Bruce Ruggeri

Animal models of human disease: Challenges in enabling translation

Animal models have historically played a critical role in the exploration and characterization of disease pathophysiology, target identification, and in the in vivo evaluation of novel therapeutic agents and treatments. In the wake of numerous clinical trial failures of new chemical entities (NCEs) with promising preclinical profiles, animal models in all therapeutic areas have been increasingly criticized for their limited ability to predict NCE efficacy, safety and toxicity in humans. The present review discusses some of the challenges associated with the evaluation and predictive validation of animal models, as well as methodological flaws in both preclinical and clinical study designs that may contribute to the current translational failure rate. The testing of disease hypotheses and NCEs in multiple disease models necessitates evaluation of pharmacokinetic/pharmacodynamic (PK/PD) relationships and the earlier development of validated disease-associated biomarkers to assess target engagement and NCE efficacy. Additionally, the transparent integration of efficacy and safety data derived from animal models into the hierarchical data sets generated preclinically is essential in order to derive a level of predictive utility consistent with the degree of validation and inherent limitations of current animal models. The predictive value of an animal model is thus only as useful as the context in which it is interpreted. Finally, rather than dismissing animal models as not very useful in the drug discovery process, additional resources, like those successfully used in the preclinical PK assessment used for the selection of lead NCEs, must be focused on improving existing and developing new animal models.

Animal models of disease: pre-clinical animal models of cancer and their applications and utility in drug discovery.

Preclinical models of human cancers are indispensable in the drug discovery and development process for new cancer drugs, small molecules and biologics. They are however imperfect facsimiles of human cancers given the genetic and epigenetic heterogeneity of the latter and the multiplicity of dysregulated survival and growth-regulatory pathways that characterize this spectrum of diseases. This review discusses pre-clinical tumor models - traditional ectopic xenografts, orthotopic xenografts, genetically engineered tumor models, primary human tumorgrafts, and various multi-stage carcinogen-induced tumor models - their advantages, limitations, physiological and pathological relevance. Collectively, these animal models represent a portfolio of test systems that should be utilized at specific stages in the drug discovery process in a pragmatic and hierarchical manner of increasing complexity, physiological relevance, and clinical predictability of the human response. Additionally, evaluating the efficacy of novel therapeutic agents emerging from drug discovery programs in a variety of pre-clinical models can better mimic the heterogeneity of human cancers and also aid in establishing dose levels, dose regimens and drug combinations for use in clinical trials. Nonetheless, despite the sophistication and physiological relevance of these human cancer models (e.g., genetically engineered tumor models and primary human tumografts), the ultimate proof of concept for efficacy and safety of novel oncology therapeutics lies in humans. The judicious interpretation and extrapolation of data derived from these models to humans, and a correspondingly greater emphasis placed on translational medical research in early stage clinical trials, are essential to improve on the current clinical attrition rates for novel oncology therapeutic agents.

Antiangiogenic and Antitumor Efficacy of EphA2 Receptor Antagonist

Tumor-associated angiogenesis is critical for tumor growth and metastasis and is controlled by various pro- and antiangiogenic factors. The Eph family of receptor tyrosine kinases has emerged as one of the pivotal regulators of angiogenesis. Here we report that interfering with EphA signaling resulted in a pronounced inhibition of angiogenesis in ex vivo and in vivo model systems. Administration of EphA2/Fc soluble receptors inhibited, in a dose-dependent manner, microvessel formation in rat aortic ring assay, with inhibition reaching 76% at the highest dose of 5000 ng/ml. These results were further confirmed in vivo in a porcine aortic endothelial cell-vascular endothelial growth factor (VEGF)/basic fibroblast growth factor Matrigel plug assay, in which administration of EphA2/Fc soluble receptors resulted in 81% inhibition of neovascularization. The additive effects of simultaneous inhibition of VEGF receptor 2 and EphA signaling pathways in aortic ring assay and antiangiogenic efficacy of EphA2/Fc soluble receptors against VEGF/basic fibroblast growth factor-mediated neovascularization in vivo indicated a critical and nonredundant role for EphA signaling in angiogenesis. Furthermore, in two independent experiments, we demonstrated that EphA2/Fc soluble receptors strongly (by ∼50% versus controls) suppressed growth of ASPC-1 human pancreatic tumor s.c. xenografts. Inhibition of tumor growth was due to decreased proliferation of tumor cells. In an orthotopic pancreatic ductal adenocarcinoma model in mice, suppression of EphA signaling by i.p. administration of EphA2/Fc (30 μg/dose, three times a week for 56 days) profoundly inhibited the growth of primary tumors and the development of peritoneal, lymphatic, and hepatic metastases. These data demonstrate a critical role of EphA signaling in tumor growth and metastasis and provide a strong rationale for targeting EphA2 receptors for anticancer therapies.

