1.25 Impact of Bone Marrow Microenvironment on 93 Apoptotic Pathway Target mRNA Expression in CLL Cells

Abstract

Chronic lymphocytic leukemia (CLL) was thought to arise due to dysregulated apoptosis rather than uncontrolled proliferation. Recent reports reveal that the CLL cell accumulation is not merely due to intrinsic defects in apoptosis, but in part due to extrinsic signals delivered by microenvironment. Although several entities within the bone marrow microenvironment have been unveiled, the critical components involved in CLL survival remain obscure. Previously, we showed that CLL primary cells co-cultured on MSCs (marrow stromal cells) are rescued from spontaneous apoptosis. At the molecular level, the mechanism of MSC-promoted CLL cell survival involves transcriptional as well as translational accumulation of antiapoptotic protein Mcl-1. In the present study, using microarray mRNA analysis, we investigated the essential apoptotic gene transcripts (n 93) that are involved in cell survival. Primary cells obtained from CLL patients (n 8) 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 incubation times(d1andd3),percentageapoptosisandmRNAmicroarrayof93 target genes 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 6-18% at d1 and d2 (n 16; p 0.001, two-tailed paired t-test) and 16-27% at d3-d6 of incubation (n 28, p 0.0001) compared to time matched controls. Next we investigated the effect of MSC on global transcription, there was a consistent increase in the global RNA synthesis (1.7 0.5 (d1, n 13, p 0.001) and 2.5 1.0 (d3, n 7, p 0.0084) fold change measured by uridine incorporation method). In order to evaluate changesinindividualtranscriptsundertheMSCmicroenvironment, we screened mRNAs using a ‘human apoptotic array micro fluidic card’ (93 targets) for d1 and d3 samples of controls and co-cultures from 8 CLL patients. Expression levels of mRNA were normalized using global normalization. Based on the functions and functional domains of target genes, the 93 genes were categorized into 7 groups and their response with respect to MSC microenvironment was elucidated. In a group that contained members ofBcl2family members, mRNA levels of pro-apoptotic members such as Bcl2l13 (MIL1), BAK1 and BIK decreased, while mRNA levels of anti-apoptotic, Bcl2, Bcl2A1 (BFL-1), Bcl-xl, and Mcl1 increased at d1 (1.36 0.32 [fold increase; mean standard deviation], p 0.0156; 1.64 0.58, p 0.0174; 2.27 0.97, p 0.04; 1.16 0.3,p 0.15 respectively). Among eight targets containing a caspase recruitment domain (CARD), pro apoptotic APAF1 mRNA level significantly decreased at d1; however, pro-apoptotic NALP1 (Card7) mRNA levelsincreasedsignificantly(1.480.59,p0.05),suggestingthis gene may have an alternate function in the apoptosis pathway. Transcript levels of HIP1 and HIPPI1 of Huntington pathway, which generally function as pro-apoptotic members, increased significantly (1.68 0.68, p 0.038; 1.43 0.50, p 0.043) in the presence ofMSCs.Inaddition,NFKB-RELcomplexfamilymembersthatare