Pevonedistat Sensitizes Diffuse Large B-Cell Lymphoma Cells to Death Receptor Ligand-Mediated Apoptosis
Authors: Paiva Cody , Godbersen Claire , Danes Christopher George , Spurgeon Stephen E , Berger Allison , Danilov Alexey V 
Published: October 2019

Abstract
Background Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma in the U.S. DLBCL can be subdivided into the activated B-cell-like (ABC) and germinal center B-cell-like (GCB) subgroups on the basis of gene expression profiling results. ABC-DLBCL is characterized by the constitutive activation of nuclear factor-κB (NFκB) transcription factor and high expression level of its target genes, including pro-survival BCL2 family members, cell cycles proteins, and chemokines. We and others have demonstrated that pevonedistat (MLN4924), an inhibitor of the NEDD8-activating enzyme, abrogates NFκB activity in DLBCL cell lines and primary chronic lymphocytic leukemia (CLL) cells. Pevonedistat has been shown to sensitize solid tumor cell lines to extrinsic apoptosis. Here we demonstrate that pevonedistat sensitizes ABC-DLBCL cells to death receptor agonists. By contrast, GCB-DLBCL and primary CLL cells are not sensitive to this combination.
Methods We employed a panel of ABC- (OCI-LY3, U2932) and GCB-DLBCL (SUDHL6, SUDHL10, VAL) cell lines. CLL B-cells were obtained from patients in the hematology clinics at the Knight Cancer Institute and were subjected to in vitro co-cultures with CD40L-expressing stroma which we have previously shown to activate NFκB and thereby mimic the lymph node microenvironment. We used the Fas agonists Apo-1-L, CH-11 and SuperFasLigand as well as KillerTRAIL. Pevonedistat was provided by Millennium Pharmaceuticals Inc., a subsidiary of Takeda Pharmaceutical Company Limited.
Results Either Fas or TRAIL agonists, as well as 0.5 µM pevonedistat alone induced modest apoptosis of ABC-DLBCL cells (<10%) upon a 24 hour incubation. Meanwhile, pre-treatment of OCI-LY3 cells with 0.5 µM pevonedistat overnight resulted in apoptosis of 72.6±6.2% cells following exposure to 100 ng/mL KillerTrail and 79.6±7.5% - 10 ng/mL SuperFasLigand (p<0.0001 compared with either agent alone). Similar findings were observed in U2932 cells. By contrast, pevonedistat had a minimal effect on death receptor ligand-mediated apoptosis in the GCB-DLBCL cell lines.
Using immunoprecipitation, we confirmed that Fas engagement led to rapid assembly of the death-inducing signaling complex in OCI-LY3 cells (within 2 hours). This was accompanied by activation of caspase-8, as manifested by generation of its active forms (p41/43). Pre-treatment with pevonedistat promoted sustained and prolonged Fas-mediated cleavage of caspase-8 (for 6 hours, as opposed to transient cleavage following isolated Fas stimulation), accompanied by caspase-mediated PARP processing. We found that pevonedistat treatment downmodulated the transcript and protein levels of cFLIP in OCI-LY3 cells, an antagonistic caspase-8 homolog, whose expression is highly dependent on NFκB transcriptional activity. This is consistent with earlier findings where abrogation of NF-κB transcriptional activity was the dominant event preceding pevodinestat-induced apoptosis in ABC-DLBCL cells. By contrast, in GCB-DLBCL cells, targeting neddylation resulted in accumulation of Cdt1, DNA re-replication and cell cycle arrest. Thus, preferential sensitivity of ABC-DLBCL to death receptor ligands in combination with pevonedistat may be dependent on the effects of the latter on NFκB signaling and cFLIP.
We previously reported that, like in ABC-DLBCL, pevonedistat abrogates NFκB in CD40-stimulated primary CLL cells. We found that CD40 upregulated Fas and TRAIL receptors, as well as cFLIP, in CLL cells. While treatment with pevonedistat downregulated cFLIP in CLL cells, it did not sensitize CLL cells to Fas or TRAIL; caspase-8 cleavage and Bid processing were not induced. Conversely, neutralizing antibodies against TNFα and TRAIL-R1/2 did not modulate the pro-apoptotic activity of pevonedistat in CLL. Hence, distinct from observations in normal monocytes, pevonedistat did not enhance death receptor-mediated apoptosis, consistent with previous reports indicating that such signaling is dysfunctional in CLL cells.
Conclusions Here we demonstrate that pevonedistat sensitizes ABC-DLBCL cells to death receptor agonists by a mechanism which includes downmodulation of cFLIP. Our data provide rationale for further development of pharmacologic agents including pevonedistat in strategies which enhance death receptor signaling in lymphoid malignancies.

Disclosures
Danes: 3Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Ltd.: Employment. Spurgeon:Gilead sciences: Honoraria, Research Funding; Bristol Meyers Squibb: Research Funding; Acerta Pharma: Research Funding; Pharmacyclics: Honoraria; Janssen: Research Funding; Genentech: Honoraria. Berger:3Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Ltd.: Employment.