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Cancer Research 66, 11214-11218, December 1, 2006. doi: 10.1158/0008-5472.CAN-06-0959
© 2006 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

Cbfß Reduces Cbfß-SMMHC–Associated Acute Myeloid Leukemia in Mice

Susan A. Heilman1, Ya-Huei Kuo1, Chantal S. Goudswaard2, Peter J. Valk2 and Lucio H. Castilla1

1 Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, Massachusetts and 2 Department of Hematology, Erasmus University Medical Center, Rotterdam, the Netherlands

Requests for reprints: Lucio H. Castilla, Program in Gene Function and Expression, University of Massachusetts Medical School, 364 Plantation Street, LRB/622, Worcester, MA 01605. Phone: 508-856-3281; Fax: 508-856-5460; E-mail: Lucio.Castilla{at}umassmed.edu.


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The gene encoding for core-binding factor ß (CBFß) is altered in acute myeloid leukemia samples with an inversion in chromosome 16, expressing the fusion protein CBFß-SMMHC. Previous studies have shown that this oncoprotein interferes with hematopoietic differentiation and proliferation and participates in leukemia development. In this study, we provide evidence that Cbfß modulates the oncogenic function of this fusion protein. We show that Cbfß plays an important role in proliferation of hematopoietic progenitors expressing Cbfß-SMMHC in vitro. In addition, Cbfß-SMMHC–mediated leukemia development is accelerated in the absence of Cbfß. These results indicate that the balance between Cbfß and Cbfß-SMMHC directly affects leukemia development, and suggest that CBF-specific therapeutic molecules should target CBFß-SMMHC function while maintaining CBFß activity. (Cancer Res 2006; 66(23): 11214-8)


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The core-binding factor (CBF) transcription factor is the most common target of chromosomal rearrangements in human acute myeloid leukemia (AML), including the fusion genes CBFB-MYH11 and RUNX1-ETO (1). Moreover, RUNX1 is frequently mutated in AML. CBF is a heterodimeric transcription factor that consists of a DNA binding {alpha}-subunit, encoded by one of three members of the RUNX family (RUNX1, RUNX2, and RUNX3), and a ß-subunit encoded by the CBFB gene that increases DNA-binding affinity to the complex. In hematopoiesis, the CBF heterodimer Cbfß:Runx1 regulates expression of genes with critical functions in differentiation of lymphoid and myeloid lineages. The Cbfß:Runx3 complex is involved in B-cell maturation and the silencing of the CD4 gene during T-cell maturation (2). Studies in the mouse have determined that Cbfb–/– and Runx1–/– embryos fail to develop embryonic definitive hematopoiesis and die at midgestation (3–6). This phenotype was rescued in Cbfb–/– mice expressing Cbfb from the hematopoietic specific promoters Tie2 or GATA1, further underscoring the key role of Cbfß during hematopoietic differentiation (7, 8).

Approximately 12% of AML patients present a chromosome 16 inversion [inv(16); ref. 9] that breaks and joins the first five exons of CBFB with the second half of the smooth muscle myosin heavy chain gene MYH11 (10). The resulting CBFB-MYH11 gene encodes the CBFß-SMMHC fusion protein, which retains the Runx-binding domain from Cbfß and multimerization domain from the myosin sequence. Studies in mice have shown that Cbfß-SMMHC is a dominant inhibitor of CBF function because Cbfb+/MYH11 heterozygous knock-in embryos expressing the fusion protein failed to develop definitive hematopoiesis (11), as was shown for the Cbfb- and Runx1-null embryos (3–6).

Induction of Cbfß-SMMHC expression or Runx1-loss in adult bone marrow does not seem to affect the maintenance of long-term hematopoietic stem cells (12–14). However, Cbfß-SMMHC expression reduces hematopoietic stem cell function by inhibiting multilineage repopulation and creating a myeloid progenitor predisposed to leukemia development (11).

