Gemma Texido

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 Ser10 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. [Mol Cancer Ther 2007;6(12):3158–68]

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 drugs 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.

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.

Abstract A243: Characterization of NMS‐E628, a small molecule inhibitor of anaplastic lymphoma kinase with antitumor efficacy in ALK‐dependent lymphoma and non‐small cell lung cancer models

The chromosomal translocation t(2;5)(p23;q35) involving the ALK tyrosine kinase gene results in expression of the NPM‐ALK fusion protein which represents the driving force for survival and proliferation of a subset of Anaplastic Large Cell Lymphoma. More recently, a distinct chromosomal rearrangement of the ALK gene leading to a new fusion variant EML4‐ALK, has been identified as a low frequency event, mutually exclusive with respect to EGFR and K‐ras mutation, in Non Small Cell Lung cancer patients. As previously found for NPM‐ALK, this new fusion variant has constitutively active ALK kinase and was demonstrated to have strong oncogenic potential. Taken together these findings support the hypothesis that ALK represents an innovative and valuable target for cancer therapy both in ALCL and NSCLC patients whose tumors harbor translocated ALK. Here we further describe the preclinical characterization of NMS‐E628, an orally available small‐molecule inhibitor of ALK kinase activity. Proliferation profiling on a wide panel of human tumor cell lines demonstrated that the compound selectively blocks proliferation of ALK‐dependent cell lines and potently inhibits ALK‐dependent signaling. In vivo, NMS‐E628 induced complete tumor regression when administered orally for ten consecutive days to SCID mice bearing Karpas‐299 or SR‐786 xenografts, with ex vivo analyses demonstrating dose‐dependent target modulation that was maintained for up to 18 hours after single treatment. NMS‐E628 was also highly efficacious in a transgenic mouse leukemia model in which human NPM‐ALK expression was targeted to T cells. In this latter model, which faithfully recapitulates pathological features of human ALCL, treatment of NPM‐ALK transgenic mice with NMS‐E628 for as little as 3 consecutive days induced complete regression of tumor masses observed in the thymus and in lymph nodes. NMS‐E628 was also highly efficacious in inhibiting the in vitro and in vivo growth of the NSCLC cell line NCI‐H2228, which bears the EML4‐ALK rearrangement. Complete regressions were also achieved in this model, and prolonged inhibition of ALK phosphorylation and downstream effector activation were observed at active doses. NMS‐E628 has favorable pharmacokinetic and toxicological properties and biodistribution analysis revealed that it is able to cross the blood‐brain barrier in different animal species. To confirm that therapeutic doses are reached in the brain, NCI‐H2228 cells were injected intracranially in nude mice and NMS‐E628 was administered orally with different schedules. Dose‐dependent increase in survival, together with inhibition of tumor growth as assessed by MRI, confirmed that NMS‐E628 does indeed possess antitumor activity in this setting, an important finding considering that a significant proportion of NSCLC patients develop brain metastases. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A244.

Efficacy of PHA-848125, a Cyclin-Dependent Kinase Inhibitor, on the K-RasG12DLA2 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-RasG12DLA2, 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-RasG12DLA2 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. Mol Cancer Ther; 9(3); 673–81

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.

Erratum: Immunological characterization of whole tumour lysate-loaded dendritic cells for cancer immunotherapy (PLoS ONE (2016) 11:1 (e0146622) DOI: 10.1371/journal.pone.0146622)

Dr. Gemma Texido should be included in the author byline. She should be listed as the fifth author, and her affiliation is #8: BU Oncology, Nerviano Medical Sciences, viale Pasteur 10, 20014 Nerviano, Milan, Italy. The contributions of this author are as follows: Conceived and designed the experiments, contributed reagents/materials/analysis tools, and specifically performed the experiments. Dr. Anna Degrassi should be included in the author byline. She should be listed as the sixth author, and her affiliation is #8: BU Oncology, Nerviano Medical Sciences, viale Pasteur 10, 20014 Nerviano, Milan, Italy. The contributions of this author are as follows: Conceived and designed the experiments, contributed reagents/materials/analysis tools, and specifically performed the experiments. The correct citation is: Rainone V, Martelli C, Ottobrini L, Biasin M, Texido G, Degrassi A, Borelli M, Lucignani G, et al. (2016) Immunological Characterization of Whole Tumour Lysate-Loaded Dendritic Cells for Cancer Immunotherapy. PLoS ONE 11(1): e0146622. doi:10.1371/journal.pone.0146622

