title: GLI2 inhibition abrogates human leukemia stem cell dormancy.
abstract: BACKGROUND
  Dormant leukemia stem cells (LSC) promote therapeutic resistance and leukemic progression as a result of unbridled activation of stem cell gene expression programs. Thus, we hypothesized that 1) deregulation of the hedgehog (Hh) stem cell self-renewal and cell cycle regulatory pathway would promote dormant human LSC generation and 2) that PF-04449913, a clinical antagonist of the GLI2 transcriptional activator, smoothened (SMO), would enhance dormant human LSC eradication.
  
  
  METHODS
  To test these postulates, whole transcriptome RNA sequencing (RNA-seq), microarray, qRT-PCR, stromal co-culture, confocal fluorescence microscopic, nanoproteomic, serial transplantation and cell cycle analyses were performed on FACS purified normal, chronic phase (CP) chronic myeloid leukemia (CML), blast crisis (BC) phase CML progenitors with or without PF-04449913 treatment.
  
  
  RESULTS
  Notably, RNA-seq analyses revealed that Hh pathway and cell cycle regulatory gene overexpression correlated with leukemic progression. While lentivirally enforced GLI2 expression enhanced leukemic progenitor dormancy in stromal co-cultures, this was not observed with a mutant GLI2 lacking a transactivation domain, suggesting that GLI2 expression prevented cell cycle transit. Selective SMO inhibition with PF-04449913 in humanized stromal co-cultures and LSC xenografts reduced downstream GLI2 protein and cell cycle regulatory gene expression. Moreover, SMO inhibition enhanced cell cycle transit and sensitized BC LSC to tyrosine kinase inhibition in vivo at doses that spare normal HSC.
  
