Targeting Autophagy As a Potential Therapeutic Strategy for the Treatment of AML

Abstract

Autophagy is a catabolic process whereby redundant organelles and proteins are digested and recycled to maintain cellular homeostasis. Malignant cells might activate autophagy in response to cellular stress, and/or to provide metabolites required for rapid cell proliferation. In addition, elevated autophagy is associated with increased chemoresistance. In normal hematopoiesis, autophagy is important for stem and progenitor cell (HSPC) maintenance. We previously observed that basal autophagy is higher in HSPCs compared more differentiated cells. Genetic interference with the autophagy machinery resulted in a strong reduction in HSPC frequencies, without interfering with the differentiation program.To study the role of autophagy in acute myeloid leukemia (AML), steady-state autophagy was measured in a panel of different myeloid leukemic cell lines (n=8). The autophagic flux was determined by the relative accumulation of cyto-ID (a marker for autophagic vesicles) measured by flow cytometry after hydroxychloroquine (HCQ) or Bafilomycin-1A treatment and LC3-II/p62 accumulation by Western blotting. Interestingly, in leukemic cell lines, a higher basal autophagy flux correlated with increased sensitivity to prolonged inhibition (6 days) of autophagy with different concentrations of HCQ (5 uM R2 =0.52 and 20 uM R2 =0.76), while a mild reduction in expansion was observed in cord blood (CB) derived normal CD34+ cells, after treatment with HCQ (5 uM). In line with the leukemic cell lines, primary sorted AML CD34+ cells (n=36) also showed decreased survival upon HCQ (20 uM) treatment compared to normal bone marrow CD34+ cells (N=6; NBM CD34+: 41.7% ± 7.1 vs. AML CD34+: 21.3% ± 3.2, p=<0.05).Gene expression analysis of autophagy genes by qPCR revealed increased expression of essential autophagy genes ATG5 and ATG7 in primary AML CD34+ compared to NBM CD34+ cells (AML vs. NBM: ATG5 10.3 fold increase, p=<0.05; ATG7 5.9 fold increase, p=<0.01). Therefore, to study the functional consequences in AML, ATG5 and ATG7 were downregulated in leukemic cell lines and primary AML CD34+ cells (n=6), using lentiviral shRNAs. Significant downregulation of ATG5 and ATG7 was confirmed by qPCR, which was also sufficient to inhibit the autophagy flux as demonstrated by reduced accumulation of LC3-GFP puncta. In line with results of HCQ treatment, downmodulation of ATG5 and ATG7 resulted in a strong reduction in expansion of leukemic cell lines. Also, transduced primary AML blasts cultured on MS5 bone marrow stromal cells for 4 weeks showed a distinct reduction in expansion (shSCR vs. shATG5 2.1 fold reduction, p=<0.05; shATG7 2.5 fold reduction p=<0.01).The genetic or pharmaceutical inhibition of autophagy triggered an apoptotic response in leukemic cellsas determined by Annexin V measurements. This coincided with increased expression of p53 and the downstream pro-apoptotic target genes BAX and PUMA and the putative AKT inhibitor PHLDA3. Moreover, HCQ induced apoptosis could be rescued by downregulation of p53 by RNAi in leukemic cell lines that express wild-type P53. Prolonged exposure to HCQ did also result in an apoptotic response in leukemic cell lines harboring p53 mutations, but without the upregulation of BAX, PUMA and PHLDA3.In conclusion, our results indicate that AML CD34+ cells are highly dependent on the autophagy machinery for growth and cell survival. Targeting autophagy might provide a new therapeutic option for treatment of this type of leukemia. DisclosuresNo relevant conflicts of interest to declare.