The Distinctive Morphogenesis of Infantile Megakaryopoiesis Is Associated with Altered P-TEFb Regulation By the Oncofetal Factor IGF2BP3

Infantile (fetal/neonatal) megakaryocytes (Mk) differ from "adult" Mk in several important aspects. They are smaller, more proliferative, less polyploid and show leaky expression of erythroid genes. Their distinctive properties contribute to multiple disease states including neonatal thrombocytopenia, poor platelet recovery post umbilical cord blood (CB) transplantation, and acute Mk leukemias (AMkL). Their leukemic propensity is highlighted by the capacity of the AMkL oncoprotein GATA1s to transform infantile but not adult Mk. Despite their altered morphogenesis, infantile megakaryocytes retain most features of the adult differentiation and signaling program. Their principal signaling perturbation has been characterized as excessive responsiveness to thrombopoietin (Tpo) particularly with regard to the mTOR pathway. Thus Tpo agonists used to treat adult thrombocytopenia may not offer appropriate therapy for neonatal thrombocytopenia.

We previously identified a signaling pathway that drives megakaryocyte morphogenesis and is disrupted by the GATA1s oncoprotein in AMkL (Elagib et al., Dev. Cell, 2013). A central feature of this pathway is the irreversible activation of the P-TEFb kinase (Cdk9/Cyclin T). This cascade is initiated by downregulation of core components of the repressive 7SK snRNP complex (MePCE, LARP7, 7SK snRNA). The resulting constitutive P-TEFb activation drives multiple features of Mk differentiation: induction of cytoskeletal morpho-genetic factors, silencing of erythroid genes, and promotion of histone H2B K120 monoubiquitiniation (H2BUb1). A critical, rate-limiting step triggering this pathway comprises MePCE proteolysis by calpain 2. GATA1s disrupts this pathway by preventing induction of calpain 2 by wild type GATA1.

We now report that infantile Mk display intrinsic defects in the Mk P-TEFb activation pathway. In repeated experiments, human CB Mk failed to upregulate P-TEFb-dependent cytoskeletal factors, exhibited global deficiency in H2BUb1, and incompletely silenced erythroid antigen expression. Their defective P-TEFb activation resulted from an inability to downregulate 7SK snRNA, despite downregulation of the key 7SK stabilizers MePCE and LARP7. The inexplicable stabilization of 7SK in CB Mk argues for the existence of an alternative, infantile 7SK snRNP complex refractory to activation by calpain. Accordingly, screening studies identified candidate 7SK binding factors preferentially expressed in CB as opposed to adult progenitors. Among these factors, the RNA binding protein IGF2BP3 showed high abundance in CB Mk but complete absence from adult peripheral blood-derived (PB) Mk. Furthermore, immunoprecipitation studies consistently showed interaction of endogenous IGF2BP3 and HEXIM1 in K562 cells. HEXIM1 is the 7SK snRNP component that mediates repression of P-TEFb. Immunoprecipitation of epitope-tagged IGF2BP3 from HEK293 cells consistently identified an association with endogenous 7SK snRNA. In addition, enforced expression of IGF2BP3 in HEK293 cells, to levels seen in CB Mk, shifted the fractionation pattern of HEXIM1 on glycerol gradient sedimentation. Notably, a similar difference in HEXIM1 fractionation was seen when comparing CB and adult Mk by glycerol gradient sedimentation. Thus, IGF2BP3 represents a fetal/neonatal factor that reconfigures the composition and/or conformation of the 7SK snRNP, potentially altering regulation of P-TEFb. Contribution of IGF2BP3 to the infantile Mk phenotype was supported by experiments in which shRNA-mediated knockdown in CB Mk consistently enhanced enlargement, polyploidization, growth arrest, and erythroid silencing. Conversely, enforced expression of IGF2BP3 in adult Mk inhibited their enlargement, polyploidization, and growth arrest. Our results thus implicate IGF2BP3 as a key contributor to the infantile Mk phenotype, interfering with morphogenesis by stabilizing 7SK and thwarting irreversible P-TEFb activation. In light of our prior published results on the inhibitory effects of GATA1s in Mk morphogenesis (Elagib et al., Dev. Cell, 2013), our current findings highlight P-TEFb regulation as a convergence point for oncogenic stimuli during megakaryopoiesis.