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Discovery of a ZIP7 inhibitor from a Notch pathway screen

Abstract

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.

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Fig. 1: Identification and characterization of molecules that inhibit Notch signaling.
Fig. 2: NVS-ZP7-3 treatment induces apoptosis and ER stress in Notch pathway-active T-ALL cell lines.
Fig. 3: Generation and characterization of a NVS-ZP7-4 compound-resistant cell line.
Fig. 4: Genetic screens reveal increased ER stress and decreased Notch signaling following ZIP7 siRNA knockdown.
Fig. 5: Genetic validation of ZIP7 as the target of NVS-ZP7-4.
Fig. 6: NVS-ZP7-4 interacts with ZIP7, increases ER Zn2+ levels in the ER and modulates Notch signaling.

Data availability

The microarray data has been deposited in GEO (GSE115690). Other datasets that were generated during the current study are provided as Supplementary Information or are available from the corresponding author upon reasonable request.

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Acknowledgements

The JAGGED1 and DLL1 expressing cell lines were kindly provide by G. Weinmaster (UCLA). HPB-ALL cells were kindly provided by A. Stasser (Walter and Eliza Hall Institute for Medical Research). The authors thank J. Paulk for his insights on the manuscript and A. Abrams for his artwork in schematic diagrams. This work was financially supported in part by NIH Director’s Pioneer Award GM114863 (to A.E.P.).

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Authors

Contributions

E.N., S.G., P.B.-E. and C.J.F. developed and/or performed cell-based assays. L.L, R.I.M., N.G., A.D., H.G., J.S., J.D., S.M.C., R.K.J. and S.M. Bushell synthesized compounds and/or directed the medicinal chemistry strategy. S.B., J.J.L., G.R., S.S. and M.B. analyzed/interpreted genomic data. S.H. performed high-throughput screens. J.L.J. and R.K.J. triaged compounds from screens for further characterization. E.N., K.P.C. and A.E.P. performed and interpreted results from zinc FRET sensor experiments. K.P.C. and A.E.P. provided input on zinc and zinc transporter biology. Z.B.K. and C.A. developed assays for zinc quantitation. K.X.X., A.C. and F.S. performed or analyzed the siRNA screen. S.M. Brittain, J.R.T. and M.S. performed or directed the photoaffinity labeling experiments. A.L., N.G. and Y.Y. designed targeting strategy for CRISPR experiments or analyzed data. J.R.-H., W.A.W., K.T., P.B.-E. and E.N. performed the variomics experiments and characterized mutants. J.A.P., O.W., D.H., E.L.G., G.B., R.K.J. and J.A.T. provided intellectual input to the mechanism of action studies. All authors contributed to writing of the work. C.J.F. and S.M. Bushell designed the experimental strategy, wrote the manuscript and held overall responsibility for the study.

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Correspondence to Simon M. Bushell or Christy J. Fryer.

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Competing interests

E.N., S.G., L.L., S.B., S.M. Brittain, P.B.-E., J.J.L., J.R.T., M.S., Y.Y., N.G., G.R., S.S., M.B., A.L., F.S., A.C., K.X.X., S.H., J.R.-H., W.A.W., K.T., D.H., R.I.M., N.G., A.D., H.G., J.S., J.D., S.M.C., G.B., E.L.G., Z.B.K., C.A., J.A.P., O.W., J.A.T., J.L.J., R.K.J., S.M. Bushell, and C.J.F. are (or were at the time the research was conducted) employees of Novartis.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–16 and Supplementary Tables 1–6

Reporting Summary

Supplementary Note 1

Chemical Synthesis

Supplementary Note 2

Chemistry Spectra

Supplementary Dataset 1

133 gene probe sets identified from microarray that are significantly changed in both TALL-1 and RPMI-8402 cells following NVS-ZP7-3 treatment (adjusted P < 0.001 and a fold change greater than two)

Supplementary Dataset 2

33 genes with single nucleotide polymorphisms (SNPs)

Supplementary Dataset 3

Chemoproteomics data

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Nolin, E., Gans, S., Llamas, L. et al. Discovery of a ZIP7 inhibitor from a Notch pathway screen. Nat Chem Biol 15, 179–188 (2019). https://doi.org/10.1038/s41589-018-0200-7

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