title: Deciphering mechanisms of drug sensitivity and resistance to Selective Inhibitor of Nuclear Export (SINE) compounds.
abstract: BACKGROUND
  Exportin 1 (XPO1) is a well-characterized nuclear export protein whose expression is up-regulated in many types of cancers and functions to transport key tumor suppressor proteins (TSPs) from the nucleus. Karyopharm Therapeutics has developed a series of small-molecule Selective Inhibitor of Nuclear Export (SINE) compounds, which have been shown to block XPO1 function both in vitro and in vivo. The drug candidate, selinexor (KPT-330), is currently in Phase-II/IIb clinical trials for treatment of both hematologic and solid tumors. The present study sought to decipher the mechanisms that render cells either sensitive or resistant to treatment with SINE compounds, represented by KPT-185, an early analogue of KPT-330.
  
  
  METHODS
  Using the human fibrosarcoma HT1080 cell line, resistance to SINE was acquired over a period of 10 months of constant incubation with increasing concentration of KPT-185. Cell viability was assayed by MTT. Immunofluorescence was used to compare nuclear export of TSPs. Fluorescence activated cell sorting (FACS), quantitative polymerase chain reaction (qPCR), and immunoblots were used to measure effects on cell cycle, gene expression, and cell death. RNA from naïve and drug treated parental and resistant cells was analyzed by Affymetrix microarrays.
  
  
  RESULTS
  Treatment of HT1080 cells with gradually increasing concentrations of SINE resulted in >100 fold decrease in sensitivity to SINE cytotoxicity. Resistant cells displayed prolonged cell cycle, reduced nuclear accumulation of TSPs, and similar changes in protein expression compared to parental cells, however the magnitude of the protein expression changes were more significant in parental cells. Microarray analyses comparing parental to resistant cells indicate that a number of key signaling pathways were altered in resistant cells including expression changes in genes involved in adhesion, apoptosis, and inflammation. While the patterns of changes in transcription following drug treatment are similar in parental and resistant cells, the extent of response was more robust in the parental cells.
  
  
  CONCLUSIONS
  These results suggest that SINE resistance is conferred by alterations in signaling pathways downstream of XPO1 inhibition. Modulation of these pathways could potentially overcome the resistance to nuclear export inhibitors.
authors.affiliation: Karyopharm Therapeutics Inc., 85 Wells Avenue, Newton, MA 02459, USA. marsha@karyopharm.com.
authors.forename: Marsha
authors.initials: M
authors.lastname: Crochiere
authors.affiliation: Karyopharm Therapeutics Inc., 85 Wells Avenue, Newton, MA 02459, USA. trinayan@karyopharm.com.
authors.forename: Trinayan
authors.initials: T
authors.lastname: Kashyap
authors.affiliation: Karyopharm Therapeutics Inc., 85 Wells Avenue, Newton, MA 02459, USA. orikalid@gmail.com.
authors.forename: Ori
authors.initials: O
authors.lastname: Kalid
authors.affiliation: Karyopharm Therapeutics Inc., 85 Wells Avenue, Newton, MA 02459, USA. sshechte@gmail.com.
authors.forename: Sharon
authors.initials: S
authors.lastname: Shechter
authors.affiliation: Karyopharm Therapeutics Inc., 85 Wells Avenue, Newton, MA 02459, USA. boris@karyopharm.com.
authors.forename: Boris
authors.initials: B
authors.lastname: Klebanov
authors.affiliation: Karyopharm Therapeutics Inc., 85 Wells Avenue, Newton, MA 02459, USA. william@karyopharm.com.
authors.forename: William
authors.initials: W
authors.lastname: Senapedis
authors.affiliation: Karyopharm Therapeutics Inc., 85 Wells Avenue, Newton, MA 02459, USA. jean@karyopharm.com.
authors.forename: Jean-Richard
authors.initials: JR
authors.lastname: Saint-Martin
authors.affiliation: Karyopharm Therapeutics Inc., 85 Wells Avenue, Newton, MA 02459, USA. yosef@karyopharm.com.
authors.forename: Yosef
authors.initials: Y
authors.lastname: Landesman
mesh_terms: D000179:Acrylates / Q000494:pharmacology*; D021581:Active Transport, Cell Nucleus / Q000187:drug effects*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D002453:Cell Cycle / Q000187:drug effects; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D019008:Drug Resistance, Neoplasm / Q000187:drug effects*; D005354:Fibrosarcoma / Q000188:drug therapy; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D028884:Karyopherins / Q000037:antagonists & inhibitors*; D018160:Receptors, Cytoplasmic and Nuclear / Q000037:antagonists & inhibitors*; D014230:Triazoles / Q000494:pharmacology*; D000097606:Exportin 1 Protein
chemical_list: D000179:Acrylates; D000970:Antineoplastic Agents; D028884:Karyopherins; D018160:Receptors, Cytoplasmic and Nuclear; D014230:Triazoles; D000097606:Exportin 1 Protein; C576639:KPT-185