Self-association mediated by the Ras association 1 domain of AF6 activates the oncogenic potential of MLL-AF6 - PubMed (original) (raw)

Self-association mediated by the Ras association 1 domain of AF6 activates the oncogenic potential of MLL-AF6

Michaela Liedtke et al. Blood. 2010.

Abstract

MLL is a common target for chromosomal translocations associated with acute leukemia resulting in its fusion with a large variety of nuclear or cytoplasmic proteins that may activate its oncogenic properties by distinct but poorly understood mechanisms. The MLL-AF6 fusion gene represents the most common leukemogenic fusion of mixed lineage leukemia (MLL) to a cytoplasmic partner protein. Here, we identified a highly conserved Ras association (RA1) domain at the amino-terminus of AF6 as the minimal region sufficient for MLL-AF6 mediated myeloid progenitor immortalization in vitro and short latency leukemogenesis in vivo. Moreover, the ability of RA1 to activate MLL oncogenesis is conserved with its Drosophila ortholog, Canoe. Although the AF6 RA1 domain has previously been defined as an interaction surface for guanosine triphosphate-bound Ras, single amino acid substitutions known to abolish the AF6-Ras interaction did not abrogate MLL-AF6-mediated oncogenesis. Furthermore, fusion of MLL to heterologous RA domains of c-Raf1 or RalGDS, or direct fusion of MLL to constitutively active K-RAS, H-RAS, or RAP1 was not sufficient for oncogenic activation of MLL. Rather, the AF6 RA1 domain efficiently mediated self-association, suggesting that constitutive MLL self-association is a more common pathogenic mechanism for MLL oncogenesis than indicated by previous studies of rare MLL fusion partners.

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Figures

Figure 1

Figure 1

The N-terminal conserved region of AF6 is sufficient for myeloid immortalization by MLL-AF6. (A) Schematic diagram showing the positions of highly conserved domains within the AF6 protein family. The degree of identity between AF6 and orthologs from Drosophila and C. elegans is indicated. RA1 and RA2 indicates RAS association domains 1 and 2, respectively; NCR, N-terminal conserved region; PDZ, PSD-95/Dlg/ZO-1 domain. (B) Schematic diagram depicting MLL, AF6, and MLL-AF6 fusion protein. ATH indicates AT hook motifs; PHD, plant homeo-domain related; TAD, transcriptional activation domain; SET, Suvar3-9/enhancer-of-zeste/trithorax motif. Total number of amino acids comprising each protein is indicated on the right. Black arrows above each protein indicate the typical position of protein fusion after chromosomal translocations. Specific AF6 protein segments fused with MLL are indicated by brackets below the schematic. (C) Myeloid immortalization assay with various retroviral constructs encoding proteins indicated on the left. Each bar represents the mean ± SD of the total number of myeloid colonies per 104 plated cells derived from at least 4 replicates. (D) Western blot analysis of MLL-AF6 and AF6 proteins expressed in transiently transfected virus-producing Phoenix cells. (E) Typical morphology of cells (top; May-Grünwald-Giemsa stain) and blast colony (bottom) in the third round of the myeloid progenitor immortalization assay after transduction with MLL-AF6NCR.

Figure 2

Figure 2

The RA1 domain within the AF6 NCR mediates myeloid immortalization by MLL-AF6. (A) Sequence alignment of the RA1 domains of AF6 family proteins. Dark shading defines residues of identity or similarity. Amino acid numbers are indicated on the left. The positions of conserved residues targeted for mutation are highlighted above the sequence. The conserved secondary structures are depicted above the sequence, where filled arrows represent β sheet and the filled box represents α helix. Brackets below indicate the RA1 amino acids present in deletion constructs identified on the left. (B) Myeloid immortalization assay resulting from transduction of various MLL-AF6 mutant proteins shown on the left. Each bar represents the mean ± SD of the total number of myeloid colonies per 104 plated cells (≥ 4 replicates). (C) Western blot analysis of MLL-AF6 fusion proteins expressed in transiently transfected virus-producing Phoenix cells. (D) Immunoprecipitation–-Western blot analysis shows that only the intact MLL-AF6RA1 is capable of coprecipitating HA-tagged RasV12 in transiently transfected 293 cells. (E) Myeloid immortalization assay with retroviral constructs encoding MLL-AF6RA1 point mutants indicated on the left. Each bar represents the mean ± SD of the total number of myeloid colonies per 104 plated cells (≥ 4 replicates). (F) Western blot analysis of MLL-AF6 mutant proteins expressed in transiently transfected virus-producing Phoenix cells.

Figure 3

Figure 3

Fusion of N-terminal MLL to heterologous Ras association domains from c-Raf1, RalGDS, or constitutively active mutants of K-Ras, H-Ras, or Rap1 does not activate the oncogenic potential of MLL. (A) Sequence alignment of the N-terminal regions of various RA domains. Dark shading defines residues of identity or similarity. Amino acid numbers are indicated on the left. The conserved secondary structures are depicted above, where arrows represent β sheets and the filled box represents an α helix. Crystal structures for c-Raf1, RalGDS, and Byr2 have been previously described.,, Putative AF6 RA1 domain secondary structure was derived from the PHD prediction algorithm. (B) Myeloid immortalization assay with various retroviral constructs encoding MLL fusions to heterologous RA domains or proteins are as indicated on the left. Each bar represents the mean ± SD of the total number of myeloid colonies per 104 plated cells (≥ 4 replicates). (C) Western blot analysis of MLL fusion proteins expressed in transiently transfected virus-producing Phoenix cells.

Figure 4

Figure 4

The RA1 domain of Canoe, the fly ortholog of AF6, activates the oncogenic potential of MLL. (A) Myeloid immortalization assay was performed with MLL fusion constructs indicated on the left. Each bar represents the mean ± SD of the total number of myeloid colonies per 104 plated cells. Data are derived from at least 4 cultures from 2 or more independent experiments. (B) Western blot analysis of MLL fusion proteins expressed in transiently transfected virus-producing Phoenix cells. (C) Survival curves are shown for sublethally irradiated C57BL/6 mice that received a transplant with various cell lines immortalized by MLL fusion genes.

Figure 5

Figure 5

The RA1 domain of AF6 mediates self-association in vitro. (A) Self-association was assessed by anti-HA Western blot analysis after anti-FLAG immunoprecipitation from lysates of 293 cells cotransfected with various HA-tagged AF6 constructs together with FLAG-tagged AF6RA1 (top). Expression levels of the relevant input proteins were determined by immunoblotting with either anti-HA (middle) or anti-FLAG (bottom) antibodies. (B) Self-association of MLL-AF6RA1 was shown by anti-HA Western blot analysis after anti-FLAG immunoprecipitation from lysates of cells cotransfected with HA-tagged MLL-AF6RA1 and FLAG-tagged MLL-AF6RA1 (top). Expression levels of the input proteins were determined with either anti-HA (middle) or anti-FLAG (bottom) antibodies.

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