The SMAC mimetic LCL-161 selectively targets JAK2V617F mutant cells.

Background:Evasion from programmed cell death is a hallmark of cancer and can be achieved in cancer cells by overexpression of inhibitor of apoptosis proteins (IAPs). Second mitochondria-derived activator of caspases (SMAC) directly bind to IAPs and promote apoptosis; thus, SMAC mimetics have been investigated in a variety of cancer types. particularly in diseases with high inflammation and NFĸB activation. Given that elevated TNFα levels and NFĸB activation is a characteristic feature of myeloproliferative neoplasms (MPN), we investigated the effect of the SMAC mimetic LCL-161 on MPN cell survival in vitro and disease development in vivo. Methods:To investigate the effect of the SMAC mimetic LCL-161 in vitro, we utilized murine and human cell lines to perform cell viability assays as well as primary bone marrow from mice or humans with JAK2V617F-driven MPN to interrogate myeloid colony formation. To elucidate the effect of the SMAC mimetic LCL-161 in vivo, we treated a JAK2V617F-driven mouse model of MPN with LCL-161 then assessed blood counts, splenomegaly, and myelofibrosis. Results:We found that JAK2V617F-mutated cells are hypersensitive to the SMAC mimetic LCL-161 in the absence of exogenous TNFα. JAK2 kinase activity and NFĸB activation is required for JAK2V617F-mediated sensitivity to LCL-161, as JAK or NFĸB inhibitors diminished the differential sensitivity of JAK2V617F mutant cells to IAP inhibition. Finally, LCL-161 reduces splenomegaly and may reduce fibrosis in a mouse model of JAK2V617F-driven MPN. Conclusion:LCL-161 may be therapeutically useful in MPN, in particular when exogenous TNFα signaling is blocked. NFĸB activation is a characteristic feature of JAK2V617F mutant cells and this sensitizes them to SMAC mimetic induced killing even in the absence of TNFα. However, when exogenous TNFα is added, NFĸB is activated in both mutant and wild-type cells, abolishing the differential sensitivity. Moreover, JAK kinase activity is required for the differential sensitivity of JAK2V617F mutant cells, suggesting that the addition of JAK2 inhibitors to SMAC mimetics would detract from the ability of SMAC mimetics to selectively target JAK2V617F mutant cells. Instead, combination therapy with other agents that reduce inflammatory cytokines but preserve JAK2 signaling in mutant cells may be a more beneficial combination therapy in MPN

Chemotherapeutic effect of a novel temozolomide analog on nasopharyngeal carcinoma in vitro and in vivo.

BackgroundMany patients with nasopharyngeal carcinoma (NPC) face poor prognosis. Due to its hidden anatomical location, the tumor is usually diagnosed quite late, and despite initially successful treatment with radiation and cisplatin, many patients will relapse and succumb to the disease. New treatment options are urgently needed. We have performed preclinical studies to evaluate the potential NPC therapeutic activity of a newly developed analog of temozolomide (TMZ), an alkylating agent that is the current chemotherapeutic standard of care for patients with malignant glioma.ResultsTMZ was covalently conjugated to the natural monoterpene perillyl alcohol (POH), creating the novel fusion compound NEO212. Its impact on two NPC cell lines was studied through colony formation assays, cell death ELISA, immunoblots, and in vivo testing in tumor-bearing mice. In vitro, NEO212 effectively triggered tumor cell death, and its potency was significantly greater than that of its individual components, TMZ or POH alone. Intriguingly, merely mixing TMZ with POH also was unable to achieve the superior potency of the conjugated compound NEO212. Treatment of NPC cells with NEO212 inactivated the chemoprotective DNA repair protein MGMT (O6-methylguanine methyltransferase), resulting in significant chemosensitization of cells to a second round of drug treatment. When tested in vivo, NEO212 reduced tumor growth in treated animals.ConclusionOur results demonstrate anticancer activity of NEO212 in preclinical NPC models, suggesting that this novel compound should be evaluated further for the treatment of patients with NPC

