Craig M. Forester

Control of mitotic exit by PP2A regulation of Cdc25C and Cdk1

Inactivation of maturation-promoting factor [(MPF) Cdk1/Cyclin B] is a key event in the exit from mitosis. Although degradation of Cyclin B is important for MPF inactivation, recent studies indicate that Cdk1 phosphorylation and inactivation occur before Cyclin B degradation and, therefore, also may be important steps in the exit from mitosis. Cdk1 activity is controlled by the Cdc25C phosphatase, which is turned on at the G2/M transition to catalyze Cdk1 activation. PP2A:B56δ is a negative regulator of Cdc25C during interphase. We show here that PP2A:B56δ also regulates Cdc25C at mitosis. Failure of PP2A:B56δ to dephosphorylate Cdc25C at mitosis results in prolonged hyperphosphorylation and activation of Cdc25C, causing persistent dephosphorylation and, hence, activation of Cdk1. This constitutive activation of Cdc25C and Cdk1 leads to a delayed exit from mitosis. Consistent with Cdk1 as a major biological target of B56δ, stable knockdown and germ-line mouse KO of B56δ leads to compensatory transcriptional up-regulation of Wee1 kinase to oppose the Cdc25C activity and permit cell survival. These observations place PP2A:B56δ as a key upstream regulator of Cdk1 activity upon exit from mitosis.

EAG2 potassium channel with evolutionarily conserved function as a brain tumor target

Over 20% of the drugs for treating human diseases target ion channels, but no cancer drug approved by the US Food and Drug Administration (FDA) is intended to target an ion channel. We found that the EAG2 (Ether-a-go-go 2) potassium channel has an evolutionarily conserved function for promoting brain tumor growth and metastasis, delineate downstream pathways, and uncover a mechanism for different potassium channels to functionally cooperate and regulate mitotic cell volume and tumor progression. EAG2 potassium channel was enriched at the trailing edge of migrating medulloblastoma (MB) cells to regulate local cell volume dynamics, thereby facilitating cell motility. We identified the FDA-approved antipsychotic drug thioridazine as an EAG2 channel blocker that reduces xenografted MB growth and metastasis, and present a case report of repurposing thioridazine for treating a human patient. Our findings illustrate the potential of targeting ion channels in cancer treatment.

Locally translated mTOR controls axonal local translation in nerve injury

Local control of localized protein synthesis Localized protein synthesis provides spatiotemporal precision for injury responses and growth decisions at remote positions in nerve axons. Terenzio et al. show that this process is controlled by local translation of preexisting axonal mRNA encoding the master regulator mTOR (see the Perspective by Riccio). mTOR controls both its own synthesis and that of most newly synthesized proteins at axonal injury sites, thereby determining the subsequent survival and growth of the injured neuron. Science, this issue p. 1416; see also p. 1331 Axonal localization of mTOR mRNA enables subcellular regulation of local protein synthesis in injured nerves. How is protein synthesis initiated locally in neurons? We found that mTOR (mechanistic target of rapamycin) was activated and then up-regulated in injured axons, owing to local translation of mTOR messenger RNA (mRNA). This mRNA was transported into axons by the cell size–regulating RNA-binding protein nucleolin. Furthermore, mTOR controlled local translation in injured axons. This included regulation of its own translation and that of retrograde injury signaling molecules such as importin β1 and STAT3 (signal transducer and activator of transcription 3). Deletion of the mTOR 3′ untranslated region (3′UTR) in mice reduced mTOR in axons and decreased local translation after nerve injury. Both pharmacological inhibition of mTOR in axons and deletion of the mTOR 3′UTR decreased proprioceptive neuronal survival after nerve injury. Thus, mRNA localization enables spatiotemporal control of mTOR pathways regulating local translation and long-range intracellular signaling.

