KLF4 and YY1 Are Resistant Factors in NHL and Potential Therapeutic Targets: The New Resistant NF-κb/YY1/KLF4/BclxL/Mcl-1 Axis

Introduction: We have recently reported that the expression of Krüppel-like factor 4 (KLF4) is upregulated in pediatric Burkitt lymphoma and it was suggested to be a prognostic biomarker for survival (Valencia-Hipolito et al., 2014)*. In addition, preliminary findings have demonstrated that KLF4 is also a resistant factor for drug-induced apoptosis.

Rationale: Analysis of the role of KLF4 in resistance may have prognostic and therapeutic implications.

Objective: To investigate the molecular mechanism underlying KLF4-induced resistance.

Hypothesis: We have reported that the transcription factor Yin Yang 1 (YY1) is overexpressed in hematological malignancies and regulates both drug resistance and immune resistance. In Non-Hodgkin lymphoma (NHL), KLF4 is overexpressed and is under the transcriptional regulation of YY1. Hence, we hypothesized that KLF4 may also regulate drug/immune resistance and its inhibition would reverse the resistance of tumor cells to drug-induced apoptosis.

Methods: We have used the NHL cell lines Ramos and DHL4 as models. The expression of KLF4 was assessed by Real Time-PCR and Western. KLF4 inhibition was achieved using the inhibitor Kenpaullone and the inhibition of YY1 and KLF4 was induced by transfection with corresponding siRNAs. Apoptosis was determined by activation of caspase 3 by flow cytometry. Tumor biopsies from pediatric NHL patients were analyzed by IHC.

Results: Several NHL human tumor cell lines showed overexpression of KLF4, although at different levels. Treatment with the KLF4 inhibitor, Kenpaullone, resulted in the inhibition of KLF4 expression and the cells were sensitized to Doxorubicin-induced apoptosis. Treatment of tumor cells with YY1 siRNA inhibited both YY1 and KLF4 expressions and the cells were sensitized to drugs-induced apoptosis. Tumor biopsies from patients were divided into two groups, namely, one group with high KLF4 and YY1 and the other group with low levels of KLF4 and YY1. Kaplan Meier analysis revealed that patients with low expression of KLF4 and YY1 responded subsequently to CHOP, whereas patients with high KLF4 and YY1 did not respond to CHOP.

Conclusions: The findings revealed the followings: (1) KLF4 overexpression is under the transcriptional regulation of YY1 (2) Inhibition of KLF4 by the chemical inhibitor Kenpaullone,  or by YY1 siRNA resulted in the sensitization of tumor cells to drug-induced apoptosis and (3) Patients with NHL whose tumors overexpressed KLF4 and YY1 did not respond to CHOP treatment.

Implications: We suggest that the overexpression of KLF4 in NHL may be a novel prognostic biomarker for response to chemotherapy and may also be a therapeutic target. In addition, a recent report by Farrugia M et al (2015)** reported that KLF4 is associated with both Bcl_xL and Mcl-1 and, thus, drug resistance is primarily under the influence of the overexpression of Bcl_xL and Mcl-1. Consequently, since KLF4 is regulated by YY1 and YY1 is in turn, regulated by NF-κB, we suggest the presence of an NF-κB/YY1/KLF4/BclxL/Mcl-1 resistant axis in NHL and  gene products in this axis maybe potential novel prognostic biomarkers and therapeutic targets.

References:
*(Valencia-Hipolito et al 2014, Lek & Lympho 55:1806-1814
** Farrugia M et al (2015_cell death and disease 19;6:e1699.