Neurotrophins and Trk receptors in human pancreatic ductal adenocarcinoma: Expression patterns and effects on In vitro invasive behavior

The aggressive and highly metastatic behavior observed in pancreatic ductal adenocarcinoma (PDAC) may be due to autocrine and/or paracrine interactions (tumor/stromal) involving altered expression of peptide growth factors and their corresponding receptors. The neurotrophin (NT) growth factor family and their cognate receptors have been demonstrated to play a role in the invasiveness, chemotactic behavior and tumor cell survival of both neuronal and non‐neuronal cancers. We hypothesized that aberrant expression of the NTs and/or the Trk receptors may contribute to the malignant phenotype of PDAC, specifically tumor cell invasiveness, through autocrine and/or paracrine interactions. In this study, we examined the expression of NTs, Trks and p75NGFR by immunohistochemical and in situ hybridization analyses in both normal (n = 14) and neoplastic pancreas (n = 47) and PDAC‐derived cell lines (n = 6). Further, we evaluated the effects of various NTs on the in vitro invasive and chemotactic behavior on 6 human PDAC‐derived cell lines in a modified Boyden chamber assay. Brain‐derived nerve growth factor (BDNF), NT‐3, NT‐4/5 and Trks A, B and C exhibited diffuse cytoplasmic and membranous immunostaining patterns in both the ducts and the acini of the exocrine pancreas and the islets of the endocrine pancreas of both normal and PDAC specimens. NT expression was primarily within the stromal compartment of the tumor, while Trk expression was weak or absent. We observed a 68%, 64% and 66% increase in the expression of Trks A, B and C, respectively, in the ductal elements of the PDAC samples examined compared with the normal adjacent tissue. Invasiveness of 4 of 6 PDAC cell lines was significantly inhibited (p < 0.05) when the cells were incubated with 100 ng/ml NT. However, when select cell lines were incubated with lower concentrations of NT‐3 and BDNF (0, 1, 5, 25 and 50 ng/ml), invasiveness was significantly stimulated (p < 0.05) through the Matrigel matrix. Collectively, our data suggest the possibility that paracrine and/or autocrine NT‐Trk interactions may influence the phenotype (possibly the invasive behavior) of PDAC. Int. J. Cancer 81:417–427, 1999.

Functional validation of the anaplastic lymphoma kinase signature identifies CEBPB and Bcl2A1 as critical target genes

Anaplastic large cell lymphomas (ALCLs) represent a subset of lymphomas in which the anaplastic lymphoma kinase (ALK) gene is frequently fused to the nucleophosmin (NPM) gene. We previously demonstrated that the constitutive phosphorylation of ALK chimeric proteins is sufficient to induce cellular transformation in vitro and in vivo and that ALK activity is strictly required for the survival of ALK-positive ALCL cells. To elucidate the signaling pathways required for ALK-mediated transformation and tumor maintenance, we analyzed the transcriptomes of multiple ALK-positive ALCL cell lines, abrogating their ALK-mediated signaling by inducible ALK RNA interference (RNAi) or with potent and cell-permeable ALK inhibitors. Transcripts derived from the gene expression profiling (GEP) analysis uncovered a reproducible signature, which included a novel group of ALK-regulated genes. Functional RNAi screening on a set of these ALK transcriptional targets revealed that the transcription factor C/EBPbeta and the antiapoptotic protein BCL2A1 are absolutely necessary to induce cell transformation and/or to sustain the growth and survival of ALK-positive ALCL cells. Thus, we proved that an experimentally controlled and functionally validated GEP analysis represents a powerful tool to identify novel pathogenetic networks and validate biologically suitable target genes for therapeutic interventions.

