NEDD8-activating Enzyme Inhibitor MLN4924 Research Articles

Inhibition of the Nedd8 System Sensitizes Cells to DNA Interstrand Cross-linking Agents

The Fanconi anemia pathway is required for repair of DNA interstrand cross-links (ICL). Fanconi anemia pathway-deficient cells are hypersensitive to DNA ICL-inducing drugs such as cisplatin. Conversely, hyperactivation of the Fanconi anemia pathway is a mechanism that may underlie cellular resistance to DNA ICL agents. Modulating FANCD2 monoubiquitination, a key step in the Fanconi anemia pathway, may be an effective therapeutic approach to conferring cellular sensitivity to ICL agents. Here, we show that inhibition of the Nedd8 conjugation system increases cellular sensitivity to DNA ICL-inducing agents. Mechanistically, the Nedd8 inhibition, either by siRNA-mediated knockdown of Nedd8-conjugating enzymes or treatment with a Nedd8-activating enzyme inhibitor MLN4924, suppressed DNA damage-induced FANCD2 monoubiquitination and CHK1 phosphorylation. Our data indicate that inhibition of the Fanconi anemia pathway is largely responsible for the heightened cellular sensitivity to DNA ICLs upon Nedd8 inhibition. These results suggest that a combination of Nedd8 inhibition with ICL-inducing agents may be an effective strategy for sensitizing a subset of drug-resistant cancer cells.

Azacitidine/Decitabine Synergism with the NEDD8-Activating Enzyme Inhibitor MLN4924 in Pre-Clinical AML Models

Abstract 578MLN4924 is an investigational small molecule inhibitor of NEDD8-activating enzyme that has shown clinical activity in a Phase I clinical trial in Acute Myelogenous Leukemia (AML). To identify potential combination partners of MLN4924 we performed a high-throughput viability screen in AML cells with 40 approved and investigational agents. In vitro characterization of AML cell lines revealed two distinct cell cycle phenotypes suggesting alternate mechanism of action following MLN4924 inhibition of NAE. One group demonstrated moderate S-phase accumulation with greater than 4N DNA content consistent with DNA-rereplication as a result of CDT1 dysregulation. The second group demonstrated distinct and rapid accumulation of subG1 cells without S-phase accumulation or DNA re-replication suggesting induction of apoptosis and cell death. These observations led us to choose two cells lines representative of each mechanism to understand potential for synergy in AML cells. Two hypomethylating agents were included in the screen (decitabine and azacitidine) and were found to be synergistic with MLN4924 by Combination Index and Blending Synergy Analysis. These data were confirmed with a second NAE inhibitor that is structurally dissimilar to MLN4924. The combination of azacitidine and MLN4924 were shown to result in significantly increased DNA-damage and cell death compared to single agent alone as measured by Western Blotting and FACS analysis of cell cycle distributions. In vivo studies were performed in HL-60 and THP-1 xenografts using MLN4924 on a clinically relevant dosing schedule twice weekly. Single agent azacitidine at its Maximum Tolerated Dose (MTD) had minimal activity in the HL-60 model and was combined with a sub-optimal dose of MLN4924 that when combined induced complete and sustained tumor regressions. The mechanism for the apparent synthetic lethality in this in vivo model is currently under evaluation; however it is supported by a dramatic elevation in DNA damage and cleaved caspase-3 in vivo in the combination arm. A second xenograft model (THP-1) that was also insensitive to single agent azacitidine treatment underwent complete and sustained tumor regressions when combined with MLN4924. Thus MLN4924 and azacitidine can combine to produce synergistic antitumor activity in pre-clinical models of AML. Coupled with their non-overlapping clinical toxicities these data suggest the potential for future combination studies in clinical trials. Disclosures:Smith:Millennium Pharmaceuticals: Employment. Traore:Millennium Pharmaceuticals: Employment. Grossman:Millennium Pharmaceuticals: Employment. Narayanan:Millennium Pharmaceuticals: Employment. Carew:Millennium Pharmaceuticals: Research Funding. Lublinksky:Millennium Pharmaceuticals: Employment. Kuranda:Millennium Pharmaceuticals: Employment. Milhollen:Millennium Pharmaceuticals: Employment.

