Therapeutic Targeting of KDM1A/LSD1 in Ewing Sarcoma with SP-2509 Engages the Endoplasmic Reticulum Stress Response

Multi-agent chemotherapeutic regimes remain the cornerstone treatment for Ewing sarcoma, the second most common bone malignancy diagnosed in pediatric and young adolescent populations. We have reached a therapeutic ceiling with conventional cytotoxic agents, highlighting the need to adopt novel approaches that specifically target the drivers of Ewing sarcoma oncogenesis. As KDM1A/lysine-specific demethylase 1 (LSD1) is highly expressed in Ewing sarcoma cell lines and tumors, with elevated expression levels associated with worse overall survival (P = 0.033), this study has examined biomarkers of sensitivity and mechanisms of cytotoxicity to targeted KDM1A inhibition using SP-2509 (reversible KDM1A inhibitor). We report, that innate resistance to SP-2509 was not observed in our Ewing sarcoma cell line cohort (n = 17; IC50 range, 81 –1,593 nmol/L), in contrast resistance to the next-generation KDM1A irreversible inhibitor GSK-LSD1 was observed across multiple cell lines (IC50 > 300 μmol/L). Although TP53/STAG2/CDKN2A status and basal KDM1A mRNA and protein levels did not correlate with SP-2509 response, induction of KDM1B following SP-2509 treatment was strongly associated with SP-2509 hypersensitivity. We show that the transcriptional profile driven by SP-2509 strongly mirrors KDM1A genetic depletion. Mechanistically, RNA-seq analysis revealed that SP-2509 imparts robust apoptosis through engagement of the endoplasmic reticulum stress pathway. In addition, ETS1/HIST1H2BM were specifically induced/repressed, respectively following SP-2509 treatment only in our hypersensitive cell lines. Together, our findings provide key insights into the mechanisms of SP-2509 cytotoxicity as well as biomarkers that can be used to predict KDM1A inhibitor sensitivity in Ewing sarcoma. Mol Cancer Ther; 17(9); 1902–16. ©2018 AACR.

[1]  Sunil Sharma,et al.  Reversible LSD1 inhibition with HCI-2509 induces the p53 gene expression signature and disrupts the MYCN signature in high-risk neuroblastoma cells , 2018, Oncotarget.

[2]  G. Armstrong,et al.  Longitudinal follow‐up of adult survivors of Ewing sarcoma: A report from the Childhood Cancer Survivor Study , 2017, Cancer.

[3]  M. Schleicher,et al.  Inactivation of Lsd1 triggers senescence in trophoblast stem cells by induction of Sirt4 , 2017, Cell Death and Disease.

[4]  S. Lessnick,et al.  Recent advances in targeted therapy for Ewing sarcoma , 2016, F1000Research.

[5]  S. Gupta,et al.  Reversible lysine-specific demethylase 1 antagonist HCI-2509 inhibits growth and decreases c-MYC in castration- and docetaxel-resistant prostate cancer cells , 2016, Prostate Cancer and Prostatic Diseases.

[6]  Matteo Benelli,et al.  Divergent clonal evolution of castration resistant neuroendocrine prostate cancer , 2016, Nature Medicine.

[7]  S. Lessnick,et al.  Therapeutic opportunities in Ewing sarcoma: EWS-FLI inhibition via LSD1 targeting , 2016, Oncotarget.

[8]  M. Nakao,et al.  Lysine‐specific demethylase‐1 contributes to malignant behavior by regulation of invasive activity and metabolic shift in esophageal cancer , 2016, International journal of cancer.

[9]  B. Vollmer,et al.  The lysine demethylase LSD1 is required for nuclear envelope formation at the end of mitosis , 2015, Journal of Cell Science.

[10]  M. Pappalardi,et al.  A DNA Hypomethylation Signature Predicts Antitumor Activity of LSD1 Inhibitors in SCLC. , 2015, Cancer cell.

[11]  X. Fang,et al.  Expression of Lysine-specific demethylase 1 in human epithelial ovarian cancer , 2015, Journal of Ovarian Research.

[12]  M. Nakao,et al.  Lysine Demethylase LSD1 Coordinates Glycolytic and Mitochondrial Metabolism in Hepatocellular Carcinoma Cells. , 2015, Cancer research.

[13]  T. Triche,et al.  Gene expression profiling of Ewing sarcoma tumours reveals the prognostic importance of tumour–stromal interactions: a report from the Children's Oncology Group , 2015, The journal of pathology. Clinical research.

[14]  R. Todorova.  Structure-Function Based Molecular Relationships in Ewing's Sarcoma , 2015, BioMed research international.

[15]  Sohrab P. Shah,et al.  Dynamics of genomic clones in breast cancer patient xenografts at single-cell resolution , 2014, Nature.

[16]  Li Ding,et al.  Genomic landscape of Ewing sarcoma defines an aggressive subtype with co-association of STAG2 and TP53 mutations. , 2014, Cancer discovery.

[17]  A. McKenna,et al.  The genomic landscape of pediatric Ewing sarcoma. , 2014, Cancer discovery.

[18]  Jun S. Wei,et al.  The Genomic Landscape of the Ewing Sarcoma Family of Tumors Reveals Recurrent STAG2 Mutation , 2014, PLoS genetics.

[19]  M. Beckerle,et al.  Reversible LSD1 Inhibition Interferes with Global EWS/ETS Transcriptional Activity and Impedes Ewing Sarcoma Tumor Growth , 2014, Clinical Cancer Research.

