Maria Tsokos

A role for the NAD-dependent deacetylase Sirt1 in the regulation of autophagy

We demonstrate a role for the NAD-dependent deacetylase Sirt1 in the regulation of autophagy. In particular, transient increased expression of Sirt1 is sufficient to stimulate basal rates of autophagy. In addition, we show that Sirt1−/− mouse embryonic fibroblasts do not fully activate autophagy under starved conditions. Reconstitution with wild-type but not a deacetylase-inactive mutant of Sirt1 restores autophagy in these cells. We further demonstrate that Sirt1 can form a molecular complex with several essential components of the autophagy machinery, including autophagy genes (Atg)5, Atg7, and Atg8. In vitro, Sirt1 can, in an NAD-dependent fashion, directly deacetylate these components. The absence of Sirt1 leads to markedly elevated acetylation of proteins known to be required for autophagy in both cultured cells and in embryonic and neonatal tissues. Finally, we show that Sirt1−/− mice partially resemble Atg5−/− mice, including the accumulation of damaged organelles, disruption of energy homeostasis, and early perinatal mortality. Furthermore, the in utero delivery of the metabolic substrate pyruvate extends the survival of Sirt1−/− pups. These results suggest that the Sirt1 deacetylase is an important in vivo regulator of autophagy and provide a link between sirtuin function and the overall cellular response to limited nutrients.

Neuroendocrine tumors of the lung with proposed criteria for large-cell neuroendocrine carcinoma. An ultrastructural, immunohistochemical, and flow cytometric study of 35 cases.

Based on our review of 35 cases and the literature, we found the spectrum of pulmonary neuroendocrine (NE) tumors to be too broad to fit into the traditional threecategory classification scheme of typical carcinoid (TC), atypical carcinoid (AC), and small-cell lung carcinoma (SCLC). We found that a spectrum of high-and low-grade tumors exist between TC and SCLC and that in the past many of these tumors have been called AC. We chose to adhere to Arrigonis definition of AC, as his original criteria characterized a low-grade tumor. For the higher grade non-small-cell tumors (NSCLC), we propose a fourth category of large-cell neuroendocrine carcinoma (LCNEC), which is characterized by: (a) light microscopic NE appearance; (b) cells of large size, polygonal shape, low nuclear-cytoplasmic ratio (N:C), coarse nuclear chromatin, and frequent nucleoli; (c) high mitotic rate [> 10/10 high-power fields (HPF)] and frequent necrosis; and (d) NE features by immunohistochemistry (IHC) or electron microscopy (EM). Thus, after deciding that a pulmonary NE tumor is high grade, the major diagnostic issue is separation of LCNEC from SCLC. This distinction is based not only on cell size, but on a variety of morphologic features. We studied 20 TC, six AC, five LCNEC, and four SCLC and characterized the clinical, light microscopic, EM, IHC, and flow cytometric features of each type of tumor. We did not find any advantage to IHC. EM, or flow cytometry over light microscopy in the subclassification or prediction of prognosis; however, these methods were useful in characterizing these four types of pulmonary NE tumors and in demonstrating their NE properties. LCNEC must be distinguished from a fifth category pulmonary NE tumor: NSCLC with NE features in which NE differentiation is not evident by light microscopy and must be demonstrated by EM or IHC. Although the prognosis of LCNEC appears to be intermediate between AC and SCLC, larger numbers of patients will be needed to demonstrate significant differences in survival.

Hepatic Failure and Lactic Acidosis Due to Fialuridine (FIAU), an Investigational Nucleoside Analogue for Chronic Hepatitis B

