The Role And Underlying Mechanisms Of Bone Marrow Stromal Cells In Drug Resistance Of Acute Lymphoblastic Leukemia

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 new therapies have been developed, which have led to a greatly improved outcome. Nevertheless, in a significant number of patients with ALL, the disease relapses and becomes resistant to treatment, causing death of the patients. Increasing evidence suggests that relapse of the disease and resistance to treatment are largely attributed to the protection of the leukemic cells by various components in the microenvironment. such as bone marrow stromal cells (BMSC). 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 evaluated the effects of BMSC on apoptosis of ALL cells induced by Ara-C and further investigated the underlying molecular mechanisms in vitro and in vivo by different molecular biologe techniques.We co-cultured human leukemia cell lines Reh, SEMK2 and RS4.11 with human and murine BMSC to mimic in vivo bone marrow microenvironment and used Ara-C to induce apoptosis. Flow cytometry analysis showed that BMSC could significantly inhibit apoptosis of ALL cells. BMSC could also provide protection of primary ALL cells from both spontaneous and Ara-C induced apoptosis. The reduced apoptosis in the co-culture was confirmed by Western blot which showed that BMSC could protect ALL cells from Caspase-3 and PARP cleavage, associated with decreased p21, Cyclin A and Cdk2 expression. In addition, BMSC could increase proliferation of ALL cells by accelerating cell cycle. We found that co-culture with BMSC resulted in phosphorylation of AKT in ALL cells and PI3K inhibitor LY294002 specifically inhibited MSC-induced activation of AKT and promoted ALL cell apoptosis. CXCR4 inhibitor AMD3100, however did not increase ALL cell apoptosis. To identify candidate molecules potentially involved in the protection of ALL cells by MSC, we performed gene expression microarray and reverse phase protein array analyses on ALL cells exposed to Ara-C in presence or absence of BMSC. Our data indicated that several signaling pathways were involved in this process, including apoptosis signaling and cell cycle checkpoint control, which was consistent with the apoptosis data described above. A group of top differentially expressed genes identified in the microarray studies were confirmed by RT-PCR.By analyzing the microarray data, we found that expression of several important members of the Wnt pathway, such as Lef-1, C-myc, and Cyclin Dl was increased in ALL cells when co-cultured with BMSC. Though (3-catenin expression was not significantly changed at mRNA level on microarray, elevated expression of?-catenin at protein level was confirmed by Western blot. These results suggested that Wnt pathway could play a role in BMSC-mediated protection. Subsequently, we found that small molecule inhibitor XAV939 could inhibit expression of?-catenin and activation of Wnt pathway, which reversed protection of BMSC and increased Ara-C induced apoptosis. Our data also suggested that the inhibitor acted on both ALL cells and BMSC, making it a potential highly effective molecule in ALL treatment in combination with chemotherapy. We tested this possibility in NOD/SCID mice. We transplanted NOD/SCID mice with Rehluc ALL cells and treated them with Ara-C with or without XAV939. Growth and development of leukemia was monitored by IVIS imaging systems. We found that combined treatment significantly slowed down leukemia development and resulted in better survival of the recipient mice.Collectively, our results demonstrated that BMSC 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 eventuallv eradicate leukemic cells.