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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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.

Author information

Author notes

    • King Pan Ng
    • , Axel M Hillmer
    • , Charles T H Chuah
    •  & Wen Chun Juan

    These authors contributed equally to this work.

Affiliations

  1. Cancer & Stem Cell Biology Signature Research Programme, Duke–National University of Singapore (NUS) Graduate Medical School, Singapore.

    • King Pan Ng
    • , Charles T H Chuah
    • , Wen Chun Juan
    • , Tun Kiat Ko
    • , Sheila Soh
    • , John W J Huang
    • , Chia-Tien Chang
    • , Shenli Zhang
    • , Dianne Poh
    • , Patrick Tan
    •  & S Tiong Ong
  2. Genome Institute of Singapore, Singapore.

    • Axel M Hillmer
    • , Audrey S M Teo
    • , Pramila N Ariyaratne
    • , Yao Fei
    • , Wah Heng Lee
    • , Xing Yi Woo
    • , Niranjan Nagarajan
    • , Vikrant Kumar
    • , Anbupalam Thalamuthu
    • , Wan Ting Poh
    • , Patrick Tan
    • , Atif Shahab
    • , Xiaoan Ruan
    • , Valère Cacheux-Rataboul
    • , Wing-Kin Sung
    •  & Yijun Ruan
  3. Department of Haematology, Singapore General Hospital, Singapore.

    • Charles T H Chuah
    • , Ai Leen Ang
    • , Hae Tha Mya
    • , Gee Fung How
    • , Li Yi Yang
    • , Hein Than
    • , Lay Cheng Lim
    • , Yeow Tee Goh
    •  & S Tiong Ong
  4. Department of Hematology, Nephrology and Rheumatology, Akita University Graduate School of Medicine, Akita, Japan.

    • Naoto Takahashi
    •  & Kenichi Sawada
  5. Department of Epidemiology and Public Health, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.

    • Yao Fei
  6. Centre for Quantitative Medicine, Duke-NUS Graduate Medical School, Singapore.

    • John C Allen Jr
  7. Department of Hematology-Oncology, National University Cancer Institute of Singapore, National University Health System, Singapore.

    • Liang Piu Koh
    • , Wee Joo Chng
    • , Ross A Soo
    •  & Tan Min Chin
  8. Clinical Pharmacology Laboratory, National Cancer Centre, Singapore.

    • Balram Chowbay
  9. University of Malaya, Kuala Lumpur, Malaysia.

    • Veera S Nadarajan
  10. Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.

    • Wee Joo Chng
  11. Cancer Science Institute of Singapore, National University of Singapore, Singapore.

    • Wee Joo Chng
    • , Patrick Tan
    •  & Ross A Soo
  12. Department of Pathology, National University Health System, Singapore.

    • Ju-Ee Seet
  13. Department of Medical Oncology, National Cancer Centre, Singapore.

    • Mei-Kim Ang
    • , Noan-Minh Chau
    • , Quan-Sing Ng
    • , Daniel S W Tan
    • , Eng Huat Tan
    • , Wan-Teck Lim
    •  & S Tiong Ong
  14. Division of Functional Genomics, Jichi Medical University, Tochigi, Japan.

    • Manabu Soda
    •  & Hiroyuki Mano
  15. Department of Respiratory Medicine, Toho University Omori Medical Center, Tokyo, Japan.

    • Kazutoshi Isobe
  16. Institute of Human Genetics, University of Bonn, Bonn, Germany.

    • Markus M Nöthen
  17. Singapore Eye Research Institute, Singapore National Eye Centre and National University Health System, Singapore.

    • Tien Y Wong
  18. Department of Pathology and Molecular Diagnostics, Aichi Cancer Center, Aichi, Japan.

    • Yasushi Yatabe
  19. Department of Medical Genomics, Graduate School of Medicine, University of Tokyo, Tokyo, Japan.

    • Hiroyuki Mano
  20. Office of Clinical Sciences, Duke-NUS Graduate Medical School, Singapore.

    • Wan-Teck Lim
  21. Department of Biochemistry, National University of Singapore, Singapore.

    • Yijun Ruan
  22. Department of Medicine, Division of Medical Oncology, Duke University Medical Center, Durham, North Carolina, USA.

    • S Tiong Ong

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Contributions

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.

Competing interests

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.

Corresponding authors

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

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    Statistics of massively parallel PET sequencing on the SOLiD platforms

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https://doi.org/10.1038/nm.2713

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