An IGF2BP3-Cdk9 Pathway Governs the Human Fetal-Adult Megakaryocyte Transition

Hematopoietic transitions that accompany fetal development, for example erythroid globin chain switching, play important roles in normal physiology and disease development. In the megakaryocyte lineage, human fetal progenitors show impaired execution of the morphogenesis program of enlargement, polyploidization, and proplatelet formation. Although these defects decline with gestational stage, they remain sufficiently severe at birth to predispose newborns to thrombocytopenia. These defects also contribute to inferior platelet recovery after cord blood stem cell transplantation and to inefficient platelet production by megakaryocytes (Mk) derived from pluripotent stem cells. In this study, comparison of neonatal versus adult human progenitors identified a blockade in the specialized positive transcription elongation factor b (P-TEFb) activation mechanism known to drive adult Mk morphogenesis. A central feature of this pathway is known to involve sustained high amplitude activation of the P-TEFb kinase (Cdk9/cyclin T1). This cascade is initiated by downregulation of core components of the repressive complex of P-TEFb: the 7SK snRNP consisting of the 7SK small nuclear RNA (7SK) and its stabilizing factors MePCE and LARP7. The resulting high amplitude activation of P-TEFb drives multiple features of Mk differentiation: induction of cytoskeletal morphogenetic factors (ACTN1, FLNA, MKL1, HIC5), silencing of erythroid genes, promotion of histone H2B K120 monoubiquitiniation (H2BUb1), and phosphorylation of Spt5 at T806 (pSpt5 T806). Critical, rate-limiting steps triggering this pathway comprise MePCE proteolysis by calpain 2 and downregulation of LARP7, both resulting in destabilization of 7SK.

In comparison to Mk derived from adult peripheral blood stem cells (adult Mk), Mk derived from umbilical cord blood stem cells (neonatal Mk) showed evidence of decreased P-TEFb activation with decreases in: 1) the expression of the cytoskeletal morphogenetic factors, 2) global H2BUb1, and 3) pSpt5 T806. In addition, neonatal Mk retained expression of the erythroid marker glycophorin A (GPA). Surprisingly, neonatal Mk failed to downregulate 7SK despite the downregulation of its stabilizers MePCE and LARP7, suggesting the existence of alternative 7SK stabilizing factor/s unique to neonatal Mk. Our screening identified the oncofetal RNA-binding protein IGF2BP3 to be expressed in neonatal but not adult Mk, and ectopic expression of IGF2BP3 in adult Mk conferred neonatal phenotypic features including reduction in size, increased proliferation and leaky erythroid antigen expression. Immunoprecipitation and glycerol gradient studies indicated the participation of IGF2BP3 in the 7SK snRNP complex. Mining of endogenous IGF2BP3 iCLIP data indicated that 7SK is one of the top direct targets of IGF2BP3 and further mapped binding to the 7SK fourth hairpin, a critical stability determinant.

In loss of function studies, the knockdown of IGF2BP3 in neonatal Mk resulted in destabilization of 7SK and upregulation of the Mk morphogenetic cytoskeletal factors, as well as increased levels of H2BUb1, pSpt5 T806, and hyperphosphorylated RNA Pol II. Phenotypically, the knockdown of IGF2BP3 in neonatal Mk elicited adult features including increased size, enhanced polyploidization, reduced proliferation and silencing of erythroid antigen expression. Collectively, these findings suggest that the block in P-TEFb activation in neonatal Mk results from ontogentic stage-specific expression of IGF2BP3 which prevents the 7SK destabilization normally associated with adult megakaryocytic P-TEFb activation. We also identified a pharmacologic approach to inhibit IGF2BP3 expression, through inhibition of bromodomain and extra- terminal (BET) proteins, which reproducibly promoted adult features in neonatal Mk including enlargement, inhibition of erythroid antigen expression, upregulation of morphogenetic cytoskeletal factors, and increased platelet formation in vitro. Enforced expression of IGF2BP3 in neonatal Mk significantly blunted the effects of BET inhibitors indicating the specificity of their action in downregulating IGF2BP3. These results identify IGF2BP3 as a human ontogenic masterswitch that restricts megakaryocyte development through modulating a lineage-specific P-TEFb activation mechanism, revealing new strategies toward enhancing platelet production.