Ablation of Autophagy-Related E1 Ligase Atg7 Primes Acute Myelogenous Leukemia Cells to Apoptosis, Sensitizes to Chemotherapy and Overcomes Stroma Mediated Protection

Background: Except for the ‘good risk’ groups identified by current cytogenetic and molecular criteria, patients with acute myelogenous leukemia (AML) largely relapse after attaining initial remissions, and outcomes of patients with early relapse is uniformly poor, highlighting the need for strategies to overcome resistance to agents used in frontline therapy. Autophagy is an ancestral adaptive mechanism to stress and can be triggered by exposure to chemotherapy. Autophagy is also believed to play an important role in tumor protection by the microenvironment. Autophagy inhibition increases chemosensitivity in several experimental models of solid tumors. The role of autophagy is not well studied in AML, but the hypoxic bone marrow niche is expected to induce autophagy in AML cells and render them chemo-resistant. Our reverse phase protein array (RPPA) analysis of over 500 newly diagnosed AML patient samples indicated that abnormal expression of several autophagy proteins in leukemic blasts (LKB1, BECLIN 1 and ATG7) is associated with poor prognosis in AML (Borthakur, G et al. ASH 2011# 2513), suggesting a therapeutic role for autophagy suppression. Atg7 is a key molecule in autophagy vesicle elongation with a role in two essential ubiquitin-like reactions (LC3 lipidation and Atg 5/12 conjugation), and its knockdown is expected to inhibit autophagy globally.

Aim: To determine the effects of autophagy inhibition on chemosensitivity of AML cells and on stroma induced resistance.

Results: We semi-quantitatively assessed ATG7 expression in AML cell lines (THP1, OCI-AML2 and 3, HL-60, MOLT-4, MOLM 13 and 16) by Western blot. HL-60 and MOLM13 cells had the lowest expression of Atg7 and were most sensitive to treatment with ara-C and idarubicin while OCI-AML3 and THP-1 had highest levels of Atg7 and were most resistant to treatment (p= .001-.004). In addition, high expression of Atg7 is associated with high levels of anti-apoptotic Mcl-1 in these lines.

As available autophagy inhibitors are non-specific, we used lentiviral shRNA to knock down Atg7 and study the role of autophagy. Atg7 knockdown cells (ATG7-KD) were treated with ara-C and idarubicin for 48-96 hours. At all time points, apoptosis was significantly higher in ATG7-KD cells compared to cells transduced with a non-silencing scrambled control (ATG7-Scr) at 96 hrs: 39.6±2.4 vs 22.4±1.3, p=.002 for ara-C 2 µM and 47.9±2.6 vs 34.7±2.3, p=.0004 for idarubicin. We co-cultured OCI-AML3 (ATG7-KD and -Scr) cells with normal bone marrow derived mesenchymal stromal cells (MSCs) to mimic the bone marrow micro-environment. Sensitivity to ara-C and idarubicin was reduced by co-culture for both cell types, but ATG7-KD cells remained more sensitive than were ATG7-Scr cells.

Western blot analysis confirmed increased p62 and decreased LC3 lipidation in ATG7-KD cells (LC3 II/I ratio= 0.15 vs 0.7 in ATG7-Scr), indicating a block in autophagy, and increased caspase 3 cleavage, indicating apoptosis. The higher expression of pro-apoptotic NOXA and BAX, and lower expression of anti-apoptotic MCL-1 and BCL-2, in ATG7-KD cells compare to ATG7-Scr indicating mitochondrial pathway apoptosis. Gene expression profiling on Illumina HT12 arrays was used to study OCI-AML3 ATG7-KD and -Scr cells at baseline and after 24 and 48 hr of araC 0.5 µM. Gene set enrichment analysis (GSEA) showed significantly lower baseline expression of interferon response genes and JAK-STAT pathway genes in OCI-AML3 ATG7-KD cells. Changes with araC treatment were largely similar, but the response to araC was different between ATG7-KD and –Scr cells for a small number of genes. In-vivo chemo-sensitivity experiments are in progress.

Conclusion: Autophagy inhibition by genetic silencing of ATG7 increases chemosensitivity of AML cells, even in the presence of bone marrow stromal cells. ATG7-KD cells appear to be more primed for apoptosis compared to their controls. Concomitant inhibition of ATG7, a potentially drugable E1 ligase, appears to be a valid strategy to enhance sensitivity to front-line treatment agents in AML.