Steve Powell

Induction of Heart Valve Lesions by Small-Molecule ALK5 Inhibitors

Aberrant signaling by transforming growth factor-β (TGF-β) and its type I (ALK5) receptor has been implicated in a number of human diseases and this pathway is considered a potential target for therapeutic intervention. Transforming growth factor-β signaling via ALK5 plays a critical role during heart development, but the role of ALK5 in the adult heart is poorly understood. In the current study, the preclinical toxicology of ALK5 inhibitors from two different chemistry scaffolds was explored. Ten-week-old female Han Wistar rats received test compounds by the oral route for three to seven days. Both compounds induced histopathologic heart valve lesions characterized by hemorrhage, inflammation, degeneration, and proliferation of valvular interstitial cells. The pathology was observed in all animals, at all doses tested, and occurred in all four heart valves. Immunohistochemical analysis of ALK5 in rat hearts revealed expression in the valves, but not in the myocardium. Compared to control animals, protein levels of ALK5 were unchanged in the heart valves of treated animals. We also observed a physeal dysplasia in the femoro-tibial joint of rats treated with ALK5 inhibitors, a finding consistent with a pharmacological effect described previously with ALK5 inhibitors. Overall, these findings suggest that TGF-β signaling via ALK5 plays a critical role in maintaining heart valve integrity.

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.

Cloning, sequencing and functional expression of a guinea pig lung bradykinin B2 receptor

Kinin receptors are classified as B1 and B2 based upon agonist and antagonist potencies and cloning and expression studies. Using sequences from human and rat bradykinin B2 receptors, polymerase chain reaction (PCR) was utilized to isolate cDNA from guinea pig lung. The receptor obtained is predicted to have 372 amino acids and shares > 80% sequence homology with human, rat, rabbit and mouse B2 receptors. In competition binding experiments in Chinese hamster ovary (CHO-K1) cells in which the guinea pig cDNA was expressed, [3H]bradykinin was displaced by kinin receptor ligands with an order of potency consistent with a B2 subtype. In CHO cells expressing the guinea pig receptor, bradykinin caused a concentration 45Ca2+ efflux. A B1 receptor agonist, desArg9-bradykinin, also caused 45Ca2+ efflux but with a potency several orders of magnitude lower than bradykinin. Curiously, several B1 and B2 receptor antagonists induced 45Ca2+ efflux, indicating that this receptor may be coupled differently in CHO cells than in native tissues.

Early phase TGFβ receptor signalling dynamics stabilised by the deubiquitinase UCH37 promotes cell migratory responses

TGFβ signals through serine/threonine kinase receptors and intracellular Smad transcription factors. An important regulatory step involves ubiquitination of Smads and/or TGFβ receptors by specific ubiquitin ligases, in a process that can be reversed by the deubiquitinating enzyme UCH37. Here, to explore the physiological role of UCH37 in TGFβ signalling we have generated stable and inducible HaCAT keratinocyte and Colo-357 pancreatic carcinoma cell lines mis-expressing UCH37. We show that UCH37 knockdown significantly inhibits the activity of a TGFβ-dependent gene reporter and selectively decreases levels of some TGFβ-dependent target genes, notably p21 and PAI-1, but only during the early phase of TGFβ receptor activation. Interestingly, UCH37 knockdown in Colo-357 cells had no effect on TGFβ-dependent cell proliferation and epithelial-mesenchymal transition, yet significantly impaired cell migration. Collectively, our data indicate that UCH37 sustains early TGFβ pathway activation kinetics that determines threshold-specific gene expression patterns, and that opposing actions of ubiquitin ligases and deubiquitinases influences distinct biological TGFβ-dependent biological responses. Moreover, we suggest that UCH37 could represent a viable target for novel and selective cancer therapeutics.

