Comprehensive Characterization of Human Lung Large Cell Carcinoma Identifies Transcriptomic Signatures with Potential Implications in Response to Immunotherapy.

Lung cancer is the leading cause of cancer mortality worldwide, with non-small cell lung cancer (NSCLC) being the most prevalent histology. While immunotherapy with checkpoint inhibitors has shown outstanding results in NSCLC, the precise identification of responders remains a major challenge. Most studies attempting to overcome this handicap have focused on adenocarcinomas or squamous cell carcinomas. Among NSCLC subtypes, the molecular and immune characteristics of lung large cell carcinoma (LCC), which represents 10% of NSCLC cases, are not well defined. We hypothesized that specific molecular aberrations may impact the immune microenvironment in LCC and, consequently, the response to immunotherapy. To that end, it is particularly relevant to thoroughly describe the molecular genotype-immunophenotype association in LCC-to identify robust predictive biomarkers and improve potential benefits from immunotherapy. We established a cohort of 18 early-stage, clinically annotated, LCC cases. Their molecular and immune features were comprehensively characterized by genomic and immune-targeted sequencing panels along with immunohistochemistry of immune cell populations. Unbiased clustering defined two novel subgroups of LCC. Pro-immunogenic tumors accumulated certain molecular alterations, showed higher immune infiltration and upregulated genes involved in potentiating immune responses when compared to pro-tumorigenic samples, which favored tumoral progression. This classification identified a set of biomarkers that could potentially predict response to immunotherapy. These results could improve patient selection and expand potential benefits from immunotherapy.

Tumor mutational burden assessment in non-small-cell lung cancer samples: results from the TMB2 harmonization project comparing three NGS panels.

BACKGROUND: Tumor mutational burden (TMB) is a recently proposed predictive biomarker for immunotherapy in solid tumors, including non-small cell lung cancer (NSCLC). Available assays for TMB determination differ in horizontal coverage, gene content and algorithms, leading to discrepancies in results, impacting patient selection. A harmonization study of TMB assessment with available assays in a cohort of patients with NSCLC is urgently needed. METHODS: We evaluated the TMB assessment obtained with two marketed next generation sequencing panels: TruSight Oncology 500 (TSO500) and Oncomine Tumor Mutation Load (OTML) versus a reference assay (Foundation One, FO) in 96 NSCLC samples. Additionally, we studied the level of agreement among the three methods with respect to PD-L1 expression in tumors, checked the level of different immune infiltrates versus TMB, and performed an inter-laboratory reproducibility study. Finally, adjusted cut-off values were determined. RESULTS: Both panels showed strong agreement with FO, with concordance correlation coefficients (CCC) of 0.933 (95% CI 0.908 to 0.959) for TSO500 and 0.881 (95% CI 0.840 to 0.922) for OTML. The corresponding CCCs were 0.951 (TSO500-FO) and 0.919 (OTML-FO) in tumors with <1% of cells expressing PD-L1 (PD-L1<1%; N=55), and 0.861 (TSO500-FO) and 0.722 (OTML-FO) in tumors with PD-L1≥1% (N=41). Inter-laboratory reproducibility analyses showed higher reproducibility with TSO500. No significant differences were found in terms of immune infiltration versus TMB. Adjusted cut-off values corresponding to 10 muts/Mb with FO needed to be lowered to 7.847 muts/Mb (TSO500) and 8.380 muts/Mb (OTML) to ensure a sensitivity >88%. With these cut-offs, the positive predictive value was 78.57% (95% CI 67.82 to 89.32) and the negative predictive value was 87.50% (95% CI 77.25 to 97.75) for TSO500, while for OTML they were 73.33% (95% CI 62.14 to 84.52) and 86.11% (95% CI 74.81 to 97.41), respectively. CONCLUSIONS: Both panels exhibited robust analytical performances for TMB assessment, with stronger concordances in patients with negative PD-L1 expression. TSO500 showed a higher inter-laboratory reproducibility. The cut-offs for each assay were lowered to optimal overlap with FO.

Clustering of Drosophila housekeeping promoters facilitates their expression.

Housekeeping genes of animal genomes cluster in the same chromosomal regions. It has long been suggested that this organization contributes to their steady expression across all the tissues of the organism. Here, we show that the activity of Drosophila housekeeping gene promoters depends on the expression of their neighbors. By measuring the expression of ∼85,000 reporters integrated in Kc167 cells, we identified the best predictors of expression as chromosomal contacts with the promoters and terminators of active genes. Surprisingly, the chromatin composition at the insertion site and the contacts with enhancers were less informative. These results are substantiated by the existence of genomic "paradoxical" domains, rich in euchromatic features and enhancers, but where the reporters are expressed at low level, concomitant with a deficit of interactions with promoters and terminators. This indicates that the proper function of housekeeping genes relies not on contacts with long distance enhancers but on spatial clustering. Overall, our results suggest that spatial proximity between genes increases their expression and that the linear architecture of the Drosophila genome contributes to this effect.

