Small Molecule Inhibitor Of NEDD8-activating Enzyme Research Articles

Neddylation in the chronically hypoperfused corpus callosum: MLN4924 reduces blood-brain barrier injury via ERK5/KLF2 signaling

Blood-brain barrier (BBB) breakdown and cerebrovascular dysfunction may contribute to the pathology in white matter lesions and consequent cognitive decline caused by cerebral hypoperfusion. Neddylation is the process of attaching a ubiquitin-like molecule NEDD8 (neuronal precursor cell-expressed developmentally downregulated protein 8) to specific targets. By modifying protein substrates, neddylation plays critical roles in various important biological processes. However, whether neddylation influences the pathogenesis of hypoperfused brain remains unclear. In the present study, cerebral hypoperfusion-induced white matter lesions were produced by bilateral common carotid artery stenosis in mice. The function of the neddylation pathway, BBB integrity, cerebrovascular dysfunction, myelin density in the corpus callosum and cognitive function were determined. We show that NEDD8 conjugation aberrantly amplified in microvascular endothelium in the corpus callosum following cerebral hypoperfusion. MLN4924, a small-molecule inhibitor of NEDD8-activating enzyme currently in clinical trials, preserved BBB integrity, attenuated glial activation and enhanced oligodendrocyte differentiation, and reduced hypoperfusion-induced white matter lesions in the corpus callosum and thus improved cognitive performance via inactivating cullin-RING E3 ligase (CRL). Administration of MLN4924 caused the accumulation of ERK5 and KLF2. The ERK5 inhibitor BIX 02189, down-regulated MLN4924-induced activation of KLF2 and reversed MLN4924-mediated increase in pericyte coverage and junctional proteins. Furthermore, BIX 02189 blocked MLN4924-afforded protection against BBB disruption and white matter lesions in the corpus callosum. Collectively, our results revealed that neddylation impairs vascular function and thus exacerbated the pathology of hypoperfused brain and that inhibition of neddylation with MLN4924 may offer novel therapeutic opportunities for cerebral hypoperfusion-associated cognitive impairment.

A Phase II Trial of Pevonedistat and Docetaxel in Patients With Previously Treated Advanced Non–Small-Cell Lung Cancer

BackgroundPostimmunotherapy (IO) treatment options for stage IV non–small-cell lung cancer (NSCLC) remain limited. Docetaxel alone or in combination with ramucirumab remains a standard of care, but response rates and survival benefit are suboptimal. Cullin-RING ligases (CRL) catalyze degradation of tumor suppressor proteins and are overactivated in NSCLC. Neddylation, which is catalyzed by the NEDD8 activating enzyme (NAE), is required for the activation of CRLs. Pevonedistat, a first-in-class small molecule NAE inhibitor, exerted antitumor activity when combined with docetaxel in preclinical studies. MethodsWe conducted a phase II, single-arm, investigator-initiated study evaluating the efficacy of pevonedistat plus docetaxel in patients with relapsed/refractory stage IV NSCLC. Patients received docetaxel 75 mg/m2 on day 1 and pevonedistat 25 mg/m2 on days 1, 3 and 5 of a 21-day cycle. The primary endpoint was objective response rate (ORR). ResultsFrom March 5, 2018 to January 26, 2021, we enrolled 31 patients. The ORR was 22% (1 CR, 5 PR), median PFS was 4.1 months, and median OS was 13.2 months. The incidence of Grade ≥3 adverse events (AE) was 53% in patients (n = 30) who received at least 1 dose of both drugs, with the most frequent being neutropenia and AST/ALT elevation. One patient was taken off study for a Grade 4 transaminase elevation. There were no Grade 5 toxicities. ConclusionOur data suggest that the combination of docetaxel and pevonedistat is safe and exerts activity in patients with relapsed NSCLC. These encouraging results suggest that the neddylation pathway is an antitumor pathway that should be further studied.

A phase II trial of pevonedistat plus docetaxel in patients with previously treated advanced non–small-cell lung cancer (NSCLC).

e21062 Background: For patients with stage IV NSCLC, treatment options are limited after progression on immunotherapy (IO) +/- platinum-based chemotherapy. Docetaxel alone or in combination with ramucirumab remains a standard of care, but response rates and survival benefit are suboptimal. Cullin-RING ligases (CRLs) catalyze the ubiquitylation and degradation of tumor suppressor proteins and are overactivated in NSCLC. Therefore, inhibition of CRLs has potential therapeutic value. Neddylation is required to activate CRLs; NAE (NEDD8 activating enzyme) catalyzes neddylation. In pre-clinical studies, pevonedistat, a first-in-class small molecule NAE inhibitor, exerts anti-tumor effects when combined with docetaxel. Methods: We conducted a phase II, single-arm, investigator-initiated study evaluating the efficacy of pevonedistat plus docetaxel in patients with stage IV NSCLC with progression on or after platinum-based chemotherapy +/- IO. Patients with tumors that had driver mutations in EGFR, ALK, ROS1, and BRAF must have also had progression on targeted inhibitors. A Simon Optimal two-stage design was utilized with 90% power and 15% type I error to detect the drug as promising when the true response rate is 25% and the historical control rate is 10%. A maximum of 37 response-evaluable (R-E) patients could be enrolled. Patients received docetaxel 75 mg/m2 on day 1 and pevonedistat 25 mg/m2 on days 1, 3 & 5 of a 21-day cycle. Response was assessed every 2 cycles based on RECIST v1.1 criteria. The primary objective was overall response rate (ORR). Those who received 2 cycles or had progression prior to the end of cycle 2 were considered R-E. Patients who received any treatment on protocol were evaluable for safety (S-E). Results: From 3/5/2018 to 1/26/2021, we enrolled 31 patients and 27 were R-E with a median follow-up of 38.7 months. The trial was terminated early due to the sponsor’s decision to discontinue the development of pevonedistat. In the R-E population, 52% were female with a median age of 63 and 85% had adenocarcinoma. The median number of prior regimens was 2, and 92% of patients had previously received IO. The ORR was 22% (95%CI 8.6-42.3%) and the disease control rate (SD+CR+PR) was 66.7% (95%IC 46.0- 83.5%). The median PFS was 4.1 (2.8-5.8) months and median OS was 13.1 (6.7-24.8) months. The median duration of response was 14.5 months. The incidence of Grade 3-4 adverse events (AE) was 53% in the 30 S-E patients. The most common grade 3-4 AEs were decrease in blood counts (anemia and neutropenia) and elevation in liver transaminases. There were no Grade 5 toxicities. Conclusions: Our data suggest that the combination of docetaxel and pevonedistat is safe and may have improved anti-tumor activity compared to historical data with docetaxel alone. These results suggest that the neddylation pathway is a targetable anti-tumor pathway that should be further studied in patients with NSCLC. Clinical trial information: NCT03228186 .

