Laura Kay Shawver

New paradigms for the treatment of cancer: The role of anti-angiogenesis agents

Angiogenesis, the sprouting of new blood vessels, plays a role in diverse disease states including cancer, diabetic retinopathy, age-related macular degeneration, rheumatoid arthritis, psoriasis, atherosclerosis, and restenosis. With regard to cancer, the clinical association of tumor vascularity with tumor aggressiveness has been clearly demonstrated in numerous tumor types. The observation of increased microvessel density in tumors not only serves as an independent prognostic indicator, but also suggests that anti-angiogenic therapy may be an important component of treatment regimens for cancer patients. The complexity of the angiogenic process, which involves both positive and negative regulators, provides a number of targets for therapy. Many positive regulators, including growth factor receptors, matrix metalloproteinases, and integrins, have been correlated with increased vascularity of tumors and poor prognosis for patient survival. Thus, these serve as ideal targets for anti-angiogenesis therapy. Many inhibitors of these targets are currently undergoing clinical evaluation as potential anti-cancer agents. In this article, we discuss the role of positive regulators in angiogenesis and tumor growth and describe the anti-angiogenic agents under development.

Targeting the AAA ATPase p97 as an Approach to Treat Cancer through Disruption of Protein Homeostasis

p97 is a AAA-ATPase with multiple cellular functions, one of which is critical regulation of protein homeostasis pathways. We describe the characterization of CB-5083, a potent, selective, and orally bioavailable inhibitor of p97. Treatment of tumor cells with CB-5083 leads to accumulation of poly-ubiquitinated proteins, retention of endoplasmic reticulum-associated degradation (ERAD) substrates, and generation of irresolvable proteotoxic stress, leading to activation of the apoptotic arm of the unfolded protein response. In xenograft models, CB-5083 causes modulation of key p97-related pathways, induces apoptosis, and has antitumor activity in a broad range of both hematological and solid tumor models. Molecular determinants of CB-5083 activity include expression of genes in the ERAD pathway, providing a potential strategy for patient selection.

Tyrosine kinases in disease: overview of kinase inhibitors as therapeutic agents and current drugs in clinical trials

Tyrosine kinases, first described as oncogenes, have been shown to play a role in normal cellular processes. Aberrations in tyrosine kinase activity lead to disease states. For fifteen years it has been postulated that the inhibition of tyrosine kinases may have therapeutic utility and the design and testing of inhibitors have been major focuses of research and development in both academic institutions and pharmaceutical companies. While early research focused on developing chemical entities that mimic phosphotyrosine, later research has focused on developing competitive adenosine triphosphate (ATP) inhibitors with various levels of selectivity on kinase targets. This review focuses on a discussion of tyrosine kinases thought to be important in disease, including platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), vascular endothelial cell growth factor (VEGF), epidermal growth factor (EGF) receptors, HER-2 and Src. In addition, the classes of inhibitors designed to affect these targets and that have overcome research and development challenges and entered clinical trials are discussed. These include isoxazole, quinazoline, substituted pyrimidines and indolinone compounds, all of which are in clinical trials or near clinical development by SUGEN, Zeneca, Novartis, Pfizer and Parke-Davis. A summary of the chemistry and activity of these agents is provided.

Receptor tyrosine kinases as targets for inhibition of angiogenesis

Anti-angiogenic agents potentially have broad applications in the clinic. Although most agents now in development are intended ultimately for use as anti-cancer drugs, patients with a range of disorders may benefit in the longer term. The signal recognition and transduction processes involved in controlling angiogenesis are complex and are likely to be dependent on the status of the target endothelial cell in a specific organ or tissue. In this review, the authors focus on signaling interactions that affect microvascular endothelium and the role of growth factors and their receptor tyrosine kinases in the regulation of microvessel physiology as they relate to the angiogenic process.

