CBFβ-SMMHC dominantly represses RUNX1 function, generates defects in definitive hematopoiesis (Castilla et al., 1996
), and predisposes mice to leukemia with cooperating gene mutations (Castilla et al., 1999
; Castilla et al., 2004
). In comparable mouse models the leukemia fusion gene RUNX1-ETO
(also known as AML1-ETO
) functions very similarly: it dominantly suppresses RUNX1 function, blocks hematopoiesis, and requires additional ‘hits’ for leukemogenesis (Yergeau et al., 1997
; Yuan et al., 2001
). Therefore the hypothesis of RUNX1 inhibition as a common leukemogenic pathway has been proposed for CBF-related leukemias (Speck and Gilliland, 2002
Previously we identified a RUNX1 high-affinity binding domain, HABD, at a proximal region of SMMHC in the CBFβ-SMMHC fusion protein (Lukasik et al., 2002
). The HABD has been considered as one of the most important domains for dominant repression of RUNX1, since the domain makes it possible for CBFβ-SMMHC to outcompete CBFβ for RUNX1 binding.
Clinically the fusion junctions between CBFB
are heterogeneous, 10 different fusion junctions have been reported (van Dongen et al., 1999
). All other CBFB-MYH11
fusion types contain the HABD except for the type I fusion. Earlier reports suggested that type I fusion is rare and tends to be associated with therapy related AML (t-AML) or myelodysplastic syndrome (t-MDS) (Dissing et al., 1998
; Grardel et al., 2002
; van der Reijden et al., 1995
; Yamamoto et al., 2006
). But a more recent publication indicated that type I fusion is potentially more frequent and can be associated with de novo AML (Monma et al., 2007
). It is noticeable that most commonly used RT-PCR primers for diagnosis of CBFB-MYH11
would not be able to detect the type I fusion, so it may have been under-diagnosed, especially in cytogenetically negative cases (Van der Reijden et al., 2001
; van Dongen et al., 1999
). The existence of the type I fusion suggests that the HABD or high-efficient RUNX1 repression is not always required for leukemogenesis.
To understand the importance of HABD for leukemogenesis, we generated mice expressing a knock-in Cbfb-MYH11 fusion gene with the HABD removed (Cbfb+/MYH11d179-221). In vitro and in vivo analyses indicated that the encoded CBFβ-SMMHCd179-221 was less efficient in binding and repressing RUNX1, as expected. Unexpectedly, leukemia development was accelerated in the Cbfb+/MYH11d179-221 knock-in mice, so that all the chimeras and F1 heterozygotes developed leukemia shortly after birth without ENU treatments. These findings cast doubts on the model that strong, dominant repression of RUNX1 is a key step in leukemogenesis.
Human CD34+ cells expressing the CBFβ-SMMHC variants behaved essentially the same as those expressing the full-length. This data is consistent with our conclusion that RUNX1 dominant inhibition may not be a critical step for leukemogenesis by CBFβ-SMMHC. However, the CBFβ-SMMHC variants did not accelerate the phenotype development in the human CD34+ cells, as compared to the accelerated leukemogenesis in the knockin mouse model. This difference is likely related to the fact that transgenic expression from retroviral vectors was used in the human CD34+ cells, while knockin technology was used in the mouse model. Both alleles of CBFB are intact in the human CD34+ cells; on the other hand, one Cbfb allele in the mouse genome is replaced by the Cbfb-MYH11 fusion gene. The endpoint of the human CD34+ cell assay system is also different from that in the mouse model, being clonal expansion rather than leukemia. All these factors could have contributed to the observed differences between these two systems.
The mechanism for the accelerated leukemogenesis in the Cbfb+/MYH11d179-221
mice is not clear. It is possible that the deletion released an anti-leukemia effect or provided a leukemia-promoting effect, such as upregulation of a cooperating gene. One potential candidate is MN1,
which was up-regulated in leukemia cells in the Cbfb+/MYH11d179-221
mice, as well as in human CD34+ cells expressing CBFβ-SMMHCd179-221
is an important cooperating gene for inv(16) leukemogenesis, as has been demonstrated by its specific upregulation in human inv(16) leukemia and its ability to accelerate leukemogenesis in our mouse Cbfb-MYH11
knock-in model (Carella et al., 2007
). It is therefore plausible that CBFβ-SMMHCd179-221
further up-regulates MN1
, which in turn cooperates with CBFβ-SMMHCd179-221
may up-regulate MN1
through multiple mechanisms. First, it appears that both full-length and d179-221 forms of CBFβ-SMMHC are able to up-regulate MN1 at the level of transcription (). Secondly, at the protein level, MN1 is recruited by p300 to act as a co-activator (van Wely et al., 2003
). It is likely that MN1 protein level/functionality is enhanced in cells expressing CBFβ-SMMHCd179-221
since there is more p300 phosphorylation in these cells than in those expressing full-length CBFβ-SMMHC (). Thirdly, hematopoietic blockage is less severe in the CBFβ-SMMHCd179-221
mice, resulting in a larger population of myeloid progenitors in the bone marrow (), which express the highest level of MN1 (Grosveld, 2007
). In summary, we believe the combined effect of a larger population of cells expressing high baseline level of MN1, more efficient p300 recruitment, and the transcriptional up-regulation contributes to higher expression level of MN1 in cells with CBFβ-SMMHCd179-221
The finding that the type I fusion and CBFβ-SMMHCd179-221 are fully capable of promoting RUNX1 and p300 phosphorylation, in contrast to CBFβ-SMMHC, is interesting. However, because this activity is similar to wildtype CBFβ, it raises the question as how this would contribute to the enhanced leukemogenic activity.