Several lines of evidence suggest that Cbfß-SMMHC may exert an incomplete block of CBF function. First, ectopic expression of the fusion protein in embryonic stem cells expressing one or both copies of Cbfb does not inhibit differentiation in vitro (15). Second, Cbfb+/MYH11 knock-in hematopoietic stem cells expressing Cbfß-SMMHC persist in the bone marrow of the chimeras (16). Third, retroviral insertional mutagenesis in Cbfb+/MYH11 knock-in chimeras identified common insertions in the Runx2 gene (17), suggesting that Cbfß-SMMHC leukemic function is affected by levels of Runx proteins.

In this study, we test the hypothesis that Cbfß modulates the Cbfß-SMMHC effect in adult hematopoiesis and leukemogenesis. We used mice with a Cbfb knock-out allele and a conditional Cbfb-MYH11 knock-in allele to study adult myeloid differentiation and leukemia progression. This study provides evidence that Cbfß modulates hematopoietic differentiation and Cbfß-SMMHC–mediated leukemia development, and suggests that CBFß up-regulation may efficiently counteract differentiation defects in human AML with inv(16).


   Materials and Methods
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 Abstract
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 Materials and Methods
Results
 Discussion
 References
 
Generation of triple Mx1Cre/Cbfb–/MYH11 transgenic mice. The design of the conditional Cbfb+/MYH11 knock-in mice has previously been described (12), with the exception that monoclonal ß-actin antibody (Sigma, St. Louis, MO) was used as western blot control. Expression of CBFß-SMMHC was induced in 3-week-old mice by activation of Cre recombinase from the Mx1Cre transgene using one to three doses of polyinosinic-polycytidylic acid (pIpC) every other day (18). Heterozygous Cbfb–/+ knockout mice were generously provided by Nancy Speck (Dartmouth Medical School, Hanover, NH; ref. 4). For this study, all mice were maintained in the 129SvEv strain. In the transplantation assays, 1 x 106 leukemic cells were transplanted into sublethally irradiated syngenic recipients as described elsewhere (19).

Molecular and cytology analysis. The Western blot, flow cytometry, and histopathology analyses were done as previously described (12). Fluorescence-activated cell-sorting analysis was done in peripheral blood of leukemic mice using FITC-c-kit and phycoerythrin-lineage antibodies (Lin+: B220, CD3, Gr1, and Mac1; all from BD Biosciences, San Jose, CA).

Colony forming assays. Mice with the genotypes Cbfb+/56M, Cre;Cbfb+/56M, Cbfb-/56M, and Cre;Cbfb-/56M were injected with pIpC at weaning every other day. Two days after the second injection, bone marrow cells were harvested, and 1 x 104 WBC were plated in duplicate in methylcellulose supplemented with cytokines interleukin (IL)-3, IL-6, and stem cell factor and erythropoietin (Methocult-3434, Stem Cell Technologies, Vancouver, Canada) in 35-mm nontreated tissue culture dishes (Corning). The number of myeloid colonies was scored at day 7. Single colonies were harvested and either cytospun for cytology analysis or placed into lysis buffer for PCR analysis.

Statistical considerations. Differences in survival functions between groups were evaluated by Kaplan-Meier product limit survival analysis using the Tarone-Ware test to test the hypothesis of overall equivalence. In the presence of significant overall differences, pairwise comparisons were made between the noncontrol groups using Tarone-Ware tests with a Sidak adjustment to compensate for the additive type I error due to multiple comparisons.