Inhibition of NEDD8 NEDDylation induced apoptosis in acute myeloid leukemia cells via p53 signaling pathway.

MLN4924 is a potent and selective small-molecule inhibitor of NEDD8-activating enzyme, which showed antitumor effect in several types of malignant tumor types. However, the mechanism of action of MLN4924 in acute myeloid leukemia (AML) requires further investigation. Real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) was conducted to detect the mRNA levels of genes. Gene expression was knocked down by short hairpin RNA (shRNA). Moreover, the protein expression was detected by Western blotting (WB) assay. The proliferation and apoptosis of AML cells were measured by Cell Counting Kit-8 (CCK8) assay and flow cytometry (FCM). In the present study, we observed that the mRNA expression levels of NEDD8, UBA3, UBE2M and RBX1 in AML patients were up-regulated compared with healthy controls, which were correlated with worse overall survival (OS) of patients. Besides, knockdown of UBA3, UBE2M and RBX1 inhibited the NEDDylation of CULs and increased the protein expression of p53 and p21 in MOLM-13 cell line. In AML cells, MLN4924 inhibited cell proliferation, promoted cell apoptosis, and induced cell cycle arrest at the G2/M phase. As revealed by experiments in vivo and in vitro, the NEDDylation of CULs was significantly inhibited and the p53 signaling pathway was activated after MLN4924 treatment. So, we concluded that NEDD8, UBA3, UBE2M and RBX1 may serve as the prognostic biomarkers and novel therapeutic targets for AML. Inhibition of the NEDDylation pathway resulted in an anti-leukemia effect by activating the p53 signaling pathway.

Celecoxib Synergistically Enhances MLN4924-Induced Cytotoxicity and EMT Inhibition Via AKT and ERK Pathways in Human Urothelial Carcinoma

MLN4924 is a specific small-molecule inhibitor of NEDD8-activating enzyme (NAE) that blocks the neddylation modification cascade. Several I/II/III clinical trials suggested that MLN4924 exerts an antitumor effect against various malignancies. However, recent studies have also found that MLN4924 activates the PI3K/AKT and MAPK/ERK signal pathways, important regulators of tumorigenesis, and drug resistance in human urothelial carcinoma (UC). This study examined the synergistic effect of celecoxib, a cyclooxygenase-2 (COX-2) selective inhibitor, on MLN4924-induced cytotoxicity and epithelial–mesenchymal transition (EMT) inhibition via AKT and ERK pathways in human UC. We performed both in vitro and in vivo experiments. Briefly, a combination of MLN4924 and celecoxib reduced the protein expression of p-AKT(S473) and p-ERK in UC cell lines. Moreover, celecoxib shifted the half-maximal inhibitory concentration (IC50) curve of MLN4924 to the left, and the combinational effect of MLN4924 and celecoxib showed significant synergism in T24 and 5637 cells. Also, celecoxib enhanced the MLN4924 antitumor effects of inhibiting UC cell growth, colony formation, migration, invasion, and inducing apoptosis. In addition, celecoxib potentiated the MLN4924-induced EMT, decreased the expression of N-cadherin and vimentin, and activated the expression of E-cadherin. Celecoxib also increased the expression of pro-apoptosis proteins PARP and BAX and reduced the expression of antiapoptosis protein Bcl2. In vivo study indicated that the combination of MLN4924 and celecoxib synergistically suppressed the tumor growth in a UC xenograft nude-mice model, which was further supported by immunohistochemistry of tumor tissues. To sum up, our study revealed that celecoxib synergistically enhanced MLN4924-induced cytotoxicity and EMT inhibition in UC. It also inhibited the activation of AKT and ERK pathways, which were activated by MLN4924. These discoveries provide a new drug combination strategy for UC treatment.

MDS-336: Phase 2 Study of Pevonedistat + Azacitidine versus Azacitidine in Patients with Higher-Risk Myelodysplastic Syndromes (MDS)/Chronic Myelomonocytic Leukemia (CMML) or Low-Blast Acute Myelogenous Leukemia (LB-AML) (NCT02610777): Subset Analysis in Higher-Risk MDS

Context: Pevonedistat, the first small-molecule inhibitor of NEDD8-activating enzyme, disrupts proteasomal degradation of select proteins. Setting: Patients with higher-risk MDS/CMML (IPSS-R >3, including intermediate [≥5% blasts], high, or very high risk) or LB-AML naive to hypomethylating agents were randomized 1:1 to receive intravenous pevonedistat 20 mg/m2 on days 1, 3, and 5 plus intravenous/subcutaneous azacitidine 75 mg/m2 on days 1–5, 8, and 9 (n=58), or azacitidine alone (n=62), in 28-day cycles. This abstract focuses on the higher-risk MDS subgroup. Main outcome measures: The study was powered on an endpoint of event-free survival (EFS). Overall survival (OS), overall response rate (ORR) and safety were also assessed. Patient-reported health-related quality of life (HRQoL) was evaluated using the European Organization for Research and Treatment of Cancer (EORTC) QoL Questionnaire-C30. Mutational profiling was performed on screening bone marrow aspirate samples. Results: In the intent-to-treat (ITT) population (n=120), EFS trended longer (median, 21.0 vs 16.6 months; hazard ratio [HR]=0.67; 95% CI, 0.42–1.05; P=.076) and median OS was 21.8 vs 19.0 months (HR=0.80; 95% CI, 0.51–1.26; P=.334) with pevonedistat+azacitidine vs azacitidine. In patients with higher-risk MDS (n=67), EFS was longer (median, 20.2 vs 14.8 months; HR=0.54; 95% CI, 0.29–1.00; P=0.045) and median OS was 23.9 vs 19.1 months (HR=0.70; 95% CI, 0.39–1.27; P=0.240) with pevonedistat+azacitidine vs azacitidine. In response-evaluable patients with higher-risk MDS (n=59), ORR was 79% (pevonedistat+azacitidine) vs 57% (azacitidine); median duration of response was 34.6 vs 13.1 months, respectively. Median azacitidine dose intensity was 98% (both arms) in patients with higher-risk MDS. With pevonedistat+azacitidine vs azacitidine, 94% vs 83% of patients with higher-risk MDS had grade ≥3 adverse events (most common: neutropenia [38% vs 37%], febrile neutropenia [22% vs 31%]). No difference was observed in patient-reported HRQoL between arms, with similar mean scores maintained from study entry to end of treatment. Clinical activity was observed with pevonedistat+azacitidine in patients with higher-risk MDS harboring poor prognostic mutations. Conclusions: Pevonedistat+azacitidine led to longer EFS vs azacitidine in higher-risk MDS, had a comparable safety profile to azacitidine alone, and azacitidine dose intensity was maintained. A randomized phase 3 trial ( NCT03268954 ) is ongoing.

