The HACE1-NRF2 Axis a Novel Target in Acute Myeloid Leukemia
Authors: Garitano-Trojaola Andoni , Teufel Eva , Rodhes Nadine , Kreckel Jennifer , Stuehmer Thorsten , Da Via' Matteo Claudio , Barrio Garcia Santiago , Haferlach Torsten , Tibes Raoul , Wajant Harald , Rosenwald Andreas , Einsele Hermann , Kortüm K. Martin 
Published: October 2019

Abstract
Acute Myeloid Leukemia (AML) is a genetically heterogenous disease characterized by clonal expansion of immature myeloid progenitors cells in the bone marrow (BM). Despite this genetic heterogeneity, AML patients share Leukemia associated oncogenes such as NF-E2-related factor 2 (Nrf2) (Rushworth SA et al.). NRF2 is a transcription factor that activates genes with antioxidant response elements (ARE)-containing promoters and protects cancer cells from apoptosis. Inhibition of NRF2 or antioxidant defense increases the level of Radical Oxygen Species (ROS), leading to tumor supression (Chio IIC et al.). Recently, the E3 Ubiquitin-Protein Ligase HACE1, a tumor suppressor in solid tumors, was demonstrated to promote the expression of NRF2 in Huntigton disease (Rotblat B et al.). Thus, we hypothesized a role for HACE1 as an oncogenic factor acting through NRF2 activation in myeloid malignancies and provide first data supporting the HACE1-NRF2 axis to be a novel target in acute myeloid leukemias.
Material and methods
The mRNA expression data from AML patients (296 samples) vs normal Hematopoietic Stem Cells (HSC) (6 samples) were exported from the bloodSpot database. HACE1 mRNA and protein expression was measured by q-RT-PCR and western blot in 12 commercially available Myeloid Malignancies cell lines. The HACE1 inducible knock down (KD) was carried out by Sleeping Beauty Transposon system in U937 and NOMO-1 cell lines. The cell viability was analyzed by Cell Titer Glo Luminescent assay. Apoptosis was measured by Annexin V (AV)/Propidium Iodide (PI) assay.
Results and discussion
HACE1 mRNA is downregulated in AML patients compared to HSC (***p<0.001, Bloodspot database). However mRNA and HACE1 protein expression do not correlate in AML cell lines, suggesting post translational modifications. High HACE1 protein expression was observed in most AML cell lines. HACE1 KD reduced drastically the cell viability of U937 cells through caspase activation and NRF2 degradation. However, no effect on cell viability was observed in NOMO-1 cells. Recently, non-programmed cell death necroptosis induction has been described by TNFR1 activation in HACE1 knock out Mouse Embrionic Fibroblast cells (Tortola L et al.). In line with this study, we observed that TNF induces strong cell death in HACE1 KD NOMO-1 cells within 48 hours.
In addition HACE1 KD promotes autophagy through p62 degradation (late autophagy marker) in U937 cells. Autophagy has recently been described to contribute to the differentiation and death of AML cells, and to the promotion of immunostimulatory signals activating immune responses against cancer cells (Chen L et al.; Pietrocola F et al.). Thus HACE1 might be a potential target to induce autophagy, providing a novel therapeutical target in the treatment of myeloid malignancies.
Finally, HACE1 KD in our hands promoted sensitization of U937 and NOMO-1 cells to cytatarabine, the backbone therapy in AML patients. This treatment promotes HACE1 protein expression at 24 and 48 hours in NOMO-1 cells, which may explain the better response rates of HACE1 KD cells to cytarabine.
Taken together, we provide first evidence of HACE1 being a novel oncogene in AML and that the HACE1-NRF2 axis is a promising target in the treatment of Acute Myeloid Leukemias.

Disclosures
Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership.