Melisa A. Martinez-Paniagua

Mcl-1 and YY1 inhibition and induction of DR5 by the BH3-mimetic Obatoclax (GX15-070) contribute in the sensitization of B-NHL cells to TRAIL apoptosis.

The pan Bcl-2 family antagonist Obatoclax (GX15-070), currently in clinical trials, was shown to sensitize TRAIL-resistant tumors to TRAIL-mediated apoptosis via the release of Bak and Bim from Mcl-1 or Bcl-2/Bcl-XL complexes or by the activation of Bax, though other mechanisms were not examined. Herein, we hypothesize that Obatoclax-mediated sensitization to TRAIL apoptosis may also result from alterations of the apoptotic pathways. The TRAIL-resistant B-cell line Ramos was used as a model for investigation. Treatment of Ramos cells with Obatoclax significantly inhibited the expression of several members of the Bcl-2 family, dissociated Bak from Mcl-1 and inhibited the NFκB activity. Cells treated with Mcl-1 siRNA were sensitized to TRAIL apoptosis. We examined whether the sensitization of Ramos to TRAIL by Obatoclax resulted from signaling of the DR4 and/or DR5. Transfection with DR5 siRNA, but not with DR4 siRNA, sensitized the cells to apoptosis following treatment with Obatoclax and TRAIL. The signaling via DR5 correlated with Obatoclax-induced inhibition of the DR5 repressor Yin Yang 1 (YY1). Transfection with YY1 siRNA sensitized the cells to TRAIL apoptosis following treatment with Obatoclax and TRAIL. Overall, the present findings reveal a new mechanism of Obatoclax-induced sensitization to TRAIL apoptosis and the involvement of the inhibition of NFκB activity and downstream Mcl-1 and YY1 expressions and activities.

Abstract B48: Resensitization to CDDP or trail-mediated apoptosis in ovarian cancer cells by Obatoclax.

Ovarian cancer is the fifth cause of death in women, annually 13,850 deaths are reported in United States. Chemo is the conventional therapy after surgery but the initial response is between 60 to 80%, however, some patients that initially respond well to the therapy suffer recurrent disease or become resistant to the treatment. As a consequence, the overall 5 year survival is only 30%. There is a necessity to identify novel therapies for ovarian cancer or to discover drugs witch re sensitize tumor cells to existence therapy. It has been suggested that the resistance of the tumor cells is because of the increased expression of antiapoptotic proteins especially from the Bcl2 family (Bcl-2, Bcl-XL, Mcl-1). Obatoclax is a small molecule that mimics the BH3 domain of the BH3 only family members, inhibiting the effect of antiapoptotic Bcl-2 members. The objective of this study was to assess the susceptibility of ovarian cancer cell lines to chemo or immune therapy mediated apoptosis by obatoclax and also to elucidate the mechanism. Some ovarian cancer cell lines were treated with different concentrations of Obatoclax, CDDP or TRAIL alone or in combinational treatment to assess the reduction of viability measured by the XTT kit (Roche). Basal and cell treated total protein expressions were assessed by western. Induction of apoptosis was measured by TUNEL. As a single agent, Obatoclax or CDDP inhibited the growth of ovarian cancer cell lines in a dose response manner, TRAIL was no having an evident response showing a plateau with the concentration used. The combinational treatment after selecting the IC50 and IC20 response showed an increased inhibition of the viability. The induction of apoptosis was mediated by a caspase dependent manner. Our data suggest that obatoclax in combination with CDDP or TRAIL can re sensitize resistance cell lines to apoptosis and also it can be an optional treatment of patients with ovarian cancer. Citation Format: Melisa A. Martinez-Paniagua, Luis A. Romero-Gonzalez, Yu-Mei Anguiano-Hernandez, Carolina Gonzalez-Torres, Mario I. Vega. Resensitization to CDDP or trail-mediated apoptosis in ovarian cancer cells by Obatoclax. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B48.

Abstract 1735: Obatoclax enhances CDDP and trail-induced apoptosis in epithelial ovarian adenocarcinoma cells.

