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

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.

Impact of Mercaptopurine Metabolites on Disease Outcome in the AIEOP‐BFM ALL 2009 Protocol for Acute Lymphoblastic Leukemia

“…Advances in ALL therapy includes the development of targeted therapy, such as tyrosine kinase inhibitors, and the introduction of immunotherapies 1 . The promise of these novel approaches is to improve cure rates, to overcome chemotherapy resistance, and, being target‐specific, to confer limited toxicity to normal tissues 33–36 . Pharmacological strategies, including the analysis of the proactive monitoring of drug metabolites and important predictive pharmacogenetic markers, could represent additional valuable tools for a rational use of “old” drugs to personalize therapy with the same purposes.…”

Hematological, clinical, immunophenotypic characterization, and treatment outcomes of prognostically significant genetic subtypes of B‐lineage acute lymphoblastic leukemia: A report of 1021 patients from India

“…BCR::ABL1 ‐like ALL is considered an entity in the 2022 World Health Organization classification of hematolymphoid neoplasms 10,11 . This entity is specified by a similar gene expression profile to that of Philadelphia (Ph)‐positive ALL cases, without expressing BCR::ABL1 fusion transcripts originating from Ph‐chromosome and additionally associated with poorer clinical outcomes 5–9,12–37 …”

Kinase Sensing Based on Protein Interactions at the Catalytic Site

“…Accordingly, abnormal kinase activity is closely connected to many diseases, including various types of cancer [2] and kinases are considered to be promising cancer biomarkers [3,4] . Many analytical methods have been developed for kinase activity quantification as prognostic tools, [5,6] including radiometric, [7,8] spectroscopic [9–11] and immunological tools [12,13] . Electrochemical methods were also developed for kinase detection, [14–18] and as all electrochemical tools they have the distinct advantages of relatively low cost, easy operation, rapid response and high sensitivity [19] .…”