Abstract A179: NMS-P113, a novel orally available JAK2 selective inhibitor

The Janus Kinases (JAK1, JAK2, JAK3, TYK2) are non-receptor tyrosine kinases that play important roles in hematopoiesis and immune response. In particular, gene ablation of JAK1 or JAK2 in the mouse is incompatible with life, due to neurological defects/immunodeficency and lack of erythropoiesis, respectively, whereas that of JAK3 or TYK2 is associated with severe immunodeficiency. Activating mutations of JAKs are found in association with malignant transformation. The best characterized gain-of-function mutation, JAK2-V617F in the pseudo-kinase domain of JAK2, is present in hematopoietic cells of patients with myeloproliferative disorders (MPD). In particular, the JAK2-V617F mutation is found in >95% of patients with polycythemia vera (PV), circa 50% of patients with essential thrombocythemia (ET), and circa 50% of myelofibrosis (MF) patients. Recently, a central role of JAK2 has been described in upregulation of the immune checkpoint component PD-L1 mediated by IFN-γ or by chromosome 9p24.1 amplification, suggesting that its inhibition might provide a new strategy to increase immune-mediated tumor inhibition in specific contexts. Although JAK inhibitors have been approved in oncological and autoimmune settings (e.g. the JAK1/JAK2 inhibitor ruxolitinib in MF and the pan-JAK inhibitor tofacitinib in rheumatoid arthritis) and multiple agents are in clinical testing, JAK2 selective compounds might be provide an advantage for long-term MPD therapy or in association with immunotherapy, given that inhibition of other JAK family members leads to immunosuppressive effects. Due to high homology amongst JAK family kinases within the ATP binding pocket, discovery of selective JAK2 inhibitors has represented a significant challenge. Here, we report the characterization of NMS-P113, a potent and selective JAK2 inhibitor. Screening of the NMS compound collection led to the identification of a pyrrole series with promising activity against JAK2. An optimization program led to identification of the potent and orally bioavailable JAK2 inhibitor NMS-P113. In biochemical assay this compound possesses low nanomolar potency against JAK2 (IC50 3 nM), with preferential activity over other JAK family members and high selectivity against a panel of 60 further kinases. In cellular assay, NMS-P113 potently inhibits proliferation of the JAK2 dependent SET-2 human megakaryoblastic leukemia line, derived from an ET patient and which harbors the JAK2-V617F mutation, as well as of Ba/F3 cells engineered to express constitutively activated JAK2 (IC50s circa 200 nM). NMS-P113 is 10-fold less active against the DERL-7 T-cell lymphoma cell line (which is dependent upon JAK1/JAK3) and has poor antiproliferative activity in JAK independent lines. Oral administration of NMS-P113 results in dose-related normalization of spleen weight and erythrocyte precursors count in an erythropoietin-induced model of PV in the mouse. Mechanism of action of NMS-P113, as assessed by inhibition of P-STAT5, was confirmed in vitro and in vivo. A favorable ADME profile with high oral bioavailability, together with permissive therapeutic safety margins in test species, indicate that NMS-P113 is suitable for further development, affording the possibility of reduced undesirable immunosuppressive activities compared to inhibitors that target multiple members of the JAK family. Citation Format: Paola Gnocchi, Maria Gabriella Brasca, Nadia Amboldi, Nilla Avanzi, Simona Bindi, Giulia Canevari, Daniele Casero, Roberta Ceruti, Marina Ciomei, Sabrina Cribioli, Cinzia Cristiani, Marcella Nesi, Wilma Pastori, Veronica Patton, Cinzia Pellizzoni, Gemma Texido, Elena Ardini, Eduard R. Felder, Antonella Isacchi, Daniele Donati, Arturo Galvani. NMS-P113, a novel orally available JAK2 selective inhibitor. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A179.