BACKGROUND We describe severe and unexpected multisystem toxicity that occurred during a study of the antiviral nucleoside analogue fialuridine (1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil, or FIAU) as therapy for chronic hepatitis B virus infection. METHODS Fifteen patients with chronic hepatitis B were randomly assigned to receive fialuridine at a dose of either 0.10 or 0.25 mg per kilogram of body weight per day for 24 weeks and were monitored every 1 to 2 weeks by means of a physical examination, blood tests, and testing for hepatitis B virus markers. RESULTS During the 13th week lactic acidosis and liver failure suddenly developed in one patient. The study was terminated on an emergency basis, and all treatment with fialuridine was discontinued. Seven patients were found to have severe hepatotoxicity, with progressive lactic acidosis, worsening jaundice, and deteriorating hepatic synthetic function despite the discontinuation of fialuridine. Three other patients had mild hepatotoxicity. Several patients also had pancreatitis, neuropathy, or myopathy. Of the seven patients with severe hepatotoxicity, five died and two survived after liver transplantation. Histologic analysis of liver tissue revealed marked accumulation of microvesicular and macrovesicular fat, with minimal necrosis of hepatocytes or architectural changes. Electron microscopy showed abnormal mitochondria and the accumulation of fat in hepatocytes. CONCLUSIONS In patients with chronic hepatitis B, treatment with fialuridine induced a severe toxic reaction characterized by hepatic failure, lactic acidosis, pancreatitis, neuropathy, and myopathy. This toxic reaction was probably caused by widespread mitochondrial damage and may occur infrequently with other nucleoside analogues.

Targeted deletion of the gene encoding iron regulatory protein-2 causes misregulation of iron metabolism and neurodegenerative disease in mice.

In mammalian cells, regulation of the expression of proteins involved in iron metabolism is achieved through interactions of iron-sensing proteins known as iron regulatory proteins (IRPs), with transcripts that contain RNA stem-loop structures referred to as iron responsive elements (IREs). Two distinct but highly homologous proteins, IRP1 and IRP2, bind IREs with high affinity when cells are depleted of iron, inhibiting translation of some transcripts, such as ferritin, or turnover of others, such as the transferrin receptor (TFRC). IRPs sense cytosolic iron levels and modify expression of proteins involved in iron uptake, export and sequestration according to the needs of individual cells. Here we generate mice with a targeted disruption of the gene encoding Irp2 (Ireb2). These mutant mice misregulate iron metabolism in the intestinal mucosa and the central nervous system. In adulthood, Ireb2−/− mice develop a movement disorder characterized by ataxia, bradykinesia and tremor. Significant accumulations of iron in white matter tracts and nuclei throughout the brain precede the onset of neurodegeneration and movement disorder symptoms by many months. Ferric iron accumulates in the cytosol of neurons and oligodendrocytes in distinctive regions of the brain. Abnormal accumulations of ferritin colocalize with iron accumulations in populations of neurons that degenerate, and iron-laden oligodendrocytes accumulate ubiquitin-positive inclusions. Thus, misregulation of iron metabolism leads to neurodegenerative disease in Ireb2−/− mice and may contribute to the pathogenesis of comparable human neurodegenerative diseases.

Ifosfamide with mesna uroprotection and etoposide: an effective regimen in the treatment of recurrent sarcomas and other tumors of children and young adults.

One hundred twenty-four children and young adults with recurrent tumors, predominantly sarcomas, were treated with the combination of ifosfamide, etoposide, and the uroprotector, mesna (2-mercaptoethane sulphonate), in a phase II trial. The treatment regimen consisted of 12 cycles of therapy administered every 3 weeks. After evaluation of the tumor response to chemotherapy alone, radiation or surgery was used to eradicate residual sites of metastatic disease where possible. At the present time, 77 patients are evaluable for response to the chemotherapy; 43 of the patients have experienced a significant reduction in the tumor size in response to the chemotherapy alone (39 partial responses [PR] and four complete responses [CR]). Sixteen of 17 patients with Ewings sarcoma, nine of 13 with rhabdomyosarcoma, four of eight with peripheral neuroepithelioma, three of eight with osteosarcoma, and 11 of 31 with other tumors have responded with a PR or CR. The toxicity of the regimen was acceptable. Moderate or severe toxicity evaluated on a per cycle basis included: neutropenia, 97%; thrombocytopenia, 32%; nephrotoxicity, less than 1%; mucositis, 1%; neurologic toxicity, 2%; nausea and vomiting, 13%; hemorrhagic cystitis, less than 1%. Fever was present after 33% of cycles and sepsis following 7%. One patient died due to sepsis and pancytopenia. At the present time, only seven of the 43 patients who responded to the chemotherapy regimen have relapsed, with a median follow-up of 10 weeks after the response. This drug combination is highly active in the treatment of recurrent sarcomas and other tumors in children and young adults.

