title: A novel signalling screen demonstrates that CALR mutations activate essential MAPK signalling and facilitate megakaryocyte differentiation.
abstract: Most myeloproliferative neoplasm (MPN) patients lacking JAK2 mutations harbour somatic CALR mutations that are thought to activate cytokine signalling although the mechanism is unclear. To identify kinases important for survival of CALR-mutant cells, we developed a novel strategy (KISMET) that utilizes the full range of kinase selectivity data available from each inhibitor and thus takes advantage of off-target noise that limits conventional small-interfering RNA or inhibitor screens. KISMET successfully identified known essential kinases in haematopoietic and non-haematopoietic cell lines and identified the mitogen activated protein kinase (MAPK) pathway as required for growth of the CALR-mutated MARIMO cells. Expression of mutant CALR in murine or human haematopoietic cell lines was accompanied by myeloproliferative leukemia protein (MPL)-dependent activation of MAPK signalling, and MPN patients with CALR mutations showed increased MAPK activity in CD34 cells, platelets and megakaryocytes. Although CALR mutations resulted in protein instability and proteosomal degradation, mutant CALR was able to enhance megakaryopoiesis and pro-platelet production from human CD34+progenitors. These data link aberrant MAPK activation to the MPN phenotype and identify it as a potential therapeutic target in CALR-mutant positive MPNs.
authors.affiliation: Cambridge Institute for Medical Research and Wellcome Trust/MRC Stem Cell Institute, University of Cambridge, Cambridge, UK.|Department of Haematology, University of Cambridge, Cambridge, UK.
authors.forename: K
authors.initials: K
authors.lastname: Kollmann
authors.affiliation: Cambridge Institute for Medical Research and Wellcome Trust/MRC Stem Cell Institute, University of Cambridge, Cambridge, UK.|Department of Haematology, University of Cambridge, Cambridge, UK.
authors.forename: W
authors.initials: W
authors.lastname: Warsch
authors.affiliation: Cambridge Institute for Medical Research and Wellcome Trust/MRC Stem Cell Institute, University of Cambridge, Cambridge, UK.|Department of Haematology, University of Cambridge, Cambridge, UK.
authors.forename: C
authors.initials: C
authors.lastname: Gonzalez-Arias
authors.affiliation: Cambridge Institute for Medical Research and Wellcome Trust/MRC Stem Cell Institute, University of Cambridge, Cambridge, UK.|Department of Haematology, University of Cambridge, Cambridge, UK.
authors.forename: F L
authors.initials: FL
authors.lastname: Nice
authors.affiliation: Cambridge Institute for Medical Research, Wellcome Trust MRC Institute of Metabolic Science and NIHR Cambridge Biomedical Research Centre, Cambridge, UK.
authors.forename: E
authors.initials: E
authors.lastname: Avezov
authors.affiliation: Cambridge Institute for Medical Research and Wellcome Trust/MRC Stem Cell Institute, University of Cambridge, Cambridge, UK.|Department of Haematology, University of Cambridge, Cambridge, UK.
authors.forename: J
authors.initials: J
authors.lastname: Milburn
authors.affiliation: Cambridge Institute for Medical Research and Wellcome Trust/MRC Stem Cell Institute, University of Cambridge, Cambridge, UK.|Department of Haematology, University of Cambridge, Cambridge, UK.
authors.forename: J
authors.initials: J
authors.lastname: Li
authors.affiliation: Cambridge Institute for Medical Research and Wellcome Trust/MRC Stem Cell Institute, University of Cambridge, Cambridge, UK.|Department of Haematology, University of Cambridge, Cambridge, UK.
authors.forename: D
authors.initials: D
authors.lastname: Dimitropoulou
authors.affiliation: Cambridge Institute for Medical Research and Wellcome Trust/MRC Stem Cell Institute, University of Cambridge, Cambridge, UK.|Department of Haematology, University of Cambridge, Cambridge, UK.
authors.forename: S
authors.initials: S
authors.lastname: Biddie
authors.affiliation: Cambridge Institute for Medical Research and Wellcome Trust/MRC Stem Cell Institute, University of Cambridge, Cambridge, UK.|Department of Haematology, University of Cambridge, Cambridge, UK.
authors.forename: M
authors.initials: M
authors.lastname: Wang
authors.affiliation: Cambridge Institute for Medical Research and Wellcome Trust/MRC Stem Cell Institute, University of Cambridge, Cambridge, UK.|Department of Haematology, University of Cambridge, Cambridge, UK.
authors.forename: E
authors.initials: E
authors.lastname: Poynton
authors.affiliation: Department of Haematology, University of Cambridge, and National Health Service Blood and Transplant, Cambridge Biomedical Campus, Cambridge, UK.
authors.forename: M
authors.initials: M
authors.lastname: Colzani
authors.affiliation: Department of Haematology, University of Cambridge, and National Health Service Blood and Transplant, Cambridge Biomedical Campus, Cambridge, UK.
authors.forename: M R
authors.initials: MR
authors.lastname: Tijssen
authors.affiliation: Cambridge Institute for Medical Research and Wellcome Trust/MRC Stem Cell Institute, University of Cambridge, Cambridge, UK.|Department of Haematology, University of Cambridge, Cambridge, UK.
authors.forename: S
authors.initials: S
authors.lastname: Anand
authors.affiliation: Cancer Genome Project, Wellcome Trust Sanger Institute, Genome Campus, Hinxton, Cambridgeshire, UK.
authors.forename: U
authors.initials: U
authors.lastname: McDermott
authors.affiliation: Cambridge Institute for Medical Research and Wellcome Trust/MRC Stem Cell Institute, University of Cambridge, Cambridge, UK.|Department of Haematology, University of Cambridge, Cambridge, UK.|Cambridge University Hospitals NHS Foundation Trust, Cambridge, UK.
authors.forename: B
authors.initials: B
authors.lastname: Huntly
authors.affiliation: Cambridge Institute for Medical Research and Wellcome Trust/MRC Stem Cell Institute, University of Cambridge, Cambridge, UK.|Department of Haematology, University of Cambridge, Cambridge, UK.|Cambridge University Hospitals NHS Foundation Trust, Cambridge, UK.
authors.forename: T
authors.initials: T
authors.lastname: Green
mesh_terms: D018952:Antigens, CD34 / Q000378:metabolism; D037282:Calreticulin / Q000037:antagonists & inhibitors / Q000235:genetics*; D002454:Cell Differentiation; D002460:Cell Line; D055808:Drug Discovery; D000066630:Ectopic Gene Expression / Q000187:drug effects; D005312:Fetal Blood / Q000166:cytology; D006801:Humans; D053614:Janus Kinase 2 / Q000037:antagonists & inhibitors / Q000235:genetics; D008533:Megakaryocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009154:Mutation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D055550:Protein Stability; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D034061:Thrombopoiesis / Q000235:genetics; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D018952:Antigens, CD34; D037282:Calreticulin; D047428:Protein Kinase Inhibitors; D053614:Janus Kinase 2; D048493:Proto-Oncogene Proteins B-raf; D020928:Mitogen-Activated Protein Kinases; D046988:Proteasome Endopeptidase Complex; D018631:ras Proteins