Treatment Of B-NHL Cell Lines Research Articles

P38 MAPK and NF-κB Survival Pathways As Targets for Action of Rituximab In Vivo in a Xenographic Model,

Treatment of B-NHL cell lines with rituximab has been shown to inhibit the p38 MAPK and NF-κB pathways, resulting in chemosensitization to drug-induced apoptosis (Vega et al., 2004 Oncogene). Chemosensitization was a direct consequence of the inhibition of the Bcl-2 / Bcl-xL anti-apoptotic proteins. It has recently shown for us (Vega, et al BMC-Cancer 2015), that activation and expression of p38 MAPK correlate with a poor prognosis in patients with DLBCL-NHL treated with CHOP alone. However, cellular signaling by rituximab in tissue from patients with B-NHL has not been described, and therefore we have examined in an in vivo model that carries a tumor xenograft for validation. Balb/c nu/nu mice were inoculated s.c. with the B-NHL Raji cell line; one group of mice was left untreated, and another group was treated with rituximab (30 mg/kg) at days 5 and 10 after implantation with 8x106 Raji cells. We monitored the tumor growth, and the animals sacrificed at day 30, the tumors collected for further analysis. Treatment with rituximab resulted in significant growth inhibition of tumor cells compared to the control. The IHC examination of the tissue tumors for the expression of phospho and nonphospho-total-p38 MAPK, NF-κB (phospho p65), and Bcl-xL revealed an overexpression of phospho-p38 MAPK, Bcl-xL, and NF-κB with a strong nuclear localization of phospho-p65. In contrast, tumor tissues derived from rituximab treated mice demonstrated a significant inhibition of phospho-p38 MAPK, Bcl-xL, NF-κB, and a reduction of nuclear localization of phospho-p65. We also analyzed the expression of CD20 in tumors and its correlation with the expression of phospho-p38 MAPK. Furthermore, we inoculated a group of mice with Ramos cells (8X106), and two weeks post-incubation the tumors were collected, and their expression of phospho-p38 MAPK and NF-κB analyzed. A section of the tumor was re-inoculated into another group of mice, and we repeated this procedure three more times. The re-inoculated tumors showed an increased phospho-p38 MAPK and NF-κB expression. These findings established for the first time, in a preclinical model, the rituximab-mediated inhibition of cell survival pathways mediated by p38 MAPK and NF-κB. Moreover, the study also corroborates the role of Bcl-xL expression in resistance and its inhibition by rituximab. It also allows establishing that p38 MAPK activation correlates with tumor malignancy in a tumor re-transplantation model. Overall, this study suggests that the survival pathways of p38 MAPK and NF-κB are targets for rituximab-mediated effects and suggests these proteins can use for intervention in cases of resistance to rituximab and p38 MAPK as well as a prognostic factor.Vega MI, et al Rituximab inhibits p38 MAPK activity in 2F7 B NHL and decreases IL-10 transcription: pivotal role of p38 MAPK in drug resistance. Oncogene. 2004;23(20):3530-40Vega GG, et al. P38 MAPK expression and activation predicts failure of response to CHOP in patients with Diffuse Large B-Cell Lymphoma. BMC Cancer. 2015;15:722. DisclosuresNo relevant conflicts of interest to declare.

Abstract 766: Galiximab disrupts the dysregulated NF-κB/YY1/Snail/BclXL circuit that regulates the resistance of B-NHL cell lines: Sensitization to chemo-immunotherapeutic drugs

