RNAi phenotype profiling of kinases identifies potential therapeutic targets in Ewing's sarcoma

<p>Abstract</p> <p>Background</p> <p>Ewing's sarcomas are aggressive musculoskeletal tumors occurring most frequently in the long and flat bones as a solitary lesion mostly during the teen-age years of life. With current treatments, significant number of patients relapse and survival is poor for those with metastatic disease. As part of novel target discovery in Ewing's sarcoma, we applied RNAi mediated phenotypic profiling to identify kinase targets involved in growth and survival of Ewing's sarcoma cells.</p> <p>Results</p> <p>Four Ewing's sarcoma cell lines TC-32, TC-71, SK-ES-1 and RD-ES were tested in high throughput-RNAi screens using a siRNA library targeting 572 kinases. Knockdown of 25 siRNAs reduced the growth of all four Ewing's sarcoma cell lines in replicate screens. Of these, 16 siRNA were specific and reduced proliferation of Ewing's sarcoma cells as compared to normal fibroblasts. Secondary validation and preliminary mechanistic studies highlighted the kinases STK10 and TNK2 as having important roles in growth and survival of Ewing's sarcoma cells. Furthermore, knockdown of STK10 and TNK2 by siRNA showed increased apoptosis.</p> <p>Conclusion</p> <p>In summary, RNAi-based phenotypic profiling proved to be a powerful gene target discovery strategy, leading to successful identification and validation of STK10 and TNK2 as two novel potential therapeutic targets for Ewing's sarcoma.</p

Selective inhibition of FLT3 by gilteritinib in relapsed or refractory acute myeloid leukaemia: a multicentre, first-in-human, open-label, phase 1-2 study.

BackgroundInternal tandem duplication mutations in FLT3 are common in acute myeloid leukaemia and are associated with rapid relapse and short overall survival. The clinical benefit of FLT3 inhibitors in patients with acute myeloid leukaemia has been limited by rapid generation of resistance mutations, particularly in codon Asp835 (D835). We aimed to assess the highly selective oral FLT3 inhibitor gilteritinib in patients with relapsed or refractory acute myeloid leukaemia.MethodsIn this phase 1-2 trial, we enrolled patients aged 18 years or older with acute myeloid leukaemia who either were refractory to induction therapy or had relapsed after achieving remission with previous treatment. Patients were enrolled into one of seven dose-escalation or dose-expansion cohorts assigned to receive once-daily doses of oral gilteritinib (20 mg, 40 mg, 80 mg, 120 mg, 200 mg, 300 mg, or 450 mg). Cohort expansion was based on safety and tolerability, FLT3 inhibition in correlative assays, and antileukaemic activity. Although the presence of an FLT3 mutation was not an inclusion criterion, we required ten or more patients with locally confirmed FLT3 mutations (FLT3mut+) to be enrolled in expansion cohorts at each dose level. On the basis of emerging findings, we further expanded the 120 mg and 200 mg dose cohorts to include FLT3mut+ patients only. The primary endpoints were the safety, tolerability, and pharmacokinetics of gilteritinib. Safety and tolerability were assessed in the safety analysis set (all patients who received at least one dose of gilteritinib). Responses were assessed in the full analysis set (all patients who received at least one dose of study drug and who had at least one datapoint post-treatment). Pharmacokinetics were assessed in a subset of the safety analysis set for which sufficient data for concentrations of gilteritinib in plasma were available to enable derivation of one or more pharmacokinetic variables. This study is registered with ClinicalTrials.gov, number NCT02014558, and is ongoing.FindingsBetween Oct 15, 2013, and Aug 27, 2015, 252 adults with relapsed or refractory acute myeloid leukaemia received oral gilteritinib once daily in one of seven dose-escalation (n=23) or dose-expansion (n=229) cohorts. Gilteritinib was well tolerated; the maximum tolerated dose was established as 300 mg/day when two of three patients enrolled in the 450 mg dose-escalation cohort had two dose-limiting toxicities (grade 3 diarrhoea and grade 3 elevated aspartate aminotransferase). The most common grade 3-4 adverse events irrespective of relation to treatment were febrile neutropenia (97 [39%] of 252), anaemia (61 [24%]), thrombocytopenia (33 [13%]), sepsis (28 [11%]), and pneumonia (27 [11%]). Commonly reported treatment-related adverse events were diarrhoea (92 [37%] of 252]), anaemia (86 [34%]), fatigue (83 [33%]), elevated aspartate aminotransferase (65 [26%]), and increased alanine aminotransferase (47 [19%]). Serious adverse events occurring in 5% or more of patients were febrile neutropenia (98 [39%] of 252; five related to treatment), progressive disease (43 [17%]), sepsis (36 [14%]; two related to treatment), pneumonia (27 [11%]), acute renal failure (25 [10%]; five related to treatment), pyrexia (21 [8%]; three related to treatment), bacteraemia (14 [6%]; one related to treatment), and respiratory failure (14 [6%]). 95 people died in the safety analysis set, of which seven deaths were judged possibly or probably related to treatment (pulmonary embolism [200 mg/day], respiratory failure [120 mg/day], haemoptysis [80 mg/day], intracranial haemorrhage [20 mg/day], ventricular fibrillation [120 mg/day], septic shock [80 mg/day], and neutropenia [120 mg/day]). An exposure-related increase in inhibition of FLT3 phosphorylation was noted with increasing concentrations in plasma of gilteritinib. In-vivo inhibition of FLT3 phosphorylation occurred at all dose levels. At least 90% of FLT3 phosphorylation inhibition was seen by day 8 in most patients receiving a daily dose of 80 mg or higher. 100 (40%) of 249 patients in the full analysis set achieved a response, with 19 (8%) achieving complete remission, ten (4%) complete remission with incomplete platelet recovery, 46 (18%) complete remission with incomplete haematological recovery, and 25 (10%) partial remission INTERPRETATION: Gilteritinib had a favourable safety profile and showed consistent FLT3 inhibition in patients with relapsed or refractory acute myeloid leukaemia. These findings confirm that FLT3 is a high-value target for treatment of relapsed or refractory acute myeloid leukaemia; based on activity data, gilteritinib at 120 mg/day is being tested in phase 3 trials.FundingAstellas Pharma, National Cancer Institute (Leukemia Specialized Program of Research Excellence grant), Associazione Italiana Ricerca sul Cancro