Immunomodulatory effects of pevonedistat, a NEDD8-activating enzyme inhibitor, in chronic lymphocytic leukemia-derived T cells.

Novel targeted agents used in therapy of lymphoid malignancies, such as inhibitors of B-cell receptor-associated kinases, are recognized to have complex immune-mediated effects. NEDD8-activating enzyme (NAE) has been identified as a tractable target in chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma. We and others have shown that pevonedistat (TAK-924), a small-molecule inhibitor of NAE, abrogates NF-κB signaling in malignant B cells. However, NF-κB pathway activity is indispensable in immune response, and T-cell function is altered in patients with CLL. Using T cells derived from patients with CLL, we demonstrate that although targeting NAE results in markedly differential expression of NF-κB-regulated genes and downregulation of interleukin (IL)-2 signaling during T-cell activation, T cells evade apoptosis. Meanwhile, NAE inhibition favorably modulates polarization of T cells in vitro, with decreased Treg differentiation and a shift toward TH1 phenotype, accompanied by increased interferon-γ production. These findings were recapitulated in vivo in immunocompetent mouse models. T cells exposed to pevonedistat in washout experiments, informed by its human pharmacokinetic profile, recover NAE activity, and maintain their response to T-cell receptor stimulation and cytotoxic potential. Our data shed light on the potential immune implications of targeting neddylation in CLL and lymphoid malignancies.

Targeting NEDD8-activating enzyme is a new approach to treat canine diffuse large B-cell lymphoma.

Canine diffuse large B-cell lymphoma (DLBCL), the most common hematologic malignancy of dogs, is associated with poor overall survival. The lack of conventional chemotherapies with sustainable efficacy warrants investigation of novel therapies. Pevonedistat (MLN4924) is a potent and selective small molecule NEDD8-activating enzyme inhibitor. In human activated B-cell-like (ABC) diffuse large B-cell lymphoma, pevonedistat induces lymphoma cell apoptosis, DNA damage and G1 cell cycle arrest by inhibiting the nuclear factor-κB (NF-κB) pathway. Genomic and transcriptomic studies showed that the NF-κB pathway is deregulated in canine DLBCL. Our results showed that pevonedistat treatment significantly reduces the viability of canine DLBCL cells by inducing G1 cell cycle arrest and apoptosis. Pevonedistat treatment inhibits NF-κB pathway activation and downregulates NF-κB target genes in canine DLBCL. Moreover, administration of pevonedistat to mice bearing canine DLBCL xenograft tumours resulted in tumour regression. Our in vivo and in vitro studies provide justification for future clinical application of pevonedistat as a potential new anti-cancer therapy that may benefit both canine and human species.

Drug Sensitivity Across Acute Myeloid Leukemia Subtypes Using an in Vitro Assay

Introduction:Acute myeloid leukemia (AML) remains a challenging malignancy to treat, with high mortality despite recent advances in cancer care. The mainstay of therapy is intensive chemotherapy including anthracyclines and antimetabolites, with or without allogeneic bone marrow transplant. The standard of care has not changed significantly in decades, and treatment options are limited for patients who do not respond to induction, or who relapse. Although the genetic and molecular diversity of AML is well recognized, the effective integration of targeted therapies into treatment regimens has been difficult. The current study evaluates the in vitro activity of an array of antineoplastic agents in AML, with the goal of identifying drug sensitivity patterns that may help guide therapies based on mutational and cytogenetic profiling.Methods:51 patients with AML were enrolled in the study from September 2013 to July 2016 under an IRB approved consenting process at Scripps Health. Samples were collected at both diagnosis and relapse if available. Both bone marrow and peripheral blood were accepted, with a requirement of significant circulating blasts if peripheral blood was used. Samples were tested for common biomarkers and cytogenetic abnormalities. 2,500 cells per well were transferred onto tissue culture treated plates. Drugs with potential antileukemic activity were added to each well in concentrations of 0.1, 1.0, and 10.0 μM. The cells were incubated with the drugs for 96 hours at 37oC. Cell viability was measured and reported as a percentage of plate-specific controls incubated with dimethylsulfoxide alone.Results:Drugs were grouped by therapeutic class. In vitro responses to anthracyclines and antimetabolites were noted across all mutational subtypes of AML. BCL-2 inhibitors and the histone deacetylase inhibitor romidepsin showed significant in vitro antileukemic activity across all subtypes. Proteasome inhibitors almost universally showed robust in vitro activity, even at the lowest drug concentration.The drug pevonedistat, a selective small-molecule inhibitor of NEDD8-activating enzyme, had significant differential activity depending on mutational status. FLT3-ITD mutations conferred sensitivity to the molecule, while mutations in NPM1 appeared to confer resistance. For the 10.0 μM concentration of pevonedistat, the average cell viability was 149.8% vs 37.3% (P=0.02) for the NPM1 mutated samples vs the FLT3-ITD mutated samples respectively.Conclusion:This in vitro assay demonstrates the ability to rapidly determine sensitivity of human AML cells to a wide variety of antileukemic drugs. Limitations include the fixed concentrations used across all medications which allowed comparisons between patients, but limits comparison of drug efficacy for an individual patient. The mechanism of some medications, such as hypomethylating agents and tretinoin, may require longer duration of exposure, thus confounding interpretation of the 96-hour viability results. Despite these limitations, we were able to find interesting patterns of responses across a wide spectrum of AML types. Anticipated applications of the assay include experimentation with novel drug combinations, directing in vivo clinical studies, and informing individualized treatment decisions in AML. DisclosuresNo relevant conflicts of interest to declare.

