Pevonedistat, a NEDD8-Activating Enzyme Inhibitor, Induces Apoptosis and Augments Efficacy of Chemotherapy and Small Molecule Inhibitors in Pre-clinical Models of Diffuse Large B-cell Lymphoma

Abstract

We studied the biological activity of pevonedistat, a first-in-class NEDD8-activating enzyme (NAE) inhibitor, in combination with various cytotoxic chemotherapy agents and small molecule inhibitors in lymphoma pre-clinical models. Pevonedistat induced cell death in activated B-cell (ABC) diffuse large B-cell lymphoma (DLBCL) cell lines and to a lesser degree in germinal center B-cell (GCB) DLBCL cell lines. In pevonedistat sensitive cells, we observed inhibition of NFκB activity by p65 co-localization studies, decreased expression of BCL-2/BCL-XL and upregulation of BAK levels. Pevonedistat enhanced the activity of cytarabine, cisplatin, doxorubicin and etoposide in ABC-, but not in the GCB-DLBCL cell lines. It also exhibited synergy with ibrutinib, selinexor, venetoclax and A-1331852 (a novel BCL-XL inhibitor). In vivo, the combination of pevonedistat and ibrutinib or pevonedistat and cytarabine prolonged survival in SCID mice xenograft models when compared with monotherapy controls. Our data suggest that targeting the neddylation pathway in DLBCL is a viable therapeutic strategy and support further clinical studies of pevonedistat as a single agent or in combination with chemotherapy or novel targeted agents.

Keywords: Apoptosis; MLN4924; NFκB; Non-Hodgkin lymphoma; Ubiquitin Proteasome System (UPS).

FIGURE 1

Pevonedistat induces a dose‐dependent and time‐dependent reduction in cell viability and cellular growth in DLBCL cell lines and exhibits a variable degree of activity in tumor cells derived from B‐NHL patients. (A) In vitro exposure of DLBCL cell lines to pevonedistat resulted in dose‐ and time‐dependent (24‐hour, 48‐hour and 72‐hour time points shown here) cell death in ABC‐DLBCL (HBL‐1, TMD8, U2932, U2932 4RH) and GCB‐DLBCL (BJAB, DB, HT, Karpas 422, NUDHL‐1, OCI‐LY2, OCI‐LY19, RL, RL4RH, SUDHL‐4, SUDHL‐6, SUDHL‐10) cells. 4RH denotes rituximab‐resistant cell lines. All cell lines were exposed to escalating doses of pevonedistat (0.313‐10 µM) for 24, 48, and 72 hours. Cell death was determined by Cell Titer Glo luminescence assay. (B) Pevonedistat IC₅₀ levels at 48 h. IC₅₀ was lower in the ABC‐DLBCL cell lines. (C) Ex vivo exposure of primary tumor cells derived from patients with B‐cell malignancies such as follicular lymphoma, small lymphocytic leukemia/chronic lymphocytic leukemia, marginal zone lymphoma, Hodgkin's lymphoma and mantle cell lymphoma to pevonedistat resulted in varying degrees of cell death. Tumor cells were obtained by B‐cell enrichment of fresh biopsy samples. (D) Ex vivo exposure of primary tumor cells derived from patients to pevonedistat lead to greater than 20% decrease in viability in two of three ABC‐DLBCL samples whereas no effect was noted in a GBC‐DLBCL sample. (E) Clinical and pathological characteristics from which primary tumor cells derived from DLBCL patients were obtained. Experiments were repeated three separate times and were reported as the median with standard deviation error bars (SE). Pevonedistat or vehicle control was utilized at 500 nM in patients with DLBCL. Cell death was determined by Cell Titer Glo luminescence assay. *P < .05

