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An allosteric inhibitor against the therapy-resistant mutant forms of EGFR in non-small cell lung cancer

Nature Cancer volume 3, pages 402–417 (2022)Cite this article

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Abstract

Epidermal growth factor receptor (EGFR) therapy using small-molecule tyrosine kinase inhibitors (TKIs) is initially efficacious in patients with EGFR-mutant lung cancer, although drug resistance eventually develops. Allosteric EGFR inhibitors, which bind to a different EGFR site than existing ATP-competitive EGFR TKIs, have been developed as a strategy to overcome therapy-resistant EGFR mutations. Here we identify and characterize JBJ-09-063, a mutant-selective allosteric EGFR inhibitor that is effective across EGFR TKI-sensitive and resistant models, including those with EGFR T790M and C797S mutations. We further uncover that EGFR homo- or heterodimerization with other ERBB family members, as well as the EGFR L747S mutation, confers resistance to JBJ-09-063, but not to ATP-competitive EGFR TKIs. Overall, our studies highlight the potential clinical utility of JBJ-09-063 as a single agent or in combination with EGFR TKIs to define more effective strategies to treat EGFR-mutant lung cancer.

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Fig. 1: Characterization of JBJ-09-063 in enzymatic assays, in vitro Ba/F3 cellular studies and in vivo xenograft models.
Fig. 2: JBJ-09-063 efficacy in human cancer cells is enhanced when combined with gefitinib.
Fig. 3: Forced dimerization of EGFR with other ERBB family members impart resistance to JBJ-09-063.
Fig. 4: Ligand-induced dimerization can render cells resistant to JBJ-09-063.
Fig. 5: JBJ-09-063 is effective in TKI-naive EGFR L858R mutant models.
Fig. 6: JBJ-09-063 is effective in a broad range of osimertinib-resistant EGFR mutant contexts.
Fig. 7: JBJ-09-063 is effective in H1975 cells exogenously expressing the osimertinib-resistant mutations.
Fig. 8: L747S mutation is an on-target resistance mechanism to JBJ-09-063, but not to osimertinib.

Data availability

All data in this study are presented in the article and Supplementary Information. The structure in Fig. 1a has been deposited in the PDB with the accession code 7JXQ. The structure depicted in Fig. 6b was accessed via PDB accession code 4ZAU. The CRISPR sequencing data in Extended Data Fig. 1g were deposited into the NCBI Biosample database (PRJNA798765) and are publicly available. All materials are available upon request and through a material transfer agreement. Source data are provided with this paper.

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Acknowledgements

We thank K. Elenius (University of Turku, Finland) for generously providing the pBABE constructs, the MGH CCI DNA Core for their next-generation sequencing services, E. Cohen from the Molecular Biology Core Facilities at the DFCI for processing the DFCI52-C797S and GR-C797S cell line validation FASTA data in Integrative Genomics Viewer and all staff at the DFCI Animal Resources Facility for their mouse husbandry services. This study was supported by National Cancer Institute grants R35CA220497 (P.A.J.), RO1 CA201049 (M.J.E., N.S.G. and P.A.J) and PO1CA154303 (M.J.E., N.S.G. and P.A.J); American Cancer Society grant CRP-17-111-01-CDD (P.A.J.); the Balassiano Family Fund for Lung Cancer Research (P.A.J.); the Gohl Family Lung Cancer Research Fund (P.A.J.); and Takeda. T.S.B. is supported by a Ruth L. Kirschstein National Research Service Award (1F32CA247198-01). Y.K. is supported in part by Japan Society for the Promotion of Science Overseas Research Fellowships and Research Fellowship from Uehara Memorial Foundation. This work used NE-CAT beamlines (P30 GM124165) and an Eiger detector (S10OD021527) at the Advanced Photon Source (DE-AC02-06CH11357).

Author information

Author notes

  1. Jaebong Jang

    Present address: College of Pharmacy, Korea University, Sejong, Korea

  2. David E. Heppner

    Present address: Department of Chemistry, University at Buffalo, Buffalo, NY, USA

  3. Yoshihisa Kobayashi

    Present address: Division of Molecular Pathology, National Cancer Center Research Institute, Tokyo, Japan

  4. Kari J. Kurppa

    Present address: Institute of Biomedicine, MediCity Research Laboratories, University of Turku, Turku, Finland

  5. Nathanael S. Gray

    Present address: Department of Medicinal Chemistry and Department of Chemistry and Systems Biology, Stanford University, Stanford, CA, USA

  6. These authors contributed equally: Ciric To, Tyler S. Beyett, Jaebong Jang.

Authors and Affiliations

  1. Lowe Center for Thoracic Oncology, Dana-Farber Cancer Institute, Boston, MA, USA

    Ciric To, William W. Feng, Magda Bahcall, Heidi M. Haikala, Bo H. Shin, Yoshihisa Kobayashi, Kari J. Kurppa, Atsuko Ogino, Mierzhati Mushajiang, Chunxiao Xu, Yanxi Zhang & Pasi A. Jänne