RPPAML/RIMS: A metadata format and an information management system for reverse phase protein arrays

<p>Abstract</p> <p>Background</p> <p>Reverse Phase Protein Arrays (RPPA) are convenient assay platforms to investigate the presence of biomarkers in tissue lysates. As with other high-throughput technologies, substantial amounts of analytical data are generated. Over 1000 samples may be printed on a single nitrocellulose slide. Up to 100 different proteins may be assessed using immunoperoxidase or immunoflorescence techniques in order to determine relative amounts of protein expression in the samples of interest.</p> <p>Results</p> <p>In this report an RPPA Information Management System (RIMS) is described and made available with open source software. In order to implement the proposed system, we propose a metadata format known as reverse phase protein array markup language (RPPAML). RPPAML would enable researchers to describe, document and disseminate RPPA data. The complexity of the data structure needed to describe the results and the graphic tools necessary to visualize them require a software deployment distributed between a client and a server application. This was achieved without sacrificing interoperability between individual deployments through the use of an open source semantic database, S3DB. This data service backbone is available to multiple client side applications that can also access other server side deployments. The RIMS platform was designed to interoperate with other data analysis and data visualization tools such as Cytoscape.</p> <p>Conclusion</p> <p>The proposed RPPAML data format hopes to standardize RPPA data. Standardization of data would result in diverse client applications being able to operate on the same set of data. Additionally, having data in a standard format would enable data dissemination and data analysis.</p

Current pre-clinical and clinical advances in the BCR-ABL1-positive and -negative chronic myeloproliferative neoplasms

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Posterior Association Networks and Functional Modules Inferred from Rich Phenotypes of Gene Perturbations

Combinatorial gene perturbations provide rich information for a systematic exploration of genetic interactions. Despite successful applications to bacteria and yeast, the scalability of this approach remains a major challenge for higher organisms such as humans. Here, we report a novel experimental and computational framework to efficiently address this challenge by limiting the ‘search space’ for important genetic interactions. We propose to integrate rich phenotypes of multiple single gene perturbations to robustly predict functional modules, which can subsequently be subjected to further experimental investigations such as combinatorial gene silencing. We present posterior association networks (PANs) to predict functional interactions between genes estimated using a Bayesian mixture modelling approach. The major advantage of this approach over conventional hypothesis tests is that prior knowledge can be incorporated to enhance predictive power. We demonstrate in a simulation study and on biological data, that integrating complementary information greatly improves prediction accuracy. To search for significant modules, we perform hierarchical clustering with multiscale bootstrap resampling. We demonstrate the power of the proposed methodologies in applications to Ewing's sarcoma and human adult stem cells using publicly available and custom generated data, respectively. In the former application, we identify a gene module including many confirmed and highly promising therapeutic targets. Genes in the module are also significantly overrepresented in signalling pathways that are known to be critical for proliferation of Ewing's sarcoma cells. In the latter application, we predict a functional network of chromatin factors controlling epidermal stem cell fate. Further examinations using ChIP-seq, ChIP-qPCR and RT-qPCR reveal that the basis of their genetic interactions may arise from transcriptional cross regulation. A Bioconductor package implementing PAN is freely available online at http://bioconductor.org/packages/release/bioc/html/PANR.html

