Abstract 2864: Inhibition of the late stages of autophagy overcomes hypoxia-induced chemoresistance and targets leukemic stem cells in acute myeloid leukemia

Abstract

Abstract Background: Acute myeloid leukemia (AML) is an aggressive hematological malignancy which resides within an inherently hypoxic bone marrow (BM) microenvironment. Hypoxia has been linked to chemoresistance, particularly of leukemia stem cells (LSCs) responsible for relapsed disease. We hypothesized that upregulation of autophagy promotes survival and chemoresistance in AML cells (specifically LSCs) within the hypoxic BM. Targeted inhibition of specific stages of autophagy therefore may represent a novel means of eradicating disease. Methods: Human AML cell lines (HEL-Luc, HL60, MOLM13) were treated with cytarabine (AraC) and multiple autophagy inhibitors (Spautin-1, MRT68921, chloroquine (CQ), bafilomycin (BafA1)) under normoxia (21% O2) or hypoxia (1% O2) and evaluated for apoptosis. MOLM13 cells were infected with lentivirus containing shRNAs against LC3B, Atg7 or control. Autophagy was measured using CytoID dye and immunoblotting for LC3 and p62. SCID mice systematically engrafted with HEL-Luc cells were treated with CQ (25 mg/kg ip for 5 days/week for 3 weeks) and evaluated for leukemia burden by bioluminescent imaging and time to morbidity. Irradiated NSG mice were injected with primary AML cells and treated with vehicle or Baf A1 (1mg/kg ip twice a week for 4 weeks). BM was then harvested and transplanted into secondary NSG recipients without additional treatment. Leukemia engraftment in mice was measured by flow cytometry for hCD45. Results: Autophagy was upregulated in multiple human AML cell lines after prolonged exposure to hypoxia as well as in a subset of primary AML samples. Inhibiting the early stages of autophagy pharmacologically with Spautin-1 or MRT68921 or shRNA knockdown of autophagy proteins Atg7 or LC3B had no effect on chemosensitivity under hypoxia. However, inhibiting fusion of the autophagosome with the lysosome (late stages) with either CQ or Baf A1 did overcome hypoxia-induced resistance to AraC. CQ also reduced tumor burden in a systemic xenograft model of AML as measured by bioluminescent imaging and resulted in a slight overall increase in survival (17.5 vs. 19 days, p=0.02). In the in vivo LSC model, Baf A1 had no effect on leukemic burden in the primary recipients; however, mice that received secondary transplants from Baf A1-treated mice demonstrated significantly reduced leukemic burden as compared to the control, consistent with LSC specific targeting. Conclusions: Our results demonstrate that treatment with the late stage autophagy inhibitors (chloroquine, bafilomycin A1) can both overcome hypoxia-induced chemoresistance and preferentially inhibit LSC growth in vivo. Taken together, these data support the development of late stage autophagy inhibitors for the treatment of AML and prevention of relapsed disease. In vivo studies to assess the efficacy of AraC/Baf A1 combination therapy are currently underway. Citation Format: Kaitlyn M. Dykstra, Dirkje W. Hanekamp, Matthew Johnson, Monica L. Guzman, Eunice S. Wang. Inhibition of the late stages of autophagy overcomes hypoxia-induced chemoresistance and targets leukemic stem cells in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2864.