Spliceosomal dysregulation dramatically affects many cellular processes, notably signal transduction, metabolism, and proliferation, and has led to the concept of targeting intracellular spliceosomal proteins to combat cancer. Here we show that a subset of lymphoma cells displays a spliceosomal complex on their surface, which we term surface spliceosomal complex (SSC). The SSC consists of at least 13 core components and was discovered as the binding target of the non-Hodgkin's lymphoma-specific aptamer C10.36. The aptamer triggers SSC internalization, causing global changes in alternative splicing patterns that eventually lead to necrotic cell death. Our study reveals an exceptional spatial arrangement of a spliceosomal complex and defines it not only as a potential target of anti-cancer drugs, but also suggests that its localization plays a fundamental role in cell survival.

Abstract Aptamers have recently gained prominence for their diagnostic and therapeutic potential. The DNA aptamer C10.36 forms a G-quadruplex and has been shown to bind the Ramos Burkitt’s lymphoma cell line. However, its binding partner on the cell surface remains unknown. Here we report on the identification of the molecular target of C10.36, which suggests its application in the therapy of B-cell lymphoma and leukaemia. Aptamer-affinity purification, followed by LC-MS/MS revealed unique proteins pulled down with C10.36 associated with Ramos cells but not Jurkat, a T-cell lymphocyte cell line. The majority of the identified target molecules were found to be associated within ribonucleoprotein complexes, of which the abundant and consistent ones belong to the nucleolin complex including nucleolin (NCL) itself and its interacting partners, i.e. nucleophosmin (NPM1), heterogeneous nuclear ribonucleoprotein (HNRNP) family members such as HNRNP C1C2 and U, rRNA 2'-O-methyltransferase fibrillarin (FBL), actin (ACTB), nucleolar RNA helicase 2 (DDX21), and proline- and glutamine-rich splicing factors (SFPQ). All proteins identified in the above ribonucleoprotein complex are aberrantly expressed on the surface of several disparate cancer cell types and have been shown to play an oncogenic role in cancer. Another G-rich anti-NCL aptamer, AS1411, has been shown to induce cell death in >100 cancer cell lines. Therefore, we tested the effect of C10.36 on viability of Ramos and other non-Hodgkin B-cell lymphoma (NHL) cancer cell lines. Our results indicate that C10.36 treatment causes specific cell death of certain lymphoma cell lines such as Ramos but not Jurkat cells. The present study identifies C10.36 as a novel anti-B cell lymphoma aptamer and underscores its potential for the development of a new targeted therapy to treat B-cell lymphomas. Citation Format: Sonal S. Tonapi, Janet E. Duncan, Matthew Rosenow, Melissa Richards, Teresa L. Tinder, Heather A. O'Neill, Mark R. Miglarese, David Spetzler, Michael Famulok, Günter Mayer. The B-cell lymphoma specific aptamer C10.36 binds a ribonucleoprotein complex on the cell surface of cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2469. doi:10.1158/1538-7445.AM2017-2469

Technologies capable of characterizing the full breadth of cellular systems need to be able to measure millions of proteins, isoforms, and complexes simultaneously. We describe an approach that fulfils this criterion: Adaptive Dynamic Artificial Poly-ligand Targeting (ADAPT). ADAPT employs an enriched library of single-stranded oligodeoxynucleotides (ssODNs) to profile complex biological samples, thus achieving an unprecedented coverage of system-wide, native biomolecules. We used ADAPT as a highly specific profiling tool that distinguishes women with or without breast cancer based on circulating exosomes in their blood. To develop ADAPT, we enriched a library of ~10 11 ssODNs for those associating with exosomes from breast cancer patients or controls. The resulting 10 6 enriched ssODNs were then profiled against plasma from independent groups of healthy and breast cancer-positive women. ssODN-mediated affinity purification and mass spectrometry identified low-abundance exosome-associated proteins and protein complexes, some with known significance in both normal homeostasis and disease. Sequencing of the recovered ssODNs provided quantitative measures that were used to build highly accurate multi-analyte signatures for patient classification. Probing plasma from 500 subjects with a smaller subset of 2000 resynthesized ssODNs stratified healthy, breast biopsy-negative, and -positive women. An AUC of 0.73 was obtained when comparing healthy donors with biopsy-positive patients.

