Potential effect of spliceosome inhibition in small cell lung cancer irrespective of the MYC status

RESEARCH ARTICLE Potential effect of spliceosome inhibition in small cell lung cancer irrespective of the MYC status Kenichi Suda1,2, Leslie Rozeboom1, Hui Yu1, Kim Ellison1, Christopher J. Rivard1, Tetsuya Mitsudomi2, Fred R. Hirsch1* 1 Division of Medical Oncology, University of Colorado Anschutz Medical Campus, Aurora, Colorado, United States of America, 2 Division of Thoracic Surgery, Department of Surgery, Kindai University Faculty of Medicine, Osaka-Sayama, Japan a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Suda K, Rozeboom L, Yu H, Ellison K, Rivard CJ, Mitsudomi T, et al. (2017) Potential effect of spliceosome inhibition in small cell lung cancer irrespective of the MYC status. PLoS ONE 12(2): e0172209. doi:10.1371/journal. pone.0172209 Editor: Aamir Ahmad, University of South Alabama Mitchell Cancer Institute, UNITED STATES Received: January 2, 2017 Accepted: February 1, 2017 Published: February 13, 2017 Copyright: © 2017 Suda et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. * Abstract Small cell lung cancer (SCLC) is a highly aggressive malignancy with few therapeutic advances in the treatment in recent decades. Based on a recent study that identified the spliceosome as a therapeutic vulnerability in MYC-driven breast cancers, we evaluated the efficacy of a spliceosome inhibitor in SCLC cell lines and analyzed the correlation with MYC status. Among 23 SCLC cell lines examined, eight showed high MYC protein expression (> 80% positive cells) by immunohistochemistry (IHC), while 10 cell lines demonstrated no staining for MYC. The remaining five cell lines showed weak staining (< 40% positive cells). All four cell lines that were previously demonstrated to have MYC gene amplification were positive for MYC by IHC. Four cell lines with high MYC expression and four with low MYC expression were used in further analysis. A spliceosome inhibitor, pladienolide B, showed high efficacy (IC50 < 12nM) in all eight cell lines tested, irrespective of the MYC IHC or MYC gene amplification status. We observed that the four cell lines with higher sensitivity to the spliceosome inhibitor were established from patients with prior chemotherapy. Therefore we chronically treated H1048 cells, that were established from a treatment-naïve patient, with cisplatin for 4 weeks, and found that H1048-cisplatin treated cells became more sensitive to pladienolide B. In conclusion, our in vitro results indicate that spliceosome inhibitors would be promising molecular target drugs in SCLC irrespective of the MYC status, especially in the second-line settings after an effective front-line chemotherapy. Data Availability Statement: All relevant data are within the paper. Introduction Funding: The present study is partly supported by International Association for the Study of Lung Cancer (IASLC) Young Investigator Award to K. Suda (2015-2017), https://www.iaslc.org/ fellowship. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. There was no additional external funding received for this study. Small cell lung cancer (SCLC) accounts for about 15–20% of lung cancer diagnoses, and is one of the most aggressive type of cancer with high mortality. Despite a recent development of molecular targeted therapy in non-small cell lung cancers [1], there have been few therapeutic advances in the treatment of SCLC in recent decades. The widely known genetic alterations in SCLC are inactivating mutation/deletion of TP53 and RB1 and amplification of MYC family genes [2–5]. Amplification of one of the MYC family genes, MYC (also known as MYCC), MYCL, or MYCN in a mutually exclusive manner, was PLOS ONE | DOI:10.1371/journal.pone.0172209 February 13, 2017 1/9 Spliceosome inhibition in small cell lung cancer Competing interests: The authors have declared that no competing interests exist. recognized in SCLC two decades ago [5]. Oncogenic MYC drives the expression of a broad number of genes with diverse functions, resulting in an increase in cell biomass [6]. However, to date, direct inhibition of the oncogenic transcriptional activity of MYC has been challenging to achieve [7]. A recent study found that the spliceosome, a dynamic macromolecular ribonucleoprotein (RNP) complex that catalyses the splicing of nuclear pre-mRNA into mRNA, is a therapeutic vulnerability in breast cancer models driven by MYC, due to MYC-induced elevation of mRNA synthesis [8]. To evaluate if this finding also applies to SCLC with MYC activation, we performed the current study to analyze the effect of a spliceosome inhibitor in SCLC cell lines with/without MYC gene amplification or MYC overexpression. Materials and methods SCLC cell lines and reagents A total of 23 human SCLC cell lines were used in this study. All cell lines were in our archive or a kind gift from our collaborators. The short tandem repeat profiles of all cell lines used have been confirmed. All cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum and 1x penicillin / streptomycin solution (Mediatech, Inc., Manassas, VA). Cells were grown at 37˚C with 5% CO2 in a cell culture incubator. A spliceosome inhibitor, Pladienolide B, was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX). Cisplatin was purchased from Selleck Chemicals (Houston, TX). TMA preparation, antibodies and Immunohistochemistry (IHC) analysis Formalin-fixed paraffin-embedded (FFPE) cell blocks were prepared to make a cell line tissue microarray (TMA). The TMA was sectioned at a thickness of 4 μm, and mounted on charged glass slides. MYC IHC staining was performed on a Ventana Discovery Ultra autostainer employing a c-MYC rabbit monoclonal antibody (clone Y69, Ventana). MYC staining was assessed by the study pathologist (H.Y.) using the H-score assessment which combines staining intensity (0–3) and the percentage of positive cells (0–100%). Cell proliferation assay Cell proliferation was measured using the PrestoBlue Cell Viability Reagent (Life Technologies, Frederick, MD), according to the manufacturer’s instructions. Briefly, tumor cells (3 x 103) were plated into each well of 96-well flat-bottomed plates and cultured for 24 hours. Pladienolide B, cisplatin, or dimethyl sulfoxide (DMSO) was added to the indicated drug concentration, and cells were incubated for an additional 72 hours. A colorimetric activity assay was performed by addition of the PrestoBlue reagent to each well and the plates incubated at 37˚C followed by fluorescence detection (560nm Ex / 590nm Em) using a Biotek Synergy II plate reader. Percent growth was calculated relative to DMSO-treated controls. Statistical differences between growth curves was assessed using the ‘compareGrowthCur (...truncated)