Role of Autophagy in Resistance of FLT3-ITD AML Stem Cells to FLT3 TKI Treatment

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. Therefore, there is considerable interest in understanding mechanisms of resistance 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 resistance of FLT3-ITD+ AML stem cells to FLT3 TKI treatment.

MV4-11 and Molm13 FLT3-ITD AML cells were transduced with GFP-LC3-RFP-LC3 ΔG constructs to measure autophagic flux. Autophagy^high AML cells showed slower growth(p<0.0001), enhanced sensitivity to the autophagy inhibitor Lys05 (p=0.01) and reduced sensitivity to the FLT3-ITD inhibitor AC220 (p=0.03) compared with autophagy^low cells. Treatment with Lys05 in combination with AC220 resulted in significantly enhanced inhibition of AML cell growth (p=0.02) and increased induction of apoptosis (p=0.024) compared to AC220 alone. We confirmed that inhibition of autophagy by shRNA-mediated knockdown of the critical autophagy gene ATG5 also enhanced sensitivity of AML cells to AC220 treatment (p=0.004). Lys05 treatment also inhibited autophagic flux in primary human FLT3-ITD+ AML CD34+ stem/progenitor cells, and resulted in significantly increased apoptosis (78% vs 58%, p=0.0245) and reduced cell growth (47% vs 68%, p=0.003) when combined with AC220 compared to AC220 alone. In contrast Lys05 treatment had minimal effect on normal CD34+ stem cells, indicating that autophagy inhibition selectively enhances sensitivity of FLT3-ITD+ AML CD34+ cells to TKI treatment while sparing normal CD34+ cells.

We tested the effect of Lys05 in vivo by treating NSG mice engrafted with primary FLT3-ITD AML cells with vehicle, AC220 (10mg/kg), Lys05 (20mg/kg) or combination for 3 weeks. Lys05 treatment reduced human CD45+ cell engraftment (8.7% vs 17%, p=0.03), and reduced CD45+CD33+ myeloid cells in BM and spleen (BM: 42% vs 81%, p<0.001, Spleen: 8% vs 14%, p=0.01), compared with vehicle. The combination of AC220 with Lys05 reduced CD45 engraftment (11% vs 37%, p=0.004) and absolute CD45+CD34+ stem cell numbers in BM compared with AC220 alone. The effect of Lys05 in vivo was also investigated in a transgenic FLT3-ITD AML mouse model (Mx1-cre-Tet2^f/f, Flt3-ITD^Ki). The ability of in vivo Lys05 treatment to inhibit autophagy was confirmed by immunofluorescence. Lys05 inhibited autophagy flux to a greater extent in c-Kit+ stem/progenitor cells compared to differentiated cells. Leukemic mice were treated with PBS, Lys05(20mg/kg), AC220(10mg/kg) or the combination for 3 weeks. The combination of AC220 and Lys05 resulted in significantly greater reduction of WBC and neutrophil counts compared to the other groups(p=0.03). The AC220 and Lys05 combination reduced spleen weight and cellularity significantly more than AC220 alone (p=0.02), and splenic ST-HSC, MPP, GMP, MEP and BM GMP, MEP significantly more than other groups. However, BM ST-HSC and MPP (LT-HSC are absent in this AML model) were similar among the different treatment groups. The proportion of mature myeloid and T cells was increased in the combination group. Ki67 and Dapi labeling indicated that Lys05 reduced the frequency of ST-HSC and MPP in G0 and increased those in G1. The ability of Lys05 to promote quiescent LSC entry into cell cycle may contribute to enhanced sensitivity to AC220 when the two are used in combination. We show that induction of autophagy in MOLM13 cells by starvation and mTOR inhibitor was associated with reduced p53 expression, whereas autophagy inhibition by chloroquine or SAR405, a PIK3C3/Vps34 inhibitor, was associated with increased p53 expression by WB. Inhibition of growth or increased apoptosis in FLT3-ITD+ AML cells following autophagy inhibition were 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 autophagy inhibition enhances sensitivity of FLT3-ITD+ AML stem cells to TKI treatment both in vitro and in vivo. We demonstrate important roles for autophagy in regulating p53 levels and cell cycle in FL3-ITD AML cells. These studies support further development of strategies to target autophagy and related pathways to enhance efficacy of TKI in eliminating AML stem cells.