Tary Traore

An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer

The clinical development of an inhibitor of cellular proteasome function suggests that compounds targeting other components of the ubiquitin–proteasome system might prove useful for the treatment of human malignancies. 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. Here we describe MLN4924, a potent and selective inhibitor of NAE. MLN4924 disrupts cullin-RING ligase-mediated protein turnover leading to apoptotic death in human tumour cells by a new mechanism of action, the deregulation of S-phase DNA synthesis. MLN4924 suppressed the growth of human tumour xenografts in mice at compound exposures that were well tolerated. Our data suggest that NAE inhibitors may hold promise for the treatment of cancer.

MLN4924, a NEDD8-activating enzyme inhibitor, is active in diffuse large B-cell lymphoma models: rationale for treatment of NF-κB–dependent lymphoma

MLN4924 is a potent and selective small molecule NEDD8-activating enzyme (NAE) inhibitor. In most cancer cells tested, inhibition of NAE leads to induction of DNA rereplication, resulting in DNA damage and cell death. However, in preclinical models of activated B cell-like (ABC) diffuse large B-cell lymphoma (DLBCL), we show that MLN4924 induces an alternative mechanism of action. Treatment of ABC DLBCL cells with MLN4924 resulted in rapid accumulation of pIkappaBalpha, decrease in nuclear p65 content, reduction of nuclear factor-kappaB (NF-kappaB) transcriptional activity, and G(1) arrest, ultimately resulting in apoptosis induction, events consistent with potent NF-kappaB pathway inhibition. Treatment of germinal-center B cell-like (GCB) DLBCL cells resulted in an increase in cellular Cdt-1 and accumulation of cells in S-phase, consistent with cells undergoing DNA rereplication. In vivo administration of MLN4924 to mice bearing human xenograft tumors of ABC- and GCB-DLBCL blocked NAE pathway biomarkers and resulted in complete tumor growth inhibition. In primary human tumor models of ABC-DLBCL, MLN4924 treatment resulted in NF-kappaB pathway inhibition accompanied by tumor regressions. This work describes a novel mechanism of targeted NF-kappaB pathway modulation in DLBCL and provides strong rationale for clinical development of MLN4924 against NF-kappaB-dependent lymphomas.

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.

A small-molecule inhibitor of the ubiquitin activating enzyme for cancer treatment

The ubiquitin–proteasome system (UPS) comprises a network of enzymes that is responsible for maintaining cellular protein homeostasis. The therapeutic potential of this pathway has been validated by the clinical successes of a number of UPS modulators, including proteasome inhibitors and immunomodulatory imide drugs (IMiDs). Here we identified TAK-243 (formerly known as MLN7243) as a potent, mechanism-based small-molecule inhibitor of the ubiquitin activating enzyme (UAE), the primary mammalian E1 enzyme that regulates the ubiquitin conjugation cascade. TAK-243 treatment caused depletion of cellular ubiquitin conjugates, resulting in disruption of signaling events, induction of proteotoxic stress, and impairment of cell cycle progression and DNA damage repair pathways. TAK-243 treatment caused death of cancer cells and, in primary human xenograft studies, demonstrated antitumor activity at tolerated doses. Due to its specificity and potency, TAK-243 allows for interrogation of ubiquitin biology and for assessment of UAE inhibition as a new approach for cancer treatment.

Abstract B196: Evaluation of the ubiquitin activating enzyme (UBA1) as a therapeutic target via inducible RNA interference in human tumor xenografts.

The successful transition of the Nedd8-activating enzyme inhibitor, MLN4924, into clinical trials has greatly increased optimism for the application of other E1-activating enzyme inhibitor therapies in cancer. UBA1 is an essential E1 enzyme that controls the first step in the activation of ubiquitin. Ubiquitin is a 9 KDa protein that is involved in multiple cellular processes through conjugation to a large range of target proteins. Several of these processes are important in cancer including ubiquitin-dependent protein turnover, cell cycle progression, apoptosis and the DNA damage response. Thus, inhibiting UBA1 with small molecule inhibitors may prove to be a viable anticancer strategy. To validate UBA1 as an oncology target we utilized a lentiviral vector-mediated inducible knockdown of UBA1 by a microRNA-based shRNA (shRNAmir) in cells and xenografts derived from human cancer cells. Our data show that shRNAmir-mediated knockdown of UBA1 is tightly regulated by the inducer doxycycline in a concentration- and time-dependent manner in HCT-116 cells. Furthermore, UBA1 expression is restored upon withdrawal of the inducing agent, which coincides with decreased shRNAmir expression as marked by turboRFP. The shRNAmir-mediated depletion of UBA1 in cells resulted in reduced abundance of ubiquitin conjugates and decreased proliferation of HCT-116 cells, ultimately resulting in apoptosis. Next, we established inducible shRNAmir HCT-116 xenograft models in nude mice to examine the in vivo efficacy of inhibiting UBA1. Our results showed knockdown of UBA1 protein levels, decreased abundance of ubiquitin conjugates and apoptosis in vivo following doxycycline administration to mice. The effects of UBA1 knockdown and inhibition of ubiquitin conjugation resulted in tumor xenograft regressions in all mice. Withdrawal of doxycycline treatment resulted in regrowth of tumors with concomitant recovery of UBA1 levels and detectable ubiquitin conjugates in tumor xenografts. This study demonstrates that UBA1 knockdown effectively inhibits tumor growth and mediates tumor regression in a human xenograft model, implicating UBA1 as a novel therapeutic target for cancer. 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 B196.

