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.

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. Krüppel-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 Krüppel-Like Factor 4 (KLF4) and its regulation by Yin Yang 1 (YY1) in non-Hodgkin's 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

Krüppel-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 Hernández-Atenógenes, 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.

Krüppel-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 Burkitt's 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-Hodgkin's lymphoma (NHL). Further analyses based on the clinical outcome revealed that KLF-4 protein expression was significantly associated with poor patient's 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&lt; 0.001). Other variables that affected survival in univariate analyses included stages and completeness of resection were shown to have statistically significant effect on survival (P=0.001), however age or sex did not have a statistically significant effect on survival. These data provide the first clinical and causal evidences that alterations of KLF-4 expression can play a critical role in the development and progression of NHL. 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.

Krüppel-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 co-expressions 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 of the expressions of KLF4 and YY1 and the correlation was markedly significant in the Burkitt subtype. These correlations were consistent with the bioinformatics analyses due by ONCOMINE in several data base. 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 putative function of KLF4 was examined by the use of chemical inhibitors for KLF4 (Kenpaullone) and inhibition of KLF4 resulted in the inhibition of cell proliferation and the spontaneous induction of apoptosis. The present findings suggest that both KLF4 and YY1 are prognostic biomarkers for pediatric lymphoma and are also potential therapeutic targets. Disclosures: No relevant conflicts of interest to declare.