Gabriel G. Vega

Galiximab signals B-NHL cells and inhibits the activities of NF-κB-induced YY1- and snail-resistant factors: mechanism of sensitization to apoptosis by chemoimmunotherapeutic drugs.

Galiximab (anti-CD80 monoclonal antibody) is a primatized (human IgG1 constant regions and cynomologus macaque variable regions) monoclonal antibody that is currently in clinical trials. Galiximab inhibits tumor cell proliferation through possibly cell signaling–mediated effects. Thus, we hypothesized that galiximab may signal the tumor cells and modify intracellular survival/antiapoptotic pathways such as the NF-κB pathway. This hypothesis was tested using various CD80+ Burkitt B-NHL (non–Hodgkin lymphomas) cell lines as models. Treatment of B-NHL cells with galiximab (25–100 μg/mL) resulted in significant inhibition of NF-κB activity and its target resistant factors such as YY1, Snail, and Bcl-2/Bcl-XL. Treatment of B-NHL cells with galiximab sensitized the tumor cells to both cis-diamminedichloroplatinum(II) (CDDP)- and TRAIL-induced apoptosis. The important roles of YY1- and Snail-induced inhibition by galiximab in the sensitization to CCDP and TRAIL were corroborated following transfection of Raji cells with YY1 or Snail short interfering RNA. The transfected cells were shown to become sensitive to both CCDP- and TRAIL-induced apoptosis in the absence of galiximab. Furthermore, knockdown of YY1 or Snail inhibited Bcl-XL. The involvement of Bcl-XL inhibition in sensitization was corroborated by the use of the pan-Bcl-2 inhibitor 2MAM-3 whereby the treated cells were sensitive to both CDDP- and TRAIL-induced apoptosis. These findings show that galiximab inhibits the NF-κB/Snail/YY1/Bcl-XL circuit that regulates drug resistance in B-NHL and in combination with cytotoxic drugs results in apoptosis. The findings also support the therapeutic application of the combination of galiximab and cytotoxic drugs in the treatment of drug-resistant CD80-positive B-cell malignancies. Mol Cancer Ther; 11(3); 572–81. ©2012 AACR.

Expression of KLF4 is a predictive marker for survival in pediatric Burkitt lymphoma.

Abstract Krüppel-like factor 4 (KLF4) is expressed in a variety of tissues with diverse physiological functions and activities. KLF4 can also function as a tumor suppressor or an oncogene, depending on the cellular context. Its role in hematological malignancies is controversial. This study examined the expression levels of KLF4 by immunohistochemistry in 73 pediatric non-Hodgkin lymphomas (NHLs) in a tissue microarray and also on several B-NHL cell lines. Elevated levels of KLF4 expression were detected in 66% of lymphoma cases and were more frequent in the Burkitt lymphoma (p = 0.05) subtype. There was a significant predictive power for outcome with low KLF4 expression, predicting a favorable overall survival compared to high levels. Multivariate analyses confirmed the association of KLF4 expression with unfavorable overall survival (p < 0.005). These findings were consistent with analyses in existing NHL microarray datasets. The present findings revealed that KLF4 is overexpressed in Burkitt pediatric lymphoma and is a potential biomarker for inferior overall survival.

Overcoming rituximab drug-resistance by the genetically engineered anti-CD20-hIFN-α fusion protein: Direct cytotoxicity and synergy with chemotherapy