Epithelial ovarian cancer comprises the majority of malignant ovarian tumors in adult women. Ovarian cancer is the fifth cause of death in women and annually 13,850 deaths are reported in United States. Effective screening strategies are lacking and the majority of cases are diagnosed with advanced stage disease. Chemo is the conventional therapy after surgery but the initial response is between 60 to 80%, however, some patients that initially responded to the therapy suffer of recurrent disease or become resistant to the treatment. As a consequence, the overall 5 year survival is only about 30%. It is important to identify novel therapies for ovarian cancer to treat or sensitize tumor cells to the existence therapy. It has been suggested that the tumor cells become resistance due to an increased expression of antiapoptotic proteins especially from the Bcl2 family (Bcl-2, Bcl-XL, Mcl-1). Obatoclax is a small molecule that mimics the BH3 domain of the BH3 only family members and inhibits the effect of antiapoptotic Bcl-2 members. The objective of this study was to assess the susceptibility of ovarian cancer cell lines to chemo or immuno therapy mediated apoptosis by obatoclax and also to elucidate the mechanism. Four different epithelial ovarian adenocarcinomas cell lines (SKOV3, NIH-OVCAR-3, TOV112D and TOV21G) were treated with different concentrations of Obatoclax (nM-μM), CDDP (5-50μg/ml) or TRAIL (50-1200ng/ml) alone or with a combination of treatment to assess the viability, measured by the XTT kit (Roche). Basal and cell treated total protein expressions were assessed by western. Induction of apoptosis was measured by TUNEL. As a single agent, Obatoclax or CDDP inhibited the viability of the cells in a dose response form; TRAIL did not show an evident response. The combination of the treatment after selecting the IC50 and IC20 response showed an increased inhibition of the viability. TUNEL positive cells were measured and the induction of apoptosis was mediated by a caspase dependent manner and PARP cleavage. Our data suggest that obatoclax in combination with CDDP or TRAIL can enhance the induction of apoptosis in resistance cell lines, suggesting to be an optional treatment of patients with ovarian cancer. Citation Format: Melisa A. Martinez-Paniagua, Luis A. Romero-Gonzalez, Yu Mei Anguiano-Hernandez, Carolina Gonzalez-Torres, Mario I. Vega. Obatoclax enhances CDDP and trail-induced apoptosis in epithelial ovarian adenocarcinoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1735. doi:10.1158/1538-7445.AM2013-1735

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

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL 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+) Burkitts 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

Abstract 4582: Regulation of Krüppel-Like Factor 4 (KLF4) expressions via Yin Yang 1 (YY1) in B Non-Hodgkin's Lymphomas (B-NHLs): KLF4 upregulation is associated with unfavorable overall survival in pediatric B-NHL

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Kruppel-like factor 4 (KLF4) is a transcription factor expressed in a variety of tissues in humans and has been implicated in several physiologic processes including development, differentiation, and tissue homeostasis. KLF4 is a bi-functional and can either activate or repress transcription depending on the target gene. For instance, KLF4 acts as a tumor suppressor gene (colon, gastric, esophageal, bladder, and NSCLC) or as an oncogene (laryngeal carcinoma, squamous cell carcinoma, ductal carcinoma of the breast). However, the role of KLF4 in hematological malignancies is still poorly understood. Studies in leukemia suggest that KLF4 may be a tumor suppressor. The goal of this study was to investigate the expression and the clinical significance of KLF4 in B cell non-Hodgkins lymphomas (B-NHLs). Both B-NHL cell lines and patient-derived tumor tissues (TMA) were examined by western blot and immunohistochemistry (IHC), respectively. Using IHC, the expression of KLF4 was calculated based on the intensity and percentage of the area stained, and scoring was corroborated by two pathologists. The complete absence of KLF4 expression was considered as negative. A significant overexpression of KLF4 in Ramos and Raji (Burkitts lymphoma) and 2F7 (AIDS lymphoma) B-NHL cell lines. However, the DHL4 (DBLCL) cell line showed a level of similar to that seen in normal cells. Among the 73 childhood lymphomas studied, 13/23 (57%) of lymphoblastic lymphoma, 7/20 (35%) of large B-cell lymphoma, 4/4 (100%) of anaplastic large cell lymphoma and 5/6 (83%) NHL not specified were KLF4 positive. Notably, 20/20 (100%) Burkitts lymphoma were KLF4 positive. Nuclear expression of KLF4 was significantly higher in Burkitts lymphoma (90%) compared to the remaining subtypes. The 3-year event-free survival rate (EFS) for the whole cohort was 67% (43% to 79%) compared to 23% (13% to 38%) in those who has tumors that were KLF4 positive, (p< 0.05). Multivariate analyses confirmed the association of KLF4 expression with unfavorable overall survival (OS; P<.005). Previous findings demonstrated overexpression of the transcription factor YY1 in B-NHL. In silico analysis of the KLF4 promoter identified the presence of four putative binding sites for YY1. We confirmed that –126 and –298 sites were binding sites for YY1 by ChIP analyses. The transcriptional regulation of KLF4 by YY1 was demonstrated following transfection with YY1 siRNA. We also found a positive correlation between the expression of YY1 and KLF4 in the NHL tissues, suggesting that YY1 regulates KLF4 in vivo. The present findings suggest that KLF4 may be considered as an oncogene in Burkitts lymphoma, and in certain subsets of other types of lymphoma, and that KLF4 may be a potential prognostic factor. We propose that KLF4 may be a therapeutic target in patients with B-NHL. 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 4582. doi:1538-7445.AM2012-4582

