Calpain Drives Megakaryopoiesis by Targeting the 7SK snRNP Complex: A Connection to Down Syndrome Megakaryocytic Neoplasia

Abstract 552

Megakaryocytic differentiation uniquely relies on a hypertrophic program involving massive cellular enlargement and nuclear polyploidization. A critical mechanism driving this program consists of global P-TEFb activation, a process involving the release of an active Cdk9-cyclin T kinase module from a larger, inactive 7SK snRNP complex. We previously implicated the protease complex calpain 2/S1 in P-TEFb activation during megakaryopoiesis and demonstrated a direct contribution of calpain activity to megakaryocytic differentiation (Elagib et al., ASH 2009). The clinical relevance of this pathway was suggested by findings in patient samples and knockin mice of defective calpain 2 upregulation in megakaryocytes bearing the leukemogenic GATA-1s mutant. Current studies address: 1) the mechanism by which calpain 2/S1 activates P-TEFb during normal megakaryopoiesis, 2) the genetic program controlled by P-TEFb during megakaryopoiesis, and 3) the relevance of calpain 2 deficiency to abnormal megakaryopoiesis associated with GATA-1s. Regarding the mechanism of P-TEFb activation by calpain, analysis of a P-TEFb interactome database identified an interaction of calpain S1 with the protein BCDIN3 (Jeronimo CD. et. al. Mol Cell 2007). BCDIN3 stabilizes the 7SK snRNA through capping activity, but also exerts enzyme-independent stabilization by forming a core complex with 7SK snRNA and the protein LARP7. This core complex provides the foundation for assembly of the 7SK snRNP involved in restraining P-TEFb activity. Using adult human CD34+ cells cultured in unilineage megakaryocytic medium, both BCDIN3 and LARP7 proteins were found to undergo dramatic downregulation during differentiation, leading to global destabilization of 7SK snRNA. By contrast, neither BCDIN3, LARP7, nor 7SK snRNA underwent downregulation during erythroid differentiation. Megakaryocytic downregulation of BCDIN3, but not LARP7, required calpain activity. Calpain inhibitors as well as shRNA knockdown of calpain 2 prevented BCDIN3 downregulation during megakaryocytic cultures. In vitro reactions with recombinant purified factors confirmed direct cleavage of BCDIN3 by calpain 2/S1. The role of BCDIN3 downregulation in megakaryopoiesis was assessed by shRNA knockdown experiments. Notably, enforcing downregulation of BCDIN3 in CD34+ cells in erythroid medium reprogrammed differentiation away from the erythroid lineage and toward the megakaryocytic lineage. Thus BCDIN3 is a critical target of calpain 2/S1 during megakaryocytic activation of P-TEFb. To identify P-TEFb target genes, a bioinformatic approach was used to screen megakaryocytic expression databases for genes coregulated with known P-TEFb targets, e.g. HEXIM1. This approach yielded a cohort of coregulated genes encoding actin associated factors: ACTN1, FLNA, TGFB1I1, and MKL1. Immunoblot analsysis validated that all four factors underwent concomitant upregulation during megakaryocytic but not erythroid differentiation. shRNA knockdowns of calpain 2 and Cdk9 confirmed that upregulation of these factors depended on calpain and P-TEFb activation. Previous studies have indicated a requirement for ACTN1 downregulation in cytokinesis. Thus, shRNA knockdowns determined whether ACTN1 upregulation contributed to megakaryocytic polyploidization. Strikingly, ACTN1 knockdown blocked the polyploidization of human CD34+ cells but allowed other features of megakaryocytic differentiation, e.g. CD41 upregulation. Thus, a key aspect of the genetic program controlled by P-TEFb in megakaryopoiesis consists of the upregulation of a cohort of actin-associated factors which contribute to a hypertrophic program of cellular enlargement and polyploidization. To address the role of calpain in the aberrant megakaryopoiesis associated with mutant GATA-1s, fetal liver (fl) cells from GATA-1s knockin (G1s ki) mice were studied. Previous studies have shown a hyperproliferative phenotype in G1s ki fl but not adult megakaryocytes. In the current study, enforced calpain 2 expression by lentiviral transduction reversed this phenotype and enhanced terminal differentiation, reflected by polyploidization and CD42 expression. Thus, the defective upregulation of calpain 2 in megakaryocytes with GATA-1s most likely plays a critical role in the abnormal megakaryopoiesis seen in Down syndrome-associated megakaryoctyic proliferative disorders.