Inhibition of PI3Kβ Signaling with AZD8186 Inhibits Growth of PTEN-Deficient Breast and Prostate Tumors Alone and in Combination with Docetaxel

Loss of PTEN protein results in upregulation of the PI3K/AKT pathway, which appears dependent on the PI3Kβ isoform. Inhibitors of PI3Kβ have potential to reduce growth of tumors in which loss of PTEN drives tumor progression. We have developed a small-molecule inhibitor of PI3Kβ and PI3Kδ (AZD8186) and assessed its antitumor activity across a panel of cell lines. We have then explored the antitumor effects as single agent and in combination with docetaxel in triple-negative breast (TNBC) and prostate cancer models. In vitro, AZD8186 inhibited growth of a range of cell lines. Sensitivity was associated with inhibition of the AKT pathway. Cells sensitive to AZD8186 (GI50 < 1 μmol/L) are enriched for, but not exclusively associated with, PTEN deficiency. In vivo, AZD8186 inhibits PI3K pathway biomarkers in prostate and TNBC tumors. Scheduling treatment with AZD8186 shows antitumor activity required only intermittent exposure, and that increased tumor control is achieved when AZD8186 is used in combination with docetaxel. AZD8186 is a potent inhibitor of PI3Kβ with activity against PI3Kδ signaling, and has potential to reduce growth of tumors dependent on dysregulated PTEN for growth. Moreover, AZD8186 can be combined with docetaxel, a chemotherapy commonly used to treat advanced TBNC and prostate tumors. The ability to schedule AZD8186 and maintain efficacy offers opportunity to combine AZD8186 more effectively with other drugs. Mol Cancer Ther; 14(1); 48–58. ©2014 AACR.

RNA-Seq Differentiates Tumour and Host mRNA Expression Changes Induced by Treatment of Human Tumour Xenografts with the VEGFR Tyrosine Kinase Inhibitor Cediranib

Pre-clinical models of tumour biology often rely on propagating human tumour cells in a mouse. In order to gain insight into the alignment of these models to human disease segments or investigate the effects of different therapeutics, approaches such as PCR or array based expression profiling are often employed despite suffering from biased transcript coverage, and a requirement for specialist experimental protocols to separate tumour and host signals. Here, we describe a computational strategy to profile transcript expression in both the tumour and host compartments of pre-clinical xenograft models from the same RNA sample using RNA-Seq. Key to this strategy is a species-specific mapping approach that removes the need for manipulation of the RNA population, customised sequencing protocols, or prior knowledge of the species component ratio. The method demonstrates comparable performance to species-specific RT-qPCR and a standard microarray platform, and allowed us to quantify gene expression changes in both the tumour and host tissue following treatment with cediranib, a potent vascular endothelial growth factor receptor tyrosine kinase inhibitor, including the reduction of multiple murine transcripts associated with endothelium or vessels, and an increase in genes associated with the inflammatory response in response to cediranib. In the human compartment, we observed a robust induction of hypoxia genes and a reduction in cell cycle associated transcripts. In conclusion, the study establishes that RNA-Seq can be applied to pre-clinical models to gain deeper understanding of model characteristics and compound mechanism of action, and to identify both tumour and host biomarkers.

Abstract 3343: The pre-clinical in vitro and in vivo activity of AZD6738: A potent and selective inhibitor of ATR kinase.

