Boni Christian, Bonifacio Massimiliano, Vezzalini Marzia, Scaffidi Luigi, Tomasello Luisa, Parker Laurie L, Boscarino Diego, Paladin Dino, Krampera Mauro, Sorio Claudio
Department of Medicine, General Pathology Section, University of Verona, Verona, Italy.
Department of Medicine, Hematology Section, University of Verona, Verona, Italy.
Front Oncol. 2022 Jun 8;12:904510. doi: 10.3389/fonc.2022.904510. eCollection 2022.
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 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.
慢性髓性白血病（CML）是一种骨髓增殖性疾病，由获得t(9;22)产生融合酪氨酸激酶BCR::ABL1所致。然而，尽管该蛋白在众多信号转导通路失调中起关键作用，但由于成熟白细胞中存在强烈的降解活性，慢性期（CP）CML中BCR::ABL1激酶活性的直接测定一直未能实现。因此，我们开发了一种程序，适用于在非变性、中性pH条件下保存原发性慢性期（CP）-CML样本中的BCR::ABL1蛋白。结果，利用对c-ABL1具有高度选择性的生物素化肽底物检测到了特异性激酶活性。此外，通过这种方法，与Ph急性淋巴细胞白血病原发性样本相比，CP-CML中几乎检测不到BCR::ABL1激酶活性，在Ph急性淋巴细胞白血病原发性样本中，激酶活性与在Ph细胞系中测得的活性相当。这些发现首次直接测定了原发性CP-CML中BCR::ABL1激酶活性，并揭示了一种仍未被表征的抑制机制的存在，该机制使CP-CML中的BCR::ABL1处于低活性状态，尽管其过度表达。
