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

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 nov...

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Autores principales:	Oksana Montecchini, Stefania Braidotti, Raffaella Franca, Giulia Zudeh, Christian Boni, Claudio Sorio, Eleonora Toffoletti, Marco Rabusin, Alberto Tommasini, Giuliana Decorti, Gabriele Stocco
Formato:	article
Lenguaje:	EN
Publicado:	Frontiers Media S.A. 2021
Materias:	ABL1-class BCR-ABL1 like acute lymphoblastic leukemia ABL1 peptide biosensor in vitro ELISA assay ABL1 tyrosin kinase inhibitors precision therapy Therapeutics. Pharmacology RM1-950
Acceso en línea:	https://doaj.org/article/b3cbf4c74ffc49329cb1727d6d00ffbb
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spelling	oai:doaj.org-article:b3cbf4c74ffc49329cb1727d6d00ffbb2021-11-19T07:53:58ZA Novel ELISA-Based Peptide Biosensor Assay for Screening ABL1 Activity in vitro: A Challenge for Precision Therapy in BCR-ABL1 and BCR-ABL1 Like Leukemias1663-981210.3389/fphar.2021.749361https://doaj.org/article/b3cbf4c74ffc49329cb1727d6d00ffbb2021-11-01T00:00:00Zhttps://www.frontiersin.org/articles/10.3389/fphar.2021.749361/fullhttps://doaj.org/toc/1663-9812The 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.Oksana MontecchiniStefania BraidottiRaffaella FrancaGiulia ZudehChristian BoniClaudio SorioEleonora ToffolettiMarco RabusinAlberto TommasiniAlberto TommasiniGiuliana DecortiGiuliana DecortiGabriele StoccoFrontiers Media S.A.articleABL1-class BCR-ABL1 like acute lymphoblastic leukemiaABL1 peptide biosensorin vitro ELISA assayABL1 tyrosin kinase inhibitorsprecision therapyTherapeutics. PharmacologyRM1-950ENFrontiers in Pharmacology, Vol 12 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	ABL1-class BCR-ABL1 like acute lymphoblastic leukemia ABL1 peptide biosensor in vitro ELISA assay ABL1 tyrosin kinase inhibitors precision therapy Therapeutics. Pharmacology RM1-950
spellingShingle	ABL1-class BCR-ABL1 like acute lymphoblastic leukemia ABL1 peptide biosensor in vitro ELISA assay ABL1 tyrosin kinase inhibitors precision therapy Therapeutics. Pharmacology RM1-950 Oksana Montecchini Stefania Braidotti Raffaella Franca Giulia Zudeh Christian Boni Claudio Sorio Eleonora Toffoletti Marco Rabusin Alberto Tommasini Alberto Tommasini Giuliana Decorti Giuliana Decorti Gabriele Stocco 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
description	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.
format	article
author	Oksana Montecchini Stefania Braidotti Raffaella Franca Giulia Zudeh Christian Boni Claudio Sorio Eleonora Toffoletti Marco Rabusin Alberto Tommasini Alberto Tommasini Giuliana Decorti Giuliana Decorti Gabriele Stocco
author_facet	Oksana Montecchini Stefania Braidotti Raffaella Franca Giulia Zudeh Christian Boni Claudio Sorio Eleonora Toffoletti Marco Rabusin Alberto Tommasini Alberto Tommasini Giuliana Decorti Giuliana Decorti Gabriele Stocco
author_sort	Oksana Montecchini
title	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
title_short	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
title_full	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
title_fullStr	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
title_full_unstemmed	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
title_sort	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
publisher	Frontiers Media S.A.
publishDate	2021
url	https://doaj.org/article/b3cbf4c74ffc49329cb1727d6d00ffbb
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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

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