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 CML cells

Abstract

Background Chronic myeloid leukemia (CML) is a biphasic hematopoietic myeloproliferative disorder characterize by a single acquired genetic abnormality, translocation t(9;22) (q34;q11) occurred in hematopoietic stem cell (HSCs) that generate the Philadelphia chromosome (Ph).Resulted BCR-ABL1 protein is able to alter different pathways including self-renewing, proliferation, and differentiation factors that contribute to commence a myeloproliferative disease. The literature highlights the BCR-ABL1 activity independence in hematopoietic stem cells (LSC) which is often associated with a leukemic burden that can not be extinguished with tyrosine kinase inhibitors (TKIs). Conventional lysis buffers are not suitable to preserve BCR-ABL1 from peripheral blood mature leukocytes due to a fast degradative activity, which rapidly and permanently destroys BCR-ABL1. Different strategies were employed to inhibit BCR-ABL1 degradation in primary samples but no alternative provided a method able to save the kinase biochemical functionality. 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 set the condition for studying BCR-ABL1 expression and activity in early phase of disease when data on the regulation of the kinase are lacking due to a fast and strong degradative activity occurring when a non-denaturing lysis protocol is applied. To achieve our purpose we set up: 1) a robust kinase assay to discern specific BCR-ABL1 protein activity and 2) a protocol suitable to protect BCR-ABL1 protein from degradation in peripheral blood CML leukocytes. We tested several formulations and procedures on a CML model made of a 1/10 ratio of K562 cells mixed in peripheral blood of healthy donor; we found a new practice successful to preserve BCR-ABL1 protein and its activity under non-denaturing, neutral pH conditions. We then applied the procedure on Ph+ CP-CML and acute lymphoblastic leukemia (ALL) primary cell samples. In this work, we demonstrate for the first time an effective 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. Conclusion Our data suggest for the first time the presence of a still uncharacterized inhibitory mechanism that maintain BCR-ABL1 in a low active state in chronic phase of chronic myeloid leukemia, a result obtained through a new useful procedure for the study of this phenomenon.