Several endocytic proteins have been identified as fusion partners of transcription factors or tyrosine kinases in hematopoietic malignancies, and it has been proposed that disruption of the endocytic process may contribute to disease progression (Lanzetti and Di Fiore, 2008
). To explore this hypothesis further, we examined the functional contribution of the CALM moiety in CALM-AF10-mediated transformation. CALM2091
mice developed disease with a similar latency (P=0.1), yet CALM2091
AF10 affects the severity of the disease, suggesting a potential role for clathrin. However, we did not identify deficits in endocytosis, proliferation or signaling in CALM-AF10+
leukemia or hematopoietic cell line models. In contrast, inclusion of the clathrin-binding domain (CALM2091
AF10) was required to inhibit transferrin endocytosis in 293T cells efficiently. As many studies of endocytic function rely heavily on the use of epithelial cell lines, our studies emphasize caution in interpreting these results in a larger biological context. For example, CALM-AF10 localized predominantly in the cytoplasm of 293T cells (where it may have transiently interacted with clathrin-coated vesicles), but was found predominantly in the nucleus of the CA2091CL1 leukemia cell line, providing a potential explanation for the different effects on endocytosis in the two cell types.
Experimental results suggest that DOT1L and H3K79me2 are regulated during progression of the cell cycle, in which H3K79me2 levels increase during M phase (Feng et al., 2002
). During interphase, clathrin plays a key role in membrane trafficking; however, when the cell enters mitosis, membrane trafficking ceases and a portion of clathrin is targeted to the mitotic spindle where it stabilizes kinetochore fibers (Royle et al., 2005
). In the CALM2091
leukemia cell line, CALM-AF10 and clathrin generally segregated to either the nucleus or cytoplasm, respectively, during interphase; however, they co-localized during metaphase (, Supplementary Fig. S5
). Therefore, we examined whether the DOT1L-sequestering effect of CALM-AF10 was influenced by interaction of the CALM moiety with clathrin. However, knock-down of clathrin had no effect on global H3K79me2 levels in a CALM AF10+
2091 cell line, leaving open the question of how CALM-AF10 may act as a dominant negative competitor of AF10 to regulate global H3K79 hypomethylation. The genomic instability observed in CALM-AF10+
cells is speculated to be associated with global H3K79 hypomethylation (Lin et al., 2009
). Given that disruption of clathrin has also been associated with genomic instability (Lemmon et al., 1990
; Royle et al., 2005
), and that CALM-AF10 colocalizes with clathrin during metaphase in the leukemia cell line tested, our data raises the intriguing possibility that clathrin/CALM-AF10 interaction may also contribute to increased genomic instability.
It has been proposed that oligomerization of chimeric transcription factors may act as a universal oncogenic amplifier that enhances the transcriptional properties by augmenting and broadening the binding affinity to interacting proteins and target DNA sequences. The nature of the endocytic machinery, characterized by their self-assembly properties and ability to form higher-order complexes, may lend itself to promoting dimerization or oligomerization of transcription factors. Moreover, previous work suggests that the homo-oligomerization of AF10 may itself enhance AF10-mediated transcription (Forissier et al., 2007
). In our mouse model, we observed a slightly more aggressive disease with a form of CALM-AF10 (CALM2091
AF10) that has a tendency to form macroscopic aggregates, regardless of cellular location, and could be shown to oligomerize by FRET analysis. The homo-oligomerization of CALM-AF10 could be envisaged to compete with endogenous AF10 for the recruitment of DOT1L, a known player in CALM-AF10-mediated transformation. Additionally, AF10 binds to GAS41 (Harborth et al., 2000
), a protein previously found to interact with the SWI/SNF complex, which in turn binds to the nuclear mitotic apparatus protein, NuMA (Bhattacharya et al., 2002
), which can be phosphorylated by Pim1. Alteration of the binding affinity of these interactions through oligomerization, could influence chromatin organization and transcriptional regulation, thereby, contributing to malignant transformation. Artificial oligomerization of AF10, as has been done for MLL (So et al., 2003
), could be used to address this hypothesis and rule out other oncogenic contributions by CALM.
In patients with CALM-AF10+
T-ALL there appears to be a correlation between the AF10 fusion and leukemic phenotype (Asnafi et al., 2003
). To date, there is no known association between CALM or AF10 fusion breakpoint and leukemic phenotype in AML patients; however, varying degrees of maturation and leukemia phenotype (AML M0-M5) have been noted (Carlson et al., 2000
). Also, CALM-AF10 fusions are found in <1% of AML cases, and only a small number of cases have been well-characterized. Nonetheless, mouse models have proven to be a powerful biological tool in deciphering the underlying mechanisms of disease. In this study, we exploited the alternate CALM fusions to decipher the functional contribution of the CALM protein in CALM-AF10-mediated transformation. Our data suggest that increased proliferation through perturbed endocytosis is not a major contributing factor to oncogenic transformation, as originally hypothesized. Instead, our findings point to an alternative mechanism involving homo-oligomerization of CALM-AF10 that could increase its binding affinity for interacting proteins, such as DOT1L or other co-activators, that influence chromatin organization and transcriptional regulation, promoting the development of overt leukemia.