A likely explanation is that RUNX1 phosphorylation is a modifying or cooperating step, which contributes to leukemogenesis only in the presence of CBFβ-SMMHC proteins. More RUNX1 and p300 phosphorylation in mice expressing CBFβ-SMMHCd179-221 is consistent with partial rescue of hematopoietic blockage in these mice. It is therefore not surprising that this activity is similar to wildtype CBFβ, since CBFβ supports rather than blocks RUNX1 function in hematopoiesis.
The implications of normal Runx1 phosphorylation and hematopoietic rescue in the CBFβ-SMMHCd179-221
mice are at least two fold. First, normal Runx function may be required for leukemogenesis. We have observed that reduction of RUNX1 activity by introducing a Runx1
dominant negative allele to the Cbfb+/MYH11
mice delayed leukemia development (LZ and PPL, unpublished observations). It was also shown recently that Runx2 cooperated with Cbfβ-SMMHC for leukemogenesis (Kuo et al., 2009
). Secondly, reduced blockage of hematopoiesis as a result of partial RUNX1 inhibition may have led to the expansion of a “leukemia-prone” cell population, which provides more “target” cells for leukemogenesis. We speculate that HSCs were the target cells in the Cbfb+/MYH11
mice while both HSCs and myeloid progenitors could be the target cells in the Cbfb+/MYH11d179-221
mice. The fact that more leukemia cells in the Cbfb+/MYH11d179-221
mice express myeloid markers () suggests that leukemia may have initiated in myeloid progenitors in these mice.
Our finding that partial inhibition of RUNX1 may be more leukemogenic than complete RUNX1 inhibition is similar to what happened with PU.1, a hematopoietic transcription factor downstream of RUNX1. Mice with homo- or heterozygous deletion of Pu.1 do not develop leukemia; on the other hand, mice carrying hypomorphic alleles of Pu.1 with reduced expression (20% of normal) developed AML rapidly and efficiently (Rosenbauer et al., 2004
Moreover, a variant of the AML1-ETO fusion protein, AML1-ETO9a that contains C-terminal truncation, was found to be a much more potent inducer of leukemia than the full-length AML1-ETO in mouse models. It has been hypothesized that the deleted region inhibits the leukemogenic potential of AML1-ETO (Peterson et al., 2007
). Interestingly AML1-ETOtr, a C-terminally truncated protein similar to AML1-ETO9a, lost the ability to inhibit cell cycle progression of myeloid cells, which may contribute to its enhanced leukemogenic potential (Yan et al., 2004
). This AML1-ETO variant is thus similar to the CBFβ-SMMHC179-221
variant reported here, in that they are both less potent in repressing RUNX1/CBFB function yet are more potent in leukemogenesis.
Recent results (Kwok et al., 2009
; Park et al., 2009
; Roudaia et al., 2009
) show that while very modest effects on the heterodimerization of CBFβ with AML1-ETO have no effect on leukemogenesis, substantial loss of binding results in a protein that is incapable of causing leukemia, likely a reflection of the relatively high concentration of CBFβ in cells. This point is echoed by recent studies on recurrent mutations in RUNX1
in patients with AML subtype M0 and familial platelet disorder with predisposition to AML. The mutations abolish DNA binding and transactivation by RUNX1 in vitro (Michaud et al., 2002
; Osato et al., 1999
) and cause hematopoietic defects and embryonic lethality in mice (Matheny et al., 2007
), suggesting that they are mostly loss-of function mutations. However, it was recently shown that these mutations also altered differentiation and increased serial replating ability (Cammenga et al., 2007
), indicating that RUNX1 has DNA-binding independent activities that play a role in leukemogenesis. As the primary role of CBFβ is to stabilize RUNX1’s interaction with DNA, these findings provide further argument for CBFB/RUNX repression independent mechanisms in leukemogenesis.
Our current model for leukemogenesis in the Cbfb+/MYH11d179-221 mice is illustrated in . More severe blockage of hematopoiesis and RUNX1 inhibition by Cbfb-MYH11 results in a smaller leukemia target cell pool, where additional mutations are needed to introduce cooperating genes and to restore RUNX1 phosphorylation. On the other hand, a larger population of leukemia target cells is available in the Cbfb+/MYH11d179-221 mice, and leukemogenesis is further accelerated by upregulation of MN1 and retention of RUNX1 phosphorylation.
Working model for leukemogenesis in Cbfb+/MYH11d179-221 mice.
In summary, loss of the HABD from CBFβ-SMMHC unexpectedly potentiated its leukemogenic activity, raising questions to the proposed dominant-negative mechanism of leukemogenesis. Partial reduction of key transcription factors such as RUNX1 and PU.1 may be a common mechanism for leukemogenesis. Moreover, CBFβ-SMMHC may contribute to leukemogenesis through pathways other than RUNX1 repression. Recent work from our group has provided evidence for such RUNX1-repression independent pathways (Hyde et al., 2010
). However, it still needs to be determined whether the leukemogeneic activities of CBFβ-SMMHC depend on its interaction with RUNX1, even for those that do not seem to require RUNX1-repression. Such studies will provide important mechanistic insight guiding the design and development of small molecule inhibitors targeting the CBFβ/AML1 interaction for leukemia treatments (Gorczynski et al., 2007