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Salinosporamide A (also called NPI-0052), recently identified from the marine bacterium Salinispora tropica, is a potent inhibitor of 20S proteasome and exhibits therapeutic potential against a wide variety of tumors through a poorly understood mechanism. Here we demonstrate that salinosporamide A potentiated the apoptosis induced by tumor necrosis factor alpha (TNF), bortezomib, and thalidomide, and this correlated with down-regulation of gene products that mediate cell proliferation (cyclin D1, cyclooxygenase-2 [COX-2], and c-Myc), cell survival (Bcl-2, Bcl-xL, cFLIP, TRAF1, IAP1, IAP2, and survivin), invasion (matrix metallopro-teinase-9 [MMP-9] and ICAM-1), and angiogenesis (vascular endothelial growth factor [VEGF]). Salinosporamide A also suppressed TNF-induced tumor cell invasion and receptor activator of nuclear factor kappaB ligand (RANKL)-induced osteoclastogenesis. We also found that it suppressed both constitutive and inducible NF-kappaB activation. Compared with bortezomib, MG-132, N-acetyl-leucyl-leucyl-norleucinal (ALLN), and lactacystin, salinosporamide A was found to be the most potent suppressor of NF-kappaB activation. Further studies showed that salinosporamide A inhibited TNF-induced inhibitory subunit of NF-kappaB alpha (IkappaBalpha) degradation, nuclear translocation of p65, and NF-kappaB-dependent reporter gene expression but had no effect on IkappaBalpha kinase activation, IkappaBalpha phosphorylation, or IkappaBalpha ubiquitination. Thus, overall, our results indicate that salinosporamide A enhances apoptosis, suppresses osteoclastogenesis, and inhibits invasion through suppression of the NF-kappaB pathway.

Indole-3-carbinol, found in Brassica species vegetables (such as cabbage, cauliflower, and brussels spouts), exhibits antitumor effects through poorly defined mechanisms. Because several genes that regulate apoptosis, proliferation, and metastasis are regulated by nuclear factor-kappaB (NF-kappaB), we postulated that indole-3-carbinol must mediate its activity through NF-kappaB modulation. We demonstrated that indole-3-carbinol suppressed constitutive NF-kappaB activation and activation induced by tumor necrosis factor (TNF), interleukin-1beta (IL-1beta), phorbol 12-myristate 13-acetate (PMA), lipopolysaccharide (LPS), and cigarette smoke; the suppression was not cell type specific, because activation was inhibited in myeloid, leukemia, and epithelial cells. This activation correlated with the sequential suppression of the IkappaBalpha kinase, IkappaBalpha phosphorylation, IkappaBalpha ubiquitination, IkappaBalpha degradation, p65 phosphorylation, p65 nuclear translocation, p65 acetylation, and NF-kappaB-dependent reporter gene expression. The NF-kappaB-regulated gene products cyclin D1, cyclooxygenase-2 (COX-2), matrix metalloproteinase-9 (MMP-9), survivin, inhibitor-of-apoptosis protein-1 (IAP1), IAP2, X chromosome-linked IAP (XIAP), Bcl-2, Bfl-1/A1, TNF receptor-associated factor-1 (TRAF1), and Fas-associated death domain protein-like interleukin-1beta-converting enzyme inhibitory protein (FLIP) were all down-regulated by indole-3-carbinol. This down-regulation led to the potentiation of apoptosis induced by cytokines and chemotherapeutic agents. Indole-3-carbinol suppressed constitutive NF-kappaB activation in mononuclear cells derived from bone marrow of acute myelogenous leukemia patients, and this correlated with inhibition of cell growth. Overall, our results indicated that indole-3-carbinol inhibits NF-kappaB and NF-kappaB-regulated gene expression and that this mechanism may provide the molecular basis for its ability to suppress tumorigenesis.

