Core-Binding Factor: a Central Player in Hematopoiesis and Leukemia 1

[CANCERRESEARCH (SUPPL.) 59, 1789s-1793s, April 1, 1999] Core-Binding Factor: A Central Player in Hematopoiesis and Leukemia 1

Nancy A. Speck, z Terry Stacy, Qing Wang, Trista North, Ting-Lei Gu, Janelle Miller, Michael Binder, and Miguel Marin-Padilla Departments of Biochemistry [N. A. S., T. S., Q. W., T.N., T-L. G., J. M.] and Anatomy and Pathology [M. B., M. M-P.], Dartmouth Medical School, Hanover, New Hampshire 03755

Abstract The Involvement of CBFs in Human Disease

Consistent chromosomal rearrangements are found in a large number CBFa2 and CBF/3 are members of a small family of transcription of hematopoietic tumors. In many cases, these rearrangements disrupt factors known as CBFs or polyomavirus enhancer binding protein 2 genes whose normal function is required for the proper development of (1). CBFs are heterodimeric transcription factors that contain one blood cells. Excellent examples are the chromosomal rearrangements subunit that binds DNA directly (CBFc0 and a second, non-DNA- t(8;21)(q22;q22), t(12;21)(p13;q22), and inv(16)(p13q22) that disrupt two binding CBF/3 subunit (Fig. 1; Refs. 2-4). Four genes encode sub- of the genes encoding a small family of heterodimeric transcription fac- units of CBF in humans: three encode CBFa subunits (CBFA1, tors, core-binding factors (CBFs). CBFs consist of a DNA-binding CBFa CBFA2, and CBFA3); and one gene encodes the common CBF/3 subunit and a non-DNA-binding CBF/3 subunit. The t(8;21), associated with de novo acute myeloid leukemias, disrupts the CBFA2 (AML1) gene, subunit, CBFB (2-7). which encodes a DNA-binding CBFa subunit. The t(12;21), the most CBFs play critical roles in both normal developmental processes common translocation in pediatric acute lymphocytic leukemias, also and disease. The CBFA1 gene, which encodes a DNA-binding disrupts CBFA2. The CBFB gene, which encodes the non-DNA-binding CBFa subunit, is required for bone development. Mice lacking subunit of the CBFs, is disrupted by the inv(16) in de novo acute myeloid both copies of the Cbfal gene are completely lacking in bone leukemias. All chromosomal rearrangements involving the CBFA2 and formation, due to a defect in osteoblast differentiation (8, 9). The CBFB genes create chimeric proteins, two of which have been unequivo- human homologue, CBFA1, is implicated in a human genetic cally demonstrated to function as transdominant negative inhibitors of disorder, cleidocranial dysplasia, an autosomal dominant trait char- CBF function. acterized by moderate skeletal malformations (10). The mutations Both the Cbfa2 and Cbfb genes are essential for normal hematopoiesis in cleidocranial dysplasia patients inactivate only one copy of the in mice, because homozygous disruption of either gene blocks definitive CBFA1 gene, indicating that the disorder is caused by haploinsuf- hematopoiesis. Recent data suggest that Cbfa2 and Cbfb are required for the emergence of definitive hematopoietic stem cells in the embryo from a ficiency of CBFA1. putative definitive hemangioblast precursor. The transdominant negative The Cbfa2 and Cbfb genes are required tor fetal liver hematopoiesis inhibitor of CBF created by the inv(16), when present from the beginning in mice and play major roles in the etiology of many human leuke- of embryogenesis, also blocks the emergence of definitive hematopoietic mias. The human homologue of Cbfa2 (CBFA2, more commonly cells in the embryo. On the other hand, chromosomal translocations known as AML1) is