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

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL 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

Abstract 664: Galiximab (anti-CD80 mAb) sensitizes TRAIL-resistant B-lymphoma to TRAIL-induced apoptosis via inhibition of the transcription repressor Yin Yang 1 (YY1)

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Treatment of B-lymphomas with rituximab, alone or in combination with chemotherapy, resulted in significant clinical responses and prolongation of survival. However, a significant subset relapses or does not respond to initial treatments. Therefore, there is an urgent need to identify new targets and develop therapies to reverse resistance. CD80 is expressed on APCs and normal B cells and most B-lymphomas express CD80 on the surface. A primatized anti-CD80 mAb, galiximab, was developed and shown to inhibit tumor cell proliferation and mediates ADCC. A phase I/II clinical study with galiximab in patients with refractory B lymphomas demonstrated its safety TRAIL is a death-inducing ligand that is not toxic to normal cells but selectively cytotoxic to sensitive tumor cells and is currently in clinical trails in patients with a variety of cancers. The objective of this study was to investigate whether galiximab can sensitize TRAIL-resistant B lymphoma to TRAIL-induced apoptosis. We hypothesized that galiximab-induced inhibition of cell proliferation must have triggered the cells and inhibited constitutively activated survival pathways, such as NF-κB, and downstream anti-apoptotic gene products, thus, reducing the threshold of resistance and facilitating TRAIL to mediate apoptosis. This hypothesis was tested in vitro using CD80+ Raji cells as model and recombinant TRAIL. Galiximab was obtained from Biogen Idec. Treatment of Raji with galiximab (5-40 μg/ml) for 18h and followed by treatment with TRAIL (2.5-10 ng/ml) resulted in significant apoptosis as assessed by activation of Caspase 3. Galiximab treatment of Raji inhibited the NF-κB pathway and its DNA-binding activity as assessed by western and EMSA, respectively. The direct role of NF-κB-induced inhibition by galiximab in sensitization was corroborated by the use of the NF-κB inhibitor DHMEQ. We have reported that the TRAIL receptor DR5 transcription repressor YY1 regulates tumor-cell sensitivity to TRAIL. We have found that galiximab inhibits YY1 expression and DNA-binding activity in Raji in parallel with upregulation of DR5. The direct role of YY1-induced inhibition by galiximab in TRAIL sensitization was corroborated in cells transfected with siRNA YY1 and which were sensitive to TRAIL apoptosis. Sensitization by galiximab was due, in part; to activation of the type II mitochondrial pathway for apoptosis. Galiximab-induced inhibition of anti-apoptotic Bcl-2 family, such as Bcl-2 and Bcl-XL, in sensitization was verified by the use of the specific Bcl-2 inhibitor, 2 MAM-A3. Altogether, these findings established that galiximab is capable to sensitize TRAIL-resistant B lymphomas to TRAIL apoptosis. The findings also suggest the potential therapeutic application of the combination of galiximab and TRAIL or agonist DR4/DR5 antibodies in the treatment of resistant B lymphomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 664.

Abstract 806: Upregulation of Krüppel-like Factor 4 (KLF-4) expression as a potential tumor progression gene product in non-Hodgkin's lymphoma

Kruppel-like factor 4 (KLF-4) is highly expressed in epithelial tissues such as gut and skin. Several studies based on clinical evidence suggest that KLF-4 functions as a tumor suppressor in cancer of colon, bladder, stomach and in leukemia. In contrast, KLF-4 expression is increased in primary breast ductal carcinomas and in oral dermal squamous cell carcinomas, suggesting that KLF-4 is important in tumor development and progression. However, KLF-4 expression in lymphomas has not been investigated. Our preliminary studies have examined KLF-4 expression in lymphoma cell lines and a TMA containing fresh tissues derived from patients with several types of lymphoma. There was a significant higher expression of KLF-4 in Burkitt9s lymphoma compared with other lymphomas such as follicular or DLBCL. These findings suggest that KLF-4 may be considered as a new biomarker in Non-Hodgkin9s lymphoma (NHL). Further analyses based on the clinical outcome revealed that KLF-4 protein expression was significantly associated with poor patient9s survival. The increased KLF-4 expression was associated with an inferior survival duration (P= 0.002). The survival for 12 patients who had a tumor with weak KLF-4 expression and 13 patients with negative KLF-4 expression was significantly longer than the 30 patients with strong KLF-4 expression (P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 806.