Generation of Organ-conditioned Media and Applications for Studying Organ-specific Influences on Breast Cancer Metastatic Behavior

Breast cancer preferentially metastasizes to the lymph node, bone, lung, brain and liver in breast cancer patients. Previous research efforts have focused on identifying factors inherent to breast cancer cells that are responsible for this observed metastatic pattern (termed organ tropism), however much less is known about factors present within specific organs that contribute to this process. This is in part because of a lack of in vitro model systems that accurately recapitulate the organ microenvironment. To address this, an ex vivo model system has been established that allows for the study of soluble factors present within different organ microenvironments. This model consists of generating conditioned media from organs (lymph node, bone, lung, and brain) isolated from normal athymic nude mice. The model system has been validated by demonstrating that different breast cancer cell lines display cell-line specific and organ-specific malignant behavior in response to organ-conditioned media that corresponds to their in vivo metastatic potential. This model system can be used to identify and evaluate specific organ-derived soluble factors that may play a role in the metastatic behavior of breast and other types of cancer cells, including influences on growth, migration, stem-like behavior, and gene expression, as well as the identification of potential new therapeutic targets for cancer. This is the first ex vivo model system that can be used to study organ-specific metastatic behavior in detail and evaluate the role of specific organ-derived soluble factors in driving the process of cancer metastasis

Organic nitrate chemistry and its implications for nitrogen budgets in an isoprene- and monoterpene-rich atmosphere: constraints from aircraft (SEAC^4RS) and ground-based (SOAS) observations in the Southeast US

Formation of organic nitrates (RONO_2) during oxidation of biogenic volatile organic compounds (BVOCs: isoprene, monoterpenes) is a significant loss pathway for atmospheric nitrogen oxide radicals (NO_x), but the chemistry of RONO_2 formation and degradation remains uncertain. Here we implement a new BVOC oxidation mechanism (including updated isoprene chemistry, new monoterpene chemistry, and particle uptake of RONO_2) in the GEOS-Chem global chemical transport model with  ∼  25  ×  25 km^2 resolution over North America. We evaluate the model using aircraft (SEAC^4RS) and ground-based (SOAS) observations of NO_x, BVOCs, and RONO_2 from the Southeast US in summer 2013. The updated simulation successfully reproduces the concentrations of individual gas- and particle-phase RONO_2 species measured during the campaigns. Gas-phase isoprene nitrates account for 25–50 % of observed RONO_2 in surface air, and we find that another 10 % is contributed by gas-phase monoterpene nitrates. Observations in the free troposphere show an important contribution from long-lived nitrates derived from anthropogenic VOCs. During both campaigns, at least 10 % of observed boundary layer RONO_2 were in the particle phase. We find that aerosol uptake followed by hydrolysis to HNO_3 accounts for 60 % of simulated gas-phase RONO_2 loss in the boundary layer. Other losses are 20 % by photolysis to recycle NO_x and 15 % by dry deposition. RONO_2 production accounts for 20 % of the net regional NO_x sink in the Southeast US in summer, limited by the spatial segregation between BVOC and NO_x emissions. This segregation implies that RONO_2 production will remain a minor sink for NO_x in the Southeast US in the future even as NO_x emissions continue to decline

Satb1 overexpression drives tumor-promoting activities in cancer-associated dendritic cells