Abstract The combination of Rituximab and chemotherapy is currently the standard treatment for several B-NHL malignancies. However, there is a subset of patients that does not initially respond and a subset that fails to respond to further treatment. Therefore, there is an urgent need to develop new therapeutic modalities for those patients. Galiximab (anti-CD80 mAb) has been developed with the objective of overcoming the resistance to rituximab and/or used in combination with rituximab to improve response rates. A Phase II double blind placebo controlled trial of rituximab+galiximab vs. rituximab+placebo in 337 subjects with relapsed or refractory, grade I-IIIa, follicular NHL in relapse were followed up of 13.8 month. The addition of Galiximab to rituximab reduced the hazard for disease progression or death by 26% compared to the rituximab+placebo group. However, the mechanisms by which Galiximab mediates its effects have not been examined. Preliminary findings demonstrated that treatment of B-NHL cell lines with Galiximab resulted in the inhibition of cell growth and sensitization of drug-resistant tumor cells to both CDDP and TRAIL-mediated apoptosis. Sensitization was a result of Galiximab-induced inhibition of the constitutively activated NF-κB pathway and downstream the resistant factors Yin Yang 1 (YY1), Snail, and BclXL. The role of each of these factors in the regulation of resistance and whether they also regulate each other were assessed by transfection with siRNAs. Treatment of Raji (CD80+) Burkitt's Lymphoma cell line with YY1 siRNA resulted in the inhibition of YY1, Snail, phospho-p65, and BclXL as assessed by western. Likewise, transfection with Snail siRNA resulted in the inhibition of Snail, YY1, phospho-p65, and BclXL. In both cases, the transfected cells resulted in the reversal of resistance and sensitization to apoptosis by both CDDP and TRAIL. These findings revealed that NF-κB regulates YY1, Snail, and BclXL and that both YY1 and Snail, in turn, regulate NF-κB and BclXL. It has been reported that YY1 regulates NF-κB via miR29 and, in turn, NF-κB regulates YY1 transcription. Also, YY1 regulates Snail transcription. However, it is not known how does Snail regulate NF-κB and YY1. We speculate that Snail regulates phospho-p65 via transcription or indirectly through the regulation of NF-κB by YY1. The present findings reveal a new dysregulated NF-κB/YY1/Snail/BclXL circuit in the regulation of resistance of B-NHL to cytotoxic drugs. Galiximab interferes with this circuit and results in the reverse of resistance. In addition, the findings revealed new targets that may be of prognostic significance as well as targets for therapy. The present findings in B-NHL cell lines may be also generalized to non-lymphoid malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 766. doi:1538-7445.AM2012-766

AT9283, a novel aurora kinase inhibitor, suppresses tumor growth in aggressive B‐cell lymphomas

Aurora kinases are oncogenic serine/threonine kinases that play key roles in regulating the mitotic phase of the eukaryotic cell cycle. Auroras are overexpressed in numerous tumors including B-cell non-Hodgkin's lymphomas and are validated oncology targets. AT9283, a pan-aurora inhibitor inhibited growth and survival of multiple solid tumors in vitro and in vivo. In this study, we demonstrated that AT9283 had potent activity against Aurora B in a variety of aggressive B-(non-Hodgkin lymphoma) B-NHL cell lines. Cells treated with AT9283 exhibited endoreduplication confirming the mechanism of action of an Aurora B inhibitor. Also, treatment of B-NHL cell lines with AT9283 induced apoptosis in a dose and time dependent manner and inhibited cell proliferation with an IC(50) < 1 μM. It is well known that inhibition of auroras (A or B) synergistically enhances the effects of microtubule targeting agents such as taxanes and vinca alkaloids to induce antiproliferation and apoptosis. We evaluated whether AT9283 in combination with docetaxel is more efficient in inducing apoptosis than AT9283 or docetaxel alone. At very low doses (5 nM) apoptosis was doubled in the combination (23%) compared to AT9283 or docetaxel alone (10%). A mouse xenograft model of mantle cell lymphoma demonstrated that AT9283 at 15 mg/kg and docetaxel (10 mg/kg) alone had modest anti-tumor activity. However, AT9283 at 20 mg/kg and AT9283 (15 or 20 mg/kg) plus docetaxel (10 mg/kg) demonstrated a statistically significant tumor growth inhibition and enhanced survival. Together, our results suggest that AT9283 plus docetaxel may represent a novel therapeutic strategy in B-cell NHL and warrant early phase clinical trial evaluation.