Investigational NEDD8-Activating Enzyme (NAE) Inhibitor, MLN4924, Demonstrates Activity Against Primary AML Blast, Progenitor and Stem Cell Populations

Abstract 1414Acute myelogenous leukemia (AML) has a poor prognosis. Most patients die of either primary refractory or relapsed disease. It has been proposed that relapse may result from ineffective ablation of leukemia stem cells (LSCs) by chemotherapy. Therefore, in order to improve therapy, it is imperative to identify agents that eliminate leukemic progenitor and stem cell populations in addition to blasts. We have previously demonstrated that proteasome inhibition can result in selective ablation of LSCs, without harming normal hematopoietic stem cells (HSCs). Thus, we sought to investigate the activity of a more specific inhibitor of the ubiquitin-proteasome pathway, an investigational NEDD8-Activating Enzyme (NAE) inhibitor MLN4924. Treatment of primary human AML and blast crisis CML samples with MLN4924 resulted in a dose-dependent decrease in the total viability of the blast-cell populations, with an average LD50 of 0.367 μM (0.203–0.530 95% CI; N=17). The average LD50 for phenotypically described LSCs was 0.645μM (0.304–0.986; 95%CI; N=12). Interestingly, when performing progenitor/stem functional assays, we observed a 64% and 86% decrease in colony formation after treatment with 0.25μM and 1 μM of MLN4924 (p< 0.001; N=5; p< 0.001 N=8 respectively). Furthermore, MLN4924 had little to no effect on colony formation of normal hematopoietic cells. Thus, MLN4924 may impair the activity of AML stem/progenitor cells, but not normal cells, in vitro. Importantly, primary AML samples that were resistant to standard chemotherapeutics such as cytarabine in vitro were sensitive to MLN4924. Treatment of primary AML samples with MLN4924 showed increased γH2AX foci, indicating activation of stress responses. Together, these data suggest that MLN4924 can eliminate AML blasts, progenitor and stem cell populations in vitro. Disclosures:Smith:Millennium Pharmaceuticals: Employment.

Treatment Emergent Mutations in NAEβ Confer Resistance to the NEDD8-Activating Enzyme Inhibitor MLN4924 in Pre-Clinical AML and DLBCL Models

Abstract 1413MLN4924 is an investigational small molecule inhibitor of NEDD8-activating enzyme (NAE) that has shown clinical activity in a Phase I clinical trial in Acute Myelogenous Leukemia (AML). MLN4924 is a mechanism-based inhibitor of NAE as demonstrated through the formation of a NEDD8-MLN4924 covalent adduct that is a tight-binding inhibitor of NAE. The antitumor activity of MLN4924 has been linked to two mechanisms in pre-clinical models: (1) the induction of DNA re-replication and cell death through dysregulation of Cdt-1 and (2) the inhibition of NF-κB signaling. Importantly, modulation of both pathways has been demonstrated in pharmacodynamic (PD) studies from patients administered MLN4924. To characterize potential mechanisms of resistance to MLN4924 xenograft models of AML and Diffuse Large-B cell Lymphoma (DLBCL) were used. Mice bearing HL-60 and THP-1 (AML) and OCI-Ly10 (DLBCL) xenografts were treated with MLN4924 twice weekly for 95–110 days and most animals achieved a complete tumor regression. However, four of ten HL-60, six of ten THP-1 and one of ten OCI-Ly10 xenografts became refractory and re-grew during the MLN4924 treatment period. These tumors were harvested and subjected to microarray analysis and DNA sequencing of genes required for NEDD8 activation by NAE. Mutations in NAEβ resulting in amino acid substitutions were detected in several xenografts in the ATP binding pocket (A171T) and NEDD8-binding cleft (E204K) suggesting that mutations in the target enzyme may play a role in emergence of refractory tumors. Tumors containing mutations in NAEβ were passaged into new mice and shown to be completely insensitive to MLN4924 confirming resistance. In addition, cell lines derived from the resistant xenografts were shown to be resistant to MLN4924 in vitro but remained sensitive to other chemotherapeutic agents. Biochemical analysis of NAEβ mutants revealed a slower rate of NEDD8-MLN4924 adduct formation and demonstrated that the adduct was no longer bound tightly by the mutant enzyme. Thus, treatment emergent mutations in NAEβ lead to MLN4924 resistance in pre-clinical models through reduced inhibitory potency. These data serve as the framework for understanding mechanisms of resistance to MLN4924, provide rationale for patient selection approaches and offer a strategy to overcome treatment emergent mutations that may arise in clinical studies. Disclosures:Milhollen:Millennium Pharmaceuticals: Employment. Thomas:Millennium Pharmaceuticals: Employment. Traore:Millennium Pharmaceuticals: Employment. Narayanan:Millennium Pharmaceuticals: Employment. Riceberg:Millennium Pharmaceuticals: Employment. Sells:Millennium Pharmaceuticals: Employment. Amidon:Millennium Pharmaceuticals: Employment. Manfredi:Millennium Pharmaceuticals: Employment. Bence:Millennium Pharmaceuticals: Employment. Brownell:Millennium Pharmaceuticals: Employment. Dick:Millennium Pharmaceuticals: Employment. Koenig:Millennium Pharmaceuticals, Inc: Employment. Gavin:Millennium Pharmaceuticals: Employment. Smith:Millennium Pharmaceuticals: Employment.

