ AML cells to TKI treatment.
Induction of autophagy by starvation or mTOR inhibitor treatment in MOLM13 and MV4-11 cells was associated with reduced p53 expression, whereas autophagy inhibition by chloroquine or SAR405, a PIK3C3/Vps34 inhibitor, was associated with increased p53 expression. Starvation-mediated autophagy in FLT3-ITD+ AML cells was significantly increased upon p53 knockdown, suggesting that p53 has an inhibitory effect on autophagy. Enhanced inhibition of FLT3-ITD+ AML cell growth by autophagy inhibition in combination with AC220 treatment was prevented by shRNA-mediated knockdown of p53, suggesting that effects of autophagy inhibition on FLT3-ITD+ AML cell growth were p53 dependent.
We conclude that FLT3-ITD expression enhances autophagic flux in FLT3-ITD+ AML cells in a kinase-independent manner, and that increased autophagy contributes to TKI resistance of FLT3-ITD+ AML cells. We show that autophagy inhibition can enhance sensitivity of FLT3-ITD+ AML stem cells to TKI treatment. Finally, we demonstrate important roles for autophagy in regulating p53 levels, and conversely for p53 in regulating autophagy and the effects of autophagy inhibition in FLT3-ITD+ AML cells. These studies support further development of strategies for targeting of autophagy and related pathways to enhance efficacy of TKI treatment in eliminating AML stem cells.

Disclosures
No relevant conflicts of interest to declare.