a-419259 and Leukemia--Myeloid--Acute

Unregulated protein-tyrosine kinase signaling is a common feature of AML, often involving mutations in Flt3 and overexpression of myeloid Src-family kinases (Hck, Fgr, Lyn). Here we show that high-level expression of these Src kinases predicts poor survival in a large cohort of AML patients. To test the therapeutic benefit of Flt3 and Src-family kinase inhibition, we used the pyrrolopyrimidine kinase inhibitor A-419259. This compound potently inhibits Hck, Fgr, and Lyn as well as Flt3 bearing an activating internal tandem duplication (ITD). Flt3-ITD expression sensitized human TF-1 myeloid cells to growth arrest by A-419259, supporting direct action on the Flt3-ITD kinase domain. Cells transformed with the Flt3-ITD mutants D835Y and F691L were resistant to A-419259, while co-expression of Hck or Fgr restored inhibitor sensitivity to Flt3-ITD D835Y. Conversely, Hck and Fgr mutants with engineered A-419259 resistance mutations decreased sensitivity of TF-1/Flt3-ITD cells. To investigate de novo resistance mechanisms, A-419259-resistant Flt3-ITD+ AML cell populations were derived via long-term dose escalation. Whole exome sequencing identified a distinct Flt3-ITD kinase domain mutation (N676S/T) among all A-419259 target kinases in each of six independent resistant cell populations. These studies show that Hck and Fgr expression influences inhibitor sensitivity and the pathway to acquired resistance in Flt3-ITD+ AML.

Topics: Amino Acid Substitution; Cell Line, Tumor; Drug Resistance, Neoplasm; Exome Sequencing; fms-Like Tyrosine Kinase 3; Gene Expression Regulation, Developmental; Gene Expression Regulation, Leukemic; Humans; Leukemia, Myeloid, Acute; Mutation, Missense; Prognosis; Protein Kinase Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-hck; Pyrimidines; Pyrroles; src-Family Kinases

A series of novel pyrrolo[2,3-d]pyrimidines were synthesized by introducing 15 different amino acids to 7-cyclohexyl-5-(4-phenoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidine-4-amine. Compounds with potent activities against HCK and FLT3-ITD were evaluated in viability studies with acute myeloid leukemia cell line MV4-11. Our structure activity relationship analyses lead to the identification of compound 31, which exhibited potent HCK and FLT3-ITD inhibition and activity against the MV4-11 cell line.

Topics: Apoptosis; Binding Sites; Cell Line, Tumor; Crystallography, X-Ray; fms-Like Tyrosine Kinase 3; Humans; Leukemia, Myeloid, Acute; Molecular Docking Simulation; Protein Kinase Inhibitors; Protein Structure, Tertiary; Proto-Oncogene Proteins c-hck; Pyrimidines; Pyrroles; Structure-Activity Relationship; Thermodynamics