Abstract A45: Investigation of efficacy of the NAE inhibitor MLN4924 across large-scale cell line panels reveals potentially sensitive cancer indications and candidate predictive biomarkers.

Abstract MLN4924 is an investigational inhibitor of NAE (Nedd8 Activating Enzyme). NAE mediates NEDD8 conjugation and thus regulates the cullin-RING ligase (CRL) proteasomal degradation pathway. MLN4924 is currently being tested in Phase 1 clinical trials of patients with hematological or solid tumors. To support future clinical development and identify potential biomarkers of tumor sensitivity or resistance, two large cancer cell line panels (Panel 1, N=653; Panel 2, N=240) were treated with MLN4924 and cell viability data (IC50, EC50, and POC - Percentage of Control) were generated. These data were used to correlate sensitivity/resistance with the cell line's tumor histology and with underlying genetic data for the specific cell lines. Overlapping cell lines from the two panels (114) showed consistent growth inhibition effects (Spearman Correlation coefficient = 0.72). Using Fisher's exact test and POC cutoffs at 25% and 50% quartiles on viability data, we identified bladder cancer and head and neck cancer lines as associated with MLN4924 sensitivity in both panels. Using median percentage of control values as cutoff, we applied Fisher's exact test to evaluate associations of individual mutations in the cell lines to MLN4924 sensitivity or resistance. Examples of mutations linked to sensitivity include KDM6A (p-value = 0.02, Panel 1; p-value = 0.06, Panel 2). APC mutation is associated with resistance to MLN4924 (p-value = 5.75 × 10−5, Panel 1; p-value = 0.04, Panel 2). Combining cell viability and baseline gene expression data from cell line Panel 2, a PLSR predictive model was developed, identifying a gene signature (102 genes) for MLN4924 sensitivity and resistance. A median cutoff of PLSR scores was applied to separate predicted sensitive/resistant cell lines. Consistent with previous association of cell viability with histology, PLSR analysis of gene expression predicted that bladder cancer cell lines are particularly sensitive to MLN4924. Two pathway analysis approaches were applied to identify MLN4924 sensitivity related pathways using either whole genome information (GSEA) or the PLSR gene signature list. The two approaches consistently indicated an over-representation of signaling pathways including cell cycle, development hedgehog signaling and ErbB signaling. The over-represented cell cycle pathway is in good agreement with previous MLN4924 mechanism of action studies. In particular, this finding was independently validated via a whole genome siRNA screen that also identified the cell cycle pathway as associated with tumor sensitivity. The consistent observations from two large cell line panels and a siRNA screen support the utility of these approaches for clarifying sensitive and resistant tumor cell types and also potentially identifying candidate biomarkers. 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 A45.

Abstract C28: Treatment emergent mutations in NAEbeta confer resistance to the investigational NEDD8-activating enzyme inhibitor MLN4924.

