Hazel M. Weir

AZD9496: An Oral Estrogen Receptor Inhibitor That Blocks the Growth of ER-Positive and ESR1-Mutant Breast Tumors in Preclinical Models.

Fulvestrant is an estrogen receptor (ER) antagonist administered to breast cancer patients by monthly intramuscular injection. Given its present limitations of dosing and route of administration, a more flexible orally available compound has been sought to pursue the potential benefits of this drug in patients with advanced metastatic disease. Here we report the identification and characterization of AZD9496, a nonsteroidal small-molecule inhibitor of ERα, which is a potent and selective antagonist and downregulator of ERα in vitro and in vivo in ER-positive models of breast cancer. Significant tumor growth inhibition was observed as low as 0.5 mg/kg dose in the estrogen-dependent MCF-7 xenograft model, where this effect was accompanied by a dose-dependent decrease in PR protein levels, demonstrating potent antagonist activity. Combining AZD9496 with PI3K pathway and CDK4/6 inhibitors led to further growth-inhibitory effects compared with monotherapy alone. Tumor regressions were also seen in a long-term estrogen-deprived breast model, where significant downregulation of ERα protein was observed. AZD9496 bound and downregulated clinically relevant ESR1 mutants in vitro and inhibited tumor growth in an ESR1-mutant patient-derived xenograft model that included a D538G mutation. Collectively, the pharmacologic evidence showed that AZD9496 is an oral, nonsteroidal, selective estrogen receptor antagonist and downregulator in ER(+) breast cells that could provide meaningful benefit to ER(+) breast cancer patients. AZD9496 is currently being evaluated in a phase I clinical trial. Cancer Res; 76(11); 3307-18. ©2016 AACR.

MEDI0639: a novel therapeutic antibody targeting Dll4 modulates endothelial cell function and angiogenesis in vivo

The Notch signaling pathway has been implicated in cell fate determination and differentiation in many tissues. Accumulating evidence points toward a pivotal role in blood vessel formation, and the importance of the Delta-like ligand (Dll) 4-Notch1 ligand–receptor interaction has been shown in both physiological and tumor angiogenesis. Disruption of this interaction leads to a reduction in tumor growth as a result of an increase in nonfunctional vasculature leading to poor perfusion of the tumor. MEDI0639 is an investigational human therapeutic antibody that targets Dll4 to inhibit the interaction between Dll4 and Notch1. The antibody cross-reacts to cynomolgus monkey but not mouse species orthologues. In vitro MEDI0639 inhibits the binding of Notch1 to Dll4, interacting via a novel epitope that has not been previously described. Binding to this epitope translates into MEDI0639 reversing Notch1-mediated suppression of human umbilical vein endothelial cell growth in vitro. MEDI0639 administration resulted in stimulation of tubule formation in a three-dimensional (3D) endothelial cell outgrowth assay, a phenotype driven by disruption of the Dll4-Notch signaling axis. In contrast, in a two-dimensional endothelial cell–fibroblast coculture model, MEDI0639 is a potent inhibitor of tubule formation. In vivo, MEDI0639 shows activity in a human endothelial cell angiogenesis assay promoting human vessel formation and reducing the number of vessels with smooth muscle actin-positive mural cells coverage. Collectively, the data show that MEDI0639 is a potent modulator of Dll4-Notch signaling pathway. Mol Cancer Ther; 11(8); 1650–60. ©2012 AACR.

Optimization of a Novel Binding Motif to (E)-3-(3,5-Difluoro-4-((1R,3R)-2-(2-Fluoro-2-Methylpropyl)-3-Methyl-2, 3,4,9-Tetrahydro-1H-Pyrido[3,4-B]Indol-1-Yl)Phenyl)Acrylic Acid (Azd9496), a Potent and Orally Bioavailable Selective Estrogen Receptor Downregulator and Antagonist.

The discovery of an orally bioavailable selective estrogen receptor downregulator (SERD) with equivalent potency and preclinical pharmacology to the intramuscular SERD fulvestrant is described. A directed screen identified the 1-aryl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole motif as a novel, druglike ER ligand. Aided by crystal structures of novel ligands bound to an ER construct, medicinal chemistry iterations led to (E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid (30b, AZD9496), a clinical candidate with high oral bioavailability across preclinical species that is currently being evaluated in phase I clinical trials for the treatment of advanced estrogen receptor (ER) positive breast cancer.

