Abstract 4014: Determination of critical residues within the CXXC domain of MLL fusions which contribute to leukemic transformation

The Mixed Lineage Leukemia (MLL) protein is a large, multi-domain protein involved in maintenance of gene expression during embryogenesis and hematopoiesis. Chimeric oncoproteins involving MLL are associated with aggressive forms of acute myeloid or lymphoid leukemia. These fusion proteins necessarily retain the N-terminal portion of MLL including the CXXC CpG-DNA-binding domain fused to the C-terminal fragment of one of more than 60 possible partner proteins. The closest homolog of MLL, MLL2 (alternatively named MLL4), cannot transform bone marrow progenitor cells in the context of a similar fusion, even though it contains similar functional domains. Knowing that the CXXC domain is a critical component of the MLL leukemic fusions, we hypothesized that specific amino acid differences between the MLL and MLL2 CXXC domains account for the differences in leukemogenic capacity. In order to test this hypothesis, the MLL2 CXXC domain was cloned into the context of the well-studied MLL-AF9 fusion to generate an artificial MLL(MLL2 CXXC)-AF9 retroviral chimera. Thirteen amino acid substitutions within the MLL2 CXXC domain were strategically introduced to restore individual residues to the MLL amino acid sequence within this synthetic chimera. Bone marrow progenitor cells were transduced with wild type or point mutant retroviral constructs to measure in vitro proliferation and replating capacity in a methylcellulose colony assay. The wild type MLL(MLL2 CXXC)-AF9 construct generated 10-fold fewer colonies than the highly transforming positive control, MLL-AF9, by the third round of replating. These colonies more closely resembled those transduced with the empty vector negative control with a more diffuse colony morphology and comprised of cells with a differentiated morphology. This was in contrast to the MLL-AF9 control which generated dense colonies comprised of blast-like cells. The thirteen point mutant MLL(MLL2 CXXC)-AF9 constructs showed variable capacities to rescue proliferation in vitro suggesting that some_but not all_of the specific residues within the CXXC domain of MLL are critical for rescuing the leukemic transformation activity of MLL2 fusion proteins. Independently, quantification of Hoxa9 expression (a direct MLL target gene) via qRT-PCR demonstrated similar results: those critical residues that rescued proliferation also showed higher expression levels of Hoxa9. The specific residues which were found to contribute to leukemogenic transformation are located both on the DNA-binding surface of the CXXC domain and on the opposite, non-DNA-contact surface. This finding suggests that there are two critical surfaces within this one domain which may be strategically targeted with therapeutics to (i) alter DNA-binding affinity or (ii) disrupt protein interactions which may ultimately allow for more successful treatment of this aggressive form of leukemia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4014. doi:10.1158/1538-7445.AM2011-4014