Ps1001 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

Abstract

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.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.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.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.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.