Expression and Prognostic Significance of the Inhibitor of Apoptosis Protein (IAPs) Family and Its Antagonists in Chronic Lymphocytic Leukemia
Authors: Grzybowska-Izydorczyk Olga , Cebula Barbara , Robak Tadeusz , Smolewski Piotr 
Published: April 2020

Abstract
The apoptotic mode of cell death is a major regulatory process in all complex organisms. The slow accumulation of malignant cells in chronic lymphocytic leukemia (CLL) suggests that this disease is caused by a defect in apoptosis regulation. Apoptosis, defined as a programmed cell death, is executed through activity of caspases, cysteine proteases which are regulated by a number of pro- and anti-apoptotic proteins. One of such checkpoints is a control of caspase activation by the family of inhibitor of apoptosis proteins (IAPs). So far eight IAP proteins have been identified in human. They were XIAP, cIAP1, cIAP2, ILP-2, NAIP, survivin and BRUCE/Apollon. Moreover, an important role in the regulation of apoptosis play also three proteins which bind to IAPs and inhibit their activity, such as Smac/DIABLO, HtrA2/Omi and XAF1. Until recently IAPs and their antagonists were poorly researched in CLL. The majority of them were even not investigated in this disease yet. The primary aim of the study was to perform a complex analysis of expression of the IAPs family proteins and their antagonists in leukemic cells (ex vivo) in untreated CLL patients in comparison to the healthy controls. In this study we have also aimed to assess differences in expression of these proteins between patients with stable(SD) and progressive disease (PD). We tried to establish a potential prognostic significance of the IAPs expression and to investigate their correlation with the clinical course of the disease. The last aim was to analyze the expression of these proteins in the cell culture (in vitro settings) in response to the drugs routinely used in CLL. One hundred untreated patients with CLL were included in the study. Patients were divided into two groups: with SD (n=52) and PD (n=48). Twenty seven healthy donors served as a control group. Expression of IAPs and their inhibitors were investigated by flow cytometry, after careful comparative studies by Western blot and fluorescence microscopy. Protein expression was assessed on the basis of the mean fluorescence intensity (MFI). Results were compared with several disease-related parameters, such as clinical stage according to Rai, lymphocyte count, ZAP-70 and CD38 expression or serum beta-2-microglobulin concentration. The secondary endpoint of the study was to compare the results with the outcome of examined patients. Our study revealed significantly higher expression of cIAP1 (median MFI level: 81.1 vs 8.2; p=0.000001) and cIAP2 (median MFI level: 313.5 vs 146.8; p=0.014) and lower expression of IAP-antagonist - Smac/DIABLO (median MFI level: 147.9 vs 301.3; p=0.010) in the group of untreated CLL patients in comparison to the control group. Furthermore, patients with PD showed significantly higher expression of all analyzed IAPs compared with those with SD. Median MFI level for cIAP1 were: 131.2 vs 60.1; p=0.002, for cIAP2: 434.3 vs 301.5; p=0.026, for XIAP: 463.7 vs 225.4; p=0.002 and for survivin: 119.6 vs 38.6; p=0.00006. We also found a positive correlation between all examined protein’s expression and clinical stage according to Rai and high lymphocytosis, but only XIAP correlated with beta-2-microglobulin concentration (p=0.017). Moreover the study showed that simultaneous expression of two or more IAPs is associated with shorter overall survival. Additionally, analyzing the expression of IAPs and their antagonists in the cell cultures (in vitro) in response to cytotoxic drugs (Cladribine, Fludarabine, Mafosfamide) we observed significantly lower level of cIAP1, cIAP2 and XIAP as well as a higher level of proapoptotic Smac/DLABLO in comparison to the control, cytostatic-free cultures. In conclusion, CLL cells are characterized by overexpression of cIAP1 and cIAP2 and downregulation of Smac/DIABLO. The anti-apoptotic pattern of these apoptosis-regulating proteins expression may be responsible for pathological accumulation of malignant cells in CLL. Moreover, expression of all IAP proteins is significantly elevated in patients with PD, what indicates a role of inhibited proclivity of CLL cells to undergo spontaneous apoptosis in the disease progression. Importantly, the co-expression of two or more IAP proteins in tumor cell seems to be unfavorable prognostic factor in CLL patients. Thus, all those data suggest that IAPs/IAP-antagonists system plays an important role in the biology of CLL and further studies to confirm their prognostic usefulness are warranted.