disrupted by the t(8;21)(q22;q22), t(12;21)(p13; involving the CBFA2 and CBFB genes in leukemias block hematopoiesis q22), and t(3;21)(q26;q22) in acute myeloid and lymphocytic leuke- at later steps. This may reflect a difference in the timing at which mias and in therapy-related leukemias and myelodysplasias (Fig. 1; translocations are acquired in the leukemias, which presumably is subse- Refs. 5 and 11-14). The CBFB gene, which encodes the non-DNA- quent to emergence of the definitive hematopoietic stem cell. The cumu- binding CBFI3 subunit, is disrupted in acute myeloid leukemias by lative data suggest that although the earliest requirement for Cbfa2 and inv(16)(p13;q22), t(16;16), and del(16)(q22) (15). Together, CBFA2 Cbfb is for emergence of definitive hematopoietic stem cells, both genes are also required at later stages in the differentiation of some hematopoietic and CBFB are disrupted in approximately a quarter of all de novo lineages. acute leukemias, making them the most frequently disrupted genes in acute human leukemias (16-20). Introduction The chromosomal translocations involving the CBFA2 and CBFB genes generate chimeric proteins that contain all or part of CBFoz2 Hematopoiesis is the process by which blood cell populations of all (AML1) or CBFI3 fused to a segment of a protein encoded on the lineages are continuously renewed through differentiation I?om a other chromosome involved in the translocation (Fig. 2). The t(8;21) common progenitor cell called the pluripotent HSC. 3 Many transcrip- fuses the NH2-terminus of CBFoz2, including its DNA-binding do- tion factors are required for blood cell development, and several, such main, to a protein called ETO (for eight twenty one), encoded on as Scl, Rbtn2, E2A, and the subjects of this review, CBFo~2 (AML1) chromosome 8. The t(3;21) fuses the NH2-terminus of CBFoz2 to one and CBFI3, also play key roles in the dysregulation of hematopoiesis of three different proteins encoded by genes closely linked on chro- manifested as leukemia. mosome 3: EAP, MDS1, or EVIl. The t(12;21) breaks the CBFA2 gene 5' to the first coding exon and fuses the NH2-terminus of a protein in the ets family called TEL to the full-length CBFoz2 protein. Received 2/5/99; accepted 2/8/99. All of these chimeric CBFol2 proteins contain the CBFoz2 (AML1) The costs of publication of this article were defrayed in part by the payment of page DNA-binding domain, and they are believed to deregulate the tran- charges. This article must therefore be hereby marked advertisement in accordance with scription of CBF target genes in a transdominant manner (21-27). 18 U.S.C. Section 1734 solely to indicate this fact. 1 Presented at the "General Motors Cancer Research Foundation Twentieth Annual The inv(16) that disrupts the CBFB gene results in a chimeric Scientific Conference: DevelopmentalBiology and Cancer," June 9-10, 1998, Bethesda, protein that contains most of the CBF~ protein fused to the COOH- MD. This work was supported by USPHS Grants CA58343 and CA75611. N. A. S. is a Leukemia Society of America Scholar. terminal c~-helical rod domain from a SMMHC (Fig. 2; Ref. 15). It is 2 To whom requests for reprints should be addressed. thought that the CBFI3-SMMHC protein blocks wild-type CBF func- 3 The abbreviations used are: HSC, hematopoietic stem cell; CBF, core-bindingfactor; SMMHC, smooth muscle myosin heavy chain; dpc, days post coitus; AGM, aorta/genital tion by assembling into multimers that sequester CBFc~ subunits into ridge/mesonephros. nonfunctional complexes (28-30). 1789s

