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Background: Cholangiocarcinoma (CC) is an aggressive bile-duct cancer that affects 8000 people per year in the US. This silent disease manifests symptoms only in advanced stages, resulting in a 5-year overall survival rate of < 10%. Mutations in the metabolic enzyme isocitrate dehydrogenase 1 (IDH1) have been observed in ~20% of CC. These mutations are implicated in promoting tumorigenesis potentially by generation of an ‘oncometabolite' resulting in changes in DNA methylation. There are currently no approved targeted therapies for CC. Ivosidenib, developed by Agios Pharmaceuticals, is a first-in-class, oral, targeted, small-molecule inhibitor of the mutant IDH1 protein currently being evaluated in patients with advanced CC with an IDH1 mutation (ClarIDHy). The Oncomine Dx Target Test (ODxTT) is an FDA approved in vitro diagnostic test for the detection of somatic alterations in human DNA and RNA isolated from FFPE non-small cell lung cancer samples. Thermo Fisher Scientific has partnered with Agios Pharmaceuticals to validate the ODxTT for the detection of IDH1 R132 mutations in CC. Here we present the results from our analytical studies which establish sensitivity, reproducibility and accuracy of the IUO ODxTT to detect 5 IDH1 R132 variants from FFPE CC specimens. Methods: Analytical studies were performed to determine the limit of detection (LOD) and reproducibility of the assay to detect the variants of interest. LOD was determined for each of the 5 variants of interest using either FFPE clinical sample or FFPE cell line blends. Reproducibility of the assay was evaluated by testing clinical samples blends for 3 of 5 variants at ~2x LOD across 3 sites, 6 operators and 3 lots of reagents. Analytical accuracy of the ODxTT was determined by testing over 340 clinical samples with both the ODxTT and a validated orthogonal method. The orthogonal method was Sanger sequencing coupled with Applied Biosystems™ Minor Variant Finder Software to allow calling minor variants down to 5%. Results: 1. LOD of the ODxTT for all 5 variants of interest (R132C, R132G, R132S, R132L and R132H) was determined to be at an allelic frequency (AF) between 3.7% - 5.5%. 2. Reproducibility of the ODxTT to detect R132C, R132G and R132L variants from clinical sample blends was 100% when each variant was tested at an AF of ~2x LOD. 3. Analytical accuracy: The positive percent agreement (PPA) of the ODxTT to Sanger sequencing was 99.4% (163/164) and the negative percent agreement (NPA) was 96.5% (164/170) when excluding unknowns (invalids and no calls). Including unknowns, the PPA of the ODxTT to Sanger sequencing was 97% (163/168) and the NPA was 90.6% (164/181). Conclusion: The data presented here demonstrated that the ODxTT is a sensitive, reproducible and accurate assay for the detection of IDH1 R132 variants from 10 ng of DNA extracted from FFPE CC specimens. Citation Format: Aparna R. Aiyer, Tristan Nater, Matthew Brentnall, Foram Rathi, Bharath Bharadwaj, Mini Manchanda, Viet Nhan, Lauren Abutin, Peter LoCoco, Bin Wu, Jay Shaw. Performance characteristics of the Oncomine™ Dx Target Test for the detection of IDH1 R132 variants from formalin-fixed, paraffin-embedded (FFPE) cholangiocarcinoma samples [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 772.

In these studies, the role of ceramide-1-phosphate (C1P) in the wound-healing process was investigated. Specifically, fibroblasts isolated from mice with the known anabolic enzyme for C1P, ceramide kinase (CERK), ablated (CERK(-/-) mice) and their wild-type littermates (CERK(+/+)) were subjected to in vitro wound-healing assays. Simulation of mechanical trauma of a wound by scratching a monolayer of fibroblasts from CERK(+/+) mice demonstrated steadily increasing levels of arachidonic acid in a time-dependent manner in stark contrast to CERK(-/-) fibroblasts. This observed difference was reflected in scratch-induced eicosanoid levels. Similar, but somewhat less intense, changes were observed in a more complex system utilizing skin biopsies obtained from CERK-null mice. Importantly, C1P levels increased during the early stages of human wound healing correlating with the transition from the inflammatory stage to the peak of the fibroplasia stage (e.g., proliferation and migration of fibroblasts). Finally, the loss of proper eicosanoid response translated into an abnormal migration pattern for the fibroblasts isolated from CERK(-/-) As the proper migration of fibroblasts is one of the necessary steps of wound healing, these studies demonstrate a novel requirement for the CERK-derived C1P in the proper healing response of wounds.

