Chromatin suspensions were immunoprecipitated with anti-Sp1 and corresponding control antibodies (IgG). nuclear factor-B (NF-B) (p65 and p50), coincided with downregulation of the major transactivator Sp1 and its dissociation from Notch1 promoter. Overexpression of the Notch1 intracellular domain name (NICD) significantly ameliorated bortezomib-induced cytotoxicity against T-ALL cells. Drug combination studies revealed that bortezomib showed synergistic or additive effects with key drugs for the treatment of T-ALL such as dexamethasone (DEX), doxorubicin and cyclophosphamide, which were readily abolished by Ruzadolane NICD overexpression. The synergy of bortezomib and DEX was confirmedin vivousing a murine xenograft model. Our findings provide a molecular basis and rationale for the inclusion of proteasome inhibitors in treatment strategies for T-ALL. Keywords:T-cell acute lymphoblastic leukemia, Notch1, proteasome inhibitor, Sp1, chemosensitization == Introduction == T-cell acute lymphoblastic leukemia Ruzadolane (T-ALL) is an aggressive hematopoietic malignancy of thymocytes. The disease represents approximately 15% of pediatric and 25% of adult ALL cases. Despite the application of rigorous multiagent chemotherapy, the treatment end result of T-ALL is not satisfactory and the prognosis of relapsed patients is still very poor.1Given the limitation of current approaches, it is important to develop new treatment strategies based on increased understanding of the pathophysiology of T-ALL. Notch1 has been recognized as one of the most important genes in T-ALL pathogenesis, because activating mutations of Notch1 are detected in >50% of T-ALL patients2,3,4and Notch1 overexpression transforms normal hematopoietic stem cells and T-cell progenitors to T-ALL in murine models.5Recent investigations suggest that Notch1 is also implicated in drug sensitivity of T-ALL cells.6,7It has been reported that this pre-treatment response to glucocorticoids is a major independent factor influencing the survival of ALL patients8and the sensitivity to glucocorticoids is negatively correlated with the expression levels of Notch1 in T-ALL cells.9 Notch1 is one of four single transmembrane Notch receptors composed of extracellular and intracellular domains that are non-covalently linked via the heterodimerization domains.10,11In response to the binding of cognate ligands, Notch receptors are subjected to proteolytic cleavage by -secretase, resulting in the liberation of the Notch1 intracellular domain (NICD). Subsequently, NICD translocates to the nuclei and activates the transcription of various genes involved in cell cycle progression, cellular differentiation and apoptosis inhibition.11,12In T-ALL, the mutations of Notch1 occur in the proline/glutamic acid/serine/threonine-rich and/or heterodimerization domains to cause constitutive activation of Notch1 via stabilization of NICD and/or ligand-independent generation of NICD.2,11,12 On the basis of these findings, -secretase inhibitors (GSIs) were expected to be effective for T-ALL because of their ability to block Ruzadolane the generation of NICD.13,14,15Unfortunately, most T-ALL cells, especially those with heterodimerization domain DIAPH2 name mutations, are resistant to GSIs.2,16Indeed, clinical trials disclosed the limitation of antitumor activity and a number of severe adverse effects of GSIs.17,18In addition, blocking antibodies for specific Notch receptors and/or ligands are also found to be of limited value for the treatment of T-ALL associated with aberrant Notch1 activation.19,20,21Therefore, the development of novel agents targeting the Notch-mediated oncogenic pathway is eagerly awaited. Proteasome inhibition is now considered a unique and effective way to kill malignancy cells that can tolerate standard chemotherapy. Bortezomib is the first proteasome inhibitor approved for clinical application and is now widely used for the treatment of multiple myeloma (MM).22Previous studies showed that bortezomib affects several molecules including nuclear factor-B (NF-B) and histone deacetylases (HDACs) in MM cells,23,24both of which could be therapeutic targets of T-ALL.25,26,27,28However, relatively little is known about the cytotoxic activity of bortezomib against T-ALL and its mechanisms of action, although a Ruzadolane preliminary report suggests the possibility.29In this study, we demonstrate that proteasome inhibitors, bortezomib and Ruzadolane K-7174, exert marked cytotoxicity against T-ALL cellsin vitroandin vivo, and also increase the sensitivity of T-ALL cells to conventional chemotherapeutic agents via transcriptional repression of Notch1 and downstream effectors such as NF-B and RUNX3. These findings provide a rationale for the inclusion of proteasome inhibitors in treatment strategies for T-ALL. == Materials and methods == == Cells and cell culture == We used fourbona fidehuman T-ALL cell lines, Jurkat, CEM, MOLT4 and KOPT-K1 (provided by Dr Takeshi Inukai, University or college of Yamanashi, Yamanashi, Japan), in this study.2Other cell lines and their origins are KMS12-BM, U266, RPMI8226 (MM), KOPM30 (B-ALL), HBL-2 (mantle cell lymphoma), Namalwa (Burkitt lymphoma), HL-60 and K562 (acute myeloid leukemia), all of which were purchased from the Health Science Research Resources Bank (Osaka, Japan). == Drugs == The drugs used in this study and their sources are bortezomib, MLN120B (Millennium Pharmaceuticals, Cambridge, MA, USA), K-7174 (Kowa, Tokyo,.