  2. Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA

    Ciric To, William W. Feng, Magda Bahcall, Heidi M. Haikala, Bo H. Shin, Yoshihisa Kobayashi, Kari J. Kurppa, Atsuko Ogino, Mierzhati Mushajiang, Chunxiao Xu, Yanxi Zhang & Pasi A. Jänne

  3. Department of Medicine, Harvard Medical School, Boston, MA, USA

    Ciric To, William W. Feng, Magda Bahcall, Heidi M. Haikala, Bo H. Shin, Yoshihisa Kobayashi, Kari J. Kurppa, Atsuko Ogino, Mierzhati Mushajiang, Chunxiao Xu, Yanxi Zhang & Pasi A. Jänne

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

    Tyler S. Beyett, Jaebong Jang, David E. Heppner, Jaimin K. Rana, Thomas W. Gero, David A. Scott, Michael J. Eck & Nathanael S. Gray

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

    Tyler S. Beyett, Jaebong Jang, David E. Heppner, Jaimin K. Rana, Thomas W. Gero, David A. Scott, Michael J. Eck & Nathanael S. Gray

  6. Experimental Therapeutics Core and Belfer Center for Applied Cancer Science, Dana-Farber Cancer Institute, Boston, MA, USA

    Brittaney A. Leeper, Kara M. Soroko, Michael J. Poitras & Prafulla C. Gokhale

  7. Institute of Biomedicine, MediCity Research Laboratories, University of Turku, Turku, Finland

    Kamal Wahid

  8. Department of Molecular Therapeutics, The Scripps Research Institute, Jupiter, FL, USA

    Michael D. Cameron

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  1. Ciric To
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Contributions

C.T. and P.A.J. conceived and designed the studies, interpreted the findings and wrote the manuscript. C.T., T.S.B., W.W.F., H.M.H., B.H.S., J.K.R., D.E.H. and M.M. performed experiments and prepared figures. B.A.L., K.M.S., M.J.P. and P.C.G. conducted and coordinated the in vivo studies. J.J., T.W.G. and D.A.S. synthesized the JBJ-09-063 compound. Y.K. generated the H3255GR-C797S and DFCI52-C797S cell lines. M.B., W.W.F., K.W. and K.J.K. generated the constructs for transfection experiments. M.C. performed the pharmacokinetic studies of JBJ-09-063. A.O., C.X. and Y.Z. established the DFCI52 cell line and DFCI52 patient-derived xenograft. M.J.E. and N.S.G. provided crucial feedback on all experimental design and data interpretation. C.T. and P.A.J. supervised the studies and coordinated the efforts of all authors. All authors reviewed the manuscript.

Corresponding authors

Correspondence to David A. Scott, Michael J. Eck, Nathanael S. Gray or Pasi A. Jänne.

Ethics declarations

Competing interests

P.A.J. has received consulting fees from AstraZeneca, Boehringer Ingelheim, Pfizer, Roche/Genentech, Takeda Oncology, ACEA Biosciences, Eli Lilly and Company, Araxes Pharma, Ignyta, Mirati Therapeutics, Novartis, LOXO Oncology, Daiichi Sankyo, Sanofi Oncology, Voronoi, SFJ Pharmaceuticals, Biocartis, Novartis Oncology, Nuvalent, Esai, Bayer, Transcenta, Silicon Therapeutics, Allorion Therapeutics, Accutar Biotech and AbbVie; receives post-marketing royalties from DFCI-owned intellectual property on EGFR mutations licensed to Lab Corp; receives or has received sponsored research funding from AstraZeneca, Astellas, Daichi-Sankyo, PUMA, Boehringer Ingelheim, Eli Lilly and Company, Revolution Medicines and Takeda and has stock ownership in Gatekeeper Pharmaceuticals. N.S.G. is a founder, science advisory board member and equity holder in Gatekeeper, Syros, Petra, C4, Allorion, Jengu, B2S, Inception, EoCys, Larkspur (board member) and Soltego (board member). The Gray lab receives or has received research funding from Novartis, Takeda, Astellas, Taiho, Jansen, Kinogen, Arbella, Deerfield and Sanofi. M.J.E. has served as a paid consultant to Novartis Institutes for Biomedical Research and H3 Biomedicine. M.J.E. receives sponsored research support from Novartis, Sanofi and Takeda. D.E.H. is a consultant for Logos Capital and the Jefferies Group. The series of compounds to which JBJ-04-125-02 and JBJ-09-063 belong is described in US patent 10,836,722 B2. All other authors declare no competing interests.