  
  CONCLUSIONS
  In summary, while GLI2, forms part of a core HH pathway transcriptional regulatory network that promotes human myeloid leukemic progression and dormant LSC generation, selective inhibition with PF-04449913 reduces the dormant LSC burden thereby providing a strong rationale for clinical trials predicated on SMO inhibition in combination with TKIs or chemotherapeutic agents with the ultimate aim of obviating leukemic therapeutic resistance, persistence and progression.
authors.affiliation: Department of Medicine, Stem Cell Program and Moores Cancer Center, University of California San Diego, 3855 Health Sciences Drive, La Jolla, 92037, CA, USA. asadarangani@ucsd.edu.|Division of Regenerative Medicine, University of California San Diego, 3855 Health Sciences Drive, La Jolla, CA, 92093-0820, USA. asadarangani@ucsd.edu.
authors.forename: Anil
authors.initials: A
authors.lastname: Sadarangani
authors.affiliation: Department of Medicine, Stem Cell Program and Moores Cancer Center, University of California San Diego, 3855 Health Sciences Drive, La Jolla, 92037, CA, USA. gpineda@ucsd.edu.
authors.forename: Gabriel
authors.initials: G
authors.lastname: Pineda
authors.affiliation: Department of Medicine, Stem Cell Program and Moores Cancer Center, University of California San Diego, 3855 Health Sciences Drive, La Jolla, 92037, CA, USA. klennon@ucsd.edu.
authors.forename: Kathleen M
authors.initials: KM
authors.lastname: Lennon
authors.affiliation: Canada's Michael Smith Genome Sciences Center, British Columbia Cancer Agency, Vancouver, BC, Canada. echun@bcgsc.ca.
authors.forename: Hye-Jung
authors.initials: HJ
authors.lastname: Chun
authors.affiliation: Department of Medicine, Stem Cell Program and Moores Cancer Center, University of California San Diego, 3855 Health Sciences Drive, La Jolla, 92037, CA, USA. ashih@ucsd.edu.
authors.forename: Alice
authors.initials: A
authors.lastname: Shih
authors.affiliation: Department of Medicine, Stem Cell Program and Moores Cancer Center, University of California San Diego, 3855 Health Sciences Drive, La Jolla, 92037, CA, USA. aschairer@ucsd.edu.
authors.forename: Annelie E
authors.initials: AE
authors.lastname: Schairer
authors.affiliation: Department of Medicine, Stem Cell Program and Moores Cancer Center, University of California San Diego, 3855 Health Sciences Drive, La Jolla, 92037, CA, USA. acourtrecart@ucsd.edu.
authors.forename: Angela C
authors.initials: AC
authors.lastname: Court
authors.affiliation: Department of Medicine, Stem Cell Program and Moores Cancer Center, University of California San Diego, 3855 Health Sciences Drive, La Jolla, 92037, CA, USA. dgoff@ucsd.edu.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Goff
authors.affiliation: Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, CA, USA. sprashad@ucla.edu.
authors.forename: Sacha L
authors.initials: SL
authors.lastname: Prashad
authors.affiliation: Department of Medicine, Stem Cell Program and Moores Cancer Center, University of California San Diego, 3855 Health Sciences Drive, La Jolla, 92037, CA, USA. igeron@ucsd.edu.
authors.forename: Ifat
authors.initials: I
authors.lastname: Geron
authors.affiliation: Department of Medicine, Stem Cell Program and Moores Cancer Center, University of California San Diego, 3855 Health Sciences Drive, La Jolla, 92037, CA, USA. rwall@ucsd.edu.
authors.forename: Russell
authors.initials: R
authors.lastname: Wall
authors.affiliation: Ontario Institute for Cancer Research, Toronto, ON, Canada. john.mcpherson@oicr.on.ca.
authors.forename: John D
authors.initials: JD
authors.lastname: McPherson
authors.affiliation: Canada's Michael Smith Genome Sciences Center, British Columbia Cancer Agency, Vancouver, BC, Canada. rmoore@bcgsc.ca.
authors.forename: Richard A
authors.initials: RA
authors.lastname: Moore
authors.affiliation: Department of Medicine, Stem Cell Program and Moores Cancer Center, University of California San Diego, 3855 Health Sciences Drive, La Jolla, 92037, CA, USA. mpu@ucsd.edu.
authors.forename: Minya
authors.initials: M
authors.lastname: Pu
authors.affiliation: Department of Medicine, Stem Cell Program and Moores Cancer Center, University of California San Diego, 3855 Health Sciences Drive, La Jolla, 92037, CA, USA. lebao@AD.UCSD.EDU.
authors.forename: Lei
authors.initials: L
authors.lastname: Bao
authors.affiliation: Pfizer, La Jolla, CA, USA. afisher@pfizer.com.
authors.forename: Amy
authors.initials: A
authors.lastname: Jackson-Fisher
authors.affiliation: Pfizer, La Jolla, CA, USA. mMunchhof@pfizer.com.
authors.forename: Michael
authors.initials: M
authors.lastname: Munchhof
authors.affiliation: Pfizer, La Jolla, CA, USA. tVanArsdale@pfizer.com.
authors.forename: Todd
authors.initials: T
authors.lastname: VanArsdale
authors.affiliation: Department of Medicine, Stem Cell Program and Moores Cancer Center, University of California San Diego, 3855 Health Sciences Drive, La Jolla, 92037, CA, USA. treya@ucsd.edu.
authors.forename: Tannishtha
authors.initials: T
authors.lastname: Reya
authors.affiliation: Department of Medicine, Stem Cell Program and Moores Cancer Center, University of California San Diego, 3855 Health Sciences Drive, La Jolla, 92037, CA, USA. shmorris@ucsd.edu.
authors.forename: Sheldon R
authors.initials: SR
authors.lastname: Morris
authors.affiliation: Department of Medicine, University of Toronto, Toronto, ON, Canada. Mark.Minden@uhn.ca.|Princess Margaret Cancer Center, University Health Network, Toronto, ON, Canada. Mark.Minden@uhn.ca.
authors.forename: Mark D
authors.initials: MD
authors.lastname: Minden
authors.affiliation: Department of Medicine, Stem Cell Program and Moores Cancer Center, University of California San Diego, 3855 Health Sciences Drive, La Jolla, 92037, CA, USA. kmesser@ucsd.edu.
authors.forename: Karen
authors.initials: K
authors.lastname: Messer
authors.affiliation: Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, CA, USA. hmikkola@mcdb.ucla.edu.
authors.forename: Hanna K A
authors.initials: HK
authors.lastname: Mikkola
authors.affiliation: Canada's Michael Smith Genome Sciences Center, British Columbia Cancer Agency, Vancouver, BC, Canada. mmarra@bcgsc.ca.
authors.forename: Marco A
authors.initials: MA
authors.lastname: Marra
authors.affiliation: Ontario Institute for Cancer Research, Toronto, ON, Canada. tom.hudson@oicr.on.ca.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Hudson
authors.affiliation: Department of Medicine, Stem Cell Program and Moores Cancer Center, University of California San Diego, 3855 Health Sciences Drive, La Jolla, 92037, CA, USA. cjamieson@ucsd.edu.|Division of Regenerative Medicine, University of California San Diego, 3855 Health Sciences Drive, La Jolla, CA, 92093-0820, USA. cjamieson@ucsd.edu.
authors.forename: Catriona H M
authors.initials: CH
authors.lastname: Jamieson
mesh_terms: D000818:Animals; D001483:Base Sequence; D018920:Coculture Techniques; D017931:DNA Primers; D005312:Fetal Blood / Q000166:cytology; D053823:Hedgehog Proteins / Q000378:metabolism; D006801:Humans; D051741:Kruppel-Like Transcription Factors / Q000037:antagonists & inhibitors* / Q000235:genetics; D007938:Leukemia / Q000473:pathology*; D051379:Mice; D018345:Mice, Knockout; D014411:Neoplastic Stem Cells / Q000473:pathology*; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D059467:Transcriptome; D000075746:Zinc Finger Protein Gli2
chemical_list: D017931:DNA Primers; C082593:GLI2 protein, human; D053823:Hedgehog Proteins; D051741:Kruppel-Like Transcription Factors; D009687:Nuclear Proteins; D000075746:Zinc Finger Protein Gli2