Abstract 3499: Identification of a novel mechanism by which the BH3 mimetic obatoclax sensitizes non-Hodgkin's lymphoma tumor cells to TRAIL-mediated apoptosis

Abstract The BH3 mimetic chemical antagonist obatoclax (GX15-070, Gemin X Pharmaceuticals) has been used in vitro and nowadays also in clinical trials for its potential therapeutic effects in both solid and lymph node malignancies. The effects of obatoclax treatment alone or in combination with cytotoxic drugs results in the reversal of tumor cell resistance. Studies by us and others have reported that inhibition of over expressed anti-apoptotic gene products such as Bcl-2 and Bcl-XL can reverse resistance to TRAIL. Obatoclax was shown to sensitize TRAIL-resistant solid tumors to TRAIL-apoptosis, inhibiting diverse anti-apoptotic molecules like Bcl-2, Bcl-XL and Mcl-1 through direct binding. We hypothesized that treatment of B-NHL cell lines with Obatoclax will result in tumor cell sensitization to TRAIL apoptosis by a different mechanism. B-NHL Ramos cell line were treated with different concentrations of Obatoclax (7-28 nM) and TRAIL (2.5-20 ng/ml) resulting in significant potentiation of apoptosis and synergy. Western analysis revealed that treatment with obatoclax resulted in significant inhibition of several members of the Bcl-2 family such as inhibition of Mcl-1, Bcl-XL, XIAP, and cIAP 1/2. In addition, obatoclax dissociated Bak from Mcl-1 as determined by immunoprecipitation. We found that, obatoclax inhibited the expression/activity of several members of the NF-κB pathway including phospho-p65, IKK2, and phosphor-IκB-α. The NF-κB activity inhibition by obatoclax was assessed by EMSA. We examined whether obatoclax-induced sensitization to TRAIL resulted from both signaling of DR4 and DR5. Treatment of Ramos cells with DR4 siRNA or DR5 siRNA and then treated with obatoclax and TRAIL resulted in the sensitization of cells treated with DR4 siRNA but not DR5 siRNA. We have previously reported that DR5 is negatively regulated by the transcription repressor YY1, upstream of NF-κB. We found that obatoclax inhibited YY1 transcription and expression. The direct role of YY1 inhibition by obatoclax in both DR5 upregulation and sensitization to TRAIL was determined in transfected cells with YY1 siRNA. Such cells showed upregulation of DR5 and sensitivity to TRAIL apoptosis. The inhibitory effect of obatoclax on Mcl-1 activity and the Mcl-1 direct role in sensitization was examined by treatment of cells with Mcl-1 siRNA. Such cells reversed their sensitivity to TRAIL-mediated apoptosis. Overall, the present findings reveal a new mechanism of obatoclax-induced sensitization to TRAIL by inhibiting NF-κB and downstream Mcl-1 and YY1. Both Mcl-1 and YY1 play a direct role in tumor cells sensitization to TRAIL mediated apoptosis. The present findings identify several new therapeutic targets modified by obatoclax whose modifications can reverse tumor cell resistance to TRAIL mediated apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3499. doi:10.1158/1538-7445.AM2011-3499

FNC, a novel nucleoside analogue inhibits cell proliferation and tumor growth in a variety of human cancer cells

Inhibition of cellular DNA synthesis is a strategy to block cancer cell division. Nucleoside analogues can incorporate into DNA and terminate DNA strand elongation. So far, several nucleoside analogues have been successfully used as anticancer drugs. FNC, 2′-deoxy-2′-β-fluoro-4′-azidocytidine is a novel cytidine analogue which demonstrated potent activity against hepatitis C virus (HCV). To investigate the therapeutic potential of FNC in human cancers we studied its activity in a number of cancer cells in vitro and in vivo. FNC potently inhibited cell proliferation with an IC 50 of 0.95–4.55 μM in a variety of aggressive human cancer cell lines including B-cell non-Hodgkin's lymphomas, lung adenocarcinoma and acute myeloid leukemia. Cells treated with FNC exhibited G1 and S cell cycle arrest at high and low dose, respectively, which confirms the mechanism of action of nucleoside analogues. Treatment of B-NHL cell lines with FNC induced apoptosis in a dose and time dependent manner. Finally, mouse xenograft models of hepatocarcinoma (H22), sarcoma (S180) and gastric carcinoma (SGC7901) demonstrated that FNC had significant tumor growth inhibition activity in a dose-dependent manner with low toxicity. Together, our results suggest that FNC may be a valuable therapy in cancer patients and warrant early phase clinical trial evaluation.

