James J. Garnsey

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.

Inhibition of NEDD8-activating enzyme: a novel approach for the treatment of acute myeloid leukemia.

NEDD8 activating enzyme (NAE) has been identified as an essential regulator of the NEDD8 conjugation pathway, which controls the degradation of many proteins with important roles in cell-cycle progression, DNA damage, and stress responses. Here we report that MLN4924, a novel inhibitor of NAE, has potent activity in acute myeloid leukemia (AML) models. MLN4924 induced cell death in AML cell lines and primary patient specimens independent of Fms-like tyrosine kinase 3 expression and stromal-mediated survival signaling and led to the stabilization of key NAE targets, inhibition of nuclear factor-kappaB activity, DNA damage, and reactive oxygen species generation. Disruption of cellular redox status was shown to be a key event in MLN4924-induced apoptosis. Administration of MLN4924 to mice bearing AML xenografts led to stable disease regression and inhibition of NEDDylated cullins. Our findings indicate that MLN4924 is a highly promising novel agent that has advanced into clinical trials for the treatment of AML.

Abstract B156: Regulation of tumor metabolism by the investigational proteasome inhibitor ixazomib in KRAS wild-type and KRAS mutant xenograft tumors.

The investigational proteasome inhibitor ixazomib (MLN2238) inhibits cell growth in a broad panel of solid tumor and hematological cell lines when tested in vitro. In contrast, antitumor activity in xenograft-bearing mice is model-dependent, with some solid tumor models showing no response to ixazomib treatment. A survey of 15 NSCLC and 6 colon xenograft models showed a striking relationship between the degree of antitumor activity of ixazomib and KRAS genotype. Tumors with wild-type (WT) KRAS showed higher sensitivity to ixazomib compared to the tumors harboring a KRAS activating mutation. These xenografts included cell-line-derived subcutaneous xenografts as well as patient derived primary tumors. To confirm that KRAS mutation impacts response to ixazomib, we used SW48 isogenic colon cancer cell lines, in which a KRAS-G13D mutation was introduced into KRAS-WT SW48 cells to generate stable SW48-KRAS-G13D cells. Although in vitro sensitivity to ixazomib was similar between the paired cell lines, in vivo antitumor activity was observed in only SW48 KRAS WT tumors, but not SW48-KRAS-G13D tumors. These data confirm the association of KRAS status and response to ixazomib. Since KRAS activating mutations are known to be associated with metabolic reprogramming, we performed metabolite profiling using in vivo SW48 isogenic tumor pairs treated with or without ixazomib. As expected, prior to treatment there were significant baseline differences between SW48 WT and SW48-KRAS-G13D tumor metabolite profiles in categories such as glutathione levels and glycogen breakdown intermediates, reflecting higher oxidative stress and glucose utilization in the KRAS-G13D tumors. After a single dose of ixazomib, significant metabolic regulation was noted in categories including free amino acid pools, and polyamine levels, and some of these changes were more pronounced in the SW48 KRAS WT tumors as compared to the KRAS-G13D tumors. Our data also showed activation of GCN2 mediated signaling following ixazomib treatment, possibly as a result of amino acid deprivation in the tumors. The results suggest additional non-clinical avenues to explore mechanisms of sensitivity and resistance to proteasome inhibitors in solid tumors. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B156. Citation Format: Nibedita Chattopadhyay, Allison Berger, James Garnsey, Hugues Bernard, Erik Koenig, Eric Lightcap, Ben Amidon. Regulation of tumor metabolism by the investigational proteasome inhibitor ixazomib in KRAS wild-type and KRAS mutant xenograft tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B156.

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

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 & 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 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.