[20]  S. Oesterreich,et al.  Inhibition of histone demethylase, LSD2 (KDM1B), attenuates DNA methylation and increases sensitivity to DNMT inhibitor-induced apoptosis in breast cancer cells , 2014, Breast Cancer Research and Treatment.

[21]  J. Long,et al.  LSD1 sustains pancreatic cancer growth via maintaining HIF1α-dependent glycolytic process. , 2014, Cancer letters.

[22]  Steven J. M. Jones,et al.  Comprehensive molecular characterization of urothelial bladder carcinoma , 2014, Nature.

[23]  T. Triche,et al.  Characterization and Drug Resistance Patterns of Ewing's Sarcoma Family Tumor Cell Lines , 2013, PloS one.

[24]  D. Bearss,et al.  High-throughput virtual screening identifies novel N'-(1-phenylethylidene)-benzohydrazides as potent, specific, and reversible LSD1 inhibitors. , 2013, Journal of medicinal chemistry.

[25]  Li Lin,et al.  High expression of lysine-specific demethylase 1 correlates with poor prognosis of patients with esophageal squamous cell carcinoma. , 2013, Biochemical and biophysical research communications.

[26]  A. Mai,et al.  The emerging role of histone lysine demethylases in prostate cancer , 2012, Molecular Cancer.

[27]  A. Llombart‐Bosch,et al.  Lysine-specific demethylase 1 (LSD1/KDM1A/AOF2/BHC110) is expressed and is an epigenetic drug target in chondrosarcoma, Ewing's sarcoma, osteosarcoma, and rhabdomyosarcoma. , 2012, Human pathology.

[28]  T. Lv,et al.  Over-Expression of LSD1 Promotes Proliferation, Migration and Invasion in Non-Small Cell Lung Cancer , 2012, PloS one.

[29]  Arul M Chinnaiyan,et al.  PARP-1 inhibition as a targeted strategy to treat Ewing's sarcoma. , 2012, Cancer research.

[30]  F. Clavel-Chapelon,et al.  Common variants near TARDBP and EGR2 are associated with susceptibility to Ewing sarcoma , 2012, Nature Genetics.

[31]  P. Hiemstra,et al.  A quantitative method for detection of spliced X-box binding protein-1 (XBP1) mRNA as a measure of endoplasmic reticulum (ER) stress , 2011, Cell Stress and Chaperones.

[32]  K. Robertson,et al.  Epigenetic mechanisms and genome stability , 2010, Clinical Epigenetics.

[33]  F. Lan,et al.  Human LSD2/KDM1b/AOF1 regulates gene transcription by modulating intragenic H3K4me2 methylation. , 2010, Molecular cell.

[34]  Xingzhi Xu,et al.  LSD1 is required for chromosome segregation during mitosis. , 2010, European journal of cell biology.

[35]  F. Forneris,et al.  Alternative Splicing of the Histone Demethylase LSD1/KDM1 Contributes to the Modulation of Neurite Morphogenesis in the Mammalian Nervous System , 2010, The Journal of Neuroscience.

[36]  F. Forneris,et al.  A Novel Mammalian Flavin-dependent Histone Demethylase* , 2009, The Journal of Biological Chemistry.

[37]  R. Versteeg,et al.  Lysine-specific demethylase 1 is strongly expressed in poorly differentiated neuroblastoma: implications for therapy. , 2009, Cancer research.

[38]  R. Bhargava,et al.  Identification of biomarkers associated with metastastic breast cancer to bone by immunohistochemistry on tissue microarrays. , 2009 .

[39]  J. Cigudosa,et al.  Array CGH and gene-expression profiling reveals distinct genomic instability patterns associated with DNA repair and cell-cycle checkpoint pathways in Ewing's sarcoma , 2008, Oncogene.

[40]  U. Dirksen,et al.  Microarray analysis of Ewing's sarcoma family of tumours reveals characteristic gene expression signatures associated with metastasis and resistance to chemotherapy. , 2008, European journal of cancer.

[41]  Rabindra Roy,et al.  Magnesium, Essential for Base Excision Repair Enzymes, Inhibits Substrate Binding of N-Methylpurine-DNA Glycosylase* , 2006, Journal of Biological Chemistry.

[42]  Afshin Samali,et al.  Mediators of endoplasmic reticulum stress‐induced apoptosis , 2006, EMBO reports.

[43]  Yang Shi,et al.  Histone Demethylation Mediated by the Nuclear Amine Oxidase Homolog LSD1 , 2004, Cell.

[44]  R. Ophir,et al.  Prediction of high risk Ewing's sarcoma by gene expression profiling , 2004, Oncogene.

[45]  S. Oyadomari,et al.  Roles of CHOP/GADD153 in endoplasmic reticulum stress , 2004, Cell Death and Differentiation.

[46]  P. Sorensen,et al.  A second Ewing's sarcoma translocation, t(21;22), fuses the EWS gene to another ETS–family transcription factor, ERG , 1994, Nature Genetics.

[47]  G. Thomas,et al.  Gene fusion with an ETS DNA-binding domain caused by chromosome translocation in human tumours , 1992, Nature.

[48]  B. Price,et al.  Brefeldin A, thapsigargin, and AlF  4− stimulate the accumulation of GRP78 mRNA in a cycloheximide dependent manner, whilst induction by hypoxia is independent of protein synthesis , 1992, Journal of cellular physiology.

[49]  Steven A. Roberts,et al.  Mutational heterogeneity in cancer and the search for new cancer genes , 2014 .

[50]  Trevor J Pugh,et al.  Mutational heterogeneity in cancer and the search for new cancer genes , 2014 .

[51]  Sanjeev Gupta,et al.  Assays for detecting the unfolded protein response. , 2011, Methods in enzymology.