Entrectinib, a Pan-TRK, ROS1, and ALK Inhibitor with Activity in Multiple Molecularly Defined Cancer Indications.

Activated ALK and ROS1 tyrosine kinases, resulting from chromosomal rearrangements, occur in a subset of non-small cell lung cancers (NSCLC) as well as other tumor types and their oncogenic relevance as actionable targets has been demonstrated by the efficacy of selective kinase inhibitors such as crizotinib, ceritinib, and alectinib. More recently, low-frequency rearrangements of TRK kinases have been described in NSCLC, colorectal carcinoma, glioblastoma, and Spitzoid melanoma. Entrectinib, whose discovery and preclinical characterization are reported herein, is a novel, potent inhibitor of ALK, ROS1, and, importantly, of TRK family kinases, which shows promise for therapy of tumors bearing oncogenic forms of these proteins. Proliferation profiling against over 200 human tumor cell lines revealed that entrectinib is exquisitely potent in vitro against lines that are dependent on the drug's pharmacologic targets. Oral administration of entrectinib to tumor-bearing mice induced regression in relevant human xenograft tumors, including the TRKA-dependent colorectal carcinoma KM12, ROS1-driven tumors, and several ALK-dependent models of different tissue origins, including a model of brain-localized lung cancer metastasis. Entrectinib is currently showing great promise in phase I/II clinical trials, including the first documented objective responses to a TRK inhibitor in colorectal carcinoma and in NSCLC. The drug is, thus, potentially suited to the therapy of several molecularly defined cancer settings, especially that of TRK-dependent tumors, for which no approved drugs are currently available. Mol Cancer Ther; 15(4); 628-39. ©2016 AACR.

Immunological Characterization of Whole Tumour Lysate-Loaded Dendritic Cells for Cancer Immunotherapy.

INTRODUCTION: Dendritic cells play a key role as initiators of T-cell responses, and even if tumour antigen-loaded dendritic cells can induce anti-tumour responses, their efficacy has been questioned, suggesting a need to enhance immunization strategies. MATHERIALS & METHODS: We focused on the characterization of bone marrow-derived dendritic cells pulsed with whole tumour lysate (TAA-DC), as a source of known and unknown antigens, in a mouse model of breast cancer (MMTV-Ras). Dendritic cells were evaluated for antigen uptake and for the expression of MHC class I/II and costimulatory molecules and markers associated with maturation. RESULTS: Results showed that antigen-loaded dendritic cells are characterized by a phenotypically semi-mature/mature profile and by the upregulation of genes involved in antigen presentation and T-cell priming. Activated dendritic cells stimulated T-cell proliferation and induced the production of high concentrations of IL-12p70 and IFN-γ but only low levels of IL-10, indicating their ability to elicit a TH1-immune response. Furthermore, administration of Antigen loaded-Dendritic Cells in MMTV-Ras mice evoked a strong anti-tumour response in vivo as demonstrated by a general activation of immunocompetent cells and the release of TH1 cytokines. CONCLUSION: Data herein could be useful in the design of antitumoral DC-based therapies, showing a specific activation of immune system against breast cancer.

Direct involvement of CD56 in cytokine-induced killer-mediated lysis of CD56+ hematopoietic target cells.

Cytokine-induced killer (CIK) cells are in-vitro-expanded T lymphocytes that represent a heterogeneous population. A large majority of CIK cells are CD3(+)CD56(+), and this population has been shown to confer a cytotoxic effect against tumor targets. The scope of this work was to study whether CD56 has a direct role in CIK-mediated cytotoxicity. Blocking of CD56 with the anti-CD56 monoclonal antibody GPR165 significantly reduced CIK-mediated lysis of three CD56(+) hematopoietic tumor cell lines (AML-NS8, NB4, and KCL22), whereas no effect was observed on three CD56(-) hematopoietic tumor cell lines (K562, REH, and MOLT-4). Knockdown of CD56 in CIK cells by short interfering RNA made the cells less cytotoxic against a CD56(+) target, and knockdown of CD56 in target cells with lentiviral short hairpin RNA significantly altered their susceptibility to CIK-mediated lysis. Our data suggest that homophilic interaction between CD56 molecules may occur in tumor-cell recognition, leading to CIK-mediated cell death.

The Polo-Like Kinase 1 (PLK1) inhibitor NMS-P937 is effective in a new model of disseminated primary CD56+ acute monoblastic leukaemia.