Discovery of a Potent, Selective, and Orally Active Proteasome Inhibitor for the Treatment of Cancer

The ubiquitin-proteasome pathway plays a central role in regulation of the production and destruction of cellular proteins. These pathways mediate proliferation and cell survival, particularly in malignant cells. The successful development of the 20S human proteasome inhibitor bortezomib for the treatment of relapsed and refractory multiple myeloma has established this targeted intervention as an effective therapeutic strategy. Herein, the potent, selective, and orally bioavailable threonine-derived 20S human proteasome inhibitor that has been advanced to preclinical development, [(1R)-1-[[(2 S,3 R)-3-hydroxy-2-[(6-phenylpyridine-2-carbonyl)amino]-1-oxobutyl]amino]-3-methylbutyl]boronic acid 20 (CEP-18770), is disclosed.

Development of vascular endothelial growth factor receptor (VEGFR) kinase inhibitors as anti-angiogenic agents in cancer therapy.

Among the known angiogenic growth factors and cytokines implicated in the modulation of normal and pathological angiogenesis, the VEGF family (VEGF-A, VEGF-B, VEGF-C, VEGF-D) and their corresponding receptor tyrosine kinases [VEGFR-1 (Flt-1), VEGFR-2 (Flk-1, KDR), and VEGFR-3 (Flt-4)] play a paramount and indispensable role in regulating the multiple facets of the angiogenic and lymphangiogenic processes, as well as the induction of vascular permeability and inflammation. The receptor VEGFR-2/KDR is the principal one through which VEGFs exert their mitogenic, chemotactic, and vascular permeabilizing effects on the host vasculature. Increased expression of VEGFs by tumor cells and VEGFR-2/KDR and VEGFR-1/Flt-1 by the tumor-associated vasculature are a hallmark of a variety of human and rodent tumors in vivo and correlates with tumor growth rate, micro-vessel density/proliferation, tumor metastatic potential, and poorer patient prognosis in a variety of malignancies. Approaches to disrupting the VEGF/VEGFR signaling cascade range from biological agents (soluble receptors, anti-VEGF and anti-VEGFR-2 antibodies, and VEGF transcription inhibitors) to small molecule ATP competitive VEGFR inhibitors. Examples from this latter class that are currently in clinical development include compounds from distinct chemical classes such as: indolin-2-ones, anilinoquinazolines, anilinophthalazines, isothiazoles, indolo- and indenocarbazoles. The structure activity relationships, biochemical and pharmacological profile of optimized representatives from each of these classes constitute the subject matter of this review.

Cholecystokinin A and B receptors are differentially expressed in normal pancreas and pancreatic adenocarcinoma.

Cholecystokinin (CCK) plays an important role in pancreatic carcinogenesis. While human CCK-A and -B receptors have been fully characterized, their relative roles in human pancreatic adenocarcinoma remain unclear. Thus, expression of CCK-A and -B receptors in normal human pancreas, pancreatic adenocarcinomas, and other human extrapancreatic tissues and malignancies was examined, using reverse transcription followed by the polymerase chain reaction (RT-PCR). mRNA isolated from 15 normal pancreas specimens, 22 pancreatic adenocarcinomas, and 58 extrapancreatic tissues and tumors was subjected to RT-PCR using primers specific for human CCK-A and -B receptors. Expression of CCK-B receptors was detected in all tissues arising from pancreas and in most extrapancreatic tissues and tumors. In contrast, CCK-A receptors exhibited a more selective pattern of expression in gall bladder, intestine, brain, ovary, spleen, and thymus. Of significance, CCK-A receptors were expressed selectively in all pancreatic adenocarcinomas, but not in any normal pancreas specimens. In situ hybridization, using receptor-specific riboprobes, localized CCK-A receptor expression to ductal cells, the presumed origin of most human pancreatic adenocarcinomas. Southern blot analysis revealed no evidence of CCK-A receptor gene amplification or rearrangement in pancreatic adenocarcinomas. Because of its selective expression, the CCK-A receptor may serve as selective biomarker for pancreatic adenocarcinoma.