Abstract MLN4924 is an investigational small molecule inhibitor of NEDD8-activating enzyme (NAE) that has shown clinical activity in Phase I clinical trials. MLN4924 is a mechanism-based inhibitor of NAE as demonstrated through the formation of a NEDD8-MLN4924 covalent adduct that is a tight-binding inhibitor of NAE. The antitumor activity of MLN4924 has been linked to two mechanisms in pre-clinical models: (1) the induction of DNA re-replication and cell death through dysregulation of Cdt-1 and (2) the inhibition of NF- B signaling. Importantly, modulation of both pathways has been demonstrated in pharmacodynamic (PD) studies from patients administered MLN4924. To characterize potential mechanisms of resistance to MLN4924, cell line and xenograft models of solid tumors were used. HCT-116 (colon), NCI-H460 (lung) and Calu-6 (lung) cells were treated with high concentrations of MLN4924 for four days after which the remaining cells were isolated and cultured in drug free media. Six HCT-116, two NCI-H460 and one Calu-6 clone were isolated and found to be at least 10-fold less sensitive to MLN4924, in most cases the isolated clones were completely insensitive indicating they were now resistant to MLN4924. However, the MLN4924 resistant clones were still sensitive to other chemotherapies including bortezomib (proteasome inhibitor), SN-38 (topoisomerase I inhibitor) and doxorubicin (anthracycline) suggesting a resistance mechanism unique to MLN4924. Co-incubation with Pgp, BCRP or MRP2 transporter inhibitors did not re-sensitize cells to MLN4924 indicating that the resistance mechanism was not related to increased drug efflux. DNA sequencing of NAE pathway genes identified heterozygous mutations in NAEbeta resulting in amino acid substitutions in the ATP binding pocket or NEDD8-binding cleft. Nude rats bearing HCT-116 xenografts were treated with the maximum tolerated dose of MLN4924 on a clinically relevant dosing regimen of Days 1, 4, 8 and 11 of a 21 day schedule. In the first cycle of therapy tumor regressions were observed but ultimately tumors regrew over subsequent cycles and were found to contain heterozygous mutations in NAEbeta. Tumors containing mutations in NAEbeta were transplanted into nude rats and shown to be insensitive to MLN4924 confirming resistance. In addition, cell lines derived from the resistant xenografts were shown to be resistant to MLN4924 in vitro but remained sensitive to other chemotherapeutic agents. Biochemical analysis of NAEbeta mutants revealed a slower rate of NEDD8-MLN4924 adduct formation and demonstrated that the adduct was no longer bound tightly by the mutant enzyme. Thus, in pre-clinical models of solid tumors treatment emergent mutations in NAEbeta lead to MLN4924 resistance. DNA sequencing of samples obtained from MLN4924 clinical trials is ongoing. These data may provide rationales for patient selection approaches with MLN4924 and the development of next generation NAE inhibitors that can be designed to overcome treatment emergent mutations found in clinical studies. 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 C28.

Inhibition of NEDD8-Activating Enzyme Induces Rereplication and Apoptosis in Human Tumor Cells Consistent with Deregulating CDT1 Turnover

Loss of NEDD8-activating enzyme (NAE) function by siRNA knockdown or inhibition by the small molecule NAE inhibitor MLN4924 leads to increased steady-state levels of direct Cullin-RING ligase (CRL) substrates by preventing their ubiquitination and proteasome-dependent degradation. Many of these CRL substrates are involved in cell cycle progression, including a critical DNA replication licensing factor CDT1. Cell cycle analysis of asynchronous and synchronous cultures after NAE inhibition revealed effects on cell cycle distribution and activation of DNA break repair signaling pathways similar to that reported for CDT1 overexpression. The siRNA knockdown of cullins critical for the turnover of CDT1 recapitulated the aberrant rereplication phenotype while CDT1 knockdown was suppressing. Although NAE inhibition leads to deregulation of many CRL substrates, these data demonstrate that CDT1 accumulation mediates the DNA rereplication phenotype resulting from loss of NAE function. DNA rereplication is an unrecoverable cellular insult and the small molecule inhibitor MLN4924, currently in phase I trials, represents an unprecedented opportunity to explore this mechanism of cytotoxicity for the treatment of cancer.

Disrupting NEDD8-Mediated Protein Turnover with MLN4924 Significantly Augments the Efficacy of Cytarabine

