Zena Wilson

CXCR2 Inhibition Profoundly Suppresses Metastases and Augments Immunotherapy in Pancreatic Ductal Adenocarcinoma.

Summary CXCR2 has been suggested to have both tumor-promoting and tumor-suppressive properties. Here we show that CXCR2 signaling is upregulated in human pancreatic cancer, predominantly in neutrophil/myeloid-derived suppressor cells, but rarely in tumor cells. Genetic ablation or inhibition of CXCR2 abrogated metastasis, but only inhibition slowed tumorigenesis. Depletion of neutrophils/myeloid-derived suppressor cells also suppressed metastasis suggesting a key role for CXCR2 in establishing and maintaining the metastatic niche. Importantly, loss or inhibition of CXCR2 improved T cell entry, and combined inhibition of CXCR2 and PD1 in mice with established disease significantly extended survival. We show that CXCR2 signaling in the myeloid compartment can promote pancreatic tumorigenesis and is required for pancreatic cancer metastasis, making it an excellent therapeutic target.

Assessing the Activity of Cediranib, a VEGFR-2/-3 tyrosine kinase inhibitor, against VEGFR-1 and members of the structurally related PDGFR-family

Cediranib is a potent inhibitor of the VEGF receptor (VEGFR)-2 and VEGFR-3 tyrosine kinases. This study assessed the activity of cediranib against the VEGFR-1 tyrosine kinase and the platelet-derived growth factor receptor (PDGFR)-associated kinases c-Kit, PDGFR-α, and PDGFR-β. Cediranib inhibited VEGF-A–stimulated VEGFR-1 activation in AG1-G1-Flt1 cells (IC50 = 1.2 nmol/L). VEGF-A induced greatest phosphorylation of VEGFR-1 at tyrosine residues Y1048 and Y1053; this was reversed by cediranib. Potency against VEGFR-1 was comparable with that previously observed versus VEGFR-2 and VEGFR-3. Cediranib also showed significant activity against wild-type c-Kit in cellular phosphorylation assays (IC50 = 1–3 nmol/L) and in a stem cell factor–induced proliferation assay (IC50 = 13 nmol/L). Furthermore, phosphorylation of wild-type c-Kit in NCI-H526 tumor xenografts was reduced markedly following oral administration of cediranib (≥1.5 mg/kg/d) to tumor-bearing nude mice. The activity of cediranib against PDGFR-β and PDGFR-α was studied in tumor cell lines, vascular smooth muscle cells (VSMC), and a fibroblast line using PDGF-AA and PDGF-BB ligands. Both receptor phosphorylation (IC50 = 12–32 nmol/L) and PDGF-BB–stimulated cellular proliferation (IC50 = 32 nmol/L in human VSMCs; 64 nmol/L in osteosarcoma cells) were inhibited. In vivo, ligand-induced PDGFR-β phosphorylation in murine lung tissue was inhibited by 55% following treatment with cediranib at 6 mg/kg but not at 3 mg/kg or less. In contrast, in C6 rat glial tumor xenografts in mice, ligand-induced phosphorylation of both PDGFR-α and PDGFR-β was reduced by 46% to 61% with 0.75 mg/kg cediranib. Additional selectivity was showed versus Flt-3, CSF-1R, EGFR, FGFR1, and FGFR4. Collectively, these data indicate that cediranib is a potent pan-VEGFR kinase inhibitor with similar activity against c-Kit but is significantly less potent than PDGFR-α and PDGFR-β. Mol Cancer Ther; 10(5); 861–73. ©2011 AACR.

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.

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.

PDGFR Inhibition Results in Pericyte Depletion and Hemorrhage into the Corpus Luteum of the Rat Ovary

