An inherited duplication at the gene p21 Protein-Activated Kinase 7 (PAK7) is a risk factor for psychosis

Identifying rare, highly penetrant risk mutations may be an important step in dissecting the molecular etiology of schizophrenia. We conducted a gene-based analysis of large (>100kb), rare copy number variants (CNVs) in the Wellcome Trust Case Control Consortium 2 (WTCCC2) schizophrenia sample of 1,564 cases and 1,748 controls all from Ireland, and further extended the analysis to include an additional 5,196 UK controls. We found association with duplications at chr20p12.2 (P=0.007) and evidence of replication in large independent European schizophrenia (P=0.052) and UK bipolar disorder case-control cohorts (P=0.047). A combined analysis of Irish/UK subjects including additional psychosis cases (schizophrenia and bipolar disorder) identified 22 carriers in 11,707 cases and 10 carriers in 21,204 controls (meta-analysis CMH P value=2x10(-4) (odds ratio (OR)=11.3, 95% CI=3.7, ∞)). Nineteen of the 22 cases and 8 of the 10 controls carried duplications starting at 9.68Mb with similar breakpoints across samples. By haplotype analysis and sequencing we identified a tandem ∼149kb duplication overlapping the gene p21 Protein-Activated Kinase 7 (PAK7, also called PAK5) which was in linkage disequilibrium with local haplotypes (P=2.5x10(-21)), indicative of a single ancestral duplication event. We confirmed the breakpoints in 8/8 carriers tested and found co-segregation of the duplication with illness in two additional family members of one of the affected probands. We demonstrate that PAK7 is developmentally co-expressed with another known psychosis risk gene (DISC1) suggesting a potential molecular mechanism involving aberrant synapse development and plasticity.

Synergistic effects of proteasome inhibitor carfilzomib in combination with tyrosine kinase inhibitors in imatinib-sensitive and -resistant chronic myeloid leukemia models

The tyrosine kinase inhibitor (TKI) imatinib has transformed the treatment and outlook of chronic myeloid leukemia (CML); however, the development of drug resistance and the persistence of TKI-resistant stem cells remain obstacles to eradicating the disease. Inhibition of proteasome activity with bortezomib has been shown to effectively induce apoptosis in TKI-resistant cells. In this study, we show that exposure to the next generation proteasome inhibitor carfilzomib is associated with a decrease in ERK signaling and increased expression of Abelson interactor proteins 1 and 2 (ABI-1/2). We also investigate the effect of carfilzomib in models of imatinib-sensitive and -resistant CML and demonstrate a potent reduction in proliferation and induction of apoptosis in a variety of models of imatinib-resistant CML, including primitive CML stem cells. Carfilzomib acts synergistically with the TKIs imatinib and nilotinib, even in imatinib-resistant cell lines. In addition, we found that the presence of immunoproteasome subunits is associated with an increased sensitivity to carfilzomib. The present findings provide a rational basis to examine the potential of carfilzomib in combination with TKIs as a potential therapy for CML, particularly in imatinib-resistant disease.

Selective induction of apoptosis by the pyrrolo-1,5-benzoxazepine 7-[[dimethylcarbamoyl]oxy]-6-(2-naphthyl)pyrrolo-[2,1-d] (1,5)-benzoxazepine (PBOX-6) in Leukemia cells occurs via the c-Jun NH2-terminal kinase-dependent phosphorylation and inactivation of Bcl-2 and Bcl-XL

Overexpression of the Bcl-2 proto-oncogene in tumor cells confers resistance against chemotherapeutic drugs. In this study, we describe how the novel pyrrolo-1,5-benzoxazepine compound 7-[[dimethylcarbamoyl]oxy]-6-(2-naphthyl)pyrrolo-[2,1-d] (1,5)-benzoxazepine (PBOX-6) selectively induces apoptosis in Bcl-2-overexpressing cancer cells, whereas it shows no cytotoxic effect on normal peripheral blood mononuclear cells. PBOX-6 overcomes Bcl-2-mediated resistance to apoptosis in chronic myelogenous leukemia (CML) K562 cells by the time- and dose-dependent phosphorylation and inactivation of antiapoptotic Bcl-2 family members Bcl-2 and Bcl-XL. PBOX-6 also induces Bcl-2 phosphorylation and apoptosis in wild-type T leukemia CEM cells and cells overexpressing Bcl-2. This is in contrast to chemotherapeutic agents such as etoposide, actinomycin D, and ultraviolet irradiation, whereby overexpression of Bcl-2 confers resistance against apoptosis. In addition, PBOX-6 induces Bcl-2 phosphorylation and apoptosis in wild-type Jurkat acute lymphoblastic leukemia cells and cells overexpressing Bcl-2. However, Jurkat cells containing a Bcl-2 triple mutant, whereby the principal Bcl-2 phosphorylation sites are mutated to alanine, demonstrate resistance against Bcl-2 phosphorylation and apoptosis. PBOX-6 also induces the early and transient activation of c-Jun NH2-terminal kinase (JNK) in CEM cells. Inhibition of JNK activity prevents Bcl-2 phosphorylation and apoptosis, implicating JNK in the upstream signaling pathway leading to Bcl-2 phosphorylation. Collectively, these findings identify Bcl-2 phosphorylation and inactivation as a critical step in the apoptotic pathway induced by PBOX-6 and highlight its potential as an effective antileukemic agent.

