Exosomes Secreted by Apoptosis-Resistant Acute Myeloid Leukemia (AML) Blasts Harbor Regulatory Network Proteins Potentially Involved in Antagonism of Apoptosis

Abstract

myeloid leukemia (AML) blasts at diagnosis is associated with disease-free survival. We previously found that the initially high apoptosis-resistance of AML cells decreased after therapy, while regaining high levels at relapse. Herein, we further explored this aspect of dynamic apoptosis regulation in AML. First, we showed that the intraindividual ex vivo apoptosis-related profiles of normal lymphocytes and AML blasts within the bone marrow of AML patients were highly correlated. The expression values of apoptosisregulating proteins were far beyond healthy control lymphocytes, which implicates the influence of microenvironmental factors. Second, we demonstrated that apoptosis-resistant primary AML blasts, as opposed to apoptosis-sensitive cells, were able to up-regulate BCL-2 expression in sensitive AML blasts in contact cultures (p ‫؍‬ 0.0067 and p ‫؍‬ 1.0, respectively). Using secretome proteomics, we identified novel proteins possibly engaged in apoptosis regulation. Intriguingly, this analysis revealed that major functional protein clusters engaged in global gene regulation, including mRNA splicing, protein translation, and chromatin remodeling, were more abundant (p ‫؍‬ 4.01E-06) in secretomes of apoptosis-resistant AML. These findings were confirmed by subsequent extracellular vesicle proteomics. Despite good remission rates observed in acute myeloid leukemia (AML) patients, the 5-year event-free survival rates reach only 35-40% in adults and 60 -70% in children (1, 2). Apoptosis is one of the crucial mechanisms influencing survival of AML cells, and its deregulation can possibly lead to chemotherapy resistance and eventually relapse (3-5). The ability of cells to undergo apoptosis is largely defined by the relative expression of anti-(i.e. BCL-2, BCL-X long isoform -BCL-XL, or MCL-1) and proapoptotic (i.e. BAX, BH3 interacting domain death agonist -BID, caspases) proteins. Several studies have shown that the levels of BCL-2 and BCL-2/BAX ratio are a determinant of apoptosis-resistance in AML blasts and are associated with survival in AML patients (3,6). We have previously demonstrated that the expression of several apoptosis-related proteins, such as BCL-2, BCL-XL, MCL-1, From the ‡Dept.

Vesicle-MaNiA: extracellular vesicles in liquid biopsy and cancer

“…Differences in EV-protein content from cancer patients have been described in several tumor types [3, 7, 24, 31, 56, 57]. The diagnostic potential of EV-protein content is well-illustrated in pancreatic ductal adenocarcinoma (PDAC) and melanoma.…”

Selecting Sample Preparation Workflows for Mass Spectrometry-Based Proteomic and Phosphoproteomic Analysis of Patient Samples with Acute Myeloid Leukemia

“…The authors used 1DE with polyacrylamide gels to resolve protein bands before in-gel digestion with trypsin [14]. The analysis of whole secretome from 11 (five with high anti-apoptosis index (AAI), six with low AAI) bone marrow samples of AML patients identified 1492 proteins whereas 4232 proteins were identified from extracellular vesicles prepared from the bone marrow samples of only two AML patients (one with high AAI, one with low AAI) from the first cohort using LTQ-FTMS and Q Exactive mass spectrometers (Thermo Fisher Scientific), respectively.…”

“…Optimized in-solution methods using trifluoroethanol as a protein denaturant [27] or guanidinium hydrochloride in the presence of TCEP and CAA [28] are attractive in-solution digestion alternatives that do not require additional protease and phosphatase inhibitors during cell lysis. Although 1DE is still of use for many MS-based proteomic studies (see [14] as an example in AML MS-based proteomics), an in-gel protocol that copolymerizes the entire protein sample with monomeric acrylamide and allows the depletion of denaturant agents used in the lysis buffer before proteolysis has been recently introduced as gel-aided sample preparation (GASP) [29]. The analogue acronym to FASP reflects similar working principles and workflows of both approaches.…”

“…However, proteomic and phosphoproteomic workflows are not universal and they might work differently depending on the characteristics of the sample and the experience of the researcher. Published global MS proteome and phosphoproteome analysis of AML patient samples have used the standard one-dimensional electrophoresis (1DE), 2DE and the in-solution digestion method with urea in the lysis buffer to digest AML proteins prior to MS analysis or phosphorylation enrichment [10,11,12,13,14]. We have recently tested several methods of sample preparation for the MS analysis of the AML blasts that included in-solution digestion methods with urea and guanidinium hydrochloride as denaturant reagents in the lysis buffer and filter-aided sample preparation (FASP) [15,16].…”

Extracelluláris vesiculák és hematológiai malignitásokban játszott szerepük

“…Az EV-k malignitásokban betöltött funkciójáról számos publikáció született, azonban ezek nagy része szolid tumoros kórképeket (például glioblastoma, medulloblastoma, melanoma) vizsgált [14,21]. Egészséges kontroll-…”