Leukaemic transformation by CALM–AF10 involves upregulation of Hoxa5 by hDOT1L

Abstract

Chromosomal translocation is a common cause of leukaemia 1 and the most common chromosome translocations found in leukaemia patients involve the mixed lineage leukaemia (MLL) gene 2,3 . AF10 is one of more than 30 MLL fusion partners in leukaemia 4 . We have recently demonstrated that the H3K79 methyltransferase hDOT1L contributes to MLL-AF10-mediated leukaemogenesis through its interaction with AF10 (ref. 5). In addition to MLL, AF10 has also been reported to fuse to CALM (clathrin-assembly protein-like lymphoid-myeloid) in patients with T-cell acute lymphoblastic leukaemia (T-ALL) and acute myeloid leukaemia (AML) 6,7 . Here, we analysed the molecular mechanism of leukaemogenesis by CALM-AF10. We demonstrate that CALM-AF10 fusion is both necessary and sufficient for leukaemic transformation. Additionally, we provide evidence that hDOT1L has an important role in the transformation process. hDOT1L contributes to CALM-AF10-mediated leukaemic transformation by preventing nuclear export of CALM-AF10 and by upregulating the Hoxa5 gene through H3K79 methylation. Thus, our study establishes CALM-AF10 fusion as a cause of leukaemia and reveals that mistargeting of hDOT1L and upregulation of Hoxa5 through H3K79 methylation is the underlying mechanism behind leukaemia caused by CALM-AF10 fusion. © 2006 Nature Publishing GroupReprints and permissions information is available online at http://npg.nature.com/naturecellbiology/ 6 Correspondence should be addressed to Y.Z. (yi_zhang@med.unc.edu).Note: Supplementary Information is available on the Nature Cell Biology website. COMPETING FINANCIAL INTERESTSThe authors declare that they have no competing financial interests. HHS Public Access Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptOur interest in CALM-AF10 fusion protein stems from our recent demonstration that hDOT1L, a histone H3K79 methyltransferase 8 , has an important role in MLL-AF10-mediated leukaemogenesis 5 . We demonstrated that mistargeting of hDOT1L to the Hoxa9 gene by MLL-AF10 results in H3K79 methylation and Hoxa9 upregulation, which contributes to leukaemic transformation 5 . We further demonstrated that the hDOT1L and MLL-AF10 interaction involves the octapeptide motif-leucine zipper (OM-LZ) region of AF10, this is also required for MLL-AF10-mediated leukaemic transformation 9 . The observation that the OM-LZ region is retained in the CALM-AF10 fusion protein raises the possibility that hDOT1L may also function in CALM-AF10-mediated leukaemia. To examine the potential role for hDOT1L in CALM-AF10-mediated leukaemia, we first attempted to establish a causal relationship between the CALM-AF10 fusion protein and leukaemia.Previous studies have established that the human monocytic leukemia cell line U937 expresses the CALM-AF10 fusion protein 10,11 . To determine whether CALM-AF10 is relevant for cell proliferation and transformation, CALM-AF10 was knocked down in U937 cells using a vector-based RNA interference (RNAi) approach 5 . Results shown in Fig. 1a indicate that we were...

The role of CALM–AF10 gene fusion in acute leukemia

“…70 In addition, U937 cells (which express a CALM-AF10 fusion) transduced with a siRNA directed to the CALM-AF10 fusion showed a modest decrease in HOXA5, HOXA7, HOXA9 and HOXA10 expression. 51 Consistent with those findings, transgenic mice that expressed a CALM-AF10 transgene showed eightfold or greater upregulation of Hoxa5, Hoxa7, Hoxa10, Hoxa11 and Meis1 in bone marrow from clinically healthy CALM-AF10 mice, and further upregulation (as much as 500-fold) of these genes in myeloid leukemia cells from CALM-AF10 mice. 52 Summary CALM-AF10 fusions result from a rare but recurring t(10;11) chromosomal translocation seen in both adult and pediatric patients with AML or ALL, and are often associated with a poor prognosis.…”

“…The 'knockdown' clones proliferated less rapidly in vitro, and showed a modest survival benefit following xenotransplantation into non-obese diabetic/severe combined immunodeficiency mice (median survival 27 vs 19.5 days post transplant). 51 A second study used retroviral transduction and bone marrow transplantation to generate acute leukemia in mice. 46 Recipient mice transplanted with bone marrow cells that expressed a CALM-AF10 fusion gene, developed disease a median of 110 days following transplantation.…”

Hox genes in hematopoiesis and leukemogenesis

“…Although the level of Hox gene expression was not reported in these lineage-positive LSCs, it would be of interest to determine if their novel ability to self-renew is a result of dysregulation of Hox gene expression, more precisely, genes of the Hoxa cluster. Indeed, CALM/ AF10 is a known upstream regulator of Hoxa gene expression (Dik et al, 2005;Bergeron et al, 2006;Okada et al, 2006).…”

Flickin’ the ubiquitin switch

“…40,84 Methylation of H3K4 and H3K79 may be pivotal in this process, as hDot1L mis-targeting to Hoxa9 is implicated in leukaemogenesis. 85,86 More directly, RNF20 knockdown in a breast cancer cell line reduces cell proliferation. 22 However, it is also important to note that RNF20 also ubiquitylates a variant form of the transcriptional co-regulator Ebp1, thereby marking it for degradation.…”