Marlyse A. Debrincat

Megakaryocytes possess a functional intrinsic apoptosis pathway that must be restrained to survive and produce platelets

Deletion of Bak and Bax, the effectors of mitochondrial apoptosis, does not affect platelet production, however, loss of prosurvival Bcl-xL results in megakaryocyte apoptosis and failure of platelet shedding.

Caspase-9 mediates the apoptotic death of megakaryocytes and platelets, but is dispensable for their generation and function

Apoptotic caspases, including caspase-9, are thought to facilitate platelet shedding by megakaryocytes. They are known to be activated during platelet apoptosis, and have also been implicated in platelet hemostatic responses. However, the precise requirement for, and the regulation of, apoptotic caspases have never been defined in either megakaryocytes or platelets. To establish the role of caspases in platelet production and function, we generated mice lacking caspase-9 in their hematopoietic system. We demonstrate that both megakaryocytes and platelets possess a functional apoptotic caspase cascade downstream of Bcl-2 family-mediated mitochondrial damage. Caspase-9 is the initiator caspase, and its loss blocks effector caspase activation. Surprisingly, steady-state thrombopoiesis is unperturbed in the absence of caspase-9, indicating that the apoptotic caspase cascade is not required for platelet production. In platelets, loss of caspase-9 confers resistance to the BH3 mimetic ABT-737, blocking phosphatidylserine (PS) exposure and delaying ABT-737-induced thrombocytopenia in vivo. Despite this, steady-state platelet lifespan is normal. Casp9(-/-) platelets are fully capable of physiologic hemostatic responses and functional regulation of adhesive integrins in response to agonist. These studies demonstrate that the apoptotic caspase cascade is required for the efficient death of megakaryocytes and platelets, but is dispensable for their generation and function.

Mcl-1 and Bcl-xL coordinately regulate megakaryocyte survival

Mature megakaryocytes depend on the function of Bcl-x(L), a member of the Bcl-2 family of prosurvival proteins, to proceed safely through the process of platelet shedding. Despite this, loss of Bcl-x(L) does not prevent the growth and maturation of megakaryocytes, suggesting redundancy with other prosurvival proteins. We therefore generated mice with a megakaryocyte-specific deletion of Mcl-1, which is known to be expressed in megakaryocytes. Megakaryopoiesis, platelet production, and platelet lifespan were unperturbed in Mcl-1(Pf4Δ/Pf4Δ) animals. However, treatment with ABT-737, a BH3 mimetic compound that inhibits the prosurvival proteins Bcl-2, Bcl-x(L), and Bcl-w resulted in the complete ablation of megakaryocytes and platelets. Genetic deletion of both Mcl-1 and Bcl-x(L) in megakaryocytes resulted in preweaning lethality. Megakaryopoiesis in Bcl-x(Pf4Δ/Pf4Δ) Mcl-1(Pf4Δ/Pf4Δ) embryos was severely compromised, and these animals exhibited ectopic bleeding. Our studies indicate that the combination of Bcl-x(L) and Mcl-1 is essential for the viability of the megakaryocyte lineage.

Mcl-1 and Bcl-xL co-ordinately regulate megakaryocyte survival

Mature megakaryocytes depend on the function of Bcl-x(L), a member of the Bcl-2 family of prosurvival proteins, to proceed safely through the process of platelet shedding. Despite this, loss of Bcl-x(L) does not prevent the growth and maturation of megakaryocytes, suggesting redundancy with other prosurvival proteins. We therefore generated mice with a megakaryocyte-specific deletion of Mcl-1, which is known to be expressed in megakaryocytes. Megakaryopoiesis, platelet production, and platelet lifespan were unperturbed in Mcl-1(Pf4Δ/Pf4Δ) animals. However, treatment with ABT-737, a BH3 mimetic compound that inhibits the prosurvival proteins Bcl-2, Bcl-x(L), and Bcl-w resulted in the complete ablation of megakaryocytes and platelets. Genetic deletion of both Mcl-1 and Bcl-x(L) in megakaryocytes resulted in preweaning lethality. Megakaryopoiesis in Bcl-x(Pf4Δ/Pf4Δ) Mcl-1(Pf4Δ/Pf4Δ) embryos was severely compromised, and these animals exhibited ectopic bleeding. Our studies indicate that the combination of Bcl-x(L) and Mcl-1 is essential for the viability of the megakaryocyte lineage.

