All living organisms sense and respond to harmful changes in their intracellular and extracellular environment through complex signaling pathways that lead to changes in gene expression and cellular function in order to maintain homeostasis. Long non-coding RNAs (lncRNAs), a large and heterogeneous group of functional RNAs, play important roles in cellular response to stressful conditions. lncRNAs constitute a significant fraction of the genes differentially expressed in response to diverse stressful stimuli and, once induced, contribute to the regulation of downstream cellular processes, including feedback regulation of key stress response proteins. While many lncRNAs seem to be induced in response to a specific stress, there is significant overlap between lncRNAs induced in response to different stressful stimuli. In addition to stress-induced RNAs, several constitutively expressed lncRNAs also exert a strong regulatory impact on the stress response. Although our understanding of the contribution of lncRNAs to the cellular stress response is still highly rudimentary, the existing data point to the presence of a complex network of lncRNAs, miRNAs, and proteins in regulation of the cellular response to stress.

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.

Krüppel-Like Factor 4 (KLF4) is a member of the KLF transcription factor family, and evidence suggests that KLF4 is either an oncogene or a tumor suppressor. The regulatory mechanism underlying KLF4 expression in cancer, and specifically in lymphoma, is still not understood. Bioinformatics analysis revealed two YY1 putative binding sites in the KLF4 promoter region (-950 bp and -105 bp). Here, the potential regulation of KLF4 by YY1 in NHL was analyzed. Mutation of the putative YY1 binding sites in a previously reported system containing the KLF4 promoter region and CHIP analysis confirmed that these binding sites are important for KLF4 regulation. B-NHL cell lines showed that both KLF4 and YY1 are co-expressed, and transfection with siRNA-YY1 resulted in significant inhibition of KLF4. The clinical implications of YY1 in the transcriptional regulation of KLF4 were investigated by IHC in a TMA with 43 samples of subtypes DLBCL and FL, and all tumor tissues expressing YY1 demonstrated a correlation with KLF4 expression, which was consistent with bioinformatics analyses in several databases. Our findings demonstrated that KLF4 can be transcriptionally regulated by YY1 in B-NHL, and a correlation between YY1 expression and KLF4 was found in clinical samples. Hence, both YY1 and KLF4 may be possible therapeutic biomarkers of NHL.

Multiple myeloma (MM) patients initially respond to conventional therapy, however, many develop resistance and have recurrences. We have reported in other tumors that the transcription factor Yin Yang 1 (YY1) is a resistant factor and, thus, we hypothesized that YY1 may be over-expressed in MM. Significantly, higher expression (staining intensity and cell frequency) of YY1 in MM cell lines and in bone marrow-derived (BM) MM from 22 MM patients was observed as compared to expression in normal BM. Higher nuclear YY1 staining was associated with disease progression. Bioinformatic analyses of mRNA in data sets corroborated the above findings and showed significant overexpression of YY1 in MM compared to normal tissues and other hematopoietic disorders. The role of YY1 expression in the regulation of drug resistance was exemplified in a drug-resistant MM cell line transfected with YY1 siRNA and which was shown to be sensitized to bortezomib-induced apoptosis. These findings highlight the potential prognostic significance of YY1 expression level in MM patients and as a therapeutic target.

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 (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-Hodgkin's 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 (Burkitt's 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%) Burkitt's lymphoma were KLF4 positive. Nuclear expression of KLF4 was significantly higher in Burkitt's 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&lt; 0.05). Multivariate analyses confirmed the association of KLF4 expression with unfavorable overall survival (OS; P&lt;.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 Burkitt's 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

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.