A MEIS1 Dependent Genetic Program in Leukemia Associated with Cell Cycle Entry and ‘Stemness’

Abstract The HOX co-factor MEIS1 is expressed in several leukemias, especially those involving MLL-gene rearrangements. Experimental data have demonstrated that MLL-fusion proteins induce the expression of MEIS1 in hematopoietic cells along with increased self-renewal and recent murine experiments indicate that MEIS1 is central to the growth-promoting effects of MLL fusion proteins. However, the cellular and molecular pathways that are regulated by MEIS1 are unknown. We studied the effect of MEIS1 knock-down in a cell line derived from a leukemic MLL-AF9 knock-in mouse. Transduction of this cell line (4166) with MEIS1-shRNA bearing lentivirus led to significant reduction in MEIS1 expression compared to cells transduced with control virus. The MEIS1 knock-down cells displayed decreased cell cycle entry, while terminal myeloid differentiation and apoptosis were enhanced. To characterize the molecular effects of MEIS1 knock-down, we performed gene expression profiling of leukemia cells with and without MEIS1 expression. We extracted RNA from 5 separate experiments where 4166 cells were transduced with vector control or MEIS1 shRNA for 48 hours and analyzed gene expression profiles using Affymetrix 430 2.0 whole genome arrays. We used a regularized two-sample paired t-test to select genes that were differentially expressed among the two groups. At a false discovery rate (FDR) of ≤ 5%, 1053 probe sets displayed decreased expression with MEIS1 knockdown, while 296 probe sets showed increased expression. Analysis of gene ontology (GO) terms by DAVID (Database for Annotation, Visualization and Integrated Discovery) revealed that the list of genes down-regulated with MEIS1 knock-down was significantly enriched in genes associated with the cell cycle and its regulation (Cdk2, Cdk6, Cdkn3, Ccna2, Cdc7, Cdc42, Rbl1, Wee1) and DNA replication (Brca1, Cdc6, Cdt1, Gmnn, Mcm4, Mcm5, Mcm8). Conversely, the genes displaying increased expression with MEIS1 knockdown were associated with inhibition of proliferation eg. Cdkn1a (p21), Btg2, Btg3 and pro-apoptotic effects such as Bax. A search of the Molecular Signatures Database for previously published profiles that overlap with our list of MEIS1-dependent genes revealed that the profile of MEIS1 knockdown in our murine leukemia cells significantly overlapped with that of neural stem cells. Specifically, of the 1838 genes expressed highly in neural stem cells compared to differentiated brain and bone marrow cells (Ramalho-Santos et al, Science 2002), 155 showed an overlap with the 594 genes in our MEIS1-dependent set (594 gene identifiers contained in 1053 probe sets; p = 3.27 e−28, hypergeometric distribution). This list of 155 genes included MEIS1 and several of the cell cycle and DNA replication-associated genes. These results reveal a core self-renewal genetic program shared by leukemia and neural stem cells that is regulated by MEIS1. Activation of MEIS1 in leukemia and possibly brain tumors could thus enhance self-renewal via the up-regulation of the above common genes. Overall, our results show that MEIS1 regulates cell cycle entry in murine MLL-AF9 leukemia, an effect that enhances self-renewal in other cells as well.