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The pathogenic role of the overactivated ABL1 tyrosine kinase (TK) pathway is well recognized in some forms of  BCR-ABL1  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 ABL )-ELISA assay to investigate ABL1 activity in four immortalized leukemic cell lines with different genetic background. The P ABL  sequence comprises an ABL1 tyrosine (Y) phosphorylation site and a targeting sequence that increases the specificity for ABL1; additional peptides (Y-site-mutated (P ABL - F ) and fully-phosphorylated (P PHOSPHO - ABL ) biosensors) were included in the assay. After incubation with whole cell lysates, average P ABL  phosphorylation was significantly increased (basal vs. P ABL  phosphorylation: 6.84 ± 1.46% vs. 32.44 ± 3.25%,  p -value < 0.0001, two-way ANOVA, Bonferroni post-test, percentages relative to P PHOSPHO - ABL  in each cell line). Cell lines expressing ABL1-chimeric proteins (K562, ALL-SIL) presented the higher TK activity on P ABL ; a lower signal was instead observed for NALM6 and REH ( p  < 0.001 and  p  < 0.05 vs. K562, respectively). Phosphorylation was ABL1-mediated, as demonstrated by the specific inhibition of imatinib ( p  < 0.001 for K562, NALM6, ALL-SIL and  p  < 0.01 for REH) in contrast to ruxolitinib (JAK2-inhibitor), and occurred on the ABL1 Y-site, as demonstrated by P ABL-F  whose phosphorylation was comparable to basal levels. In order to validate this novel P ABL -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 ( BCR-ABL1  positive) and a child affected by ALL ( BCR-ABL1  negative) were performed. Phosphorylation of P ABL  was specifically inhibited after the incubation of  BCR-ABL1  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 ABL -based ELISA assay provides a novel  in vitro  tool for screening both the aberrant ABL1 activity in  BCR-ABL1  like ALL leukemic cells and their potential response to TK inhibitors.

frontiers in pharmacology

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