Genetic variants of kinase suppressors of Ras (KSR1) to predict survival in patients with ERα-positive advanced breast cancer

In patients with breast cancer (BC), deregulation of estrogen receptor (ERα) activity may account for most resistance to endocrine therapies. Our previous study used a whole-human kinome siRNA screen to identify functional actors in ERα modulation and showed the implication of proteins kinase suppressors of ras (KSR1). From those findings we evaluated the clinical impact of KSR1 variants in patients with ERα+ BC treated with TAM. DNA was obtained from 222 patients with advanced ERα+ BC treated with TAM who had undergone surgery from 1981 to 2003. We selected three potentially functional relevant KSR1 polymorphisms; two within the 3'UTR (rs224190, rs1075952) and one in the coding exon 7 (rs2293180). The primary end points were overall survival (OS) and disease-free survival (DFS). After a 6.4-year median follow-up, patients carrying the rs2241906 TT genotype showed shorter DFS (2.1 vs 7.1 years, P=0.005) and OS (2.6 vs 8.4 years P=0.002) than those with the TC or TT genotypes. Those associations remained significant in the multivariable analysis adjusting age, lymph node status, LMTK3 and IGFR variants and HER2 status. The polymorphisms rs2241906 and rs1075952 were in linkage disequilibrium. No association was shown between rs2293180 and survival. Among the actors of ERα signaling, KSR1 rs2241906 variants may predict survival in patients with advanced ERα+ BC treated with adjuvant TAM.

The dual EGFR/HER-2 tyrosine kinase inhibitor lapatinib sensitizes colon and gastric cancer cells to the irinotecan active metabolite SN-38

Members of the human epidermal receptor (HER) family are frequently associated with aggressive disease and poor prognosis in multiple malignancies. Lapatinib is a dual tyrosine kinase inhibitor targeting the epidermal growth factor receptor (EGFR) and HER-2. This study evaluated the therapeutic potential of lapatinib, alone and in combination with SN-38, the active metabolite of irinotecan (CPT-11), in colon and gastric cancer cell lines. Concentration-dependent antiproliferative effects of both lapatinib and SN-38 were observed in all colon and gastric cancer cell lines tested but varied significantly between individual cell lines (lapatinib range 0.08-11.7 muM; SN-38 range 3.6-256 nM). Lapatinib potently inhibited the growth of a HER-2 overexpressing gastric cancer cell line and demonstrated moderate activity in gastric and colon cancer cells with detectable HER-2 expression. The combination of lapatinib and SN-38 interacted synergistically to inhibit cell proliferation in all colon and gastric cancer cell lines tested. Cotreatment with lapatinib and SN-38 also resulted in enhanced cell cycle arrest and the induction of apoptosis with subsequent cellular pharmacokinetic analysis demonstrating that lapatinib promoted the increased intracellular accumulation and retention of SN-38 when compared to SN-38 treatment alone. Finally, the combination of lapatinib and CPT-11 demonstrated synergistic antitumor efficacy in the LoVo colon cancer mouse xenograft model with no apparent increase in toxicity compared to CPT-11 monotherapy. These results provide compelling preclinical rationale indicating lapatinib to be a potentially efficacious chemotherapeutic combination partner for irinotecan in the treatment of gastrointestinal carcinomas.

