A Novel ELISA-Based Peptide Biosensor Assay for Screening ABL1 Activity : A Challenge for Precision Therapy in BCR-ABL1 and BCR-ABL1 Like Leukemias.

The pathogenic role of the overactivated ABL1 tyrosine kinase (TK) pathway is well recognized in some forms of like acute lymphoblastic leukemia (ALL); TK inhibitors represent a useful therapeutic choice in these patients who respond poorly to conventional chemotherapy. Here we report a novel peptide biosensor (P)-ELISA assay to investigate ABL1 activity in four immortalized leukemic cell lines with different genetic background. The P sequence comprises an ABL1 tyrosine (Y) phosphorylation site and a targeting sequence that increases the specificity for ABL1; additional peptides (Y-site-mutated (P-) and fully-phosphorylated (P-) biosensors) were included in the assay. After incubation with whole cell lysates, average P phosphorylation was significantly increased (basal vs. P phosphorylation: 6.84 ± 1.46% vs. 32.44 ± 3.25%, -value < 0.0001, two-way ANOVA, Bonferroni post-test, percentages relative to P- in each cell line). Cell lines expressing ABL1-chimeric proteins (K562, ALL-SIL) presented the higher TK activity on P; a lower signal was instead observed for NALM6 and REH ( < 0.001 and < 0.05 vs. K562, respectively). Phosphorylation was ABL1-mediated, as demonstrated by the specific inhibition of imatinib ( < 0.001 for K562, NALM6, ALL-SIL and < 0.01 for REH) in contrast to ruxolitinib (JAK2-inhibitor), and occurred on the ABL1 Y-site, as demonstrated by P whose phosphorylation was comparable to basal levels. In order to validate this novel P-ELISA assay on leukemic cells isolated from patient's bone marrow aspirates, preliminary analysis on blasts derived from an adult affected by chronic myeloid leukaemia ( positive) and a child affected by ALL ( negative) were performed. Phosphorylation of P was specifically inhibited after the incubation of positive cell lysates with imatinib, but not with ruxolitinib. While requiring further optimization and validation in leukemic blasts to be of clinical interest, the P-based ELISA assay provides a novel tool for screening both the aberrant ABL1 activity in like ALL leukemic cells and their potential response to TK inhibitors.