Integrin-β7 (ITGB7) mRNA is detected in multiple myeloma (MM) cells and its presence is correlated with MAF gene activation. Although the involvement of several integrin family members in MM-stoma cell interaction is well documented, the specific biologic functions regulated by integrin-β7 in MM are largely unknown. Clinically, we have correlated integrin-β7 expression in MM with poor survival outcomes post autologous stem cell transplantation and postsalvage therapy with bortezomib. Functionally, we have found that shRNA-mediated silencing of ITGB7 reduces MM-cell adhesion to extra-cellular matrix elements (fibronectin, E-cadherin) and reverses cell-adhesion–mediated drug resistance (CAM-DR) sensitizing them to bortezomib and melphalan. In addition, ITGB7 silencing abrogated MM-cell transwell migration in response to SDF1α gradients, reduced vessel density in xenografted tumors, and altered MM cells in vivo homing into the BM. Mechanistically, ITGB7 knockdown inhibited focal adhesion kinase (FAK) and Src phosphorylation, Rac1 activation, and SUMOylation, reduced VEGF production in MM–BM stem cell cocultures and attenuated p65-NF-κB activity. Our findings support a role for integrin-β7 in MM-cell adhesion, migration, and BM homing, and pave the way for a novel therapeutic approach targeting this molecule.

Caspase-3 is an effector caspase that is activated downstream of mitochondrial outer-membrane permeabilization (MOMP) during apoptosis. However, previous work has demonstrated that caspase-3-deficient mouse embryonic fibroblasts (MEFs) are resistant to mitochondrially mediated cell death and display a delay in the mitochondrial events of apoptosis, including Bax activation, MOMP and release of cytochrome c. Here, we show that caspase-3 regulates fibronectin secretion and impacts on cell morphology, adhesion and migration. Surprisingly, the catalytic activity of caspase-3 is not required for these non-apoptotic functions. Moreover, we found that caspase-3-deficient MEFs are not resistant to death by anoikis and that exogenous fibronectin protects wild-type MEFs from cell death induced by serum withdrawal. Taken together, our data indicate that procaspase-3 has a non-apoptotic function; it regulates the secretion of fibronectin and influences morphology, adhesion and migration. Furthermore, this novel procaspase-3 function might alter the apoptotic threshold of the cell.

BackgroundApoptosis is a form of programmed cell death that is regulated by the Bcl-2 family and caspase family of proteins. The caspase cascade responsible for executing cell death following cytochromecrelease is well described; however the distinct roles of caspases-9, -3 and -7 during this process are not completely defined.ResultsHere we demonstrate several unique functions for each of these caspases during cell death. Specific inhibition of caspase-9 allows for efficient release of cytochromec, but blocks changes in mitochondrial morphology and ROS production. We show that caspase-9 can cleave Bid into tBid at amino acid 59 and that this cleavage of Bid is required for ROS production following serum withdrawal. We also demonstrate that caspase-3-deficient MEFs are less sensitive to intrinsic cell death stimulation, yet have higher ROS production. In contrast, caspase-7-deficient MEFs are not resistance to intrinsic cell death, but remain attached to the ECM.ConclusionsTaken together, these data suggest that caspase-9 is required for mitochondrial morphological changes and ROS production by cleaving and activating Bid into tBid. After activation by caspase-9, caspase-3 inhibits ROS production and is required for efficient execution of apoptosis, while effector caspase-7 is required for apoptotic cell detachment.