Interleukin-21 Induces Cell Cycle Arrest and Apoptosis of Diffuse Large B-Cell Lymphomas (DLBCL) Via Activation of STAT3 and Upregulation of C-Myc

IL-21, a recently discovered member of the IL-2 cytokine family, has been shown to have diverse regulatory effects on B-cells including the induction of antibody secretion, differentiation, or apoptosis depending on the cellular milieu and activation status. However, the effects of IL-21 on B-cell neoplasms such as DLBCL are largely unknown. Our research has uncovered the widespread expression of the IL-21 receptor (IL-21R) in B-cell lymphomas including DLBCL. Our results confirmed reports that IL-21R stimulation results in potent phosphorylation of STAT-1 and -3 and weak activation of STAT-5. However, our findings also show that treatment of DLBCL cell lines with IL-21 induces cell cycle arrest and apoptosis. The cell death is caspase-dependent and evident in a majority of DLBCL cell lines. To further examine the potential therapeutic applicability of IL-21, we assessed the effects of IL-21 on primary DLBCL tumor cells and in vivo DLBCL mice models. In primary tumors, IL-21 induced apoptosis in five of five DLBCLs compared to two of three follicular lymphomas, and two of seven chronic lymphocytic leukemias. No apoptosis or cell death was induced in normal peripheral B-lymphocytes. In xenograft DLBCL tumors, in situ IL-21 injections induced tumor regression and dramatically extended the overall survival of mice (P<0.001). To elucidate the mechanism of IL-21-induced cell death, microarray analysis was performed on endogenously sensitive or resistant DLBCL cell lines as well as an RCK8 cell line that acquired IL-21 resistance following chronic exposure to the cytokine. The most striking difference between IL-21 sensitive and resistant cell lines was observed in the expression of c-Myc, a known target of STAT-3, which was only induced in cell lines undergoing apoptosis upon IL-21 treatment. We have shown that the cell death associated with IL-21 treatment was prevented by utilizing c-Myc specific siRNAs and shRNA. Interestingly, c-Myc upregulation and cell death was also prevented upon knockdown of STAT-3 with siRNAs. Stimulation of the STAT-3-cMYC signaling pathway led to decreases in levels of anti-apoptotic proteins Bcl-XL and Bcl-2, which are bone-fide targets of c-Myc. Our results delineate a new IL-21-induced pro-apoptotic signaling pathway involving STAT-3 and c-Myc, which are usually considered as anti-apoptotic effectors in cancer cells. Furthermore, our findings demonstrate that IL-21 is a highly potent anti-DLBCL agent in vitro and in xenograft DLBCL models and warrant initiation of clinical studies in patients.