The Pivotal Role of Yin Yang 1 (YY1) Inhibition (and downstream Bcl-2/Bclxl) by Galiximab (anti-CD80 mAb) In the Reversal of Resistance of B-NHL Cells to Chemotherapy

AbstractAbstract 2887Galiximab (anti-CD80 mAb) is a primatized mAb (human IgG1 constant region and Cynomogous macaque variable region) that binds CD80 on lymphoma cells. It has been shown in vitro that Galiximab inhibits tumor cell proliferation and mediates ADCC. Galiximab is currently in clinical trials for a variety of cancers. Our preliminary findings demonstrated that Galiximab treatment of B-NHL cell lines, like Raji, triggers the cells and inhibits the constitutively activated NF-κB pathway. We hypothesized that Galiximab-induced inhibition of NF-κB may result in the inhibition downstream of several anti-apoptotic gene products and sensitizes cells to drug-induced apoptosis. Raji cells were treated with Galiximab (20-100 μg/ml) for 18h and followed by treatment with the chemotherapeutic drug CDDP (5-10 μg/ml) for 24h and apoptosis was determined by flow for activation of caspase 3. The findings demonstrated that the cells treated with Galiximab were sensitized to CDDP-induced apoptosis. Analysis of the apoptotic pathway following treatment with Galiximab revealed the inhibition of anti-apoptotic gene products such as Bcl-2 and Bclxl. We have also found that Galiximab, like rituximab, inhibits the Fas and DR5 transcription repressor Yin Yang 1 (YY1) and the direct inhibition of YY1 resulted in tumor cell sensitization to both Fas-L and TRAIL. We examined whether inhibition of YY1 by Galiximab was also involved in the sensitization to CDDP apoptosis. Raji cells were treated with YY1 siRNA and, unlike control siRNA or non-treated siRNA cells, the tumor cells were sensitized to CDDP apoptosis. The inhibition of YY1 by siRNA correlated with the inhibition of Bcl-2 and Bclxl. The direct role of Bcl-2 and Bclxl in the regulation of resistance was corroborated by treatment of cells with the Bcl-2 family inhibitor, 2MMA3, and such cells mimicked Galiximab and were sensitive to CDDP-induced apoptosis. The mechanism by which treatment with YY1 siRNA resulted in the inhibition of Bcl-2 and Bclxl and the reversal of resistance is not clear. We suggest that YY1 inhibition, following Galiximab-induced inhibition of NF-κB, will result in the inhibition of Snail transcription (Palmer, MB et al., Mol cancer Res 7:221, 2009). Inhibition of the RKIP (Raf kinase inhibitor protein) repressor Snail will result in the induction of RKIP (Wu, K and Bonavida, B Crit Rev immu 29:241, 2009) and, in turn, RKIP will inhibit NF-κB and resulting downstream in the inhibition of Bcl-2 and Bclxl. In addition, it has been reported that YY1 negatively regulates p53 (Sui, G et al., Cell 117:889, 2004) and YY1 inhibition by Galiximab will upregulate p53 and which will result in the inhibition of Bcl-2 and Bclxl (see scheme below). The present findings demonstrate that Galiximab sensitizes drug-resistant B-NHL cells to drug-induced apoptosis via modulation of the NF-κB/YY1/Snail/RKIP/p53 loop. Current studies are validating the present findings with freshly-derived B-NHL cells and also examining the molecular mechanism by which YY1 regulates Bcl-2/Bclxl expression and the reversal of resistance. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Rituximab Inhibits the PI3K/Akt Survival Pathway In B-NHL Cells Via Induction of the PTEN Tumor Suppressor: Pivotal Roles of the Inhibition of the PTEN Repressors Snail and YY1