Abstract B148: Population pharmacokinetics (PK) of the investigational NEDD8-activating enzyme (NAE) inhibitor pevonedistat (TAK-924/MLN4924) administered alone or in combination with azacitidine in patients (pts) with solid or hematologic malignancies

Abstract Introduction: Pevonedistat is a first-in-class small molecule inhibitor of NAE that is currently in phase 1b clinical development across multiple solid tumors and in acute myeloid leukemia (AML). Here we report a population modeling analysis to describe pevonedistat IV PK in cancer pts and identify potential predictors of interpatient variability in pevonedistat exposures when given alone or combined with azacitidine. Methods: Plasma concentration-time data were analyzed using nonlinear mixed effects modeling (NONMEM 7.2). Two- and 3-compartment linear disposition with zero-order input models were tested. Subsequently, effects of selected demographic (age, sex, race, and body size) and physiologic (hepatic transaminases, bilirubin, blood parameters, and creatinine clearance [CRCL]) covariates were evaluated. First Order Conditional Estimation method with log transform-both-sides was used. Model goodness-of-fit and performance (visual predictive check and nonparametric bootstrapping) were also assessed. Results: A total of 250 pts (64% male; 80% white) from 5 completed or ongoing phase 1/1b dose-escalation clinical studies contributed 2882 observations. Of these, 42% were 65 yrs or older and 18% were 75 yrs or older. Half of pts had normal renal function, while 34% and 17% had mild (CRCL 60-89 mL/min) to moderate (CRCL 30-59 mL/min) renal impairment, respectively. Data were described by a 2-compartment PK model parameterized in terms of systemic clearance (CL), intercompartmental clearance (Q), and central (Vc) and peripheral (Vp) volumes of distribution with associated between-subject variability (BSV). Terms describing correlations between BSV for CL, Q, and Vp were included. Body size was described as an allometric function with the effect of body surface area (BSA, range 1.38-3.00 m2) on all parameters. Age (range 19-90 yrs) on CL was included as a power function normalized by the reference age of 65 yrs. Categorical covariates (sex and race) were modeled to estimate the proportional change in the typical value of the PK parameter. Consistent with observations of dose-proportional increases in pevonedistat exposures within each study, CL was constant across doses. Pevonedistat PK was unaffected after daily dosing with azacitidine in pts with AML. Final population mean parameter estimates (95% bootstrap confidence interval [CI]) were CL = 29.0 L/h (25.8, 32.7); Vc = 93.5 L (79.1, 110); Vp = 107 L (99.3, 115); and Q = 20.1 L/h (18.3, 22.2). Proportional residual variability was 28%. Upon exploration of patient-specific covariate effects, body size influenced CL (interindividual variability [IIV] ∼25% CV) and volume of distribution (IIV∼22% CV). Pevonedistat CL tended to gradually decrease with age (∼25% over the 30-90 yrs age range). There was no apparent effect of renal function status (CRCL >30 mL/min) on pevonedistat PK. Conclusions: The clinical PK profile of pevonedistat is comparable in pts with solid tumors, lymphomas, or AML. No changes were observed in the presence of azacitidine when compared to historical single-agent data. Elderly pts may have reduced pevonedistat CL; additional monitoring of these pts is recommended. Citation Format: Hélène M. Faessel, Diane R. Mould, Bruce J. Dezube, Sergio Santillana, Karthik Venkatakrishnan. Population pharmacokinetics (PK) of the investigational NEDD8-activating enzyme (NAE) inhibitor pevonedistat (TAK-924/MLN4924) administered alone or in combination with azacitidine in patients (pts) with solid or hematologic malignancies. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B148.

High Efficiency Apoptosis Induction in Breast Cancer Cell Lines by MLN4924/2DG Co-Treatment.

2-deoxy-D-Glucose (2DG) causes cytotoxicity in cancer cells by disrupting thiol metabolism. It is an effective component in therapeutic strategies. It targets the metabolism of cancer cells with glycolysis inhibitory activity. On the other hand, MLN4924, a newly discovered investigational small molecule inhibitor of NAE (NEDD8 activating enzyme), inactivates SCF E3 ligase and causes accumulation of its substrates which triggers apoptosis. Combination of these components might provide a more efficient approach to treatment. In this research, 2DG and MLN4924 were co-applied to breast cancer cells (MCF-7 and SKBR-3) and cytotoxic and apoptotic activity were evaluated the by Micro culture tetrazolium test (MTT), TUNEL and ELISA methods. Caspase3 and Bcl2 genes expression were evaluated by real time Q-PCR methods. The results showed that MLN4924 and MLN4924/2DG dose-dependently suppressed the proliferation of MCF7 and SKBR-3 cells. Cell survival of breast cancer cells exposed to the combination of 2DG/MLN4924 was decreased significantly compared to controls (p<0.05), while 2DG and MLN4924 alone had less pronounced effects on the cells. The obtained results suggest that 2DG/MLN4924 is much more efficient in breast cancer cell lines with enhanced cytotoxicity via inducing a apoptosis cell signaling gene, caspase-3.

