Skip to main content

A screen to identify drug resistant variants to target-directed anti-cancer agents

Abstract

The discovery of oncogenes and signal transduction pathways important for mitogenesis has triggered the development of target-specific small molecule anti-cancer compounds. As exemplified by imatinib (Gleevec), a specific inhibitor of the Chronic Myeloid Leukemia (CML)-associated Bcr-Abl kinase, these agents promise impressive activity in clinical trials, with low levels of clinical toxicity. However, such therapy is susceptible to the emergence of drug resistance due to amino acid substitutions in the target protein. Defining the spectrum of such mutations is important for patient monitoring and the design of next-generation inhibitors. Using imatinib and BCR/ABL as a paradigm for a drug-target pair, we recently reported a retroviral vector-based screening strategy to identify the spectrum of resistance-conferring mutations. Here we provide a detailed methodology for the screen, which can be generally applied to any drug-target pair.

References

  1. Druker BJ, Tamura S, Buchdunger E, Ohno S, Segal GM, Fanning S, Zimmermann J, Lydon NB. Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells.Nat Med 1996; 2(5):561–566.

    Article  PubMed  CAS  Google Scholar 

  2. Druker BJ, Sawyers CL, Kantarjian H, Resta DJ, Reese SF, Ford JM, Capdeville R, Talpaz M. Activity of a specific inhibitor of the BCR-ABL tyrosine kinase in the blast crisis of chronic myeloid leukemia and acute lymphoblastic leukemia with the Philadelphia chromosome.N Engl J Med 2001; 344:1038–1042.

    Article  PubMed  CAS  Google Scholar 

  3. Dancey J, Sausville EA. Issues and progress with protein kinase inhibitors for cancer treatment.Nat Rev Drug Discov 2003; 2:296–313.

    Article  PubMed  CAS  Google Scholar 

  4. O’Brien SG, Guilhot F, Larson RA, Gathmann I, Baccarani M, Cervantes F, Cornelissen JJ, Fischer T, Hochhaus A, Hughes T, Lechner K, Nielsen JL, Rousselot P, Reiffers J, Saglio G, Shepherd J, Simonsson B, Gratwohl A, Goldman JM, Kantarjian H, Taylor K, Verhoef G, Bolton AE, Capdeville R, Druker BJ; IRIS Investigators. Imatinib compared with interferon and low-dose cytarabine for newly diagnosed chronic-phase chronic myeloid leukemia.N Engl J Med 2003; 348:994–1004.

    Article  PubMed  CAS  Google Scholar 

  5. Gorre ME, Mohammed M, Ellwood K, Hsu N, Paquette R, Rao PN, Sawyers CL. Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification.Science 2001; 293:876–880.

    Article  PubMed  CAS  Google Scholar 

  6. Shah NP, Nicoll JM, Nagar B, Gorre ME, Paquette RL, Kuriyan J, Sawyers CL. Multiple BCR-ABL kinase domain mutations confer polyclonal resistance to the tyrosine kinase inhibitor imatinib (STI571) in chronic phase and blast crisis chronic myeloid leukemia.Cancer Cell 2002; 2:117–125.

    Article  PubMed  CAS  Google Scholar 

  7. Branford S, Rudzki Z, Walsh S, Grigg A, Arthur C, Taylor K, Herrmann R, Lynch KP, Hughes TP. High frequency of point mutations clustered within the adenosine triphosphate-binding region of BCR/ABL in patients with chronic myeloid leukemia or Ph-positive acute lymphoblastic leukemia who develop imatinib (STI571) resistance.Blood 2002; 99(9):3472–3475.

    Article  PubMed  CAS  Google Scholar 

  8. Azam M, Latek LR, Daley GQ. Mechanisms of Autoinhibition and STI-571/Imatinib Resistance Revealed by Mutagenesis of BCR-ABL.Cell 2003; 112:831–843.

    Article  PubMed  CAS  Google Scholar 

  9. Koh EY, Chen T, Daley GQ. Enhanced cDNA Rescue Strategies to Facilitate Functional Genomic/Phenotypic Screens in Mammalian Cells.Nucleic Acids Res 2002; 30:E142–2.

    Article  PubMed  Google Scholar 

  10. Daley GQ, Baltimore D. Transformation of an interleukin 3-dependent hematopoietic cell line by the chronic myelogenous leukemia-specific P210bcr/abl protein.Proc Natl Acad Sci USA 1988;85:9312–9316.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to George Q. Daley.

Additional information

Azam and Raz contributed equally to this manuscript.

Published: October 24, 2003

Rights and permissions

Reprints and permissions

About this article

Cite this article

Azam, M., Raz, T., Nardi, V. et al. A screen to identify drug resistant variants to target-directed anti-cancer agents. Biol. Proced. Online 5, 204–210 (2003). https://doi.org/10.1251/bpo63

Download citation

  • Received:

  • Revised:

  • Issue Date:

  • DOI: https://doi.org/10.1251/bpo63

Indexing terms