Clonal KEAP1 mutations with loss of heterozygosity share reduced immunotherapy efficacy and low immune cell infiltration in lung adenocarcinoma.

BACKGROUND: KEAP1 mutations have been associated with reduced survival in lung adenocarcinoma (LUAD) patients treated with immune checkpoint inhibitors (ICIs), particularly in the presence of STK11/KRAS alterations. We hypothesized that, beyond co-occurring genomic events, clonality prediction may help identify deleterious KEAP1 mutations and their counterparts with retained sensitivity to ICIs. PATIENTS AND METHODS: Beta-binomial modelling of sequencing read counts was used to infer KEAP1 clonal inactivation by combined somatic mutation and loss of heterozygosity (KEAP1 C-LOH) versus partial inactivation [KEAP1 clonal diploid-subclonal (KEAP1 CD-SC)] in the Memorial Sloan Kettering Cancer Center (MSK) MetTropism cohort (N = 2550). Clonality/LOH prediction was compared to a streamlined clinical classifier that relies on variant allele frequencies (VAFs) and tumor purity (TP) (VAF/TP ratio). The impact of this classification on survival outcomes was tested in two independent cohorts of LUAD patients treated with immunotherapy (MSK/Rome N = 237; DFCI N = 461). Immune-related features were studied by exploiting RNA-sequencing data (TCGA) and multiplexed immunofluorescence (DFCI mIF cohort). RESULTS: Clonality/LOH inference in the MSK MetTropism cohort overlapped with a clinical classification model defined by the VAF/TP ratio. In the ICI-treated MSK/Rome discovery cohort, predicted KEAP1 C-LOH mutations were associated with shorter progression-free survival (PFS) and overall survival (OS) compared to KEAP1 wild-type cases (PFS log-rank P = 0.001; OS log-rank P < 0.001). Similar results were obtained in the DFCI validation cohort (PFS log-rank P = 0.006; OS log-rank P = 0.014). In both cohorts, we did not observe any significant difference in survival outcomes when comparing KEAP1 CD-SC and wild-type tumors. Immune deconvolution and multiplexed immunofluorescence revealed that KEAP1 C-LOH and KEAP1 CD-SC differed for immune-related features. CONCLUSIONS: KEAP1 C-LOH mutations are associated with an immune-excluded phenotype and worse clinical outcomes among advanced LUAD patients treated with ICIs. By contrast, survival outcomes of patients whose tumors harbored KEAP1 CD-SC mutations were similar to those with KEAP1 wild-type LUADs.

KEAP1-driven co-mutations in lung adenocarcinoma unresponsive to immunotherapy despite high tumor mutational burden.

BACKGROUND: Immune checkpoint inhibitors (ICIs) have demonstrated significant overall survival (OS) benefit in lung adenocarcinoma (LUAD). Nevertheless, a remarkable interpatient heterogeneity characterizes immunotherapy efficacy, regardless of programmed death-ligand 1 (PD-L1) expression and tumor mutational burden (TMB). KEAP1 mutations are associated with shorter survival in LUAD patients receiving chemotherapy. We hypothesized that the pattern of KEAP1 co-mutations and mutual exclusivity may identify LUAD patients unresponsive to immunotherapy. PATIENTS AND METHODS: KEAP1 mutational co-occurrences and somatic interactions were studied in the whole MSKCC LUAD dataset. The impact of coexisting alterations on survival outcomes in ICI-treated LUAD patients was verified in the randomized phase II/III POPLAR/OAK trials (blood-based sequencing, bNGS cohort, N = 253). Three tissue-based sequencing studies (Rome, MSKCC and DFCI) were used for independent validation (tNGS cohort, N = 289). Immunogenomic features were analyzed using The Cancer Genome Atlas (TCGA) LUAD study. RESULTS: On the basis of KEAP1 mutational co-occurrences, we identified four genes potentially associated with reduced efficacy of immunotherapy (KEAP1, PBRM1, SMARCA4 and STK11). Independent of the nature of co-occurring alterations, tumors with coexisting mutations (CoMut) had inferior survival as compared with single-mutant (SM) and wild-type (WT) tumors (bNGS cohort: CoMut versus SM log-rank P = 0.048, CoMut versus WT log-rank P < 0.001; tNGS cohort: CoMut versus SM log-rank P = 0.037, CoMut versus WT log-rank P = 0.006). The CoMut subset harbored higher TMB than the WT disease and the adverse significance of coexisting alterations was maintained in LUAD with high TMB. Significant immunogenomic differences were observed between the CoMut and WT groups in terms of core immune signatures, T-cell receptor repertoire, T helper cell signatures and immunomodulatory genes. CONCLUSIONS: This study indicates that coexisting alterations in a limited set of genes characterize a subset of LUAD unresponsive to immunotherapy and with high TMB. An immune-cold microenvironment may account for the clinical course of the disease.