Introduction: The transcription factor NF-κB plays a major role in various cellular functions such as apoptosis, proliferation, and inflammation and is often deregulated in cancer. It is known to regulate the expression of anti-apoptotic genes (e.g. TRAF1, Mcl-1) and to be constitutively active in CLL. Different mouse models indicate a role for NF-κB in CLL pathogenesisand p65 (RelA), a subunit of NF-κB, appears to have prognostic relevance in CLL. Indeed, high binding activity of p65 to its DNA-binding site is predictive of a short time to first treatment, time to subsequent treatment, and overall survival. These data suggest NF-κB as promising treatment target in CLL. Indeed, several NF-κB inhibitors induced apoptosis of mono-cultured CLL cells in vitro. However, the efficacy of NF-κB inhibition has not been tested on CLL cells in the presence of protective microenvironment or in combination with B-cell receptor (BCR) pathway inhibitors. Methods: The specific NF-κB inhibitor Dehydroxymethylepoxyquinomicin (DHMEQ) was used alone (2µg/ml, 5µg/ml) or in combination with the Btk-inhibitor Ibrutinib (10µM), the PI3K-inhibitor Idelalisib (10µM), or the Syk-inhibitor R406 (4µM) on primary CLL cells in mono- or in co-culture with stroma cells (M2-10B4- or HS-5-cell lines, ratio CLL cells : stroma cells = 20:1). Apoptosis measurements were performed using AnnexinV/PI stainings. Protein expression was analyzed by western blot using standard protocols. For measuring the activity of the different NF-κB subunits (p65, p50, c-Rel, p52, RelB) with a DNA-binding ELISA, protein lysates were prepared after 8h of treatment with DHMEQ (5µg/ml). Results: DHMEQ significantly induced apoptosis of freshly isolated CLL cells in monoculture compared with untreated cells (19% vs. 70% live cells, 5µg/ml, n=6) but surprisingly this effect was suppressed when co-cultured with HS-5 cells (81% vs. 85%). This observation was confirmed with M2-10B4 cells. DHMEQ treatment of mono-cultured CLL cells for 48h significantly downregulated the protein expression of the anti-apoptotic factors Mcl-1 and TRAF1 by 43% and 68% respectively (n=9) while increasing PARP-cleavage four-fold (n=9). However, in co-culture with HS-5 cells no change in the expression of these proteins was observed. A time course analysis of protein expression showed that TRAF1 down-regulation occured after 2h before the increase in PARP-cleavage after 8h (n=6). Although the activity of all NF-κB subunits was reduced in both mono- and co-culture with M2-10B4 cells upon DHMEQ treatment, this effect was attenuated by stroma cell presence: the latter reduced DNA-binding activity of p50 and p65 by 29% and 60% in mono-cultured and 13% and 35% in M2-10B4 co-cultured CLL cells (c-Rel: 25% vs. 15% ; p52: 34% vs. 17% ; RelB: 32% vs. 19% ; n=3). Finally, the combined use of DHMEQ (5µg/ml) with Ibrutinib (n=10) or R406 (n=12) on CLL cells co-cultured with M2-10B4 cells led to a significantly higher rate of apoptosis than DHMEQ, Ibrutinib, or R406 alone. A similar tendency was observed with Idelalisib but it did not reach statistical significance (n=10). Conclusions: In contrast to mono-cultured CLL cells, the specific NF-κB inhibitor DHMEQ did not induce apoptosis in stroma-supported CLL cells, suggesting that apoptosis induction by NF-κB inhibition may be impaired by the microenvironment. As seen in the NF-kB activity assay, this could in part be due to maintained high NF-κB activity in CLL cells co-cultured with stroma cells even in the presence of DHMEQ. However, we could also show that the effect of BCR-pathway inhibitors, which are highly effective in the treatment of CLL, was further increased by the use of DHMEQ. To further evaluate these encouraging results, we are currently investigating the molecular basis of these observations. We are also planning to verify the efficacy of DHMEQ combination treatments in a CLL mouse model. Disclosures No relevant conflicts of interest to declare.

Recently, several novel mechanisms of tumor progression were characterized in chronic lymphocytic leukemia (CLL), stressing an important role of tumor microenvironment. Direct cell-cell interactions as well as soluble factors might support tumor growth. Pro-survival signals might be supported by the tumor necrosis factor (TNF) or some distinctive members of TNF family as well as angiogenic factors. Thalidomide has been shown to be a promising immunomodulatory drug (IMiD) that targets not only leukemic cells but also the tumor microenvironment and by inhibiting angiogenesis. However, so far little is known about the molecular effects of IMiDs in-vivo. Therefore, we investigated cellular and molecular changes induced by a 7 day thalidomide mono-treatment of CLL patients. In forty cases TNF expression levels were assessed using a high sensitivity ELISA test, and T-cell subpopulations were analyzed by flow cytometry. To evaluate the influence of thalidomide on gene expression (GEP), 20 paired pre-treatment and thalidomide-treated samples were analyzed using Affymetrix U133plus2.0 microarrays. Thalidomide therapy was effective in decreasing the number of CLL cells (CD5+CD19+) from 51.75 G/L to 31.7 G/L after treatment. The number of CD3 lymphocytes showed no significant change during thalidomide therapy. No effect on CD4+ as well as CD8+ T cells was observed. Interestingly, we found significant decrease in the number of CD4+CD25hiFOXP3+ T regulatory cells (Tregs) after thalidomide (p=0.05). Thalidomide therapy did not reduce the number of other T-cell subpopulations reported to possess regulatory properties such as CD8+CD28−, CD4+GITR+, CD4+CD62L+ and CD3+TCRγδ+. The changes in Tregs during thalidomide therapy differed in cases who responded to thalidomide with a WBC reduction compared to non-responders. In cases with a WBC reduction there was a greater fold of Tregs reduction. With regard to TNF, we observed no significant changes in the TNF plasma levels after thalidomide treatment. However, the expression of TNF R1 was significantly higher in patients without WBC reduction following thalidomide (p=0.03). Gene expression profiling revealed a thalidomide-induced signature comprising 123 differentially expressed genes (paired t-test with random variance model, p

