Abstract
Expression of apoptosis-regulating proteins (B-cell CLL/lymphoma 2 - BCL-2, Myeloid Cell Leukemia 1 - MCL-1, BCL-2 like 1 - BCL-X and BCL-2-associated X protein - BAX) in acute 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 intraindividualex vivoapoptosis-related profiles of normal lymphocytes and AML blasts within the bone marrow of AML patients were highly correlated. The expression values of apoptosis-regulating 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 andp= 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. Finally, confocal-microscopy-based colocalization studies show that splicing factors-containing vesicles secreted by high AAI cells are taken up by low AAI cells. The current results constitute the first comprehensive analysis of proteins released by apoptosis-resistant and sensitive primary AML cells. Together, the data point to vesicle-mediated release of global gene regulatory protein clusters as a plausible novel mechanism of induction of apoptosis resistance. Deciphering the modes of communication between apoptosis-resistant blasts may in perspective lead to the discovery of prognostic tools and development of novel therapeutic interventions, aimed at limiting or overcoming therapy resistance.
Highlights
From the ‡Dept. of Pediatric Oncology/Hematology, §Dept. of Hematology, ¶OncoProteomics Laboratory, Dept. of Medical Oncology, Dept. of Rheumatology, VUmc-Cancer Center Amsterdam, VU University Medical Center, De Boelelaan 1117, 1081HV Amsterdam, The Netherlands; **Dept. of Physiology, ICaR-VU, VU University Medical Center, De Boelelaan 1117, 1081HV Amsterdam, The Netherlands; ‡‡Dept. of Biology, Fred Wyszkowski Cancer Research Laboratory, Faculty of Biology, Technion-Israel, Institute of Technology, Haifa 3200003, Israel
The expression levels and subsequent anti-apoptosis index (AAI) were examined for correlation between lymphocytes and blasts from 61 acute myeloid leukemia (AML) specimens as well as in 10 normal bone marrow samples (Fig. 1A)
This observation is supported by our earlier findings, which prove that elevated expression of BCL-2 and BCL-X is associated with higher rates of spontaneous apoptosis in vitro [39]
Summary
MRD can be detected at a low frequency in bone marrow (BM) at the time of remission and is thought to contain the relapse-initiating cells (8 –10) These observations imply that leukemic cells that harbor an apoptosis-resistant protein profile at diagnosis can better survive chemotherapy, thereby eventually causing a relapse. The values of the AAI profile increased again at relapse, indicating apoptosis-resistance [11] Based on these unexpected findings, we hypothesized that expression of apoptosis-related proteins in AML blasts, and possibly in bystander cells in the bone marrow, is regulated by extracellular factors present in the AML microenvironment. Tumor cell communication with its microenvironment is emerging as an important determinant playing multiple roles in cancer In this respect, both soluble factors and extracellular vesicles (EVs), most notably exosomes, have been shown to influence cellular processes of malignant and normal cells in the tumor microenvironment [12,13,14]. The aim of our current study was to characterize the microenvironment produced by apoptosisresistant AML blasts in terms of its capacity to influence apoptosis regulation in neighboring cells and protein content
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