Gain of Function MLL1 Model Systems Distinguish Hematopoietic from Leukemogenic Pathways

Hematopoiesis represents an ongoing developmental process in which hematopoietic stem (HSCs) or progenitor cells (HPCs) differentiate into various blood lineages in response to intra- and extra-cellular signals. Recent work has illuminated the centrality of epigenetic regulators in controlling both normal and malignant hematopoiesis. Among them, MLL1 (Mixed Lineage Leukemia-1), is a paradigmatic histone H3 lysine 4 methyltransferase that is essential for HSC maintenance and is closely associated with hematologic disorders and acute leukemia when disrupted by chromosomal translocations or rearrangements. Previous studies successfully employed loss-of-function alleles to demonstrate a critical role for MLL1 in HSC function, suggesting increased MLL1 may expand HSCs or HPCs. However, this hypothesis has never been tested due to the lack of a gain-of-function (transgenic) model. Furthermore, it has remained unclear how increased wild-type MLL1 might differ from leukemogenic MLL1 fusion proteins in influencing normal or aberrant hematopoiesis. Here we present mouse models in which increasing wild-type MLL1, but not MLL-ENL, in developing hematopoietic progenitors promotes hematopoietic colony frequency (CFU).

To investigate the impact of increasing MLL1 levels in hematopoietic populations, we generated a Tet-inducible transgene by integrating a single copy of human wild-type MLL1 into the Col1a1 locus using the system developed by the Jaenisch group (Beard C. et al. 2006). Targeted embryonic stem cells (ESCs) were identified and induced expression of MLL1 was confirmed to be within physiologic levels. This mild induction of MLL1 protein did not increase histone H3, lysine 4 methylation levels, but did influence gene expression. The ESCs were then differentiated in vitro to form embryoid bodies (EBs) that recapitulate early development of all three germ layers. MLL1 induction generally did not affect EB proliferation or morphology. Representative gene expression patterns characteristic of each of the germ layers were not significantly changed by inducing MLL1 expression during all or defined periods of EB differentiation. Induction of MLL1 from day 4-6 did not affect the numbers of c-Kit+/CD41+ hematopoietic progenitors produced. However, MLL1-induced, c-Kit+/CD41+ hematopoietic progenitors exhibited increased myelo-erythroid colonies. Surprisingly, this increase did not occur through protection from apoptosis.

Side-by-side comparison of MLL1 versus MLL-ENL induction (Ugale A. et al. 2014) illustrated that the promotion of hematopoietic colonies is a function of wild-type MLL1 but not the oncogenic MLL-ENL. MLL-ENL induction did not transform EB-derived hematopoietic progenitors to form leukemic colonies or acquire serial replating activity. Instead, progenitors in which MLL-ENL was induced ceased to replate. Comparison of hematopoietic progenitors cultured from MLL1 and MLL-ENL induced EBs showed distinct immunophenotypes, gene expression patterns and cellular processes affected.

We report the first model in which the induction of wild-type MLL1 expression is achieved and demonstrate that enhancing MLL1 protein levels can selectively increase hematopoietic potential in developing EBs. MLL1 and MLL-ENL regulate distinct gene sets in hematopoietic progenitors that result in different outcomes in developing hematopoietic cells. These findings provide novel insight into how HSC regulators can be subverted by leukemogenic programs. In addition, the enhanced hematopoiesis resulting from increased wild-type MLL1 illustrates the potential for expanding HSCs for transplantation.