Special AT-rich sequence-binding protein 1 (Satb1) governs genome-wide transcriptional programs. Using a conditional knockout mouse, we find that Satb1 is required for normal differentiation of conventional dendritic cells (DCs). Furthermore, Satb1 governs the differentiation of inflammatory DCs by regulating major histocompatibility complex class II (MHC II) expression through Notch1 signaling. Mechanistically, Satb1 binds to the Notch1 promoter, activating Notch expression and driving RBPJ occupancy of the H2-Ab1 promoter, which activates MHC II transcription. However, tumor-driven, unremitting expression of Satb1 in activated Zbtb46(+) inflammatory DCs that infiltrate ovarian tumors results in an immunosuppressive phenotype characterized by increased secretion of tumor-promoting Galectin-1 and IL-6. In vivo silencing of Satb1 in tumor-associated DCs reverses their tumorigenic activity and boosts protective immunity. Therefore, dynamic fluctuations in Satb1 expression govern the generation and immunostimulatory activity of steady-state and inflammatory DCs, but continuous Satb1 overexpression in differentiated DCs converts them into tolerogenic/pro-inflammatory cells that contribute to malignant progression.Fil: Tesone, Amelia J.. The Wistar Institute. Tumor Microenvironment and Metastasis Program; Estados UnidosFil: Rutkowski, Melanie R.. The Wistar Institute. Tumor Microenvironment and Metastasis Program; Estados UnidosFil: Brencicova, Eva. The Wistar Institute. Tumor Microenvironment and Metastasis Program; Estados UnidosFil: Svoronos, Nikolaos. The Wistar Institute. Tumor Microenvironment and Metastasis Program; Estados UnidosFil: Perales Puchal, Alfredo. The Wistar Institute. Tumor Microenvironment and Metastasis Program; Estados UnidosFil: Stephen, Tom L.. The Wistar Institute. Tumor Microenvironment and Metastasis Program; Estados UnidosFil: Allegrezza, Michael J.. The Wistar Institute. Tumor Microenvironment and Metastasis Program; Estados UnidosFil: Payne, Kyle K.. The Wistar Institute. Tumor Microenvironment and Metastasis Program; Estados UnidosFil: Nguyen, Jenny M.. The Wistar Institute. Tumor Microenvironment and Metastasis Program; Estados UnidosFil: Wickramasinghe, Jayamanna. The Wistar Institute. Center for Systems and Computational Biology; Estados UnidosFil: Tchou, Julia. University of Pennsylvania; Estados UnidosFil: Borowsky, Mark E.. Christiana Care Health System. Helen F. Graham Cancer Center; Estados UnidosFil: Rabinovich, Gabriel Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Kossenkov, Andrew V.. The Wistar Institute. Center for Systems and Computational Biology; Estados UnidosFil: Conejo Garcia, José R.. The Wistar Institute. Tumor Microenvironment and Metastasis Program; Estados Unido

From Entitlement to Experiment: Industry Report on Case Studies of high performing providers

The abstract is included in the text

The MBHBM Project-I: measurement of the central black hole mass in spiral galaxy NGC 3504 using molecular gas kinematics

We present a dynamical mass measurement of the supermassive black hole (SMBH) in the nearby double-barred spiral galaxy NGC 3504 as part of the Measuring Black Holes in below Milky Way (M sstarf) Mass Galaxies Project. Our analysis is based on Atacama Large Millimeter/submillimeter Array cycle 5 observations of the ${}^{12}\mathrm{CO}(2-1)$ emission line. These observations probe NGC 3504's circumnuclear gas disk (CND). Our dynamical model of the CND simultaneously constrains a black hole (BH) mass of ${1.6}_{-0.4}^{+0.6}\times {10}^{7}$ M ⊙, which is consistent with the empirical BH–galaxy scaling relations and a mass-to-light ratio in the H band of 0.44 ± 0.12 (M ⊙/${L}_{\odot }$). This measurement also relies on our new estimation of the distance to the galaxy of 32.4 ± 2.1 Mpc using the surface brightness fluctuation method, which is much further than the existing distance estimates. Additionally, our observations detect a central deficit in the ${}^{12}\mathrm{CO}(2-1)$ integrated intensity map with a diameter of 6.3 pc at the putative position of the SMBH. However, we find that a dense gas tracer CS(5 − 4) peaks at the galaxy center, filling in the 12CO(2 − 1)-attenuated hole. Holes like this one are observed in other galaxies, and our observations suggest these may be caused by changing excitation conditions rather than a true absence of molecular gas around the nucleus