Pediatric Aplastic Anemia and Refractory Cytopenia: A retrospective analysis assessing outcomes and histomorphologic predictors

Pediatric acquired aplastic anemia (AA) is a bone marrow disorder that is difficult to distinguish from inherited bone marrow failure syndromes and hypocellular refractory cytopenia of childhood (RCC). Historically, patients with hypocellular RCC have been given the diagnosis of AA. To assess the clinical and histologic distinction between RCC and AA, we performed a retrospective analysis of 149 patients previously diagnosed with AA between 1976 and 2010. We evaluated event free survival (EFS), overall survival (OS), response rates to immunosuppressive therapy, treatment‐related toxicities and clonal evolution. The 5‐year EFS and OS were 50.8% ± 5.5% and 73.1% ± 4.7%, respectively. Patients with very severe AA had worse OS compared to patients with severe and moderately severe AA. Seventy‐two patients had diagnostic pathology specimens available for review. Three pediatric hematopathologists reviewed and reclassified these specimens as AA, RCC or Other based on 2008 WHO Criteria. The concordance between pathologists in the diagnosis of AA or RCC was modest. RCC was associated with a trend toward improved OS and EFS and was not prognostic of immunosuppression therapy treatment failure. There was a low rate of clonal evolution exclusively associated with moderately severe AA. Our findings indicate that a diagnosis of RCC is difficult to establish with certainty and does not predict outcomes, calling into question the reproducibility and clinical significance of the RCC classification and warranting further studies. Am. J. Hematol. 90:320–326, 2015.

Rat Cytomegalovirus Major Immediate-Early Enhancer Switching Results in Altered Growth Characteristics

ABSTRACT It has been hypothesized that the major immediate-early (MIE) enhancer of cytomegalovirus (CMV) is important in determining virus tropism and latency because of its essential role in initiating the cascade of early gene expression necessary for virus replication. Although rat CMV (RCMV) and murine CMV (MCMV) exhibit extreme species specificity in vivo, they differ in their ability to replicate in tissue culture. MCMV can replicate in a rat embryo fibroblast (REF) cell line while RCMV does not grow in murine fibroblasts. The tropism is not due to a block in virus entry into the cell. We have constructed a recombinant RCMV in which the RCMV MIE enhancer has been replaced with that of MCMV. Growth of the recombinant virus in tissue culture remains restricted to rat cells, suggesting that other viral and/or host factors are more important in determining in vitro tropism. Unlike findings using recombinant MCMV in which the human CMV (HCMV) MIE enhancer substitutes for the native one (A. Angulo, M. Messerle, U. H. Koszinowski, and P. Ghazal, J. Virol. 72:8502–8509, 1998), infection with our recombinant virus at a low multiplicity of infection resulted in a substantial decrease in virus replication. This occurred despite comparable or increased MIE transcription from the recombinant virus. In vivo experiments showed that the recombinant virus replicates normally in the spleen during acute infection. Notably, the recombinant virus appears to be deficient in spreading to the salivary gland, suggesting a role for the MIE enhancer in tropism for certain tissues involved in virus dissemination. Four months after infection, recombinant virus with the foreign MIE enhancer was reactivated from spleen explants.

Osteosarcoma presenting as hemorrhagic cerebellar metastasis

PurposeOsteosarcoma is the most common malignant bone tumor in children and adolescents. Brain metastasis from osteosarcoma was once uncommon; however, with the advent of chemotherapeutic agents and improved imaging modalities, it has become a more common and recognized finding. Brain metastases are, rarely, the initial presenting symptom, but instead are a late and preterminal event in the disease process. When osteosarcomas manifest in the central nervous system, they tend to occur in the gray–white junction in the anterior circulation akin to other metastatic lesions in the brain.Case reportThe authors report a case of a 16-year-old boy who presented with acute neurological deterioration due to a posterior fossa hemorrhage and was subsequently found to have a primary site localizing to the metaphysis of the right femur with florid metastatic disease.ConclusionsThis is the first case reported in the literature in which an osteosarcoma initially presented as cerebral metastasis in the form of a posterior fossa hemorrhage with a rapidly deteriorating course.