Treatment of patients with B-NHL with rituximab and CHOP has resulted in significant clinical responses. However, a subset of patients develops resistance to further treatments. The mechanism of unresponsiveness in vivo is not known. We have reported the development of rituximab-resistant clones derived from B-NHL cell lines as models to investigate the mechanism of resistance. The resistant clones exhibit hyper-activated survival/anti-apoptotic pathways and no longer respond to a combination of rituximab and drugs. Recent studies reported the therapeutic efficacy in mice bearing B-cell lymphoma xenografts following treatment with the anti-CD20-hIFNα fusion protein. We hypothesized that the fusion protein may bypass rituximab resistance and inhibit survival signaling pathways. Treatment of the rituximab-resistant clones with anti-CD20-hIFNα, but not with rituximab, IFNα, or rituximab+IFNα resulted in significant inhibition of cell proliferation and induction of cell death. Treatment with anti-CD20-hIFNα sensitized the cells to apoptosis by CDDP, doxorubicin and Treanda. Treatment with anti-CD20-hIFNα inhibited the NF-κB and p38 MAPK activities and induced the activation of PKC-δ and Stat-1. These effects were corroborated by the use of the inhibitors SB203580 (p38 MAPK) and Rottlerin (PKC-δ). Treatment with SB203580 enhanced the sensitization of the resistant clone by anti-CD20-hIFNα to CDDP apoptosis. In contrast, treatment with Rotterin inhibited significantly the sensitization induced by anti-CD20-hIFNα. Overall, the findings demonstrate that treatment with anti-CD20-hIFNα reverses resistance of B-NHL. These findings suggest the potential application of anti-CD20-hIFNα in combination with drugs in patients unresponsive to rituximab-containing regimens.

Cytotoxic effect caspase activation dependent of a genetically engineered fusion protein with a CD154 peptide mimetic (OmpC-CD154p) on B-NHL cell lines is mediated by the inhibition of bcl-6 and YY1 through MAPK p38 activation

Abstract The interaction between CD40, and its ligand, CD154, is essential for the development of humoral and cellular immune responses. The selective inhibition or activation of this pathway forms the basis for the development of new therapeutics against immunologically based diseases and malignancies. We are developing a gene fusion of Salmonella typhi OmpC protein expressing the CD154 Tyr140-Ser-149 amino acid strand. This OmpC-CD154 binds CD40 and activates B cells. In this study, we demonstrate that OmpC-CD154p treatment inhibits cell growth, proliferation and induced apoptosis in the B-NHL cell lines Raji and Ramos. The Bcl-2 family proteins were regulated and the Bcl-6 and YY1 oncoproteins were inhibited. p38 MAPK activation is an important mechanism underlying the effect on proliferation and apoptosis mediated by this fusion protein. This study establishes a basis for the possible use of fusion protein OmpC-CD154 as an alternative treatment for B-NHL.

Abstract 3617: Inhibition of KLF4 expression in resistant B-NHL cell lines inhibited cell growth and sensitized the cells to drug-induced apoptosis

