BACKGROUND Chronic myeloid leukemia (CML) is a myeloproliferative disease caused by acquisition of t(9;22) translocation (resulting in Philadelphia chromosome) occurring in a hematopoietic stem cell and transforming it into a leukemic stem cell (LSC) giving rise to a neoplastic clone. The genetic rearrangement lead to the constitutive expression of the fusion tyrosine kinase BCR-ABL1 that alters numerous signal transduction usually governed by growth factors and cytokines. Literature bring back a fast degradative activity present in peripheral blood mature leukocytes, which massively destroys BCR-ABL1 when non-denaturing lysis conditions are applied thus preventing an accurate protein analysis. Data derived from a direct measure of kinase activity in chronic phase (CP) CML are not present in the literature therefore data on the regulation of its enzymatic activity in early phase of the disorder are incomplete. METHODS Cell lysis, Immunoprecipitation, Kinase Assay using a phosphopeptide and an anti-phosphotyosine assay (ELISA format). Western blotting with anti-ABL and pABL antibodies. RESULTS We aim studying BCR-ABL1 kinase activity in early phase of the disease when data are limited. To achieve our purpose we needed to solve the fast degradation activity present in peripheral blood CML leukocytes and set up a robust kinase assay enabling us to discern specific BCR-ABL1 protein activity. We addressed the first issue creating a CML model obtained mixing healthy donor blood cell with K562 cell line (ratio 10:1) to simulate CML in a peripheral blood context. We then tested several formulations and procedures before lysis and eventually succeeded to preserve BCR-ABL1 protein under non-denaturing, neutral pH conditions. The result was confirmed by western blot and immunoprecipitation techniques. Kinase assay was performed utilizing ELISA format assay based on a biotinylated peptidesubstrate highly selective for c-Abl1. We then applied whole procedure on Ph+ CP-CML and Ph+ acute lymphoblastic leukemia (ALL) primary cell samples. In this work, I demonstrate for the first time a successful protection of BCR-ABL1 from degradation in the peripheral blood of leukemic patients and the parallel measurement of protein kinase activity in the same samples. Surprisingly we found that tyrosine kinase activity is strongly impaired in CML while activity was readily detectable in Ph+ ALL-derived leukocytes. CONCLUSIONS We have established a new protocol permitting for the first time the direct measure of BCR/ABL1 enzymatic activity in chronic phase CML and applicable in other cellular contexts. Thanks to this original approach we provide the evidence of a novel, still uncharacterized, inhibitory mechanism that maintain BCR-ABL1 in a low active state in chronic phase of chronic myeloid leukemia.
|Titolo:||Successful preservation of BCR-ABL1 protein and direct measure of kinase activity in peripheral blood of CML and Ph+ ALL patients unveil a kinase inhibitory activity present in chronic phase CML cells|
BONI, CHRISTIAN (Corresponding)
|Data di pubblicazione:||2021|
|Appare nelle tipologie:||07.13 Doctoral Thesis|