Transcriptional up-Regulation of IL-21 Receptor-α On CLL Cells by CpG685 through An NF-κB Mediated Pathway Provides a Rationale for Combination Strategies with IL-21.
Authors: Browning Rebekah L. , Wang Shu-Huei , Ramanunni Asha , Muthusamy Natarajan , Byrd John C. 
Published: October 2019

Abstract
Abstract 2761
Poster Board II-737
IL-21 is a cytokine produced by CD4 cells that has diverse effects on the immune system. In CLL, IL-21 has been shown to promote apoptosis when tumor cells express the IL-21 receptor-α (IL21R-α). A phase I trial with IL-21 + Rituximab had promising clinical activity in a small number of relapsed CLL patients, including one patient who attained a sustained molecular complete remission, sustained for 4+ years. Expression of IL21R-α is variable between CLL patients, and this expression has been shown to correlate with IL-21-mediated apoptosis. Both CpG oligonucleotides working via TLR9 and CD40 ligand have been shown to up-regulate the IL21R-α in CLL cells. We have systematically interrogated IL21R-α promoter regulation to facilitate novel pharmacologic combination strategies with IL-21. Initial studies were initiated with CpG685, a novel CpG oligonucleotide currently in clinical trials for CLL at our institution. CpG685 promotes immune activation of CLL cells with enhanced NF-κB DNA binding concurrent with up-regulation of CD40, CD80, CD86, and HLA-DR antigen expression. A similar activation phenotype on CLL cells has been noted with CD40 ligand treatment. Interestingly, CpG685 induced a time-dependent induction of IL21R-α in CLL cells at as early as six hours. Quantitative real time PCR revealed that this up-regulation was due to enhanced transcriptional activity, showing an increase in IL21R-α mRNA with peak levels occurring 3 hours post treatment with CpG 685. IL21R-α transcript levels showed a mean 16-fold change over media control at 3 hours of CpG685 treatment, with variability between patients. Up-regulated IL21R-α was functionally competent as evidenced by increased phosphorylation of downstream targets of IL-21 such as STAT1, STAT3, and JAK1, following treatment with IL-21, as compared to media controls. The functional competence of CpG685-induced IL21R-α to mediate IL-21-induced apoptosis was evaluated. For a subset of patients that showed upregulation of IL21R-α but no cytotoxicity in response to CpG685 or IL-21 alone, the combination of CpG685 and IL-21 enhanced IL-21 mediated cytotoxicity. We next explored how CpG685 influences the promoter of IL21R-α. Luciferase promoter constructs containing the proximal SP1 binding site, previously reported to be important in transcriptional activation of IL21R-α in T-cells, did not show increased activity in CpG685 treated CLL cells as compared to control. We therefore hypothesized that alternative cis elements and trans-factors distal to this site may be involved in regulation. Given that both CpG685 and CD40 ligand enhance NF-κB DNA binding and promote IL21R-α expression on CLL cells, we next determined if early inhibition of NF-κB DNA binding by the IKK-β inhibitor Bay11 prevented IL21R-α induction. Indeed, Bay11 treatment of CLL cells prevents CpG685 induction of IL21R-α; preliminary data from a single experiment confirmed concurrent diminished NF-κB DNA binding. Ongoing studies are focused on identifying the specific NF-κB DNA binding site responsible for enhanced IL21R-α expression. Collectively, these studies demonstrate that therapeutic agents such as CpG685 that activate CLL cells through enhanced transcriptional activity of NF-κB have unique potential for combination strategies with other non-immunosuppressive agents such as IL-21. This work was supported by D. Warren Brown Foundation, Specialized Center of Research from the Leukemia and Lymphoma Society, and P01 CA95426 and P01 CA101956 from the National Cancer Institute. RB is a Howard Hughes Medical Institute Research Training Fellow.

Disclosures:
No relevant conflicts of interest to declare.