The selective poly(ADP-ribose) polymerase-1(2) inhibitor, CEP-8983, increases the sensitivity of chemoresistant tumor cells to temozolomide and irinotecan but does not potentiate myelotoxicity

The effect of the potent and selective poly(ADP-ribose) (PAR) polymerase-1 [and PAR polymerase-2] inhibitor CEP-8983 on the ability to sensitize chemoresistant glioblastoma (RG2), rhabdomyosarcoma (RH18), neuroblastoma (NB1691), and colon carcinoma (HT29) tumor cells to temozolomide- and camptothecin-induced cytotoxicity, DNA damage, and G2-M arrest and on the potentiation of chemotherapy-induced myelotoxicity was evaluated using in vitro assays. In addition, the effect of the prodrug CEP-9722 in combination with temozolomide and/or irinotecan on PAR accumulation and tumor growth was also determined using glioblastoma and/or colon carcinoma xenografts relative to chemotherapy alone. CEP-8983 sensitized carcinoma cells to the growth-inhibitory effects of temozolomide and/or SN38 increased the fraction of and/or lengthened duration of time tumor cells accumulated in chemotherapy-induced G2-M arrest and sensitized tumor cells to chemotherapy-induced DNA damage and apoptosis. A granulocyte-macrophage colony-forming unit colony formation assay showed that coincubation of CEP-8983 with temozolomide or topotecan did not potentiate chemotherapy-associated myelotoxicity. CEP-9722 (136 mg/kg) administered with temozolomide (68 mg/kg for 5 days) or irinotecan (10 mg/kg for 5 days) inhibited significantly the growth of RG2 tumors (60%) or HT29 tumors (80%) compared with temozolomide or irinotecan monotherapy, respectively. In addition, CEP-9722 showed “stand alone” antitumor efficacy in these preclinical xenografts. In vivo biochemical efficacy studies showed that CEP-9722 attenuated PAR accumulation in glioma xenografts in a dose- and time-related manner. These data indicate that CEP-8983 and its prodrug are effective chemosensitizing agents when administered in combination with select chemotherapeutic agents against chemoresistant tumors. [Mol Cancer Ther 2007;6(8):2290–302]

PDGFR blockade is a rational and effective therapy for NPM-ALK-driven lymphomas

Anaplastic large cell lymphoma (ALCL) is an aggressive non-Hodgkins lymphoma found in children and young adults. ALCLs frequently carry a chromosomal translocation that results in expression of the oncoprotein nucleophosmin–anaplastic lymphoma kinase (NPM-ALK). The key molecular downstream events required for NPM-ALK–triggered lymphoma growth have been only partly unveiled. Here we show that the activator protein 1 family members JUN and JUNB promote lymphoma development and tumor dissemination through transcriptional regulation of platelet-derived growth factor receptor-β (PDGFRB) in a mouse model of NPM-ALK–triggered lymphomagenesis. Therapeutic inhibition of PDGFRB markedly prolonged survival of NPM-ALK transgenic mice and increased the efficacy of an ALK-specific inhibitor in transplanted NPM-ALK tumors. Notably, inhibition of PDGFRA and PDGFRB in a patient with refractory late-stage NPM-ALK+ ALCL resulted in rapid, complete and sustained remission. Together, our data identify PDGFRB as a previously unknown JUN and JUNB target that could be a highly effective therapy for ALCL.