Molecular Delineation and Targeting of a Megakaryocyte Ontogenic Switch for Therapeutic Applications

In contrast to adult-type megakaryocytes (Mk), fetal-type Mk display increased progenitor proliferation, decreased morphogenesis, and decreased platelet release. Mk morphogenesis comprises a coordinated sequence of events that primes the cells for intravascular platelet release: endomitosis, polyploidization, cellular enlargement, and polarized process extension. Impairment in this program occurs most prominently during fetal development but persists in normal infants for up to a year and strongly manifests in iPSC-derived megakaryocytes. These ontogenically controlled features ensure functional adaptation to host needs. Thus, overall development of the fetus is best supported by rapid Mk expansion and restrained production of platelets. However, fetal-type megakaryopoiesis lacks the capacity for stress thrombopoiesis, leading to clinical problems in situations requiring rapidly augmented platelet production, and is also uniquely susceptible to neoplastic transformation. Pharmacologic interventions to induce adult-type megakaryopoiesis, i.e. ontogenic switching, do not exist but would greatly benefit the following clinical problems: 1) neonatal thrombocytopenia, 2) platelet recovery post umbilical cord blood (UCB) transplant, 3) Down syndrome-associated megakaryocyte (Mk) neoplasms, and 4) large scale ex vivo platelet production. Our lab over the past 15 years has characterized several of the molecular determinants of Mk ontogeny. We recently found that Dyrk kinase signaling, by repressing Mkl1 co-activator function, maintains the fetal program in human neonatal Mk (Elagib et al. J. Clin. Invest., 2022). While Dyrk inhibition induced ontogenic switching in vitro, in vivo application was precluded by broad toxicity. In pursuing a downstream target of Dyrk signaling, we have discovered a novel ontogenic control factor, Kifc3, a minus-end-directed kinesin involved in delivery of proteins to the centrosome. Therapeutically attractive features of this protein include a unique, structurally characterized ATP-binding pocket and absence of abnormalities in knockout mice. Our functional analysis of Kifc3 in human progenitors has revealed: 1) 2-fold lower levels of protein in adult vs neonatal cells (P < 0.005); 2) induction of adult morphogenesis in neonatal cells subjected to knockdown (kd), with 2.5-fold enhanced polyploidization (P < 0.005); 3) 3-fold enhancement in functional platelet production by neonatal cells subjected to kd (P < 0.005). In silico modelling using motor domain structures of Kifc3 and of Kifc1 bound to an inhibitor has enabled identification of candidate Kifc3 small molecule inhibitors that elicit the same phenotype as Kifc3 kd, including 1.7-fold enhancement in platelet production by neonatal Mk (P < 0.05). Our recent studies have also identified a key target of Kifc3 in megakaryocyte ontogeny, Cep192. Cep192 is known to be a scaffold for aurora (Aurk) and polo-like (Plk) kinases, recruiting them to centrosomes to coordinate mitotic maturation. A recent publication shows that Cep192 also recruits these kinases to cortical actin to promote cytoskeletal remodeling and process extension (Luo et al., Nat. Commun., 2022). Elevated levels of Cep192 are known to sequester these kinases away from mitotic scaffolds that coordinate cytokinesis. Comparison of neonatal and adult Mk by immunofluorescence showed marked differences in Cep192: focal-centriolar in > 80% neonatal Mk and broadly dispersed in 60% adult Mk (P < 0.01); by immunoblot, adult Mk expressed 3-fold higher levels (P < 0.01). Knocking down or inhibiting Kifc3 in neonatal Mk caused Cep192 to increase 2-fold in amount (P < 0.01)) and disperse from centrosomes in 60% of cells (P < 0.05)). Knocking down Cep192 in adult Mk blocked morphogenesis (2-fold decreased polyploidization, P < 0.005), and a CRISPR hypomorph of Cep192 in mice caused a 17% decrease in platelet counts (P = 0.015) with no effect on red cells or white cells. Overall, our findings provide a novel, therapeutically relevant, mechanistic pathway in which Kifc3 levels dictate Mk ontogeny via Cep192. We propose a model in which Kifc3-Cep192 signaling acts on cytoskeletal kinases (Aurk and Plk) to coordinate loss of cytokinesis with process extension. Clinically, these findings provide proof of principle for therapeutic induction of Mk ontogenic switching to enable stress thrombopoiesis in settings of infantile hematopoiesis.