is one of the most frequent targets of somatic mutations in leukemia and is mutated in an autosomal dominant disorder affecting platelets and predisposing to leukemia development. Better characterization of its in vivo
function is likely to give insight into the mechanisms leading to the development of leukemia, and will provide new candidate genes for leukemogenesis. We do not believe that as a transcription factor and master regulator of hematological cancers, RUNX1 will alter the function of only one oncogenic molecule, but multiple molecules in the same pathways, and our analyses and functional assays are carefully designed to study these effects. We have described a combination of genomic and bioinformatic approaches to identify the biological pathways and genes regulated by RUNX1, an overview of which is in Figure . Each approach independently provides a large source of data to identify RUNX1 targets according to RUNX1
gene dosage. However, the combination of them is powerful because of their convergence. Although the approaches described here are not the ideal models to study myeloid leukemia, each of them has their own advantages and their integration compensates for their limitations: 1) The use of cells derived from patients harbouring a RUNX1
mutation but who have not yet developed leukemia allow us to observe effects, largely due to changes in RUNX1 dosage. However, it should be kept in mind that due to the difficulties of obtaining myeloid cell lines, these studies were performed in lymphoid cells. 2) The overexpression system using HeLa cells provided a highly homogenous cell population, which is necessary to perform gene expression profiling. 3) The knockout mouse embryos represent various cell types, however they give us global information of the complete absence of RUNX1, which is difficult to obtain using cell lines. Efficient and homogenous knockdown levels are indeed difficult to obtain using siRNA especially in hematopoietic cells [28
Figure 7 Part of the networks downsteam of RUNX1. Additional data from the literature and our studies were used to update the standard Ingenuity Pathway System (Ingenuity® Systems, http://www.ingenuity.com) network analyses. Genes up-regulated (red) or (more ...)
The highly significant correlation observed between the genes identified in the FPD-AML cells and the overexpression system and clinical data on AML samples supports the hypothesis that large number of genes would be broadly regulated by RUNX1 in our various approaches disregarding of the cell type. Genes identified as differentially expressed following disregulation of RUNX1 expression level and/or in these AML samples are good candidates for targets of secondary hits during leukemogenesis downstream of RUNX1 mutation. The various approaches described in this study, including conserved binding sites and co-expression studies, will also help to further prioritize genes that might sustain secondary hits. For example, the gene encoding the Cyclin D3 (CCND3) was differentially expressed following overexpression of the CBF complex and mutations in this gene have been described in acute myeloid leukemia patients [29
In order to generate insights into the in vivo
role of RUNX1, we employed bioinformatics tools to identify processes that were changed following alteration of RUNX1 expression level. We have shown that genes involved in megakaryopoiesis tend to be differentially expressed in the FPD and CBF datasets, demonstrating that a large number of the differentially expressed genes may play a role in platelet formation. Enrichment for genes involved in cell proliferation was also observed in both the FPD and CBF datasets, and functional assays on the FPD-AML cell lines showed that heterozygous mutation of RUNX1
reduced proliferation of lymphoblasts. These data validate our integrative approach as they confirm studies in transgenic mice expressing the fusion proteins CBFβ-MYH11 [30
] and RUNX1-ETO [13
], which both act in a dominant negative fashion over the wild-type protein. These mice show a decrease in both lymphoid and myeloid cell proliferation. This observation also correlates with mouse data showing that Runx1 promotes cell cycle progression from G1 to S phase [31
]. An anti-proliferative effect of a RUNX1 mutant protein may have an oncogenic effect due to an improper balance between proliferation and differentiation. For example, overexpression of RUNX1 usually results in ALL while complete or partial loss of RUNX1 results in AML development.
