Amplification of Histone Methyltransferase, Mixed-Lineage Leukemia (MLL), Perturbs Normal Hematopoiesis and Induces Hematological Disorder.

The Mixed-Lineage Leukemia Gene, MLL, encoding a SET domain with an H3K4 methyltransferase, is frequently mutated in a wide range of hematological malignancies. Apart from formation of chimeric fusion genes, MLL is amplified (MLLn) or partially tandem duplicated (MLL-PTD) in approximately 1% of acute myeloid leukemia (AML) and 4-7% of cytogenetically normal AML patients, respectively. MLLn is also observed in myelodysplastic syndrome and rare cases of pre-B ALL. While the roles of MLL fusion proteins in leukemogenesis are relatively well defined, very little is known about MLLn and MLL-PTD. In contrast to MLL fusions, MLL-PTD appears to be a weak oncoprotein, which could perturb normal hematopoiesis but was insufficient to induce leukemia in a murine model. MLLn is the only reported MLL aberration resulting in structural amplification of the SET domain, but also is the least characterized one with no animal models available. Its role and the underlying mechanisms for transformation are completely unknown.

Although animal models have been established for MLL fusions using retroviral transduction/transplantation approaches, it has not been possible to model MLLn using similar approaches due to the size of the cDNA (>10kb). Similarly, knockin approach used for MLL-PTD will not be feasible for introducing any significant change of number copy. To this end, we have adopted a transgenic approach where human MLL cDNA under the control of SCL promoter/enhancer protected by globin insulator elements (MLLn). The employed SCL elements could drive expression of the transgene in HSCs and early progenitors, while the insulators could ensure proper transgene expression and protect any interference due to positional effect of random integration. MLL-PTD mice generated by a knockin approach were also analyzed and compared side-by-side with the MLLn mice.

Two independent MLLn mice with 10-15 and 80-100 copies of MLL transgene, which are in line with the corresponding ranges identified in AML patients, were obtained and characterized. Over-expression of MLL in various hematological organs including bone marrow, spleen and thymus positively correlated with the copy number of the transgene, confirming the molecular features of the models. Although these mice exhibited normal numbers and ratios of phenotypically defined hematopoietic compartments including HSCs and early progenitors (CMP, CLP, GMP, MEP) at young ages, their functional properties were clearly perturbed. In both myeloid and lymphoid CFC assays, a significant increase in colonies was observed in bone marrow cells and splenocytes from both MLLn and MLL-PTD mice. An enhanced replating ability was also observed in splenocytes although only MLL-PTD cells could form colonies after the third round. Bone marrow cells from both models exhibited a higher cell proliferation rate, and had an increased proportion of cycling cells in S phase. In contrast to MLL-PTD mice, a decrease in cellularity was found in the spleen of MLLn mice, which had however an expansion of Mac1+Gr1+ myeloid cells. Similar to MLL-PTD mice, most of the MLLn mice did not develop leukemia after 18 months of observation. However, about 10% of MLL(80-100) mice developed tumor(s) at about 12 months, while none were found in the littermate controls or MLL-PTD mice. The tumors were hematopoietic in origin and composed of mostly lymphoid (80%) and some myeloid (20%) cells. Most of the B-cells were in the pre-B fraction with oligo-clonal IgH rearrangement, suggesting a lymphoproliferative disorder (LPD).

The initial discovery of enzymatic activity associated with oncogenic transcription factors has fuelled the enthusiasm of developing inhibitors for these classically untraceable targets, however the functional significance of H3K4 methyltransferase activity in MLL mediated pathogenesis remains elusive. We have now developed novel MLLn disease models where full-length MLL, including the catalytic SET domain, was amplified between 10-100 times. MLL amplification triggered various degrees of perturbation in different hematopoietic tissues, promoted cell growth and enhanced replating ability in vitro. Also in vivo, a small percentage of mice went on to develop a LPD. Together, these results reveal a critical function of MLLn and potentially its associated H3K4 methyltransferase activity in disease progression.

No relevant conflicts of interest to declare.