Total mutational load and clinical features as predictors of the metastatic status in lung adenocarcinoma and squamous cell carcinoma patients.

BACKGROUND: Recently, extensive cancer genomic studies have revealed mutational and clinical data of large cohorts of cancer patients. For example, the Pan-Lung Cancer 2016 dataset (part of The Cancer Genome Atlas project), summarises the mutational and clinical profiles of different subtypes of Lung Cancer (LC). Mutational and clinical signatures have been used independently for tumour typification and prediction of metastasis in LC patients. Is it then possible to achieve better typifications and predictions when combining both data streams? METHODS: In a cohort of 1144 Lung Adenocarcinoma (LUAD) and Lung Squamous Cell Carcinoma (LSCC) patients, we studied the number of missense mutations (hereafter, the Total Mutational Load TML) and distribution of clinical variables, for different classes of patients. Using the TML and different sets of clinical variables (tumour stage, age, sex, smoking status, and packs of cigarettes smoked per year), we built Random Forest classification models that calculate the likelihood of developing metastasis. RESULTS: We found that LC patients different in age, smoking status, and tumour type had significantly different mean TMLs. Although TML was an informative feature, its effect was secondary to the "tumour stage" feature. However, its contribution to the classification is not redundant with the latter; models trained using both TML and tumour stage performed better than models trained using only one of these variables. We found that models trained in the entire dataset (i.e., without using dimensionality reduction techniques) and without resampling achieved the highest performance, with an F1 score of 0.64 (95%CrI [0.62, 0.66]). CONCLUSIONS: Clinical variables and TML should be considered together when assessing the likelihood of LC patients progressing to metastatic states, as the information these encode is not redundant. Altogether, we provide new evidence of the need for comprehensive diagnostic tools for metastasis.

Beneficial effects of mineralocorticoid receptor blockade in experimental non-alcoholic steatohepatitis.

BACKGROUND: Therapeutic options to treat Non-alcoholic steatohepatitis (NASH) are limited. Mineralocorticoid receptor (MR) activation could play a role in hepatic fibrogenesis and its modulation could be beneficial for NASH. AIM: To investigate whether eplerenone, a specific MR antagonist, ameliorates liver damage in experimental NASH. METHODS: C57bl6 mice were fed a choline-deficient and amino acid-defined (CDAA) diet for 22 weeks with or without eplerenone supplementation. Serum levels of aminotransferases and aldosterone were measured and hepatic steatosis, inflammation and fibrosis scored histologically. Hepatic triglyceride content (HTC) and hepatic mRNA levels of pro-inflammatory pro-fibrotic, oxidative stress-associated genes and of MR were also assessed. RESULTS: CDAA diet effectively induced fibrotic NASH, and increased the hepatic expression of pro-inflammatory, pro-fibrotic and oxidative stress-associated genes. Hepatic MR mRNA levels significantly correlated with the expression of pro-inflammatory and pro-fibrotic genes and were significantly increased in hepatic stellate cells obtained from CDAA-fed animals. Eplerenone administration was associated to a reduction in histological steatosis and attenuation of liver fibrosis development, which was associated to a significant decrease in the expression of collagen-α1, collagen type III, alpha 1 and Matrix metalloproteinase-2. CONCLUSION: The expression of MR correlates with inflammation and fibrosis development in experimental NASH. Specific MR blockade with eplerenone has hepatic anti-steatotic and anti-fibrotic effects. These data identify eplerenone as a potential novel therapy for NASH. Considering its safety and FDA-approved status, human studies are warranted.

LINE-1 ORF1p as a candidate biomarker in high grade serous ovarian carcinoma.

Long interspersed element 1 (LINE-1) open reading frame 1 protein (ORF1p) expression is a common feature of many cancer types, including high-grade serous ovarian carcinoma (HGSOC). Here, we report that ORF1p is not only expressed but also released by ovarian cancer and primary tumor cells. Immuno-multiple reaction monitoring-mass spectrometry assays showed that released ORF1p is confidently detectable in conditioned media, ascites, and patients' plasma, implicating ORF1p as a potential biomarker. Interestingly, ORF1p expression is detectable in fallopian tube (FT) epithelial precursors of HGSOC but not in benign FT, suggesting that ORF1p expression in an early event in HGSOC development. Finally, treatment of FT cells with DNA methyltransferase inhibitors led to robust expression and release of ORF1p, validating the regulatory role of DNA methylation in LINE-1 repression in non-tumorigenic tissue.

Increase in ADAR1p110 activates the canonical Wnt signaling pathway associated with aggressive phenotype in triple negative breast cancer cells.

