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

NCI60 Cancer Cell Line Panel Data and RNAi Analysis Help Identify EAF2 as a Modulator of Simvastatin and Lovastatin Response in HCT-116 Cells

Simvastatin and lovastatin are statins traditionally used for lowering serum cholesterol levels. However, there exists evidence indicating their potential chemotherapeutic characteristics in cancer. In this study, we used bioinformatic analysis of publicly available data in order to systematically identify the genes involved in resistance to cytotoxic effects of these two drugs in the NCI60 cell line panel. We used the pharmacological data available for all the NCI60 cell lines to classify simvastatin or lovastatin resistant and sensitive cell lines, respectively. Next, we performed whole-genome single marker case-control association tests for the lovastatin and simvastatin resistant and sensitive cells using their publicly available Affymetrix 125K SNP genomic data. The results were then evaluated using RNAi methodology. After correction of the p-values for multiple testing using False Discovery Rate, our results identified three genes (NRP1, COL13A1, MRPS31) and six genes (EAF2, ANK2, AKAP7, STEAP2, LPIN2, PARVB) associated with resistance to simvastatin and lovastatin, respectively. Functional validation using RNAi confirmed that silencing of EAF2 expression modulated the response of HCT-116 colon cancer cells to both statins. In summary, we have successfully utilized the publicly available data on the NCI60 cell lines to perform whole-genome association studies for simvastatin and lovastatin. Our results indicated genes involved in the cellular response to these statins and siRNA studies confirmed the role of the EAF2 in response to these drugs in HCT-116 colon cancer cells

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

549. Clinical Characteristics and Outcomes of Patients with COVID-19 treated with Convalescent Plasma in Miami, Florida

AbstractBackgroundThe Coronavirus disease of 2019 (COVID-19) global health crisis caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in unprecedented mortality, impacted society, and strained healthcare systems, yet sufficient data regarding treatment options are lacking. Convalescent plasma, used since 1895 for infectious disease outbreaks, offers promise as a treatment option for COVID-19.MethodsThis is a retrospective study of patients diagnosed by a nasopharyngeal swab SARS-CoV-2 reverse transcriptase–polymerase chain reaction (RT-PCR), who received convalescent plasma between April to June 2020 at two large hospitals in Miami, Florida, as part of the US FDA Expanded Access Program for COVID-19 convalescent plasma (CCP).ResultsA total of 23 patients received CCP, 13 (57%) had severe COVID-19 disease, while 8 (35%) had critical or critical with multiorgan dysfunction. Median time of follow up was 26 (range, 7–79) days. Overall, 11 (48%) survived to discharge, 6 (26%) died, while 6 (26%) are currently hospitalized. All deaths reported were due to septic shock from secondary infections. 15 (65%) showed improvement in oxygen requirements 7 days post CCP transfusion. Measured inflammatory markers, c-reactive protein, lactate dehydrogenase, ferritin and d-dimer improved 7 days post transfusion in 13 (57%) patients. No adverse events due to the transfusion were reported. 10 (43.4%) patients had a negative SARS-CoV-2 RT-PCR at a median of 14.5 (range, 4–31) days after receiving convalescent plasma.ConclusionAdministration of convalescent plasma was found to be safe, with favorable outcomes in this small cohort of relatively high acuity patients. Larger studies including control arms are needed to establish the efficacy of convalescent plasma on clinical and virologic outcomes for patients with COVID-19.TableDisclosuresAll Authors: No reported disclosure