Defects in apoptosis regulation are known to impact chemotherapy resistance and consequently refractoriness and relapse of acute myeloid leukemia (AML). We previously showed that apoptosis-resistant protein profile of AML blasts at diagnosis is associated with shorter disease-free survival. Specifically, by flow cytometry, we measured the expression of Bcl-2, Bcl-xL, Mcl-1 and Bax in leukemic cells and combined these parameters to define their anti-apoptosis index (AAI). Interestingly, the AAI of normal lymphocytes in the AML patients corresponded to the AAI of AML blasts obtained from the same patient, reaching values far outside the normal AAI range of lymphocytes. In addition, the AAI in both cell types displayed parallel changes during the course of therapy. This points to a role of microenvironment in regulation of apoptosis in bone marrow cells of AML patients. Therefore, the aim of the current study was to assess if apoptosis-resistant AML cells are able to regulate the AAI of apoptosis-sensitive cells by influencing the microenvironment, as well as to perform molecular dissection of microenvironment, to identify novel proteins that regulate apoptosis. First, we showed that apoptosis-resistant AML blasts (high AAI) release factors that modulate sensitive AML blasts (low AAI) to upregulate Bcl-2 and become apoptosis-resistant. In the majority of cases (10 out of 14), Bcl-2 expression was significantly increased in apoptosis-sensitive AML blasts upon contact culture with apoptosis-resistant AML blasts (1.7-fold; p=0.0067). To characterize the AML microenvironment, conditioned medium (18 hrs) from patient samples displaying either apoptosis-resistant (n=5) or apoptosis-sensitive profile (n=6) were collected. Using mass spectrometry-based proteomics, comparative analysis was performed on these secretomes. Strikingly, we found that the major functional protein clusters upregulated in secretomes of the apoptosis-resistant AML were involved in mRNA splicing, protein translation and chromatin remodeling/chromosome organization. We further compared protein profiles of the soluble secretome and the extracellular vesicle fraction of a high AAI patient to those of a low AAI patient. Proteomic analysis of these fractions of the conditioned medium showed that the functional protein networks found in the whole secretome are well-represented in extracellular vesicles that are enriched for exosome markers. Transfer of functional proteins between cells by extracellular vesicles is a well documented phenomenon. Therefore, it is conceivable that the regulatory protein networks detected in the vesicles excreted by AML blasts are involved in regulation of apoptosis-related proteins in recipient AML blasts and other cells residing in the bone marrow, thereby contributing to therapy resistance.
|Publication status||Published - 2014|