Combined PDGFR and HDAC inhibition overcomes PTEN disruption in Chordoma

Background: The majority of chordomas show activation of the platelet-derived growth factor receptor (PDGFR). Based on in vitro intertumoral variation in response to recombinant PDGF protein and PDGFR inhibition, and variable tumor response to imatinib, we hypothesized that chordomas resistant to PDGFR inhibition may possess downstream activation of the pathway. Methods: Molecular profiling was performed on 23 consecutive chordoma primary tissue specimens. Primary cultures established from 20 of the 23 specimens, and chordoma cell lines, UCH-1 and UCH-2, were used for in vitro experiments. Results: Loss of heterozygosity (LOH) at the phosphatase and tensin homolog (PTEN) locus was observed in 6 specimens (26%). PTEN disruption statistically correlated with increased Ki-67 proliferation index, an established marker of poor outcome for chordoma. Compared to wild type, PTEN deficient chordomas displayed increased proliferative rate, and responded less favorably to PDGFR inhibition. PTEN gene restoration abrogated this growth advantage. Chordomas are characterized by intratumoral hypoxia and local invasion, and histone deacetylase (HDAC) inhibitors are capable of attenuating both hypoxic signaling and cell migration. The combination of PDGFR and HDAC inhibition effectively disrupted growth and invasion of PTEN deficient chordoma cells. Conclusions: Loss of heterozygosity of the PTEN gene seen in a subset of chordomas is associated with aggressive in vitro behavior and strongly correlates with increased Ki-67 proliferative index. Combined inhibition of PDGFR and HDAC attenuates proliferation and invasion in chordoma cells deficient for PTEN

Use of radiolabelled choline as a pharmacodynamic marker for the signal transduction inhibitor geldanamycin

There is an urgent need to develop non-invasive pharmacodynamic endpoints for the evaluation of new molecular therapeutics that inhibit signal transduction. We hypothesised that, when labelled appropriately, changes in choline kinetics could be used to assess geldanamycin pharmacodynamics, which involves inhibition of the HSP90 molecular chaperone→Raf1→Mitogenic Extracellular Kinase→Extracellular Signal-Regulated Kinase 1 and 2 signal transduction pathway. Towards identifying a potential pharmacodynamic marker response, we have studied radiolabelled choline metabolism in HT29 human colon carcinoma cells following treatment with geldanamycin. We studied the effects of geldanamycin, on net cellular accumulation of (methyl-14C)choline and (methyl-14C)phosphocholine production. In parallel experiments, the effects of geldanamycin on extracellular signal-regulated kinase 1 and 2 phosphorylation and cell viability were also assessed. Additional validation studies were carried out with the mitogenic extracellular kinase inhibitor U0126 as a positive control; a cyclin-dependent kinase-2 inhibitor roscovitine and the phosphatidylinositol 3-kinase inhibitor LY294002 as negative controls. Hemicholinium-3, an inhibitor of choline transport and choline kinase activity was included as an additional control. In exponentially growing HT29 cells, geldanamycin inhibited extracellular signal-regulated kinase 1 and 2 phosphorylation in a concentration- and time-dependent manner. These changes were associated with a reduction in (methyl-14C)choline uptake, (methyl-14C) phosphocholine production and cell viability. Brief exposure to U0126, suppressed phosphocholine production to the same extent as Hemicholinium-3. In contrast to geldanamycin and U0126, which act upstream of extracellular signal-regulated kinase 1 and 2, roscovitine and LY294002 failed to suppress phosphocholine production. Our results suggest that when labelled with carbon-11 isotope, (methyl-11C)choline may be a useful pharmacodynamic marker for the non-invasive evaluation of geldanamycin analogues

Chronic treatment with 17-DMAG improves balance and coordination in a new mouse model of Machado-Joseph disease

Machado-Joseph disease (MJD) or spinocerebellar ataxia type 3 (SCA3) is a neurodegenerative disease currently with no treatment. We describe a novel mouse model of MJD which expresses mutant human ataxin-3 at near endogenous levels and manifests MJD-like motor symptoms that appear gradually and progress over time. CMVMJD135 mice show ataxin-3 intranuclear inclusions in the CNS and neurodegenerative changes in key disease regions, such as the pontine and dentate nuclei. Hsp90 inhibition has shown promising outcomes in some neurodegenerative diseases, but nothing is known about its effects in MJD. Chronic treatment of CMVMJD mice with Hsp90 inhibitor 17-DMAG resulted in a delay in the progression of their motor coordination deficits and, at 22 and 24 weeks of age, was able to rescue the uncoordination phenotype to wild-type levels; in parallel, a reduction in neuropathology was observed in treated animals. We observed limited induction of heat-shock proteins with treatment, but found evidence that 17-DMAG may be acting through autophagy, as LC3-II (both at mRNA and protein levels) and beclin-1 were induced in the brain of treated animals. This resulted in decreased levels of the mutant ataxin-3 and reduced intranuclear aggregation of this protein. Our data validate this novel mouse model as a relevant tool for the study of MJD pathogenesis and for pre-clinical studies, and show that Hsp90 inhibition is a promising therapeutic strategy for MJD.We would like to thank to Dr. Henry Paulson for providing the anti-ataxin-3 serum, Dr. Monica Sousa for the pCMV vector and to Eng. Lucilia Goreti Pinto, Lu s Martins, Miguel Carneiro and Celina Barros for technical assistance. This work was supported by Fundacao para a Ciencia e Tecnologia through the projects FEDER/FCT, POCI/SAU-MMO/60412/2004 and PTDC/SAU-GMG/64076/2006. This work was supported by Fundacao para a Ciencia e Tecnologia through fellowships SFRH/BPD/91562/2012 to A.S-F., SFRH/BD/78388/2011 to S. D-S., SFRH/BD/51059/2010 to A.N-C., and SFRH/BPD/79469/2011 to A.T-C.