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Kruppel-like factor 4 (KLF4) is a member of the KLF4 zinc-finger containing transcription factor family. Reported studies have indicated the involvement of KLF4 in the regulation of proliferation, apoptosis, and differentiation of B-cells and B-cell malignancies. We have recently reported that KLF4 is overexpressed in pediatric Burkitt lymphomas and is a predictive biomarker for survival. In addition, overexpression of KLF4 predicted unresponsiveness to CHOP treatment (Valencia-Hipolito A et al Leuk Lymphoma. 2014;55:1806-14). Preliminary findings demonstrated that the transcription factor Yin Yang 1 (YY1) plays, in part, a role in the regulation of KLF4 expression in B-NHL. We hypothesized that (1) the chemical inhibition of KLF4 may result in the inhibition of proliferation, induction of apoptosis, and sensitization to drug-induced apoptosis in B-NHL cell lines and (2) the inhibition of YY1 would mimic the chemical inhibition of KLF4. Analysis of two B-NHL cell lines revealed that the expression of KLF4 was high in Ramos and low Raji as compared to normal B-cells. Treatment with increasing concentrations (0.5-10 μM) of Kenpaullone (a KLF4 inhibitor) induced in Ramos cells both the inhibition of proliferation and cell survival and the induction of apoptosis; however, there was no effect on the treatment of Raji cells. Treatment of Ramos cells with the combination of Kenpaullone and CDDP potentiated apoptosis as compared to treatment with either the chemical inhibitor or CDDP used alone. The finding with the chemical inhibitor was validated with the transfection of Ramos cells with siRNA-KLF4. In addition, treatment with Kenpaullone inhibited the transcriptional expression of KLF4 (using a reporter assay system with pKLF4-GFP). Additional studies demonstrated that the transfection of Ramos cells with siRNA-YY1 resulted in significant inhibition of KLF4 expression and correlated with the inhibition of proliferation and the induction of apoptosis. The latter is due, in part, as KLF4 suppresses the extrinsic apoptotic pathway by inhibiting the activation and cleavage of caspases 7, 9, and 3.. Our previous analysis in TMA of pediatric lymphomas demonstrated, in all of the tumor tissues, the presence of a positive correlation between the expression of KLF4 and YY1 and that this correlation was markedly significant in the Burkitt subtype. In conclusion, the overexpression of KLF4 may be responsible, in part, in the pathogenesis, malignancy, and drug resistance of B-NHL lymphomas. In addition, the present findings suggest that the chemical inhibition of KLF4 by Kenpaullone treatment or the inhibition of YY1 may be considered as targets for therapeutic intervention in the treatment of B-lymphoma overexpressing KLF4, when used alone or in combination with sub-toxic chemo/immune-drugs. Current studies are evaluating the role of KLF4 inhibition in vivo using B-NHL tumor xenografts models. Citation Format: Mayra R. Montecillo-Aguado, Gabriel G. Vega, Hector Mayani, Sara Huerta-Yepez, Rogelio Hernandez-Pando, Otoniel Martinez-Maza, Benjamin Bonavida, Mario I. Vega. Inhibition of KLF4 expression in resistant B-NHL cell lines inhibited cell growth and sensitized the cells to drug-induced apoptosis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3617. doi:10.1158/1538-7445.AM2015-3617

Abstract B199: Transcriptional regulation of KLF4 by Yin Yang 1 (YY1) in pediatric B-NHL and clinical significance.

Kruppel-like factor 4 (KLF4) is a member of the KLF zinc-finger containing transcription factor family. Supporting evidence has established that KLF4 is either an oncogene or a tumor suppressor. Reported studies have indicated the involvement of KLF4 in the regulation of apoptosis, proliferation, and differentiation of B cells and B-cell malignancies. In contrast to adult lymphomas and solid tumors, recently, we have shown in a TMA the overexpression of KLF4 in pediatric NHL tumor tissues. The KLF4 overexpresion predicted unresponsiveness to CHOP treatment. In addition, we have also reported that the transcription factor Yin Yang 1 (YY1) is overexpressed in B-NHL and is a prognostic factor. We hypothesized that the coexpression of KLF4 and YY1 may result from the transcriptional regulation of KLF4 by YY1. This hypothesis was tested in various experimental designs both in cell lines and tumor tissues derived from patients. Analysis of the B-NHL cell line Ramos revealed that both KLF4 and YY1 are overexpressed compared to normal B cells. The transfection of Ramos with siRNA YY1 showed significant inhibition of KLF4. In silico analyses of the KLF4 promoter identified the presence of four putative binding sites for YY1. We confirmed that the -126 site as the binding site for YY1 by CHIP analysis. We also used a reporter system of the KLF4 promoter and mutated the putative binding site for YY1 (-126) and confirmed it as an important site for the regulation of KLF4. The coexpressions of KLF4 and YY1 were examined in TMA of pediatric lymphomas and showed by IHC that all of the tumor tissues exhibited a positive correlation between the expression of KLF4 and YY1 and the correlation was markedly significant in the Burkitt subtype. KLF4 acts as a transcriptional activator of epithelial genes and as a repressor of mesenchymal genes. In addition, KLF4 suppresses the extrinsic apoptotic pathway by inhibiting the activation and cleavage of caspases (7, 9, and 3). Thus, the overexpression of KLF4 in lymphoma may be responsible, in part, in the pathogenesis, malignancy, and drug resistance. The present findings suggest that both KLF4 and YY1 are prognostic biomarkers in pediatric lymphoma. Further, inhibitors of KLF4 are being clinically tested and may be applicable in the treatment of lymphoma. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B199. Citation Format: Mario I. Vega, Alberto Valencia-Hipolito, Miriam Hernandez-Atenogenes, Gabriel G. Vega, Hector Mayani, Sara Huerta-Yepez, Benjamin Bonavida. Transcriptional regulation of KLF4 by Yin Yang 1 (YY1) in pediatric B-NHL and clinical significance. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B199.

