Absence Of Marrow Stromal Cells Research Articles

Abstract 1758: Influence of stromal microenvironment on rGel/BLyS-induced cytotoxicity in diffuse large B-cell lymphoma cells

Abstract Diffuse large B cell lymphoma (DLBCL) is an aggressive sub-type accounting for 30-40% of non-Hodgkin's lymphoma. Despite recent advances in the understanding of the molecular and cellular basis for their pathogenesis, these tumors are still associated with poor response to treatment and a fatal outcome. The interactions of marrow stromal cells (MSCs) with tumor cells appear to be important in the development of tumor drug resistance through complex cytokine and adhesion-mediated mechanisms. We previously generated a highly cytotoxic growth factor fusion toxin designated rGel/BLyS for receptor-mediated delivery of the rGel toxin to malignant B cells. This fusion toxin specifically targets malignant B cells expressing the BLyS receptors BAFF-R, TACI, and BCMA. In this study, we examined the impact of marrow stromal cells (MSCs) on rGel/BLyS-induced cytotoxicity in DLBCL cells. Treatment of tumor cell lines with dexamethasone and doxorubicine in the presence of MSCs was found to suppress the cytotoxic effect. In contrast, in vitro treatment with rGel/BLyS showed specific cytotoxic effects to DLBCL cell lines (IC50 ∼ 1-10 nM) in absence of MSCs or in presence of MSCs. At these doses, we found no cytotoxicity to MSCs. Treatment with rGel/BLyS induced down-regulation of Mcl-1, c-Myc, Bcl-xL, X-IAP, and Bcl-2 in DLBCL cells in absence of MSCs or in presence of MSCs. We also found that rGel/BLyS induced down-regulation of interleukin-6 receptor in DLBCL cells in absence of MSCs or in presence of MSCs. In addition, treatment of cells with rGel/BLyS induced apoptosis through caspase-9 activation, caspase-3 activation, and PARP cleavage. Our results demonstrate that rGel/BLyS can overcome stromal cell-mediated drug resistance which is a factor reducing the effectiveness of other conventionally-used agents for the treatment of this disease. This suggests that rGel/BLyS may be an excellent candidate for the treatment of DLBCLs which are resistant to conventional chemotherapeutic regimens. This research was conducted, in part, by the Clayton Foundation for Research and by Translational Research Award (LLS 6234-07) from the Leukemia and Lymphoma Society. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1758. doi:10.1158/1538-7445.AM2011-1758

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

AbstractAbstract 2587Acute 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. Disclosures:No relevant conflicts of interest to declare.

Inhibition of PI3K or Integrin-Linked Kinase (ILK) Target Primary AML Cells within the Bone Marrow Microenvironment in the In Vitro Co-Culture System.

In hematological malignancies, there are reciprocal interactions between leukemic cells and cells of the bone marrow microenvironment such as marrow stromal cells (MSC). It is proposed that specific niches within the bone marrow microenvironment provide a sanctuary for subpopulations of leukemic cells to evade chemotherapy-induced death, and we indeed demonstrated that MSC protect primary AML cells from Ara-C induced apoptosis in vitro (Konopleva, Leukemia 2002). Integrin-linked kinase (ILK) has been shown to directly interact with β integrins and phosphorylate AKT in a PI3-kinase(PI3K)-dependent manner to promote cell survival and proliferation. In this study, we tested the hypothesis that selective inhibition of ILK signaling will provide a novel approach for targeting both leukemic cells and cells in their surrounding microenvironment. Direct co-culture of human MSC and leukemic NB4 cells results in activation of PI3K/ILK/AKT signaling as evidenced by enhanced ILK kinase activity, elevated phospho(p)-Akt, p-GSK3β and nuclear translocation of β-catenin. Both, PI3K inhibitor LY294002 (10μM) and specific ILK inhibitor QLT0267 (10μM) inhibited stroma-induced activation of AKT and suppressed GSK phosphorylation. This resulted in massive induction of apoptosis which was not abrogated by stromal co-culture (AnnexinV positivity %, MSC(−) vs MSC(+); 51.4+2.5 vs 55.8+3.5 p=0.26, LY 47.0+8.1 vs 47.9+6.1 p=0.85, 48hrs). In contrast, MSC co-culture effectively blocked apoptosis induced by MEK inhibitor PD98059 despite activation of pERK (62.5+3.2% vs 45.6+2.3%, p=0.02). We next examined anti-leukemia effects of PI3K and ILK inhibitors in the co-culture system of primary AML and human MSC. AML blasts from 7 primary AML samples with high (>54%) blast count were co-cultured with MSC for 24 hours, after which they were exposed to 10μM LY294002 or QLT0267 for 4–8 days. After this period, induction of apoptosis was analyzed in non-adherent AML cells by Annexin V flow cytometry after gating on the CD90-negative (non-MSC) population. To control for differences in spontaneous apoptosis, we calculated % specific apoptosis as (test - control) x 100 / (100 - control). MSCs protected leukemic blasts from spontaneous apoptosis in all 7 samples studied (mean annexin V positivity, 49.5±7.2% vs 25.3±4.8%, p<0.001). In contrast, inhibition of PI3K/ILK signaling induced unopposed apoptosis even in MSC co-cultures (% specific apoptosis, LY294002, 30.3±4.8%; LY+MSC, 28.3±7.7%; QLT0267, 26.9±9.8%; QLT+MSC, 33.1±9.3%, p>0.3 comparing cell death in the presence or absence of MSC). This resulted in corresponding loss of viability (% of control, LY294002, 66.0±11.0%; LY+MSC, 57.6±11.2%; QLT0267, 66.4±7.28%; QLT+MSC, 50.4±11.3%, p>0.1 comparing viability in the presence or absence of MSC). These observations indicate that disruption of leukemia/stroma interactions by specific PI3K/ILK inhibitors represents a novel therapeutic approach to eradicate leukemia in the bone marrow microenvironment. Further studies are aimed at the elucidation of the role of the BM microenvironment and its ability to activate specific signaling pathways in the pathogenesis of leukemias. Focus on this stroma-leukemia crosstalk may result in the development of strategies that alleviate the acquisition of a chemoresistant phenotype and enhance the efficacy of therapies in hematological malignancies.