Abstract 1490: Impact of bone marrow microenvironment on mRNA expression of genes in the apoptotic pathway in CLL cells
Authors: Viralkumar M. Patel, Lisa S. Chen, William G. Wierda, Kumudha Balakrishnan, Varsha Gandhi
Journal: Cancer Research (2012)

Abstract Chronic lymphocytic leukemia (CLL) is thought to arise due to dysregulated apoptosis rather than uncontrolled proliferation. Recent reports revealed that CLL cells accumulate due to both an intrinsic defect in apoptosis and extrinsic signals delivered by the microenvironment. Although several entities of bone marrow stroma cell (MSC) have been unveiled, the critical components involved in CLL survival remain obscure. In the present study, we used low density microarray analysis to identify the essential apoptotic genes (n=93) that are involved in cell survival. Primary cells obtained from CLL patients (n=12) were co-cultured with MSC (Nktert, human stromal cell line that mimics bone marrow microenvironment) in a ratio of 100:1. At the end of the incubation (12hr, d1, and d3), the expression profile of 93 genes as well as the percent apoptosis were evaluated. When CLL lymphocytes were co-cultured on MSC for 6 days, the viability of CLL cells, measured by Annexin/PI staining, increased by 6-27% at d1-d6 (n=9, p&amp;lt;0.001) compared to time matched controls. Next, we investigated the effect of MSC on global transcription. There was a 1.7+0.5 (d1, n=13) and 2.5+1.0 (d3, n=7) fold increase in global RNA synthesis as measured by [3H]uridine incorporation (p&amp;lt;0.001). Examination of mRNA profile of 93 genes showed that among the BCL2 family, the mRNA levels of the pro-apoptotic members such as BCL2L13, BAK1 and BIK decreased, while mRNA levels of anti-apoptotic, BCL2, BFL-1, BCL-XL, increased at d1 (1.46+0.56, p=0.015; 1.68+0.67, p=0.005; 2.17+1.46, p=0.018; respectively). Among caspase recruitment domain targets, pro-apoptotic APAF1 mRNA level significantly decreased at d1 but NALP1 mRNA level increased significantly (1.53+0.42, p=0.001), suggesting this gene may have alternate function in apoptosis pathway. In addition, NFKB-REL complex family members demonstrated decrease with MSC; in addition, member of IKB family, inhibitor of NFKB complex, such as NFKBIA increased significantly (1.56+0.7, p=0.018) at d1. Furthermore, in tumor necrosis factor receptor super family, TNFRSF1B expression significantly decreased (d1,0.74+0.21, p=0.001; d3,0.75+0.18, p=0.005), while TNFRSF10A and TNFRSF21 mRNA expression levels significantly increased (1.85+0.72, p=0.002; 2.81+2.08, p=0.016) at d1 but returned to baseline at d3. Additionally, 4 out of 6 DED domain containing pro-apoptotic proteins, (FADD, TRADD-adaptor proteins interacting with TNFRSF proteins, DEDD, DEDD2) demonstrated increase in mRNA levels compared to control. Moreover, mRNA levels of anti-apoptotic FLIP, which inhibits Caspase8 and Caspase10, increased significantly (1.50+0.59, p=0.013) after incubation with MSC. In summary, our data shows that MSC promoted survival to CLL lymphocytes is associated with transcriptional regulation of several genes including TNFRSF and BCL2 anti-apoptotic family members. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1490. doi:1538-7445.AM2012-1490