tbuckley/cabot-search huggingface dataset:
Alternate-locus aware variant calling in whole genome sequencing
Authors: Marten Jäger, Max Schubach, Tomasz Zemojtel, Knut Reinert, Deanna M. Church, Peter N. Robinson
Journal: Genome Medicine (2016)

The last two human genome assemblies have extended the previous linear golden-path paradigm of the human genome to a graph-like model to better represent regions with a high degree of structural variability. The new model offers opportunities to improve the technical validity of variant calling in whole-genome sequencing (WGS).We developed an algorithm that analyzes the patterns of variant calls in the 178 structurally variable regions of the GRCh38 genome assembly, and infers whether a given sample is most likely to contain sequences from the primary assembly, an alternate locus, or their heterozygous combination at each of these 178 regions. We investigate 121 in-house WGS datasets that have been aligned to the GRCh37 and GRCh38 assemblies.We show that stretches of sequences that are largely but not entirely identical between the primary assembly and an alternate locus can result in multiple variant calls against regions of the primary assembly. In WGS analysis, this results in characteristic and recognizable patterns of variant calls at positions that we term alignable scaffold-discrepant positions (ASDPs). In 121 in-house genomes, on average 51.8±3.8 of the 178 regions were found to correspond best to an alternate locus rather than the primary assembly sequence, and filtering these genomes with our algorithm led to the identification of 7863 variant calls per genome that colocalized with ASDPs. Additionally, we found that 437 of 791 genome-wide association study hits located within one of the regions corresponded to ASDPs.Our algorithm uses the information contained in the 178 structurally variable regions of the GRCh38 genome assembly to avoid spurious variant calls in cases where samples contain an alternate locus rather than the corresponding segment of the primary assembly. These results suggest the great potential of fully incorporating the resources of graph-like genome assemblies into variant calling, but also underscore the importance of developing computational resources that will allow a full reconstruction of the genotype in personal genomes. Our algorithm is freely available at https://github.com/charite/asdpex .
Differential expression of BRCA1 and genes involved in the nuclear factor kappa B (NFκB) and notch signalling pathways in non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) patients (p).
Authors: I. Magri, M. Santarpia, M. Sanchez-Ronco, S. Viteri, C. Costa, A. Gasco, N. Mederos, M. Gonzalez Cao, F. Salazar, J. Bertran-Alamillo, A. Gimenez Capitan, M. A. Molina, Z. Yeste, E. Aldeguer, T. Moran, G. Altavilla, S. Benlloch, M. Taron, R. Rosell
Journal: Journal of Clinical Oncology (2011)

e21025 Background: Genetic diversity in lung cancer according to histological subtype has not been fully explored. BRCA1 and RAP80 influence response to chemotherapy. Musashi 2 activates HES-1 in the Notch pathway, and HES-1 can abrogate CYLD. A20, AEG-1, EZH2 and TRAF6 are also involved in NFkB activation. We have examined mRNA expression of BRCA1, RAP80 and components of the NFkB and Notch pathways in lung cancer p. Methods: mRNA expression of Musashi 2, CYLD, HES-1, A20, EZH2, AEG-1, TRAF6, NFKBIA, RelA, BRCA1 and RAP80 was analyzed by quantitative RT-PCR in tumor samples from 85 lung cancer p (77 NSCLC, 8 SCLC). Results: p characteristics: 51 males; median age, 59; 33 smokers, 26 ex-smokers; 49 adenocarcinoma, 10 large cell carcinoma (LCC), 18 squamous cell carcinoma (SCC), 8 SCLC. Source of tumor sample: 36 primary tumor, metastasis, 15. 11 p had K-ras mutations. There were differences in the expression levels of Musashi 2, BRCA1, EZH2 and RAP80 according to histological subtype. The median Musashi 2 mRNA expression was 4 times higher in SCLC than in NSCLC (adenocarcinoma, SCC, LCC) (P<0.001); BRCA1 expression was 3 times higher (P<0.001); EZH2 was 7 times higher (P<0.001); RAP80 was 2 times higher (P=0.011). There were no differences in expression levels of any of the 11 genes analyzed according to p age, smoking history or source of the tumor sample. NSCLC p with K-ras mutations had higher expression of both AEG-1 and NFKBIA than p with wild-type K-ras (P=0.04). Conclusions: Musashi 2, BRCA1, EZH2 and RAP80 expression was significantly higher in SCLC p. Further investigation is warranted to examine the potential association between expression of these genes and response to platinum regimens and radiotherapy in SCLC. Elevated AEG-1 levels associated with K-ras-mutant tumors could identify a subgroup of NSCLC p with poor prognosis.
Abstract 1990: The key role of MECOM complex locus and its products in high-grade ovarian carcinoma pathogenesis and clinical outcomes
Authors: Arsen O. Batagov, Surya Pavan Yenamandra, Piroon Jenjaroenpun, Anna V. Ivshina, Vladimir A. Kuznetsov
Journal: Cancer Research (2015)

