bay-11-7082 and Myelodysplastic-Syndromes

The death domain containing TNF receptor 6 (CD95/Fas) is a direct target for the NF-κB transcription factor and is repressed in solid tumors such as colon carcinomas. Previously, we reported that the Fas death receptor, while overexpressed in low-risk myelodysplastic syndromes (MDS), becomes undetectable on CD34(+) progenitors when the disease progresses to secondary acute myeloid leukemia (AML). This study determined the interplay between NF-κB and Fas during MDS progression. We first observed that Fas was induced by TNF-α in the HL60 cell line. In these cells, p65 (RELA) was associated with the FAS promoter, and inhibition of the NF-κB pathway by an IKKα inhibitor (BAY11-7082) or lentiviral expression of a nondegradable mutant of IκBα (IκSR) blocked Fas expression. In contrast, TNF-α failed to induce Fas expression in the colon carcinoma cell line SW480, due to hypermethylation of the FAS promoter. Azacitidine rescued p65 binding on FAS promoter in vitro, and subsequently Fas expression in SW480 cells. Furthermore, inhibition of the NF-κB pathway decreased the expression of Fas in MDS CD45(lo)CD34(+) bone marrow cells. However, despite the nuclear expression of p65, Fas was often low on CD45(lo)CD34(+) AML cells. TNF-α failed to stimulate its expression, while azacitidine efficiently rescued p65 binding and Fas reexpression. Overall, these data suggest that DNA methylation at NF-κB sites is responsible for FAS gene silencing.

Topics: Azacitidine; Base Sequence; Bone Marrow Cells; Cell Line, Tumor; Disease Progression; DNA Methylation; Epigenesis, Genetic; fas Receptor; Humans; Leukocytes, Mononuclear; Molecular Sequence Data; Myelodysplastic Syndromes; NF-kappa B; Nitriles; Promoter Regions, Genetic; Protein Binding; Sulfones; Transcription, Genetic

CD34(+) bone marrow blasts from high-risk myelodysplastic syndrome (MDS) patients as well as MDS patient-derived cell lines (P39 and MOLM13) constitutively activate the nuclear factor-kappaB (NF-kappaB) pathway and undergo apoptosis when NF-kappaB is inhibited. Here, we show that the combination of conventional chemotherapeutic agents (daunorubicin, mitoxantrone, 5-azacytidine or camptothecin) with the NF-kappaB inhibitor BAY11-7082 did not yield a synergistic cytotoxicity. In contrast, BAY11-7082 (which targets the NF-kappaB-activating I-kappaB kinase (IKK) complex) or knockdown of essential components of the NF-kappaB system (such as the IKK1 and IKK2 subunits of the IKK complex and the p65 subunit of NF-kappaB), by small interfering RNAs sensitized MDS cell lines to starvation-induced apoptosis. The combination of BAY11-7082 and nutrient depletion synergistically killed the acute myeloid leukemia (AML) cell line U937 as well as primary CD34(+) bone marrow blasts from AML and high-risk MDS patients. The synergistic killing by BAY11-7082, combined with nutrient depletion, led to cell death accompanied by all hallmarks of apoptosis, including an early loss of the mitochondrial transmembrane potential, the release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria, activation of caspase-3, phosphatidylserine exposure on the plasma membrane surface and nuclear chromatin condensation. Transmission electron microscopy revealed the presence of numerous autophagic vacuoles in the cytoplasm before cells underwent nuclear apoptosis. Nonetheless, cell death was neither inhibited by the pan-caspase inhibitor z-VAD-fmk nor by knockdown of AIF or of essential components of the autophagy pathway (ATG5, ATG6/Beclin-1, ATG10, ATG12). In contrast, external supply of glucose, insulin or insulin-like growth factor-I could retard the cell death induced by BAY11-7082 combined with starvation. These results suggest that in MDS cells, NF-kappaB inhibition can precipitate a bioenergetic crisis that leads to an autophagic stress response followed by apoptotic cell death.

Topics: Acute Disease; Antineoplastic Combined Chemotherapy Protocols; Cell Death; Fluorescent Antibody Technique; Humans; Leukemia, Myeloid; Myelodysplastic Syndromes; NF-kappa B; Nitriles; Sulfones