Functional characterization of human cancer-derived TRKB mutations.

Cancer originates from cells that have acquired mutations in genes critical for controlling cell proliferation, survival and differentiation. Often, tumors continue to depend on these so-called driver mutations, providing the rationale for targeted anticancer therapies. To date, large-scale sequencing analyses have revealed hundreds of mutations in human tumors. However, without their functional validation it remains unclear which mutations correspond to driver, or rather bystander, mutations and, therefore, whether the mutated gene represents a target for therapeutic intervention. In human colorectal tumors, the neurotrophic receptor TRKB has been found mutated on two different sites in its kinase domain (TRKB(T695I) and TRKB(D751N)). Another site, in the extracellular part of TRKB, is mutated in a human lung adenocarcinoma cell line (TRKB(L138F)). Lastly, our own analysis has identified one additional TRKB point mutation proximal to the kinase domain (TRKB(P507L)) in a human melanoma cell line. The functional consequences of all these point mutations, however, have so far remained elusive. Previously, we have shown that TRKB is a potent suppressor of anoikis and that TRKB-expressing cells form highly invasive and metastatic tumors in nude mice. To assess the functional consequences of these four TRKB mutations, we determined their potential to suppress anoikis and to form tumors in nude mice. Unexpectedly, both colon cancer-derived mutants, TRKB(T695I) and TRKB(D751N), displayed reduced activity compared to that of wild-type TRKB. Consistently, upon stimulation with the TRKB ligand BDNF, these mutants were impaired in activating TRKB and its downstream effectors AKT and ERK. The two mutants derived from human tumor cell lines (TRKB(L138F) and TRKB(P507L)) were functionally indistinguishable from wild-type TRKB in both in-vitro and in-vivo assays. In conclusion, we fail to detect any gain-of-function of four cancer-derived TRKB point mutations.

Using multiplexed regulation of luciferase activity and GFP translocation to screen for FOXO modulators.

BACKGROUND: Independent luciferase reporter assays and fluorescent translocation assays have been successfully used in drug discovery for several molecular targets. We developed U2transLUC, an assay system in which luciferase and fluorescent read-outs can be multiplexed to provide a powerful cell-based high content screening method. RESULTS: The U2transLUC system is based on a stable cell line expressing a GFP-tagged FOXO transcription factor and a luciferase reporter gene under the control of human FOXO-responsive enhancers. The U2transLUC assay measures nuclear-cytoplasmic FOXO shuttling and FOXO-driven transcription, providing a means to analyze these two key features of FOXO regulation in the same experiment. We challenged the U2transLUC system with chemical probes with known biological activities and we were able to identify compounds with translocation and/or transactivation capacity. CONCLUSION: Combining different biological read-outs in a single cell line offers significant advantages over conventional cell-based assays. The U2transLUC assay facilitates the maintenance and monitoring of homogeneous FOXO transcription factor expression and allows the reporter gene activity measured to be normalized with respect to cell viability. U2transLUC is suitable for high throughput screening and can identify small molecules that interfere with FOXO signaling at different levels.

Chemical genetic analysis of FOXO nuclear-cytoplasmic shuttling by using image-based cell screening.

FOXO proteins are direct targets of PI3K/Akt signaling and they integrate the signals of several other transduction pathways at the transcriptional level. FOXO transcription factors are involved in normal cell homeostasis and neoplasia, and they are regulated by multiple post-transcriptional modifications. In cancer research, the regulation of the FOXO factors is receiving increasing attention as their activation has been linked to cell-cycle arrest and apoptosis. Hence, FOXO proteins have been proposed to act as tumor suppressors. Here, we applied a chemical biology approach to study the mechanisms that influence the intracellular localization of the FOXO family member FOXO3a. We established a high-throughput cellular-imaging assay that monitors the nuclear-cytoplasmic translocation of a GFP-FOXO3a fusion protein in tumor cells. Nuclear accumulation of fluorescent signals upon treatment with the known PI3K inhibitors LY294002, wortmannin, PIK-75, and PI-103 was dose dependent and agreed well with the IC(50) values reported for PI3Kalpha inhibition in vitro. Additionally, we identified 17 compounds from a panel of 73 low-molecular-weight compounds capable of inducing the nuclear accumulation of GFP-FOXO. These compounds include chemicals known to interfere with components of the PI3K/Akt signaling pathway, as well as with nuclear export and Ca(2+)/calmodulin (CaM)-dependent signaling events. Interestingly, the therapeutic agent vinblastine induced efficient nuclear translocation of the FOXO reporter protein. Our data illustrate the potential of chemical genetics when combined with robust and sensitive high-content-screening technology.