An Antibody Targeted to VEGFR-2 Ig Domains 4-7 Inhibits VEGFR-2 Activation and VEGFR-2–Dependent Angiogenesis without Affecting Ligand Binding

Inhibition of VEGFR-2 signaling reduces angiogenesis and retards tumor growth. Current biotherapeutics that inhibit VEGFR-2 signaling by either sequestering VEGF ligand or inhibiting VEGF binding to VEGFR-2 may be compromised by high VEGF concentrations. Here we describe a biotherapeutic that targets VEGFR-2 signaling by binding to Ig domains 4-7 of VEGFR-2 and therefore has the potential to work independently of ligand concentration. 33C3, a fully human VEGFR-2 antibody, was generated using XenoMouse technology. To elucidate the mechanism of action of 33C3, we have used a number of competition and binding assays. We show that 33C3 binds VEGFR-2 Ig domains 4-7, has no impact on VEGF-A binding to VEGFR-2, and does not compete with an antibody that interacts at the ligand binding site. 33C3 has a high affinity for VEGFR-2 (KD < 1 nmol/L) and inhibits VEGF-A induced phosphorylation of VEGFR-2 with an IC50 of 99 ± 3 ng/mL. In vitro, in a 2D angiogenesis assay, 33C3 potently inhibits both tube length and number of branch points, and endothelial tubule formation in a 3D assay. In vivo, 33C3 is a very effective inhibitor of angiogenesis in both a human endothelial angiogenesis assay and in a human skin chimera model. These data show targeting VEGFR-2 outside of the ligand binding domain results in potent inhibition of VEGFR-2 signaling and inhibition of angiogenesis in vitro and in vivo. Mol Cancer Ther; 10(5); 770–83. ©2011 AACR.

Imidazoles: Sar and Development of a Potent Class of Cyclin-Dependent Kinase Inhibitors.

An imidazole series of cyclin-dependent kinase (CDK) inhibitors has been developed. Protein inhibitor structure determination has provided an understanding of the emerging structure activity trends for the imidazole series. The introduction of a methyl sulfone at the aniline terminus led to a more orally bioavailable CDK inhibitor that was progressed into clinical development.

Imidazole pyrimidine amides as potent, orally bioavailable cyclin-dependent kinase inhibitors

The development of a novel series of imidazole pyrimidine amides as cyclin-dependent kinase (CDK) inhibitors is described. The series was found to have much improved CDK2 inhibition and potent in vitro anti-proliferative effects against cancer cell lines. Control of overall lipophilicity was important to achieve good in vitro potency along with acceptable physiochemical properties and margins against inhibition of both CYP isoforms and the hERG potassium ion channel. A compound with an attractive overall balance of properties was profiled in vivo and possessed suitable physiochemical and pharmacokinetic profiles for oral dosing.

Imidazole Piperazines: Sar and Development of a Potent Class of Cyclin-Dependent Kinase Inhibitors with a Novel Binding Mode.

A piperazine series of cyclin-dependent kinase (CDK) inhibitors have been identified. The compounds exhibit excellent physiochemical properties and a novel binding mode, whereby a bridging interaction via a water molecule with Asp 86 of CDK2, leads to selectivity for the CDK family of enzymes over other kinases. Piperazines 2e and 2i were subsequently shown to inhibit tumour growth when dosed orally in a nude mouse xenograft study. Additional chemical series that exploit this unexpected interaction with Asp 86 are also described.

Effective combination therapies in preclinical endocrine resistant breast cancer models harboring ER mutations

Although endocrine therapy is successfully used to treat patients with estrogen receptor (ER) positive breast cancer, a substantial proportion of this population will relapse. Several mechanisms of acquired resistance have been described including activation of the mTOR pathway, increased activity of CDK4 and activating mutations in ER. Using a patient derived xenograft model harboring a common activating ER ligand binding domain mutation (D538G), we evaluated several combinatorial strategies using the selective estrogen receptor degrader (SERD) fulvestrant in combination with chromatin modifying agents, and CDK4/6 and mTOR inhibitors. In this model, fulvestrant binds WT and MT ER, reduces ER protein levels, and downregulated ER target gene expression. Addition of JQ1 or vorinostat to fulvestrant resulted in tumor regression (41% and 22% regression, respectively) though no efficacy was seen when either agent was given alone. Interestingly, although the CDK4/6 inhibitor palbociclib and mTOR inhibitor everolimus were efficacious as monotherapies, long-term delayed tumor growth was only observed when co-administered with fulvestrant. This observation was consistent with a greater inhibition of compensatory signaling when palbociclib and everolimus were co-dosed with fulvestrant. The addition of fulvestrant to JQ1, vorinostat, everolimus and palbociclib also significantly reduced lung metastatic burden as compared to monotherapy. The combination potential of fulvestrant with palbociclib or everolimus were confirmed in an MCF7 CRISPR model harboring the Y537S ER activating mutation. Taken together, these data suggest that fulvestrant may have an important role in the treatment of ER positive breast cancer with acquired ER mutations.

