Julie Zhang

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.

Abstract PR12: MLN0264, an investigational, first-in-class antibody-drug conjugate (ADC) targeting guanylyl cyclase C (GCC), demonstrates antitumor activity alone and in combination with gemcitabine in human pancreatic cancer xenograft models expressing GCC.

Background: MLN0264 consists of a fully human anti-GCC monoclonal antibody linked to the microtubule-disrupting agent monomethyl auristatin E (MMAE) via a protease-cleavable linker (MMAE and linker technology licensed from Seattle Genetics). The transmembrane cell surface receptor GCC is expressed by ∼95% of primary and metastatic colorectal cancer (mCRC) tumors. MLN0264 has shown selective binding and antitumor activity in mouse xenograft models of mCRC expressing GCC, and is currently being investigated in a phase 1 study in patients with advanced gastrointestinal malignancies. GCC is also expressed in subsets of pancreatic cancers. Further to the findings in mCRC, we investigated GCC expression in human pancreatic tumors and evaluated MLN0264 activity in mouse xenograft models of GCC-expressing human pancreatic cancer. Methods: GCC expression in multiple human pancreatic cancer samples including tissue microarrays (TMAs) was evaluated by immunohistochemistry (IHC). For in vivo studies, 7 mouse xenograft primary human tumor explant (PHTX) models of pancreatic cancer were developed, including tumor tissue from patients with wild-type and mutant KRAS . Animals were treated when the tumor reached ∼230 mm3. In single-agent studies, animals were administered vehicle, MMAE 0.135 mg/kg once weekly (QW), or MLN0264 3.75 or 7.5 mg/kg QW. In combination studies, animals received vehicle, or MLN0264 7.5 mg/kg QW alone or in combination with gemcitabine 15 or 20 mg/kg twice weekly (BIW), or gemcitabine 15 mg/kg on days 1, 3 each week. Average tumor volume was determined at multiple time points following the start of treatment using vernier callipers. Results: In the GCC-expressing PHTX-249 mouse xenograft model ( KRAS mutant G12), single-agent MLN0264 showed significant tumor growth inhibition (TGI) versus vehicle or free MMAE by day 21, with the 7.5 mg/kg dose significantly better than 3.75 mg/kg by day 20-22. Similarly, in the GCC-expressing PHTX-215 model ( KRAS wild-type), MLN0264 7.5 mg/kg resulted in significantly greater TGI (79%) versus free MMAE or MLN0264 3.75 mg/kg by day 22, including some tumor regression. Across the 7 models (variable apical GCC expression; KRAS wild-type and mutant), TGI ranged from 24% (p=0.17) to 79% (p<0.001) with single-agent MLN0264. In the PHTX-249 model, MLN0264 7.5 mg/kg plus gemcitabine 15/20 mg/kg BIW or 15 mg/kg, days 1, 3, showed significantly greater TGI than either agent alone. At day 20-21, TGI was 46-47% for single-agent MLN0264, 66-79% for gemcitabine 15 mg/kg BIW and gemcitabine days 1, 3, and 84-88% for the respective combinations; 3 of 7 animals in the latter combination group had a smaller tumor volume at day 20 versus day 0 (TGI, 93%). Conclusions: These findings indicate that MLN0264 has antitumor activity as a single agent and in combination with gemcitabine in GCC-expressing pancreatic cancer xenograft models, and support clinical evaluation of MLN0264 in patients with pancreatic cancer. Data from additional models will be presented. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):PR12. Citation Format: Julie Zhang, Melissa Gallery, Tim Wyant, Brad Stringer, Mark Manfredi, Hadi Danaee, Petter Veiby. MLN0264, an investigational, first-in-class antibody-drug conjugate (ADC) targeting guanylyl cyclase C (GCC), demonstrates antitumor activity alone and in combination with gemcitabine in human pancreatic cancer xenograft models expressing GCC. [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 PR12.

A monomethyl auristatin E-conjugated antibody to guanylyl cyclase C is cytotoxic to target-expressing cells in vitro and in vivo

Guanylyl cyclase C (GCC) is a cell-surface protein that is expressed by normal intestinal epithelial cells, more than 95% of metastatic colorectal cancers (mCRC), and the majority of gastric and pancreatic cancers. Due to strict apical localization, systemically delivered GCC-targeting agents should not reach GCC in normal intestinal tissue, while accessing antigen in tumor. We generated an investigational antibody-drug conjugate (TAK-264, formerly MLN0264) comprising a fully human anti-GCC monoclonal antibody conjugated to monomethyl auristatin E via a protease-cleavable peptide linker. TAK-264 specifically bound, was internalized by, and killed GCC-expressing cells in vitro in an antigen-density-dependent manner. In GCC-expressing xenograft models with similar GCC expression levels/patterns observed in human mCRC samples, TAK-264 induced cell death, leading to tumor regressions and long-term tumor growth inhibition. TAK-264 antitumor activity was generally antigen-density-dependent, although some GCC-expressing tumors were refractory to TAK-264-targeted high local concentrations of payload. These data support further evaluation of TAK-264 in the treatment of GCC-expressing tumors.

Abstract C146: Combination treatment with the investigational RAF kinase inhibitor MLN2480 and the investigational MEK kinase inhibitor TAK-733 inhibits the growth of BRAF mutant and RAS mutant preclinical models of melanoma and CRC.