Abstract The ecotropic virus integration site 1(Evi1) transcription factor, encoded in the MECOM complex locus, is implicated in several cancers, including ovarian cancer(OC). High-grade serous OC(HG-SOC) represents its most common and aggressive form. The pathobiological and clinical roles of MECOM and its products in HG-SOC are poorly understood. Here, across all HG-SOC stages and grades, our meta-analysis demonstrated MECOM amplification in about 80% and EVI1 overexpression in 100% HG-SOC samples, relative to normal controls. We observed that Evi1 directly promotes double-strand DNA breaks repair via binding to DNA recombination/repair protein complexes, including a novel Rad50 isoform. To elucidate the molecular basis and clinical significance of these findings, we studied genome-wide genome-wide Evi1-DNA binding, DNA copy number alterations, gene expression and proteome-scale Evi1 interactions, combining cell line and clinical meta-data and network/pathway analyses. Our experiments revealed major, previously unknown Evi1-binding DNA motifs specifying expression of 309 target genes, whose protein products were classified into eight functional and prognosis-significant modules implicated in embryogenesis, EMT, RNA metabolism, cancer cell survival, retinoic acid, anti-viral and therapeutic responses. The Evi1 pathway modules can collectively stratify the HG-SOC patients into four high-confidence post-surgery survival subgroups(P&amp;lt;1.0×10-50), over-performing previously reported HG-SOC predictors and clinical markers. This tumor stratification correlates with chemotherapy sensitivity and treatment outcomes. Starting from early stages of HG-SOC, MECOM/Evi1 amplifies genomic instability and activates the Evi1 pathway that promotes most cancer hallmarks. Thus, MECOM/Evi1 and the Evi1 pathway provide mechanistic means for HG-SOC re-classification, introduces high-confidence early diagnostic and prognostic biomarkers and perspective therapeutic targets. Citation Format: Arsen O. Batagov, Surya Pavan Yenamandra, Piroon Jenjaroenpun, Anna V. Ivshina, Vladimir A. Kuznetsov. The key role of MECOM complex locus and its products in high-grade ovarian carcinoma pathogenesis and clinical outcomes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1990. doi:10.1158/1538-7445.AM2015-1990
Influence of ZAP-70 Expression On Migration of Chronic Lymphocytic Leukemia (CLL) and Lymphoma Cells.
Authors: Eva Calpe, Carles Codony, Maria Joao Baptista, Eva Fernandez, Marta Crespo, Eva Gine, Pau Abrisqueta, Emili Montserrat, Francesc Bosch
Journal: Blood (2009)
Abstract 3914: IGF-II causes translocation of estrogen receptor beta to the mitochondria in triple negative breast cancer cell lines
Authors: Chane' O'Bannon-Joseph, Daisy D. DeLeon
Journal: Cancer Research (2015)