CD56 is expressed in 15-20% of acute myeloid leukaemias (AML) and is associated with extramedullary diffusion, multidrug resistance and poor prognosis. We describe the establishment and characterisation of a novel disseminated model of AML (AML-NS8), generated by injection into mice of leukaemic blasts freshly isolated from a patient with an aggressive CD56(+) monoblastic AML (M5a). The model reproduced typical manifestations of this leukaemia, including presence of extramedullary masses and central nervous system involvement, and the original phenotype, karyotype and genotype of leukaemic cells were retained in vivo. Recently Polo-Like Kinase 1 (PLK1) has emerged as a new candidate drug target in AML. We therefore tested our PLK1 inhibitor NMS-P937 in this model either in the engraftment or in the established disease settings. Both schedules showed good efficacy compared to standard therapies, with a significant increase in median survival time (MST) expecially in the established disease setting (MST = 28, 36, 62 days for vehicle, cytarabine and NMS-P937, respectively). Importantly, we could also demonstrate that NMS-P937 induced specific biomarker modulation in extramedullary tissues. This new in vivo model of CD56(+) AML that recapitulates the human tumour lends support for the therapeutic use of PLK1 inhibitors in AML.

Genetically engineered animal models for in vivo target identification and validation in oncology.

In vitro approaches using human cancer cell lines aimed to identify and validate oncology targets, have pinpointed a number of key targets and signalling pathways which control cell growth and cell death. However, tumors are more than insular masses of proliferating cancer cells. Instead they are complex tissues composed of multiple distinct cell types that participate in homotypic and heterotypic interactions and depend upon each other for their growth. Therefore, many targets in oncology need to be validated in the context of the whole animal. This review provides an overview on how animal models can be generated and used for target identification and validation in vivo.

In vivo target validation by inducible RNAi in human xenograft mouse models.

Proper target selection and validation are crucial to the discovery of new anti-cancer agents. Since tumors depend on a suitable microenvironment for their growth, once a putative target has been identified, its validation should be performed whenever possible in vivo. This chapter deals with the generation of human xenograft mouse models genetically modified to induce the modulation of cancer-related genes as an approach to validate oncology targets.

In vivo imaging of early stage apoptosis by measuring real-time caspase-3/7 activation.

In vivo imaging of apoptosis in a preclinical setting in anticancer drug development could provide remarkable advantages in terms of translational medicine. So far, several imaging technologies with different probes have been used to achieve this goal. Here we describe a bioluminescence imaging approach that uses a new formulation of Z-DEVD-aminoluciferin, a caspase 3/7 substrate, to monitor in vivo apoptosis in tumor cells engineered to express luciferase. Upon apoptosis induction, Z-DEVD-aminoluciferin is cleaved by caspase 3/7 releasing aminoluciferin that is now free to react with luciferase generating measurable light. Thus, the activation of caspase 3/7 can be measured by quantifying the bioluminescent signal. Using this approach, we have been able to monitor caspase-3 activation and subsequent apoptosis induction after camptothecin and temozolomide treatment on xenograft mouse models of colon cancer and glioblastoma, respectively. Treated mice showed more than 2-fold induction of Z-DEVD-aminoluciferin luminescent signal when compared to the untreated group. Combining D: -luciferin that measures the total tumor burden, with Z-DEVD-aminoluciferin that assesses apoptosis induction via caspase activation, we confirmed that it is possible to follow non-invasively tumor growth inhibition and induction of apoptosis after treatment in the same animal over time. Moreover, here we have proved that following early apoptosis induction by caspase 3 activation is a good biomarker that accurately predicts tumor growth inhibition by anti-cancer drugs in engineered colon cancer and glioblastoma cell lines and in their respective mouse xenograft models.

Efficacy of PHA-848125, a cyclin-dependent kinase inhibitor, on the K-Ras(G12D)LA2 lung adenocarcinoma transgenic mouse model: evaluation by multimodality imaging.

