Sheila J. Miknyoczki

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.

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.

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]

Synthesis and Biological Profile of the pan-Vascular Endothelial Growth Factor Receptor/Tyrosine Kinase with Immunoglobulin and Epidermal Growth Factor-Like Homology Domains 2 (VEGF-R/TIE-2) Inhibitor 11-(2-Methylpropyl)-12,13-dihydro-2-methyl-8-(pyrimidin-2-ylamino)-4H-indazolo[5,4-a]pyrrolo[3,4-c]carbazol-4-one (CEP-11981): A Novel Oncology Therapeutic Agent

A substantial body of evidence supports the utility of antiangiogenesis inhibitors as a strategy to block or attenuate tumor-induced angiogenesis and inhibition of primary and metastatic tumor growth in a variety of solid and hematopoietic tumors. Given the requirement of tumors for different cytokine and growth factors at distinct stages of their growth and dissemination, optimal antiangiogenic therapy necessitates inhibition of multiple, complementary, and nonredundant angiogenic targets. 11-(2-Methylpropyl)-12,13-dihydro-2-methyl-8-(pyrimidin-2-ylamino)-4H-indazolo[5,4-a]pyrrolo[3,4-c]carbazol-4-one (11b, CEP-11981) is a potent orally active inhibitor of multiple targets (TIE-2, VEGF-R1, 2, and 3, and FGF-R1) having essential and nonredundant roles in tumor angiogenesis and vascular maintenance. Outlined in this article are the design strategy, synthesis, and biochemical and pharmacological profile for 11b, which completed Phase I clinical assessing safety and pharmacokinetics allowing for the initiation of proof of concept studies.

Abstract C272: Antitumor activity of the dual AXL/c-Met inhibitor CEP-40783 in Champions primary TumorGraft™ models of human non-small cell lung cancer (NSCLC).

The oncogenic receptor tyrosine kinases (RTK) AXL and c-Met are over-expressed and constitutively activated in a variety of human cancers. Activation of these receptors contributes to multiple steps in tumor progression, including the epithelial-mesenchymal transition (EMT) by which cancer cells escape to form metastases and frequently develop resistance to a variety of agents, most notably to epidermal growth factor receptor (EGFR) targeted therapies. CEP-40783 is an orally-active, nanomolar potent and highly kinase-selective Type II inhibitor of the AXL and c-Met RTK in drug development. In the current study, five Champions human TumorGraft™ models of NSCLC expressing constitutively activated AXL and/or c-Met were used to determine the anti-tumor efficacy of single agent CEP-40783 compared to paclitaxel and the EGFR inhibitor, erlotinib (Genentech-Roche; OSI Pharmaceuticals). Champions Oncology has developed an innovative platform that utilizes the implantation of primary human tumors in immune-deficient mice in a manner that preserves the biological properties of the original human tumor and results in more rigorous and clinically relevant models for drug discovery. Previous reports have shown the correlation between responsiveness observed in Champions TumorGraft™ models and clinical responses of the patients from which the models were derived. Mice bearing established Champions TumorGrafts™ were treated orally with 10 mg/kg and 30 mg/kg qd of CEP-40783 for 10 to 34 days (until control tumors achieved >1000 mm3 volume) and anti-tumor efficacy and tolerability were evaluated. In 3/5 (60%) of the tumor models, CEP-40783 showed in vivo efficacy, including tumor regressions, significantly superior to that achieved with an optimal regimen of paclitaxel. In 4/4 (100%) of the erlotinib-insensitive tumor models, CEP-40783 demonstrated significant efficacy (66 to 118% TGI) compared to the control group at the 30 mg/kg dose. Additionally, CEP-40783 in combination with erlotinib demonstrated superior anti-tumor efficacy compared to CEP-40783 and erlotinib single agents in the one erlotinib-sensitive model evaluated. CEP-40783 as a single agent and in combination with erlotinib was well tolerated. Retrospective bioinformatics analyses are planned to determine potential signatures of response and resistance to CEP-40783. These results demonstrate that CEP-40783 shows superior efficacy alone and in combination with erlotinib in Champions TumorGraft™ models of NSCLC compared to SOC agents and supports the development of CEP-40783 for the treatment of erlotinib-resistant NSCLC. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C272. Citation Format: Jay A. Friedman, Rodney Donaldson, Sheila Miknyoczki, Mangeng Cheng, Robert Hudkins, Bruce Dorsey, Mark Ator, Thelma Angeles, Bruce Ruggeri, Elizabeth Bruckheimer. Antitumor activity of the dual AXL/c-Met inhibitor CEP-40783 in Champions primary TumorGraft™ models of human non-small cell lung cancer (NSCLC). [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 C272.