Immunomodulatory Drugs Are Efficacious Against Primary Effusion Lymphoma By Targeting IKZF1, IRF4 and MYC in a CRBN-Dependent Manner and Are Synergistic with BRD4 Inhibitors

Primary effusion lymphoma (PEL) is an aggressive type of non-Hodgkin lymphoma localized predominantly in body cavities. Kaposi’s sarcoma-associated herpes virus is the causative agent of PEL. PEL is an incurable malignancy and has extremely poor prognosis when treated with conventional chemotherapy. PEL cells universally express interferon regulatory factor 4 (IRF4). We found that shRNA-mediated knockdown of IRF4 is toxic to PEL. Further, we observed that FDA-approved immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide show selective cytotoxicity towards PEL in a panel of cell lines comprising 11 hematological malignancies. The cytotoxic effect of IMiDs towards PEL was observed within their physiologically achievable concentrations and involved the downregulation of IRF4 and MYC. Genome-wide microarray examination (illumina) of PEL cells treated with lenalidomide followed by Gene Set Enrichment Analysis (http://www.broadinstitute.org/gsea) revealed activation of interferon (IFN) signaling. Further, recombinant IFNs α, β and γ are very effective and selective against PEL in a panel of cell lines tested. However, blocking the IFN pathway using specific blocking antibodies did not block the anti-proliferative potential of IMiDs towards PEL suggesting that activation of IFN pathway is not responsible for the cytotoxicity of IMiDs against PEL. Ikaros family proteins IKZF1 and IKZF3 are transcription factors which play crucial roles in immunity and cell-fate decisions. Recently, it has been shown that IMiDs selectively degrade these transcription factors (Kronke et al., and Lu et al., Science, 2014). In PEL, IKZF1 is strongly downregulated upon treatment with IMiDs. Additionally, shRNA-mediated knockdown of IKZF1 is toxic to PEL. Cereblon (CRBN) is a direct cellular binding target of IMiDs. IMiDs have no significant effect on the expression of CRBN in PEL and shRNA-mediated knockdown of CRBN (shCRBN) is not toxic to PEL. However, all the anti-PEL effects of IMiDs were blunted in cells expressing shCRBN. CRBN is a component and substrate receptor for Culling-RING ubiquitin ligase complex (CRL) (Ito et al., Science, 2010) and CRL activity depends on NEDDylation (Soucy et al., Nature, 2009). MLN4924, small molecule NEDD8-activating enzyme inhibitor phenocopied the effects of shCRBN on the anti-PEL activities of IMiDs suggesting that CRBN and its CRL activity is essential for the anti-proliferative activity of IMiDs in PEL. MYC is a downstream target of IMiDs in PEL but the role of MYC on the activity of IMiDs is not known. While investigating the role of MYC in IMiDs activity against PEL we found that shRNA-mediated knockdown of MYC enhanced the cytotoxicity of IMiDs suggesting a potential synergism between IMiDs and MYC inhibition. BRD4 inhibitors have been shown to block MYC expression (Delmore et al., Cell, 2011; Mertz et al., PNAS, 2011). We observed that lenalidomide is synergistic with all the BRD4 inhibitors (JQ-1, IBET151 and PFI-1) tested in PEL. Combined treatment of IMiDs with JQ-1 at low doses resulted in comprehensive downregulation of MYC and significant increase in the number of cells undergoing apoptosis as observed by AnnexinV staining and PARP cleavage. Knocking down BRD4 using specific shRNA also enhanced the cytotoxicity of IMiDs towards PEL suggesting that the synergism observed between IMiDs and BRD4 inhibition may not be limited only to the BRD4 inhibitors used in our study. Furthermore, combined administration of lenalidomide and JQ-1 significantly increased the survival of PEL bearing NOD.SCID mice in an orthotopic xenograft model. These results provide the basis for clinical testing of IMiDs alone and in combination with BRD4 inhibitors for PEL. DisclosuresGopalakrishnan:University of Southern California: Inventor on a patent application relevant to this work filed to US patent office (No. 62/031,053). Patents & Royalties. Chaudhary:University of Southern California: Inventor on a patent application relevant to this work filed to US patent office (No. 62/031,053). Patents & Royalties.

Neddylation pathway is up-regulated in human intrahepatic cholangiocarcinoma and serves as a potential therapeutic target.

Therapeutic intervention in neddylation pathway is an emerging area for cancer treatment. Herein, we evaluated the clinical relevance and therapeutic potential of targeting this pathway in intrahepatic cholangiocarcinoma (ICC). Immunohistochemistry of neddylation pathway components in a cohort of 322 cases showed that E1 (NAE1 and UBA3) and E2 (UBC12) enzymes, as well as global NEDD8 conjugation, were upregulated in over 2/3 of human ICC. Notably, NAE1 was identified as an independent prognosticator for postoperative recurrence (P=0.009) and a combination of NEDD8 and NAE1 provided a better power for predicting patient clinical outcomes. In vitro treatment with MLN4924, a small-molecule NEDD8-activating enzyme inhibitor, led to a dose-dependent decrease of viability in both established and primary cholangiocarcinoma cell lines. Additionally, MLN4924 exhibited at least additive effect when combined with cisplatin. By blocking cullins neddylation, MLN4924 inactivated Cullin-Ring ligase (CRL) and caused the accumulation of CRL substrates that triggered cell cycle arrest, senescence or apoptosis. Meanwhile, MLN4924 was well-tolerated and significantly inhibited tumor growth in xenograft model of cholangiocarcinoma. Taken together, our findings indicated that upregulated neddylation pathway was involved in ICC progression and interference in this pathway could be a promising target for ICC therapy.

Targeting Neddylation pathways to inactivate cullin-RING ligases for anticancer therapy.

Protein neddylation is catalyzed by an E1 NEDD8-activating enzyme (NAE), an E2 NEDD8-conjugating enzyme, and an E3 NEDD8 ligase. Known physiological substrates of neddylation are cullin family members. Cullin neddylation leads to activation of cullin-RING ligases (CRLs), the largest family of E3 ubiquitin ligases responsible for ubiquitylation and degradation of many key signaling/regulatory proteins. Thus, through modulating CRLs, neddylation regulates many biological processes, including cell cycle progression, signal transduction, and tumorigenesis. Given that NEDD8 is overexpressed and CRLs are abnormally activated in many human cancers, targeting protein neddylation, in general, and cullin neddylation, in particular, appears to be an attractive anticancer approach. MLN4924, a small molecule inhibitor of NAE, was discovered that inactivates CRLs and causes accumulation of CRL substrates to suppress tumor cell growth both in vitro and in vivo. Promising preclinical results advanced MLN4924 to several clinical trials for anticancer therapy. In preclinical settings, MLN4924 effectively suppresses tumor cell growth by inducing apoptosis, senescence, and autophagy, and causes sensitization to chemoradiation therapies in a cellular context-dependent manner. Signal molecules that determine the cell fate upon MLN4924 treatment, however, remain elusive. Cancer cells develop MLN4924 resistance by selecting target mutations. In the clinical side, several Phase 1b trials are under way to determine the safety and efficacy of MLN4924, acting alone or in combination with conventional chemotherapy, against human solid tumors. In the preclinical side, the efforts are being made to develop additional neddylation inhibitors by targeting NEDD8 E2s and E3s.

