title: Combined PDK1 and CHK1 inhibition is required to kill glioblastoma stem-like cells in vitro and in vivo.
abstract: Glioblastoma (GBM) is the most common and deadly adult brain tumor. Despite aggressive surgery, radiation, and chemotherapy, the life expectancy of patients diagnosed with GBM is ∼14 months. The extremely aggressive nature of GBM results from glioblastoma stem-like cells (GSCs) that sustain GBM growth, survive intensive chemotherapy, and give rise to tumor recurrence. There is accumulating evidence revealing that GSC resilience is because of concomitant activation of multiple survival pathways. In order to decode the signal transduction networks responsible for the malignant properties of GSCs, we analyzed a collection of GSC lines using a dual, but complementary, experimental approach, that is, reverse-phase protein microarrays (RPPMs) and kinase inhibitor library screening. We treated GSCs in vitro with clinically relevant concentrations of temozolomide (TMZ) and performed RPPM to detect changes in phosphorylation patterns that could be associated with resistance. In addition, we screened GSCs in vitro with a library of protein and lipid kinase inhibitors to identify specific targets involved in GSC survival and proliferation. We show that GSCs are relatively insensitive to TMZ treatment in terms of pathway activation and, although displaying heterogeneous individual phospho-proteomic profiles, most GSCs are resistant to specific inhibition of the major signaling pathways involved in cell survival and proliferation. However, simultaneous multipathway inhibition by the staurosporin derivative UCN-01 results in remarkable inhibition of GSC growth in vitro. The activity of UCN-01 on GSCs was confirmed in two in vivo models of GBM growth. Finally, we used RPPM to study the molecular and functional effects of UCN-01 and demonstrated that the sensitivity to UCN-01 correlates with activation of survival signals mediated by PDK1 and the DNA damage response initiated by CHK1. Taken together, our results suggest that a combined inhibition of PDK1 and CHK1 represents a potentially effective therapeutic approach to reduce the growth of human GBM.
authors.affiliation: Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Signore
authors.affiliation: Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy.
authors.forename: F
authors.initials: F
authors.lastname: Pelacchi
authors.affiliation: Italian National Cancer Institute 'Regina Elena', Rome, Italy.
authors.forename: S
authors.initials: S
authors.lastname: di Martino
authors.affiliation: Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy.
authors.forename: D
authors.initials: D
authors.lastname: Runci
authors.affiliation: Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Biffoni
authors.affiliation: Institute of Human Anatomy, Università Cattolica del Sacro Cuore, Rome, Italy.
authors.forename: S
authors.initials: S
authors.lastname: Giannetti
authors.affiliation: Institute of Human Anatomy, Università Cattolica del Sacro Cuore, Rome, Italy.
authors.forename: L
authors.initials: L
authors.lastname: Morgante
authors.affiliation: Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy.
authors.forename: M
authors.initials: M
authors.lastname: De Majo
authors.affiliation: Center for Applied Proteomics and Molecular Medicine, George Mason University, Manassas, VA, USA.
authors.forename: E F
authors.initials: EF
authors.lastname: Petricoin
authors.affiliation: Cancer Biology and Patient Tailoring, Discovery Oncology, Eli Lilly and Company, Indianapolis, IN, USA.
authors.forename: L
authors.initials: L
authors.lastname: Stancato
authors.affiliation: Institute of Pathology, Università Cattolica del Sacro Cuore, Rome, Italy.
authors.forename: L M
authors.initials: LM
authors.lastname: Larocca
authors.affiliation: Italian National Cancer Institute 'Regina Elena', Rome, Italy.
authors.forename: R
authors.initials: R
authors.lastname: De Maria
authors.affiliation: Institute of Neurosurgery, Università Cattolica del Sacro Cuore, Rome, Italy.
authors.forename: R
authors.initials: R
authors.lastname: Pallini
authors.affiliation: Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy.
authors.forename: L
authors.initials: L
authors.lastname: Ricci-Vitiani
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001932:Brain Neoplasms / Q000188:drug therapy* / Q000201:enzymology / Q000473:pathology; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D000071877:Checkpoint Kinase 1; D003606:Dacarbazine / Q000031:analogs & derivatives / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D019008:Drug Resistance, Neoplasm; D005909:Glioblastoma / Q000188:drug therapy* / Q000201:enzymology / Q000473:pathology; D006801:Humans; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D058990:Molecular Targeted Therapy; D014411:Neoplastic Stem Cells / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D040081:Protein Array Analysis; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D015398:Signal Transduction / Q000187:drug effects; D054852:Small Molecule Libraries; D019311:Staurosporine / Q000031:analogs & derivatives / Q000494:pharmacology; D000077204:Temozolomide; D013997:Time Factors; D047368:Tumor Burden / Q000187:drug effects; D014407:Tumor Cells, Cultured; D023041:Xenograft Model Antitumor Assays
chemical_list: D000970:Antineoplastic Agents; C000631429:PDK1 protein, human; C000631433:Pdk1 protein, mouse; D047428:Protein Kinase Inhibitors; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D054852:Small Molecule Libraries; C054852:7-hydroxystaurosporine; D003606:Dacarbazine; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; C000605869:Chek1 protein, mouse; D017346:Protein Serine-Threonine Kinases; D019311:Staurosporine; D000077204:Temozolomide