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Izar, Benjamin

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Izar

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Benjamin

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Izar, Benjamin
Author's Publications: search.filters.isAuthorOfPublication.159b60e9-4b7d-46b8-bc18-71ef3171062b

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    Evolution of Delayed Resistance to Immunotherapy in a Melanoma Responder
    (Springer Nature, 2021-05-03) Liu, David; Lin, Jia-Ren; Robitschek, Emily; Kasumova, Gyulnara; Heyde, Alexander; Shi, Alvin; Kraya, Adam; Zhang, Gao; Moll, Tabea; Frederick, Dennie; Chen, Yu-An; Schapiro, Denis; Ho, Li-Lun; Bi, Kevin; Sahu, Avinash; Mei, Shaolin; Miao, Benchun; Sharova, Tatyana; Alvarez-Breckenridge, Christopher; Stocking, Jackson; Kim, Tommy; Fadden, Riley; Lawrence, Donald; Hoang, Mai; Cahill, Daniel; Maleh Mir, Mohsen; Nowak, Martin; Brastianos, Priscilla; Lian, Christine; Ruppin, Eytan; Izar, Benjamin; Herlyn, Meenhard; Van Allen, Eliezer; Nathanson, Katherine; Flaherty, Keith; Sullivan, Ryan; Kellis, Manolis; Sorger, Peter; Boland, Genevieve
    Despite initial responses, most melanoma patients develop resistance to immune checkpoint blockade (ICB). To understand the evolution of resistance, we studied 37 tumor samples over 9 years from a metastatic melanoma patient with exceptional response followed by delayed recurrence and death. Phylogenetic analysis revealed co-evolution of 7 lineages with multiple convergent, but independent resistance-associated alterations (RAAs). All recurrent tumors emerged from a lineage characterized by loss of chromosome 15q, with post-treatment clones acquiring additional genomic driver events. Deconvolution of bulk RNAseq and highly-multiplexed immunofluorescence (t-CyCIF) revealed differences in immune composition amongst different lineages. Imaging revealed a vasculogenic mimicry phenotype in NGFR-High tumor cells with high PD-L1 expression in close proximity to immune cells. Rapid autopsy demonstrated 2 distinct NGFR spatial patterns with high polarity and proximity to immune cells in subcutaneous tumors versus a diffuse spatial pattern in lung tumors, suggesting different roles of this neural crest-like program in different tumor microenvironments. Broadly, this study establishes a high-resolution map of the evolutionary dynamics of resistance to ICB, characterizes a de-differentiated, neural crest tumor population in melanoma immunotherapy resistance, and describes site specific differences in tumor-immune interactions via longitudinal analysis of a melanoma patient with an unusual clinical course.
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    A first‐in‐human phase I, multicenter, open‐label, dose‐escalation study of the oral RAF/VEGFR‐2 inhibitor (RAF265) in locally advanced or metastatic melanoma independent from BRAF mutation status
    (John Wiley and Sons Inc., 2017) Izar, Benjamin; Sharfman, William; Hodi, F. Stephen; Lawrence, Donald; Flaherty, Keith; Amaravadi, Ravi; Kim, Kevin B.; Puzanov, Igor; Sosman, Jeffrey; Dummer, Reinhard; Goldinger, Simone M.; Lam, Lyhping; Kakar, Shefali; Tang, Zhongwen; Krieter, Oliver; McDermott, David; Atkins, Michael B.
    Abstract To establish the maximum tolerated dose (MTD), dose‐limiting toxicities (DLT), safety profile, and anti‐tumor efficacy of RAF265. We conducted a multicenter, open‐label, phase‐I, dose‐escalation trial of RAF265, an orally available RAF kinase/VEGFR‐2 inhibitor, in patients with advanced or metastatic melanoma. Pharmacokinetic (PK) analysis, pharmacodynamics (PD) and tumor response assessment were conducted. We evaluated metabolic tumor response by 18[F]‐fluorodeoxyglucose‐positron‐emission tomography (FDG‐PET), tissue biomarkers using immunohistochemistry (IHC), and modulators of angiogenesis. RAF265 has a serum half‐life of approximately 200 h. The MTD was 48 mg once daily given continuously. Among 77 patients, most common treatment‐related adverse effects were fatigue (52%), diarrhea (34%), weight loss (31%) and vitreous floaters (27%). Eight of 66 evaluable patients (12.1%) had an objective response, including seven partial and one complete response. Responses occurred in BRAF‐mutant and BRAF wild‐type (WT) patients. Twelve of 58 (20.7%) evaluable patients had a partial metabolic response. On‐treatment versus pretreatment IHC staining in 23 patients showed dose‐dependent p‐ERK inhibition. We observed a significant temporal increase in placental growth factor levels and decrease in soluble vascular endothelial growth factor receptor 2 (sVEGFR‐2) levels in all dose levels. RAF265 is an oral RAF/VEGFR‐2 inhibitor that produced antitumor responses, metabolic responses, and modulated angiogenic growth factor levels. Antitumor activity occurred in patients with BRAF‐mutant and BRAF‐WT disease. Despite low activity at tolerable doses, this study provides a framework for the development of pan‐RAF inhibitors and modulators of angiogenesis for the treatment of melanoma.
