Abstract
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 (PABL)-ELISA assay to investigate ABL1 activity in four immortalized leukemic cell lines with different genetic background. The PABL sequence comprises an ABL1 tyrosine (Y) phosphorylation site and a targeting sequence that increases the specificity for ABL1; additional peptides (Y-site-mutated (PABL-F) and fully-phosphorylated (PPHOSPHO-ABL) biosensors) were included in the assay. After incubation with whole cell lysates, average PABL phosphorylation was significantly increased (basal vs. PABL phosphorylation: 6.84 ± 1.46% vs. 32.44 ± 3.25%, p-value < 0.0001, two-way ANOVA, Bonferroni post-test, percentages relative to PPHOSPHO-ABL in each cell line). Cell lines expressing ABL1-chimeric proteins (K562, ALL-SIL) presented the higher TK activity on PABL; 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 PABL-F whose phosphorylation was comparable to basal levels. In order to validate this novel PABL-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 PABL 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 PABL-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.
Highlights
In clinics, a sensitive, quick, convenient and versatile detection method to measure aberrant kinase activity is desirable for many pathologies, including various forms of leukemias, and could be important to improve diagnosis and treatment
Four human leukemia cell lines with different genetic background were selected and initially characterized by western blot to confirm the presence of candidate chimeric proteins: NALM6 and acute lymphoblastic leukemia (ALL)-SIL cells were chosen because they harbor the gene fusions ETV6-PDGFRB (Matheson and Hall, 2003) and NUP214ABL1 respectively, both belonging to the BCR-ABL1 like aberrations and both leading to ABL1 pathway overactivation (Zhou and Yang, 2014)
Because of the t(9;22) rearrangement encoding BCR-ABL1, K562 was used as control model for the PABL-based ELISA assay
Summary
A sensitive, quick, convenient and versatile detection method to measure aberrant kinase activity is desirable for many pathologies, including various forms of leukemias, and could be important to improve diagnosis and treatment. In ALL, BCRABL1 like forms exist [10–15% of B-ALL pediatric cases, incidence increased with age (Roberts et al, 2014a)], presenting blasts that are transcriptionally related to BCRABL1 expressing cells althought they lack the t(9;22) transloctation. These forms are recognized as a high-risk ALL subtype across all clinical studies, being characterized by an unfavorable prognosis and a higher relapse rate in comparison to other B-ALL subtypes (Roberts et al, 2014a; Boer et al, 2015). A third, smaller group is characterized by involvement of genetic fusions that lead to altered TK in the RAS pathways (Ofran and Izraeli, 2017)
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