Perineuronal Net Formation and the Critical Period for Neuronal Maturation in the Hypothalamic Arcuate Nucleus

In leptin-deficient ob/ob mice, obesity and diabetes are associated with abnormal development of neurocircuits in the hypothalamic arcuate nucleus (ARC)1, a critical brain area for energy and glucose homoeostasis2,3. Because this developmental defect can be remedied by systemic leptin administration, but only if given before postnatal day 28, a critical period for leptin-dependent development of ARC neurocircuits has been proposed4. In other brain areas, critical-period closure coincides with the appearance of perineuronal nets (PNNs), extracellular matrix specializations that restrict the plasticity of neurons that they enmesh5. Here we report that in humans and rodents, subsets of neurons in the mediobasal aspect of the ARC are enmeshed in PNN-like structures. In mice, these neurons are densely packed into a continuous ring that encircles the junction of the ARC and median eminence, which facilitates exposure of ARC neurons to the circulation. Most of the enmeshed neurons are both γ-aminobutyric acid-ergic and leptin-receptor positive, including a majority of Agouti-related-peptide neurons. Postnatal formation of the PNN-like structures coincides precisely with closure of the critical period for maturation of Agouti-related-peptide neurons and is dependent on input from circulating leptin, because postnatal ob/ob mice have reduced ARC PNN-like material that is restored by leptin administration during the critical period. We conclude that neurons crucial to metabolic homoeostasis are enmeshed in PNN-like structures and organized into a densely packed cluster situated circumferentially at the ARC–median eminence junction, where metabolically relevant humoral signals are sensed

Biomarker discovery for colon cancer using a 761 gene RT-PCR assay

<p>Abstract</p> <p>Background</p> <p>Reverse transcription PCR (RT-PCR) is widely recognized to be the gold standard method for quantifying gene expression. Studies using RT-PCR technology as a discovery tool have historically been limited to relatively small gene sets compared to other gene expression platforms such as microarrays. We have recently shown that TaqMan^®RT-PCR can be scaled up to profile expression for 192 genes in fixed paraffin-embedded (FPE) clinical study tumor specimens. This technology has also been used to develop and commercialize a widely used clinical test for breast cancer prognosis and prediction, the Onco <it>type</it>DX™ assay. A similar need exists in colon cancer for a test that provides information on the likelihood of disease recurrence in colon cancer (prognosis) and the likelihood of tumor response to standard chemotherapy regimens (prediction). We have now scaled our RT-PCR assay to efficiently screen 761 biomarkers across hundreds of patient samples and applied this process to biomarker discovery in colon cancer. This screening strategy remains attractive due to the inherent advantages of maintaining platform consistency from discovery through clinical application.</p> <p>Results</p> <p>RNA was extracted from formalin fixed paraffin embedded (FPE) tissue, as old as 28 years, from 354 patients enrolled in NSABP C-01 and C-02 colon cancer studies. Multiplexed reverse transcription reactions were performed using a gene specific primer pool containing 761 unique primers. PCR was performed as independent TaqMan^®reactions for each candidate gene. Hierarchal clustering demonstrates that genes expected to co-express form obvious, distinct and in certain cases very tightly correlated clusters, validating the reliability of this technical approach to biomarker discovery.</p> <p>Conclusion</p> <p>We have developed a high throughput, quantitatively precise multi-analyte gene expression platform for biomarker discovery that approaches low density DNA arrays in numbers of genes analyzed while maintaining the high specificity, sensitivity and reproducibility that are characteristics of RT-PCR. Biomarkers discovered using this approach can be transferred to a clinical reference laboratory setting without having to re-validate the assay on a second technology platform.</p