Equivalent benefit of mTORC1 blockade and combined PI3K-mTOR blockade in a mouse model of tuberous sclerosis

<p>Abstract</p> <p>Background</p> <p>Tuberous sclerosis (TSC) is a hamartoma syndrome in which renal and lung tumors cause the greatest morbidity. Loss of either TSC1 or TSC2 in TSC hamartomas leads to activation of mTORC1 and suppression of AKT. Recent studies indicate that inhibition of mTORC1 with RAD001 (everolimus) leads to rebound activation of AKT, which could protect tumors from drug-induced cell death. Here we examine the potential benefit of inhibition of both mTOR and AKT signaling in a mouse model of TSC, using a dual pan class I PI3K/mTOR catalytic small molecule inhibitor NVP-BEZ235.</p> <p>Results</p> <p>Using ENU to enhance <it>Tsc2</it>^+-kidney tumor development, both RAD001 (10 mg/kg PO 5 d/week) and NVP-BEZ235 (45 mg/kg PO QD) had equivalent effects in suppressing tumor development during a 4 week treatment period, with a 99% reduction in tumor cell mass. Marked reduction in activation of mTORC1, induction of cell cycle arrest, and absence of apoptotic cell death was seen in mice treated with either drug. However, when either was discontinued, there was prompt recovery of tumor growth, with extensive proliferation.</p> <p>Conclusion</p> <p>Both mTORC1 blockade alone and combined PI3K-mTOR blockade lead to suppression of tumor development but not tumor elimination in this TSC model.</p

Response of a neuronal model of tuberous sclerosis to mammalian target of rapamycin (mTOR) inhibitors: Effects on mTORC1 and Akt signaling lead to improved survival and function

Tuberous sclerosis (TSC) is a hamartoma syndrome due to mutations in either TSC1 or TSC2 in which brain involvement causes epilepsy, mental retardation, and autism. We have recently reported (Meikle et al., J Neurosci 2007) a mouse neuronal model of TSC in which Tsc1 is ablated in most neurons during cortical development. We have tested rapamycin and RAD001, both mTORC1 inhibitors, as potential therapeutic agents in this model. Median survival is improved from 33 days to over 100 days; behavior, phenotype, and weight gain are all also markedly improved. There is brain penetration of both drugs, with accumulation over time with repetitive treatment, and effective reduction of levels of phospho-S6, a downstream target of mTORC1. In addition, there is restoration of phospho-Akt and phospho-GSK3 levels in the treated mice, consistent with restoration of Akt function. Neurofilament abnormalities, myelination, and cell enlargement are all improved by the treatment. However, dysplastic neuronal features persist, and there are only modest changes in dendritic spine density and length. Strikingly, mice treated with rapamycin or RAD001 for 23 days only (P7 — P30) displayed a persistent improvement in phenotype with median survival of 78 days. In summary, rapamycin/RAD001 are highly effective therapies for this neuronal model of TSC, with benefit apparently due to effects on mTORC1 and Akt signaling, and consequently cell size and myelination. Although caution is appropriate, the results suggest the possibility that rapamycin/RAD001 may have benefit in the treatment of TSC brain disease, including infantile spasms

Anti-PD-1 monoclonal antibody MEDI0680 in a phase I study of patients with advanced solid malignancies.

BACKGROUND: The safety, efficacy, pharmacokinetics, and pharmacodynamics of the anti-programmed cell death-1 antibody MEDI0680 were evaluated in a phase I, multicenter, dose-escalation study in advanced solid malignancies. METHODS: MEDI0680 was administered intravenously once every 2 weeks (Q2W) or once every 3 weeks at 0.1, 0.5, 2.5, 10 or 20 mg/kg. Two cohorts received 20 mg/kg once a week for 2 or 4 weeks, then 20 mg/kg Q2W. All were treated for 12 months or until progression. The primary endpoint was safety. Secondary endpoints were efficacy and pharmacokinetics. Exploratory endpoints included pharmacodynamics. RESULTS: Fifty-eight patients were treated. Median age was 62.5 years and 81% were male. Most had kidney cancer (n = 36) or melanoma (n = 9). There were no dose-limiting toxicities. Treatment-related adverse events occurred in 83% and were grade ≥ 3 in 21%. Objective clinical responses occurred in 8/58 patients (14%): 5 with kidney cancer, including 1 with a complete response, and 3 with melanoma. The relationship between dose and serum levels was predictable and linear, with apparent receptor saturation at 10 mg/kg Q2W and all 20 mg/kg cohorts. CONCLUSIONS: MEDI0680 induced peripheral T-cell proliferation and increased plasma IFNγ and associated chemokines regardless of clinical response. CD8+ T-cell tumor infiltration and tumoral gene expression of IFNG, CD8A, CXCL9, and granzyme K (GZMK) were also increased following MEDI0680 administration. TRIAL REGISTRATION: NCT02013804 ; date of registration December 12, 2013

