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

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 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 two of 40 tumors examined show active NOTCH3 signaling. We also identified evidence of NOTCH3 activation in 12 of 24 patient-derived orthotopic xenograft models, two 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

Discovery of a ZIP7 inhibitor from a Notch pathway screen

The identification of activating mutations in NOTCH1 in 50% of T cell acute lymphoblastic leukemia has generated interest in elucidating how these mutations contribute to oncogenic transformation and in targeting the pathway. A phenotypic screen identified compounds that interfere with trafficking of Notch and induce apoptosis via an endoplasmic reticulum (ER) stress mechanism. Target identification approaches revealed a role for SLC39A7 (ZIP7), a zinc transport family member, in governing Notch trafficking and signaling. Generation and sequencing of a compound-resistant cell line identified a V430E mutation in ZIP7 that confers transferable resistance to the compound NVS-ZP7-4. NVS-ZP7-4 altered zinc in the ER, and an analog of the compound photoaffinity labeled ZIP7 in cells, suggesting a direct interaction between the compound and ZIP7. NVS-ZP7-4 is the first reported chemical tool to probe the impact of modulating ER zinc levels and investigate ZIP7 as a novel druggable node in the Notch pathway

Discovery of a ZIP7 Inhibitor from a Notch Pathway Screen

The identification of activating mutations in NOTCH1 in over 50% of T-cell acute lymphoblastic leukemia has generated interest in elucidating how these mutations contribute to oncogenic transformation and in targeting the Notch signaling pathway in this disease. The receptors and ligands of the Notch signaling pathway must be trafficked to the cell surface where they interact and activate signaling. A phenotypic screen for inhibitors of the Notch signaling pathway identified compounds that interfere with trafficking of Notch to the cell surface, and induce apoptosis in T-ALL via an ER stress mechanism. Comprehensive target identification approaches revealed a role for SLC39A7 / ZIP7, a Zrt-, Irt-like protein zinc transport family member, in governing Notch trafficking and signaling. Generation and sequencing of a compound resistant cell line identified a V430E mutation in ZIP7 that confers transferable resistance to the compound NVS-ZP7-4. NVS-ZP7-4 altered zinc in the ER, but not the cytosol, suggesting ER zinc homeostasis is critical to both Notch signaling and ER stress. A diazirine-containing analog of the compound photoaffinity labeled ZIP7 in cells, suggesting a direct interaction between the compound and ZIP7. The ZIP and the Zn transporter (ZnT) family of zinc transporters are found in all aspects of life, and both families of transporters play critical roles in cellular and physiological functions. NVS-ZP7-4 is the first reported chemical tool to probe the impact of modulating ER zinc levels and to further investigate ZIP7 as a novel druggable node in the Notch pathway