Toll like receptor (TLR) signaling has been suggested to play an important role in the inflammatory microenvironment of solid tumors and through this inflammation-mediated tumor growth. Here, we studied the role of tumor cells in their process of self-maintaining TLR expression independent of inflammatory cells and cytokine milieu for autoregulative tumor growth signaling in pancreatic cancer. We analyzed the expression of TLR2, -4, and -9 in primary human cancers and their impact on tumor growth via induced activation in several established pancreatic cancers. TLR-stimulated pancreatic cancer cells were specifically investigated for activated signaling pathways of VEGF/PDGF and anti-apoptotic Bcl-xL expression as well as tumor cell growth. The primary pancreatic cancers and cell lines expressed TLR2, -4, and -9. TLR-specific stimulation resulted in activated MAP-kinase signaling, most likely via autoregulative stimulation of demonstrated TLR-induced VEGF and PDGF expression. Moreover, TLR activation prompted the expression of Bcl-xL and has been demonstrated for the first time to induce tumor cell proliferation in pancreatic cancer. These findings strongly suggest that pancreatic cancer cells use specific Toll like receptor signaling to promote tumor cell proliferation and emphasize the particular role of TLR2, -4, and -9 in this autoregulative process of tumor cell activation and proliferation in pancreatic cancer.

In patients with peritoneal carcinomatosis cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy (HIPEC) represents a promising treatment strategy. Here, we studied the role of hyperthermic chemotherapy on heat shock protein (HSP) expression and induction of tumor cell death and survival. HSP27, HSP70, and HSP90 combined with effects on tumor cell proliferation and chemosensitivity were analyzed in human colon cancer. Hyperthermic chemotherapy resulted in significant HSP27/HSP70 and HSP90 gene/protein overexpression in analyzed HT-29/SW480/SW620 colon cancer cells and peritoneal metastases from patients displaying amplified expression of proliferation markers, proliferating cell nuclear antigen and antiapoptotic protein Bcl-xL. Moreover, functionally increased chemoresistance against 5-fluorouracil/mitomycin C and oxaliplatin after hyperthermic chemotherapy points to induced survival mechanisms in cancer cells. In conclusion, the results indicate that intracellular HSP-associated antiapoptotic and proliferative effects after hyperthermic chemotherapy negatively influence beneficial effects of hyperthermic chemotherapy-induced cell death. Therefore, blocking HSPs could be a promising strategy to further improve the rate of tumor cell death and outcome of patients undergoing HIPEC therapy.

TNF-like weak inducer of apoptosis (TWEAK) and inhibition of protein synthesis with cycloheximide (CHX) sensitize for poly(I:C)-induced cell death. Notably, although CHX preferentially enhanced poly(I:C)-induced apoptosis, TWEAK enhanced primarily poly(I:C)-induced necroptosis. Both sensitizers of poly(I:C)-induced cell death, however, showed no major effect on proinflammatory poly(I:C) signaling. Analysis of a panel of HeLa-RIPK3 variants lacking TRADD, RIPK1, FADD, or caspase-8 expression revealed furthermore similarities and differences in the way how poly(I:C)/TWEAK, TNF, and TRAIL utilize these molecules for signaling. RIPK1 turned out to be essential for poly(I:C)/TWEAK-induced caspase-8-mediated apoptosis but was dispensable for this response in TNF and TRAIL signaling. TRADD-RIPK1-double deficiency differentially affected poly(I:C)-triggered gene induction but abrogated gene induction by TNF completely. FADD deficiency abrogated TRAIL- but not TNF- and poly(I:C)-induced necroptosis, whereas TRADD elicited protective activity against all three death inducers. A general protective activity against poly(I:C)-, TRAIL-, and TNF-induced cell death was also observed in FLIPL and FLIPS transfectrants.

Tumor necrosis factor (TNF) receptor associated factor-2 (TRAF2) knockout (KO) cells were generated to investigate the role of TRAF2 in signaling by TNFR1 and the CD95-type death receptors (DRs) TRAILR1/2 and CD95. To prevent negative selection effects arising from the increased cell death sensitivity of TRAF2-deficient cells, cell lines were used for the generation of the TRAF2 KO variants that were protected from DR-induced apoptosis downstream of caspase-8 activation. As already described in the literature, TRAF2 KO cells displayed enhanced constitutive alternative NFκB signaling and reduced TNFR1-induced activation of the classical NFκB pathway. There was furthermore a significant but only partial reduction in CD95-type DR-induced upregulation of the proinflammatory NFκB-regulated cytokine interleukin-8 (IL8), which could be reversed by reexpression of TRAF2. In contrast, expression of the TRAF2-related TRAF1 protein failed to functionally restore TRAF2 deficiency. TRAF2 deficiency resulted furthermore in enhanced procaspase-8 processing by DRs, but this surprisingly came along with a reduction in net caspase-8 activity. In sum, our data argue for (i) a non-obligate promoting function of TRAF2 in proinflammatory DR signaling and (ii) a yet unrecognized stabilizing effect of TRAF2 on caspase-8 activity.

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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.

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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.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.