Characterization of activating mutations of NOTCH3 in T-cell acute lymphoblastic leukemia and anti-leukemic activity of NOTCH3 inhibitory antibodies

Oncogene. 2016 Nov 24;35(47):6077-6086. doi: 10.1038/onc.2016.133. Epub 2016 May 9.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Substitution
  • Animals
  • Antibodies, Monoclonal / pharmacology*
  • Antineoplastic Agents / pharmacology*
  • Cell Line, Tumor
  • Codon
  • Disease Models, Animal
  • Epitopes / chemistry
  • Epitopes / immunology
  • Female
  • Humans
  • Mice
  • Models, Molecular
  • Mutation*
  • Precursor T-Cell Lymphoblastic Leukemia-Lymphoma / drug therapy
  • Precursor T-Cell Lymphoblastic Leukemia-Lymphoma / genetics*
  • Precursor T-Cell Lymphoblastic Leukemia-Lymphoma / metabolism
  • Precursor T-Cell Lymphoblastic Leukemia-Lymphoma / pathology
  • Protein Conformation
  • Receptor, Notch3 / antagonists & inhibitors*
  • Receptor, Notch3 / chemistry
  • Receptor, Notch3 / genetics*
  • Receptor, Notch3 / metabolism
  • Signal Transduction / drug effects
  • Xenograft Model Antitumor Assays

Substances

  • Antibodies, Monoclonal
  • Antineoplastic Agents
  • Codon
  • Epitopes
  • Receptor, Notch3