A dual-color fluorescence-based platform to identify selective inhibitors of Akt signaling.

BACKGROUND: Inhibition of Akt signaling is considered one of the most promising therapeutic strategies for many cancers. However, rational target-orientated approaches to cell based drug screens for anti-cancer agents have historically been compromised by the notorious absence of suitable control cells. METHODOLOGY/PRINCIPAL FINDINGS: In order to address this fundamental problem, we have developed BaFiso, a live-cell screening platform to identify specific inhibitors of this pathway. BaFiso relies on the co-culture of isogenic cell lines that have been engineered to sustain interleukin-3 independent survival of the parental Ba/F3 cells, and that are individually tagged with different fluorescent proteins. Whilst in the first of these two lines cell survival in the absence of IL-3 is dependent on the expression of activated Akt, the cells expressing constitutively-activated Stat5 signaling display IL-3 independent growth and survival in an Akt-independent manner. Small molecules can then be screened in these lines to identify inhibitors that rescue IL-3 dependence. CONCLUSIONS/SIGNIFICANCE: BaFiso measures differential cell survival using multiparametric live cell imaging and permits selective inhibitors of Akt signaling to be identified. BaFiso is a platform technology suitable for the identification of small molecule inhibitors of IL-3 mediated survival signaling.

MAP17 inhibits Myc-induced apoptosis through PI3K/AKT pathway activation.

MAP17 is a non-glycosylated membrane-associated protein that has been shown to be over-expressed in human carcinomas, suggesting a possible role of this protein in tumorigenesis. However, very little is known about the molecular mechanism mediating the possible tumor promoting properties of MAP17. To analyze the effect of MAP17 on cell survival, we used Rat1 fibroblasts model where Myc over-expression promotes apoptosis in low serum conditions. In the present work, we report that over-expression of MAP17 protects Rat1a fibroblasts from Myc-induced apoptosis through reactive oxygen species (ROS)-mediated activation of the PI3K/AKT signaling pathway. MAP17-mediated survival was associated with absence of Bax translocation to the mitochondria and reduced caspase-3 activation. We show that a fraction of PTEN undergoes oxidation in MAP17-over-expressing cells. Furthermore, activation of AKT by MAP17 as measured by Thr308 phosphorylation was independent of PI3K activity. Importantly, modulation of ROS by antioxidant treatment prevented activation of AKT, restoring the level of apoptosis in serum-starved Rat1/c-Myc fibroblasts. Finally, over-expression of a dominant-negative mutant of AKT in MAP17-expressing clones makes them sensitive to serum depletion. Our data indicate that MAP17 protein activates AKT through ROS and this is determinant to confer resistance to Myc-induced apoptosis in the absence of serum.

An HTS approach to screen for antagonists of the nuclear export machinery using high content cell-based assays.

Intracellular localization is essential for the regulated activity of many signaling molecules associated with disease-relevant pathways. High content screening is a powerful technology to monitor the impact of small molecules or interfering RNAs on translocation of proteins within intact cells. Several assays have been developed to measure the nucleocytoplasmic shuttling of proteins like nuclear factor kappaB, FoxO, or nuclear factor of activated T-cells involved in distinct signaling networks. However, since all these proteins bear a leucine-rich nuclear export signal (NES), modulators of the NES-dependent export machinery can lead to misinterpretation of the assay readout. Here we report the generation of U2nesRELOC, a cell-based system for the identification of nuclear export inhibitors and specific silencers of the nuclear export machinery, and its adaptation to high throughput screening. The assay is based on mammalian cells stably expressing green fluorescent protein (GFP)-labeled Rev protein, which contains a strong heterologous NES. The fluorescent signal of untreated U2nesRELOC cells localizes exclusively to the cytoplasm. Upon treatment with the nuclear export inhibitor leptomycin B the GFP-labeled reporter protein accumulates rapidly in the cell nucleus. The assay has been adapted to 96-multiwell format and fully automated. Pilot experiments with a panel of 50 test compounds using three different concentrations per compound resulted in very consistent data sets with excellent reproducibility and an average Z' value of 0.76. In summary, U2nesRELOC is a cell-based nuclear export assay suitable for high throughput screening, providing counterscreens for pathway deconvolution.

Albumin prevents mitochondrial depolarization and apoptosis elicited by endoplasmic reticulum calcium depletion of neuroblastoma cells.