Abstract Breast cancer is the most frequently diagnosed cancer and is the leading cause of death among women worldwide. Breast cancer is classified by the presence or absence of the estrogen receptor (ER), progesterone receptor (PR), and the human epidermal growth factor receptor 2 (HER2). Triple negative breast cancers (TNBCs) lack all 3 receptors and account for approximately 15-20% of all breast cancers. Due to lack of targeted treatments TNBCs become aggressive and can metastasize to other parts of the body. The insulin-like growth factor-II (IGF-II) has an important role in fetal and cancer development. IGF-II signals through the IGF-I receptor (IGF-IR), insulin receptor (Ins-R), and has been associated in cross-talk with the estrogen receptors alpha and beta (ERα, ERß). Our lab has demonstrated that IGF-II activates the translocation of estrogen receptor alpha (ER-α) and estrogen receptor beta (ER-β) in the absence of the estrogen ligand. When co-expressed, ER-β counteracts the proliferative function of ER-α, however the role of ER-β in ER-α negative breast cancers is unclear. Although TNBC accounts for 20% of all BC, 50-90% of all TNBC express high levels of ER-β. Triple negative breast cancer can be more aggressive and difficult to treat. Also, the cancer is more likely to spread and recur. With the elevated levels of expression of the ER-β found in TNBC our lab demonstrated through western blotting and confocal microscopy that not only did treatment with IGF-II increase the expression of ER-β in both ER positive (MCF-7) and TNBC cell lines (CRL-231, 2335, and HS578-T) but it also stimulated translocation predominantly to the mitochondria in the TNBC cell lines. The presence of ER-β in the mitochondria suggests a need for further study on the action of ER-β in the mitochondria of all BC cell lines, especially TNBC cell lines. Citation Format: Chane' O'Bannon-Joseph, Daisy D. DeLeon, Center for Health Disparities and Molecular Medicine. IGF-II causes translocation of estrogen receptor beta to the mitochondria in triple negative breast cancer cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3914. doi:10.1158/1538-7445.AM2015-3914
Decreased Expression of Diphthamide Genes Is Responsible for the Resistance to the Anti-CD22 Immunotoxin Moxetumomab Pasudotox (m. pasudotox) in Acute Lymphoblastic Leukemia (ALL)/Non-Hodgkin Lymphoma (NHL) Cell Lines.
Authors: Hui Wei, Laiman Xiang, Alan S Wayne, Ira Pastan
Journal: Blood (2012)
28. Mouse and Pig Photoreceptor Transduction Mediated by Triple AAV Vectors
Authors: Andrea Maddalena, Patrizia Tornabene, Fabio Dell'Aquila, Ivana Trapani, Alberto Auricchio
Journal: Molecular Therapy (2016)

Purpose: The majority of inherited blinding diseases are caused by mutations in genes expressed specifically in photoreceptors (PRs). However the size of several of these genes exceeds the DNA cargo capacity of AAV vectors, to date the safest and most effective gene therapy vectors in the retina. We have previously demonstrated that dual AAV vectors expand AAV PR transfer capacity to about 9 kb. Our aim is to further expand this to around 13.5 kb using a triple AAV vector system. Methods: To assess the percentage of PR transduction mediated by three independent vectors, we initially injected subretinally in 4 week-old C57/BL6 mice single AAV8 vectors encoding either EGFP, dsRed or beta-gal reporter proteins. Then, in order to evaluate the PR transduction efficiency of triple AAV vectors we generated a reporter EGFP-dsRed fusion protein under the control of either the ubiquitous CMV or the PR-specific IRBP(interphotoreceptor retinoid-binding protein) promoters. The corresponding EGFP-dsRed expression cassettes were either included in single or triple AAV8 vectors (Fig1) which were administered by subretinal injection to C57/BL6 mice or large white pigs. Direct fluorescence, Western blot, and ELISA analysis were used to evaluate transgene expression. Results: In mice injected with three independent AAV8 vectors encoding for the different EGFP, dsRed and beta-gal reporter proteins, the percentage of PR co-transduced was 12%. In mouse and pig eyes injected with triple and single AAV8 vectors encoding EGFP-dsRed, full length protein expression was confirmed by Western Blot analysis independently of the CMV or IRBP promoters used; importantly, 75% of mouse and 65% of pig eyes injected with triple IRBP vectors showed bands of the expected size. Quantification in mice of EGFP-dsRed protein expression from triple AAV8 vectors mediated by either the CMV or the IRBP promoter shows 30% and 1%, respectively, of the levels observed with a single AAV8. Interestingly, in pigs, the levels of EGFP-dsRed protein expression from triple AAV8-IRBP vectors were 40% ratio of those observed with a single AAV8. Conclusions: Our results show that triple AAV vectors, which increase AAV transfer capacity up to 13.5 kb, transduce mouse and pig PR at levels that are 1% and 40% respectively of those obtained with single AAV vectors. This bode well for further testing this platform in animal models of inherited retinal degenerations.
The Final Cellular Output in Human Erythroid Massive Amplification Culture (HEMA) Is Determined by Dynamic Interactions Between Immature and Mature Cell Populations.
Authors: Massimo Sanchez, Francesca Masiello, Valentina Tirelli, Barbara Ghinassi, Carolyn Whitsett, Anna Rita Migliaccio, Giovanni Migliaccio
Journal: Blood (2009)
Abstract 687: Compound screen identifies PIM kinases as therapeutic targets for melanoma
Authors: Adina M. Vultur, Batool Shannan, Quan Chen, Andrea Watters, Stefan Mollin, Eric Meggers, Clemens Krepler, Michela Perego, Ling Li, Phyllis A. Gimotty, Xiaowei Xu, Meenhard Herlyn
Journal: Cancer Research (2015)