MLN4924, an Investigational Small Molecule Inhibitor of NEDD8-Activating Enzyme (NAE), Inhibits NF-κB Activity and Induces G1 Cell Cycle Arrest and Apoptosis in Pre-Clinical Models of Hodgkin Lymphoma (HL)

Abstract 3717The incorporation of combined-modality therapy, risk-stratified chemotherapy selection, high-dose chemotherapy and autologous stem cell support (HDC-ASCS), and monitoring treatment response by functional imaging are factors that have contributed to the improvement in clinical outcomes in HL patients. Unfortunately, those patients not eligible for or that have failed HDC-ASCS remain a challenge for the treating oncologist, stressing the need for novel therapeutic strategies. Significant improvements in the understanding of the biology of HL have been achieved, including cellular pathways altered in HL (e.g. the ubiquitin-proteasome system) and the role of the tumor microenvironment. MLN4924 is an investigational small-molecule inhibitor of NEDD8-activating enzyme (NAE). NAE is an enzyme responsible for activating NEDD8, an ubiquitin-like molecule in the neddylation cascade that is responsible for cullin-ring ligase (CRL) mediated polyubiquitination of proteins targeting them for proteasomal degradation. In order to better understand the activity of MLN4924 in HL, we performed pre-clinical testing in IkB wild type (L-1236), IkB mutated (KM-H2 and L-428) HL cell lines, and in primary tumor cells derived from a HL patient. Malignant cells were exposed to escalating doses of MLN4924 and changes in cell viability were quantified at different time periods by alamar Blue reduction assay. Patient tumor cells were incubated with MLN4924 for 48 hrs and cell viability was determined using the CellTiterGlo assay. Induction of apoptosis in HL cell lines following exposure to MLN4924 was determined by flow cytometry for Annexin-V and propidium iodide (PI) staining and western blot for caspase-3 and PARP cleavage. Cell cycle analysis was performed by flow cytometry using PI staining. Inhibition of NAE by MLN4924 in HL cell lines was measured by western blot for NEDD8-cullin. Finally, changes in NF-kB activity following MLN4924 exposure were determined by p65 nuclear localization using Image stream technology. MLN4924 exhibited a dose- and time-dependent decrease in cell viability in all HL cell lines at nM concentrations. No differences in anti-tumor activity were observed between IkB-wild type (L-1236 IC50 = 250nM) and IkB–mutated HL cell lines (KM-H2 IC50 = 250nM and L-428 IC50 = 300nM). MLN4924 induced apoptosis in a dose-dependent manner in all cell lines tested. In addition, MLN4924 induced cell cycle arrest in G1 phase and inhibition of NAE was demonstrated by a decrease in NEDD8 conjugated CRL. L1236 cells exposed to MLN4924 also demonstrated a decrease in degradation of IκBα as evidenced by increased levels of p-IκBα following exposure to MLN4924 with a corresponding decrease in p65 nuclear translocation. Surprisingly KMH-2 cells, which carry a mutated IκBα protein that is truncated and non-functional, had a decrease in nuclear p65 following exposure to MLN4924, suggesting an alternative mechanism of NF-kB inhibitory activity by MLN4924. In summary, MLN4924 demonstrates activity against HL cells in vitro through inhibition of NF-kB, and is a promising novel agent for the treatment of HL. We continue to investigate the pre-clinical activity of MLN4924 both as a single-agent and in combination with traditional chemotherapy and other novel agents. Disclosures:No relevant conflicts of interest to declare.

The p21-Dependent Radiosensitization of Human Breast Cancer Cells by MLN4924, an Investigational Inhibitor of NEDD8 Activating Enzyme

Radiotherapy is a treatment choice for local control of breast cancer. However, intrinsic radioresistance of cancer cells limits therapeutic efficacy. We have recently validated that SCF (SKP1, Cullins, and F-box protein) E3 ubiquitin ligase is an attractive radiosensitizing target. Here we tested our hypothesis that MLN4924, a newly discovered investigational small molecule inhibitor of NAE (NEDD8 Activating Enzyme) that inactivates SCF E3 ligase, could act as a novel radiosensitizing agent in breast cancer cells. Indeed, we found that MLN4924 effectively inhibited cullin neddylation, and sensitized breast cancer cells to radiation with a sensitivity enhancement ratio (SER) of 1.75 for SK-BR-3 cells and 1.32 for MCF7 cells, respectively. Mechanistically, MLN4924 significantly enhanced radiation-induced G2/M arrest in SK-BR-3 cells, but not in MCF7 cells at early time point, and enhanced radiation-induced apoptosis in both lines at later time point. However, blockage of apoptosis by Z-VAD failed to abrogate MLN4924 radiosensitization, suggesting that apoptosis was not causally related. We further showed that MLN4924 failed to enhance radiation-induced DNA damage response, but did cause minor delay in DNA damage repair. Among a number of tested SCF E3 substrates known to regulate growth arrest, apoptosis and DNA damage response, p21 was the only one showing an enhanced accumulation in MLN4924-radiation combination group, as compared to the single treatment groups. Importantly, p21 knockdown via siRNA partialy inhibited MLN4924-induced G2/M arrest and radiosensitization, indicating a causal role played by p21. Our study suggested that MLN4924 could be further developed as a novel class of radiosensitizer for the treatment of breast cancer.