Our integrative approach unraveled a novel process that may play an important role in RUNX1 function, involving the cytoskeletal dynamics. Indeed following the finding that an enrichment of microtubule and cytoskeleton related molecules was observed when the CBF complex was overexpressed, functional assay using the FPD-AML cells demonstrated an increase of polymerized microtubules in FPD-AML affected cells compared to cells from unaffected individuals. Microtubules are important in many processes such as cell migration, cell division, cellular transport and signal transduction [32
] and microtubule remodeling is essential during the cell cycle, especially during mitosis when a correct microtubule network is essential for proper chromosomal segregation [33
]. Interestingly, the fusion protein, CBFβ-MYH11 that results from inv(16), co-localizes with the actin cytoskeleton and disorganizes stress fibers and F-actin structures [34
]. A mild microtubule defect might partially explain the platelet defect observed in FPD-AML patients, as microtubules are necessary at several different stages of megakaryopoiesis including endomitosis, production of platelets from mature polyploid megakaryocytes, and release of the content of platelet granules [35
]. Moreover, mutations in the actin-binding protein WASP and the myosin heavy chain MYH9 cause the Wiskott-Aldrich [36
] and May-Hegglin [37
] syndromes of thrombocytopenia, respectively. However, RUNX1 is likely to regulate only specific tubulin isoforms or tissue-specific cytoskeleton-associated proteins as a strong cytoskeleton defect would be more detrimental to the whole organism. In addition, the dosage of normal RUNX1 activity necessary for normal function might differ according to cell type, and some cell types may be more susceptible than others to perturbation in RUNX1 levels. Interestingly, Taxol resistant leukemic cells have been shown to have a reduced total level of tubulin and an increased level of polymerized tubulin [38
], similar to the results seen in the FPD-AML cells. Furthermore, a high level of survivin (BIRC5), which was down-regulated following overexpression of the CBF complex, is associated with resistance to Taxol [39
]. This is the first evidence demonstrating a relationship between RUNX1 and microtubule dynamics.
Finally, we showed that the predisposition of FPD-AML to develop leukemia may be due to an increased rate of mutation in RUNX1
heterozygous cells. Every dataset showed significant correspondence with genes involved in DNA damage response. Although not conclusive, the glycophorin A assay, which measures the frequency of the progeny of mutated erythrocyte precursors in blood, showed a mild increase in mutation frequency in FPD-AML patients compared to unaffected individuals. Recently, it was shown that the RUNX1-ETO fusion protein induces mutations in transfected U937 myeloid cells [40
]. This study demonstrated that the fusion protein regulates many genes involved in the base excision repair pathway, which mainly corrects for point mutations. Furthermore, a higher incidence of leukemia in CBFβ-MYH11 chimeras compared to normal chimeras when exposed to ENU mutagenesis has also been observed [41
]. This demonstrates that alteration of RUNX1 function may increase the rate of mutation and lead to an accumulation of mutated cells.
The three processes described here (proliferation, cytoskeleton stability and genomic instability) are tightly interconnected and may explain the phenotype observed in FDP-AML patients. Indeed, a proliferation defect would have an impact on megakaryopoiesis and cytoskeleton remodeling. In turn, a cytoskeleton defect could also affect proliferation and trigger chromosomal aberrations. The necessary threshold level of RUNX1 expression is likely to be cell-specific, explaining why RUNX1 heterozygous mutation affects only hematopoietic cells; nevertheless, our observations could conceivably suggest possible involvement of RUNX1 in solid-tissue tumor.
We also identified new potential RUNX1 target genes by analyzing the regulatory regions and the expression pattern of the differentially expressed genes present in the overlaps between the different platforms. Many RUNX1 target genes have already been described in the literature, mainly from in vitro
studies and in mouse cells [43
]. Four of the published target genes, CSF1R, MYB, MPO and TIMP1, were differentially expressed in the Runx1
knockout embryos. In addition, target genes that were described more recently, including CCND3 [45
] and IGFBP3 [46
], were identified following overexpression of the CBF complex. That there was not more correlation may be due to incomplete microarray platforms, but more importantly is likely to reflect the bias present in the published RUNX1 target genes that were identified because of their primary role in hematopoiesis and these may not represent the most common RUNX1 target genes. Interesing candidates were among the 16 genes differentially expressed in every dataset, such as Annexin I (ANXA1), which was shown to reduce inflammation, by inhibiting neutrophil recruitment [47
] and has an anti-proliferative effect by inducing aberrant cytoskeleton formation [48
]. This gene is likely to play an important role downstream of RUNX1.