Serum albumin protects against cell death elicited by various cytotoxic agents; however, conflicting views on the protective mechanism still remain. Hence, we have studied the ability of serum albumin to prevent apoptosis of human neuroblastoma SH-SY 5 Y cells elicited by four compounds known to release Ca(2+) from the endoplasmic reticulum, i.e. dotarizine, flunarizine, thapsigargin and cyclopiazonic acid. Spontaneous basal apoptosis, after 24 h incubation in Dulbecco's Modified Eagle Medium (DMEM) containing 10% serum, was 5%. Dotarizine (30--50 microM) enhanced basal apoptosis to 18--43%, flunarizine (30--50 microM) to 15%, thapsigargin (1--10 microM) to 21--35%, and cyclopiazonic acid (100 microM) to 10%. Serum deprivation augmented basal apoptosis to 20%. Under serum-free medium, 30 microM dotarizine or flunarizine drastically enhanced apoptosis to 63% and 68%, respectively; the increase was milder with 1 microM thapsigargin (37%) and 30 microM cyclopiazonic acid (27%). In serum-free medium, albumin (29 or 49 mg/ml) fully prevented the apoptotic effects of dotarizine, flunarizine and cyclopiazonic acid. The four compounds increased the cytosolic Ca(2+) concentration ([Ca(2+)](c)) in fluo-4 loaded cells; such increase developed slowly to reach a plateau after several minutes, followed by a slow decline. Albumin did not modify the kinetic parameters of such increase. In the absence of serum, dotarizine, flunarizine, thapsigargin, and cyclopiazonic acid caused mitochondrial depolarization in tetramethylrhodamine ethyl ester (TMRE)-loaded cells; depolarization was inhibited by cytoprotective concentrations of albumin. These results suggest that albumin protects cells from entering into apoptosis by preventing mitochondrial depolarization. They also suggest that inhibition of mitochondrial depolarization might become a target to develop new anti-apoptotic compounds with therapeutic neuroprotective potential in stroke, Alzheimer's disease, and other neurodegenerative diseases.

Membrane localization of all class I PI 3-kinase isoforms suppresses c-Myc-induced apoptosis in Rat1 fibroblasts via Akt.

Phosphoinositide 3'-kinases (PI3Ks) constitute a family of lipid kinases implicated in signal transduction through tyrosine kinase receptors and heterotrimeric G protein-linked receptors. PI3Ks are heterodimers made up of four different 110-kDa catalytic subunits (p110alpha, p110beta, p110gamma, and p110delta) and a smaller regulatory subunit. Despite a clear implication of PI3Ks in survival signaling, the contribution of the individual PI3K isoforms has not been elucidated. To address this issue, we generated Rat1 fibroblasts that co-express c-Myc and membrane targeted derivates of the different p110 isoforms. Here we present data for the first time showing that activation of PI3-kinase signaling through membrane localization of p110beta, p110gamma, and p110delta protects c-Myc overexpressing Rat1 fibroblasts from apoptosis caused by serum deprivation like it has been described for p110alpha. Expression of each p110 isoform reduces significantly caspase-3 like activity in this apoptosis model. Decreased caspase-3 activity correlates with the increase in Akt phosphorylation in cells that contain one of the myristoylated p110 isoforms. p110 isoform-mediated protection from cell death was abrogated upon expression of a kinase-negative version of Akt.

Effect of ibuprofen on cyclooxygenase and nitric oxide synthase of gastric mucosa: correlation with endoscopic lesions and adverse reactions.

The aim of this study was to evaluate the effect of ibuprofen on gastric mucosa and enzymes involved in gastroprotection in healthy volunteers. Twenty-four Helicobacter pylori-negative subjects were randomized to treatment with ibuprofen or ibuprofen-arginate (each 600 mg/6 hr during 3 days). Endoscopies were performed 1 week before and after treatment. Biopsies were taken from the gastric antrum and corpus for determination of prostaglandin E2 (PGE2) by ELISA and cyclooxygenase (COX-1 and COX-2) and nitric oxide synthase (eNOS and iNOS) by western blot. All subjects had at least one gastric lesion except for two individuals taking ibuprofen-arginate. Ibuprofen-arginate caused a lower rate of clinical adverse reactions than ibuprofen. Subjects with gastric lesions or adverse reactions had lower PGE2 levels. COX-1, COX-2, eNOS, and iNOS were detectable in all subjects. The constitutive enzymes (COX-1 and eNOS) did not change after treatment. COX-2 was higher in corpus than antrum and it increased after ibuprofen treatment. iNOS tended to increase mildly in the corpus in subjects with adverse reactions or endoscopic lesions. There were no significant differences between ibuprofen and ibuprofen-arginate in PGE2, or enzymes.