Malignant lymphoma and erythrophagocytosis simulating malignant histiocytosis

Six patients with malignant lymphoma are described, in whom a syndrome that mimicked malignant histiocytosis clinically and pathologically developed as a terminal event. The clinical course was characterized by hepatosplenomegaly, fever, and a rapid fall in all hematologic counts. Survival from onset of this syndrome was less than six weeks. Pathologically, activation of benign histiocytes with marked erythrophagocytosis was seen throughout the reticuloendothelial system. Disseminated lymphoma was also evident but was grossly and microscopically segregated from the erythrophagocytic process. Immunologic and cytochemical studies in two cases indicated the lymphomas to be of peripheral T cell origin, whereas the phagocytic cells had markers characteristic of histiocytes. A likely mechanism for this syndrome is the production of lymphokines (macrophage-activating factors) by neoplastic T lymphocytes.

Nelfinavir, A lead HIV protease inhibitor, is a broad-spectrum, anticancer agent that induces endoplasmic reticulum stress, autophagy, and apoptosis in vitro and in vivo

Purpose: The development of new cancer drugs is slow and costly. HIV protease inhibitors are Food and Drug Administration approved for HIV patients. Because these drugs cause toxicities that can be associated with inhibition of Akt, an emerging target in cancer, we assessed the potential of HIV protease inhibitors as anticancer agents. Experimental Design: HIV protease inhibitors were screened in vitro using assays that measure cellular proliferation, apoptotic and nonapoptotic cell death, endoplasmic reticulum (ER) stress, autophagy, and activation of Akt. Nelfinavir was tested in non–small cell lung carcinoma (NSCLC) xenografts with biomarker assessment. Results: Three of six HIV protease inhibitors, nelfinavir, ritonavir, and saquinavir, inhibited proliferation of NSCLC cells, as well as every cell line in the NCI60 cell line panel. Nelfinavir was most potent with a mean 50% growth inhibition of 5.2 μmol/L, a concentration achievable in HIV patients. Nelfinavir caused two types of cell death, caspase-dependent apoptosis and caspase-independent death that was characterized by induction of ER stress and autophagy. Autophagy was protective because an inhibitor of autophagy increased nelfinavir-induced death. Akt was variably inhibited by HIV protease inhibitors, but nelfinavir caused the greatest inhibition of endogenous and growth factor–induced Akt activation. Nelfinavir decreased the viability of a panel of drug-resistant breast cancer cell lines and inhibited the growth of NSCLC xenografts that was associated with induction of ER stress, autophagy, and apoptosis. Conclusions: Nelfinavir is a lead HIV protease inhibitor with pleiotropic effects in cancer cells. Given its wide spectrum of activity, oral availability, and familiarity of administration, nelfinavir is a Food and Drug Administration–approved drug that could be repositioned as a cancer therapeutic.

Phosphoprotein Pathway Mapping: Akt/Mammalian Target of Rapamycin Activation Is Negatively Associated with Childhood Rhabdomyosarcoma Survival