Triple-negative breast cancer (TNBC) represents a challenge in the search for new therapeutic targets. TNBCs are aggressive and generate resistance to chemotherapy. Tumors of TNBC patients with poor prognosis present a high level of adenosine deaminase acting on RNA1 (ADAR1). We explore the connection of ADAR1 with the canonical Wnt signaling pathway and the effect of modulation of its expression in TNBC. Expression data from cell line sequencing (DepMap) and TCGA samples were downloaded and analyzed. We lentivirally generated an MDA-MB-231 breast cancer cell line that overexpress (OE) ADAR1p110 or an ADAR knockdown. Abundance of different proteins related to Wnt/ß-catenin pathway and activity of nuclear ß-catenin were analyzed by Western blot and luciferase TOP/FOP reporter assay, respectively. Cell invasion was analyzed by matrigel assay. In mice, we study the behavior of tumors generated from ADAR1p110 (OE) cells and tumor vascularization immunostaining were analyzed. ADAR1 connects to the canonical Wnt pathway in TNBC. ADAR1p110 overexpression decreased GSK-3ß, while increasing active ß-catenin. It also increased the activity of nuclear ß-catenin and increased its target levels. ADAR1 knockdown has the opposite effect. MDA-MB-231 ADAR1 (OE) cells showed increased capacity of invasion. Subsequently, we observed that tumors derived from ADAR1p110 (OE) cells showed increased invasion towards the epithelium, and increased levels of Survivin and CD-31 expressed in vascular endothelial cells. These results indicate that ADAR1 overexpression alters the expression of some key components of the canonical Wnt pathway, favoring invasion and neovascularization, possibly through activation of the ß-catenin, which suggests an unknown role of ADAR1p110 in aggressiveness of TNBC tumors.

Immune-related IncRNA LINC00944 responds to variations in ADAR1 levels and it is associated with breast cancer prognosis.

AIMS: Breast cancer is one of the leading causes of woman deaths worldwide, being a major public health problem. It has been reported that the expression of the RNA-editing enzyme Adenosine Deaminase Acting on RNAs 1 (ADAR1) is upregulated in breast cancer, predicting poor prognosis in patients. A few reports in literature examine ADAR1 and long non-coding RNAs (lncRNAs) interplay in cancer and suggest key roles in cancer-related pathways. This study aimed to investigate whether ADAR1 could alter the expression levels of lncRNAs and explore how those changes are related to breast cancer biology. MAIN METHODS: ADAR1 overexpression and knockdown studies were performed in breast cancer cell lines to analyze the effects over lncRNAs expression. Guilt-by-Association correlation analysis of the TCGA-BRCA cohort was performed to predict the function of the lncRNA LINC00944. KEY FINDINGS: Here, we show that LINC00944 is responsive to ADAR1 up- and downregulation in breast cancer cells. We found that LINC00944 expression has a strong relationship with immune signaling pathways. Further assessment of the TCGA-BRCA cohort showed that LINC00944 expression was positively correlated to tumor-infiltrating T lymphocytes and pro-apoptotic markers. Moreover, we found that LINC00944 expression was correlated to the age at diagnosis, tumor size, and estrogen and progesterone receptor expression. Finally, we show that low expression of LINC00944 is correlated to poor prognosis in breast cancer patients. SIGNIFICANCE: Our study provides further evidence of the effect of ADAR1 over lncRNA expression levels, and on the participation of LINC00944 in breast cancer, suggesting to further investigate its potential role as prognostic biomarker.

Combinatorial immunotherapy induces tumor-infiltrating CD8+ T cells with distinct functional, migratory, and stem-like properties.

BACKGROUND: Programmed death (ligand) 1 (PD-(L)1) blockade and OX40/4-1BB costimulation have been separately evaluated in the clinic to elicit potent antitumor T cell responses. The precise mechanisms underlying single agent activity are incompletely understood. It also remains unclear if combining individual therapies leads to synergism, elicits novel immune mechanisms, or invokes additive effects. METHODS: We performed high-dimensional flow cytometry and single-cell RNA sequencing-based immunoprofiling of murine tumor-infiltrating lymphocytes (TILs) isolated from hosts bearing B16 or MC38 syngeneic tumors. This baseline infiltrate was compared to TILs after treatment with either anti-PD-(L)1, anti-OX40, or anti-4-1BB as single agents or as double and triple combinatorial therapies. Fingolimod treatment and CXCR3 blockade were used to evaluate the contribution of intratumoral versus peripheral CD8+ T cells to therapeutic efficacy. RESULTS: We identified CD8+ T cell subtypes with distinct functional and migratory signatures highly predictive of tumor rejection upon treatment with single agent versus combination therapies. Rather than reinvigorating terminally exhausted CD8+ T cells, OX40/4-1BB agonism expanded a stem-like PD-1loKLRG-1+Ki-67+CD8+ T cell subpopulation, which PD-(L)1 blockade alone did not. However, PD-(L)1 blockade synergized with OX40/4-1BB costimulation by dramatically enhancing stem-like TIL presence via a CXCR3-dependent mechanism. CONCLUSIONS: Our findings provide new mechanistic insights into the interplay between components of combinatorial immunotherapy, where agonism of select costimulatory pathways seeds a pool of stem-like CD8+ T cells more responsive to immune checkpoint blockade (ICB).