The use of erythropoiesis-stimulating agents with ruxolitinib in patients with myelofibrosis in COMFORT-II: an open-label, phase 3 study assessing efficacy and safety of ruxolitinib versus best available therapy in the treatment of myelofibrosis

BACKGROUND: Anemia is considered a negative prognostic risk factor for survival in patients with myelofibrosis. Most patients with myelofibrosis are anemic, and 35-54 % present with anemia at diagnosis. Ruxolitinib, a potent inhibitor of Janus kinase (JAK) 1 and JAK2, was associated with an overall survival benefit and improvements in splenomegaly and patient-reported outcomes in patients with myelofibrosis in the two phase 3 COMFORT studies. Consistent with the ruxolitinib mechanism of action, anemia was a frequently reported adverse event. In clinical practice, anemia is sometimes managed with erythropoiesis-stimulating agents (ESAs). This post hoc analysis evaluated the safety and efficacy of concomitant ruxolitinib and ESA administration in patients enrolled in COMFORT-II, an open-label, phase 3 study comparing the efficacy and safety of ruxolitinib with best available therapy for treatment of myelofibrosis. Patients were randomized (2:1) to receive ruxolitinib 15 or 20 mg twice daily or best available therapy. Spleen volume was assessed by magnetic resonance imaging or computed tomography scan.

RESULTS: Thirteen of 146 ruxolitinib-treated patients had concomitant ESA administration (+ESA). The median exposure to ruxolitinib was 114 weeks in the +ESA group and 111 weeks in the overall ruxolitinib arm; the median ruxolitinib dose intensity was 33 mg/day for each group. Six weeks before the first ESA administration, 10 of the 13 patients had grade 3/4 hemoglobin abnormalities. These had improved to grade 2 in 7 of the 13 patients by 6 weeks after the first ESA administration. The rate of packed red blood cell transfusions per month within 12 weeks before and after first ESA administration remained the same in 1 patient, decreased in 2 patients, and increased in 3 patients; 7 patients remained transfusion independent. Reductions in splenomegaly were observed in 69 % of evaluable patients (9/13) following first ESA administration.

CONCLUSIONS: Concomitant use of an ESA with ruxolitinib was well tolerated and did not affect the efficacy of ruxolitinib. Further investigations evaluating the effects of ESAs to alleviate anemia in ruxolitinib-treated patients are warranted (ClinicalTrials.gov identifier, NCT00934544; July 6, 2009).

Epirubicin, oxaliplatin, and capecitabine with or without panitumumab for patients with previously untreated advanced oesophagogastric cancer (REAL3): a randomised, open-label phase 3 trial.

BACKGROUND: EGFR overexpression occurs in 27-55% of oesophagogastric adenocarcinomas, and correlates with poor prognosis. We aimed to assess addition of the anti-EGFR antibody panitumumab to epirubicin, oxaliplatin, and capecitabine (EOC) in patients with advanced oesophagogastric adenocarcinoma. METHODS: In this randomised, open-label phase 3 trial (REAL3), we enrolled patients with untreated, metastatic, or locally advanced oesophagogastric adenocarcinoma at 63 centres (tertiary referral centres, teaching hospitals, and district general hospitals) in the UK. Eligible patients were randomly allocated (1:1) to receive up to eight 21-day cycles of open-label EOC (epirubicin 50 mg/m(2) and oxaliplatin 130 mg/m(2) on day 1 and capecitabine 1250 mg/m(2) per day on days 1-21) or modified-dose EOC plus panitumumab (mEOC+P; epirubicin 50 mg/m(2) and oxaliplatin 100 mg/m(2) on day 1, capecitabine 1000 mg/m(2) per day on days 1-21, and panitumumab 9 mg/kg on day 1). Randomisation was blocked and stratified for centre region, extent of disease, and performance status. The primary endpoint was overall survival in the intention-to-treat population. We assessed safety in all patients who received at least one dose of study drug. After a preplanned independent data monitoring committee review in October, 2011, trial recruitment was halted and panitumumab withdrawn. Data for patients on treatment were censored at this timepoint. This study is registered with ClinicalTrials.gov, number NCT00824785. FINDINGS: Between June 2, 2008, and Oct 17, 2011, we enrolled 553 eligible patients. Median overall survival in 275 patients allocated EOC was 11.3 months (95% CI 9.6-13.0) compared with 8.8 months (7.7-9.8) in 278 patients allocated mEOC+P (hazard ratio [HR] 1.37, 95% CI 1.07-1.76; p=0.013). mEOC+P was associated with increased incidence of grade 3-4 diarrhoea (48 [17%] of 276 patients allocated mEOC+P vs 29 [11%] of 266 patients allocated EOC), rash (29 [11%] vs two [1%]), mucositis (14 [5%] vs none), and hypomagnesaemia (13 [5%] vs none) but reduced incidence of haematological toxicity (grade ≥ 3 neutropenia 35 [13%] vs 74 [28%]). INTERPRETATION: Addition of panitumumab to EOC chemotherapy does not increase overall survival and cannot be recommended for use in an unselected population with advanced oesophagogastric adenocarcinoma. FUNDING: Amgen, UK National Institute for Health Research Biomedical Research Centre.