GENES DISEASE MUTATIONS

CBFB AML M4Eo inv(16)(p13;q22) Fig. 1. The CBF complex and its association with leukemias. CBF binds the sequence CBFA 1 CCD Haploinsufficiency PyGPyGGT. The CBF/3 subunit forms a 1:1 complex with CBF~ and increases the binding affinity of CBFc~ for DNA (2, 4). Three genes encode CBFA2 ~ML1) AML M2 t(8;21)(q22;q22) CBFc~ subunits, and one of these genes, the CBFA2 gene, along with the CBFB gene, are the most frequently disrupted genes in acute human leuke- t-MDS t mias. t-AML t(3;21 )(q26.2;q22) CML-BC PyGPyGGT A L L t(12;21 )(p13 ;q22)

CBFA3

Cbfa2 andCbfb Are Required for the Emergence of Definitive and are primarily primitive nucleated erythrocytes and small numbers Hematopoietic Stem Cells of granulocytes and macrophages (31). This first, transient wave of "primitive" yolk sac hematopoiesis is followed shortly thereafter by Gene disruption experiments in mice demonstrated that both Cbfa2 the appearance of "definitive" hematopoietic progenitor cells, which and Cbfb are required for the differentiation of all hematopoietic cells derived from the HSCs. Before describing the hematopoietic defect in are capable of differentiating into all adult blood cell lineages. De- Cbfa2- and Cbfb-deficient mice in more detail, it is worthwhile to finitive hematopoietic cells emerge from several sites in the embryo: briefly review the early steps of hematopoiesis in the developing from the extraembryonic yolk sac; the omphalomesenteric and um- embryo. bilical arteries; the embryonic splanchnopleure; and from the AGM Pluripotent HSCs are the source of all blood cells in the adult. region (32-37). The first-long term repopulating HSCs, which are However, paradoxically, they are not the first blood cells to develop cells capable of reconstituting an intact hematopoietic system when in the embryo. The first blood cells, which arise from mesoderm, transplanted into a lethally irradiated adult mouse, appear at 10.0 dpc appear in the extraembryonic yolk sac of mouse embryos at -8 dpc in the AGM region (36, 37). After their emergence, definitive hema-

t(8;21) A t(12;21) t(3;21)

CBF~2 (AML1)

AML1-ETO ~....{:!...~:.~.:::~.:~.::::~...:~.:::~...::~.~:::.~::~.::~:..:2:~.~:.7:~:.~:..~:::~:.~:...~.:~.::.~::::~.:~:....~:.:~.::....:~.::.~.::~.:::~:~::~.] t(8;21)

AML1-EAP t(3;21) AML1 -MDS1 / // / t(3;21) AML1 -EVIl t(3;21)

TEL-AML1 I ~ ] t(12;21)

inv(16)

B CBF~

CBF~-SMMHC inv(16)

Fig. 2. CBFot2 (AMLI) and CBF/3 fusion proteins. A, chimeric proteins resulting from chromosomal translocations involving the CBFA2 (AMLI) gene. Black rectangle, the DNA-binding domain. Sequences derived from fusion partners are indicated by the stippled, dashed, and lined rectangles. White or black regions represent sequences derived from the CBFa2 (AML1) protein. B, the CBF/3 protein and the chimeric CBF/3-SMMHC protein formed by the inv(16). Black rectangle, the sequences responsible for heterodimerizing with CBFo~. Stippled region, SMMHC sequences. 1790s

Myeloid ]_. ~ Mast Cell Progenitor ~ ~.~ ....

Cbfb HSC f Erythrocytes

Fig. 3. Hematopoietic blocks associated with loss of CBFc~2(AML1):CBF/3 function. Cbfa2 and Cbfb are required for the emergence of definitive HSCs in the embryo. The transdominant inhibitors of CBF (AML1-ETO and ~k~ @ --)~ =I~ Platelets CBF/3-SMMHC) are associated with acute myeloid leukemias. TEL-AML1, which has not yet been shown geneti- Lymphoid cally to mimic loss of Cbfa2 or Cbfb function during em- Progenitor bryogenesis, is found in pre-B-cell leukemias. Eosinophil TEL-AML1 / @ @ @ ~ Neutrophi, Primitive B Cell T Cell Erythrocyte Monocyte

