Critical Functions Specified by the MLL CXXC Domain Determine Leukemogenic Capacity

TheMixed Lineage Leukemia(MLL) gene can participate in chromosomal translocations which generate a fusion protein leading to acute leukemia. A better understanding of how MLL fusion proteins contribute to leukemia is necessary in order to develop more effective treatments. In my dissertation project, I investigated the functional role of amino acids within the MLL CXXC domain to determine how specific residues contribute to leukemogenic capacity. MLL fusion proteins retain the amino-terminal portion of MLL including the CXXC DNA-binding domain while the carboxy-terminal portion is comprised of a fusion partner. The closest homolog of MLL, MLL2 (alternatively named MLL4), also contains a similar CXXC domain, yet an artificial MLL2 fusion protein is unable to transform cellsin vitro. I hypothesized that specific amino acid differences between the MLL and MLL2 CXXC domains account for differences in leukemogenic capacity. To test this hypothesis, the MLL2 CXXC domain was cloned into the context of the well-studied MLL-AF9 fusion protein to generate an artificial MLL/MLL2-AF9 chimera. Amino acid substitutions were then introduced within the MLL2 CXXC domain of this synthetic chimera to restore residues to the MLL sequence. By comparing residues of the MLL and MLL2 CXXC domains in colony formation and protein binding assays, critical amino acids were identified on both the DNA-binding surface and on the opposite, non-DNA-contact surface of the CXXC. Cysteine 1188 of the MLL CXXC domain is the only non-zinc-coordinating cysteine residue within the CXXC domain. This residue is critically positioned on the DNA-binding surface with susceptibility to post-translational modification. I hypothesized that the Cys1188 may be physiologically altered to regulate DNA-binding affinity. Transformed MLL-AF9 progenitor cells were treated with modifying agents or grown under conditions of varying oxygen concentration. The MLL-AF9 cells showed a modest susceptibility to parthenolide treatmentin vitrobut showed no significant differences in proliferation when grown under conditions of varying oxygen. The results from these studies build on the initial work conducted in our laboratory on the MLL CXXC domain and Cys1188 providing valuable direction for future investigations which may eventually allow for therapeutic targeting of the CXXC domain in MLL-associated leukemia