Xia Zhen-Biao, Popovic Relja, Chen Jing, Theisler Catherine, Stuart Tara, Santillan Donna A, Erfurth Frank, Diaz Manuel O, Zeleznik-Le Nancy J
Department of Medicine, Molecular Biology Program, and Oncology Institute, Cardinal Bernardin Cancer Center, Loyola University Medical Center, Maywood, IL 60153, USA.
Proc Natl Acad Sci U S A. 2005 Sep 27;102(39):14028-33. doi: 10.1073/pnas.0506464102. Epub 2005 Sep 16.
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 (p27kip1) 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.
MLL参与了许多与急性髓系和淋巴细胞白血病相关的染色体易位，已知有超过50个伙伴基因，它能够与这些基因形成读码框内融合。鉴定MLL及MLL融合蛋白的重要下游靶基因可能为治疗MLL相关白血病提供合理的治疗策略。我们探索了最常见的MLL融合蛋白MLL-AF4的下游靶基因。为此，我们在不同背景下构建了可诱导的MLL-AF4融合细胞系。MLL-AF4的过表达在两种细胞系中均未导致增殖增加，相反，与可诱导表达截短MLL的类似细胞系相比，细胞生长减缓。我们发现，在MLL-AF4诱导的细胞系中，细胞周期蛋白依赖性激酶抑制剂基因CDKN1B在RNA和蛋白质（p27kip1）水平上均发生了显著变化。相比之下，CDKN1A（p21）和CDKN2A（p16）的表达水平未发生改变。为了探究CDKN1B是否可能是MLL和MLL-AF4的直接靶标，我们使用了染色质免疫沉淀（ChIP）分析和荧光素酶报告基因分析。MLL-AF4在体内与CDKN1B启动子结合并调节CDKN1B启动子活性。此外，我们通过ChIP证实了MLL-AF4以及MLL-AF9白血病细胞系中CDKN1B启动子的结合。我们的结果表明，CDKN1B是MLL和MLL-AF4的下游靶标，并且根据背景细胞类型，MLL-AF4抑制或激活CDKN1B的表达。这一发现可能对MLL-AF4白血病中白血病干细胞对化疗的耐药性具有影响。
