Abstract
Abstract Introduction: Relapse and drug resistance of acute myeloid leukemia (AML) are the main challenges in clinical treatment, and the root cause lies in the survival advantage and drug resistance protection of leukemia cells anchoring to the bone marrow microenvironment (BMME) through adhesion molecules. ICAM-2 (CD102) is an important adhesion molecule of immunoglobulin superfamily, which mediates cell-cell and cell-matrix interactions. Our previous study showed that ICAM-2 was significantly highly expressed in bone marrow blasts of AML patients and a variety of AML cell lines, and its expression level was associated with poor patient prognosis. The aim of this study was to investigate whether ICAM-2 activates downstream survival signaling pathways by mediating the adhesion of AML cells to BMME components (especially bone marrow stromal cells), ultimately leading to chemoresistance.
Methods: ICAM-2 expression was stably knocked down in Molm-13 and OCI-AML3 cells using lentiviral shRNA. An in vitro co-culture model of AML cells with bone marrow stromal cell lines was constructed to mimic BMME. The effect of ICAM-2 knockdown on the adhesion ability of AML cells to stromal cells was assessed by flow cytometry and fluorescence microscopy. CCK-8 and flow cytometry (Annexin V/PI staining) were used to detect changes in the sensitivity of AML cells to cytarabine (Ara-C) and daunorubicin after ICAM-2 knockdown under monoculture and co-culture conditions. The expression levels of key pro-survival/resistance signaling pathways (PI3K/AKT/mTOR, NF-κB) and anti-apoptotic proteinsin AML cells under ICAM-2 knockdown and co-culture conditions were analyzed by Western blot. Specific inhibitors were utilized to validate the role of the relevant pathways in ICAM-2 mediated resistance.
Results: Successful knockdown of ICAM-2 significantly attenuated the adhesion ability of AML cells to bone marrow stromal cells HS-5 (p < 0.01). Control AML cells exhibited significant resistance to Ara-C and Daunorubicin under co-culture conditions (mimicking BMME protection). ICAM-2 knockdown, on the other hand, significantly reversed this microenvironment-mediated resistance and significantly increased the rate of chemotherapy-induced cell death and apoptosis. ICAM-2 knockdown also enhanced chemosensitivity in mono-cultures, but the increase was less than in co-culture conditions. Western blot results showed that co-culture with stromal cells could activate PI3K/AKT/mTOR and NF-κB signaling pathways (as shown by increased levels of p-AKT, p-mTOR, p-p65) in AML cells, and up-regulate the expression of anti-apoptotic proteins Bcl-2 and MCL-1. Knockdown of ICAM-2 effectively suppressed coculture-induced PI3K/AKT/mTOR and NF-κB pathway activation and significantly decreased BCL-2 and MCL-1 expression. Treatment with PI3K inhibitor was able to mimic the effect of ICAM-2 knockdown and enhance the sensitivity of co-cultured AML cells to chemotherapy.
Conclusion:ICAM-2 is highly expressed in AML cells and is a key adhesion molecule mediating its anchoring to the bone marrow stromal microenvironment. ICAM-2 eventually confers strong chemoresistance to AML cells by promoting the tight adhesion of AML cells to BMME, activating PI3K/AKT/mTOR and NF-κB, two key pro-survival/resistance signaling pathways, and then up-regulating the expression of anti-apoptotic proteins such as Bcl-2 and MCL-1. Targeted inhibition of ICAM-2 or its mediated downstream signaling pathway can effectively disrupt the connection between AML cells and the protective microenvironment and reverse microenvironment-mediated chemoresistance. Therefore, ICAM-2 is a potential therapeutic target to overcome AML drug resistance and improve the efficacy of chemotherapy.