Analysis of human AML samples. Patients had a diagnosis of primary AML, confirmed by cytologic examination of blood and bone marrow. After informed consent, bone marrow aspirates or peripheral blood samples were taken at diagnosis (n = 285) and processed for Affymetrix U133A GeneChip analysis (20). For PCR and sequence analyses, cDNA prepared from 50 ng of RNA was used for all PCR amplifications. The CBFB coding region was sequenced for 27 inv(16)+ AML samples by cDNA amplification using the primers CBFB-FOR 5'-CAGAGAAGCAAGTTCGAGAACG-3' with CBFB-REV 5'-GTTTGAGGTCATCACCACCAC-3' and CBFB-FOR with CBFB6 5'-GTCTTGTTGTCTTCTTGCCAG-3' (25 mmol/L deoxynucleotide triphosphate, 15 pmol primers, 2 mmol/L MgCl2, Taq polymerase and 10x buffer; Invitrogen Life Technologies, Breda, the Netherlands). Cycling conditions for both primer sets consisted of a denaturing cycle for 5 minutes at 94°C followed by 30 cycles for amplification (1 minute 94°C, 1 minute 62°C, 1 minute 72°C), and a final extension cycle for 7 minutes at 72°C. PCR products were purified using the Multiscreen-PCR 96-well system (Millipore, Bedford, MA) followed by direct sequencing with CBFB-FOR, CBFB-REV, and CBFB6 using an ABI-PRISM3100 genetic analyzer (Applied Biosytems, Foster City, CA).


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cbfß is essential to maintain proliferation capacity of myeloid progenitor cells expressing Cbfß-SMMHC. We have recently shown that bone marrow cells expressing Cbfß-SMMHC accumulate abnormal myeloid progenitors able to form myeloid colonies in vitro (12). To assess whether this effect is dependent on the presence of Cbfß, colony-forming unit (CFU) assays were done with bone marrow cells from heterozygous floxed (Cbfb+/56M), hemizygous floxed (Cbfb-/56M), heterozygous restored (Cbfb+/MYH11), and hemizygous restored (Cbfb–/MYH11) mice (Fig. 1 ). The switch from Cbfß to Cbfß-SMMHC expression (switching floxed to restored allele) was induced by pIpC-mediated Cre activation using the Mx1Cre transgene.


Figure 1
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  		Figure 1. Cbfb alleles used in this study. Exons 1 to 6 of the Cbfb gene are shown in boxes, and the encoded protein is shown on the right. The Cbfb knock-out allele includes a neomycin (N) gene fused to the 3'-end of exon 5 (4). The floxed Cbfb56M allele includes exons 5 and 6 and a hygromycin gene (H) between loxP sites (triangle), followed by exon 5 fused to the 3' MYH11 sequence and a neomycin gene (12). Upon Cre-mediated loxP deletion, Cbfß-SMMHC is induced in the CbfbMYH11 restored allele.

 
The number of myeloid and erythroid colonies was significantly reduced in hemizygous restored bone marrow progenitor cells expressing Cbfß-SMMHC when compared with control groups (Fig. 2A ). Considering that the loss of one Cbfb allele did not affect CFU formation (heterozygous floxed versus hemizygous floxed), these results indicate that the number of CFUs is markedly reduced at Cbfb levels below 50%. In addition, the size of the colonies was markedly reduced in hemizygous restored progenitors when compared with controls (Fig. 2B). The Cre-lox–mediated excision in CFUs from hemizygous restored plates was confirmed by PCR analysis (data not shown). Cytology analysis of day-7 CFUs revealed the presence of all myeloid forms, with a small but consistent increase of blastlike immature cells in hemizygous restored colonies (Fig. 2C). These results revealed that the loss of Cbfß in the context of Cbfß-SMMHC significantly reduces the proliferation capacity of bone marrow myeloid progenitors in vitro.


Figure 2
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  		Figure 2. Cbfß modulates bone marrow myeloid proliferation in vitro. Colony forming assays in methylcellulose cultures using 1 x 104 bone marrow progenitor cells with heterozygous floxed (+/56M), heterozygous restored (+/MYH11), hemizygous floxed (–/56M), or hemizygous restored (–/MYH11) Cbfb genotypes. A, erythroid colonies were scored at day 4 (gray) and myeloid colonies at day 4 (black) and day 7 (white). Columns, number of colonies from three independent experiments, each in duplicate. B, representative images of colony size (amplification, x50). C, histogram representation of cytology analysis of CFU-GEMM, CFU-Blast, CFU-GM, CFU-M, and CFU-G.

