In Vitro Evolution Reveals a Single Mutation as Sole Source of Src-Family Kinase C-Helix-out Inhibitor Resistance.
Ravi K PatelYash K PatelThomas E SmithgallPublished in: ACS chemical biology (2020)
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.
Keyphrases
- tyrosine kinase
- epidermal growth factor receptor
- protein kinase
- acute myeloid leukemia
- crispr cas
- dna binding
- single cell
- working memory
- stem cells
- oxidative stress
- computed tomography
- gene expression
- mesenchymal stem cells
- molecular dynamics simulations
- transcription factor
- allogeneic hematopoietic stem cell transplantation
- magnetic resonance imaging
- magnetic resonance
- cell therapy
- bone marrow
- dna methylation
- climate change
- acute lymphoblastic leukemia
- small molecule
- crystal structure