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

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

Author information

1
Developmental and Molecular Pathways, Novartis Institutes for Biomedical Research, Cambridge, MA, USA.
2
Center for Proteomic Chemistry, Novartis Institutes for Biomedical Research, Cambridge, MA, USA.
3
Department of Oncology, Novartis Institutes for Biomedical Research, Cambridge, MA, USA.
4
Discovery Alliances and Technologies, MorphoSys AG, Martinsried, Germany.
5
Department of Cancer Biology, Dana Farber Cancer Institute, Boston, MA, USA.
6
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA.
7
Department of Biologics, Novartis Institutes for Biomedical Research, Cambridge, MA, USA.
8
Department of Pathology, Brigham and Women's Hospital, Boston, MA, USA.
9
Pathology Children Hospital Boston, Boston, MA, USA.

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.

PMID:
27157619
PMCID:
PMC5102827
DOI:
10.1038/onc.2016.133
[Indexed for MEDLINE]
Free PMC Article

Conflict of interest statement

of Potential Conflicts of Interest: P. Bernasconi-Elias, T. Hu, B. Firestone, S. Gans, E. Kurth, P. Capodieci, P. LeMotte, A. London, E. Nolin, M. Jones, K. Slocum, are employees of Novartis Institutes for Biomedical Research. J. Deplazes-Lauber, K. Petropoulos, J. Jaehrling are employees of MorphoSys AG. S. Blacklow serves as a consultants for Novartis and receives research support through the Dana Farber - Novartis DDP. No other authors declare any conflicts of interest.

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