Abstract
Chronic Myeloid Leukemia (CML) is a stem cell disease sustained by a rare population of kinetic quiescent cells, frequently resistant to tyrosine kinase inhibitors (TKIs). The Bcr-Abl oncogene and the resulting fusion protein, in fact, activates multiple cross-talking signal transduction pathways (STP), such as RAS/MEK/ERK, PI3K/Akt, Wnt and STAT5, potentially contributing to TKIs drug resistance. Since increasing evidence reports the cooperation of numerous STP in the control of cell proliferation and survival in CML, the aim of this project was to analyze, at the protein level, the expression and activation profile of various STP involved in the mechanisms of cell proliferation and survival of CML CD34+ cells, as compared to different sources of normal CD34+ cells. CD34+ cells were purified by immunomagnetic separation from peripheral blood (PB) of 7 newly diagnosed chronic phase (CP) CML patients and compared to the normal counterpart obtained from normal bone marrow of three healthy donors (NBM) and/or from umbilical cord blood (CB) of three donors. The phosphorylation status of 46 different proteins belonging to numerous STP and the expression of 32 proteins of the apoptotic machinery were assessed by using a customized direct phase proteome profiler antibody array. The resulting dots were visualised using ECL and quantified by densitometric analysis. CP-CML samples were obtained from patients with WBC counts ranging between 41,900 to 421,400; Sokal score resulted intermediate in six patients and low in one. The comparison between the phospho-proteomic profile of CP-CML CD34+ cells and NBM CD34+ cells showed that the former are characterized by: 1) lower phosphorylation of STAT2 (p=0.023), Chk-2 (p=0.036), a serine/threonine-protein kinase required for checkpoint-mediated cell cycle arrest, and tyrosine kinases of the Src family - Lck, Fyn, Src, particularly Yes (p=0.04) - involved in the regulation of growth and cell survival; 2) higher phosphorylation of p53, both at Ser15 (p=0.047) and at Ser46 (p=0.039), p70S6 kinase (p=0.035), RSK (p=0.046), a mediator of mitogens- and stress-induced activation of several transcription factors, and Pyk-2 (p=0.036), a tyrosine kinase involved in cell adhesion and migration. The analysis of the 32 apoptotic proteins revealed that CD34+ cells from CP-CML, compared to CD34+ cells from NBM, are characterized by: 1) lower expression of the catalase (p=0.044), an enzyme that protects cells from the toxic effects of hydrogen peroxide and promotes growth of normal and neoplastic cells including myeloid leukemia cells; 2) higher expression of some members of the heat shock proteins family - HSP60 and HSP70 (p=0.033). We then compared CD34+ cells obtained from CP-CML with the other normal CD34+ cell source represented by the CB. The proteomic profile indicated a remarkable similarity between the CD34+ from CP-CML and those from CB. Accordingly, the two normal CD34+ cells sources showed some differences: in particular, as for CP-CML CD34+ cells, those from CB had significantly lower phosphorylation of STAT2 (p=0.026), of Chk-2 (p=0.014) and higher phosphorylation of p53 at Ser15 (p=0.05), compared to NBM CD34+ cells. In summary, we reported that CD34+ cells from CP-CML are characterized by a proteomic and phospho-proteomic profile that promotes quiescence status through the inhibition of proliferation. A striking similarity was found between CD34+ cells obtained from CP-CML and those from CB. The two normal sources of CD34 displayed differences in the activation status of selected proteins. The presence of these additional and complex changes in the signaling network of CP-CML must be taken into account for the investigation on novel targeted therapies.
Disclosures:
Castagnetti: Novartis Farma : Consultancy, Honoraria; Bristol Myers Squibb : Consultancy, Honoraria. Rosti:Bristol Myers Squibb : Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; Novartis Pharma: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding, Speakers Bureau; Roche: Speakers Bureau; Pfizer: Speakers Bureau.
Knockdown of ADORA2A antisense RNA 1 inhibits cell proliferation and enhances imatinib sensitivity in chronic myeloid leukemia