AbstractAbstract 3255Cytarabine-based therapy has been utilized in acute myeloid leukemia (AML) therapy for more than 30 years. However, the complete response (CR) rates are markedly inferior in older compared to younger patients with AML (45% versus 75%, respectively) due, in part, to the reduced ability of elderly patients to tolerate intensive therapy. Improving the outcomes for patients treated with cytarabine-based regimens represents a major clinical challenge in this disease. A randomized study of elderly patients with AML demonstrated that low dose cytarabine (LDAC) is superior to best supportive care. However, this regimen was not associated with any CRs in patients with adverse karyotype disease and/or poor baseline performance scores. Novel approaches are urgently needed to increase the efficacy of LDAC therapy for these patients. Timed protein destruction plays a crucial role in cellular homeostasis and is essential for many critical functions including cell cycle progression, signal transduction, and apoptosis. The processes that govern protein degradation frequently become dysregulated in cancer cells. Aberrant protein turnover contributes to disease progression, metastasis, and therapeutic resistance and therefore is an attractive target for selective pharmacological inhibition. The cullin-RING ubiquitin ligases (CRLs) are a subset of E3 ubiquitin ligases whose activity is regulated by modification with the ubiquitin-like molecule NEDD8. The CRLs control the ubiquitination and subsequent degradation of many proteins with important roles in cell cycle progression, DNA damage, stress responses, and signal transduction. MLN4924 is a potent and selective small molecule inhibitor of NEDD8 activating enzyme (NAE), the proximal regulator of the NEDD8 conjugation pathway, and has entered Phase I clinical trials for AML and other forms of cancer. Our earlier preclinical studies demonstrated that MLN4924 induced cell death in AML cell lines and primary patient specimens independent of FLT3 expression and stromal-mediated survival signaling and led to the stabilization of key NAE targets, inhibition of NF-kB activity, DNA damage, and reactive oxygen species generation. Notably, administration of MLN4924 to mice bearing AML xenografts was very well tolerated, led to stable disease regression and inhibition of NEDDylated cullins. Based on the high tolerability, potency, and multifaceted mechanism of action of MLN4924, we hypothesized that it may significantly augment the efficacy of the standard agent cytarabine. To test our hypothesis, we first investigated the effects of this therapeutic combination on cell viability, clonogenic survival, and apoptosis induction in a panel of AML cell lines. MLN4924 cooperated with cytarabine to significantly reduce cell viability, inhibit clonogenic survival, and induce mitochondrial-dependent apoptosis. The addition of MLN4924 did not significantly alter the sensitivity of normal peripheral blood mononuclear cells from healthy donors to cytarabine, indicating that this combination may have therapeutic selectivity. Immunoblotting analyses revealed that MLN4924 enhanced cytarabine-induced stabilization of the NEDD8 target and cell cycle regulator, p27. The MLN4924/cytarabine combination also promoted increased phosphorylation of the DNA damage response regulator Chk1. Targeted knockdown of Chk1 demonstrated a critical role for Chk1 as a mediator of the pro-apoptotic effects of this combination. In vivo examining the combination is in progress and will be presented. Our collective findings suggest that combining the novel NAE inhibitor MLN4924 with cytarabine is a promising strategy for AML therapy that warrants further investigation. Disclosures:Smith:Millennium Pharmaceuticals, Inc.: Employment.

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.

409 The novel, investigational Nedd8-activating enzyme inhibitor MLN4924 in patients with metastatic melanoma: a phase 1 study

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and the third leading cause of cancer death. The current view of cancer progression and malignancy supports the notion that cancer cells must undergo through a post-translational modification (PTM) regulation and a metabolic switch or reprogramming in order to progress in an unfriendly environment. NEDDylation is a post-translational modification of the proteins involved in several processes such as cell growth, viability and development. A ground-breaking knowledge on a new critical aspect of HCC research has been to identify that NEDDylation plays a role in HCC by regulating the liver oncogenic driver Hu antigen R (HuR). HuR is a RNA-binding protein that stabilizes target mRNAs involved in cell dedifferentiation, proliferation, and survival, all well-established hallmarks of cancer. And importantly, HuR levels were found to be highly representative in liver and colon cancer.These findings open a completely new area of research, exploring the impact that NEDDylation plays in liver diseases and paving the way for novel therapeutical approaches.

Abstract A31: Development of an immunohistochemistry-based pharmacodynamic biomarker program for phase I trials of MLN4924, an inhibitor of the NEDD8 activating enzyme