The growth plate, ovary, adrenal gland, and rodent incisor tooth are sentinel organs for antiangiogenic effects since they respond reliably, quantitatively, and sensitively to inhibition of the vascular endothelial growth factor receptor (VEGFR). Here we report that treatment of rats with platelet-derived growth factor receptor beta (PDGFRβ) inhibitors that target pericytes results in severe ovarian hemorrhage with degeneration and eventual rupture of the corpus luteum. Evaluation of the growth plate, adrenal gland, and incisor tooth that are typical target organs for antiangiogenic treatment in the rodent revealed no abnormalities. Histologically, the changes in the ovary were characterized by sinusoidal dilatation, increased vessel fragility, and hemorrhage into the corpus luteum. Immunocytochemical staining of vessels with alpha smooth muscle actin and CD31 that recognize pericytes and vascular endothelium, respectively, demonstrated that this effect was due to selective pericyte deficiency within corpora lutea. Further experiments in which rats were treated concurrently with both PDGFRβ and VEGFR inhibitors ablated the hemorrhagic response, resulting instead in corpus luteum necrosis. These changes are consistent with the notion that selective pericyte loss in the primitive capillary network resulted in increased vessel fragility and hemorrhage, whereas concomitant VEGFR inhibition resulted in vessel regression and reduced vascular perfusion that restricted development of the hemorrhagic vessels. These results also highlight the utility of the rodent ovary to respond differentially to VEGFR and PDGFR inhibitors, which may provide useful information during routine safety assessment for determining target organ toxicity.

Discovery and characterization of AZD6738, a potent inhibitor of ataxia telangiectasia mutated and rad3 related (ATR) kinase with application as an anti-cancer agent

The kinase ataxia telangiectasia mutated and rad3 related (ATR) is a key regulator of the DNA-damage response and the apical kinase which orchestrates the cellular processes that repair stalled replication forks (replication stress) and associated DNA double-strand breaks. Inhibition of repair pathways mediated by ATR in a context where alternative pathways are less active is expected to aid clinical response by increasing replication stress. Here we describe the development of the clinical candidate 2 (AZD6738), a potent and selective sulfoximine morpholinopyrimidine ATR inhibitor with excellent preclinical physicochemical and pharmacokinetic (PK) characteristics. Compound 2 was developed improving aqueous solubility and eliminating CYP3A4 time-dependent inhibition starting from the earlier described inhibitor 1 (AZ20). The clinical candidate 2 has favorable human PK suitable for once or twice daily dosing and achieves biologically effective exposure at moderate doses. Compound 2 is currently being tested in multiple phase I/II trials as an anticancer agent.

Abstract 1828: Are callipers obsolute? A novel 3D scanning technology to measure subcutaneous tumor volume

Most preclinical oncology studies (xenograft, PDX, GEMMS) involve monitoring tumour growth rates, measuring them with callipers, and calculating the volume. Volume is calculated from the width and the length to estimate a 3D volume and is directly used to assess treatment efficacy. Although this technique is useful, it is unable to accurately assess non-uniformly shaped or very small tumours and introduces a systematic bias by assuming that tumours present with spheroid shape. Furthermore callipers do not inform of the tumour condition, which is dependent upon a visual estimation. Here we describe the development and validation of a 3D scanner as an alternative method to callipers to monitor tumour progression in rodents. The resulting 3D scanner solution made up of hardware and software, has the potential to impact on the 3Rs guiding principles underpinning the humane use of animals in oncology research. The 3Rs benefits identified are primarily through reduction of animals via improved data accuracy allowing reduction in group sizes or the ability to include irregularly shaped tumours to test. In addition the scanner system described will make it possible to record tumour measurements in a rapid, minimally invasive, morphology-independent, and human-bias-free way, removing interoperator variability. This photo-based technique captures external symptoms of redness, paleness, ulceration of tumours, etc., which could ultimately be used to detect early toxicities of compounds or determine scales of animal welfare. We describe the development and early validation of the scanner system within our laboratories. Using the 3D scanner alongside tumour callipers to monitor tumour growth of Oncology tumour studies we demonstrated that we can accurately measure tumour size parameters (length, width and volume) in multiple mouse strains and across a range of tumour models. 3D scanning tumour data is comparable to tumour measures generated from tumour callipers If successful the introduction of this system to replace tumour callipers could have a large impact for groups running oncology in-vivo tumour studies. Citation Format: Zena Wilson, Juan Delgado, Michael Davies, Rebecca Whiteley, Jennifer Hare, Amar Rahi, Stephen Marshall, Andrew Smith, Stephen Atkinson, Jarno Ralli, Adeala Zabair, Adeala Zabair, Jane Kendrew. Are callipers obsolute? A novel 3D scanning technology to measure subcutaneous tumor volume [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1828. doi:10.1158/1538-7445.AM2017-1828

