Identification of Inducers of Megakaryocyte Polyploidization and Their Use as Targeted Differentiation Therapy for Acute Megakaryocytic Leukemia.
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 
Published: October 2019

Abstract
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 differentiation of GATA-1 deficient and GATA-1s knock-in fetal liver megakaryocytes, which mimic the aberrant megakaryopoiesis seen in infants with DS. Importantly, diMF did not induce polyploidization of the CD41-negative fraction of primary murine or human ex vivo cultures, revealing its specificity for megakaryocytes. When administered to mice by oral gavage, diMF selectively affected megakaryocytes within the bone marrow and did not alter peripheral blood counts. diMF also effectively inhibited AMKL development in mice transplanted with MPL mutant, OTT-MAL expressing cells: 50% and 30% of the recipients treated with 66 mg/kg and 33 mg/kg diMF bid respectively, remain disease free after three months whereas all placebo treated mice succumbed to AMKL within 3 weeks. To identify the targets of diMF, we used a combination of the SILAC (stable isotope labeling with amino acids in cell culture) proteomic approach and Ambit's KinomeScan. These unbiased assays identified multiple binding targets of diMF, including JAK3, MAP4K2, and Rho kinase 1 (ROCK1). Using shRNA mediated knock-down, we found that decreased expression of ROCK1, but not ROCK2, led to increased sensitivity of CMK cells to diMF induced polyploidization. Knockdown of ROCK1 alone also induced features of megakaryocyte differentiation such as enhanced CD41 expression. These studies implicate ROCK1 in regulation of the switch between proliferation and differentiation of megakaryocytes and show that diMF acts through inhibition of ROCK1. Taken together, our work demonstrates that diMF and other polyploidy inducing agents discovered in our screen represent potential targeted agents for AMKL. Furthermore, since diMF acted similarly on megakaryoblasts with the various genetic lesions associated with each of the different classes of AMKL, we predict that this approach will be an effective therapeutic strategy for all categories of megakaryocytic neoplasms.

Disclosures:
No relevant conflicts of interest to declare.