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    Listeria monocytogenes Induces a Virulence-Dependent microRNA Signature That Regulates the Immune Response in Galleria mellonella
    (Frontiers Media S.A., 2017) Mannala, Gopala K.; Izar, Benjamin; Rupp, Oliver; Schultze, Tilman; Goesmann, Alexander; Chakraborty, Trinad; Hain, Torsten
    microRNAs (miRNAs) coordinate several physiological and pathological processes by regulating the fate of mRNAs. Studies conducted in vitro indicate a role of microRNAs in the control of host-microbe interactions. However, there is limited understanding of miRNA functions in in vivo models of bacterial infections. In this study, we systematically explored changes in miRNA expression levels of Galleria mellonella larvae (greater-wax moth), a model system that recapitulates the vertebrate innate immunity, following infection with L. monocytogenes. Using an insect-specific miRNA microarray with more than 2000 probes, we found differential expression of 90 miRNAs (39 upregulated and 51 downregulated) in response to infection with L. monocytogenes. We validated the expression of a subset of miRNAs which have mammalian homologs of known or predicted function. In contrast, non-pathogenic L. innocua failed to induce these miRNAs, indicating a virulence-dependent miRNA deregulation. To predict miRNA targets using established algorithms, we generated a publically available G. mellonella transcriptome database. We identified miRNA targets involved in innate immunity, signal transduction and autophagy, including spätzle, MAP kinase, and optineurin, respectively, which exhibited a virulence-specific differential expression. Finally, in silico estimation of minimum free energy of miRNA-mRNA duplexes of validated microRNAs and target transcripts revealed a regulatory network of the host immune response to L. monocytogenes. In conclusion, this study provides evidence for a role of miRNAs in the regulation of the innate immune response following bacterial infection in a simple, rapid and scalable in vivo model that may predict host-microbe interactions in higher vertebrates.
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    Current status of antisense RNA-mediated gene regulation in Listeria monocytogenes
    (Frontiers Media S.A., 2014) Schultze, Tilman; Izar, Benjamin; Qing, Xiaoxing; Mannala, Gopala K.; Hain, Torsten
    Listeria monocytogenes is a Gram-positive human-pathogen bacterium that served as an experimental model for investigating fundamental processes of adaptive immunity and virulence. Recent novel technologies allowed the identification of several hundred non-coding RNAs (ncRNAs) in the Listeria genome and provided insight into an unexpected complex transcriptional machinery. In this review, we discuss ncRNAs that are encoded on the opposite strand of the target gene and are therefore termed antisense RNAs (asRNAs). We highlight mechanistic and functional concepts of asRNAs in L. monocytogenes and put these in context of asRNAs in other bacteria. Understanding asRNAs will further broaden our knowledge of RNA-mediated gene regulation and may provide targets for diagnostic and antimicrobial development.
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    Opposing Immune and Genetic Mechanisms Shape Oncogenic Programs in Synovial Sarcoma
    (Cold Spring Harbor Laboratory, 2021-01-25) Jerby-Arnon, Livnat; Neftel, Cyril; Shore, Marni E.; Weisman, Hannah R.; Mathewson, Nathan; McBride, Matthew J.; Haas, Brian; Izar, Benjamin; Volorio, Angela; Boulay, Gaylor; Cironi, Luisa; Richman, Alyssa R.; Broye, Liliane C.; Gurski, Joseph M.; Luo, Christina; Mylvaganam, Ravindra; Nguyen, Lan; Mei, Shaolin; Melms, Johannes; Georgescu, Christophe; Cohen, Ofir; Buendia Buendia, Jorge Eduardo; Segerstolpe, Asa; Sud, Malika; Cuoco, Michael; Labes, Danny; Zollinger, Daniel R.; Ortogero, Nicole; Beechem, Joseph M.; Nielsen, G. Petur; Chebib, Ivan; Nguyen-Ngoc, Tu; Montemurro, Michael; Cote, Gregory; Choy, Edwin; Letovanec, Igor; Cherix, Stéphane; Wagle, Nikhil; Sorger, Peter; Haynes, Alex; Mullen, John; Stamenkovic, Ivan; Rivera, Miguel; Kadoch, Cigall; Wucherpfennig, Kai; Rozenblatt-Rosen, Orit; Suvà, Mario L.; Riggi, Nicolò; Regev, Aviv
    ABSTRACTSynovial sarcoma is an aggressive mesenchymal neoplasm, driven by the SS18-SSX fusion, and characterized by immunogenic antigens expression and exceptionally low T cell infiltration levels. To study the cancer-immune interplay in this disease, we profiled 16,872 cells from 12 human synovial sarcoma tumors using single-cell RNA-sequencing (scRNA-Seq). Synovial sarcoma manifests antitumor immunity, high cellular plasticity and a core oncogenic program, which is predictive of low immune levels and poor clinical outcomes. Using genetic and pharmacological perturbations, we demonstrate that the program is controlled by the SS18-SSX driver and repressed by cytokines secreted by macrophages and T cells in the tumor microenvironment. Network modeling predicted that SS18-SSX promotes the program through HDAC1 and CDK6. Indeed, the combination of HDAC and CDK4/6 inhibitors represses the program, induces immunogenic cell states, and selectively targets synovial sarcoma cells. Our study demonstrates that immune evasion, cellular plasticity, and cell cycle are co-regulated and can be co-targeted in synovial sarcoma and potentially in other malignancies.