Growth inhibition by tyrosine kinase inhibitors in mesothelioma cell lines.

Clinical outcome following chemotherapy for malignant pleural mesothelioma is poor and improvements are needed. This preclinical study investigates the effect of five tyrosine kinase inhibitors (PTK787, ZD6474, ZD1839, SU6668 and SU11248) on the growth of three mesothelioma cell lines (NCI H226, NCI H28 and MSTO 211H), the presence of growth factor receptors and inhibition of their downstream signalling pathways. GI50 values were determined: ZD6474 and SU11248, mainly VEGFR2 inhibitors, gave the lowest GI50 across all cell lines (3.5-6.9 microM) whereas ZD1839 gave a GI50 in this range only in H28 cells. All cell lines were positive for EGFR, but only H226 cells were positive for VEGFR2 by Western blotting. ZD6474 and ZD1839 inhibited EGF-induced phosphorylation of EGFR, AKT and ERK, whereas VEGF-induced phosphorylation of VEGFR2 was completely inhibited with 0.1 microM SU11248. VEGFR2 was detected in tumour samples by immunohistochemistry. VEGFR2 tyrosine kinase inhibitors warrant further investigation in mesothelioma.

LtpD is a novel legionella pneumophila effector that binds phosphatidylinositol 3-phosphate and inositol monophosphatase IMPA1

The Dot/Icm type IV secretion system (T4SS) of Legionella pneumophila is crucial for the pathogen to survive in protozoa and cause human disease. Although more than 275 effector proteins are delivered into the host cell by the T4SS, the function of the majority is unknown. Here we have characterized the Dot/Icm effector LtpD. During infection, LtpD localized to the cytoplasmic face of the membrane of the Legionella-containing vacuole (LCV). In A549 lung epithelial cells, ectopically expressed LtpD localized to large vesicular structures that contained markers of endosomal compartments. Systematic analysis of LtpD fragments identified an internal 17-kDa fragment, LtpD_471-626, which was essential for targeting ectopically expressed LtpD to vesicular structures and for the association of translocated LtpD with the LCV. LtpD_471-626 bound directly to phosphatidylinositol 3-phosphate [PtdIns(3)P] in vitro and colocalized with the PtdIns(3)P markers FYVE and SetA in cotransfected cells. LtpD was also found to bind the host cell enzyme inositol (myo)-1 (or 4)-monophosphatase 1, an important phosphatase involved in phosphoinositide production. Analysis of the role of LtpD in infection showed that LtpD is involved in bacterial replication in THP-1 macrophages, the larvae of Galleria mellonella, and mouse lungs. Together, these data suggest that LtpD is a novel phosphoinositide- binding L. pneumophila effector that has a role in intracellular bacterial replication. © 2013, American Society for Microbiology.

A phase I pharmacokinetic and pharmacodynamic study of the oral mitogen-activated protein kinase kinase (MEK) inhibitor, WX-554, in patients with advanced solid tumours

Purpose: We performed a multi-centre phase I study to assess the safety, pharmacokinetics (PK) and pharmacodynamics (PD) of the orally available small molecule mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor, WX-554, and to determine the optimal biological dose for subsequent trials.
Experimental design: Patients with treatment-refractory, advanced solid tumours, with adequate performance status and organ function were recruited to a dose-escalation study in a standard 3 + 3 design. The starting dose was 25 mg orally once weekly with toxicity, PK and PD guided dose-escalation with potential to explore alternative schedules.
Results: Forty-one patients with advanced solid tumours refractory to standard therapies and with adequate organ function were recruited in eight cohorts up to doses of 150 mg once weekly and 75 mg twice weekly. No dose-limiting toxicities were observed during the study, and a maximum tolerated dose (MTD) was not established. The highest dose cohorts demonstrated sustained inhibition of extracellular signal-regulated kinase (ERK) phosphorylation in peripheral blood mononuclear cells following ex-vivo phorbol 12-myristate 13-acetate stimulation. There was a decrease of 70 ± 26% in mean phosphorylated (p)ERK in C1 day 8 tumour biopsies when compared with pre-treatment tumour levels in the 75 mg twice a week cohort. Prolonged stable disease (>6 months) was seen in two patients, one with cervical cancer and one with ampullary carcinoma.
Conclusions: WX-554 was well tolerated, and an optimal biological dose was established for further investigation in either a once or twice weekly regimens. The recommended phase 2 dose is 75 mg twice weekly.