MKL1 Enhances Megakaryocytic Differentiation of Primary CD34+ Cells.

Acute Megakaryoblastic Leukemia (AMKL or AML variant M7), which occurs most often in infants and young children, is characterized by a failure of megakaryocyte (MK) differentiation, bone marrow fibrosis, cytogenetic abnormalities, and a poor prognosis. We are particularly interested in AMKL that is associated with the translocation t(1;22)(p13;q13), which yields an in-frame fusion of RBM15 (OTT) and MKL1 (MAL) on chromosomes 1 and 22, respectively. The resultant fusion, RBM15-MKL1 is believed to include all of the functional domains of each component. In order to better understand the role of RBM15-MKL1 in AMKL, it is necessary to understand the roles of the constituent genes, RBM15 and MKL1, in hematopoiesis. We have studied the role of human MKL1 in megakaryopoiesis using primary human CD34+ cells purified from G-CSF mobilized PBMC from healthy donors (n=4). To optimize the CD34+ model, we tested the ability of TPO vs. TPO+SCF vs. TPO+SCF+IL–3 to induce megakaryocytopoiesis. TPO and TPO+SCF gave the highest percentages of MK (12% and 7%, respectively) on day 9. However, due to enhanced cell proliferation with TPO+SCF, the absolute number of MK was highest using this cytokine combination. To test the effect of MKL1 overexpression on megakaryopoiesis, we generated VSVG-pseudotyped lentiviral vectors containing human MKL1 and tested the effect of retronectin on viral transduction of CD34 cells. Surprisingly, retronectin decreased the level of transduction when compared to no retronectin (12% vs. 15% transduction respectively). We also found that polybrene enhanced transduction compared to lipofectamine 2000 (20% vs. 6% transduction, respectively). Using our optimized protocols, we examined the effect of MKL1 overexpression on megakaryocytopoiesis. One million CD34+ cells were thawed, infected the following two days with either empty lentivirus (pCCL) or lentivirus containing human MKL1 (pCCL-MKL), and cultured in TPO+SCF for 9 days. Since both lentiviral vectors included GFP driven by the PGK promoter, we measured the levels of CD41a, CD42d, and CD61 in GFP+ cells at day 9. In a representative experiment (of 4), CD41a levels increased in cells containing pCCL-MKL1 vs. pCCL (50% vs. 40%). Moreover, CD42d levels (22% vs. 7%) and CD61 levels (53% vs. 44%) were increased in cells containing pCCL-MKL1 virus when compared to cells containing pCCL virus. We also tested the ability of MKL1 to increase megakaryocyte differentiation using a semisolid Megacult assay from Stem Cell Technologies. CD34+ cells were cultured and infected as described above with either pCCL or pCCL-MKL1 virus. Two days post infection, GFP+ cells were FACS sorted and plated at two different concentrations in semisolid Megacult medium containing collagen, TPO, IL-6, and IL-3. Eleven days post plating, cells were stained for CD41/CD61. Cells infected with pCCL-MKL1 cells gave approximately 2 fold more MK colonies than pCCL infected cells at both low cell concentration plating (395 vs. 182 colonies, respectively) and high concentration plating (900 vs. 389 colonies, respectively). These data suggest that overexpression of human MKL1 enhances megakaryocyte differentiation of primary human CD34+ cells. A further understanding of the normal roles of RBM15 and MKL1 in megakaryopoiesis will allow us to better understand the role of the RBM15-MKL1 fusion in AMKL, and aid in the development of treatments for this disease.