No abstract

Cytopenias are key prognostic indicators of life-threatening infection, contributing to immunosuppression and mortality. Here we define a role for Caspase-1-dependent death, known as pyroptosis, in infection-induced cytopenias by studying inflammasome activation in hematopoietic progenitor cells. The NLRP1a inflammasome is expressed in hematopoietic progenitor cells and its activation triggers their pyroptotic death. Active NLRP1a induced a lethal systemic inflammatory disease that was driven by Caspase-1 and IL-1β but was independent of apoptosis-associated speck-like protein containing a CARD (ASC) and ameliorated by IL-18. Surprisingly, in the absence of IL-1β-driven inflammation, active NLRP1a triggered pyroptosis of hematopoietic progenitor cells resulting in leukopenia in the steady state. During periods of hematopoietic stress induced by chemotherapy or lymphocytic choriomeningitis virus (LCMV) infection, active NLRP1a caused prolonged cytopenia, bone marrow hypoplasia and immunosuppression. Conversely, NLRP1-deficient mice showed enhanced recovery from chemotherapy and LCMV infection, demonstrating that NLRP1 acts as a cellular sentinel to alert Caspase-1 to hematopoietic and infectious stress.

Ets-related gene (ERG), which encodes a member of the Ets family of transcription factors, is a potent oncogene. Chromosomal rearrangements involving ERG are found in acute myeloid leukemia, acute lymphoblastic leukemia, Ewing's sarcoma and more than half of all prostate cancers; however, the normal physiological function of Erg is unknown. We did a sensitized genetic screen of the mouse for regulators of hematopoietic stem cell function and report here a germline mutation of Erg. We show that Erg is required for definitive hematopoiesis, adult hematopoietic stem cell function and the maintenance of normal peripheral blood platelet numbers.

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.

A pivotal mediator of actin dynamics is the protein cofilin, which promotes filament severing and depolymerization, facilitating the breakdown of existing filaments, and the enhancement of filament growth from newly created barbed ends. It does so in concert with actin interacting protein 1 (Aip1), which serves to accelerate cofilin's activity. While progress has been made in understanding its biochemical functions, the physiologic processes the cofilin/Aip1 complex regulates, particularly in higher organisms, are yet to be determined. We have generated an allelic series for WD40 repeat protein 1 (Wdr1), the mammalian homolog of Aip1, and report that reductions in Wdr1 function produce a dramatic phenotype gradient. While severe loss of function at the Wdr1 locus causes embryonic lethality, macrothrombocytopenia and autoinflammatory disease develop in mice carrying hypomorphic alleles. Macrothrombocytopenia is the result of megakaryocyte maturation defects, which lead to a failure of normal platelet shedding. Autoinflammatory disease, which is bone marrow-derived yet nonlymphoid in origin, is characterized by a massive infiltration of neutrophils into inflammatory lesions. Cytoskeletal responses are impaired in Wdr1 mutant neutrophils. These studies establish an essential requirement for Wdr1 in megakaryocytes and neutrophils, indicating that cofilin-mediated actin dynamics are critically important to the development and function of both cell types.

Histone deacetylase inhibitor (HDACI)-induced thrombocytopenia (TCP) is a major dose-limiting toxicity of this new class of drugs. Using preclinical models to study the molecular and biologic events that underpin this effect of HDACI, we found that C57BL/6 mice treated with both the HDAC1/ 2-selective HDACI romidepsin and the pan-HDACI panobinostat developed significant TCP. HDACI-induced TCP was not due to myelosuppression or reduced platelet lifespan, but to decreased platelet release from megakaryocytes. Cultured primary murine megakaryocytes showed reductions in proplatelet extensions after HDACI exposure and a dose-dependent increase in the phosphorylation of myosin light chain 2 (MLC2). Phosphorylation of MLC to phospho-MLC (pMLC) and subsequent proplatelet formation in megakaryocytes is regulated by the Rho-GTPase proteins Rac1, CDC42, and RhoA. Primary mouse megakaryocytes and the human megakaryoblastic cell line Meg-01 showed reductions in Rac1, CDC42, and RhoA protein levels after treatment with HDACIs. We were able to overcome HDACIinduced TCP by administering the mousespecific thrombopoietin (TPO) mimetic AMP-4, which improved platelet numbers to levels similar to untreated controls. Our report provides the first detailed account of the molecular and biologic processes involved in HDACI-mediated TCP. Moreover, our preclinical studies provide evidence that dose-limiting TCP induced by HDACIs may be circumvented using a TPO mimetic. (Blood. 2011;117(13):3658-3668) IntroductionCovalent posttranslational modifications to specific sites within histone proteins, including acetylation, methylation, and phosphorylation, are able to affect gene transcription in cells. 1 Increased acetylation of histones is associated with open DNA and increased transcription, whereas deacetylation is associated with transcriptional repression. 2 Disruption of this balance is associated with cancer onset and progression. 3 The histone deacetylase (HDAC) enzymes control the structural conformation of chromatin via deacetylation of core nucleosomal histones. To date, a total of 18 HDACs have been described and are divided into 4 general classes. Class I HDACs are thought to be located within the cell nucleus only, whereas class II and class IV HDACs shuttle between the cell cytoplasm and the nucleus. Class III HDACs comprise the NAD ϩ -dependent sirtuin family proteins. 4 HDAC inhibitors (HDACIs) are structurally diverse antineoplastic agents distinguished both by their chemical structure and by their target specificity. 5 HDACIs induce chromatin remodeling and altered gene expression, and the function of nonhistone proteins may also be affected by direct acetylation. 6,7 Panobinostat is a cinnamic hydroxamic acid with inhibitory effects against all class I, II, and IV HDAC enzymes and marked antitumor activity across a broad range of hematologic cancers, including Hodgkin lymphoma. [8][9][10] Romidepsin is a bicyclic tetrapeptide that preferentially interacts with class I enzymes, and has activity in cutaneous T-cell lymphoma, ...