 
Hemizygous bone marrow cells expressing Cbfß-SMMHC show higher susceptibility to AML. The Cbfb+/MYH11 restored mice succumb to AML with a median latency of 5 months (12). This latency is dose dependent as mice induced with three pIpC doses developed AML with a median latency of 5 months, mice induced with one pIpC dose had AML with a median latency of 8 months, and uninduced mice remained disease-free.

To test whether the presence of Cbfß has an effect in Cbfß-SMMHC–mediated AML, we compared heterozygous restored and hemizygous restored mice after treatment with three doses of pIpC. In the absence of Cbfß, 100% of mice with bone marrow cells expressing Cbfß-SMMHC developed AML with a significant acceleration of disease onset (median latency of 1.5 ± 0.5 months; P < 0.00001; Fig. 3A ). Surprisingly, uninduced Mx1Cre/Cbfb-/56M mice also developed AML with similar latency to that of induced group (Fig. 3A; red dashed line, uninduced; red solid line, induced). It has previously been reported that Cre expression from the Mx1Cre transgene is leaky in mice not treated with pIpC (21). In our study, all AML samples from induced and uninduced groups exhibited deletion of the floxed sequence by PCR analysis (data not shown) and Cbfß-SMMHC expression was detected by Western blot analysis (Fig. 3B). Furthermore, secondary transplantation of Cbfb–/MYH11 AML cells derived from induced or uninduced mice produced leukemia in sublethally irradiated recipients with a median latency of 6 weeks (data not shown). These results indicate that Cbfß-SMMHC–induced AML development is accelerated in the absence of Cbfß.


Figure 3
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  		Figure 3. Loss of Cbfß accelerates Cbfß-SMMHC–mediated AML. A, Kaplan-Meier survival curve of mice expressing Cbfß-SMMHC in the presence or absence of Cbfß. Mice induced with pIpC (+) heterozygous restored [red dotted line, +/MYH11;Cre (+); n = 38], uninduced (–) heterozygous floxed [black line with star mark, +/56M (–); n = 20], uninduced hemizygous floxed [black line with circle mark, –/56M (–); n = 15], untreated hemizygous restored [red dashed line, –/MYH11;Cre (–); n = 20], or treated hemizygous restored [red solid line, –/MYH11;Cre (+); n = 16]. B, Western blot analysis of Cbfß-SMMHC and ß-actin in AML samples derived from restored Cbfb mice induced (+) with pIpC or uninduced (–). The Cbfb genotype of the AML cells is shown on the top. C, disease pathology analysis depicting an increase of immature leukemic cells (top row; triangle, blastlike; arrow, myeloid form; magnification, x1,000), disruption of spleen architecture (middle row; magnification, x100), and the presence of infiltrating leukemia cells (white arrow) in the liver (bottom row; magnification, x100). Cells analyzed from wild-type control (left column) and leukemic mice expressing Cbfß-SMMHC in the presence (middle column) or absence (right column) of Cbfß. D, FACS analysis of leukemic cells from hemizygous restored mice (bottom) compared with wild type control (top) using lineage markers (Gr1, B220, Mac1, CD3) and a progenitor marker (c-kit).

 
Disease pathology is dictated by Cbfß-SMMHC. The hemizygous restored leukemic Cbfb–/MYH11 mice presented an increase in WBC count (mean, 41.7 ± 74.7 x 106/mL) when compared with healthy littermates (mean, 4 x 106/mL). As previously described in heterozygous restored leukemic mice, the leukemic cells from the hemizygous mice included predominant blastlike and myelomonocytic cells (Fig. 3C, top row) with the characteristic cell-surface profile c-kit+/Lin– (Fig. 3D). Disease was also marked by progressive anemia and infiltration into other organs, including the spleen and liver. Histology analysis of these organs confirmed the disruption of splenic architecture marked by infiltration of leukemic cells (Fig. 3C, middle row) and focal infiltrations of leukemic cells into the interstitial spaces of the liver (Fig. 3C, bottom row). Compromise of the thymus and lymph nodes was not observed. Taken together, the pathology of disease in hemizygous and heterozygous mice was similar, suggesting that the AML phenotype was determined by Cbfß-SMMHC.

