title: Characterization of activating mutations of NOTCH3 in T-cell acute lymphoblastic leukemia and anti-leukemic activity of NOTCH3 inhibitory antibodies.
abstract: Notch receptors have been implicated as oncogenic drivers in several cancers, the most notable example being NOTCH1 in T-cell acute lymphoblastic leukemia (T-ALL). To characterize the role of activated NOTCH3 in cancer, we generated an antibody that detects the neo-epitope created upon gamma-secretase cleavage of NOTCH3 to release its intracellular domain (ICD3), and sequenced the negative regulatory region (NRR) and PEST (proline, glutamate, serine, threonine) domain coding regions of NOTCH3 in a panel of cell lines. We also characterize NOTCH3 tumor-associated mutations that result in activation of signaling and report new inhibitory antibodies. We determined the structural basis for receptor inhibition by obtaining the first co-crystal structure of a NOTCH3 antibody with the NRR protein and defined two distinct epitopes for NRR antibodies. The antibodies exhibit potent anti-leukemic activity in cell lines and tumor xenografts harboring NOTCH3 activating mutations. Screening of primary T-ALL samples reveals that 2 of 40 tumors examined show active NOTCH3 signaling. We also identified evidence of NOTCH3 activation in 12 of 24 patient-derived orthotopic xenograft models, 2 of which exhibit activation of NOTCH3 without activation of NOTCH1. Our studies provide additional insights into NOTCH3 activation and offer a path forward for identification of cancers that are likely to respond to therapy with NOTCH3 selective inhibitory antibodies.
authors.affiliation: Developmental and Molecular Pathways, Novartis Institutes for Biomedical Research, Cambridge, MA, USA.
authors.forename: P
authors.initials: P
authors.lastname: Bernasconi-Elias
authors.affiliation: Center for Proteomic Chemistry, Novartis Institutes for Biomedical Research, Cambridge, MA, USA.
authors.forename: T
authors.initials: T
authors.lastname: Hu
authors.affiliation: Department of Oncology, Novartis Institutes for Biomedical Research, Cambridge, MA, USA.
authors.forename: D
authors.initials: D
authors.lastname: Jenkins
authors.affiliation: Department of Oncology, Novartis Institutes for Biomedical Research, Cambridge, MA, USA.
authors.forename: B
authors.initials: B
authors.lastname: Firestone
authors.affiliation: Developmental and Molecular Pathways, Novartis Institutes for Biomedical Research, Cambridge, MA, USA.
authors.forename: S
authors.initials: S
authors.lastname: Gans
authors.affiliation: Department of Oncology, Novartis Institutes for Biomedical Research, Cambridge, MA, USA.
authors.forename: E
authors.initials: E
authors.lastname: Kurth
authors.affiliation: Developmental and Molecular Pathways, Novartis Institutes for Biomedical Research, Cambridge, MA, USA.
authors.forename: P
authors.initials: P
authors.lastname: Capodieci
authors.affiliation: Discovery Alliances and Technologies, MorphoSys AG, Martinsried, Germany.
authors.forename: J
authors.initials: J
authors.lastname: Deplazes-Lauber
authors.affiliation: Discovery Alliances and Technologies, MorphoSys AG, Martinsried, Germany.
authors.forename: K
authors.initials: K
authors.lastname: Petropoulos
authors.affiliation: Discovery Alliances and Technologies, MorphoSys AG, Martinsried, Germany.
authors.forename: P
authors.initials: P
authors.lastname: Thiel
authors.affiliation: Discovery Alliances and Technologies, MorphoSys AG, Martinsried, Germany.
authors.forename: D
authors.initials: D
authors.lastname: Ponsel
authors.affiliation: Department of Cancer Biology, Dana Farber Cancer Institute, Boston, MA, USA.|Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA.
authors.forename: S
authors.initials: S
authors.lastname: Hee Choi
authors.affiliation: Department of Biologics, Novartis Institutes for Biomedical Research, Cambridge, MA, USA.
authors.forename: P
authors.initials: P
authors.lastname: LeMotte
authors.affiliation: Department of Biologics, Novartis Institutes for Biomedical Research, Cambridge, MA, USA.
authors.forename: A
authors.initials: A
authors.lastname: London
authors.affiliation: Developmental and Molecular Pathways, Novartis Institutes for Biomedical Research, Cambridge, MA, USA.
authors.forename: M
authors.initials: M
authors.lastname: Goetcshkes
authors.affiliation: Developmental and Molecular Pathways, Novartis Institutes for Biomedical Research, Cambridge, MA, USA.
authors.forename: E
authors.initials: E
authors.lastname: Nolin
authors.affiliation: Department of Oncology, Novartis Institutes for Biomedical Research, Cambridge, MA, USA.
authors.forename: M D
authors.initials: MD
authors.lastname: Jones
authors.affiliation: Department of Oncology, Novartis Institutes for Biomedical Research, Cambridge, MA, USA.
authors.forename: K
authors.initials: K
authors.lastname: Slocum
authors.affiliation: Department of Pathology, Brigham and Women's Hospital, Boston, MA, USA.
authors.forename: M J
authors.initials: MJ
authors.lastname: Kluk
authors.affiliation: Department of Cancer Biology, Dana Farber Cancer Institute, Boston, MA, USA.
authors.forename: D M
authors.initials: DM
authors.lastname: Weinstock
authors.affiliation: Department of Cancer Biology, Dana Farber Cancer Institute, Boston, MA, USA.
authors.forename: A
authors.initials: A
authors.lastname: Christodoulou
authors.affiliation: Pathology Children Hospital Boston, Boston, MA, USA.
authors.forename: O
authors.initials: O
authors.lastname: Weinberg
authors.affiliation: Discovery Alliances and Technologies, MorphoSys AG, Martinsried, Germany.
authors.forename: J
authors.initials: J
authors.lastname: Jaehrling
authors.affiliation: Department of Oncology, Novartis Institutes for Biomedical Research, Cambridge, MA, USA.
authors.forename: S A
authors.initials: SA
authors.lastname: Ettenberg
authors.affiliation: Developmental and Molecular Pathways, Novartis Institutes for Biomedical Research, Cambridge, MA, USA.
authors.forename: A
authors.initials: A
authors.lastname: Buckler
authors.affiliation: Department of Cancer Biology, Dana Farber Cancer Institute, Boston, MA, USA.|Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA.|Department of Pathology, Brigham and Women's Hospital, Boston, MA, USA.
authors.forename: S C
authors.initials: SC
authors.lastname: Blacklow
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA.|Department of Pathology, Brigham and Women's Hospital, Boston, MA, USA.
authors.forename: J C
authors.initials: JC
authors.lastname: Aster
authors.affiliation: Developmental and Molecular Pathways, Novartis Institutes for Biomedical Research, Cambridge, MA, USA.
authors.forename: C J
authors.initials: CJ
authors.lastname: Fryer
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D045744:Cell Line, Tumor; D003062:Codon; D004195:Disease Models, Animal; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D005260:Female; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D009154:Mutation; D054218:Precursor T-Cell Lymphoblastic Leukemia-Lymphoma / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D011487:Protein Conformation; D000071656:Receptor, Notch3 / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D023041:Xenograft Model Antitumor Assays
chemical_list: D000911:Antibodies, Monoclonal; D000970:Antineoplastic Agents; D003062:Codon; D000939:Epitopes; D000071656:Receptor, Notch3