Design of an orally efficacious hydroxyethylamine (HEA) BACE-1 inhibitor in a preclinical animal model.

In this Letter, we describe our efforts to design HEA BACE-1 inhibitors that are highly permeable coupled with negligible levels of permeability-glycoprotein activity. These efforts culminate in producing 16 which lowers Αß by 28% and 32% in the cortex and CSF, respectively, in the preclinical wild type Hartley guinea pig animal model when dosed orally at 30mpk BID for 2.5days.

ADAR1 Transcriptome editing promotes breast cancer progression through the regulation of cell cycle and DNA damage response.

RNA editing has emerged as a novel mechanism in cancer progression. The double stranded RNA-specific adenosine deaminase (ADAR) modifies the expression of an important proportion of genes involved in cell cycle control, DNA damage response (DDR) and transcriptional processing, suggesting an important role of ADAR in transcriptome regulation. Despite the phenotypic implications of ADAR deregulation in several cancer models, the role of ADAR on DDR and proliferation in breast cancer has not been fully addressed. Here, we show that ADAR expression correlates significantly with clinical outcomes and DDR, cell cycle and proliferation mRNAs of previously reported edited transcripts in breast cancer patients. ADAR's knock-down in a breast cancer cell line produces stability changes of mRNAs involved in DDR and DNA replication. Breast cancer cells with reduced levels of ADAR show a decreased viability and an increase in apoptosis, displaying a significant decrease of their DDR activation, compared to control cells. These results suggest that ADAR plays an important role in breast cancer progression through the regulation of mRNA stability and expression of those genes involved in proliferation and DDR impacting the viability of breast cancer cells.

Ligand-Blocking and Membrane-Proximal Domain Targeting Anti-OX40 Antibodies Mediate Potent T Cell-Stimulatory and Anti-Tumor Activity.

Agonistic antibodies targeting the tumor necrosis factor (TNF) superfamily of co-stimulatory receptors (TNFRSF) are progressing through various stages of clinical development for cancer treatment, but the desired and defining features of these agents for optimal biological activity remain controversial. One idea, based on recent studies with CD40, is that non-ligand-blocking antibodies targeting membrane-distal cysteine-rich domain 1 (CRD1) have superior agonistic activities compared with ligand-blocking antibodies targeting more membrane-proximal CRDs. Here, we determined the binding and functional characteristics of a panel of antibodies targeting CRDs 1-4 of OX40 (also known as TNFRSF4 or CD134). In striking contrast to CD40, we found that ligand-blocking CRD2-binding and membrane-proximal CRD4-binding anti-OX40 antibodies have the strongest agonistic and anti-tumor activities. These findings have important translational implications and further highlight that the relationship between epitope specificity and agonistic activity will be an important issue to resolve on a case-by-case basis when optimizing antibodies targeting different co-stimulatory tumor necrosis factor receptors (TNFRs).

Partial ablation of the orexin field induces a sub-narcoleptic phenotype in a conditional mouse model of orexin neurodegeneration.

Narcolepsy type 1 (Na-1) and 2 (Na-2) are characterized by an inability to sustain wakefulness and are likely caused by degeneration of orexin neurons. Near complete orexin neurodegeneration depletes orexin-A from the cerebrospinal fluid and produces Na-1. The pathophysiology of Na-2 is less understood but has been hypothesized to be due to less extensive loss of orexin neurotransmission. The orexin-tTA; TetO diphtheria toxin A mouse allows conditional control over the extent and timing of orexin neurodegeneration. To evaluate partial ablation of the orexin field as a model of Na-2, orexin-A positive cell counts and sleep/wake phenotypes (determined by piezoelectric monitoring) were correlated within individual mice after different protocols of diet-controlled neurodegeneration. Partial ablations that began during the first 8 days of study were 14% larger than partial ablations induced during the last 8 days of study, 6 weeks later and prior to sacrifice of all mice, suggesting orexin-A positive cell death continued despite the resumption of conditions intended to keep orexin neurons intact. Sleep/wake of mice with 71.0% orexin-A positive cell loss, initiated at the beginning of study, resembled that of orexin-intact controls more than mice with near complete neurodegeneration. Conversely, mice with 56.6% orexin-A positive cell loss, created at the end of study, had sleep/wake phenotypes that were similar to those of mice with near complete orexin-A positive cell loss. Collectively, these results suggest that compensatory wake-promotion develops in mice that have some critical portion of their orexinergic system remaining after partial ablation.