Testing a novel biotechnological passive sampler for monitoring atmospheric PAH pollution.

In this study we evaluated a new type of passive air sampler, the "mossphere" device, filled with a Sphagnum palustre clone. For this purpose, we compared the atmospheric levels of polyaromatic hydrocarbons (PAHs) collected using this device and those collected in conventional bulk deposition and particulate matter (PM10) samplers. All three types of samplers were exposed at 10 sites affected by different levels of pollution and located in two different climate zones. The bulk deposition/ mossphere comparison yielded a greater number of significant regressions with higher coefficients of determination than the PM10/ mossphere comparison. No significant regressions were observed for 3-ring PAHs in either comparison. The mosspheres explain ca. 50% of the variability of the concentrations of 4-, 5- and 6-ring PAHs and total PAHs detected in PM10 and ca. 70% of the corresponding concentrations detected in the bulk deposition. The use of the Sphagnum clone enables standardization of the set-up, thus making the mossphere device a good sampling tool for monitoring 4-, 5- and 6-ring and total PAHs, especially those associated with bulk deposition. The findings indicate the potential usefulness of this innovative technology for mapping PAH levels.

Tracking the route of phenanthrene uptake in mosses: An experimental trial.

In recent decades, mosses have been used as native species or as transplants in monitoring a wide range of pollutants from inorganic (i.e. metals and metalloids) to organic contaminants (mainly polycyclic aromatic hydrocarbons-PAHs). To implement the use of mosses as biomonitors of PAHs, one important issue is the study of the interactions between these compounds and moss tissues. In this study we investigated the mode of phenanthrene uptake in four moss species (Amblystegium humile, Plagiomnium affine, Hypnum cupressiforme and a clone of Sphagnum palustre) and its movements from air to plant surface and within the biomonitors, using fluorescent and confocal microscopy. The target compound, partitioned between gas and particulate phase depending on air conditions, was selected since it is one of the most abundant PAHs released into the atmosphere. Our findings support the hypothesis that phenanthrene aggregates in particles and in this form it is chiefly intercepted and uptaken onto moss surfaces, albeit with different frequency in the four species, with S. palustre>H. cupressiforme>P. affine=A. humile. Phenanthrene enters the dead, empty hyalocysts of S. palustre. Specific surface area and composition, frequency and distribution of binding groups may also explain the different ability of phenanthrene uptake by the four moss species.

PAH detection in Quercus robur leaves and Pinus pinaster needles: A fast method for biomonitoring purpose.

Due to the complexity and heterogeneity of plant matrices, new procedure should be standardized for each single biomonitor. Thus, here is described a matrix solid-phase dispersion extraction method, previously used for moss samples, improved and modified for the analyses of PAHs in Quercus robur leaves and Pinus pinaster needles, species widely used in biomonitoring studies across Europe. The improvements compared to the previous procedure are the use of Florisil added with further clean-up sorbents, 10% deactivated silica for pine needles and PSA for oak leaves, being these matrices rich in interfering compounds, as shown by the gas chromatography-mass spectrometry analyses acquired in full scan mode. Good trueness, with values in the range 90-120% for the most of compounds, high precision (intermediate precision between 2% and 12%) and good sensitivity using only 250mg of samples (limits of quantification lower than 3 and 1.5ngg(-1), respectively for pine and oak) were achieved by the selected procedures. These methods proved to be reliable for PAH analyses and, having advantage of fastness, can be used in biomonitoring studies of PAH air contamination.

Biomonitoring of atmospheric pollution by moss bags: Discriminating urban-rural structure in a fragmented landscape.