AZD6738 is a potent and selective orally bioavailable kinase inhibitor of ataxia telangiectasia and rad3 related (ATR). Here we report the pre-clinical in vitro and in vivo and biological profile of AZD6738. ATR is a serine/threonine protein kinase involved in DNA damage response signalling caused by DNA replication associated stress. Activation of ATR at stalled replication forks leads to suppression of replication fork origin firing, promotes repair and S/G2-cell cycle checkpoints to prevent premature mitosis and maintain genomic integrity. Failure to resolve damage leads to genomic instability and if sufficiently high, cell death. Stalled replication forks may collapse leading to formation of DNA double stranded breaks and activation of the ataxia telangiectasia mutated (ATM) kinase. ATM works in conjunction with ATR to efficiently resolve replication associated DNA damage creating a co-dependency with loss of one leading to a greater reliance on the other to maintain genomic stability. ATM is frequently inactivated across B-cell malignancies, head and neck, breast and lung cancers through chromosomal deletion, promoter hypermethylation or mutation. ATM-deficient tumours are hypothesised to be more reliant on ATR for survival and specific inhibition of ATR may lead enhanced anti-tumour activity while minimizing normal tissue toxicity. AZD6738 demonstrates this preclinical profile. AZD6738 inhibits the phosphorylation of direct downstream substrate CHK1 while increasing the phosphorylation of ATM-dependent substrate CHK2 and DNA damage marker γH2AX. This is associated with impaired S-phase cell cycle progression with prolonged inhibition causing cell death, indicative of replication fork stalling, collapse and irreversible damage. AZD6738 is active as a single agent across cancer cell line panels but shows enhanced sensitivity in cell lines with ATM-pathway defects. AZD6738 when used in combination with DNA damaging inducing agents’ gemcitabine, cisplatin or ionising radiation (IR) shows enhanced synergistic cell killing activity. In vivo, AZD6738 monotherapy treatment leads to significant anti-tumour activity in ATM-deficient but not ATM-proficient xenograft models at equivalent, tolerated doses. When AZD6738 is used in combination with carboplatin or IR anti-tumour growth inhibitory activity or regression is observed. AZD6738 in vivo activity is also associated with a persistent increased γH2AX staining in tumour tissue but only a transient increase in normal bone marrow or gut tissue suggesting a favourable therapeutic index can be achieved. Early pre-clinical safety studies support these findings with target related bone marrow suppression, neutropenia and GI tract impact only at high-doses. These data merit further investigation of AZD6738 as a monotherapy or in combination with chemo or radiotherapy. Citation Format: Sylvie M. Guichard, Elaine Brown, Rajesh Odedra, Adina Hughes, Dan Heathcote, Jen Barnes, Alan Lau, Steve Powell, Clifford D. Jones, Willem Nissink, Kevin M. Foote, Philip J. Jewsbury, Martin Pass. The pre-clinical in vitro and in vivo activity of AZD6738: A potent and selective inhibitor of ATR kinase. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3343. doi:10.1158/1538-7445.AM2013-3343

Irreversible Inhibition of EGFR: Modeling the Combined Pharmacokinetic–Pharmacodynamic Relationship of Osimertinib and Its Active Metabolite AZ5104

Osimertinib (AZD9291) is a potent, selective, irreversible inhibitor of EGFR-sensitizing (exon 19 and L858R) and T790M-resistant mutation. In vivo, in the mouse, it is metabolized to an active des-methyl metabolite, AZ5104. To understand the therapeutic potential in patients, this study aimed to assess the relationship between osimertinib pharmacokinetics, the pharmacokinetics of the active metabolite, the pharmacodynamics of phosphorylated EGFR reduction, and efficacy in mouse xenograft models of EGFR-driven cancers, including two NSCLC lines. Osimertinib was dosed in xenografted models of EGFR-driven cancers. In one set of experiments, changes in phosphorylated EGFR were measured to confirm target engagement. In a second set of efficacy studies, the resulting changes in tumor volume over time after repeat dosing of osimertinib were observed. To account for the contributions of both molecules, a mathematical modeling approach was taken to integrate the resulting datasets. The model was able to describe the pharmacokinetics, pharmacodynamics, and efficacy in A431, PC9, and NCI-H1975 xenografts, with the differences in sensitivity described by the varying potency against wild-type, sensitizing, and T790M-mutant EGFR and the phosphorylated EGFR reduction required to reduce tumor volume. It was inferred that recovery of pEGFR is slower after chronic dosing due to reduced resynthesis. It was predicted and further demonstrated that although inhibition is irreversible, the resynthesis of EGFR is such that infrequent intermittent dosing is not as efficacious as once daily dosing. Mol Cancer Ther; 15(10); 2378–87. ©2016 AACR.

Abstract A109: AZD9291: an irreversible, potent and selective third generation tyrosine kinase inhibitor (TKI) targeting EGFR activating (EGFRm+) and resistance (T790M) mutations in advanced lung adenocarcinoma.