AbstractAbstract 3152We have reported that treatment of B-NHL cell lines with rituximab resulted in the inhibition of the constitutively activated PI3K-AKT pathway (Suzuki et al., Oncogene 26:6184, 2007). Examination of the mechanism by which rituximab inhibits the PI3K/Akt pathway revealed that it induces the expression of the PI3K/Akt inhibitor PTEN (phosphatase and tensin homolog detected on chromosome 10). Time kinetic analysis indicated that the induction of PTEN occurs as early as 6 h post-rituximab treatment. The objective of this study is to delineate the molecular mechanism by which PTEN is induced by rituximab. We hypothesized that rituximab-induced inhibition of the constitutively activated NF-κB pathway, directly and indirectly through inhibition of the PI3K/Akt pathway, may result in the inhibition downstream of the PTEN transcription factors and repressors, Snail and Yin Yang 1 (YY1). Snail has been reported to repress the transcription of PTEN (Escriva, M et al., Mol Cell Biol 28:1528, 2008). Also, YY1 has been reported to positively regulate Snail transcription and expression (Palmer, MB et al., Mol Cancer Res 7:221, 2009). In addition, the induction of PTEN by rituximab also results, in a feed-back loop, in the suppression of YY1 and Snail and potentiates the induction of PTEN (Petriella et al, Cancer Biology Therapy, 8, 1389, 2009). This hypothesis was tested using the B-NHL Ramos cells, as model, for these studies. Treatment of Ramos with rituximab (20ug/ml for 16 hours) resulted in the inhibition of NF-κB, Snail, and YY1 and induction of PTEN expression as assessed by western. The direct role of Snail and YY1 in the suppression of PTEN expression was demonstrated in cells transfected with Snail or YY1 siRNA. The treated cells demonstrated significant induction of PTEN and, concomitantly, inhibition of the PI3K/Akt pathway. We have reported that rituximab sensitizes B-NHL cells to apoptosis by various chemotherapeutic drugs and demonstrated that inhibition of the PI3K/Akt pathway by various inhibitors mimics rituximab in the sensitization of the tumor cells to apoptosis by chemotherapeutic drugs (Suzuki et al., Oncogene 26:6184, 2007). The role of PTEN induction by rituximab in the sensitization of resistanr B-NHL cells to drug-apoptosis was demonstrated in cells pre-treated with rituximab (to induce PTEN) and then transfected with PTEN siRNA. The transfected cells were resistant to drug-induced apoptosis compared to the control siRNA treated cells. Altogether, the above findings demonstrate that rituximab-induced inhibition of the PI3K/Akt pathway is due, in part, to the induction of PTEN through rituximab-induced inhibition of the PTEN repressors Snail and YY1, downstream of NF-κB. Thus, the induction of PTEN by rituximab plays a major role in the reversal of tumor cell resistance to chemotherapeutic drugs. Further, the findings reveal that the dysregulated PI3K/Akt/NF-κB/Snail/YY1/PTEN loop in B-NHL cells can be interfered by rituximab. This interference leads to the inhibition of cell survival and reversal of resistance through sensitization to drugs. We propose that the gene products in this loop are potential novel therapeutic targets in the treatment of lymphoma. Disclosures:No relevant conflicts of interest to declare.

Clinical Significance of the Inhibition of YY1 Activity and Expression by Therapeutic Anti-Cancer Antibodies

ABSTRACT: A large number of hematologic malignancies of B-cell origin express the CD20 receptor on the cell surface. CD20 is expressed in the B-cell lineage and is absent in plasma cells. Antibodies directed against CD20 result in the elimination of normal and malignant B cells and, hence, led to the development of a therapeutic antibody, rituximab, that has been approved by the U.S. Food and Drug Administration for the treatment of B-non-Hodgkin’s lymphoma (NHL). Antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), in part, cause depletion of B cells by rituximab in vivo. In addition, we have found that rituximab and several novel anti-CD20 monoclonal antibodies signal the cells and inhibit several intracellular pathways. For instance, we have demonstrated that treatment of B-NHL cell lines with anti-CD20 monoclonal antibody (mAb) resulted in the inhibition of constitutively activated transcription factors NF-κB and Yin Yang 1 (YY1). Inhibition of YY1 by anti-CD20 mAb was caused, in part, by the inhibition of NF-κB and induction of the Raf-1 kinase inhibitor protein (RKIP). Inhibition of YY1 with siRNA YY1 resulted in sensitization of B-NHL to apoptosis by drugs. These findings demonstrate that anti-CD20 mAb-induced cell sensitization is the result of inhibition of YY1 activity and expression. Similar findings were observed with the humanized anti-CD20 mAb, BM-ca, and anti-CD80 mAb, galiximab. Hence, YY1 is suggested to be a therapeutic target that can be used in conjunction with anti-CD20 antibodies and other therapeutic antibodies in the reversal of resistance in B-NHL and other malignancies.