Abstract 5450: High expression of Krüppel-Like Factor 4 (KLF4) and its regulation by Yin Yang 1 (YY1) in non-Hodgkin's B-cell lymphomas: clinical implication.

Non-Hodgkin Lymphoma (NHL) is a group of different kinds of cancer rising on the basis of monoclonal expansion of B and T Lymphocytes. Over 90% of NHL has its origin in B lymphocytes. The causes of NHL are poorly understood. Diffuse large B-cell lymphoma (DLBCL), the most common lymphoma in adults, and Burkitt s lymphoma are highly aggressive forms of NHL. It is of interest to characterize diverse transcription factors that are involved in the development of lymphoid progenitors because of their potential therapeutic applications. Kruppel-like factor 4 (KLF4) is a member of the KLF zinc-finger-containing transcription factor family. Evidence has established KLF4 as an oncogene or a tumor suppressor. Recent studies indicated the involvement of KLF4 in the regulation of apoptosis, proliferation, and differentiation of B cells and B-cell malignancies. By using a Tissue Micro Array (TMA), we have recently shown that NHLs have high expression of KLF4. The underlying mechanisms of the regulation of the expression of KLF4 in NHL are not yet understood. We have performed a bioinformatics analysis of the KLF4 promoter region and found that the transcription factor Yin Yang 1 (YY1) has putative binding sites on the KLF4 promoter, thus, hypothesizing the potential regulation of KLF4 by means of YY1 in NHL. This hypothesis was tested by various means: (1) In silico analyses by the CHIP (Chromatin Immunoprecipitation) experimental technique demonstrated that the KLF4 promoter contains four putative binding sites for YY1. We confirmed that the -126 site was a binding site for YY1 by CHIP analysis. (2) Using a reporter system with the KLF4 promoter, we have found that mutation of the putative binding site for YY1 (at -126) inhibited KLF4 expression and (3) Treatment of the Ramos cell line with siRNA for YY1 resulted in dowregulation of KLF4 expression. The clinical implication of YY1 in the transcriptional regulation of KLF4 was correlated by IHC in a TMA with 73 B-NHL samples and by western in B-NHL cell lines. The analyses showed that, in all of the tumor tissues, KLF4 expression had a positive correlation with YY1 expression and this correlation was markedly observed in the Burkitt subtype. Overall, our findings demonstrated that KLF4 can be transcriptionally regulated by YY1 in B-NHL. Previous findings demonstrated that YY1 regulates drug resistance in many cancers including B-NHL. Hence, the present findings suggest that both KLF4 and YY1 are prognostic biomarkers and therapeutic targets in NHL. Citation Format: Mario I. Vega, Alberto Valencia-Hipolito, Miriam Hernandez-Atenogenes, Gabriel G. Vega, Hector Mayani, Alfonso Mendez-Tenorio, Otoniel Martinez-Maza, Sara Huerta-Yepez, Benjamin Bonavida. High expression of Kruppel-Like Factor 4 (KLF4) and its regulation by Yin Yang 1 (YY1) in non-Hodgkin9s B-cell lymphomas: clinical implication. [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 5450. doi:10.1158/1538-7445.AM2013-5450

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