topoietic progenitors and long-term repopulating HSCs migrate to the for the activity of this heterodimer in vivo, the Cbfb gene appears fetal liver, where they further expand and differentiate into definitive to be expressed ubiquitously (2, 4). Thus, we reasoned that Cbfa2 enucleated erythrocytes and cells of the myeloid lineage. expression would more accurately identify the cells in which the Mutations in several genes encoding transcription factors, such as active heterodimer is found. We find that Cbfa2 is expressed in Scl, Rbtn2, Gatal, and Gata2, have profound effects on the develop- sites where definitive hematopoietic cells emerge, including the ment of both primitive and definitive blood cell lineages (reviewed in yolk sac, vitelline and umbilical arteries, and in clusters of hema- Ref. 38). Other genes (Pu.1, Myb, Eklf, Ikaros, Pax5, E2A, and topoietic cells that appear to bud from the endothelium into the NF-E2) seem to be required for the development of a subset of lumina of these arteries. In the AGM region where HSCs first definitive hematopoietic lineages but not for the development of emerge, Cbfa2 is expressed in endothelial cells and in the paraaor- primitive blood cells in the yolk sac. Mutations in the Cbfa2 and Cbfb tic mesenchyme of the dorsal aorta in a ventral to dorsal gradient. genes specifically impair the development of all definitive hemato- Hematopoietic cells expressing Cbfa2 are also found in the fetal poietic lineages but not the primitive erythroid lineage (39-43). liver by 10.5 dpc. Cbfa2- and Cbfb-deficient mouse embryonic stem cells are unable to What happens to cells expressing Cbfa2 in embryos lacking the contribute to definitive hematopoietic lineages in chimeric mice, functional CBFoz2:CBF/3 heterodimer? We find that Cbfa2 deficient indicating that the hematopoietic block caused by loss of Cbfa2 and embryos contain almost no definitive hematopoietic progenitors, ei- Cbfb, at least in part, is cell autonomous (39, 41). ther in their fetal livers or in circulating blood (44). Furthermore, Analyses of Cbfa2- and CbJb-deficient mice demonstrated a lack of when we examine the sites where definitive hematopoietic progenitors definitive hematopoietic elements in histological sections of fetal emerge, such as the vitelline and umbilical arteries, the yolk sac, and livers. Cells isolated from the yolk sac or fetal liver were also unable the AGM region, we find no evidence of hematopoietic cells budding to differentiate into definitive erythroid, myeloid, or mixed lineage from hemogenic endothelium at these sites. On the basis of these and colonies in vitro. These results, plus the inability of Cbfa2- and previous genetic data, we hypothesize that Cbfa2 is required for the Cbfb-deficient mouse embryonic stem cells to undergo lymphopoiesis emergence of definitive hematopoietic cells from hemogenic endo- in chimeric mice, placed the requirement for Cbfa2 and Cbfb at the thelia in the developing embryo. level of a definitive hematopoietic stem cell. However, the precise nature of the hematopoietic stem cell defect in Cbfa2- and Cbfb- Chromosomal Translocations and Leukemia deficient mice could not be resolved from these initial studies, because an impairment of fetal liver hematopoiesis could result from one of Functional assays, as well as more recent genetic studies in mice, multiple defects. For example, Cbfa2 and Cbfb could be required for established that chromosomal translocations involving the CBFA2 and the emergence of definitive hematopoietic progenitors and HSCs from CBFB genes create transdominant negative alleles of these genes an HSC precursor. Alternatively, HSCs and definitive hematopoietic (21-27, 45-47). In mice, both Yergeau et al. (26) and Okuda et al. progenitors could emerge but fail to migrate from their site of origin (27) modified the Cbfa2 gene in such a way that the chimeric protein to the fetal liver, or once in the liver be unable to further expand and created by the t(8;21) was expressed from the locus. Using a similar differentiate. strategy, Castilla et al. (47) recreated the inv(16) in mice by intro- To more precisely define the step at which Cbfa2 and Cbfb are ducing a cDNA encoding the CBF/3-SMMHC fusion protein into the required, we have identified cells that express the Cbfa2 gene and Cbfb locus. In all three studies, chimeric mice containing the determined their fate in animals lacking the functional CBFa2: "knocked in" t(8;21) and inv(16) alleles were generated, but these CBF/3 heterodimer (44). We chose to track only Cbfa2 expression, mice failed to produce offspring heterozygous for the knocked-in because although both the CBFa2 and CBF/3 subunits are required alleles. Further analysis established that embryos heterozygous for the 1791s