Abstract 2494: ATR inhibitor AZD6738 as monotherapy and in combination with olaparib or chemotherapy: defining pre-clinical dose-schedules and efficacy modelling

Introduction: AZD6738 is a potent and selective oral inhibitor of the ataxia telangiectasia and rad3 related (ATR) protein kinase. ATR has a key role in the DNA replication stress response (RSR) pathway of DNA repairby facilitating the recovery and repair of potentially cytotoxic persistent, stalled DNA replication forks. Inhibition of ATR leads to the inability to resolve replication associated damage and the accumulation of DNA strand breaks, which if remains unrepaired leads to cell death. AZD6738 is currently in Phase I/II clinical trials being evaluated as a monotherapy and in combination with novel agents olaparib / Lynparza (PARP DNA damage response inhibitor), durvalumab (PD-L1 immune checkpoint inhibitor) and DNA-damaging agents such as carboplatin and ionising radiation. Critical in helping to guide the clinical usage of AZD6738 and maximise patient benefit, pre-clinical studies were performed to determine optimal doses and schedules as monotherapy and in combination with olaparib and carboplatin. Experimental procedures: Human cancer cell lines, xenograft and patient-derived explant (PDX) models of non-small cell lung cancer (NSCLC), head & neck squamous cell carcinoma (HNSCC), and triple-negative breast cancer (TNBC) were tested comparing once daily versus twice daily dosing, the number of consecutive days dosing (3 days/week, 5 days/week, continuous) and co-dosing versus sequential or intermittent dosing with AZD6738 alone or in combination with olaparib or carboplatin. The magnitude and duration of anti-tumour responses were then compared with AZD6738 mouse pharmacokinetic (PK), pharmacodynamic (PD) and in vitro target (IC) / growth inhibition (GI) profiles. Results: A mathematical model was derived which adequately described the AZD6738 PK/PD-efficacy relationship. This modelling confirms that duration of cover (time) above cellular ATR target inhibition thresholds (IC90 pCHK1 / GI90) per day, rather than Cmax or exposures per se, is the major determinant of anti-tumour responses. As monotherapy, in sensitive ATM-deficient models, it is necessary to inhibit ATR continuously to give tumour stabilisation, which can be achieved through repeat daily of AZD6738 over several weeks. Co-dosing AZD6738 in combination with olaparib or carboplatin gives best efficacy compared to sequential dosing and PK cover over the first 48-72 hours is necessary to give tumour regressions. The models predict that extending the duration of ATR cover, achieved through repeat daily dosing, further increases efficacy. These pre-clinical dose-schedules were compared to human free plasma AZD6738 PK data and predicted efficacious exposures found to be clinically achievable. Conclusions: Together these data further support the clinical evaluation of AZD6738 and suggest optimal dosing schedules for ATR inhibitors. Citation Format: Alan Y. Lau, James Yates, Zena Wilson, Lucy A. Young, Adina M. Hughes, Alienor Berges, Amy Cheung, Rajesh Odedra, Elaine Brown, Mark J. O9Connor, Simon Hollingsworth. ATR inhibitor AZD6738 as monotherapy and in combination with olaparib or chemotherapy: defining pre-clinical dose-schedules and efficacy modelling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2494. doi:10.1158/1538-7445.AM2017-2494

Abstract DDT01-03: Discovery and pre-clinical pharmacology of AZD9496: An oral, selective estrogen receptor down-regulator (SERD)

