Veronica Patton

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]

A Cdc7 kinase inhibitor restricts initiation of DNA replication and has antitumor activity.

Cdc7 is an essential kinase that promotes DNA replication by activating origins of replication. Here, we characterized the potent Cdc7 inhibitor PHA-767491 (1) in biochemical and cell-based assays, and we tested its antitumor activity in rodents. We found that the compound blocks DNA synthesis and affects the phosphorylation of the replicative DNA helicase at Cdc7-dependent phosphorylation sites. Unlike current DNA synthesis inhibitors, PHA-767491 prevents the activation of replication origins but does not impede replication fork progression, and it does not trigger a sustained DNA damage response. Treatment with PHA-767491 results in apoptotic cell death in multiple cancer cell types and tumor growth inhibition in preclinical cancer models. To our knowledge, PHA-767491 is the first molecule that directly affects the mechanisms controlling initiation as opposed to elongation in DNA replication, and its activities suggest that Cdc7 kinase inhibition could be a new strategy for the development of anticancer therapeutics.

Vascular endothelial growth factor C promotes ovarian carcinoma progression through paracrine and autocrine mechanisms

Vascular endothelial growth factor C (VEGFC) has been reported to promote tumor progression in several tumor types, mainly through the stimulation of lymphangiogenesis and lymphatic metastasis. However, the expression and biological significance of the VEGFC/VEGF receptor (VEGFR)-3 pathway in ovarian cancer growth and dissemination are unclear, and have been investigated in this study. Soluble VEGFC was detected in the plasma and ascites of patients with ovarian carcinoma, and VEGFR3 expression was found in their tumor tissues. In human ovarian carcinoma xenograft models, high levels of soluble VEGFC in ascites and serum were detected, in association with disease progression, tumor burden, and volume of ascites. Peak VEGFC expression preceded para-aortic lymph node infiltration by HOC8 neoplastic cells. Histological detection of tumor cells in blood and lymphatic vessels indicated both hematogenous and lymphatic dissemination. Overexpression of VEGFC in the VEGFR3-positive and luciferase-expressing IGROV1 cells promoted carcinoma dissemination after orthotopic transplantation in the ovary of immunodeficient mice. In vitro, VEGFC released by the tumor cells stimulated tumor cell migration in an autocrine manner. Cediranib, an inhibitor of VEGFR1-3 and c-kit, inhibited in vivo metastasis of VEGFC-overexpressing IGROV1 and in vitro autocrine effects. These findings suggest that the VEGFC/VEGFR3 pathway acts as an enhancer of ovarian cancer progression through autocrine and paracrine mechanisms, hence offering a potential target for therapy.

Dual Targeting of CDK and Tropomyosin Receptor Kinase Families by the Oral Inhibitor PHA-848125, an Agent with Broad-Spectrum Antitumor Efficacy

Altered expression and activity of cyclin-dependent kinase (CDK) and tropomyosin receptor kinase (TRK) families are observed in a wide variety of tumors. In those malignancies with aberrant CDK activation, the retinoblastoma protein (pRb) pathway is deregulated, leading to uncontrolled cell proliferation. Constitutive activation of TRKs is instead linked to cancer cell survival and dissemination. Here, we show that the novel small-molecule PHA-848125, a potent dual inhibitor of CDKs and TRKs, possesses significant antitumor activity. The compound inhibits cell proliferation of a wide panel of tumoral cell lines with submicromolar IC50. PHA-848125–treated cells show cell cycle arrest in G1 and reduced DNA synthesis, accompanied by inhibition of pRb phosphorylation and modulation of other CDK-dependent markers. The compound additionally inhibits phosphorylation of TRKA and its substrates in cells, which functionally express this receptor. Following oral administration, PHA-848125 has significant antitumor activity in various human xenografts and carcinogen-induced tumors as well as in disseminated primary leukemia models, with plasma concentrations in rodents in the same range as those found active in inhibiting cancer cell proliferation. Mechanism of action was also confirmed in vivo as assessed in tumor biopsies from treated mice. These results show that the dual CDK-TRK inhibitor PHA-848125 has the potential for being a novel and efficacious targeted drug for cancer treatment. Mol Cancer Ther; 9(8); 2243–54. ©2010 AACR.

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

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.

Therapeutic efficacy of the pan-cdk inhibitor PHA-793887 in vitro and in vivo in engraftment and high-burden leukemia models

OBJECTIVE The aim of the work was to determine and characterize, in vitro and in vivo, the therapeutic activity of PHA-793887, a new potent pan-cdk inhibitor, in the context of hematopoietic neoplasms. MATERIALS AND METHODS Thirteen leukemic cell lines bearing different cytogenetic abnormalities and normal hematopoietic cells were used in cytotoxicity and colony assays. The drug activity at the molecular level was analyzed by Western blotting. PHA-793887 was also tested in vivo in several leukemia xenograft models. RESULTS PHA-793887 was cytotoxic for leukemic cell lines in vitro, with IC(50) ranging from 0.3 to 7 microM (mean: 2.9 microM), regardless of any specific chromosomal aberration. At these doses, the drug was not cytotoxic for normal unstimulated peripheral blood mononuclear cells or CD34(+) hematopoietic stem cells. Interestingly, in colony assays PHA-793887 showed very high activity against leukemia cell lines, with an IC(50) <0.1 microM (mean: 0.08 microM), indicating that it has efficient and prolonged antiproliferative activity. PHA-793887 induced cell-cycle arrest, inhibited Rb and nucleophosmin phosphorylation, and modulated cyclin E and cdc6 expression at low doses (0.2-1 microM) and induced apoptosis at the highest dose (5 microM). It was also effective in vivo in both subcutaneous xenograft and primary leukemic disseminated models that better mimic naturally occurring human disease. Interestingly, in one disseminated model derived from a relapsed Philadelphia-positive acute lymphoid leukemia patient, PHA-793887 showed strong therapeutic activity also when treatment was started after establishment of high disease burden. CONCLUSIONS We conclude that PHA-793887 has promising therapeutic activity against acute leukemias in vitro and in vivo.

Diagnostic exercise: sudden death in a mouse with experimentally induced acute myeloid leukemia.

A 22-week-old female 129/SvEv mouse suddenly died in the context of an experiment aimed at defining the efficacy of valproic acid in a mouse model of PML/RARα-induced acute myeloid leukemia. Histologic analysis confirmed the mouse as being affected by a progressive myeloid leukemia, with infiltration of the spleen, bone marrow, liver, kidneys, and lungs. Variably sized intravascular clumps (emboli) of dense basophilic material admixed with necrotic or lytic neoplastic cells were also observed in multiple organs. A positive reaction to Feulgen and Hoechst stain confirmed the high content in chromatin of these basophilic emboli. Cleaved caspase-3 activity was demonstrated both in the leukemic infiltrates and among the intravascular necrotic or lytic neoplastic cells accompanying the basophilic emboli. A diagnosis of acute tumor lysis syndrome related to therapy-induced massive necrosis and/or apoptosis of leukemic cells with subsequent dissemination of emboli of chromatin was proposed.

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.