mTOR Pathway Links Suppressed Autophagy to HDAC Inhibitor-Induced Apoptosis in Myeloid Leukemia,

Abstract 3614

Despite well-described anti-cancer effects and promising results in clinical trials, little is known regarding the selectivity of histone deacetylase (HDAC) inhibitors in killing malignant and sparing normal cells. Using Down syndrome associated myeloid leukemia (DS-AMKL) as a model for an exceptionally sensitive form of acute myeloid leukemia (AML), we elucidated a role of HDAC inhibitors in blocking autophagy, which exploits a specific vulnerability of several types of cancer.

We could show that HDAC1/2 inhibition efficiently inversed the DS-AMKL gene signature, resulting in p53-independent cell cycle arrest (BrdU incorporation and CFSE assay) and apoptosis (Annexin V/ 7-AAD and Nicoletti staining, caspase 3/7 activity assay) of DS-AMKL cell lines (CMK and CMY) and primary DS-AMKL leukemic blast. In contrast, control cell lines K562 and M07 were resistant to VPA-induced apoptosis. DS-AMKL is characterized by the high activation of the insulin-like growth factor (IGF) signaling cascade, which in turn results in the constitutive activation of mammalian target of rapamycin (mTOR). mTOR has been characterized as a major suppressor of autophagy, a lysosomal degradation process that takes place constitutively at a basal level and protects cells against damaged or unnecessary organelles and protein aggregates. Measuring endogenous LC3-I to LC3-II conversion levels by Western blot, a posttranslational modification during autophagy, we confirmed a lower basal autophagic activity in DS-AMKL cells compared to the control cell lines, K562 and M07 (LC3B-II/actin [RU]: 1.5 CMK, 1.0 CMY, 6.0 K562 and 3.0 M07).

Studying the effect of HDAC inhibition on autophagy, we detected an initial, dose-dependent increase in LC3-II band formation, punctae formation and decrease in total LC3-GFP cellular abundance (i.e., autophagy activation) 12h after VPA treatment in all cell lines (CMK, CMY, K562 and M07), indicative for induction of autophagy. However, prolonged exposure to VPA for up to 24h led to a block of autophagic flux, as shown by a gradual accumulation of LC3-GFP (fold increase in LC3-GFP MFI compared to untreated control: 1.3 CMK**, 1.1 CMY*, 2.0 K562** and 2.0 M07** at 2mM VPA; **p<0.01; *p<0.05).

Autophagy participates in the removal of damaged mitochondria, which prevents the initiation of intrinsic apoptotic pathway or DNA damage by increased ROS generation. Our results demonstrated a dose-dependent relationship between VPA-associated accumulation of mitochondrial mass and the intrinsic autophagic activity of the respective leukemic cell lines (fold increase in MitoTracker green MFI: 4.7 CMK**, 4.8 CMY**, 1.3 K562 and 1.2 M07 at 10mM VPA; **p<0.01). Likewise, the VPA-mediated mitochondrial mass accumulation correlated with ROS formation (fold increase in CM-H₂DCFDA MFI: 3.3 CMK**, 2.7 CMY**, 1.5 K562** and 1.2 M07** at 10mM VPA; **p<0.01) and reflects the sensitivity to VPA induced apoptosis. In addition, we detected DNA-double strand breaks as indicated by an increase of phosphorylated H2AX.

The effects of HDAC inhibitors on DS-AMKL cells could be recapitulated by pharmacologic (autophagosome-lysosome fusion using vinblastine and nocodazole; autophagolysosomal degradation using ammonium chloride, chloroquine and hydroxychloroquine) and genetic (shRNA-mediated knockdown of ATG5 or ATG7) inhibition of autophagy, underlining dependency on their low basal level of autophagy. Inversely, induction of autophagy by starvation reversed the effects of HDAC inhibition on cell lines and primary DS-AMKL cells (i.e. induction of apoptosis, accumulation of mitochondria and ROS-production).

Thus, our study identified the role of HDAC inhibitors in blocking autophagy, exploiting the specific vulnerability of DS-AMKL cells with suppressed basic autophagy due to high mTOR activation. This was unexpected as previous studies proposed HDAC inhibitors as autophagy activators. VPA treatment of DS-AMKL cell lines and primary cells repressed autophagy below a critical threshold leading to accumulation of mitochondria, production of ROS, DNA-damage and apoptosis, which could be reverted upon autophagy activation. Our findings suggest repression of autophagy by HDAC inhibition as a novel treatment strategy in leukemic cells with highly active mTOR pathway.

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