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A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors
Published on: December 7, 2014
In Vitro Evolution Reveals a Single Mutation as Sole Source of Src-Family Kinase C-Helix-out Inhibitor Resistance
Ravi K Patel1, Yash K Patel1, Thomas E Smithgall1
1Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, 450 Technology Drive, Pittsburgh, Pennsylvania 15219, United States.
Abstract:
Understanding cancer cell drug resistance to protein-tyrosine kinase inhibitors, which often arises from acquired mutations in the target kinase, is central to the development of more durable therapies. Experimental systems that reveal potential paths to resistance for a given inhibitor and kinase target have an important role in preclinical development of kinase inhibitor drugs. Here, we employed a codon mutagenesis strategy to define the mutational landscape of acquired resistance in HCK, a member of the SRC tyrosine kinase family and therapeutic target in acute myeloid leukemia (AML). Using PCR-based saturation mutagenesis, we created a cDNA library designed to replace each codon in the HCK open reading frame with all possible codons. This HCK mutant library was used to transform Rat-2 fibroblasts, followed by selection for resistant colonies with A-419259, a pyrrolopyrimidine HCK inhibitor and drug lead for AML. X-ray crystallography has shown that A-419259 binding induces outward rotation of the kinase domain αC-helix, a conformation incompatible with phosphotransfer. Remarkably, only a single resistance mutation evolved during A-419259 selection: histidine substitution for threonine at the gatekeeper position in the kinase domain. Deep sequencing confirmed representation of nearly all other missense mutations across the entire HCK open reading frame. This observation suggests that A-419259 and other C-helix-out Src-family kinase inhibitors may have a narrow path to acquired resistance in the context of AML cases where Hck is an oncogenic driver.
Insights
Developing durable cancer therapies requires understanding drug resistance. Researchers identified a single key mutation conferring resistance to an HCK inhibitor in acute myeloid leukemia (AML), suggesting a narrow resistance pathway for this drug class.
Area of Science:
- Oncology
- Molecular Biology
- Drug Discovery
Background:
- Acquired mutations in target kinases are a primary cause of cancer drug resistance to protein-tyrosine kinase inhibitors.
- Developing durable therapies necessitates understanding resistance mechanisms and creating experimental systems to predict them.
- HCK (hematopoietic cell kinase) is a SRC family tyrosine kinase and a therapeutic target in acute myeloid leukemia (AML).
Purpose of the Study:
- To define the mutational landscape of acquired resistance in HCK using a codon mutagenesis strategy.
- To identify resistance mutations against A-419259, a pyrrolopyrimidine HCK inhibitor and drug lead for AML.
- To assess the potential resistance pathways for HCK inhibitors in AML.
Main Methods:
- Employed PCR-based saturation mutagenesis to create a HCK mutant cDNA library.
- Transformed Rat-2 fibroblasts with the HCK mutant library.
- Selected for resistant colonies using the HCK inhibitor A-419259, followed by deep sequencing.
Main Results:
- A single resistance mutation, a threonine-to-histidine substitution at the gatekeeper position, evolved during A-419259 selection.
- X-ray crystallography revealed A-419259 binding induces an outward rotation of the kinase domain αC-helix, crucial for its inhibitory mechanism.
- Deep sequencing confirmed the representation of numerous other missense mutations across the HCK open reading frame, indicating broad library coverage.
Conclusions:
- A-419259 and related Src-family kinase inhibitors may possess a limited acquired resistance profile in AML.
- The gatekeeper mutation is the predominant resistance mechanism identified for this HCK inhibitor.
- Further investigation into HCK inhibitor resistance is warranted for optimizing AML treatment strategies.
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