Protection of ALL Cells by Bone Marrow Stromal Cells and the Underlying Molecular Mechanism.

Abstract 2587

Acute lymphoblastic leukemia (ALL) is one of the fastest-growing hematological malignancies affecting patients with all ages, particularly children. Significant advances have been made in recent years in our understanding of the disease and the development of new therapies, which have led to a greatly improved outcome. Nevertheless, in a significant number of patients with ALL, the disease relapse and become resistant to treatment, causing death of the patients. Increasing evidence suggests that relapse of the disease and resistant to treatment are largely attributed to the protection of the leukemic cells by various components in the microenvironment, such as bone marrow stromal cells. However, the cross-talk between leukemic cells and their microenvironment remains poorly understood. Therefore, better understanding the mechanisms underlying the protection of ALL cells by the microenvironment is of ultimate importance in developing new therapies targeting such protection and eventually eradicating all the leukemic cells to cure the disease. In this study, we used a coculture system with leukemic cells and bone marrow stromal cells (MSC) to mimic the in vivo interaction between the two cell types to explore the molecular events that might be responsible for the protection of ALL cells from Ara-C induced apoptosis.

We cocultured human primary ALL cells with hTERT-immortalized normal human MSC and evaluated ALL cell apoptosis by FACS after staining with Annexin V and propidium iodide. In all 8 cases, the MSC provided significant protection of ALL cells from both spontaneous and Ara-C induced apoptosis. For example, the mean Ara-C induced apoptosis of ALL cells cultured without MCS was 42.7% (range, 27–54%), whereas it was 19.1% (range, 8–27%) with MSC. Similar results were obtained with human leukemia cell lines Reh, SEMK2 and RS4.11. We also found that the murine MSC line M210B4 could provide similar protection to ALL cells, whether the ALL cells are primary or cell lines. The reduced apoptosis in the coculture were confirmed by Western blot which showed that MSC could protect ALL cells from Caspase-3 and PARP cleavage. Furthermore, our results showed no significant Ara-C induced reduction in S phase when cocultured with MSC. This phenomenon was associated with decreased cyclinA and CDK2 expression. In addition, we found that cocultured with MSC resulted in phosphorylation of AKT in ALL cells and PI3K inhibitor LY294002 specifically inhibited MSC-induced activation of AKT and promoted ALL cell apoptosis. In addition, beta-catenin and c-myc had increased expression in ALL cells cocultured with MSC, suggesting that Wnt pathway could play a role in MSC-mediated protection. To identify candidate molecules potentially involved in the protection of ALL cells by MSC, we performed gene expression microarray analyses with ALL cells exposed to Ara-C in presence or absence of MSC. Our data indicated that several signaling pathways might be involved in this process, including apoptosis signaling and cell cycle checkpoint control, which confirmed above findings. The top expressed genes identified in the microarray studies were confirmed by RT-PCR.

Collectively, our results demonstrated that MSC can protect ALL cells from Ara-C induced apoptosis by multiple signaling pathways, such as those involving PI3K/AKT and Wnt signaling. Hence, targeting these pathways may become potential novel therapeutic strategies to disrupt the support of the microenvironment to ALL cells and to eventually eradicate leukemic cells.