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HACE1, A NOVEL TUMOR SUPPRESSOR GENE WHICH REGULATE THE RESPONSE OF VENETOCLAX, GANT61 AND NECROPTOSIS ACTIVATORS BY CONTROLLING C-FLIP, GLI2 AND BCL2 GENES IN ACUTE MYELOID LEUKEMIA

PS1001

Garitano, A.1; Teufel, E.1; Kreckel, J.1; Nadine, R.1; Stühmer, T.2; Da Via, M.1; Barrio, S.1; Nerreter, S.1; Haertle, L.1; Vogt, C.1; Düll, J.1; Rasche, L.1; Wajant, H.1; Rosenwald, A.3; Einsele, H.1; Kortüm, M.1

doi: 10.1097/01.HS9.0000562300.73603.50
Poster Session II: Acute myeloid leukemia - Biology - translational research
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1Medizin II

2Comprehensive Cancer Center Mainfranken

3Pathology, University Hospital Würzburg, Würzburg, Germany

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Background:

Acute Myeloid Leukemia (AML) is a genetically heterogenous disease characterized by clonal expansion of immature myeloid progenitors cells in the bone marrow (BM). The identification of mutations in FLT3 and NPM1&FLT3, as well as rearrangements including MLL (MLL-r) were identified as biomarkers of poor outcome causing disease relapse, often characterized by an apoptosis resistant phenotype. Necroptosis (non-programmed cell death) is a mechanism to overcome apoptosis resistance and the E3 Ubiquitine ligase HACE1 was recently identified to regulates necroptosis in Mouse Embrionic Fibroblast cells.

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Aims:

Thus, we aimed to determine the role of HACE1 in AML pathogenesis and to assess its impact on the response to anti-apoptosis inhibitors.

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Methods:

We analyzed the HACE1 mRNA and protein expression in primary pts samples and cell lines and compared with CD34+ peripheral blood mononuclear cells (PBMCs) from healthy individuals.

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Results:

We found the HACE1 protein expression to be decreased in MLL-r, NPM1 and FLT-3 mutated AML cell lines (NOMO-1/THP1/MV4-11/MOLM-13) versus non-MLL-r/non-NPM1/non-FLT3 mutated cell lines (U937/HL60/KG1/HEL/SET-2) and healthy CD34+ PBMCs. This was confirmed by IHC on 34 BM samples obtained from newly diagnosed AML cases. Of these, 8 harbored a MML-r, 3 a NPM1&FLT3 mutation and 7 a mutation in FLT3. HACE1 expression was generally low in our cohort with an average IHC score (S) of 0.67 (0 = absent, 3 = highest), in FLT3 mutated patients (S:0.71), NPM1&FLT3 mutated patients (S:0.66) or patients with an MLL-r (S:0.5). In concordance, mRNA expression from the blood spot database confirmed low HACE1 expression in AML patients with MLL-r compared to healthy CD34+ BM hematopoietic stem cells (***p < 0,001). Next, we re-expressed HACE1 (MCSV retrovirus) in NOMO-1 and MV4-11 and observed signficant activation of apoptosis and reduced cell viability (72,54%). Conversely, HACE1 knock down (KD) (by Sleeping Beauty) potentiated anti-apoptosis related proteins such as c-FLIP (long/small iso-forms), GLI2 and BCL2. Of note, HACE1 KD increased the response to Venetoclax, a BCL2 inhibitor in clincial use (WT IC50 75,04 nM vs HACE1 KD IC50 36,94 nM) as well as to GANT61, a specific GLI1/2 inhibitor (WT IC50 16,65 μM vs HACE1 KD IC50 9,3 μM). In addition, synergistic effect on cell death were observed by the combination of Venetoclax and GANT61 in NOMO-1, MV4-11 and MOLM-13 cells. Our observations suggest that HACE1 KD induces an apoptosis resistant phenotype in AML, thus, we next investigated the role of necroptosis, which provides an non-programmed alternative cell death mechanism in tumor cells: Strikingly, sensitivity to treatment with the necroptosis activators TNF (34,64% more cell death in HACE1 KD vs WT), emricasan (54,16% more cell death in HACE1 KD vs WT) was signficantly increased in HACE1 KD NOMO1 cells compared to WT. In addition, combinations between the caspase 8 inhibitor emricasan and GANT61 and TNF demonstrated synergistic effects in both NOMO-1 and MOLM-13 cells. These results suggest that necroptosis activation may provide a worth investigating treatment strategy in patients with low levels of HACE1.

Finally, we investigated the impact of HACE1 KD in NOMO-1 cells on the treatment response to gold-standard cyatarabine and found increased sensitivity (WT IC50 412,5 nM vs HACE1 KD IC50 157,2 nM), which is in line with published data about a particularly well responsiveness of MLL-r patients in the clinic.

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Summary/Conclusion:

Taken together, we provide first data for a pivotal role of HACE1 as a novel tumor suppressor in AML. Further research is highly warranted.

Copyright © 2019 The Authors. Published by Wolters Kluwer Health Inc., on behalf of the European Hematology Association.