Mechanism of Obatoclax (GX15-070; Bcl-2 Family Inhibitor)-Induced-Sensitization of B-NHL Cells to TRAIL-Mediated Apoptosis: Inhibition of the NF-κB Survival Pathway and the DR5 Transcription Repressor Yin Yang1 (YY1)

Patients with B-NHL respond initially to treatment with rituximab (chimeric anti- CD20 monoclonal antibody) in combination with CHOP. However, a subset of patients does not respond or develop refractoriness to further treatments. Therefore, there is an urgent need to develop novel therapeutic strategies to treat unresponsive patients. We have explored the potential therapeutic efficacy of TRAIL though, most tumors and cell lines are resistant to TRAIL-induced apoptosis. Our previous findings and those of others have demonstrated that the overexpression of anti-apoptotic gene products such as Bcl-2, BclXL, and Mcl-1 regulates resistance to TRAIL and thus, inhibition of these gene products reverses resistance. Hence, we hypothesized that treatment of B-NHL cell lines with the Bcl-2 family inhibitor, Obatoclax (GX15-070; Gemin X Pharmaceuticals, Malvern, PA) will result in tumor cell sensitization to TRAIL apoptosis. We have used the B-NHL Ramos cell line as model. Treatment of Ramos cells with various concentrations of Obatoclax (7–28 nM) and TRAIL (2.5–20 ng/ml) resulted in significant potentiation of apoptosis and the combination treatment was synergistic. We then explored the mechanism of Obatoclax-induced sensitization to TRAIL. Treatment of Ramos cells with Obatoclax inhibited NF-κB activity and downstream anti-apoptotic gene products regulated by NF- κB (example Bcl-xl, Mcl-1 and XIAP) as assessed by western. Since Obatoclax inhibited NF-κB activity, we explored its effect on the transcription repressor YY1 and DR5 expression. Treatment of Ramos with Obatoclax significantly inhibited YY1 expression concomitantly with upregulation of total and surface DR5 expression that are regulated by NF-κB. The direct role of YY1 in the regulation of resistance to TRAIL was demonstrated by treatment of Ramos with siRNA YY1. Such treated cells showed upregulation of DR5 expression and sensitization to TRAIL apoptosis. The sensitization by Obatoclax resulted in activation of both Type I and Type II apoptotic pathways when used in combination with TRAIL. These findings establish a novel mechanism of Obatoclax-induced gene modification aside from its direct inhibition of Bcl-2 family. Further, our findings with Obatoclax are different from those recently reported by Song et al., [JBC 2008; July 3 (Epub ahead of print)] demonstrating that ABT-737, a small molecule Bcl-2 inhibitor, potentiated TRAIL-induced apoptosis via activation of NF-κB and NF-κB-induced upregulation of DR5 transcription via NF-κB DNA binding site on the DR5 promoter. It is possible that Obatoclax and ABT-737 mediate their sensitization to TRAIL via distinct mechanisms. In summary, our findings demonstrate the potential therapeutic application of Obatoclax in combination with TRAIL or agonist DR4/DR5 antibodies in the reversal of tumor cell resistance to TRAIL.

Direct and Enhanced Cytotoxicity of the Bcl-2 Family Inhibitor GX15-070 on Rituximab-Sensitive and Rituximab-Resistant B-NHL Clones.