Development of a stress response therapy targeting aggressive prostate cancer

The PERK-eIF2α pathway is activated in aggressive prostate cancer and associated with patient outcome, providing a therapeutic target for the disease. Stressing out prostate cancer As tumors grow, they undergo a variety of metabolic changes that facilitate their proliferation. Protein synthesis is one of the cellular processes that is altered in cancer cells, because its continued activation helps drive cancer growth. This is not a benign adaptation, however, and unchecked up-regulation of protein synthesis can be toxic to the cells because it promotes cellular stress. As Nguyen et al. discovered, prostate cancer cells with a specific combination of mutations can override this stress by activating a protein called eIF2α, which protects them from excessive protein synthesis. To target this pathway, the authors identified an inhibitor of eIF2α that blocks this protective mechanism and has therapeutic activity against aggressive and otherwise untreatable prostate cancer. Oncogenic lesions up-regulate bioenergetically demanding cellular processes, such as protein synthesis, to drive cancer cell growth and continued proliferation. However, the hijacking of these key processes by oncogenic pathways imposes onerous cell stress that must be mitigated by adaptive responses for cell survival. The mechanism by which these adaptive responses are established, their functional consequences for tumor development, and their implications for therapeutic interventions remain largely unknown. Using murine and humanized models of prostate cancer (PCa), we show that one of the three branches of the unfolded protein response is selectively activated in advanced PCa. This adaptive response activates the phosphorylation of the eukaryotic initiation factor 2–α (P-eIF2α) to reset global protein synthesis to a level that fosters aggressive tumor development and is a marker of poor patient survival upon the acquisition of multiple oncogenic lesions. Using patient-derived xenograft models and an inhibitor of P-eIF2α activity, ISRIB, our data show that targeting this adaptive brake for protein synthesis selectively triggers cytotoxicity against aggressive metastatic PCa, a disease for which presently there is no cure.

Revealing nascent proteomics in signaling pathways and cell differentiation

Significance Deciphering which networks of genes allow cells to rapidly respond to environmental cues is key to understanding processes such as proliferation, differentiation, and death. Here, we describe a technique to quantitatively identify nascent protein synthesis using O-propargyl-puromycin (OPP), a specific covalent label of growing polypeptides, which are then isolated and measured by mass spectrometry. This technique, named OPP-mediated identification (OPP-ID), is applicable across a wide array of cell types for identification of proteins in situ. We illustrate the capability of OPP-ID by identifying a constellation of mammalian target of rapamycin (mTOR)-dependent gene networks and uncovering an array of genes involved in early erythroid progenitor expansion. OPP-ID is a powerful technique to study how cells modulate nascent protein expression to respond to stresses and stimuli. Regulation of gene expression at the level of protein synthesis is a crucial element in driving how the genetic landscape is expressed. However, we are still limited in technologies that can quantitatively capture the immediate proteomic changes that allow cells to respond to specific stimuli. Here, we present a method to capture and identify nascent proteomes in situ across different cell types without disturbing normal growth conditions, using O-propargyl-puromycin (OPP). Cell-permeable OPP rapidly labels nascent elongating polypeptides, which are subsequently conjugated to biotin-azide, using click chemistry, and captured with streptavidin beads, followed by digestion and analysis, using liquid chromatography–tandem mass spectrometry. Our technique of OPP-mediated identification (OPP-ID) allows detection of widespread proteomic changes within a short 2-hour pulse of OPP. We illustrate our technique by recapitulating alterations of proteomic networks induced by a potent mammalian target of rapamycin inhibitor, MLN128. In addition, by employing OPP-ID, we identify more than 2,100 proteins and uncover distinct protein networks underlying early erythroid progenitor and differentiation states not amenable to alternative approaches such as amino acid analog labeling. We present OPP-ID as a method to quantitatively identify nascent proteomes across an array of biological contexts while preserving the subtleties directing signaling in the native cellular environment.

Tumefactive intracranial presentation of precursor B-cell acute lymphoblastic leukemia

In children, leukemia is the most common malignancy, and approximately 75% of leukemias are acute lymphoblastic leukemia (ALL). Central nervous system leukemia is found at diagnosis in fewer than 5% of children with ALL. Leukemic intracranial masses have been described with acute myeloid leukemia, but ALL presenting as a mass lesion is rare. We describe a unique case of an intracranial confirmed precursor B cell (pre-B) ALL mass in a 13-year-old girl that was diagnosed by brain CT, MRI and cerebral angiography, and confirmed by biopsy. This report details pertinent history and distinguishing imaging features of an intracranial ALL tumefaction.

Transfusion practices and complications in thalassemia: Transfusion practices and complications in thalassemia

The severe forms of thalassemia are the most common inherited anemias managed with regular blood transfusion therapy. Transfusion policies and complications are critical to quality of life and survival, but there is a lack of standardized care.