Treatment-Emergent Mutations in NAEβ Confer Resistance to the NEDD8-Activating Enzyme Inhibitor MLN4924

MLN4924 is an investigational small-molecule inhibitor of NEDD8-activating enzyme (NAE) in clinical trials for the treatment of cancer. MLN4924 is a mechanism-based inhibitor, with enzyme inhibition occurring through the formation of a tight-binding NEDD8-MLN4924 adduct. In cell and xenograft models of cancer, we identified treatment-emergent heterozygous mutations in the adenosine triphosphate binding pocket and NEDD8-binding cleft of NAEβ as the primary mechanism of resistance to MLN4924. Biochemical analyses of NAEβ mutants revealed slower rates of adduct formation and reduced adduct affinity for the mutant enzymes. A compound with tighter binding properties was able to potently inhibit mutant enzymes in cells. These data provide rationales for patient selection and the development of next-generation NAE inhibitors designed to overcome treatment-emergent NAEβ mutations.

Abstract A196: Development, validation, and clinical implementation of a peripheral blood RT-PCR pharmacodynamic assay for MLN4924, an investigational small molecule inhibitor of NEDD8-activating enzyme (NAE).

Abstract The ubiquitin proteasome system regulates the degradation of intracellular proteins that are involved in many cellular processes. These processes, such as cell cycle, DNA replication, and response to oxidative stress play a key role in tumor growth and survival. The ubiquitination and proteasomal degradation of a subset of proteins involved in these processes is controlled by Cullin-RING ubiquitin ligases (CRLs). NEDD8-activating enzyme (NAE) activates the ubiquitin-like protein NEDD8 for conjugation to CRLs and therefore regulates the proteasomal destruction of CRL substrate proteins. NAE inhibition prevents degradation of CRL substrates (e.g. NRF2, CDT1), leading to their accumulation, subsequent apoptosis and cell death. MLN4924 is an investigational small molecule NAE inhibitor with antitumor activity in preclinical models of several tumor types that is currently in Phase I clinical development. MLN4924 is the first NAE inhibitor to enter clinical development, and evidence of target inhibition and/or downstream pathway modulation were key objectives of the Phase I studies. Here we describe the development, validation and clinical implementation of a pharmacodynamic (PD) RT-PCR assay that quantifies levels of gene transcripts regulated in response to NAE inhibition in human whole blood. To develop a prototype PD assay, Affymetrix gene expression profiling studies on HCT116 cells treated in-vitro with an NAE inhibitor were performed. Genes regulated greater than two-fold by NAE inhibition were identified. The list of genes was observed to be enriched for NRF2 regulated transcripts, consistent with the mechanism of action for MLN4924. Twenty-seven of the most robustly regulated genes were tested by RT-PCR (TaqMan) using RNA samples extracted from MLN4924 treated HCT-116 cells and human PBMCs treated ex-vivo with MLN4924 in order to validate the microarray results and develop a RT-PCR based PD assay in human blood. Transcripts showing a robust response to NAE inhibition in both HCT116 cells and PBMCs were identified for further evaluation in whole blood as a preferred sample matrix for clinical studies. To explore the relationship of MLN4924 regulated transcripts with time and drug concentration, blood samples from multiple healthy volunteers were treated ex-vivo with a range of MLN4924 concentrations for three durations (4, 8, 24 hours). Eight genes (NQO1, SLC7A11, TXNRD1, SRXN1, GSR, GCLM, ATF3, and MAG1) that displayed a robust induction (&amp;gt;3-fold) in whole blood were selected for the PD biomarker panel. Inter- and intra-assay variability was established via testing two pools of cDNA control samples (RNA from MLN4924 ex-vivo treated and untreated blood) in replicates on the same plate, and on multiple plates run separately. All genes had inter- and intra-assay CV values of &amp;lt;5%. This RT-PCR panel has been implemented in four Phase I clinical trials with MLN4924 in both solid tumor and hematological malignancies. Each study demonstrates induction of the NAE-regulated transcripts following MLN4924 administration starting at drug doses below the maximum tolerated dose. These results demonstrate the successful development and implementation of a RT-PCR assay that detects downstream effects of NAE inhibition in human whole blood. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A196.

Abstract 740: Pediatric Preclinical Testing Program (PPTP) evaluation of the NEDD8-activating enzyme (NAE) inhibitor MLN4924

Abstract Background: MLN4924 is a potent and selective small molecule NAE inhibitor. In most cancer cells tested, inhibition of NAE leads to induction of DNA rereplication, resulting in DNA damage and cell death. NEDD8-activating enzyme (NAE) is an essential component of the NEDD8 conjugation pathway that controls the activity of the cullin-RING subtype of ubiquitin ligases, thereby regulating the turnover of a subset of proteins upstream of the proteasome. Substrates of cullin-RING ligases have important roles in cellular processes associated with cancer cell growth and survival pathways. The activity of MLN4924 was evaluated against the PPTP's in vitro and in vivo panels. Methods: The PPTP includes a molecularly characterized in vitro panel of cell lines (n=27) and in vivo panel of xenografts (n=61) representing most of the common types of childhood solid tumors and childhood acute lymphoblastic leukemia (ALL). MLN4294 (provided by Millennium Pharmaceuticals) was tested in vitro at concentrations from 1.0 nM to 10.0 µM and was tested against the PPTP in vivo panel using a 100 mg/kg dose (66mg/kg for ALL panel) BID administered SQ daily × 5 for 3 weeks, with observation for 6 weeks. Three measures of antitumor activity were used: 1) an objective response measure modeled after the clinical setting; 2) a treated to control (T/C) tumor volume measure; and 3) a time to event (4-fold increase in tumor volume) measure based on the median event-free survival (EFS) of treated and control animals for each xenograft. Results: The median relative IC50 for MLN4924 against the PPTP cell lines was 143 nM, with a range from 15 nM to 678 nM. The median relative IC50 value for the Ewing panel (31 nM) was significantly lower than that of the remaining PPTP cell lines. MLN4924 demonstrated an activity pattern consistent with cytotoxic activity. MLN4924 was well tolerated, with a toxicity rate of 4.6%. 42 of 45 xenograft models were considered evaluable for efficacy. MLN4924 induced significant differences in EFS distribution compared to control in 20 of 34 (59%) solid tumor xenografts. MLN4924 induced intermediate activity (EFS T/C values &amp;gt; 2) in 9 of the 33 evaluable xenografts (27%), including 4 of 4 glioblastoma xenografts, 2 of 3 Wilms tumor xenografts, 2 of 5 rhabdomyosarcoma xenografts, and 1 of 4 neuroblastoma xenografts. For the ALL panel, 5 of 8 evaluable xenografts showed intermediate activity for the EFS T/C measure. The maximum shift in time to event for treated animals was 3.8-fold (ALL-3), and two other xenografts had shifts in time to event of 3.0-fold (ALL-2 and ALL-17). MLN4924 did not induce objective responses in the PPTP solid tumor or ALL panels, with the best response being stable disease for 1 ALL xenograft. Conclusions: MLN4924 potently inhibits the cell lines of the PPTP in vitro panel and shows an interesting pattern of in vivo activity. Pharmacokinetic and pharmacodynamic studies are ongoing. (Supported by NCI NO1CM42216) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 740. doi:10.1158/1538-7445.AM2011-740