Rituximab (chimeric anti-CD20 mAb) has been used for the treatment of Non Hodgkin B cell lymphomas (B-NHL), alone or in combination with CHOP. However, a subset of patients does not respond to treatment or develops refractoriness to further treatments. Therefore, there is an urgent need to develop new alternatives to treat these patients. We have reported that treatment of B-NHL cell lines with rituximab inhibits anti-apoptotic survival pathways and down regulates the expression of anti-apoptotic Bcl-2 family proteins (i.e. Bcl-2/Bcl-xl) resulting in sensitization to chemotherapeutic drugs. Further, rituximab-resistant clones showed over-expression of anti-apoptotic gene products (Jazirehi et al., Cancer Research 1:1270–81, 2007). Therefore, we hypothesize that inhibitors of anti-apoptotic Bcl-2 family may reverse the resistance to apoptotic stimuli. We examined chemical inhibitors that mimic natural ligands of the anti-apoptotic BH3-only proteins. GX15–070 (Gemin X Biotechnologies, Inc., Canada) inhibits Bcl-2 protein-protein interactions resulting in Bak and Bax oligomerization, release of cytochrome C, and activation of caspases (Shore and Viallet, Hematology , 2005; ASH, 226–230). Treatment of B-NHL cell lines (Raji, Ramos, 2F7, DHL-4) with subtoxic concentrations of GX15–070 (20–50 uM) resulted in inhibition of cell proliferation and subsequently induction of apoptosis as determined by TUNEL. There was a time and concentration-dependent effect of GX15–070 on cytostasis and apoptosis. Analysis of cells treated with GX15–070 by western blotting revealed that Bcl-2, Bcl-xl, and Mcl-1 protein expressions were significantly inhibited as compared to controls. The protein inhibition by GX15–070 was not expected and needs further investigation. We also examined the effect of combination treatment of GX15–070 with the chemotherapeutic drug CDDP and there was an additive or synergistic cytotoxic effect. Treatment of rituximab-resistant clones (generated from 2F7, Raji and Ramos) treated with GX15–070 resulted in significant inhibition of cell growth and apoptosis. The cytotoxicity of GX15–070 in the B-NHL cell lines was tumor specific, because treatment of human peripheral blood leukocytes from different donors did not show any cytotoxic effect. Likewise, treatment of nude mice with different concentrations of GX15–070 did not show any detectable toxicity. These findings demonstrate that GX15–070 is cytotoxic to various drug/rituximab-resistant B-NHL cell lines and is not toxic to normal human leukocytes. This study suggests that combination of GX15–070 with subtoxic concentrations of chemotherapeutic drugs may have additive/synergistic effects. The present findings support the potential therapeutic application of GX15–070 in the treatment of patients with B-NHL that are resistant to current therapies.

Rituximab-Mediated Inhibition of the p38 MAPK and NF-κB Survival Pathways In Vitro and Validation in Nude Mice Bearing the Raji B-NHL Tumor.

Treatment of B-NHL cell lines with rituximab inhibited the p38 MAPK and NF-κB pathways, resulting in chemosensitization to drug-induced apoptosis (Vega et al., 2004 Oncogene, 23:3530–40; Jazirehi et al., 2005 Cancer Res., 65:264–76). Chemosensitization was the direct result of inhibition by these pathways of the anti-apoptotic gene products Bcl-2/Bcl-xL. Cell signaling by rituximab in B-NHL patients has not been investigated and thus, we have examined an in vivo model bearing a tumor xenograft for validation. Balb/c nude mice were transplanted s.c. with the B-NHL cell line Raji; a group of mice was left untreated and another group was treated with rituximab (1000 μg) at days 5 and 10 following implantation of 8x106 tumor cells. The animals were monitored for tumor cell growth and sacrificed at day 30 and tumors were obtained for further analysis. Treatment with rituximab resulted in significant inhibition of tumor cell growth compared to control. Tumor tissues were examined by immunohistochemistry for phospho and non-phospho p38 MAPK and NF-κB (p50), Bcl-2 and Bcl-xL expression. Analysis of tumor-derived tissues from control mice revealed overexpression of phospho-p38 MAPK and NF-κB (p50) and strong nuclear localization of phospho-p50. In addition, there was overexpression of Bcl-2 and Bcl-xL. In contrast, tumor tissues derived from rituximab-treated mice demonstrated significant inhibition of phospho-p38 MAPK, phospho-p50-NF-κB and reduced nuclear localization of phospho-p50. In addition, Bcl-2 and Bcl-xL expression was also significantly reduced. These findings established for the first time, in a pre-clinical model, rituximab-mediated inhibition of the cell survival pathways mediated by p38 MAPK and NF-κB. In addition, the study also corroborates the role of Bcl-2 and Bcl-xL expression in resistance and their inhibition by rituximab. In a separate abstract, tumor tissues derived from many untreated B-NHL patients were analyzed by immunohistochemistry for the activation of both phospho-p38 MAPK and NF-κB and compared to biopsies derived from control individuals. Overall, this study suggests that the p38 MAPK and NF-κB survival pathways are targets for rituximab-mediated effects and also suggests that such targets can be used for intervention in cases of rituximab resistance.

Rituximab-Mediated Upregulation of PTEN Expression and Inhibition of Both the PI3K/AKT Pathway and Pleitrophin (PTN) Expression in B-NHL: Roles in Cell Growth Inhibition and Chemosensitization.

