Abstract 1666: Targeting autophagy-related E1 ligase Atg7 increases chemosensitivity in acute myelogenous leukemia cells and overcomes stroma mediated resistance.
Authors: Borthakur Gautam , Duvvuri Seshagiri , Lan Yongsheng , Ruvolo Vivian , Ruvolo Peter , Qiu Yihua , Konopleva Marina , Kornblau Steven , Andreeff Michael 
Published: August 2014

In the context of chemotherapy (CT), autophagy is a resistance mechanism. Atg7 is an key autophagy-related E1 ligase that can switch transcription downstream of p53 between cell cycle arrest and apoptosis induction (Lee, IH et al. Science 2012;336: 225). p53 mutations are infrequent in newly diagnosed normal karyotype acute myelogenous leukemias (NK-AML) and thus we used a genetic model to evaluate the potential of targeting Atg7 in AML therapy in the context of wild type p53. As autophagy is critical for the mutual metabolic dependence between tumor and stroma (Martinez-Outschroom et al. Cell Cycle 2010;9:4297), we evaluated, in a co-culture model, whether ATG7 knockdown in AML can overcome ‘stroma-mediated’ chemoresistance. We also correlated expression of Atg7 protein in primary AML blasts to clinical outcome in 511 newly diagnosed patients (pts).

Methods: In a syngeneic background we studied the effect of stable Atg7 knockdown (shRNA mediated) in p53 wild type (WT) AML cells on apoptosis (Annexin binding by flowcytometry) after CT and nutrient deprivation. Role of ATG7 in stroma mediated chemoresistance was tested by co-culturing AML cells (WT and Atg7 KD) with normal bone marrow stromal cells and measuring apoptosis after treatment with CT. Finally we correlated expression of Atg7 protein in the AML blasts of 511 newly diagnosed pts with AML by reverse phase protein array (RPPA) with clinical outcomes.

Results: Stable knockdown (KD) of Atg7 in OCI-AML3 cells increased apoptosis upon exposure to two CT agents most commonly used in AML therapy: cytarabine (1 μmol for 48 hrs) (33.6% apoptosis in WT vs 66.2% in Atg7 KD, p=.04) and idarubicin (50 nG/ml for 48 hrs) (33% apoptosis in WT vs 54% in Atg7 KD, p=.001) as well as serum deprivation (8.5% apoptosis in WT vs 28% in Atg7 KD, p=.00005). Treatment with cytarabine was associated with increased induction of pro-apoptotic protein PUMA and decreased induction of cell cycle arrest mediator p21 in Atg7 KD OCI-AML3 cells, as compared to WT. In co-culture model, co-culture with normal marrow stromal cells failed to protect Atg7 KD cells from apoptosis after treatment with cytarabine (5 μmol for 48 hrs) while the WT cells were protected (27% apoptosis in WT vs 57% in Atg7 KD, p=0.0007).

Finally, reverse-phase protein array demonstrated that abnormal expression of Atg7 in primary AML blasts (higher or lower compared to normal CD34+ cells) among 511 newly diagnosed pts was associated with shorter overall survival (p=.007) and this effect was most prominent among pts with both good-risk (p=.02) and poor-risk cytogenetics AML (p=.0005).

Conclusion: Our results highlight the role of autophagy as chemo-resistance mechanism in AML, identify Atg7 as a potential therapeutic target to enhance CT induced apoptosis and overcome stroma-mediated resistance to CT.

Citation Format: Gautam Borthakur, Seshagiri Duvvuri, Yongsheng Lan, Vivian Ruvolo, Peter Ruvolo, Yihua Qiu, Marina Konopleva, Steven Kornblau, Michael Andreeff. Targeting autophagy-related E1 ligase Atg7 increases chemosensitivity in acute myelogenous leukemia cells and overcomes stroma mediated resistance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1666. doi:10.1158/1538-7445.AM2013-1666