The NFκB transcription factors p50/p65 regulate the expression of genes encoding various growth-promoting factors and anti-apoptotic proteins, such as the cellular inhibitors of apoptosis (c-IAPs), Caspase-8/Flice-inhibitory protein (FLIP), A1 (also known as Bfl1), tumor necrosis factor receptor (TNFR)-associated factor 1 (TRAF1) and 2 (TRAF2). Furthermore, constitutive activation of NFκB has been observed in many tumor types, supporting the notion that activation of NFκB can play a causal role in tumor development and/or progression. Studies have shown chronic lymphocytic leukemia (CLL) cells experience activation of NFκB in vitro upon ligation of their surface immunoglobulin (Ig), which commonly possesses polyreactive-binding activity for many self-antigens. Other studies also have found that CLL cells from different patients vary in their capacity to undergo B-cell-receptor signaling following ligation of their surface Ig receptors, a capacity that appears associated with leukemia-B-cell expression of the zeta-associated protein of 70 kD (ZAP-70). We examined whether CLL B cell expression of ZAP-70 also was associated with the capacity to activate NFκB upon surface Ig ligation. For this we used CLL B cells of 8 different patients that expressed ZAP-70 and CLL B cells from 8 other patients that had negligible expression of this tyrosine kinase (as assessed by immunoblot and flow cytometric analysis). The CLL B cells of these two groups of patients had similar expression levels of surface, allowing us to use a F(ab’)2 anti-human IgM (anti-μ) to effect comparable surface Ig receptor ligation. Following treatment with anti-μ, we observed early and sustained degradation of IκB-α, thereby releasing cytoplasmic p50/p65 to the nucleus - the hallmark of NFκB activation. Moreover, this was associated with subsequent increased expression of NFκB target genes. In contrast, similar events were not observed following treatment with anti-μ in the cases lacking expression of ZAP-70. Also, activation of NFκB in ZAP-70+ cases was associated with a greater release of intracellular calcium and calcium flux following treatment with anti-μ than observed in ZAP-70-negative cases. Both calcium flux and activation of NFκB induced by anti-μ in these leukemia cells could be inhibited by Cyclosporine-A, indicating that these responses were mediated via a calmodulin-calcineurin-dependent pathway. These studies reveal that expression of ZAP-70 in B cell CLL is associated with a greater capacity to induce activation of NFκB following ligation of surface Ig, a characteristic that might account for the more aggressive clinical behavior of patients with leukemia B cells that express this tyrosine kinase. Moreover, if constitutive activation via ligation of surface Ig with self-antigen in vivo leads to activation of NFκB, then targeting the calmodulin-calcineurin-dependent pathway might have therapeutic potential for this subset of patients with this disease.

LPS activates immature DC via TLR4 and induces maturation of DC for initiating antigen presenting activity (Medzhitov; Nat Rev Immunol 2001). We have previously demonstrated decreased gene expression and protein production of IL-12, IL-15, IL-18 in activated CB MNC and decreased DC MLR (Lee/Cairo, Blood 1996; Qian/Cairo, Blood 1997; Wu/Cairo, Blood 100:3668 p51b 2002). Recently, we have identified differential gene expression patterns including differential immunoregulatory and chemokine genes in LPS-CB vs APB Mo by microarray (Jiang/Cairo, J. Immunol 2004). Since the myeloid lineage DC is derived from Mo, we sought to determine in LPS activated CB vs. APB DC, differential expressed genes that associate with TLR4-mediated signaling pathway. Briefly, Mo were purified from fresh CB or APB and cultured for 7 days with GM-CSF & IL-4 [immature DC (iDC)] and LPS [mature DC (mDC)]. Aliquots from iDC and mDC were analyzed for DC immunophenotype, morphology and DC allogeneic antigen activity. mRNA was isolated, reverse transcripted to cDNA, labeled & hybridized to oligonucleotides (Affymetrix, U133A). Data was analyzed by MAS 5.0 (Affymetrix) and GeneSpring 5.0 software (Silicon Genetics). Several genes were analyzed by RT-PCR (One-Step SuperScript, Invitrogen) and protein expression was analyzed by Western Blot (Bio-Rad). Inverted microscopy demonstrated DC mature morphology at day 8 and flow cytometry demonstrated decreased CD14 and increased CD83 expression in CB & APB mDC. We also demonstrated significant increase in the allogeneic stimulatory effects on CD4+ T cells in APB vs. CB mDC. The microarray analysis demonstrated a significant decreased gene expression of TLR4 [3 fold (F)] and CD14 (2.1 F) (p