Background: RAS mutant melanoma and colorectal cancer represent areas of significant unmet medical need. MLN2480 is an investigational class II RAF kinase inhibitor and TAK-733 is an investigational allosteric MEK kinase inhibitor; each of which is the subject of a single agent phase I clinical trial. The present studies have characterized the combination activity of these agents in BRAF mutant and RAS mutant preclinical models of melanoma and colorectal cancer. Methods: Combination effects of MLN2480 and TAK-733 on cell viability were studied using an ATP-based cell viability assay across a panel of BRAF and RAS mutant melanoma and CRC cell lines. Western blot analysis was used to compare effects on MAPK pathway signaling and response markers in cell lines showing a range of sensitivity to this combination. Pharmacodynamic responses and growth inhibitory effects of the combination were studied in xenografts of the same cell lines, as well as in primary human tumor xenografts, of RAS mutant melanoma and CRC. Results: MLN2480 inhibits MAPK pathway signaling in BRAF mutant and some RAS mutant preclinical cancer models at concentrations that are tolerated in vivo. MLN2480 is most potent in BRAF mutant melanoma models but also has single agent activity in some RAS mutant models. The combination of MLN2480 with TAK-733 inhibits the growth of a broader range of RAS mutant tumor models than single agent MLN2480, including primary human tumor xenograft models of melanoma and CRC. In vitro analysis of this drug combination in cell proliferation assays demonstrates synergistic activity. Western blot analysis demonstrated the effect of MLN2480 in reversing feedback activation of MEK in response to TAK-733, leading to more concerted MAPK pathway inhibition. Conclusions: The activity of the RAF kinase inhibitor MLN2480 in preclinical models of BRAF and RAS mutant melanoma and CRC provides a rationale for clinical testing. The combination of MLN2480 with the MEK inhibitor TAK-733 represents an additional strategy for clinical research within these tumor types. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C146. Citation Format: Elizabeth Grace Carideo Cunniff, Julie Zhang, Jouhara Chouitar, Jerome Mettetal, Kazuhide Nakamura, Takeo Arita, Akito Nakamura, Masanori Okaniwa, Tomoyasu Ishikawa, Sei Yoshida, Robyn Fabrey, Patrick Vincent, Kurt Eng, Khristofer Garcia, Deanna Borelli, Teena Vagrhese, Steve Stroud, Saurabh Menon, Mike Kuranda, Katherine Galvin. Combination treatment with the investigational RAF kinase inhibitor MLN2480 and the investigational MEK kinase inhibitor TAK-733 inhibits the growth of BRAF mutant and RAS mutant preclinical models of melanoma and CRC. [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 C146.

Abstract B40: Development of a pharmacodynamic assay for the second‐generation proteasome inhibitor MLN9708 in clinical trials of multiple myeloma

The ubiquitin‐proteasome system processes the majority of cellular proteins and is the principal pathway by which cells regulate protein homeostasis. The successful development of VELCADE® (bortezomib) for Injection for multiple myeloma and previously treated mantle cell lymphoma has validated the proteasome as a therapeutic target for the treatment of malignancies. MLN9708 is a second‐generation reversible proteasome inhibitor developed to achieve greater oral bioavailability, improved pharmacokinetics and greater antitumor activity than bortezomib. MLN9708 is currently in human clinical development for both hematological and non‐hematological malignancies. MLN9708 immediately hydrolyzes to the biologically active form MLN2238 upon exposure to aqueous solutions or plasma, and MLN2238 was used for all preclinical studies described below. In vitro, MLN2238 inhibited 20S proteasome activity, preferentially binding the 20S 5 site with an IC50 of 3.4 nM and demonstrated potent activity against cultured cancer cells in cell viability assays. In vivo, MLN2238 achieved exposures that resulted in significant blood and tumor proteasome inhibition in xenograft‐bearing mice and had increased plasma and tumor exposure compared to bortezomib when dosed at their respective maximum tolerated doses (MTD). MLN2238 also elicited a stronger pharmacodynamic (PD) response than bortezomib in xenograft tumors, as measured by tumor 20S 5 site‐specific activity and expression levels of GADD34 and ATF‐3, two genes involved in the unfolded protein response (UPR) pathway shown to be upregulated in response to proteasome inhibition. Here we describe the development of an ATF‐3 IHC assay suitable for use in bone marrow samples isolated from multiple myeloma patients enrolled in Phase I trials of MLN9708. Using both quantitativeWestern blotting and IHC assays, we demonstrated that treatment of cultured myeloma cell lines with MLN2238 results in a dose‐dependent increase in ATF‐3 levels. Antibody specificity was confirmed by IHC analysis of HCT‐116 cells following knockdown of ATF‐3 by siRNA. An ATF‐3 PD response is detectable by IHC in several xenograft models and in selected normal tissues from mice dosed with MLN2238. The elevation in ATF‐3 is delayed by several hours compared to 20S proteasome inhibition in cells and tissues, consistent with it being a downstream effect of proteasome inhibition. To test the use of the ATF‐3 IHC assay on clinical samples from multiple myeloma patients, we developed a dual staining assay with CD38, a marker expressed on multiple myeloma cells. We show baseline staining of ATF‐3 and CD38 in formalin‐fixed paraffin embedded bone marrow aspirates and biopsies from patients who have not received MLN9708. This assay has potential for use in evaluating the levels of CD38 and ATF‐3 in pre‐ and post‐treatment bone marrow samples from patients treated with MLN9708. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B40.

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.