Abstract C82: Identification and preclinical characterization of inhibitors of the ubiquitin-activating enzymes UBA1 and UBA6.

Millennium Pharmaceuticals, Inc. is dedicated to the discovery and development of novel oncology therapeutics in the area of protein homeostasis. Here we report the identification and characterization of compounds that target the ubiquitin activating enzymes, UBA1 and UBA6. These compounds are mechanism based inhibitors that inactivate the ubiquitin E1 enzymes by forming a ubiquitin compound adduct that remains tightly associated with the E1 adenylate binding site. Treatment of cells with these inhibitors results in cellular effects consistent with known Uba1 biology including rapid loss of E2 ubiquitin thioesters, loss of total ubiquitin conjugates, and accumulation of many ubiquitin proteasome system substrates. Following prolonged treatment, cells primarily arrest in the G2 phase of the cell cycle and ultimately undergo apoptosis. Reflecting the extensive cellular roles of ubiquitin, the compounds also impact global protein turnover, ER stress and DNA damage repair. UBA1 inhibition impairs ubiquitination of PCNA and the Fanconia Anemia protein FANCD2 leading to defective repair of UV induced DNA damage. UBA1 inhibition impacts numerous biological pathways relevant to cancer, results in apoptosis in vitro and is capable of inhibiting tumor growth in mouse xenografts in vivo. These data implicate UBA1 as a target for the treatment of cancer. 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 C82.

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

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.

Abstract B34: Clinical pharmacodynamic assay development for the first small molecule inhibitor of Nedd8‐activating enzyme (NAE), MLN4924

NEDD8‐Activating Enzyme (NAE) initiates the conjugation of the ubiquitin‐like protein NEDD8 to its cellular targets, members of the cullin protein family. NEDD8 conjugation to cullins is known to be essential for the ubiquitination activity of cullin‐RING ubiquitin ligases (CRLs). CRLs control the timely ubiquitination and subsequent degradation of many proteins with important roles in cell cycle progression and signal transduction. MLN4924 is a first in class, small molecule inhibitor of NAE. Inhibition of NAE with MLN4924 disrupts the conjugation of NEDD8 to CRLs. This subsequently prevents ubiquitination and proteasomal degradation of CDL substrates involved in cell cycle regulation (p27), signal transduction (pIkBa), DNA replication (Cdt‐1), stress response (Nrf‐2), and other processes crucial to tumor cell growth and survival. The regulation of these markers of NAE inhibition is well characterized across of a variety of cancer cell types grown in culture and as xenografts implanted in immunocompromised mice. Here we describe the development of clinical pharmacodynamic (PD) assays to evaluate two markers of NAE inhibition in the blood compartment, measurement of neddylated cullins and pIkBa levels in peripheral blood mononuclear cells (PBMCs). In vivo administration of a single dose of MLN4924 to nude mice harboring subcutaneous HCT‐116 colon carcinoma xenografts results in a dose‐dependent decrease in neddylated cullin (CUL‐N8) levels and elevation of the CRL substrate pIkBa. In addition, we demonstrate a dose‐dependent inhibition of CUL‐N8 levels in PBMCs isolated from the same mice. We expanded our PD analysis to human PBMCs treated ex vivo with MLN4924 to assess CUL‐N8 levels by quantitativeWestern blot and pIKBa levels by ELISA. PBMCs isolated from healthy volunteers were used to assess the technical and biological variability of theWestern blot and ELISA assays. In these experiments, whole blood was treated ex vivo with increasing concentrations of MLN4924 and PBMCs were subsequently isolated with VACUTAINER® CPT™ tubes that are often utilized in Phase I trials. Repeated CUL‐N8 or pIkBa measurements of replicate samples on different days demonstrated coefficient of variation values of less than 12% for both assays. Biological variability of baseline (i.e. untreated) and MLN4924‐regulated levels of CUL‐N8 and pIKBa in PBMCs was assessed by performing ex vivo treatment of whole blood obtained from the same donors on three separate occasions. Statistical analysis of this data demonstrated good biological reproducibility for both assays. Plasma concentrations measured from the ex vivo treated blood samples indicate that both the CUL‐N8 Western and pIkBa ELISA assays detect MLN4924‐induced regulation within the predicted range for human plasma exposures in a clinical setting. The demonstrated PD response and anti‐tumor activity of MLN4924 in preclinical models has supported its ongoing evaluation for safety and PD in patients with hematological and solid tumor disease in multiple Phase I clinical trials. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B34.

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

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‐Hodgkin9s 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>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= 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.