Immunomodulatory Effects of Pevonedistat, a NEDD8-Activating Enzyme (NAE) Inhibitor, in Chronic Lymphocytic Leukemia (CLL)
Authors: Best Scott R , Kittai Adam , Rowland Taylor , Bruss Nur , Spurgeon Stephen E , Berger Allison , Moran Amy , Danilov Alexey V 
Published: October 2019

Abstract
Introduction: T cells from patients with CLL and lymphoma show highly impaired immune synapse formation, cytotoxic function, and adhesion and migration capabilities. Recent advances in immunooncology led to the emergence of therapeutic agents that permit reversal of T-cell exhaustion in cancer. However, rational development of novel combination approaches in immunotherapy requires detailed understanding of how targeted therapies influence T-cell function.
We have shown that pevonedistat (TAK-924), an investigational NAE inhibitor, abrogates NFκB activation in CLL cells. Pevonedistat forms a covalent adduct with NEDD8, a ubiquitin-like modifier, thereby disrupting its interaction with NAE. This leads to reduced activity of Cullin-RING ligases (CRLs), a group of ubiquitin ligases that require modification by NEDD8 for their function. Ultimately, a decrease in CRL activity leads to reduced ubiquitination and proteasomal degradation of CRL substrates, extending the half-life of these proteins, including inhibitor of NFκB (IκB). Moreover, NFκB is critical in T-cell function. However, limited data exist on the effects of targeting neddylation on T-cell response. Here, we demonstrate that targeting neddylation in vitro preserves T-cell functionality and may lead to favorable T-cell population shifts in CLL.
Methods: Peripheral blood mononuclear cells were isolated from patients with CLL (n=50), and T cells were purified using Dynabeads. Pevonedistat was obtained from Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited (Cambridge, MA).
Results: In vitro T-cell receptor (TCR; CD3/CD28) stimulation induced T-cell activation and proliferation. Continuous treatment of T cells with pevonedistat led to rapid (2 hour) disruption of cullin neddylation, followed by a significant reduction in activity of NFκB and NFAT as assessed by immunoblotting and immunofluorescence. Despite this reduction, CD4 and CD8 T cells continued to respond to TCR stimulation, with relative abundance of early markers of activation (CD40L, CD69). However, we observed reduced expression of CD25 and PD-1 at 72 hours. Continuous treatment with pevonedistat led to a dose-dependent decrease in IL-2 secretion and reduced proliferation of the CD4 T-cell subset (CFSE, Ki-67) but did not induce apoptosis. Unlike CLL cells, CD4 T cells did not undergo DNA re-replication and G2/M arrest in response to pevonedistat.
We further analyzed T-cell subsets following TCR stimulation. Concurrent treatment with pevonedistat led to an increase in IFNγ-secreting CD4 T cells, whereas IL-4 production decreased, suggesting a shift toward the Th1 phenotype. Furthermore, we observed a robust decrease of the iTreg population, accompanied by downregulation of FoxP3 mRNA and protein within the CD4 T-cell subset, indicating that targeting neddylation may help to reverse the immunosuppressive phenotype in CLL.
To mimic the in vivo pharmacokinetics of pevonedistat, we performed drug washouts where CLL-derived T cells were exposed to 2-hour pulse treatment with 1 µM pevonedistat prior to TCR stimulation. Under these conditions, cullin neddylation and NFκB activity began to recover by 8 hours, with near complete recovery by 24 hours. Moreover, pevonedistat did not disrupt allogeneic (OCI-LY19 cells) or autologous (CD40L-stimulated CLL cells) T-cell cytotoxicity. Meanwhile, CD8 T cells continued to produce perforin and granzyme B.
Conclusions: Our data suggest that pharmacologic targeting of NAE preserves T-cell cytotoxic function and may enhance anti-tumor immunity in CLL. Combined with our earlier reports that targeting NAE kills CLL cells under lymph node-mimicking conditions, these data provide a strong rationale for continued investigation of pevonedistat in CLL and lymphoid malignancies.

Disclosures
Spurgeon: Bristol Myers Squibb: Research Funding; Gilead Sciences, Inc.: Consultancy, Research Funding; Oncternal: Research Funding; Acerta: Research Funding; Genentech: Research Funding; Janssen: Research Funding; Pharmacyclics: Consultancy, Research Funding; MEI Pharma: Consultancy. Berger:Takeda Pharmaceuticals International Co.: Employment. Danilov:Gilead Sciences: Consultancy, Research Funding; Astra Zeneca: Consultancy; Verastem: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Aptose Biosciences: Research Funding; Takeda Oncology: Research Funding; TG Therapeutics: Consultancy; Bayer Oncology: Consultancy, Research Funding.