The first generation EGFR TKIs gefitinib and erlotinib provide significant clinical benefit in patients with advanced lung adenocarcinoma harbouring activating EGFR mutants (EGFRm+), but patients will ultimately develop disease progression due to acquired resistance. Acquisition of the EGFR T790M mutation is the most common mechanism of drug resistance, detected in more than 50% of gefitinib/erlotinib resistant patients. Current therapeutic strategies are limited for advanced lung adenocarcinoma patients with EGFR T790M (EGFRm+/T790M), so this remains a key area of unmet need. AZD9291 (structure to be disclosed at meeting) is an oral, irreversible, third generation, selective inhibitor of both EGFR activating (EGFRm+) and resistance (EGFRm+/T790M) mutations. The mechanistic and functional activity of AZD9291 was characterised in vitro and in vivo across a number of cell lines harbouring various EGFR-mutations or wild type EGFR. Presented data shows AZD9291 potently inhibits EGFR phosphorylation in EGFRm+ (e.g. PC9; 500nM). Consistently, AZD9291 showed significantly more potent inhibition of proliferation in mutant EGFR cell lines compared to wild-type in vitro. In addition, AZD9291 administered once daily orally at 5mg/kg caused profound regression of tumours across EGFRm+ (PC9; 178% growth inhibition) and EGFRm+/T790M (H1975; 119% growth inhibition) tumour models in vivo, after 14 days dosing. Furthermore 5mg/kg AZD9291 was sufficient to cause significant shrinkage of EGFRm+ and EGFRm+/T790M transgenic mouse lung tumours. Tumour growth inhibition was associated with profound inhibition of EGFR phosphorylation and key downstream signaling pathways such as AKT and ERK. Chronic long-term treatment of PC9 and H1975 xenograft tumours with AZD9291 led to a complete and sustained macroscopic response, with no visible tumours after 40 days dosing, and being maintained beyond 100 days. Furthermore, pre-clinical data also indicates that AZD9291 could target tumours that have acquired resistance to the more recently identified HER2-amplification mechanism, thus potentially extending its benefit in TKI resistant patients. Taken together, preclinical data demonstrates that AZD9291 is a potent and effective inhibitor of both EGFR activating (EGFRm+) and resistance (EGFRm+/T790M) mutations whilst sparing wild-type EGFR. These data support the further clinical investigation of AZD9291 in advanced EGFR mutant lung adenocarcinoma. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A109. Citation Format: Darren Cross, Sue Ashton, Caroline Nebhan, Cath Eberlein, M. Raymond V. Finlay, Gareth Hughes, Vivien Jacobs, Martine Mellor, Monica Red Brewer, Catherine Meador, Jonathon Orme, Paula Spitzler, Steve Powell, Amar Rahi, Paula Taylor, Richard A. Ward, Paula Daunt, Anne Galer, Teresa Klinowska, Graham Richmond, William Pao. AZD9291: an irreversible, potent and selective third generation tyrosine kinase inhibitor (TKI) targeting EGFR activating (EGFRm+) and resistance (T790M) mutations in advanced lung adenocarcinoma. [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 A109.

Expression of stromal genes associated with the angiogenic response are not differentiated between human tumour xenografts with divergent vascular morphologies.

Human tumour xenografts have commonly been used to explore the mechanisms of tumour angiogenesis and the interaction of tumour cells with their microenvironment, as well as predict potential utility of anti-angiogenic inhibitors across different tumour types. To investigate how well human tumour xenografts can be used to differentiate the effects of stromal targeting agents we performed a comparative assessment of the murine angiogenic response across a panel of pre-clinical tumour xenografts. By analysing a panel of 22 tumour xenografts with a range of vascular morphologies, micro-vessel densities and levels of fibroblast and inflammatory infiltrate, we have examined the relationship between angiogenic stroma and human tumour models. These models were studied using a combination of immunohistochemistry and species specific mRNA profiling to differentiate the tumour and stromal transcript mRNA profiles. Principal Component Analysis (PCA) and regression analysis was used to investigate the transcriptional relationships between the individual models and the correlation with the stromal architecture. We found the human tumour cell expressed factors to be independent of the murine host responses such as microvessel density, and fibroblast or macrophage cellular infiltrate. Moreover mRNA profiling of the mouse stroma suggested that the host response to the different tumours was relatively uniform despite differences in stromal structures within the tumour. Supporting this, models with different stromal compositions responded similarly to cediranib, a small molecule inhibitor of VEGF signalling. The data indicate that although the angiogenic response to the tumour results in reproducible stromal architectures, these responses are not differentiated at the level of gene expression.