Abstract 863: Differential activity and SERD sensitivity of clinical ESR1 mutations

Background: Mutations in the ligand binding domain of ESR1 have been identified as recurrent alterations in ER+ metastatic breast cancer patients previously treated with aromatase inhibitors. Selective estrogen receptor degraders (SERDs) such as fulvestrant have been speculated to be a rational therapeutic approach to inhibiting these mutants by promoting receptor inhibition and degradation. Whether such drugs can potently and durably inhibit the distinct ESR1 LBD mutants both in vitro and in vivo has not been well characterized. In this study, we investigated the activities of different SERDs against a set of ESR1 LBD mutants identified in the clinic. Methods: The diversity of ESR1 LBD mutations was analyzed by next generation sequencing of metastatic breast tumors from over 900 patients treated at MSKCC. Laboratory models of these different mutants were generated to interrogate their activities and drug sensitivity. We utilized in vitro reporters of ER driven transcription, proliferation assays, and both cell line derived and patient derived models to characterize the different mutants. Results: In addition to previously characterized mutations, D538G, Y537S/N/C, L536R, and E380Q, we identified a number of novel ESR1 LBD mutations in this series such as S432L, L469V, and Y537D among others. We found that while most ESR1 LBD mutations promoted estrogen independent ER function, some did not. We also found that different activating ER mutants had significant differences in the degree to which they induced estrogen independent signaling. Consistent with these findings, we found that while most SERDs could antagonize all of the mutant receptors, there were significant differences in potency. Whereas certain ESR1 mutants such as E380Q were inhibited at similar concentrations as wild type receptors, other mutants such as Y537S required higher concentrations. These differences appeared to impact the in vivo efficacy of the FDA approved SERD, fulvestrant, which is known to have poor pharmacokinetic properties. However, a SERD with high bioavailability and potency, AZD9496, was found to be sufficient to fully inhibit mutant driven tumor growth from WT, D538G and Y537S expressing tumors in vivo. Conclusions: Altogether, the data suggest that clinical ESR1 LBD mutations have distinct effects in activating the receptor and differentially impact the efficacy of ER antagonists. In order to be broadly effective against different ESR1 LBD mutants, SERDs may require highly optimized pharmacokinetic properties. Citation Format: Weiyi Toy, Hazel Weir, Pedram Razavi, Michael Berger, Wai Lin Wong, Elisa De Stanchina, Joœe Baselga, Sarat Chandarlapaty. Differential activity and SERD sensitivity of clinical ESR1 mutations. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 863.

Abstract A152: Novel autotaxin inhibitory antibodies block lysophosphatidic acid production in plasma and tumour cell proliferation in vitro

Autotaxin (ATX) is a secreted phosphodiesterase that cleaves lysophosphatidylcholine (LPC) in serum to produce lysophosphatidic acid (LPA). LPA can signal through a family of GPCR receptors to mediate cell proliferation, migration and survival in cancer cells. Blocking the enzymatic activity of ATX is proposed to have anti-tumour activity by reducing levels of LPA and preventing signaling through the LPA receptor family. A number of synthetic inhibitors have been developed to understand the role in ATX in driving carcinogenesis and these encompass lipid substrate mimetics as well as inhibitors identified from small molecule library screens. Some of these compounds demonstrate low nM activity in vitro and a subset have shown some in vivo activity although none have progressed into the clinic. We sought to identify novel selective ATX antibodies with low nM affinity, which fully inhibited ATX enzyme activity of human, cyno and mouse ATX protein. Using an ATX enzyme inhibition assay we identified and characterised two ATX antibodies, 9E10 and 18B7 which, interestingly, had different binding mechanisms of inhibition and showed potent cross-species inhibition of ATX in vitro. We measured human and mouse ATX expression levels across of panel of xenograft models and verified ATX activity by measuring cleavage of an ATX substrate using conditioned media from these cells. 9E10 and 18B7 antibodies were shown to inhibit the activity of secreted ATX and prevent cleavage of an ATX substrate. 9E10 and 18B7 showed significant inhibition of LPC stimulated cell growth and IL-8 secretion in a range of ATX and/or LPA responsive tumour cell lines. In ex vivo plasma ATX activity assays, treatment of mouse or human plasma with the ATX antibodies inhibited the increase in LPA levels that are observed over time when serum samples are incubated at 37°C. In summary we have identified highly selective ATX antibody inhibitors which will be useful tools to explore the role of ATX in tumour growth and invasion. Citation Format: Sarah Ross, Scott Collins, Jelena Jovanovic, Jane Kendrew, Simon Barry, Swami Rathanaswami, Blakey David, Hazel Weir. Novel autotaxin inhibitory antibodies block lysophosphatidic acid production in plasma and tumour cell proliferation in vitro. [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 A152.