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1999 American Association for Cancer Research. CBF IN HEMATOPOIESIS AND LEUKEMIA t(8 ;21) and inv(l 6) alleles died in midgestation, exhibiting a spectrum partner of a novel Drosophila runt-related DNA binding protein PEBP2a. Virology, of phenotypes very similar to that seen in Cbfa2- and Cbfb-deficient 194: 314-331, 1993. 3. Ogawa, E., Maruyama, M., Kagoshima, H., inuzuka, M., Lu, J., Satake, M., Shige- mice, including a severe impairment of fetal liver hematopoiesis. sada, K., and Ito, Y. PEBP2/PEA2 represents a new family of transcription factor These experiments unequivocally demonstrated that the t(8;21) and homologous to the products of the Drosophila runt mad the human AML1 gene. Proc. inv(16) create transdominant negative alleles of Cbfa2 and Cbfb, that Natl. Acad. Sci. USA, 90: 6859-6863, 1993. 4. Wang, S., Wang, Q., Crute, B. E., Melnikova, I. N., Keller, S. R., and Speck, N. A. when present from the beginning of embryogenesis, block hemato- Cloning and characterization of subunits of the T-cell receptor and murine leukemia poiesis at essentially the same point as homozygous loss of Cbfa2 or virus enhancer core-binding factor. Mol. Cell. Biol., 13: 3324-3339, 1993. 5. Miyoshi, H., Shimizu, K., Kozu, T., Maseki, N., Kaneko, Y., and Ohki, M. t(8;21) cbfb. breakpoints on chromosome 21 in acute myeloid leukemia are clustered within a Although it is clear from the genetic data that t(8 ;21) and inv(16) limited region of a single gene, AML1. Proc. Natl. Acad. Sci. USA, 88: 10431-10434, create transdominant negative alleles of Cbfa2 and Cbfb, how these 1991. alleles contribute to leukemogenesis remains one of the outstand- 6. Levanon, D., Negreanu, V., Bernstein, Y., Bar-Am, I., Avivi, L., and Groner, Y. AML1, AML2, and AML3, the human members of the runt domain gene-family: ing questions in the CBF feld. The t(8;21) and inv(16) are asso- cDNA structure, expression, and chromosomal localization. Genomics, 23: 425-432, ciated with acute myeloid leukemias in humans, indicating a dif- 1994. ferentiation block at a much later stage of hematopoiesis than seen 7. Bae, S-C., Takahashi, E., Zhang, Y. W., Ogawa, E., Shigesada, K., Namba, Y., Satake, M., and Ito, Y. Cloning, mapping and expression of PEBP2aC, a third gene in the mouse knock-in models (Fig. 3). Furthermore, conspicuously encoding the mammalian Runt domain. Gene (Amst.), 159: 245-248, 1995. absent in the chimeric mice heterozygous for the t(8;21) and 8. Otto, F., Thomell, A. P., Crompton, T., Denzel, A., Gilmour, K. C., Rosewell, I. R., inv(16) alleles were signs of overt leukemia. What accounts for Stamp, G. W. H., Beddington, R. S. P., Mundlos, S., Olsen, B. R., Selby, P. B., and Owen, M. J. Cbfal, a candidate gene for cleidocranial dysplasia syndrome, is these discrepancies? essential for osteoblast differentiation and bone development. Cell, 89: 765-772, At this time, we can only speculate on the differences in the 1997. behavior of these transdominant negative alleles in the developing 9. Komori, T., Yagi, H., Nomura, S., Yamaguchi, A., Sasaki, K., Deguchi, K., Shimizu, Y., Bronson, R. T., Gao, Y-H., Inada, M., Sato, M., Okanaoto, R., Kitamura, Y., embryo and the leukemia patient, but there are several possibilities to Yoshiki, S., and Kishimoto, T. Targeted disruption of Cbfal results in a complete lack consider. One difference is timing; in the mouse knock-in experi- of bone formation owing to maturationai arrest of osteoblasts. Cell, 89: 755-764, ments, the t(8;21) allele and the inv(16) allele were present in all cells 1997. 10. Mundlos, S., Otto, F., Mundlos, C., Mulliken, J. B., Aylsworth, A. S., Albright, S., in the embryo, from the very beginning of development, and before Lindhout, D., Cote, W. G., Henn, W., Knoll, J. H. M., Owen, M. J., Mertelsmann, R., the emergence of HSCs. In contrast, in the leukemia patient, the Zabel, B. U., and Olsen, B. R. Mutations involving the transcription factor CBFAl translocations are presumably acquired somatically in a single cell cause cleidocranial dysplasia. Cell, 89: 773-780, 1997. 11. Golub, T. R., Barker, G. F., Bohlander, S. K., Hiebert, S., Ward, D. C., Bray-Ward, that had differentiated to a more committed stage in the hematopoietic P., Morgan, E., Raimondi, S. C., Rowley, J. D., and Gilliland, D. G. Fusion of the TEL pathway. Whether Cbfa2 and Cbfb are required for both the emer- gene on 12p13 to the AML1 gene on 21q22 in acute lymphoblastic leukemia. Proc. gence of the HSC, and the further differentiation of some hematopoi- Natl. Acad. Sci. USA, 92: 4917-4921, 1995. 12. Romana, S. 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1793s

Nancy A. Speck, Terry Stacy, Qing Wang, et al.

Cancer Res 1999;59:1789s-1793s.

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