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

Oncogene volume 35, pages 6077–6086 (2016)Cite this article

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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.

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Acknowledgements

The JAGGED1 and DLL1 expressing cell lines were kindly provide by Dr Gerry Weinmaster (UCLA). HPB-ALL cells were kindly provided by Andreas Stasser (Walter and Eliza Hall Institute for Medical Research, Australia). We thank the Novartis Biologics Center including Thomas Pietzonka, Janine Shulok, Nadine Charara, Bill Tschantz and Tony Fleming. We also thank many senior Novartis leaders for supporting this project, including Jeff Porter and William Sellers. We thank Rajiv Chopra and Kirk Clark of Novartis Institutes for BioMedical Research for the instructive discussion on crystallography experiments. Use of the IMCA-CAT beamline 17-ID at the Advanced Photon Source was supported by the companies of the Industrial Macromolecular Crystallography Association through a contract with Hauptman–Woodward Medical Research Institute. Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-06CH11357. This work was financially supported in part by the Dana Farber–Novartis DDP, and grants from the National Institutes of Health (P01 CA119070) and the Leukemia and Lymphoma Society (7003-13).

Author information

Author notes

  1. D Jenkins

    Present address: 10present address: Tesaro, Waltham, MA, USA.,

  2. P Thiel

    Present address: 11present address: 1stQ Deutschland GmbH & Co KG, Mannheim, Germany.,

  3. D Ponsel

    Present address: 12present address: Roche AG, Penzberg, Germany.,

  4. M Goetcshkes & A Buckler

    Present address: 13present address: Biogen, Cambridge, MA, USA.,

  5. M J Kluk

    Present address: 14present address: Cornell-Weill Medical College, New York, NY, USA.,

  6. S A Ettenberg

    Present address: 15present address: Unum Therapeutics, Cambridge, MA, USA.,

Authors and Affiliations

  1. Developmental and Molecular Pathways, Novartis Institutes for Biomedical Research, Cambridge, MA, USA

    P Bernasconi-Elias, S Gans, P Capodieci, M Goetcshkes, E Nolin, A Buckler & C J Fryer

  2. Center for Proteomic Chemistry, Novartis Institutes for Biomedical Research, Cambridge, MA, USA

    T Hu

  3. Department of Oncology, Novartis Institutes for Biomedical Research, Cambridge, MA, USA

    D Jenkins, B Firestone, E Kurth, M D Jones, K Slocum & S A Ettenberg

  4. Discovery Alliances and Technologies, MorphoSys AG, Martinsried, Germany

    J Deplazes-Lauber, K Petropoulos, P Thiel, D Ponsel & J Jaehrling

  5. Department of Cancer Biology, Dana Farber Cancer Institute, Boston, MA, USA

    S Hee Choi, D M Weinstock, A Christodoulou & S C Blacklow

  6. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA

    S Hee Choi, S C Blacklow & J C Aster

  7. Department of Biologics, Novartis Institutes for Biomedical Research, Cambridge, MA, USA

    P LeMotte & A London

  8. Department of Pathology, Brigham and Women’s Hospital, Boston, MA, USA

    M J Kluk, S C Blacklow & J C Aster

  9. Pathology Children Hospital Boston, Boston, MA, USA

    O Weinberg

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Correspondence to C J Fryer.

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Competing interests

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. The remaining authors declare no conflict of interest.

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Bernasconi-Elias, P., Hu, T., Jenkins, D. et al. Characterization of activating mutations of NOTCH3 in T-cell acute lymphoblastic leukemia and anti-leukemic activity of NOTCH3 inhibitory antibodies. Oncogene 35, 6077–6086 (2016). https://doi.org/10.1038/onc.2016.133

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