Expression Modulation of Immune Inhibitory Molecules by Small Molecule Inhibitor Drugs in Leukemic Cells of Chronic Lymphocytic Leukemia

Abstract

Background: Targeted therapy with small molecule inhibitors (SMIs) has been considered a highly effective therapeutic strategy for chronic lymphocytic leukemia (CLL). However, there is little information on the detailed mechanisms and association of SMIs with immune evasion mechanisms. Objectives: This study examined the effects of signaling pathway inhibitors ibrutinib, idelalisib, duvelisib, and venetoclax on the expression of immune checkpoint ligands: Programmed death ligand 1 (PD-L1), galectin-9 (Gal-9), cluster of differentiation (CD)200, CD155, and herpes virus entry mediator (HVEM) in CLL leukemic cells. Methods: Leukemic cells were isolated from fifteen CLL patients using the magnetic activated cell sorting method, confirmed by flow cytometry, and then cultured and treated with different SMIs for 72 hours. The optimal doses of the applied SMIs for in-vitro treatment were determined by MTT assay. The mRNA expression was measured by real-time PCR assay using β-actin as a housekeeping gene. Results: The purity of isolated CLL leukemic cells was determined to be more than 97%, as confirmed by dual-color flow cytometry. Based on the IC50 results obtained from the MTT assay, the optimal doses of 5, 15.03, 1.07 μM, and 0.5 nM were determined for ibrutinib, idelalisib, duvelisib, and venetoclax, respectively. None of the SMI drugs showed changes in PD-L1 expression levels compared to the untreated group. Additionally, the level of Gal-9 mRNA expression was slightly decreased in all treated groups. The expression level of CD155 was downregulated only after treatment with venetoclax, and an upregulation was observed in the other treated groups. Finally, ibrutinib and idelalisib indicated a mild non-significant upregulation in HVEM gene expression. Conclusions: Altogether, the treatment of leukemic cells with different SMIs in this study indicated increased or decreased variations in the expression level of immune checkpoint inhibitory ligands in CLL. Therefore, these mechanisms should be considered for further treatment approaches, especially for combinational strategies.