Deterministic Effects Propagation Networks for reconstructing protein signaling networks from multiple interventions

<p>Abstract</p> <p>Background</p> <p>Modern gene perturbation techniques, like RNA interference (RNAi), enable us to study effects of targeted interventions in cells efficiently. In combination with mRNA or protein expression data this allows to gain insights into the behavior of complex biological systems.</p> <p>Results</p> <p>In this paper, we propose Deterministic Effects Propagation Networks (DEPNs) as a special Bayesian Network approach to reverse engineer signaling networks from a combination of protein expression and perturbation data. DEPNs allow to reconstruct protein networks based on combinatorial intervention effects, which are monitored via changes of the protein expression or activation over one or a few time points. Our implementation of DEPNs allows for latent network nodes (i.e. proteins without measurements) and has a built in mechanism to impute missing data. The robustness of our approach was tested on simulated data. We applied DEPNs to reconstruct the <it>ERBB </it>signaling network in <it>de novo </it>trastuzumab resistant human breast cancer cells, where protein expression was monitored on Reverse Phase Protein Arrays (RPPAs) after knockdown of network proteins using RNAi.</p> <p>Conclusion</p> <p>DEPNs offer a robust, efficient and simple approach to infer protein signaling networks from multiple interventions. The method as well as the data have been made part of the latest version of the R package "nem" available as a supplement to this paper and via the Bioconductor repository.</p

Abortive Autophagy Induces Endoplasmic Reticulum Stress and Cell Death in Cancer Cells

Autophagic cell death or abortive autophagy has been proposed to eliminate damaged as well as cancer cells, but there remains a critical gap in our knowledge in how this process is regulated. The goal of this study was to identify modulators of the autophagic cell death pathway and elucidate their effects on cellular signaling and function. The result of our siRNA library screenings show that an intact coatomer complex I (COPI) is obligatory for productive autophagy. Depletion of COPI complex members decreased cell survival and impaired productive autophagy which preceded endoplasmic reticulum stress. Further, abortive autophagy provoked by COPI depletion significantly altered growth factor signaling in multiple cancer cell lines. Finally, we show that COPI complex members are overexpressed in an array of cancer cell lines and several types of cancer tissues as compared to normal cell lines or tissues. In cancer tissues, overexpression of COPI members is associated with poor prognosis. Our results demonstrate that the coatomer complex is essential for productive autophagy and cellular survival, and thus inhibition of COPI members may promote cell death of cancer cells when apoptosis is compromised

Randomized multicenter phase II study of flavopiridol (alvocidib), cytarabine, and mitoxantrone (FLAM) versus cytarabine/daunorubicin (7+3) in newly diagnosed acute myeloid leukemia

Serial studies have demonstrated that induction therapy with FLAM [flavopiridol (alvocidib) 50 mg/m2 days 1–3, cytarabine 667 mg/m2/day continuous infusion days 6–8, and mitoxantrone (FLAM) 40 mg/m2 day 9] yields complete remission rates of nearly 70% in newly diagnosed poor-risk acute myeloid leukemia. Between May 2011–July 2013, 165 newly diagnosed acute myeloid leukemia patients (age 18–70 years) with intermediate/adverse-risk cytogenetics were randomized 2:1 to receive FLAM or 7+3 (cytarabine 100 mg/m2/day continuous infusion days 1–7 and daunorubicin 90 mg/m2 days 1–3), across 10 institutions. Some patients on 7+3 with residual leukemia on day 14 received 5+2 (cytarabine 100 mg/m2/day continuous infusion days 1–5 and daunorubicin 45 mg/m2 days 1–2), whereas patients on FLAM were not re-treated based on day 14 bone marrow findings. The primary objective was to compare complete remission rates between one cycle of FLAM and one cycle of 7+3. Secondary end points included safety, overall survival and event-free survival. FLAM led to higher complete remission rates than 7+3 alone (70% vs. 46%; P=0.003) without an increase in toxicity, and this improvement persisted after 7+3+/−5+2 (70% vs. 57%; P=0.08). There were no significant differences in overall survival and event-free survival in both arms but post-induction strategies were not standardized. These results substantiate the efficacy of FLAM induction in newly diagnosed AML. A phase III study is currently in development. This study is registered with clinicaltrials.gov identifier: 01349972