The wild-type CBFB allele is not a frequent target of mutations in inv(16) AML. To assess whether CBFB is frequently altered in human CBF AML samples, expression and mutation analyses of CBFB were undertaken. Sequence analysis of the CBFB coding region in a panel of 29 inv(16) AML samples identified no mutations. Expression analysis of CBFB in a panel of 285 human AML samples indicated that inv(16) AML samples had a 40% reduction in CBFB transcript when compared with CD34+ bone marrow cells (relative value, 0.4 ± 0.08), as expected by the expression of one CBFB allele. The CBFB levels in t(8;21) and non-CBF cytogenetic groups were unchanged [t(8;21) relative value, 0.9 ± 0.23; non-CBF relative value, 1.0 ± 0.32]. These results indicate that the remaining CBFB allele is not frequently altered in inv(16) AMLs.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Endogenous expression of Cbfß and CbfFormula-SMMHC from the Cbfb allele of conditional knock-in mice creates a leukemia precursor that progresses to AML in a multistep process (12). Although the fusion protein is thought to act as a dominant factor in differentiation and transformation (11, 12, 16), the role of Cbfß in Cbfß-SMMHC–mediated leukemia is not clear. Here we showed that the capacity of Cbfß-SMMHC to induce AML in mice is modulated by Cbfß.

The presence of Cbfß is critical for embryonic definitive hematopoiesis (4) and for in vitro myeloid differentiation from Cbfb–/– embryonic stem cells (15). Our study indicates that Cbfß is necessary for in vitro myeloid-erythoid differentiation of bone marrow hematopoietic progenitors. In addition, because colonies were drastically reduced in the absence of Cbfß but not in the presence of Cbfß-SMMHC, our results support the hypothesis that Cbfß-SMMHC may have an incomplete effect in differentiation. As Cbfß and Cbfß-SMMHC compete for binding with Runx proteins in bone marrow cells, basal levels of Cbfß:Runx1 complex in hematopoietic progenitors expressing Cbfß-SMMHC may be critical for proliferation of myeloid progenitors and delayed transformation.

Endogenous expression of Cbfß-SMMHC and Cbfß in bone marrow induces AML with a median latency of 5 months (12). We observed that upon Cre-lox–mediated switch from Cbfß to Cbfß-SMMHC expression in progenitor cells lacking a wild-type Cbfb allele, AML latency was shortened to 6 weeks. These results strongly suggest that Cbfß-SMMHC function is enhanced by Cbfß loss. Surprisingly, a similar AML latency was observed between induced and uninduced groups. Probably, a small progenitor population may have undergone Cre/lox deletion due to "leaky" Cre expression from the Mx1Cre transgene (21), and thus becoming a leukemia precursor. Importantly, all AML samples presented Cre-mediated deletion, suggesting that transformation is due to the Cbfß to Cbfß-SMMHC switch. Furthermore, the finding that CBFb is not frequently lost in human AML argues against its role as an inv(16) cooperating tumor suppressor in AML. Rather, our results suggest that increase in the Cbfß-SMMHC-to-Cbfß ratio reduced proliferation of myeloid progenitors while increasing their susceptibility to neoplastic transformation, although the underlying mechanism is unclear. However, we cannot rule out the possibility that Cbfb loss in bone marrow could induce AML. The generation of conditional Cbfb knock-out alleles will provide a critical tool to directly address this possibility using a genetic approach.

Finally, these findings have important implications on the design of targeted therapies. One potential avenue is the identification of drugs that inhibit the fusion protein. Although candidate molecules should act to disrupt Cbfß-SMMHC:Runx1 binding, it will be critical that CBFß:Runx1 binding remains unaltered.


   Acknowledgments
 
Grant support: NIH grant CA096983 (L.H. Castilla) and the Ruth L. Kirschstein National Research Service postdoctoral award F32CA101571 (Y-H. Kuo).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 4/ 6/06. Revised 7/26/06. Accepted 8/28/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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