K-ras is the most frequently mutated oncogene in non-small cell lung cancer (NSCLC), the most common form of lung cancer. Recent studies indicate that NSCLC patients with mutant K-ras do not respond to epidermal growth factor receptor inhibitors. In the attempt to find alternative therapeutic regimes for such patients, we tested PHA-848125, an oral pan cyclin-dependent kinase inhibitor currently under evaluation in phase II clinical trial, on a transgenic mouse model, K-Ras(G12D)LA2, which develops pulmonary cancerous lesions reminiscent of human lung adenocarcinomas. We used magnetic resonance imaging and positron emission tomography to follow longitudinally disease progression and evaluate therapeutic efficacy in this model. Treatment of K-Ras(G12D)LA2 mice with 40 mg/kg twice daily for 10 days with PHA-848125 induced a significant tumor growth inhibition at the end of treatment (P < 0.005) and this was accompanied by a reduction in the cell membrane turnover, as seen by 11C-Choline-positron emission tomography (P < 0.05). Magnetic resonance imaging data were validated versus histology and the mechanism of action of the compound was verified by immunohistochemistry, using cyclin-dependent kinase-related biomarkers phospho-Retinoblastoma and cyclin A. In this study, multimodality imaging was successfully used for the preclinical assessment of PHA-848125 therapeutic efficacy on a lung adenocarcinoma mouse model. This compound induced a volumetric and metabolic anticancer effect and could represent a valid therapeutic approach for NSCLC patients with mutant K-ras.

PHA-739358, a potent inhibitor of Aurora kinases with a selective target inhibition profile relevant to cancer.

PHA-739358 is a small-molecule 3-aminopyrazole derivative with strong activity against Aurora kinases and cross-reactivities with some receptor tyrosine kinases relevant for cancer. PHA-739358 inhibits all Aurora kinase family members and shows a dominant Aurora B kinase inhibition-related cellular phenotype and mechanism of action in cells in vitro and in vivo. p53 status-dependent endoreduplication is observed upon treatment of cells with PHA-739358, and phosphorylation of histone H3 in Ser(10) is inhibited. The compound has significant antitumor activity in different xenografts and spontaneous and transgenic animal tumor models and shows a favorable pharmacokinetic and safety profile. In vivo target modulation is observed as assessed by the inhibition of the phosphorylation of histone H3, which has been validated preclinically as a candidate biomarker for the clinical phase. Pharmacokinetics/pharmacodynamics modeling was used to define drug potency and to support the prediction of active clinical doses and schedules. We conclude that PHA-739358, which is currently tested in clinical trials, has great therapeutic potential in anticancer therapy in a wide range of cancers.

Magnetic resonance imaging and histopathological characterization of prostate tumors in TRAMP mice as model for pre-clinical trials.

BACKGROUND: The Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) develops progressive forms of prostate cancer. Due to the lack of a validated non-invasive methodology, pathology has been so far the most common parameter evaluated in efficacy studies. METHODS: We studied by magnetic resonance imaging (MRI) 210 mice that were repeatedly measured up to 33 weeks of age in order to stage prostate tumors and follow pathological progression in single animals. A pre-clinical trial with doxorubicin was also performed. RESULTS: Progressive forms of cancer (well and poorly differentiated (PD) adenocarcinomas) were easily recognized on MR images and MRI findings were validated against histopathological analysis. Age at tumor onset was different for the two tumoral forms. Doxorubicin treatment caused a strong reduction in tumor volume. CONCLUSIONS: Prostate cancer in TRAMP mice is multifocal and heterogeneous: a non-invasive methodology such as MRI facilitates the rational design of translational pre-clinical trials in this widely used animal model.

FGF2 signaling is required for the development of neuronal circuits regulating blood pressure.

Fibroblast growth factor 2 (FGF2) signaling is involved in angiogenesis, vascular contractility, and cardiac hypertrophy. Mice lacking a functional FGF2 gene (FGF2(-/-)) are hypotensive, but the primary physiological role of FGF2 in cardiovascular homeostasis remained unknown. Using a chicken FGF2 (cFGF2) transgene under control of the Wnt-1 promotor, we selectively re-expressed FGF2 in the developing nervous system of FGF2(-/-) (transgenic FGF2 mutant) embryos. Expression of the cFGF2 transgene in the developing nervous system, including its autonomic region, was limited to the period between embryonic day 9.5 and 14.5. Significantly, no FGF2 re-expression was detected in developing heart and blood vessels. Pharmacological analysis revealed a normalization of the blood pressure response to isoproterenol-induced vasodilation in adult transgenic FGF2 mutant mice. In addition, the hypotensive phenotype was rescued in 1 line (of 2) transgenic FGF2 mutant adult mice having expressed higher levels of cFGF2 proteins during nervous system development. These genetic studies indicate that FGF2 signaling is essential for complete development of the neural circuitry required for central regulation of blood pressure, whereas it appears dispensable for blood pressure control in the healthy adult. The full text of this article is available at http://www.circresaha.org.