FIGURE 2

Pevonedistat in vitro exposure altered the balance of Bcl‐2 family member proteins favoring the induction of apoptosis. (A) DLBCL cell lines were exposed to pevonedistat (SUDHL4 100 nM, OCI‐LY2 125 nM, TMD8 25 nM, and U2932 50 nM), Sytox Blue and PE‐Cyanine7 Annexin‐V, fixed and analyzed by flow cytometry. The ABC‐DLBCL cell lines (TMD8 and U2932) were more sensitive to pevonedistat compared to the GCB‐DLBCL cell lines (SUDHL4 and OCI‐LY2). Experiments were repeated three separate times and were reported as the median. (B) In vitro exposure of two ABC‐DLBCL cell lines (TMD8 and U2932) and GCB‐DLBCL cell lines (SUDHL‐4 and OCI‐LY2) to pevonedistat for 48 hours altered the balance of pro‐apoptotic (Bak) and anti‐apoptotic (BCL‐2 and Bcl‐XL) Bcl‐2 family members leading to PARP cleavage. Cells were treated with Pevonedistat (IC₂₅ and IC₅₀, derived from Annexin PI staining) for 48 hours followed by protein extraction. Protein was separated using a 12% sodium dodecyl sulfate (SDS) PAGE gel and transferred to a polyvinylidene difluoride (PVDF) membrane. Primary rabbit anti‐human antibodies were used a 1:1000 while secondary anti‐rabbit antibodies were used at 1:10000

FIGURE 3

In vitro exposure of DLBCL cell lines to pevonedistat decreases NFκB activity. (A) A panel of DLBCL cell lines were exposed to pevonedistat (0.5‐5 µM) for 1 and 4 h. Changes in NFκB activation were analyzed using ImageStream technology. Nuclear p65 translocation into the nuclei is reported as similarity score (SS) of Dapi and p65 images. The RD metric reports the changes in SS by measuring the shift between two distributions. (B) Changes in NFκB p65 expression were analyzed by western blotting following pevonedistat in vitro exposure. NFκB p65 expression was decreased in U2932 and DHL‐4, but not in TMD8 and OCI‐LY2

FIGURE 4

In vitro effects of pevonedistat on the antitumor activity of chemotherapy agents. TMD8, U2932, SUDHL‐4 and OCI‐LY2 cells were pretreated with pevonedistat for 48 h at 0.5 and 1 µM respectively. Cells were then washed and exposed to Cisplatin (1.56‐50µM) (A), Cytarabine (1.56‐50 µM) (B), Doxorubicin (0.125‐4µM) (C), or Etoposide (1.56‐50µM) (D) for 48 h. Viability was determined by Cell Titer Glo luminescence assay. Experiments were performed in triplicates. All four chemotherapeutic agents showed synergy with pevonedistat in TMD8 and U2932. Cisplatin, doxorubicin and etoposide also showed synergy with pevonedistat pretreatment in OCI‐LY2 cells, but to a lesser extent

FIGURE 5

In vitro effects on pevonedistat on the anti‐tumor activity of small molecule inhibitors. Pevonedistat demonstrated significant synergy with A‐1331852 (A), venetoclax (B), ibrutinib (C) and selinexor (D). DLBCL cells (0.25 × 10⁶/mL) were exposed to pevonedistat (0.977‐4 µM) and/or ibrutinib (0.39‐250 nM), selinexor (0.0625‐10 µM), A‐1331852 (0.0977‐6.25 µM) and venetoclax (0.1‐1000 nM) for 72 h and cell viability assessed by PrestoBlue®. Synergistic activity between pevonedistat and other agents was evaluated using the CalcuSyn Software Version 2.11 (Biosoft, Great Shelford, Cambridge, United Kingdom)

FIGURE 5

In vitro effects on pevonedistat on the anti‐tumor activity of small molecule inhibitors. Pevonedistat demonstrated significant synergy with A‐1331852 (A), venetoclax (B), ibrutinib (C) and selinexor (D). DLBCL cells (0.25 × 10⁶/mL) were exposed to pevonedistat (0.977‐4 µM) and/or ibrutinib (0.39‐250 nM), selinexor (0.0625‐10 µM), A‐1331852 (0.0977‐6.25 µM) and venetoclax (0.1‐1000 nM) for 72 h and cell viability assessed by PrestoBlue®. Synergistic activity between pevonedistat and other agents was evaluated using the CalcuSyn Software Version 2.11 (Biosoft, Great Shelford, Cambridge, United Kingdom)

FIGURE 6

Effect of Pevonedistat on the anti‐tumor activity of cytarabine, ibrutinib and rituximab in vivo. In vivo, pevonedistat in combination with cytarabine or ibrutinib improved the median survival (not reached at the time of sacking the TMD8‐bearing SCID mice on 214th day post‐treatment) compared to cytarabine (41 days) (A) or ibrutinib (47 days) (B) monotherapy (P < .001). The combination of pevonedistat with rituximab did not improve survival compared to rituximab alone (C). Survival differences between groups were compared using log rank analysis. P values are of combination compared to single agent treatment. Experiments were repeated three separate times. (n/a = Not reached)