Mapping of protein signaling networks within tumors can identify new targets for therapy and provide a means to stratify patients for individualized therapy. Despite advances in combination chemotherapy, the overall survival for childhood rhabdomyosarcoma remains approximately 60%. A critical goal is to identify functionally important protein signaling defects associated with treatment failure for the 40% nonresponder cohort. Here, we show, by phosphoproteomic network analysis of microdissected tumor cells, that interlinked components of the Akt/mammalian target of rapamycin (mTOR) pathway exhibited increased levels of phosphorylation for tumors of patients with short-term survival. Specimens (n = 59) were obtained from the Childrens Oncology Group Intergroup Rhabdomyosarcoma Study (IRS) IV, D9502 and D9803, with 12-year follow-up. High phosphorylation levels were associated with poor overall and poor disease-free survival: Akt Ser(473) (overall survival P < 0.001, recurrence-free survival P < 0.0009), 4EBP1 Thr(37/46) (overall survival P < 0.0110, recurrence-free survival P < 0.0106), eIF4G Ser(1108) (overall survival P < 0.0017, recurrence-free survival P < 0.0072), and p70S6 Thr(389) (overall survival P < 0.0085, recurrence-free survival P < 0.0296). Moreover, the findings support an altered interrelationship between the insulin receptor substrate (IRS-1) and Akt/mTOR pathway proteins (P < 0.0027) for tumors from patients with poor survival. The functional significance of this pathway was tested using CCI-779 in a mouse xenograft model. CCI-779 suppressed phosphorylation of mTOR downstream proteins and greatly reduced the growth of two different rhabdomyosarcoma (RD embryonal P = 0.00008; Rh30 alveolar P = 0.0002) cell lines compared with controls. These results suggest that phosphoprotein mapping of the Akt/mTOR pathway should be studied further as a means to select patients to receive mTOR/IRS pathway inhibitors before administration of chemotherapy.

Succinate Dehydrogenase Mutation Underlies Global Epigenomic Divergence in Gastrointestinal Stromal Tumor

Gastrointestinal stromal tumors (GIST) harbor driver mutations of signal transduction kinases such as KIT, or, alternatively, manifest loss-of-function defects in the mitochondrial succinate dehydrogenase (SDH) complex, a component of the Krebs cycle and electron transport chain. We have uncovered a striking divergence between the DNA methylation profiles of SDH-deficient GIST (n = 24) versus KIT tyrosine kinase pathway-mutated GIST (n = 39). Infinium 450K methylation array analysis of formalin-fixed paraffin-embedded tissues disclosed an order of magnitude greater genomic hypermethylation relative to SDH-deficient GIST versus the KIT-mutant group (84.9 K vs. 8.4 K targets). Epigenomic divergence was further found among SDH-mutant paraganglioma/pheochromocytoma (n = 29), a developmentally distinct SDH-deficient tumor system. Comparison of SDH-mutant GIST with isocitrate dehydrogenase-mutant glioma, another Krebs cycle-defective tumor type, revealed comparable measures of global hypo- and hypermethylation. These data expose a vital connection between succinate metabolism and genomic DNA methylation during tumorigenesis, and generally implicate the mitochondrial Krebs cycle in nuclear epigenomic maintenance.

Total-body irradiation and autologous bone marrow transplant in the treatment of high-risk Ewing's sarcoma and rhabdomyosarcoma.

PURPOSE In an effort to improve outcome in patients with metastatic or high-risk localized Ewings sarcoma family of tumors (ESF) and rhabdomyosarcoma (RMS), we explored the role of consolidation therapy with total-body irradiation (TBI) plus autologous bone marrow transplantation (ABMT). PATIENTS AND METHODS Ninety-one patients were entered onto one of three consecutive protocols from 1981 to 1986. Induction therapy consisted of four or five cycles of vincristine, doxorubicin, and cyclophosphamide (VAdriaC); in the earlier series, patients received one or two cycles with dactinomycin instead of doxorubicin. Irradiation of the primary site was used for local control. Patients who attained a complete response (CR) to induction therapy were eligible for consolidation with 8 Gy TBI plus VAdriaC and ABMT. RESULTS Nineteen patients were ineligible for consolidation after failing to achieve or maintain a CR following induction therapy; all 19 are dead of disease. Seven eligible patients elected to forgo consolidation; three of seven are long-term event-free survivors. Sixty-five patients received consolidation therapy; 20 of 65 are long-term event-free survivors. A local control rate of 83% was achieved using radiation therapy as the primary modality of local control. Patients with metastatic disease at diagnosis fared substantially worse than did patients with localized tumors (6-year event-free survival [EFS] rate, 14% v 38%; two-sided P [P2] = .008). CONCLUSIONS Consolidation of patients with metastatic or high-risk localized pediatric sarcomas with 8 Gy TBI plus ABMT has failed to improve the outcome of this group of patients. Metastatic disease at diagnosis continues to confer the poorest prognosis. New therapeutic strategies are needed to consolidate more effectively the remissions that can be achieved in the majority of these patients.