Abstract The novel small molecule MLN4924 is an inhibitor of the NEDD8 activating enzyme (NAE) and is currently in Phase I trials. As part of the ubiquitin-proteasome system, NAE is required for activity of cullin ring ligases (CRLs), a category of ubiquitin E3 ligases. When NAE is inhibited, levels of CRL substrates increase due to impaired ubiquitination and proteasomal degradation; CRL substrates can therefore serve as pharmacodynamic (PD) biomarkers of NAE pathway inhibition. We describe the development of Immunohistochemistry (IHC) assays for two CRL substrates: NRF2 (a transcription factor involved in the oxidative stress response) and CDT1 (a protein that regulates DNA replication). CDT1 and NRF2 IHC assays were initially evaluated in PD studies of xenograft tumors grown in immunocompromised mice and showed dose-dependent regulation. The IHC assays were then adapted for use in clinical samples, including skin and tumor biopsies from patients in Phase I trials of MLN4924. Sample quality and morphology were assessed by pathologist review of hematoxylin and eosin pathology stain. A set of formalin-fixed, paraffin-embedded cell pellets was evaluated with each IHC run to detect technical variability and establish pass/fail criteria for each experimental IHC run. The QC samples were generated from the HCT116 cell line treated with various concentrations of MLN4924. The QC controls were assayed multiple times to generate an expected range as a percent positive area above a threshold for each concentration of MLN4924. The ranges were then applied to each experimental run to pass or fail the experiment. In cases of assay failure, the clinical samples were re-stained. Skin and melanoma tumors are inherently challenging for IHC analysis due to the population variability of melanin pigment, which can be indistinguishable from brown DAB chromagen. Adaption of the assays to a blue chromagen (NBT/BCIP) enabled a contrast between the chromagen stain and endogenous pigments. A region of interest (ROI) for skin was manually drawn to select the epidermal area. Semi-automated image analysis was used to measure percent positive area for CDT1 and NRF2 within the total epidermal region. To determine the baseline levels for CDT1 and NRF2 signal, 10 benign skin samples were studied. Staining of six sections at 10-micron increments was performed on each sample to investigate both the inter- and intra-patient variability. Overall, the percent positive area of both substrates was below 1% in benign skin and variation from section to section was minimal. To survey the baseline levels of NRF2 and CDT1 across a variety of tumor types, we evaluated archival tumors of various types including melanoma, head &amp; neck squamous cell carcinoma, prostate, lung, and breast cancer. A manual ROI was identified by pathologist review to select for tumor area, and semi-automated image analysis was used to measure the percent positive area for NRF2 and CDT1 within the total tumor area. Unlike skin, the tumor samples showed variable levels of NRF2 and CDT1. However, most samples showed a percent positive area less than 40%, indicating the feasibility of detecting a PD effect by comparing pre- and post-dose tumor biopsies. In summary, reliable IHC PD biomarker assays have been established for the NAE inhibitor MLN4924. The assays are currently being used to measure PD effects of NAE inhibition in skin and solid tumors from Phase I trials of MLN4924. Citation Information: Clin Cancer Res 2010;16(7 Suppl):A31

Abstract B189: The Nedd8-activating enzyme inhibitor MLN4924 combined with rituximab induces additive/synergistic anti-tumor activity in preclinical non-Hodgkin's lymphoma models

Abstract MLN4924 is a novel, small molecule inhibitor of Nedd8-activating enzyme (NAE) that is currently in Phase I clinical trials in hematologic malignancies. We have previously described the pre-clinical anti-tumor activity of MLN4924 in human xenograft models of hematological and solid tumors. Rituximab is an anti-CD20 monoclonal antibody that has been established as part of the standard of care for the treatment of non-Hodgkin's lymphoma (NHL). In the present study, we investigated whether a combination of MLN4924 and Rituximab may act in a complementary manner in pre-clinical models of NHL. We utilized models of ABC-like Diffuse Large B-cell Lymphoma (ABC-like DLBCL, OCI-Ly10 cells) dependent on NF- B signaling for survival and Germinal Center B-cell like DLBCL (GCB-like DLBCL, OCI-Ly19 cells) that are not dependent on NF-κB signaling for survival. In vivo administration of MLN4924 to mice bearing xenograft tumors of OCI-Ly10 and OCI-Ly19 resulted in a pharmacodynamic response of NAE pathway inhibition. In both models, a single dose of MLN4924 resulted in a dose-dependent inhibition of NAE and stabilization of Cullin Dependent Ligase substrates including Nrf-2 and pI Bα. MLN4924 induced antitumor activity that was dose-dependent in both models. In the OCI-Ly10 model co-administration of MLN4924 (10 or 3 mg/kg) and Rituximab (0.3 or 1 mg/kg) induced either synergistic or additive anti-tumor activity (P&amp;gt;0.05). The combination of 10 mg/kg MLN4924 and 1 mg/kg Rituximab resulted in 3 partial and 7 complete responses. In the OCI-Ly19-luc disseminated lymphoma model combining MLN4924 (10–30 mg/kg) with Rituximab resulted in additive tumor growth inhibition. The mean survival endpoint was significantly longer (p=&amp;lt;0.01) in the combination group when compared to the single agent group. Importantly, all animals in the MLN4924 (60 mg/kg) and Rituximab (10 mg/kg) combination group were still alive 100 days after treatment ended. All doses and regimens were well tolerated. The results of these studies demonstrate that combination treatment with MLN4924 and Rituximab resulted in additive and/or synergistic reduction in tumor burden in the OCI-Ly10 and OCI-Ly19 models. In addition, these data provide the rationale for future clinical evaluation of MLN4924 in combination with Rituximab in lymphoma. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B189.