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 IntroductionMegakaryocytes are large polyploid cells responsible for the production of blood platelets. They develop primarily in the BM and spleen. On reaching maturity, megakaryocytes extend long pseudopodial projections called proplatelets into the circulation, and it is from these structures that platelets are released. [1][2][3] Once born, platelets have a brief lifespan in the circulation: 10 days in humans, 5 days in mice. 4,5 In recent years, it has become apparent that this finite existence is governed by the interplay between members of the Bcl-2 protein family, the critical regulators of the "intrinsic" or "mitochondrial" apoptosis pathway. 6 Platelets depend on the prosurvival protein Bcl-x L to maintain their viability. 7 Mutations in murine Bcl-x L cause dose-dependent cell-intrinsic reductions in platelet lifespan. 7-9 Pharmacologic blockade of Bcl-x L with the BH3 mimetic drugs ABT-737 10 or ABT-263 11 triggers platelet death 7,12-15 and thrombocytopenia in mice, 7,16 dogs, 15 and humans. 17,18 The function of Bcl-x L is to restrain the prodeath proteins Bak and Bax. When activated, Bak and Bax induce mitochondrial outer membrane permeabilization (MOMP), resulting in platelet apoptosis, which, at least in vitro, is characterized by cytochrome c release, caspase activation, and phosphatidylserine exposure. [12][13][14][15] At steady state in vivo, aged platelets that have escaped hemostatic consumption undergo Bak-mediated apoptosis and clearance from the circulation. Genetic deletion of Bak and Bax almost doubles platelet lifespan, 9 rescues the thrombocytopenia caused by loss of Bcl-x L , 9 and renders platelets refractory to the effects of ABT-737. 13 We recently demonstrated that Bcl-x L is also essential for mature megakaryocytes to proceed safely through the process of platelet shedding. 9 In vitro, loss of Bcl-x L triggers Bak-and Bax-mediated mitochondrial damage, caspase activation, and failure of proplatelet formation. In vivo, mature, shedding megakaryocytes lacking Bcl-x L exhibit severe dysmorphology and produce grossly abnormal platelets that survive only hours in the circulation. Bcl-x L is, however, dispensable for the growth and development of megakaryocytes, suggesting that other Bcl-2 family prosurvival proteins are necessary to promote survival during this process. Of the 5 prosurvival members of the Bcl-2 family, 6 3 are known to be expressed in megakaryocytes: Bcl-2, Bcl-x L , and Mcl-1. 9 Given its critical role in hematopoietic progenitors and lymphocy...

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 phospha- IntroductionApoptotic caspases are a family of aspartate-specific cysteinyl proteases that are activated during, and facilitate the execution of, programmed cell death. They cleave a range of cellular substrates, thereby causing the morphologic and biochemical signatures of apoptosis, such as DNA fragmentation, membrane blebbing, and phosphatidylserine (PS) externalization. In addition to their role in cell death, apoptotic caspases have been ascribed an increasing number of functions. These include critical roles in the differentiation of erythroid cells, 1 osteoblasts, 2 keratinocytes, 3 lens fiber cells, 4 skeletal muscle, 5 and embryonic stem (ES) cells. 6 Apoptotic caspases have also been suggested to regulate B-lymphocyte proliferation, 7 HSC quiescence, 8 activation of microglia, 9 and the reprogramming of fibroblasts into iPS (induced pluripotent stem cells). 10 There are 2 pathways to apoptosis, the intrinsic and the extrinsic. Both ultimately converge on the apoptotic effector caspases, caspase-3 and caspase-7. The intrinsic (or mitochondrial) apoptosis pathway is regulated by the interaction between Bcl-2 family proteins, which are divided into prosurvival and prodeath subsets. The critical mediators of the pathway are the prodeath proteins Bak and Bax, which, in viable cells are restrained by one or more of the 5 prosurvivals: Bcl-2, Bcl-x L , Mcl-1, Bcl-w, and A1. In response to apoptotic signals such as DNA damage or growth factor deprivation, a third group of Bcl-2 family members, the "BH3-only" proteins, trigger the activation of Bak and Bax. 11,12 Active Bak and Bax cause mitochondrial outer membrane permeabilization (MOMP), which allows apoptogenic factors, including cytochrome c, to enter the cytosol. 13 Cytochrome c interacts with the scaffolding protein Apaf-1. Subsequently, the initiator caspase, caspase-9, is recruited to the cytochrome c/Apaf-1 complex to form the apoptosome. 14 This leads to the activation of caspase-9 and triggering of the caspase cascade. The importance of the caspas...