The MLL fusion gene, MLL-AF4 , regulates cyclin-dependent kinase inhibitor CDKN1B (p27 ^kip1 ) expression

Abstract

MLL, involved in many chromosomal translocations associated with acute myeloid and lymphoid leukemia, has >50 known partner genes with which it is able to form in-frame fusions. Characterizing important downstream target genes of MLL and of MLL fusion proteins may provide rational therapeutic strategies for the treatment of MLL-associated leukemia. We explored downstream target genes of the most prevalent MLL fusion protein, MLL-AF4. To this end, we developed inducible MLL-AF4 fusion cell lines in different backgrounds. Overexpression of MLL-AF4 does not lead to increased proliferation in either cell line, but rather, cell growth was slowed compared with similar cell lines inducibly expressing truncated MLL. We found that in the MLL-AF4-induced cell lines, the expression of the cyclin-dependent kinase inhibitor gene CDKN1B was dramatically changed at both the RNA and protein (p27 kip1 ) levels. In contrast, the expression levels of CDKN1A (p21) and CDKN2A (p16) were unchanged. To explore whether CDKN1B might be a direct target of MLL and of MLL-AF4, we used chromatin immunoprecipitation (ChIP) assays and luciferase reporter gene assays. MLL-AF4 binds to the CDKN1B promoter in vivo and regulates CDKN1B promoter activity. Further, we confirmed CDKN1B promoter binding by ChIP in MLL-AF4 as well as in MLL-AF9 leukemia cell lines. Our results suggest that CDKN1B is a downstream target of MLL and of MLL-AF4, and that, depending on the background cell type, MLL-AF4 inhibits or activates CDKN1B expression. This finding may have implications in terms of leukemia stem cell resistance to chemotherapy in MLL-AF4 leukemias.leukemia ͉ acute lymphoblastic leukemias ͉ cell cycle M LL is involved in chromosomal translocations associated with leukemia. Remarkably, MLL is involved in translocations with Ͼ50 different genes (1, 2). MLL is specifically cleaved shortly after translation into two peptides that noncovalently associate with each other (3, 4). The amino-terminal portion of MLL contains a region with AT-hooks that binds DNA, as well as a region with transcriptional repression activity (5) that binds CpG-rich DNA (6) and recruits histone deacetylases, the corepressor CtBP1, and polycomb group proteins (7). The carboxyl-terminal portion contains a transcriptional activation domain (5), which interacts with CBP (2), and a SET domain, with histone methyltransferase activity (3,8). Different MLL fusion partners are associated with leukemias producing blast cells of various lineages. MLL-AF9 results mainly in acute myeloid leukemia (AML), whereas MLL-AF4 causes almost exclusively B-cell lineage acute lymphoblastic leukemias. These findings suggest that MLL chimeras affect the phenotype of the leukemia by influencing differentiation pathways of uncommitted cells or early progenitors. MLL-AF4, an MLL fusion protein that is associated with infant pro-B acute lymphoblastic leukemias, is the most prevalent of the numerous MLL fusion proteins (9), and it is usually associated with a poor prognosis (10). Numerous data show that MLL fu...

Combined effects of the two reciprocal t(4;11) fusion proteins MLL·AF4 and AF4·MLL confer resistance to apoptosis, cell cycling capacity and growth transformation

“…It has already been shown that the MLL . AF4 fusion protein directly binds to the promoter regions of two cell cycle inhibitors, p18 and p27, and regulates their gene expression (Xia et al, 2005). Recently, functional data were obtained from murine embryonic fibroblasts derived from Taspase1 knockout mice (Takeda et al, 2006).…”

Biphasic MLL takes helm at cell cycle control: Implications in human mixed lineage leukemia

“…71 Additionally, several recent reports also identified MLL as a direct activator of Cyclins and CDKIs. 44,45,72,73 To further investigate how MLL regulates the cell cycle, we examined protein level of MLL through the cell cycle and observed a tightly controlled biphasic expression of MLL in all cell types. 74 This unique expression is conferred by defined windows of degradation mediated by specialized cell cycle E3 ligases, SCF Skp2 and APC Cdc20 (Fig.…”

The heterodimerization domains of MLL—FYRN and FYRC—are potential target structures in t(4;11) leukemia

“…Two transgenic mouse knock-in model systems, expressing the MLL-AF4 fusion protein, developed B-cell lymphomas (50 or 70%, respectively) after a long latency (540 or 720 days, respectively). 5,6 The lack of the ALL phenotype in these animal models could be explained by the finding that the MLL-AF4 fusion protein binds to the promoter of the cell cycle inhibitors CDKN1B (p27 kip1 ) and CDKN4C (p18INK4c), 7,8 and thus interferes with cell cycle progression. Recently, an Mll-AF4 knock-in mouse model system has been established demonstrating that the MLL-AF4 allele is able to cause predominantly acute myeloid leukemia, but also ALL and MLL.…”