Platelet production proceeds independently of the intrinsic and extrinsic apoptosis pathways

BH3 mimetic drugs that target BCL-2 family pro-survival proteins to induce tumour cell apoptosis represent a new era in cancer therapy. Clinical trials of navitoclax (ABT-263, which targets BCL-2, BCL-XL and BCL-W) have shown great promise, but encountered dose-limiting thrombocytopenia. Recent work has demonstrated that this is due to the inhibition of BCL-XL, which is essential for platelet survival. These findings raise new questions about the established model of platelet shedding by megakaryocytes, which is thought to be an apoptotic process. Here we generate mice with megakaryocyte-specific deletions of the essential mediators of extrinsic (Caspase-8) and intrinsic (BAK/BAX) apoptosis. We show that megakaryocytes possess a Fas ligand-inducible extrinsic apoptosis pathway. However, Fas activation does not stimulate platelet production, rather, it triggers Caspase-8-mediated killing. Combined loss of Caspase-8/BAK/BAX does not impair thrombopoiesis, but can protect megakaryocytes from death in mice infected with lymphocytic choriomeningitis virus. Thus, apoptosis is dispensable for platelet biogenesis.

Expansion of the neonatal platelet mass is achieved via an extension of platelet lifespan.

The fetal/neonatal hematopoietic system must generate enough blood cells to meet the demands of rapid growth. This unique challenge might underlie the high incidence of thrombocytopenia among preterm neonates. In this study, neonatal platelet production and turnover were investigated in newborn mice. Based on a combination of blood volume expansion and increasing platelet counts, the platelet mass increased sevenfold during the first 2 weeks of murine life, a time during which thrombopoiesis shifted from liver to bone marrow. Studies applying in vivo biotinylation and mathematical modeling showed that newborn and adult mice had similar platelet production rates, but neonatal platelets survived 1 day longer in circulation. This prolonged lifespan fully accounted for the rise in platelet counts observed during the second week of murine postnatal life. A study of pro-apoptotic and anti-apoptotic Bcl-2 family proteins showed that neonatal platelets had higher levels of the anti-apoptotic protein Bcl-2 and were more resistant to apoptosis induced by the Bcl-2/Bcl-xL inhibitor ABT-737 than adult platelets. However, genetic ablation or pharmacologic inhibition of Bcl-2 alone did not shorten neonatal platelet survival or reduce platelet counts in newborn mice, indicating the existence of redundant or alternative mechanisms mediating the prolonged lifespan of neonatal platelets.

Ankyrin repeat and suppressors of cytokine signaling box protein asb-9 targets creatine kinase B for degradation.

The suppressors of cytokine signaling (SOCS) proteins inhibit cytokine action by direct interaction with Janus kinases or activated cytokine receptors. In addition to the N-terminal and Src homology 2 domains that mediate these interactions, SOCS proteins contain a C-terminal SOCS box. DNA data base searches have identified a number of other protein families that possess a SOCS box, of which the ankyrin repeat and SOCS box-containing (Asb) proteins constitute the largest. Although it is known that the SOCS proteins are involved in the negative regulation of cytokine signaling, the biological and biochemical functions of the Asbs are largely undefined. Using a proteomics approach, we demonstrate that creatine kinase B (CKB) interacts with Asb-9 in a specific, SOCS box-independent manner. This interaction increases the polyubiquitylation of CKB and decreases total CKB levels within the cell. The targeting of CKB for degradation by Asb-9 was primarily SOCS box-dependent and suggests that Asb-9 acts as a specific ubiquitin ligase regulating levels of this evolutionarily conserved enzyme.