With over 70% of breast cancers expressing estrogen receptor alpha protein (ERα), treatment with either anti-hormonal therapies that directly block ERα function (e.g. tamoxifen) or therapies that block the production of estrogen itself (e.g. anastrozole) have proven to be effective treatments for the disease. Following the discovery of the ERα antagonist tamoxifen in the 1960s, identification of the selective estrogen receptor down-regulator (SERD) fulvestrant represented a further step forward in the treatment of advanced ER+ breast cancer, especially in the endocrine resistance setting where ERα appears to be activated by a ligand independent route through other growth factor signaling pathways. In addition, fulvestrant has also shown significant overall survival (OS) results in the FIRST trial comparing 500 mg fulvestrant with anastrozole in first line advanced ER+ve patients where the majority of patients had not received prior endocrine therapy. Given fulvestrant9s low bioavailability following intramuscular injection and the levels of ERα protein in clinical samples after treatment, the question remains as to whether an agent that could achieve higher steady state levels of drug more rapidly and drive further decreases in ERα levels would give enhanced clinical benefit. We have identified a novel, potent, non-steroidal SERD that can be administered orally and could yield improved exposure and clinical benefit. This presentation will describe the discovery and pre-clinical pharmacology of AZD9496, a small molecule that can antagonise ERα and induce receptor degradation in breast cancer cell lines at picomolar concentrations. The good oral pharmacokinetic properties of the compound in pre-clinical species led to significant tumor growth inhibition in an endocrine sensitive MCF-7 xenograft model at a dose of 5 mg/kg and >90% reduction in ER-regulated, progesterone receptor (PR) levels. Tumor regressions were seen in a long term estrogen deprived (LTED) in vivo model, representing the aromatase resistant setting, and corresponded with significant reductions in ERα protein levels, >90% at 5 mg/kg dose. AZD9496 also showed antagonist and down-regulation activity against ERα mutant protein both in vitro and in vivo. These findings strongly supported selection of AZD9496 as a clinical candidate for the treatment of ER+ve breast cancer and the drug is now under evaluation in a Phase 1 clinical trial. Citation Format: Hazel Weir, Mandy Lawson, Rowena Callis, Michael Hulse, Michael Tonge, Gareth Davies, Graeme Walker, Rachel Rowlinson, Jon Curwen, Zena Wilson, Steve Powell, Robert Bradbury, Alfred Rabow, Craig Donald, David Buttar, Richard Norman, Camila de Almeida, Peter Ballard, Gordon Currie, David Andrews, Graham Richmond, Anne Marie Mazzola, Ermira Pazolli, Brendon Ladd, Celina D9Cruz, Chris De Savi. Discovery and pre-clinical pharmacology of AZD9496: An oral, selective estrogen receptor down-regulator (SERD). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr DDT01-03. doi:10.1158/1538-7445.AM2015-DDT01-03

Abstract P5-09-20: Development of a novel selective estrogen receptor down-regulator which shows efficacy in pre-clinical models of endocrine resistance

With over 70% of breast cancers expressing the estrogen receptor alpha (ERa), treatment with either anti-hormonal therapies that directly block ER function (e.g.Tamoxifen) or therapies that block the production of estrogen itself (e.g. aromatase inhibitors) are key to management of the disease. Despite initial efficacy seen with endocrine therapies, development of either de novo or acquired resistance ultimately limits the use of these agents although the majority of tumours continue to depend on ERα for growth. The ability to down regulate ER is particularly important in the endocrine resistance setting where ER appears to be activated in a ligand independent fashion by other growth factor signaling pathways. Agents such as Fulvestrant have been shown to offer additional benefit in the advanced setting due to its unique mechanism of binding and degradation of the ER receptor by the ubiquitin-proteosome complex. Given its low level of bioavailability and metabolic profile, Fulvestrant has been formulated as an intramuscular injection with 2 × 250mg monthly doses currently being tested clinically. We have sought to identify a novel, non-steroidal ERα antagonist and down-regulator that can be administered orally and could yield improved bioavailability and clinical benefit through enhanced biomarker modulation. We have identified a novel compound that can induce ERα degradation in breast cancer cell lines at picomolar concentrations with a similar half-life to Fulvestrant and is clearly differentiated from Tamoxifen which appears to stabilise ER protein. Due to good oral pharmacokinetic properties of the compound we have seen significant tumour efficacy in both tamoxifen-sensitive and -resistant models of breast cancer in vivo. In an MCF-7 model we have shown >90% reduction in PR levels in a pharmacodynamic study and complete tumour growth inhibition (TGI) at 5mg/kg dose. Fulvestrant in the same model setting was unable to achieve >60% PR inhibition at dose levels 3.8 fold greater than those achieved clinically with 500mg. We have also seen tumour regressions in a pre-clinical long term estrogen deprived (LTED) model, which mimic the aromatase resistance setting, and efficacy correlates with a significant decrease in ERα levels (>60%). This orally bioavailable compound is progressing towards clinical evaluation and shows promise as a new SERD agent for the treatment of ER+ breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-09-20.