A common BIM deletion polymorphism mediates intrinsic resistance and inferior responses to tyrosine kinase inhibitors in cancer

Abstract

Tyrosine kinase inhibitors (TKIs) elicit high response rates among individuals with kinase-driven malignancies, including chronic myeloid leukemia (CML) and epidermal growth factor receptor–mutated non–small-cell lung cancer (EGFR NSCLC). However, the extent and duration of these responses are heterogeneous, suggesting the existence of genetic modifiers affecting an individual's response to TKIs. Using paired-end DNA sequencing, we discovered a common intronic deletion polymorphism in the gene encoding BCL2-like 11 (BIM). BIM is a pro-apoptotic member of the B-cell CLL/lymphoma 2 (BCL2) family of proteins, and its upregulation is required for TKIs to induce apoptosis in kinase-driven cancers. The polymorphism switched BIM splicing from exon 4 to exon 3, which resulted in expression of BIM isoforms lacking the pro-apoptotic BCL2-homology domain 3 (BH3). The polymorphism was sufficient to confer intrinsic TKI resistance in CML and EGFR NSCLC cell lines, but this resistance could be overcome with BH3-mimetic drugs. Notably, individuals with CML and EGFR NSCLC harboring the polymorphism experienced significantly inferior responses to TKIs than did individuals without the polymorphism (P = 0.02 for CML and P = 0.027 for EGFR NSCLC). Our results offer an explanation for the heterogeneity of TKI responses across individuals and suggest the possibility of personalizing therapy with BH3 mimetics to overcome BIM-polymorphism–associated TKI resistance.

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Figure 1: A 2,903-bp deletion polymorphism in intron 2 of BIM is present in TKI-resistant CML samples.
Figure 2: Effects of the deletion polymorphism on BIM gene function.
Figure 3: De novo generation and analysis of CML cell lines with the BIM deletion polymorphism.
Figure 4: The BIM deletion polymorphism is sufficient to cause intrinsic TKI resistance in EGFR NSCLC cell lines.
Figure 5: The BIM deletion polymorphism predicts shorter PFS in individuals with EGFR-mutant NSCLC treated with EGFR TKI therapy.

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Acknowledgements

This study was supported by grants from the National Medical Research Council of Singapore and the Biomedical Research Council (BMRC) of the Agency for Science, Technology and Research (A*STAR), Singapore. Additional support was also provided by the Genome Institute of Singapore internal research funds from the BMRC and the Department of Clinical Research, Singapore General Hospital. We are grateful for insightful conversations regarding this study with G. Bourque, M. Garcia-Blanco, E. Liu, X. Roca, S. Rosen, S. Shenolikar, D. Virshup and M. Voorhoeve. We thank C.-L. Wei and H. Thoreau for management of the sequencing platform, S.T. Leong, S.C. Neo and P.S. Choi for sequencing, J. Chen and C.S. Chan for help in data processing, H.P. Lim, Y.Y. Sia and Y.H. Choy for PCR validation and A. Lim and T.H. Lim for assistance in the fluorescence in situ hybridization (FISH) analysis. We also thank M. Garcia-Blanco (Duke University), K. Itahana (Duke-NUS), A. Vazquez (Institut National de la Santé et de la Recherche Médicale U.1014, Villejuif, France and Université Paris-Sud, Paris, France) and P. Koeffler (Cedars-Sinai Medical Center, Los Angeles, California, USA and Cancer Science Institute of Singapore, Singapore) for the kind gifts of the pl-12 vector, pcDNA3-FLAG3 plasmid, BIM expression vectors and NSCLC cell lines, respectively. Finally, we are grateful to the patients and physicians at the Department of Haematology, Singapore General Hospital, the Department of Hematology-Oncology, Akita University Hospital, Japan, the Toho University Omori Medical Center, Japan, the Aichi Cancer Center, Japan, the National University Cancer Institute, National University Health System, Singapore, National Cancer Centre, Singapore and the University of Malaya Medical Centre, Kuala Lumpur, Malaysia who contributed patient material.

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K.P.N. and A.M.H. performed data analyses, generated the list of structural variations, validated the paired-end ditag data and wrote the first draft of the manuscript. C.T.H.C. provided CML clinical input and generated and analyzed the clinical data in Table 1. W.C.J. and T.K.K. devised and performed the experiments in Figures 2–4. C.-T.C. performed the experiments in Figures 3 and 4. J.W.J.H. performed FISH and PCR analysis on patient and normal control samples. A.S.M.T. and Y.F. constructed DNA-PET libraries for high-throughput sequencing. P.N.A., W.H.L. and W.-K.S. developed the bioinformatics pipeline for the DNA-PET analysis, N.N. contributed to the pipeline development, and X.Y.W. developed the copy number analysis. W.T.P. ran the bioinformatics pipeline. V.K. and A.T. performed BIM deletion screening in the HapMap samples, and A.T. performed the population-level genetic statistical analysis. X.R. managed the high-throughput sequencing, and A.S. managed the bioinformatics infrastructure. C.T.H.C., N.T., K.S., A.L.A., H.T.M., G.F.H., L.Y.Y., L.P.K., B.C., V.S.N., W.J.C., H.T., L.C.L. and Y.T.G. provided samples from patients with CML, as well as clinical data from the same patients. M.M.N. and T.Y.W. provided samples from normal individuals. K.P.N., J.W.J.H. and W.C.J. analyzed CML samples for the BIM deletion polymorphism. J.C.A. Jr. performed the statistical analysis of the CML clinical data. V.C.-R. performed and interpreted FISH data and provided scientific advice. S.S. compiled the clinical data and, together with J.C.A. Jr., performed the statistical analyses for Figure 5a. K.P.N., J.W.J.H., S.Z., D.P., P.T. and M.S. analyzed samples for EGFR mutations and the BIM deletion polymorphism. J.-E.S., M.-K.A., N.-M.C., Q.-S.N., D.S.W.T., K.I., Y.Y., H.M., E.H.T., R.A.S., T.M.C. and W.-T.L. provided samples from subjects with EGFR NSCLC, as well as the accompanying clinical data. Y.R. and S.T.O. designed and directed the study and analyzed data. S.T.O. wrote the final draft of the manuscript, which was reviewed by K.P.N., A.M.H., C.T.H.C., W.C.J., T.K.K., W.-T.L. and Y.R.

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Correspondence to Wan-Teck Lim or Yijun Ruan or S Tiong Ong.

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K.P.N., A.M.H., C.T.H.C., W.C.J., Y.R. and S.T.O. hold a National University of Singapore, Singapore Health Services Pte Ltd and the Agency for Science, Technology and Research, Singapore patent (BRC/P/06094/01/PCT) for a method to detect resistance to cancer therapy and guide therapy to overcome resistance.

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Supplementary Text and Figures

Supplementary Figures 1–4, Supplementary Tables 1, 3, 4 and 7–12 and Supplementary Note (PDF 1842 kb)

Supplementary Tables 2, 5, 6 and 13

Statistics of massively parallel PET sequencing on the SOLiD platforms (XLS 261 kb)

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Ng, K., Hillmer, A., Chuah, C. et al. A common BIM deletion polymorphism mediates intrinsic resistance and inferior responses to tyrosine kinase inhibitors in cancer. Nat Med 18, 521–528 (2012). https://doi.org/10.1038/nm.2713

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