Abstract C275: CEP-40783: A potent and selective AXL/c-Met inhibitor for use in breast, non-small cell lung (NSCLC), and pancreatic cancers.

The oncogenic receptor tyrosine kinases AXL and c-Met are over-expressed and constitutively activated in a variety of human cancers. Activation of these receptors contributes to multiple steps in tumor progression by promoting cancer cell migration and invasion, enhancing tumor angiogenesis, facilitating cancer cell survival and tumor growth, and contributing to tumor resistance to several chemotherapy and molecular-targeted therapeutic agents. CEP-40783 is an orally active, potent and selective AXL and c-Met kinase inhibitor, with enzyme IC50 values of 7 nM and 12 nM, respectively. In AXL-transfected 293GT cells, CEP-40783 was 27-fold more active compared to recombinant enzyme with an IC50 value of 0.26 nM. Comparably high cellular potency was observed in NCI-H1299 human NSCL cells. CEP-40783 also demonstrated superior activity against c-Met in GTL-16 cells (IC50 = 6 nM). The increased inhibitory activity of CEP-40783 in cells could be attributed to its extended residence time on both AXL and c-Met, consistent with a Type II mechanism. The prolonged residence time of CEP-40783 at the target may provide for improved in vivo efficacy, selectivity and therapeutic index. Additionally, CEP-40783 showed high kinome selectivity against 298 kinases with an S90 of 0.04 (fraction of kinases showing >90% inhibition at 1 µM). In PK/PD studies, CEP-40783 showed dose- and time-dependent inhibition of AXL phosphorylation using NCI-H1299 NSCL xenografts with ∼80% target inhibition at 0.3 mg/kg 6 h post dose and complete target inhibition to >90% inhibition at 1 mg/kg between 6-24 h, while a 10 mg/kg po dose resulted in complete AXL inhibition up to 48 h post dosing. In AXL/NIH3T3 xenografts, 0.3 mg/kg po resulted in complete tumor regressions. CEP-40783 was also efficacious in reducing spontaneous lymph node and pulmonary metastatic tumor burden in the MDA-MB-231-luc and 4T1-luc orthotopic breast cancer models, respectively, at 10 and 30 mg/kg po. PK/PD evaluation of the c-Met activity of CEP-40783 (10, 30, 55 mg/kg po qdX5d) showed significant to complete inhibition of c-Met phosphorylation in GTL-16 gastric carcinoma xenografts. Efficacy studies in GTL-16 xenografts demonstrated significant anti-tumor efficacy (tumor stasis and regressions) at 10 and 30 mg/kg po. In EBC-1 NSCL xenografts, administration of CEP-40783 (3, 10 and 30 mg/kg, po qd) resulted in dose-related efficacy, with tumor stasis at 3 mg/kg, tumor regressions and >96% TGI at 10 mg/kg. In all studies CEP-40783 was well tolerated with no compound-related body weight loss. CEP-40783 demonstrated potent AXL and c-Met pharmacodynamic and anti-tumor efficacy in established tumor xenograft models, having potential therapeutic utility in multiple human tumor types in which c-Met and AXL activity play a critical role in tumor formation, local invasion and metastasis. Studies in primary human tumorgrafts are in progress. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C275. Citation Format: Sheila Miknyoczki, Mangeng Cheng, Robert Hudkins, Thelma Angeles, Lisa Aimone, Jean Husten, Jie Qian, Seetha Murthy, Thomas Conners, Robert Bendesky, Amy Landis, Jennifer Grobelny, Hong Chang, Bruce Dorsey, Mark Ator, Bruce Ruggeri. CEP-40783: A potent and selective AXL/c-Met inhibitor for use in breast, non-small cell lung (NSCLC), and pancreatic cancers. [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 C275.