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    A single-cell and single-nucleus RNA-Seq toolbox for fresh and frozen human tumors
    (Springer Science and Business Media LLC, 2020-05-01) Slyper, Michal; Porter, Caroline; Ashenberg, Orr; Waldman, Julia; Drokhlyansky, Eugene; Wakiro, Isaac; Smilie, Christopher; Smith-Rosario, Gabriela; Wu, Jingyi; Dionne, Danielle; Vigneau, Sebastien; Jane-Valbuena, Judit; Tickle, Timothy; Napolitano, Sara; Su, Mei-Ju; Patel, Anand; Karlstrom, Asa; Gristch, Simon; Nomura, Masashi; Waghray, Avinash; Gohil, Satyen; Tsankov, Alexander; Jerby-Arnon, Livnat; Cohen, Ofir; Klughammer, Johanna; Rosen, Yanay; Gould, Joshua; Nguyen, Lan; Hofree, Matan; Tramontozzi, Peter; Levy, Rachel; Li, Bo; Wu, Catherine; Izar, Benjamin; Haq, Rizwan; Hodi, Stephen; Yoon, Charles; Hata, Aaron; Baker, Suzanne; Suva, Mario; Bueno, Raphael; Stover, Elizabeth; Clay, Michael; Dyer, M Aiven; Collins, Natalie; Matulonis, Ursula; Wagle, Nikhil; Johnson, Bruce; Rotem, Asaf; Rozenblatt-Rosen, Orit; Regev, Aviv
    Single-cell genomics is essential to chart tumor ecosystems. Although single-cell RNA-Seq (scRNA-Seq) profiles RNA from cells dissociated from fresh tumors, single-nucleus RNA-Seq (snRNA-Seq) is needed to profile frozen or hard-to-dissociate tumors. Each requires customization to different tissue and tumor types, posing a barrier to adoption. Here, we have developed a systematic toolbox for profiling fresh and frozen clinical tumor samples using scRNA-Seq and snRNA-Seq, respectively. We analyzed 216,490 cells and nuclei from 40 samples across 23 specimens spanning eight tumor types of varying tissue and sample characteristics. We evaluated protocols by cell and nucleus quality, recovery rate and cellular composition. scRNA-Seq and snRNA-Seq from matched samples recovered the same cell types, but at different proportions. Our work provides guidance for studies in a broad range of tumors, including criteria for testing and selecting methods from the toolbox for other tumors, thus paving the way for charting tumor atlases.
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    Qualifying Antibodies for Image-Based Immune Profiling and Multiplexed Tissue Imaging
    (Springer Science and Business Media LLC, 2019-09-18) Lin, Jia-Ren; Rashid, Rumana; Maliga, Zoltan; Izar, Benjamin; Santagata, Sandro; Du, Ziming; Wang, Shu; Aster, Jon; Songer, Peter
    Multiplexed tissue imaging enables precise, spatially resolved enumeration and characterization of cell types and states in human resection specimens. A growing number of methods applicable to formalin-fixed, paraffin-embedded (FFPE) tissue sections have been described, the majority of which rely on antibodies for antigen detection and mapping. This protocol provides step-by-step procedures for confirming the selectivity and specificity of antibodies used in fluorescence-based tissue imaging and for the construction and validation of antibody panels. Although the protocol is implemented using tissue-based cyclic immunofluorescence (t-CyCIF) as an imaging platform, these antibody-testing methods are broadly applicable. We demonstrate assembly of a 16-antibody panel for enumerating and localizing T cells and B cells, macrophages, and cells expressing immune checkpoint regulators. The protocol is accessible to individuals with experience in microscopy and immunofluorescence; some experience in computation is required for data analysis. A typical 30-antibody dataset for 20 FFPE slides can be generated within 2 weeks.