In humans, the CBF complex containing RUNX1 as the α-subunit is one of the most frequent targets of chromosomal and genetic alterations in leukemia. Chromosomal rearrangements involving RUNX1 or CBFβ [6], somatic point mutations in RUNX1 [7] and amplification of RUNX1 [8] have all been described in acute leukemia. In addition to somatic alterations, germ-line point mutations in RUNX1 are responsible for an autosomal dominant platelet disorder with a propensity to develop leukemia (FPD-AML, OMIM 601399) [9,10]. Interestingly, the dosage of RUNX1 protein seems to play a role in the determination of the leukemic phenotype. Indeed, low dosage of RUNX1, resulting from haploinsufficient or dominant negative mutations, lead to the development of myeloid leukemia [9-11], whereas amplification of RUNX1 gene is more often observed in lymphoid leukemia, particularly pediatric ALL [12]. A number of observations also suggest that although RUNX1 is involved in the first steps of leukemia development, additional somatic mutations are necessary and probably determinant for the leukemic phenotype: 1) The predisposition to develop leukemia in FPD-AML patients shows that germline RUNX1 mutations are not sufficient for the development of the disease [10]. 2) Somatic translocations are not able to induce leukemia in mouse cells on their own [13]. 3) The translocation t(12;21), which fuses ETV6 (TEL) to RUNX1, can arise in utero but does not trigger leukemia until later in childhood, with as much as nine years latency [14]. These additional mutations are likely to occur in molecules involved in the same biological pathways as RUNX1, as hemizygous loss of several molecules in the same biological pathway (e.g. RUNX1 and SPI1) is thought to be almost as tumorigenic as homozygous loss of one molecule (e.g. homozygous RUNX1 mutation in AML-M0) [15]. Therefore the identification of downstream targets of RUNX1, with care to the model systems including species and cell type of origin, is of great interest in order to identify novel candidate molecules involved in leukemogenesis.

The identification of the biological pathways regulated by RUNX1 is also of importance to shed light on its in vivo function and role in leukemia development. The observation that Runx1 knockout mice show a lack of definitive hematopoietic maturation and die at embryonic stage 12 from hemorrhages in the central nervous system demonstrates that RUNX1 plays a critical role during development of the hematopoietic system [16,17]. In addition, RUNX1 might also play a role in other systems as it is expressed in many other embryonic tissues [18-20] and in epithelial cells [19,20]. It is furthermore overexpressed in endometrioid carcinoma [21] and down-regulated in gastric cancer [22]. The in vivo function of RUNX1 is therefore yet to be fully understood.

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 [41,42]. 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.