We have recently reported that rituximab treatment of B-NHL cell lines, like Ramos, inhibited the PI3K/AKT signaling pathway and downregulated Bcl-xL expression. The role of the AKT pathway in chemosensitization was corroborated by treating Ramos cells with the AKT inhibitor, LY294002, and resulted in sensitization of the cells to drug-induced apoptosis. We have investigated a potential underlying mechanism responsible for the rituximab-mediated inhibition of the AKT pathway. PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a tumor suppressor phosphatase and functions as a negative regulator of the PI3K pathway through its phosphatase activity. We hypothesized that rituximab may upregulate PTEN expression and thus, inhibiting the AKT pathway. Treatment of Ramos cells with rituximab (20μg/ml for 20h) resulted in significant upregulation of PTEN expression (as assessed by both Western and RT-PCR). Time kinetic analysis showed that PTEN is upregulated as early as 6–9h post rituximab treatment. In addition, since rituximab inhibits cell proliferation and cell growth, we have examined the effect of rituximab on the expression of the growth factor pleitrophin (PTN). PTN is a heparin-binding and secreted growth differentiation factor that mediates various functions such as cell motility and migration, survival, growth and differentiation. Treatment of Ramos cells with rituximab inhibited PTN expression as early as 12h post treatment. The present findings suggested that rituximab-mediated induction of PTEN expression and inhibition of both the AKT pathway and PTN epxression may be interrelated and play an important role in rituximab-mediated cell growth inhibition and chemosensitization. A recent report by Li et al., (JBC, 281:10663,2006) demonstrated that PTEN null cells exhibited upregulation of PTN expression and activation of the PI3K/AKT pathway. Further, inhibition of PTN resulted in inhibition of the AKT pathway, thus establishing a feedback mechanism. Our findings with rituximab are consistent with the Li's findings'. The mechanism by which rituximab inhibits PTN expression is not clear. Reported studies have implicated the role of AP-1 in PTN transcription and in agreement, our findings have also demonstrated that rituximab inhibits AP-1. Overall, the present studies suggest novel targets modified by rituximab namely, PTN and PTEN, which can be considered for therapeutic intervention in the treatment of both rituximab-sensitive and rituximab-resistant tumors.

Regulation of Chemoresistance and Immune Resistance of B-NHL Cell Lines by Overexpression of YY1 and Bcl-xL, Respectively: Reversal of Resistance by Rituximab.

We have recently reported that treatment of B-NHL cell lines with rituximab sensitizes the tumor cells to both chemotherapy and Fas-induced apoptosis (Jazirehi and Bonavida, 2005, Oncogene , 24:2121–2145). This study investigated the underlying molecular mechanism of rituximab-mediated reversal of resistance. Treatment of B-NHL cell lines inhibited the constitutively activated NF- κB. Cells expressing dominant active IκB or treated with NF-κB specific inhibitors were sensitized to both drugs and FasL agonist mAb (CH-11)-induced apoptosis. Downregulation of Bcl-xL expression via inhibition of NF-κB activity correlated with chemosensitivity. The direct role of Bcl-xL in chemoresistance was demonstrated by the use of Bcl-xL overexpressing Ramos cells, Ramos HA-BclxL (gift from Genhong Cheng, UCLA), which were not sensitized by rituximab to drug-induced apoptosis. However, inhibition of Bcl-xL in Ramos HA-Bcl-x resulted in sensitization to drug-induced apoptosis. The role of Bcl-xL expression in the regulation of Fas resistance was not apparent as Ramos HA-Bcl cells were as sensitive as the wild type cells to CH-11-induced apoptosis. Several lines of evidence support the direct role of the transcription repressor Yin-Yang 1 (YY1) in the regulation of resistance to CH-11-induced apoptosis. Inhibition of YY1 activity by either rituximab, the NO donor DETANONOate, or following transfection with YY1 siRNA all resulted in upregulation of Fas expression and sensitization to CH-11-induced apoptosis. These findings suggest two complementary mechanisms underlying the chemo-sensitization and immuno-sensitization of B NHL cells by rituximab via inhibition of NF-κB. The regulation of chemoresistance by NF-κB is mediated via Bcl-xL expression whereas the regulation of Fas resistance by NF-κB is mediated via YY1 expression and activity. These findings suggest that drug-resistant NHL tumor cells may be sensitive to immune-mediated therapeutics.