Constitutive activity of the melanocortin-4 receptor is maintained by its N-terminal domain and plays a role in energy homeostasis in humans.

The melanocortin-4 receptor (MC4R), a centrally expressed G protein-coupled receptor (GPCR), is essential for the maintenance of long-term energy balance in humans. Mutations in MC4R are the most common genetic cause of obesity. Since activation of this receptor leads to a decrease in food intake, MC4R is also a major therapeutic target for the treatment of obesity. Control of MC4R activity in vivo is modulated by the opposing effects of the anorexigenic agonist alpha-melanocyte-stimulating hormone (alpha-MSH) and the orexigenic antagonist agouti-related protein (AGRP). In addition, experiments in vitro have demonstrated that the human MC4R has an intrinsic constitutive activity on which AGRP also acts as an inverse agonist. The physiological role of this constitutive activity in the control of energy balance as well as the domain of the protein implicated in its maintenance are unknown. By systematically studying functional defects in naturally occurring MC4R mutations from obese patients, we defined a cluster of N-terminal mutations that selectively impair the constitutive activity of the receptor. Further characterization of this domain demonstrated that it functions as a tethered intramolecular ligand that maintains the constitutive activity of MC4R and may provide novel avenues for the design of drugs targeting this receptor. Our results also suggest that the tonic satiety signal provided by the constitutive activity of MC4R may be required for maintaining long-term energy homeostasis in humans.

Discovery of (R)-4-cyclopropyl-7,8-difluoro-5-(4-(trifluoromethyl)phenylsulfonyl)-4,5-dihydro-1H-pyrazolo[4,3-c]quinoline (ELND006) and (R)-4-cyclopropyl-8-fluoro-5-(6-(trifluoromethyl)pyridin-3-ylsulfonyl)-4,5-dihydro-2H-pyrazolo[4,3-c]quinoline (ELND007): metabolically stable γ-secretase Inhibitors that selectively inhibit the production of amyloid-ß over Notch.

Herein, we describe our strategy to design metabolically stable γ-secretase inhibitors which are selective for inhibition of Aß generation over Notch. We highlight our synthetic strategy to incorporate diversity and chirality. Compounds 30 (ELND006) and 34 (ELND007) both entered human clinical trials. The in vitro and in vivo characteristics for these two compounds are described. A comparison of inhibition of Aß generation in vivo between 30, 34, Semagacestat 41, Begacestat 42, and Avagacestat 43 in mice is made. 30 lowered Aß in the CSF of healthy human volunteers.

Polo-like kinase 2 (PLK2) phosphorylates alpha-synuclein at serine 129 in central nervous system.

Several neurological diseases, including Parkinson disease and dementia with Lewy bodies, are characterized by the accumulation of alpha-synuclein phosphorylated at Ser-129 (p-Ser-129). The kinase or kinases responsible for this phosphorylation have been the subject of intense investigation. Here we submit evidence that polo-like kinase 2 (PLK2, also known as serum-inducible kinase or SNK) is a principle contributor to alpha-synuclein phosphorylation at Ser-129 in neurons. PLK2 directly phosphorylates alpha-synuclein at Ser-129 in an in vitro biochemical assay. Inhibitors of PLK kinases inhibited alpha-synuclein phosphorylation both in primary cortical cell cultures and in mouse brain in vivo. Finally, specific knockdown of PLK2 expression by transduction with short hairpin RNA constructs or by knock-out of the plk2 gene reduced p-Ser-129 levels. These results indicate that PLK2 plays a critical role in alpha-synuclein phosphorylation in central nervous system.

Engineering the melanocortin-4 receptor to control constitutive and ligand-mediated G(S) signaling in vivo.

The molecular and functional diversity of G protein-coupled receptors is essential to many physiological processes. However, this diversity presents a significant challenge to understanding the G protein-mediated signaling events that underlie a specific physiological response. To increase our understanding of these processes, we sought to gain control of the timing and specificity of G(s) signaling in vivo. We used naturally occurring human mutations to develop two G(s)-coupled engineered receptors that respond solely to a synthetic ligand (RASSLs). Our G(s)-coupled RASSLs are based on the melanocortin-4 receptor, a centrally expressed receptor that plays an important role in the regulation of body weight. These RASSLs are not activated by the endogenous hormone alpha-melanocyte-stimulating hormone but respond potently to a selective synthetic ligand, tetrahydroisoquinoline. The RASSL variants reported here differ in their intrinsic basal activities, allowing the separation of the effects of basal signaling from ligand-mediated activation of the G(s) pathway in vivo. These RASSLs can be used to activate G(s) signaling in any tissue, but would be particularly useful for analyzing downstream events that mediate body weight regulation in mice. Our study also demonstrates the use of human genetic variation for protein engineering.