A tumor-derived protein which provides T-cell costimulation through accessory cell activation

A recently described tumor-derived glycoprotein, designated 90K, has been shown to have positive effects on the generation of cytotoxic effector cells (NK/LAK) from human PBMC. To determine the mechanism of these effects, we have examined the effects of 90K on cytokine production by human PBMC. A culture of normal PBMC with 90K alone did not result in IL-2 secretion; however, in coculture with suboptimal doses of ConA, 90K increased IL-2 secretion by PBMC. Coculture of PBMC with 90K and ConA also resulted in increased production of the cytokines IL-1, IL-6, GM-CSF, and TNF alpha. T cells depleted of accessory cells failed to respond to ConA alone, 90K alone, or the combination of ConA and 90K, suggesting that this protein does not have a direct effect on T cells. However, 90K alone was sufficient to induce cytokine production by unfractionated PBMC (IL-1, IL-6, GM-CSF, and TNF alpha) or by CD14-enriched PBMC (IL-1 and IL-6). In addition, expression of ICAM-1 was increased on a human monocytic cell line cultured with purified 90K in the absence of any other stimulus. This 90K-induced upregulation of ICAM-1 expression was accompanied by an increased accessory function of the monocytes, demonstrated by their ability to support ConA-induced activation of peripheral blood T cells. Based on the current data, we propose a model in which 90K activates accessory cells, resulting in the secretion of cytokines and the expression of adhesion molecules, which in turn act as costimulatory signals for T-cell activation. Activated T cells then produce cytokines such as IL-2, which lead to a more vigorous cell-mediated immune response to tumor cells and virus-infected cells. Thus, 90K shows promise as an immunotherapeutic reagent for diseases such as cancer and viral infection.

Pharmacokinetics and interspecies scaling of a novel VEGF receptor inhibitor, SU5416.

The pharmacokinetics and allometric relationships of SU5416, a novel small anti‐angiogenesis agent, were studied. The pharmacokinetics of SU5416 were examined in mice, rats, dogs, and cancer patients. The in‐vitro intrinsic clearance (CLint) was estimated from the in‐vitro metabolism study in mouse, rat, dog, monkey and human liver microsomes. The parameters of interest were correlated across species as a function of bodyweight using an allometric approach. The steady‐state volume of distribution (Vdss), plasma clearance (CLs), and CLint of SU5416 were well correlated across species. The exponent of the allometric relationship (b) of the corresponding parameters was 0.92, 0.80 and 0.66, respectively. The elimination half‐life (t½) was consistent across species and independent of bodyweight. The prediction of CLs, Vdss, CLint, and t½ in humans using the data from mouse, rat, and dog, and monkey (for CLint) was reasonably good (within 4‐fold of the observed values). However, an improved prediction (within 2‐fold of the observed values) of the corresponding parameters in humans was obtained when extrapolation from only the rodent data was performed, suggesting that the rodent data are sufficient for the scale‐up of SU5416 pharmacokinetic parameters in humans. Using allometry, it was possible to achieve reasonable predictions of the pharmacokinetic parameters of SU5416 in cancer patients with various solid tumours.

Discovery of a First-in-Class, Potent, Selective, and Orally Bioavailable Inhibitor of the p97 AAA ATPase (CB-5083)

The AAA-ATPase p97 plays vital roles in mechanisms of protein homeostasis, including ubiquitin-proteasome system (UPS) mediated protein degradation, endoplasmic reticulum-associated degradation (ERAD), and autophagy. Herein we describe our lead optimization efforts focused on in vitro potency, ADME, and pharmaceutical properties that led to the discovery of a potent, ATP-competitive, D2-selective, and orally bioavailable p97 inhibitor 71, CB-5083. Treatment of tumor cells with 71 leads to significant accumulation of markers associated with inhibition of UPS and ERAD functions, which induces irresolvable proteotoxic stress and cell death. In tumor bearing mice, oral administration of 71 causes rapid accumulation of markers of the unfolded protein response (UPR) and subsequently induces apoptosis leading to sustained antitumor activity in in vivo xenograft models of both solid and hematological tumors. 71 has been taken into phase 1 clinical trials in patients with multiple myeloma and solid tumors.

