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Random Mutagenesis Reveals Residues of JAK2 Critical in Evading Inhibition by a Tyrosine Kinase Inhibitor
BACKGROUND: The non-receptor tyrosine kinase JAK2 is implicated in a group of myeloproliferative neoplasms including polycythemia vera, essential thrombocythemia, and primary myelofibrosis. JAK2-selective inhibitors are currently being evaluated in clinical trials. Data from drug-resistant chronic m...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2012
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3420867/ https://www.ncbi.nlm.nih.gov/pubmed/22916261 http://dx.doi.org/10.1371/journal.pone.0043437 |
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author | Marit, Michael R. Chohan, Manprit Matthew, Natasha Huang, Kai Kuntz, Douglas A. Rose, David R. Barber, Dwayne L. |
author_facet | Marit, Michael R. Chohan, Manprit Matthew, Natasha Huang, Kai Kuntz, Douglas A. Rose, David R. Barber, Dwayne L. |
author_sort | Marit, Michael R. |
collection | PubMed |
description | BACKGROUND: The non-receptor tyrosine kinase JAK2 is implicated in a group of myeloproliferative neoplasms including polycythemia vera, essential thrombocythemia, and primary myelofibrosis. JAK2-selective inhibitors are currently being evaluated in clinical trials. Data from drug-resistant chronic myeloid leukemia patients demonstrate that treatment with a small-molecule inhibitor generates resistance via mutation or amplification of BCR-ABL. We hypothesize that treatment with small molecule inhibitors of JAK2 will similarly generate inhibitor-resistant mutants in JAK2. METHODOLOGY: In order to identify inhibitor-resistant JAK2 mutations a priori, we utilized TEL-JAK2 to conduct an in vitro random mutagenesis screen for JAK2 alleles resistant to JAK Inhibitor-I. Isolated mutations were evaluated for their ability to sustain cellular growth, stimulate downstream signaling pathways, and phosphorylate a novel JAK2 substrate in the presence of inhibitor. CONCLUSIONS: Mutations were found exclusively in the kinase domain of JAK2. The panel of mutations conferred resistance to high concentrations of inhibitor accompanied by sustained activation of the Stat5, Erk1/2, and Akt pathways. Using a JAK2 substrate, enhanced catalytic activity of the mutant JAK2 kinase was observed in inhibitor concentrations 200-fold higher than is inhibitory to the wild-type protein. When testing the panel of mutations in the context of the Jak2 V617F allele, we observed that a subset of mutations conferred resistance to inhibitor, validating the use of TEL-JAK2 in the initial screen. These results demonstrate that small-molecule inhibitors select for JAK2 inhibitor-resistant alleles, and the design of next-generation JAK2 inhibitors should consider the location of mutations arising in inhibitor-resistant screens. |
format | Online Article Text |
id | pubmed-3420867 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2012 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-34208672012-08-22 Random Mutagenesis Reveals Residues of JAK2 Critical in Evading Inhibition by a Tyrosine Kinase Inhibitor Marit, Michael R. Chohan, Manprit Matthew, Natasha Huang, Kai Kuntz, Douglas A. Rose, David R. Barber, Dwayne L. PLoS One Research Article BACKGROUND: The non-receptor tyrosine kinase JAK2 is implicated in a group of myeloproliferative neoplasms including polycythemia vera, essential thrombocythemia, and primary myelofibrosis. JAK2-selective inhibitors are currently being evaluated in clinical trials. Data from drug-resistant chronic myeloid leukemia patients demonstrate that treatment with a small-molecule inhibitor generates resistance via mutation or amplification of BCR-ABL. We hypothesize that treatment with small molecule inhibitors of JAK2 will similarly generate inhibitor-resistant mutants in JAK2. METHODOLOGY: In order to identify inhibitor-resistant JAK2 mutations a priori, we utilized TEL-JAK2 to conduct an in vitro random mutagenesis screen for JAK2 alleles resistant to JAK Inhibitor-I. Isolated mutations were evaluated for their ability to sustain cellular growth, stimulate downstream signaling pathways, and phosphorylate a novel JAK2 substrate in the presence of inhibitor. CONCLUSIONS: Mutations were found exclusively in the kinase domain of JAK2. The panel of mutations conferred resistance to high concentrations of inhibitor accompanied by sustained activation of the Stat5, Erk1/2, and Akt pathways. Using a JAK2 substrate, enhanced catalytic activity of the mutant JAK2 kinase was observed in inhibitor concentrations 200-fold higher than is inhibitory to the wild-type protein. When testing the panel of mutations in the context of the Jak2 V617F allele, we observed that a subset of mutations conferred resistance to inhibitor, validating the use of TEL-JAK2 in the initial screen. These results demonstrate that small-molecule inhibitors select for JAK2 inhibitor-resistant alleles, and the design of next-generation JAK2 inhibitors should consider the location of mutations arising in inhibitor-resistant screens. Public Library of Science 2012-08-16 /pmc/articles/PMC3420867/ /pubmed/22916261 http://dx.doi.org/10.1371/journal.pone.0043437 Text en © 2012 Marit et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Marit, Michael R. Chohan, Manprit Matthew, Natasha Huang, Kai Kuntz, Douglas A. Rose, David R. Barber, Dwayne L. Random Mutagenesis Reveals Residues of JAK2 Critical in Evading Inhibition by a Tyrosine Kinase Inhibitor |
title | Random Mutagenesis Reveals Residues of JAK2 Critical in Evading Inhibition by a Tyrosine Kinase Inhibitor |
title_full | Random Mutagenesis Reveals Residues of JAK2 Critical in Evading Inhibition by a Tyrosine Kinase Inhibitor |
title_fullStr | Random Mutagenesis Reveals Residues of JAK2 Critical in Evading Inhibition by a Tyrosine Kinase Inhibitor |
title_full_unstemmed | Random Mutagenesis Reveals Residues of JAK2 Critical in Evading Inhibition by a Tyrosine Kinase Inhibitor |
title_short | Random Mutagenesis Reveals Residues of JAK2 Critical in Evading Inhibition by a Tyrosine Kinase Inhibitor |
title_sort | random mutagenesis reveals residues of jak2 critical in evading inhibition by a tyrosine kinase inhibitor |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3420867/ https://www.ncbi.nlm.nih.gov/pubmed/22916261 http://dx.doi.org/10.1371/journal.pone.0043437 |
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