The James Webb Space Telescope Mission

Twenty-six years ago a small committee report, building on earlier studies, expounded a compelling and poetic vision for the future of astronomy, calling for an infrared-optimized space telescope with an aperture of at least $4m$. With the support of their governments in the US, Europe, and Canada, 20,000 people realized that vision as the $6.5m$ James Webb Space Telescope. A generation of astronomers will celebrate their accomplishments for the life of the mission, potentially as long as 20 years, and beyond. This report and the scientific discoveries that follow are extended thank-you notes to the 20,000 team members. The telescope is working perfectly, with much better image quality than expected. In this and accompanying papers, we give a brief history, describe the observatory, outline its objectives and current observing program, and discuss the inventions and people who made it possible. We cite detailed reports on the design and the measured performance on orbit.Comment: Accepted by PASP for the special issue on The James Webb Space Telescope Overview, 29 pages, 4 figure

Cellular Size as a Means of Tracking mTOR Activity and Cell Fate of CD4+ T Cells upon Antigen Recognition

<div><p>mTOR is a central integrator of metabolic and immunological stimuli, dictating immune cell activation, proliferation and differentiation. In this study, we demonstrate that within a clonal population of activated T cells, there exist both mTOR^hi and mTOR^lo cells exhibiting highly divergent metabolic and immunologic functions. By taking advantage of the role of mTOR activation in controlling cellular size, we demonstrate that upon antigen recognition, mTOR^hi CD4+ T cells are destined to become highly glycolytic effector cells. Conversely, mTOR^lo T cells preferentially develop into long-lived cells that express high levels of Bcl-2, CD25, and CD62L. Furthermore, mTOR^lo T cells have a greater propensity to differentiate into suppressive Foxp3+ T regulatory cells, and this paradigm was also observed in human CD4+ T cells. Overall, these studies provide the opportunity to track the development of effector and memory T cells from naïve precursors, as well as facilitate the interrogation of immunologic and metabolic programs that inform these fates.</p></div

Cellular size (indicative of mTORC1 activity) influences the phenotype of human CD4+ T cells similar to mouse.

<p>CD4+ T cells were purified from the PBMCs of 8 human volunteers. Cells were stimulated with human anti-CD3 and anti-CD28 activator beads overnight. <b>a)</b> 24hrs after stimulation, mTORC1 activity was assessed by the pS6 expression of the 15% largest and smallest activated (HLA-DR+) CD4+ T cells. Histograms in the lower FACs plot were determined from the ‘big’ and ‘small’ gates shown in the upper plot. The pS6 MFI is shown in the upper corner of the FACs plot. <b>b-c)</b> 24hrs after stimulation, the cells were sorted from the 15% largest and smallest activated (CD38+) CD4+ T cells. Sorted populations were cultured in media supplemented with human IL-2 for 72 hrs. After 72 hrs, the phenotype of the large ‘mTOR^hi’ and small ‘mTOR^lo’ populations was assessed by flow cytometry. <b>b)</b> Human T-reg populations were determined from the CD127^lo CD25^hi gating strategy. <b>c</b>) FACs plots show percentage of Foxp3+ CTLA-4+ double positive cells gated from the ‘T-reg’ gate in b. Statistics are shown to the right. The connecting lines demonstrate the changes in percentages between the mTOR^hi and mTOR^lo sorted populations from the same individual. The data are representative of 8 distinct human samples.</p

mTORC1 activity is not required for antigenic recognition.

<p><b>a-b)</b> WT, T-Raptor^–/—and T-TSC2^–/—OT-II+ splenocytes were stimulated with OVA^323–339 for 24hrs. <b>a)</b> mTORC1 activity, as assessed by pS6^S240/244 expression was measured from the CD4+ population of each genotype. The bar graph below displays the MFI ± standard deviation (S.D.) of pS6 per genotype represented in above histogram plot. <b>b)</b> Expression of activation marker CD69, and homing marker, CD62L, are shown from the CD4+ population of each genotype. Below bar graphs indicate MFI of each molecule of interest from 3 independent experiments. <b>c)</b> 5c.c7 Rag^–/—CD4+ T cells were stimulated with PCC for 24hrs and expression of CD69 and CD62L was assessed from the 10% smallest and largest populations of activated CD4+ cells. Expression was compared against non-activated 5c.c7 Rag^–/—CD4+ T cells (CD4+ CD69–). Bar graphs below represent MFI ± S.D. of each molecule of interest from 3 independent experiments. The data are representative of (a,c) and/or are a composite of 3 independent experiments (b,c).</p

Foxp3+ CD4+ T cells generated from mTOR^hi and mTOR^lo sorted populations have distinct phenotypes.

<p>Splenocytes from 5c.c7 Rag^–/—TCR transgenic mice were stimulated with 0.5ug/ml PCC peptide for 24hrs, and then sorted into CD4+CD69+ populations with FSC/SSC “big” mTOR^hi or FSC/SSC “small” mTOR^lo profiles. Cells were cultured in media supplemented with IL-2 for 96hrs, and analyzed by flow cytometry. <b>a)</b> The percentage of CD4+ Foxp3+ cells was determined from each population. <b>b)</b> Histograms show shifts in MFI of markers associated with a T-reg phenotype. Populations were gated from the Foxp3+ or Foxp3- cells of the mTOR^hi or mTOR^lo sorted samples. The MFI of each protein is shown in the top corner of each FACs plot. The data are representative of at least 3 independent experiments.</p