Abstract Current treatments against malignant melanoma, including the use of small molecule inhibitors, are displaying encouraging results in the clinic. However, tumor resistance develops even for patients who initially respond favorably. To identify novel drug targets and compounds with anti-melanoma activity, we screened a panel of genetically distinct human-derived metastatic melanoma cell lines against structurally diverse organometallic kinase inhibitors. We observed a compound that preferentially inhibits melanoma cell proliferation compared to normal primary fibroblasts and melanocytes in adherent and three-dimensional (3D) cultures. We further showed that this compound (SM200) is pro-apoptotic and anti-invasive in 3D melanoma cell cultures. A kinome screen to identify the targets responsible for the anti-melanoma effects of SM200 revealed PIM kinases to be highly inhibited. PIM1 knockdown studies to validate this target in human metastatic melanoma indicate that PIM1 contributes to melanoma growth in 3D culture and in a xenograft model. We then evaluated the clinically available PIM kinase inhibitor SGI-1776 in our melanoma models and observed reduced cell proliferation in 2D, 3D, and in vivo melanoma models (as seen for SM200) suggesting an important role for PIM kinases in melanoma pathobiology. Taken together, our findings suggest that PIM kinases are valid targets for melanoma and PIM kinase inhibitors could enhance the effects of current therapies in the clinic. Citation Format: Adina M. Vultur, Batool Shannan, Quan Chen, Andrea Watters, Stefan Mollin, Eric Meggers, Clemens Krepler, Michela Perego, Ling Li, Phyllis A. Gimotty, Xiaowei Xu, Meenhard Herlyn. Compound screen identifies PIM kinases as therapeutic targets for melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 687. doi:10.1158/1538-7445.AM2015-687
Abstract 4304: MMP-9 as a marker of response to treatment with B-Raf inhibitors in cutaneous melanoma
Authors: Saverio Candido, Grazia Malaponte, Rossella Salemi, Franca Maria Pezzino, Aurora Scalisi, James A. McCubrey, Massimo Libra
Journal: Cancer Research (2015)

Abstract Malignant melanoma is an aggressive tumor of the skin with a poor prognosis for patients with advanced disease. In melanoma, both the Ras/Raf/MEK/ERK (MAPK) and the PI3K/AKT (AKT) signaling pathways are constitutively activated through multiple mechanisms. Although, at present it has been demonstrated that the treatment with B-Raf enzyme inhibitors improve survival of melanoma patients, still these patients may relapse or become resistant to treatment. Therefore, there is a need to identify novel factors involved in melanoma development and/or progression. Matrix metalloproteinases (MMP) have been regarded as critical molecules in promoting tumor cell metastasis. Prognostic and diagnostic significance of MMP-9 overexpression has been shown in several cancer types. However, their role in melanoma was not fully investigated. Thus, we hypothesized that MMP-9 overexpression in melanoma may be involved in transformation and act as a prognostic biomarker. On this light, MMP-9 circulating levels, and MMP-9 gelatinase activity were measured by ELISA and zymography in a total of 148 melanoma samples with and without BRAFV600E mutation compared to healthy controls. The results showed that highest circulating levels of MMP-9 and MMP-9 activity were associated with advanced stages of disease. We have also demonstrated that higher levels of MMP-9 were detected among melanoma samples harboring BRAFV600E mutation compared to those BRAFWT. We further investigated if MMP-9 may be associated with acquired resistance to B-Raf inhibitors, such as Dabrafenib. Functional in vitro experiments were performed using commercial available melanoma cell lines. The results showed that MMP-9 was down-regulated in melanoma cells after treatment with Dabrafenib. While, higher MMP-9 expression levels were observed in the resistant clones compared to those detected in the parental cells. Intriguingly, similar trend was observed for pERK expression. Overall, these data indicate that MMP-9 may play a role for the evaluation of response to treatment with B-Raf inhibitors in melanoma patients. Note: This abstract was not presented at the meeting. Citation Format: Saverio Candido, Grazia Malaponte, Rossella Salemi, Franca Maria Pezzino, Aurora Scalisi, James A. McCubrey, Massimo Libra. MMP-9 as a marker of response to treatment with B-Raf inhibitors in cutaneous melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4304. doi:10.1158/1538-7445.AM2015-4304
Abstract 405: Kindlin-2 regulates integrin function and sensitivity to docetaxel in prostate cancer cells
Authors: Edward F. Plow, Mitali Das, Jamila Hirbawi, Khalid Sossey-Alaoui
Journal: Cancer Research (2015)