The Novel, Investigational NEDD8-Activating Enzyme Inhibitor MLN4924 In Adult Patients with Acute Myeloid Leukemia (AML) or High-Grade Myelodysplastic Syndromes (MDS): A Phase 1 Study

AbstractAbstract 658 Background:NEDD8-activating enzyme (NAE) regulates the NEDD8 conjugation pathway, and is required for the activity of the cullin-RING E3 ligases (CRLs). CRLs control the timed degradation of several substrates involved in cell-cycle regulation, signal transduction, DNA replication, and stress response, including proteins important for the survival of AML cells. We evaluated the preclinical anti-leukemic activity of MLN4924, a novel, investigational, first-in-class small molecule inhibitor of NAE, and based on the activity of MLN4924 in preclinical AML models (Swords RT et al, Blood 2010) we conducted a phase 1 study to evaluate the safety and tolerability of this agent in patients with AML and advanced MDS. Methods:The primary objectives of this study were to evaluate the safety and tolerability of MLN4924, to establish the maximum tolerated dose (MTD), and to determine the recommended phase 2 dose of MLN4924 in patients with AML and high-grade MDS. Secondary objectives included a preliminary assessment of efficacy, and analysis of pharmacokinetics and pharmacodynamics (via NAE-regulated proteins in peripheral blood mononuclear cells). Patients aged ≥18 years, with ECOG performance status 0–2, who had AML or high-grade MDS, and who were not candidates for potentially curative therapy, were eligible. MLN4924 was administered as a 60-minute IV infusion on days 1, 3, and 5 of a 21-day cycle for up to 12 months or until documented disease progression. Dose escalation was commenced at 25 mg/m2 and proceeded using a standard 3+3′ escalation method until the MTD was established. Response assessment was based on recently published guidelines (Döhner H et al, Blood 2010) and adverse events (AEs) were graded per National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events v3.0 (NCI Cancer Therapy Evaluation Program, 2006). Results:To date, 15 patients (9 males, 6 females; 14 AML, 1 high-grade MDS) have been enrolled and treated, including 3, 4, 3, 3, and 2 at dose levels of 25, 33, 44, 59, and 78 mg/m2, respectively. Median age was 62.3 years (range 29.3–84.0 years). By cytogenetics, 1 (7%), 5 (33%), and 7 (47%) patients had good-, intermediate-, and poor-risk disease (not available in 2). Prior antineoplastic therapies included cytarabine (n=7), azacitidine, daunorubicin (n=3 each), decitabine, etoposide, gemtuzumab, idarubicin, and mitoxantrone (n=2 each). To date, 3 patients have received ≥8 cycles; 6 remain on treatment. Two dose-limiting toxicities have been reported at the 78 mg/m2 dose level: one patient with multi-organ failure in Cycle 2, and one with reversible elevation of alanine aminotransferase in Cycle 1. The most common AEs were pneumonia (n=6), atelectasis, constipation, diarrhea, and febrile neutropenia (each n=4); most common grade ≥3 AEs were febrile neutropenia (n=4), elevated aspartate aminotransferase, and pneumonia (each n=3). Three patients have achieved a complete response (CR) to date. A 29-year-old woman with relapsed AML following allogeneic stem cell transplantation achieved a CR after cycle 1 at 25 mg/m2 before developing progressive disease at an extramedullary site during cycle 8. An 82-year-old man with history of high-risk MDS, which was unresponsive to azacitidine, that evolved into AML had a partial response in cycle 8 and a CR with incomplete recovery of blood counts (CRi) in cycle 10 at 33 mg/m2; the patient is currently in cycle 12 and has become transfusion-independent. A 71-year-old man with de-novo AML refractory to standard cytarabine plus daunorubicin induction achieved a CRi during cycle 1 at 44 mg/m2; although this was not maintained, the patient continued to benefit from treatment and is currently in cycle 11 with reduced transfusion dependence. Pharmacodynamic data are available for 9 patients; 7 show evidence of target inhibition in peripheral blood by changes in NAE-regulated proteins. Conclusion:The preliminary findings of this study indicate that the novel mechanism of action of MLN4924 through NAE inhibition results in observed activity in patients with relapsed or refractory AML, and suggest the successful translation of preclinical research in AML models into the clinic. Enrollment continues in expanded cohorts of AML and MDS patients at 59 mg/m2. Updated efficacy and safety data will be presented, together with data on MLN4924 pharmacokinetics and pharmacodynamics. Disclosures:Off Label Use: Investigational agent in clinical development for the treatment of acute myeloid leukemia or myelodysplastic syndromes. Erba:Millennium Pharmaceuticals, Inc.: Research Funding. DeAngelo:Deminimus: Consultancy. Smith:Millennium Pharmaceuticals, Inc.: Employment. Pickard:Millennium Pharmaceuticals, Inc.: Employment. Dezube:Millennium Pharmaceuticals: Employment, Equity Ownership. Giles:Millennium Pharmaceuticals, Inc.: Research Funding. Medeiros:Millennium Pharmaceuticals, Inc.: Consultancy, Research Funding.