Application of LC/MS/MS in the quantitation of SU101 and SU0020 uptake by 3T3/PDGFr cells

SU101 or leflunomide, has been studied extensively because of its anti-cancer and immunomodulating properties. The parent isoxazole compound is converted in vitro and metabolized in vivo to an open ring isomeric form, SU0020. Several pharmacological activities have been reported for the parent and metabolite compounds including inhibition of platelet-derived growth factor (PDGF)-mediated signaling for the parent compound and inhibition of de novo pyrimidine biosynthesis for the metabolite. The inhibition of PDGF-mediated signaling and the anti-tumor properties have been ascribed to the parent compound. In spite of its short plasma half-life of the parent molecule, SU101 can be administered intermittently in animal tumor models and retain efficacy. Therefore, the relationship between plasma levels of SU101 and its efficacy in tumor-implanted immuno-compromised mice is not well established. This study was conducted to assess the concentration of SU101 in 3T3/PDGFr alpha and beta cells (NIH3T3 mouse fibroblasts engineered to overexpress human PDGFr alpha or beta) to better understand the cellular levels of SU101 and SU0020. Two strains of 3T3/PDGFr cells (alpha and beta) were incubated with 1, 25, and 100 microM concentrations of SU101 for 1, 6, 24, and 48 hours. Quantitation of SU101 and SU0020 in these cell lines was achieved by a specific and sensitive liquid chromatography-tandem mass spectrometry (LC/MS/MS) method. Interestingly, in both alpha and beta cell lysates SU101 was much more concentrated than SU0020. The greater concentration of SU101 versus SU0020 that was observed may be due to the preferential partitioning of SU101 into the cells and this shows that significant levels of the parent drug can reach the pharmacological site of action for inhibition of PDGF receptors. The data suggest that the conversion of SU101 to SU0020 is much slower in these cells than in the incubation media.

The p97 inhibitor CB-5083 is a unique disrupter of protein homeostasis in models of Multiple Myeloma.

Inhibition of the AAA ATPase, p97, was recently shown to be a novel method for targeting the ubiquitin proteasome system, and CB-5083, a first-in-class inhibitor of p97, has demonstrated broad antitumor activity in a range of both hematologic and solid tumor models. Here, we show that CB-5083 has robust activity against multiple myeloma cell lines and a number of in vivo multiple myeloma models. Treatment with CB-5083 is associated with accumulation of ubiquitinated proteins, induction of the unfolded protein response, and apoptosis. CB-5083 decreases viability in multiple myeloma cell lines and patient-derived multiple myeloma cells, including those with background proteasome inhibitor (PI) resistance. CB-5083 has a unique mechanism of action that combines well with PIs, which is likely owing to the p97-dependent retro-translocation of the transcription factor, Nrf1, which transcribes proteasome subunit genes following exposure to a PI. In vivo studies using clinically relevant multiple myeloma models demonstrate that single-agent CB-5083 inhibits tumor growth and combines well with multiple myeloma standard-of-care agents. Our preclinical data demonstrate the efficacy of CB-5083 in several multiple myeloma disease models and provide the rationale for clinical evaluation as monotherapy and in combination in multiple myeloma. Mol Cancer Ther; 16(11); 2375–86. ©2017 AACR.

Receptor Tyrosine Kinases in Angiogenesis

Cell to cell communication is imperative for physiological and pathological processes associated with multicellular organisms. This is demonstrated in the process of angiogenesis where the blood vessel endothelial cells require signals generated from cells in the hypoxic tissue in order to initiate the process of forming new capillaries. The transduction of signals from the extracellular milieu of the endothelial cell to the nucleus are receptor-mediated events. It is clear, from the body of research, that some of the receptors that mediate critical events in angiogenesis are receptor tyrosine kinases. Receptor tyrosine kinases (RTKs), also known as growth factor receptors, are a family of transmembrane proteins with a large extracellular ligand-binding domain, a transmembrane domain, and an intracellular domain with intrinsic tyrosine kinase activity (for review, see ref. 1).