Phenylbutyrate Counteracts Shigella Mediated Downregulation of Cathelicidin in Rabbit Lung and Intestinal Epithelia: A Potential Therapeutic Strategy

BACKGROUND: Cathelicidins and defensins are endogenous antimicrobial peptides (AMPs) that are downregulated in the mucosal epithelia of the large intestine in shigellosis. Oral treatment of Shigella infected rabbits with sodium butyrate (NaB) reduces clinical severity and counteracts the downregulation of cathelicidin (CAP-18) in the large intestinal epithelia. AIMS: To develop novel regimen for treating infectious diseases by inducing innate immunity, we selected sodium 4-phenylbutyrate (PB), a registered drug for a metabolic disorder as a potential therapeutic candidate in a rabbit model of shigellosis. Since acute respiratory infections often cause secondary complications during shigellosis, the systemic effect of PB and NaB on CAP-18 expression in respiratory epithelia was also evaluated. METHODS: The readouts were clinical outcomes, CAP-18 expression in mucosa of colon, rectum, lung and trachea (immunohistochemistry and real-time PCR) and release of the CAP-18 peptide/protein in stool (Western blot). PRINCIPAL FINDINGS: Significant downregulation of CAP-18 expression in the epithelia of rectum and colon, the site of Shigella infection was confirmed. Interestingly, reduced expression of CAP-18 was also noticed in the epithelia of lung and trachea, indicating a systemic effect of the infection. This suggests a causative link to acute respiratory infections during shigellosis. Oral treatment with PB resulted in reduced clinical illness and upregulation of CAP-18 in the epithelium of rectum. Both PB and NaB counteracted the downregulation of CAP-18 in lung epithelium. The drug effect is suggested to be systemic as intravenous administration of NaB could also upregulate CAP-18 in the epithelia of lung, rectum and colon. CONCLUSION: Our results suggest that PB has treatment potential in human shigellosis. Enhancement of CAP-18 in the mucosal epithelia of the respiratory tract by PB or NaB is a novel discovery. This could mediate protection from secondary respiratory infections that frequently are the lethal causes in dysentery

A Switching Mechanism in Doxorubicin Bioactivation Can Be Exploited to Control Doxorubicin Toxicity

Although doxorubicin toxicity in cancer cells is multifactorial, the enzymatic bioactivation of the drug can significantly contribute to its cytotoxicity. Previous research has identified most of the components that comprise the doxorubicin bioactivation network; however, adaptation of the network to changes in doxorubicin treatment or to patient-specific changes in network components is much less understood. To investigate the properties of the coupled reduction/oxidation reactions of the doxorubicin bioactivation network, we analyzed metabolic differences between two patient-derived acute lymphoblastic leukemia (ALL) cell lines exhibiting varied doxorubicin sensitivities. We developed computational models that accurately predicted doxorubicin bioactivation in both ALL cell lines at high and low doxorubicin concentrations. Oxygen-dependent redox cycling promoted superoxide accumulation while NADPH-dependent reductive conversion promoted semiquinone doxorubicin. This fundamental switch in control is observed between doxorubicin sensitive and insensitive ALL cells and between high and low doxorubicin concentrations. We demonstrate that pharmacological intervention strategies can be employed to either enhance or impede doxorubicin cytotoxicity in ALL cells due to the switching that occurs between oxygen-dependent superoxide generation and NADPH-dependent doxorubicin semiquinone formation

A phase II study of amrubicin and carboplatin for previously untreated patients with extensive-disease small cell lung cancer

Background: Amrubicin is active in the treatment of extensive-disease small cell lung cancer (ED-SCLC), and carboplatin is an analogue of cisplatin with less non-hematological toxicity. Purpose: The purpose of this study was to determine the efficacy and toxicity of amrubicin and carboplatin combination chemotherapy for previously untreated patients with ED-SCLC. Patients and methods: Eligibility criteria were chemotherapy-naive ED-SCLC patients, performance status 0-1, age ?75, and adequate hematological, hepatic and renal function. Based on the phase I study, the patients received amrubicin 35 mg/m2 i.v. infusion on days 1, 2, and 3, and carboplatin AUC 5 i.v. infusion on day 1. Four cycles of chemotherapy were repeated every 3 weeks. Results: Thirty-five patients were enrolled, and 34 patients were eligible and assessable for response, toxicity, and survival. Patients\u27 characteristics were as follows: male/female = 26/8; performance status 0/1 = 4/30; median age (range) = 64 (41-75); stage IV = 34. Evaluation of responses was 6 complete response, 21 partial response, and 7 stable disease (response rate 79.4 %, 95 % CI 63.6-88.5 %). Grade 3 and 4 leukopenia, neutropenia, and thrombocytopenia occurred in 59, 82, and 26 %, respectively. There were no treatment-related deaths or pneumonitis. Three patients experienced hypotension as an amrubicin infusion reaction. The median progression-free survival time was 6.5 months. The median overall survival time and 1-, 2-, and 3-year survival rates were 15.6 months, and 63, 28, and 7 %, respectively. Conclusions: Amrubicin and carboplatin were effective and tolerable as chemotherapy for previously untreated patients with ED-SCLC. Further investigation of amrubicin and carboplatin is warranted