Constitutive activation of the nuclear factor-kappaB (NF-kappaB) pathway has been shown to be involved in the resistance of tumor cells to apoptosis in several human malignancies of the hematopoietic lineage. By using electrophoretic mobility shift assay (EMSA) and confocal microscopic analysis, we demonstrate that NF-kappaB is constitutively activated in cutaneous T-cell lymphoma (CTCL) cell lines HuT-78, MyLa, and SeAx and in peripheral blood lymphocytes (PBLs) from patients with Sézary syndrome (SS) presenting a high ratio of tumor cells, with evidence of p50 and RelA/p65 in DNA-linked complexes. Transfection of SeAx line with a kappaB/luciferase reporter plasmid showed that translocated NF-kappaB complexes were functional. Selective inhibition of NF-kappaB, by transfecting CTCL cell lines with a super-repressor form of IkappaB alpha, led to apoptosis. We evidenced down-regulation of NF-kappaB activation and induction of CTCL cell apoptosis in the presence of proteasome 26S inhibitors ALLN, MG132, and bortezomib. Bortezomib at nanomolar concentrations inhibited constitutive activation of NF-kappaB and induced apoptosis of CTCL cells, with evidence of an upregulation of Bax expression. These results demonstrate the key role played by NF-kappaB in the resistance of CTCL to apoptosis and suggest that bortezomib might be useful for the treatment of patients with advanced stages of CTCL refractory to standard antineoplastic chemotherapy.

Purpose: A key molecular feature of cutaneous T-cell lymphomas (CTCL) is the constitutive activation of the nuclear factor-nB (NF-nB) transcription factor. We investigated in vitro the effects on CTCL survival and chemoresistance of a specific inhibition of InB kinase subunit 2 (IKK2). Experimental Design: Selective IKK2 inhibition was carried out by transfection of SeAx and MyLa CTCL lines with an inactive form of IKK2 and by exposing these lines and tumor cells from 10 patients with Se¤ zary syndrome (SS) to AS602868, a new IKK2 inhibitor. The constitutive nuclear translocation of NF-nB was analyzed by electrophoretic mobility shift assay and confocal microscopy. Apoptosis was determined by Annexin V/propidium iodide^positive staining and mitochondrial transmembrane potential alterations as well as poly(ADP-ribose)polymerase cleavage. The expression of Bcl-2 family oncoproteins and survivin was studied by immunoblotting. Results: Specific IKK2 inhibition resulting from transfection or from incubation with AS602868 allowed a down-regulation of NF-nB transcriptional activity. As shown by electrophoretic mobility shift assay and apoptosis assays, AS602868 down-regulated the nuclear translocation of NF-nB and induced a potent apoptotic response in CTCL lines and in tumor cells from patients with SS while preserving the viability of both peripheral blood lymphocytes from healthy donors and of nonmalignant T cells from SS patients. Moreover, CTCL death induction by conventional antineoplastic agents etoposide and vincristine was potentiated by AS602868. Finally, AS602868-induced apoptosis of CTCL cells was associated with an up-regulation of Bax dimers and a decrease of survivin. Conclusion: These results indicate that IKK2 inhibition represents a promising strategy for the treatment of advanced stages of CTCL.