title: PI3K/AKT/mTOR inhibition in combination with doxorubicin is an effective therapy for leiomyosarcoma.
abstract: BACKGROUND
  Leiomyosarcoma (LMS) is a common type of soft tissue sarcoma that responds poorly to standard chemotherapy. Thus the goal of this study was to identify novel selective therapies that may be effective in leiomyosarcoma by screening cell lines with a small molecule library comprised of 480 kinase inhibitors to functionally determine which signalling pathways may be critical for LMS growth.
  
  
  METHODS
  LMS cell lines were screened with the OICR kinase library and a cell viability assay was used to identify potentially effective compounds. The top 10 % of hits underwent secondary validation to determine their EC50 and immunoblots were performed to confirm selective drug action. The efficacy of combination drug therapy with doxorubicin (Dox) in vitro was analyzed using the Calcusyn program after treatment with one of three dosing schedules: concurrent treatment, initial treatment with a selective compound followed by Dox, or initial treatment with Dox followed by the selective compound. Single and combination drug therapy were then validated in vivo using LMS xenografts.
  
  
  RESULTS
  Compounds that targeted PI3K/AKT/mTOR pathways (52 %) were most effective. EC50s were determined to validate these initial hits, and of the 11 confirmed hits, 10 targeted PI3K and/or mTOR pathways with EC50 values <1 μM. We therefore examined if BEZ235 and BKM120, two selective compounds in these pathways, would inhibit leiomyosarcoma growth in vitro. Immunoblots confirmed on-target effects of these compounds in the PI3K and/or mTOR pathways. We next investigated if there was synergy with these agents and first line chemotherapy doxorubicin (Dox), which would allow for earlier introduction into patient care. Only combined treatment of BEZ235 and Dox was synergistic in vitro. To validate these findings in pre-clinical models, leiomyosarcoma xenografts were treated with single agent and combination therapy. BEZ235 treated xenografts (n = 8) demonstrated a decrease in tumor volume of 42 % whereas combining BEZ235 with Dox (n = 8) decreased tumor volume 68 % compared to vehicle alone.
  
  
  CONCLUSIONS
  In summary, this study supports further investigation into the use of PI3K and mTOR inhibitors alone and in combination with standard treatment in leiomyosarcoma patients.
authors.affiliation: Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, M5G 1X5, Canada. babichev@lunenfeld.ca.
authors.forename: Yael
authors.initials: Y
authors.lastname: Babichev
authors.affiliation: Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, M5G 1X5, Canada. kabaroff@lunenfeld.ca.
authors.forename: Leah
authors.initials: L
authors.lastname: Kabaroff
authors.affiliation: Sinai-McLaughlin Assay and Robotic Technologies Facility, Lunenfeld-Tanenbaum Research Institute, Toronto, M5G 1X5, Canada. datti@lunenfeld.ca.|Department of Agricultural, Food, and Environmental Sciences, University of Perugia, 06121, Perugia, Italy. datti@lunenfeld.ca.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Datti
authors.affiliation: Drug Discovery Group, Ontario Institute for Cancer Research, Toronto, M5G 0A3, Canada. David.Uehling@oicr.on.ca.
authors.forename: David
authors.initials: D
authors.lastname: Uehling
authors.affiliation: Drug Discovery Group, Ontario Institute for Cancer Research, Toronto, M5G 0A3, Canada. Methvin.Isaac@oicr.on.ca.
authors.forename: Methvin
authors.initials: M
authors.lastname: Isaac
authors.affiliation: Drug Discovery Group, Ontario Institute for Cancer Research, Toronto, M5G 0A3, Canada. Rima.AlAwar@oicr.on.ca.|Department of Pharmacology and Toxicology, University of Toronto, Toronto, M5S 1A8, Canada. Rima.AlAwar@oicr.on.ca.
authors.forename: Rima
authors.initials: R
authors.lastname: Al-Awar
authors.affiliation: Drug Discovery Group, Ontario Institute for Cancer Research, Toronto, M5G 0A3, Canada. Michael.Prakesch@oicr.on.ca.
authors.forename: Michael
authors.initials: M
authors.lastname: Prakesch
authors.affiliation: Department of Pharmacology and Toxicology, University of Toronto, Toronto, M5S 1A8, Canada. Ren.Sun@oicr.on.ca.|Informatics and Biocomputing Program, Ontario Institute for Cancer Research, Toronto, M5G 0A3, ON, Canada. Ren.Sun@oicr.on.ca.
authors.forename: Ren X
authors.initials: RX
authors.lastname: Sun
authors.affiliation: Department of Pharmacology and Toxicology, University of Toronto, Toronto, M5S 1A8, Canada. Paul.Boutros@oicr.on.ca.|Informatics and Biocomputing Program, Ontario Institute for Cancer Research, Toronto, M5G 0A3, ON, Canada. Paul.Boutros@oicr.on.ca.|Department of Medical Biophysics, University of Toronto, Toronto, M5S 1A1, ON, Canada. Paul.Boutros@oicr.on.ca.
authors.forename: Paul C
authors.initials: PC
authors.lastname: Boutros
authors.affiliation: Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, M5G 1X5, Canada. venier@lunenfeld.ca.
authors.forename: Rosemarie
authors.initials: R
authors.lastname: Venier
authors.affiliation: Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, M5G 1X5, ON, Canada. BDickson@mtsinai.on.ca.
authors.forename: Brendan C
authors.initials: BC
authors.lastname: Dickson
authors.affiliation: Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, M5G 1X5, Canada. gladdy@lunenfeld.ca.|Department of Surgery, University of Toronto, Toronto, M5S 1A1, Canada. gladdy@lunenfeld.ca.|Institute of Medical Science, University of Toronto, Toronto, M5S 1A1, Canada. gladdy@lunenfeld.ca.|Cancer Stem Cell Program, Ontario Institute for Cancer Research, Toronto, M5G 0A3, ON, Canada. gladdy@lunenfeld.ca.|Lunenfeld-Tanenbaum Research Institute, 25 Orde Street, Room 5-1015-2, Toronto, ON, M5T 3H7, Canada. gladdy@lunenfeld.ca.
authors.forename: Rebecca A
authors.initials: RA
authors.lastname: Gladdy
mesh_terms: D000631:Aminopyridines / Q000494:pharmacology / Q000627:therapeutic use; D000818:Animals; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology / Q000627:therapeutic use*; D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004317:Doxorubicin / Q000494:pharmacology / Q000627:therapeutic use*; D004353:Drug Evaluation, Preclinical; D004357:Drug Synergism; D005260:Female; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology / Q000627:therapeutic use; D007890:Leiomyosarcoma / Q000188:drug therapy* / Q000473:pathology; D016688:Mice, Inbred NOD; D009025:Morpholines / Q000494:pharmacology / Q000627:therapeutic use; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors / Q000494:pharmacology / Q000627:therapeutic use*; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011804:Quinolines / Q000494:pharmacology / Q000627:therapeutic use; D015203:Reproducibility of Results; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D000631:Aminopyridines; D004317:Doxorubicin; D007093:Imidazoles; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt; D011804:Quinolines; D058570:TOR Serine-Threonine Kinases; C571178:NVP-BKM120; C531198:dactolisib