Abstract Over the past decade, many lines of evidence have emerged to implicate the three members of the kindlin family of FERM domain proteins as important regulators of integrin adhesion receptors. Specifically, kindlins appear to cooperate with talin to be essential regulators of the ligand binding function of integrins and thereby enhance the adhesive and migratory responses of cells. Kindlin-2 (FERMT2) is the most broadly distributed of the kindlins. To explore the relationship between integrin activation and kindlin-2, prostate cancer cell lines were screened and LNCaP prostate cancer cells were found to express relatively low levels of kindlin-2 and endogenous levels of beta-1 integrin on their surface. A portion of this integrin was expressed in an activated state as detected with an activation specific antibody, HUTS-4. These cells were transfected with vectors for kindlin-2, with or without talin-head domain, a combination that leads to activation of β3 integrins in model cells. Transfections with kindlin-2 suppressed β1 integrin activation on the surface of the LNCaP cells. This inhibitory effect was abolished by mutation of kindlin-2 that prevented its binding to integrin cytoplasmic tail. This inhibitory response to kindlin-2 on β1 integrin was in stark contract to the effects of kindlin-2 plus talin-head expression on β3 function, where co-expression of the same constructs enhanced integrin activation. A mutation in kindlin-2, deletion of its last 15 amino acids, that blocks activation of β3 integrins without preventing its binding to the integrin, also prevents its inhibition of β1 function. We next looked at the effects of kindlin-2 on docetaxel induced apoptosis as monitored with Annexin V staining and cell death as monitored by propidium iodide staining (both by flow cytometry). Compared to LNCaP cells expressing EGFP alone, cells overexpressing EGFP-kindlin-2 were protected against apoptosis and cell death. This effect was dependent upon binding of kindlin-2 to the cytoplasmic tail of integrins. Hence, the capacity of kindlin-2 to influence integrin function depends upon the integrin subfamily and can range from activation to suppression of function. Kindlin-2, which is often overexpressed in transformed cells, may desensitize cells towards chemotherapeutic agents. Citation Format: Edward F. Plow, Mitali Das, Jamila Hirbawi, Khalid Sossey-Alaoui. Kindlin-2 regulates integrin function and sensitivity to docetaxel in prostate cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 405. doi:10.1158/1538-7445.AM2015-405
538. Promoter Compatibility Enables Viral Vectors to Deliver Antiviral RNA Genes
Authors: Carolina Camargo, Margaret J. Lange, Kyle J. Hill, Donald H. Burke
Journal: Molecular Therapy (2015)

HIV-1 reverse transcriptase (RT) copies the viral RNA genome into double-stranded DNA during viral replication. Nucleic acid aptamers that bind RT inhibit its enzymatic activity in biochemical and biological assays and can be delivered to target cells by lentiviral vectors. We have demonstrated that RNA aptamers efficiently suppress viral replication; however, using lentiviral vectors to deliver anti-HIV genes might give rise to self-targeting, in which expression of the cargo gene during vector production interferes with efficient gene delivery. We hypothesize that promoter strength will predict the degree of viral suppression, and that regulated promoters are more suitable than constitutive promoters for delivering anti-RT aptamer genes by viral vectors. Specifically, promoters that are strong in T cells are expected to yield the most effective suppression of replicating virus, while promoters that are weak in HEK293FT are expected to yield the highest transduction efficiencies by minimizing self-targeting during particle production. To establish relative promoter strength in producer HEK293FT cells, we first cloned constitutive and regulated promoters (CMV, CMV-TO, EF1α, UbC, CD4 and U6) to drive the expression of a fluorescent aptamer (stabilized dimeric Broccoli) and EGFP. Baseline promoter strength (EGFP MFI) varied widely in HEK293FT cells, with strong expression under CMV, intermediate expression under EF1α and UbC, almost no expression under CD4. Regulated expression was achievable under CMV-TO by co-transfecting a plasmid expressing TetR Repressor. We next evaluated the effect of each promoter/aptamer combination on viral infectivity for which constructs were transiently transfected into producer cells (HEK293FT) along with proviral and helper plasmids. Virus was harvested after 48h and used in subsequent infections. Virus produced in aptamer-expressing HEK293 FT cells showed decrease in viral infectivity both in epithelial (HEK293FT) and T cell lines (CEM-T4), in each case correlating with promoter strength in the producer cells. These data strongly suggest that promoters with lower levels of expression in producer cells, such as CD4 and CMV-TO, might overcome self-targeting and become optimal for production of lentiviral vectors, which is an important goal in order to move aptamer-based strategies into pre-clinical contexts. Additionally, EGFP and stabilized dimeric Broccoli expression are informative surrogates for viral inhibition.