Introduction: Aptamers are valuable tools for identifying novel therapeutic targets due to their high affinity and specificity. The relative ease of selection of aptamers binding to complex targets and their lack of immunogenicity have led to the incorporation of aptamers into drug transport vehicles and cell labeling tools. However, their potential as direct anti-cancer therapies remains to be explored. Recent advances in aptamer therapeutics have underscored their potential to meet the need for better therapies in B-cell non-Hodgkin lymphomas (NHL). For example, the aptamer AS1411 binds nucleolin and inhibits the proliferation of multiple leukemia and lymphoma cancer cell lines. The DNA aptamer C10.36 has been shown to selectively bind to Ramos Burkitt lymphoma cells and to be internalized via clathrin-dependent endocytosis. Here we identify the molecular target of C10.36 and explore its potential role as a targeted therapy in NHL. Methods: The protein targets of C10.36 on the surface of tumor cells were identified by LC-MS/MS following affinity purification using biotin labeled aptamers. Direct molecular targets of C10.36 were identified by aptamer-based affinity labeling of target proteins after photo-crosslinking and subsequent affinity purification. Bands were excised from polyacrylamide gels, reduced, alkylated with an Iodo-TMT126/129 label, digested, and analyzed by LC-MS/MS. Targets were confirmed by the presence of the precursor mass of the peptide with the TMT-SDAD label and both the 126 and 129 reporter ions in the MS/MS spectrum. To confirm extracellular expression of proteins on intact cells, we carried out selective biotin labeling of surface proteins followed by subsequent purification and LC-MS/MS detection. In vitro anti-proliferative activity of C10.36 was assessed by CellTiter-Glo® assay. Preferential binding and internalization of C10.36 were assessed by flow cytometry and immunofluorescence, respectively. Results: C10.36 affinity pull-downs revealed that it associates with a ribonucleoprotein complex on the surface of Ramos cells. Gene Ontology enrichment analysis revealed proteins over-represented in chromatin organization and RNA metabolic process gene sets. Covalent crosslinking identified heterogeneous nuclear ribonucleoprotein U (hnRNP U) as a direct binding target of C10.36 within the complex. We next surveyed additional lymphoma and leukemia cell lines for C10.36 binding. Interestingly, C10.36 binding to MEC-1 and SU-DHL-1 cells was readily observed, whereas binding to a normal B cell derivative, SKW6.4, was greatly reduced. Although hnRNP U was detected on the surface of all four cell lines by cell surface protein labeling, affinity purification of hnRNP U by C10.36 was only observed in the fully transformed cell lines and not in SKW6.4 cells. This observation indicates that C10.36 binding may be determined by conformation, context or post-translational modification. Treatment with C10.36 led to a loss of viability and IC50 values for sensitive cell lines ranged from 100 to 400 nM. Importantly, a single point mutation in C10.36 (G24A) abrogated both binding and anti-proliferative activity. Conclusion: The present study identifies cell surface hnRNP U and its ribonucleoprotein interaction partners as potential therapeutic targets for NHL and highlights the potential for the development of C10.36 as a novel anti-B cell lymphoma targeted therapy. Disclosures Tonapi: Caris Life Sciences: Employment. Duncan: Caris Life Sciences: Employment. Vaishali: Caris Life Sciences: Employment. Rosenow: Caris Life Sciences: Employment. Richards: Caris Life Sciences: Employment. Tinder: Caris Life Sciences: Employment. O9Neill: Caris Life Sciences: Employment. Miglarese: Caris Life Sciences: Employment. Spetzler: Caris Life Sciences: Employment. Famulok: Caris Life Sciences: Consultancy. Mayer: Caris Life Sciences: Consultancy.