Identification of Inducers of Megakaryocyte Polyploidization and Their Use as Targeted Differentiation Therapy for Acute Megakaryocytic Leukemia

• Published in: Blood 2009-11, Vol.114 (22), p.792-792
• Main Authors: Wen, Qiang, Huang, Zan, Small, Sara, Bubna, Niket, An, Frank, Bliss-Moreau, Meghan, Verplank, Lynn, Lewis, Tim, Schenone, Monica, Tolliday, Nicola, Moore, Chris, Carpenter, Ann, Mercher, Thomas, Levine, Ross L., Izraeli, Shai, Stern, Andrew, Gould, Robert, Bradner, James E., Crispino, John
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V114.22.792.792

Abstract 792 Acute megakaryoblastic leukemia (AMKL) is a rare subtype of acute myeloid leukemia characterized by expansion of immature megakaryocytes and bone marrow myelofibrosis. AMKL is frequently associated with chromosomal abnormalities, such as trisomy 21 or t(1;22), which leads to expression of the OTT-MAL fusion protein. Mutations in GATA1 are present in nearly all cases of Down syndrome AMKL, while mutations in JAK3, MPL, KIT, and FLT3 are associated with a smaller subset of patients. Since most patients with AMKL face a very poor prognosis, new therapies are desperately needed. Given that megakaryocytes undergo polyploidization during the normal course of differentiation, we hypothesized that small molecule inducers of polyploidization would drive megakaryoblasts to exit the proliferative cell cycle and induce terminal differentiation. These agents would thus serve as targeted differentiation therapeutics for AMKL in a manner analogous to ATRA for treatment of acute promyelocytic leukemia. To identify small molecules that induce megakaryocyte polyploidization, we incubated the CMK megakaryoblastic cell line, which harbors both GATA1 and JAK3 mutations, with libraries of kinase or histone deacetylase inhibitors, natural products, and small molecules derived from diverse oriented synthesis. After three days, we stained the cells with Hoechst dye, imaged plates with the ImageXpress Micro microscope and converted image files into quantitative values of DNA content with Cell Profiler software. By screening 10,000 compounds, we identified 207 molecules that significantly and reproducibly increased polyploidization. Among the compounds scoring in this screen were microtubule disrupting and stabilizing agents and actin disrupting agents, which are expected to cause alterations in spindle formation or cytokinesis and result in polyploidization. A more interesting subset of compounds led to robust polyploidization, with DNA contents reaching 32N and 64N, and also to induction of megakaryocyte differentiation. We have focused our efforts on the most potent compound of the latter class, dimethylfasudil (diMF), a known Rho kinase inhibitor. diMF blocked proliferation and simultaneously induced marked polyploidization, differentiation and apoptosis of human and murine megakaryoblastic leukemia cell lines as well as primary murine and human megakaryocyte progenitors. diMF also inhibited megakaryocyte colony formation and induced polyploidization and differentia