BCL-2 is dispensable for thrombopoiesis and platelet survival

Navitoclax (ABT-263), an inhibitor of the pro-survival BCL-2 family proteins BCL-2, BCL-XL and BCL-W, has shown clinical efficacy in certain BCL-2-dependent haematological cancers, but causes dose-limiting thrombocytopaenia. The latter effect is caused by Navitoclax directly inducing the apoptotic death of platelets, which are dependent on BCL-XL for survival. Recently, ABT-199, a selective BCL-2 antagonist, was developed. It has shown promising anti-leukaemia activity in patients whilst sparing platelets, suggesting that the megakaryocyte lineage does not require BCL-2. In order to elucidate the role of BCL-2 in megakaryocyte and platelet survival, we generated mice with a lineage-specific deletion of Bcl2, alone or in combination with loss of Mcl1 or Bclx. Platelet production and platelet survival were analysed. Additionally, we made use of BH3 mimetics that selectively inhibit BCL-2 or BCL-XL. We show that the deletion of BCL-2, on its own or in concert with MCL-1, does not affect platelet production or platelet lifespan. Thrombocytopaenia in Bclx-deficient mice was not affected by additional genetic loss or pharmacological inhibition of BCL-2. Thus, BCL-2 is dispensable for thrombopoiesis and platelet survival in mice.

Physiological restraint of Bak by Bcl-xL is essential for cell survival

Due to the myriad interactions between prosurvival and proapoptotic members of the Bcl-2 family of proteins, establishing the mechanisms that regulate the intrinsic apoptotic pathway has proven challenging. Mechanistic insights have primarily been gleaned from in vitro studies because genetic approaches in mammals that produce unambiguous data are difficult to design. Here we describe a mutation in mouse and human Bak that specifically disrupts its interaction with the prosurvival protein Bcl-xL Substitution of Glu75 in mBak (hBAK Q77) for leucine does not affect the three-dimensional structure of Bak or killing activity but reduces its affinity for Bcl-xL via loss of a single hydrogen bond. Using this mutant, we investigated the requirement for physical restraint of Bak by Bcl-xL in apoptotic regulation. In vitro, Bak(Q75L) cells were significantly more sensitive to various apoptotic stimuli. In vivo, loss of Bcl-xL binding to Bak led to significant defects in T-cell and blood platelet survival. Thus, we provide the first definitive in vivo evidence that prosurvival proteins maintain cellular viability by interacting with and inhibiting Bak.

Mice Haploinsufficient for Ets1 and Fli1 Display Middle Ear Abnormalities and Model Aspects of Jacobsen Syndrome

E26 transformation-specific 1 (ETS1) and friend leukemia integration 1 (FLI1) are members of the ETS family of transcription factors, of which there are 28 in humans. Both genes are hemizygous in Jacobsen syndrome, an 11q contiguous gene deletion disorder involving thrombocytopenia, facial dysmorphism, growth and mental retardation, malformation of the heart and other organs, and hearing impairment associated with recurrent ear infections. To determine whether any of these defects are because of hemizygosity for ETS1 and FLI1, we characterized the phenotype of mice heterozygous for mutant alleles of Ets1 and Fli1. Fli1(+/-) mice displayed mild thrombocytopenia, as did Ets1(+/-)Fli1(+/-) animals. Fli1(+/-) and Ets1(+/-)Fli1(+/-) mice also displayed craniofacial abnormalities, including a small middle ear cavity, short nasal bone, and malformed interface between the nasal bone process and cartilaginous nasal septum. They exhibited hearing impairment, otitis media, fusions of ossicles to the middle ear wall, and deformed stapes. Hearing impairment was more penetrant and stapes malformations were more severe in Ets1(+/-)Fli1(+/-) mice than in Fli1(+/-) mice, indicating partial functional redundancy of these transcription factors during auditory development. Our findings indicate that the short nose, otitis media, and hearing impairment in Jacobsen syndrome are likely because of hemizygosity for ETS1 and FLI1.