We have isolated a human cDNA for the signaling adapter molecule FRS-2/suc1-associated neurotrophic factor target and shown that it is tyrosine-phosphorylated in response to nerve growth factor (NGF) stimulation. Importantly, we demonstrate that the phosphotyrosine binding domain of FRS-2 directly binds the Trk receptors at the same phosphotyrosine residue that binds the signaling adapter Shc, suggesting a model in which competitive binding between FRS-2 and Shc regulates differentiation versus proliferation. Consistent with this model, FRS-2 binds Grb-2, Crk, the SH2 domain containing tyrosine phosphatase SH-PTP-2, the cyclindependent kinase substrate p13 suc1 , and the Src homology 3 (SH3) domain of Src, providing a functional link between TrkA, cell cycle, and multiple NGF signaling effectors. Importantly, overexpression of FRS-2 in cells expressing an NGF nonresponsive TrkA receptor mutant reconstitutes the ability of NGF to stop cell cycle progression and to stimulate neuronal differentiation.

We have investigated the signaling properties of the fibroblast growth factor (FGF) receptor substrate 3 (FRS3), also known as SNT-2 or FRS2beta, in neurotrophin-dependent differentiation in comparison with the related adapter FRS2 (SNT1 or FRS2alpha). We demonstrate that FRS3 binds all neurotrophin Trk receptor tyrosine kinases and becomes tyrosine phosphorylated in response to NGF, BDNF, NT-3 and FGF stimulation in transfected cells and/or primary cortical neurons. Second, the signaling molecules Grb2 and Shp2 bind FRS3 at consensus sites that are highly conserved among FRS family members and that Shp2, in turn, becomes tyrosine phosphorylated. While FRS3 over-expression in PC12 cells neither increases NGF-induced neuritogenesis nor activation of Map kinase/AKT, comparable to previous reports on FRS2, over-expression of a chimeric adapter containing the PH/PTB domains of the insulin receptor substrate (IRS) 2, in place of the PTB domain of FRS3 (IRS2-FRS3) supports insulin-dependent Map kinase activation and neurite outgrowth in PC12 cells. Collectively, these data demonstrate that FRS3 supports ligand-induced Map kinase activation and that the chimeric IRS2-FRS3 adapter is stimulating sufficient levels of activated MapK to support neurite outgrowth in PC12 cells.

The signaling adapter proteins FRS2 and FRS3 are implicated in the transmission of extracellular signals from nerve growth factor (NGF) or fibroblast growth factor (FGF) receptors to the Ras/mitogen-activated protein kinase signaling cascade. This study presents the genomic sequence and exon-intron organization of the mouse FRS2 and FRS3 loci as well as their evolutionary conservation with their human counterparts. Both FRS2 and FRS3 contain 5 coding exons spanning over 7 kb of genomic sequence with similar exon sizes and organization. Comparative genomic sequence analyses show a highly conserved genomic organization between mouse and human in both FRS2 and FRS3 genes. Non-coding sequences, highly conserved between mouse and human, were identified in the FRS3 introns that may potentially function as regulatory elements. To assay potential differences in their patterns of expression, RT-PCR analysis was used to assay FRS2 and FRS3 expression in the developing embryo and neural tube (NT) during the time of neurogenesis.

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Introduction: RNA fusion transcripts result from genomic rearrangements where two distinct genes become juxtaposed and fused. Fusion transcripts are an important class of somatic alterations because they have the potential to create chimeric proteins with altered function, contributing to oncogene activation. These aberrant proteins are expressed in a tumor-specific manner and are thus excellent targets for therapeutic intervention. We have designed an amplicon-based library preparation (LP) kit that targets over 350 fusion gene pairs, representing over 1,000 unique fusion breakpoint events from both solid tumor and hematologic cancers. This panel is specifically designed to run on a newly developed microfluidic integrated fluidic circuit (IFC) that supports flexible amplicon library preparation of 1-6 unique LP panels on a single IFC. Samples can be run against the LP panels in groups of 8, allowing for up to 48 samples per IFC. In this poster we present the results of our analytical validation tests, demonstrating the performance of our comprehensive gene fusion panel on synthetic targets, reference FFPE samples, and subject FFPE samples for use in research studies. Methods: Primers for amplicon library generation targeting known fusion breakpoints were designed in collaboration with Q2 Solutions®. The panel is comprised of over 1,000 amplicons with an average insert size of 170 bp. Primers were divided into 8 assay pools using informatic parameters that minimize generation of off-target products. Assay pools and cDNA from an off-IFC reverse transcription reaction, along with sample barcodes, were dispensed into designated inlets on an IFC and placed in a Juno™ targeted DNA sequencing library preparation system for mixing the nanoscale reactions, thermal cycling, and amplicon harvesting. Harvested amplicons were collected from the IFC, pooled, and prepared for sequencing on an Illumina® NextSeq™. Results: The panel was tested against synthetic targets representing all targeted amplicons to demonstrate that each assay reliably detects the intended target of interest. Reference standards and subject FFPE samples were used to assess performance, including positive predictive agreement (PPA), positive predictive value (PPV), and limit of detection. Using these samples at inputs as low as 10 ng of total RNA, PPA and PPV were greater than 99% for the fusion events tested. In addition, fusion events could be reliably detected with as little as 250 copies of target fusion material loaded onto the IFC. Conclusions: A comprehensive fusion gene panel for targeted next-generation sequencing has been developed using nanoliter-scale PCR based enrichment on a newly developed IFC. Generation of high-quality libraries from a minimum of 10 ng total RNA from FFPE samples for use in research studies has been demonstrated. Citation Format: Michael L. Gonzales, Jian Qin, Xiaohui Wang, Sangpen Chamnongpol, Jeff Jasper, Thomas Goralski, Christopher Kubu. Development of a comprehensive gene fusion NGS panel using an integrated microfluidic circuit enabling highly efficient, multiplex PCR enrichment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 745.