Peer review

Peer review information

Nature Cancer thanks Rafael Rosell, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Structure, potency, pharmacodynamic studies of JBJ-09-063 and the development and validation of H3255GR-C797S and DFIC52-C797S model in vitro.

a, Positive Fo-Fc electron density at 3 sigma for JBJ-09-063. b, Cell viability assay and c, Western Blot analyses of EGFRL858R/C797S Ba/F3 cells treated with increasing concentrations of JBJ-09-063, osimertinib and gefitinib. Data shown in panel b is a representative figure from N=3 independent experiments with 3 technical replicates in each experiment. d, Pharmacodynamic study of JBJ-09-063 in lung tumor tissues of H1975 xenograft mice dosed with 50 mg/kg of the compound. Samples were collected at 0 or 2, 8, 16 and 24h after dosing and samples were processed for Western Blotting analyses. e, Treatment history of patient who was given the first-generation TKI erlotinib (pink bar). Pleural effusion was subsequently drawn (orange arrow with PDX) to establish patient-derived xenograft (PDX) model. DFCI52 cell line was derived from PDX tumors (mouse #135 derived from the effusion). f, Workflow of EGFRL858R/T790M/C797S cell line generation: H3255GR and DFCI52 cells, which already harbor the EGFRL858R/T790M mutation, were transfected by nucleofection with a sgRNA designed to incorporate the C797S mutation in cis with EGFRT790M. 100 nM osimertinib was used for selection for 1 week and samples were sent for next-generation sequencing to determine the presence and frequency of C797S mutation. g, Visualization of the presence and frequency of T790M and C797S allele variants in cis in H3255GR-C797S and DFCI52-C797S cells using Integrated Genome Viewer (IGV). h, Cell viability assays and i, Western Blot analyses of H3255GR vs. H3255GR-C797S and DFCI52 vs. DFCI52-C797S cells treated with increasing concentrations of osimertinib. Data shown in panel h is a representative figure from N=3 independent experiments with 3 technical replicates in each experiment. All cell viability assays shown in this figure were graphed as a percentage of activity relative to DMSO control (mean ± SD) over indicated concentrations.

Source data

Extended Data Fig. 2 JBJ-09-063 efficacy is reduced in vitro unless combined with gefitinib in H3255GR and DFCI52 cells or with osimertinib in H3255GR-C797S and DFCI52-C797S cells.

a, Cell viability and b, Western blot analyses of H3255GR cells treated with indicated concentrations of osimertinib, JBJ-09-063 and JBJ-04-125-02. c, Cell viability, d, apoptosis and e, Western Blot analyses of DFCI52 cells treated with indicated concentrations of gefitinib, JBJ-09-063 or the combination of both agents. Cell viability of H3255GR-C797S cells in panel f, and DFIC52-C797S cells in panel g treated with indicated concentrations of JBJ-09-063 and osimertinib as a single agent or in combination of both compounds. h, Western Blot analyses of H3255GR-C797S and DFIC52-C797S cells treated with indicated concentrations of JBJ-09-063 and osimertinib as a single agent or in combination of both compounds. All cell viability assays shown in this figure were graphed as a percentage of activity relative to DMSO control (mean ± SD) over indicated concentrations. Data shown in panels a, c, f, g are representative figures from N=3 independent experiments with 3 technical replicates in each experiment. The apoptosis experiments were graphed as normalized caspase-3/7 activity (in average arbitrary units ± SEM)) over time. Data shown in panel f is a representative figure from N=3 independent experiments with 12 technical replicates in each experiment.

Source data

Supplementary information

Reporting Summary

Supplementary Tables

Supplementary Table 1. Data collection and refinement statistics (molecular replacement) of JBJ-09-063. Supplementary Table 2. IC50 of JBJ-04-125-02 and JBJ-09-063 in enzymatic assays, Ba/F3 cells, H1975 cells, and human cancer cells. Supplementary Table 3. Pharmacokinetic profile of JBJ-09-063. Supplementary Table 4. Sequences and primers for plasmids and cell lines generation.

Source data

Source Data Fig. 1

Unprocessed western blots with molecular markers.

Source Data Fig. 1

Quantitation of statistical data.

Source Data Fig. 2

Unprocessed western blots with molecular markers.

Source Data Fig. 2

Quantitation for statistical data.

Source Data Fig. 3

Unprocessed western blots with molecular markers.

Source Data Fig. 3

Quantitation of statistical data.

Source Data Fig. 4

Unprocessed western blots with molecular markers.

Source Data Fig. 4

Quantitation for statistical data

Source Data Fig. 5

Unprocessed western blots with molecular markers.

Source Data Fig. 5

Quantitation of statistical data.

Source Data Fig. 6

Unprocessed western blots with molecular markers.

Source Data Fig. 6

Quantitation of statistical data.

Source Data Fig. 7

Unprocessed western blots with molecular markers.

Source Data Fig. 7

Quantitation for statistical data.

Source Data Fig. 8

Unprocessed western blots with molecular markers.

Source Data Fig. 8

Quantitation for statistical data.

Source Data Extended Data Fig. 1

Unprocessed western blots with molecular markers.

Source Data Extended Data Fig. 1

Quantitation of statistical data

Source Data Extended Data Fig. 2

Unprocessed western blots with molecular markers..

Source Data Extended Data Fig. 2

Quantitation of statistical data.

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To, C., Beyett, T.S., Jang, J. et al. An allosteric inhibitor against the therapy-resistant mutant forms of EGFR in non-small cell lung cancer. Nat Cancer 3, 402–417 (2022). https://doi.org/10.1038/s43018-022-00351-8

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