Successful Preservation of Native BCR::ABL1 in Chronic Myeloid Leukemia Primary Leukocytes Reveals a Reduced Kinase Activity

Christian Boni; Massimiliano Bonifacio; Marzia Vezzalini; Luigi Scaffidi; Luisa Tomasello; Laurie L Parker; Diego Boscarino; Dino Paladin; Mauro Krampera; Claudio Sorio

doi:10.3389/fonc.2022.904510

Successful Preservation of Native BCR::ABL1 in Chronic Myeloid Leukemia Primary Leukocytes Reveals a Reduced Kinase Activity

Front Oncol. 2022 Jun 8:12:904510. doi: 10.3389/fonc.2022.904510. eCollection 2022.

Affiliations

¹ Department of Medicine, General Pathology Section, University of Verona, Verona, Italy.
² Department of Medicine, Hematology Section, University of Verona, Verona, Italy.
³ Department of Biochemistry, Molecular Biology and Biophysics, Masonic Cancer Center, University of Minnesota, Minneapolis, United States.
⁴ AB Analitica, Padova, Italy.

Abstract

Chronic myeloid leukemia (CML) is a myeloproliferative disease caused by the acquisition of t(9;22) generating the fusion tyrosine kinase BCR::ABL1. However, despite the crucial role of this protein in the dysregulation of numerous signal transduction pathways, a direct measure of BCR::ABL1 kinase activity in chronic phase (CP) CML was never accomplished due to intense degradative activity present in mature leukocytes. Therefore, we developed a procedure suitable to preserve BCR::ABL1 protein under non-denaturing, neutral pH conditions in primary, chronic phase (CP)-CML samples. As a result, specific kinase activity was detected utilizing a biotinylated peptide substrate highly selective for c-ABL1. Furthermore, through this approach, BCR::ABL1 kinase activity was barely detectable in CP-CML compared to Ph⁺ acute lymphoblastic leukemia primary samples, where kinase activity is comparable to those measured in Ph⁺ cell lines. These in vitro findings provide the first direct measure of BCR::ABL1 kinase activity in primary CP-CML and reveal the presence of a still uncharacterized inhibitory mechanism that maintains BCR::ABL1 in a low activity state in CP-CML despite its overexpression.

Keywords: Bcr Abl; acute lymphocytic leukemia; chronic myelogenous leukemia; imatinib (Gleevec); kinase activity; kinase assay; philadelphia chromosome; tyrosine kinase.