Calpain 2 Activation of P-TEFb Drives Megakaryocyte Morphogenesis and Is Disrupted by Leukemogenic GATA1 Mutation

Abstract

SUMMARY Megakaryocyte morphogenesis employs a “hypertrophy-like” developmental program, dependent on P-TEFb kinase activation and cytoskeletal remodeling. P-TEFb activation classically occurs by a feedback regulated process of signal-induced, reversible release of active Cdk9-cyclin T modules from large inactive 7SK snRNP complexes. Here we have identified an alternative pathway of irreversible P-TEFb activation in megakaryopoiesis, mediated by dissolution of the 7SK snRNP complex. In this pathway calpain 2 cleavage of the core 7SK snRNP component MePCE promoted P-TEFb release and consequent upregulation of a cohort of cytoskeleton remodeling factors, including α-actinin-1. In a subset of human megakaryocytic leukemias, the transcription factor GATA1 undergoes truncating mutation (GATA1s). Here we linked the GATA1s mutation to defects in megakaryocytic upregulation of calpain 2 and of P-TEFb-dependent cytoskeletal remodeling factors. Restoring calpain 2 expression in GATA1s-mutant megakaryocytes rescued normal development, implicating this morphogenetic pathway as a target in human leukemogenesis.

Neonatal expression of RNA-binding protein IGF2BP3 regulates the human fetal-adult megakaryocyte transition

“…In adult megakaryopoiesis, constitutive P-TEFb activation drives upregulation of a cohort of cytoskeletal remodeling factors that function as critical effectors in morphogenesis (11). In neonatal megakaryopoiesis, in contrast, minimal lineage consolidation of megakaryocytes emerging from bipotent megakaryocyte-erythroid progenitors, a notion further supported by the hybrid CD41 + glycophorin A + (GPA) cells observed in pluripotent stem cell (PSC) hematopoiesis (17).…”

“…For lentiviral shRNA-mediated knockdown, pLKO.1-derived constructs were purchased from Open Biosystems GE-Dharmacon. Lentiviral packaging by cotransfection of HEK293T cells was conducted as described (11), except that experiments after 2014 used the hCMV-BaevRless envelope construct (57). This construct uses the CMV promoter to express during packaging the baboon retroviral envelope glycoprotein, which provides superior transduction efficiency for human CD34 + progenitors.…”

“…Their results suggest an ontogenic influence on Recently, megakaryocyte morphogenesis was found to depend on sustained, high-amplitude activation of the positive transcription elongation factor b (P-TEFb) kinase complex, consisting of CDK9 and cyclin T (11,12). P-TEFb promotes RNA polymerase II (RNAPII) elongation on stimulus-responsive genes through phosphorylation of pausing factors (5,6-dichloro-1-β-dribofuranosylbenzimidazole [DRB] sensitivity-inducing factor [DSIF] and negative elongation factor [NELF]) and the carboxy terminal domain of RNAPII large subunit (RNAPII CTD) (13).…”

“…The specialized pathway of P-TEFb activation in adult megakaryocytes involves destabilization of 7SK snRNA through downregulation of its binding partners, the stabilizing factors LARP7 and MePCE (11). In this process, MePCE undergoes proteolysis due to megakaryocytic upregulation of calpain 2, which also cleaves the cytoskeletal remodeling factor FLNA.…”

“…The megakaryocytic differentiation program, in contrast, employs a specialized activation pathway initiated by MePCE and LARP7 downregulation, leading to 7SK destruction and global, irreversible P-TEFb release (11). This mode of activation upregulates a cohort of cytoskeletal remodeling factors, such as megakaryoblastic leukemia (translocation) 1 (MKL1), filamin A (FLNA), and α-actinin 1 (ACTN1), which function as morphogenesis effectors during differentiation (11,14,15).…”