Novel IL-21 signaling pathway up-regulates c-Myc and induces apoptosis of diffuse large B-cell lymphomas

Abstract

IntroductionInterleukin-21 (IL-21) is a member of type I cytokine family that uses the shared ␥-common receptor chain for signaling. 1,2 It is predominantly secreted by activated CD4 ϩ T and natural killer (NK) T cells and induces pleiotropic effects on the immune system by regulating functions of T, B, NK, and myeloid cells. 1,3,4 The IL-21 receptor (IL-21R) has been reported to be present on almost all mature lymphocytes, with the highest expression on activated B cells. 5,6 The nature of IL-21's effects on B cells depends on the organism, specific cellular context (eg, activation and developmental stages), and presence of costimulatory factors. 7,8 IL-21 increases growth and differentiation of murine B lymphocytes that received both B-cell receptor (BCR) and T-cell help mediating signals while inducing apoptosis in cells lacking the concomitant BCR activation. 5 Although there have been several reports of IL-21 inducing apoptosis in murine B cells, the effects of IL-21 on human nonneoplastic B cells have been confined to regulation of B-cell activation and differentiation. Specifically, IL-21 has been shown to costimulate human B-cell proliferation induced by anti-CD40 antibody, yet inhibit proliferation induced by IL-4 and BCR stimulation. 1,6 IL-21 was also reported to have a central role in the differentiation of human primary B cells into plasma cells 9 and in promoting class-switch recombination and secretion of immunoglobulin G (IgG) and IgA in postswitch IgM ϩ memory B cells. 10 Since IL-21 was shown to stimulate the immune system, its effects on some tumors have been explored. IL-21 was reported to have potent antitumor activity in a variety of solid tumor models in mice. 11 Because solid tumors do not express IL-21R, these effects are likely to be indirect and mediated by IL-21-induced terminal differentiation of NK cells, regulation of T-cell differentiation and proliferation, and induction of cytotoxic T-cell responses. 12 In contrast to indirect immune-mediating effects of IL-21 on solid tumors, IL-21 may have direct effects on IL-21R-expressing malignancies originating from B lymphocytes. It was reported that IL-21 enhanced growth of multiple myeloma (MM) cells 13 yet induced apoptosis in chronic lymphocytic leukemia (CLL) B cells. 14,15 Diffuse large B-cell lymphoma (DLBCL), the most common subtype of non-Hodgkin lymphoma, is characterized by heterogeneity in clinical course and response to therapy. With the recent introduction of the anti-CD20 antibody rituximab into clinical practice, the "gold standard" therapy of DLBCL has evolved to include rituximab with cyclophosphamide, doxorubicin, vincristine, and prednisone. This has resulted in significant improvement in patient outcome, with 5-year survival reaching 50% to 60%. However, a significant proportion of patients still succumb to DLBCL and an urgent need for new therapies exists.Because IL-21 may be proapoptotic for certain B cells, we have examined the direct effects of IL-21 on DLBCL cell lines and primary tumors. We show that IL-21R is ...

Anti-CD20-interleukin-21 fusokine targets malignant B cells via direct apoptosis and NK-cell–dependent cytotoxicity

“…Furthermore, the cell death induced by the fusokine was higher in 5 of the 6 tested primary tumors in comparison with combination of IL-21 with parent aCD20-IgG1 Ab, suggesting superior cytotoxic efficacy of the fusokine. Of note, similar to our previous observations with IL-21, 35 in vitro treatment with the aCD20-IL-21 fusokine did not induce apoptosis of normal B-cell (supplemental Figure 3B). …”

“…21,34 Apart from its immunostimulatory effects, IL-21 exerts direct cytotoxicity on a variety of IL-21R-expressing NHLs, including diffuse large B-cell lymphoma (DLBCL), MCL, chronic lymphocytic leukemia, and follicular lymphoma. 32,[35][36][37][38][39][40][41][42] We found that the direct apoptotic effects of IL-21 in DLBCL and MCL were mediated via activation of an IL-21R-dependent signaling pathway that leads to phosphorylation of STATs followed by upregulation of cMyc and Bax and downregulation of BCL-2 and BCL-XL. 32,35 These studies suggest IL-21 may become a potent adjuvant to cancer immunotherapy against B-cell lymphoma.…”

“…32,[35][36][37][38][39][40][41][42] We found that the direct apoptotic effects of IL-21 in DLBCL and MCL were mediated via activation of an IL-21R-dependent signaling pathway that leads to phosphorylation of STATs followed by upregulation of cMyc and Bax and downregulation of BCL-2 and BCL-XL. 32,35 These studies suggest IL-21 may become a potent adjuvant to cancer immunotherapy against B-cell lymphoma. 37 Indeed, a recent clinical trial of IL-21 in combination with rituximab for relapsed or refractory low-grade B-cell lymphomas showed clinical responses in 8 (42%) of 19 patients, with a response rate of 33% in rituximab-resistant patients.…”

“…We have previously demonstrated that IL-21-induced apoptosis is dependent on STAT3, cMYC, and Bax, and is independent of STAT1 in DLBCL and MCL cell lines. 32,35 Treatment with aCD20-IL-21 fusokine resulted in upregulation of pSTAT1, pSTAT3, and pSTAT5, similar to IL-21 alone, and was not observed in cells treated with the parent aCD20-IgG1 Ab ( Figure 3A). Furthermore, we also found upregulation in cMyc, BAX, and downregulation of the antiapoptotic proteins BCL-xL and Bcl-2, which was more pronounced in the fusokine-treated cells compared with native IL-21 ( Figure 3B; supplemental Figure 4A).…”

“…To compare the efficacy of the fusokine to native IL-21, we selected an IL-21 dose that led to maximal killing (50 ng/mL), based on our prior studies and reports in the literature. 20,35 Cells were exposed to equimolar doses of the aCD20-IL-21 fusokine and IL-21. aCD20-IL-21 induced nearly equivalent or slightly higher cell death compared with native IL-21 or its combination with the parental Ab, suggesting its biological activity is retained (Figure 2A; supplemental Figure 3A).…”