In this paper we investigated the possibility to use moss bags to detect pollution inputs - metals, metalloids and polycyclic aromatic hydrocarbons (PAHs) - in sites chosen for their different land use (agricultural, urban/residential scenarios) and proximity to roads (sub-scenarios), in a fragmented conurbation of Campania (southern Italy). We focused on thirty-nine elements including rare earths. For most of them, moss uptake was higher in agricultural than in urban scenarios and in front road sites. Twenty PAHs were analyzed in a subset of agricultural sites; 4- and 5-ringed PAHs were the most abundant, particularly chrysene, fluoranthene and pyrene. Overall results indicated that investigated pollutants have a similar spatial distribution pattern over the entire study area, with road traffic and agricultural practices as the major diffuse pollution sources. Moss bags proved a very sensitive tool, able to discriminate between different land use scenarios and proximity to roads in a mixed rural-urban landscape.

Che-1 modulates the decision between cell cycle arrest and apoptosis by its binding to p53.

The tumor suppressor p53 is mainly involved in the transcriptional regulation of a large number of growth-arrest- and apoptosis-related genes. However, a clear understanding of which factor/s influences the choice between these two opposing p53-dependent outcomes remains largely elusive. We have previously described that in response to DNA damage, the RNA polymerase II-binding protein Che-1/AATF transcriptionally activates p53. Here, we show that Che-1 binds directly to p53. This interaction essentially occurs in the first hours of DNA damage, whereas it is lost when cells undergo apoptosis in response to posttranscriptional modifications. Moreover, Che-1 sits in a ternary complex with p53 and the oncosuppressor Brca1. Accordingly, our analysis of genome-wide chromatin occupancy by p53 revealed that p53/Che1 interaction results in preferential transactivation of growth arrest p53 target genes over its pro-apoptotic target genes. Notably, exposure of Che-1(+/-) mice to ionizing radiations resulted in enhanced apoptosis of thymocytes, compared with WT mice. These results confirm Che-1 as an important regulator of p53 activity and suggest Che-1 to be a promising yet attractive drug target for cancer therapy.

Steering the efficiency of carbon nanotube-silicon photovoltaic cells by acid vapor exposure: a real-time spectroscopic tracking.

Hybrid carbon nanotube-silicon (CNT-Si) junctions have been investigated by angle resolved photoemission spectroscopy (AR-XPS) with the aim to clarify the effects of a nonstoichiometric silicon oxide buried interface on the overall cell efficiency. A complex silicon oxide interface has been clearly identified and its origin and role in the heterojunction have been probed by exposing the cells to hydrofluoric (HF) and nitric (HNO3) acid. Real-time monitoring of the cell efficiencies during the steps following acid exposure (up to 1 week after etching) revealed a correlation between the thickness and chemical state of the oxide layer and the cell efficiencies. By matching the AR-XPS and Raman spectroscopy with the electrical response data it has been possible to discriminate the effects on the cell efficiency of the buried SiO(x) interface from those related to CNT acid doping. The overall cell behavior recorded for different thicknesses of the SiO(x) interface indicates that the buried oxide layer is likely acting as a passivating/inversion layer in a metal-insulator-semiconductor junction.

HIPK2 sustains apoptotic response by phosphorylating Che-1/AATF and promoting its degradation.

Che-1/AATF is an RNA polymerase II-binding protein that is involved in the regulation of gene transcription, which undergoes stabilization and accumulation in response to DNA damage. We have previously demonstrated that following apoptotic induction, Che-1 protein levels are downregulated through its interaction with the E3 ligase HDM2, which leads to Che-1 degradation by ubiquitylation. This interaction is mediated by Pin1, which determines a phosphorylation-dependent conformational change. Here we demonstrate that HIPK2, a proapoptotic kinase, is involved in Che-1 degradation. HIPK2 interacts with Che-1 and, upon genotoxic stress, phosphorylates it at specific residues. This event strongly increases HDM2/Che-1 interaction and degradation of Che-1 protein via ubiquitin-dependent proteasomal system. In agreement with these findings, we found that HIPK2 depletion strongly decreases Che-1 ubiquitylation and degradation. Notably, Che-1 overexpression strongly counteracts HIPK2-induced apoptosis. Our results establish Che-1 as a new HIPK2 target and confirm its important role in the cellular response to DNA damage.

PAHs in decaying Quercus ilex leaf litter: mutual effects on litter decomposition and PAH dynamics.

The investigation of the relationships between litter decomposition and polycyclic aromatic hydrocarbons (PAHs) is important to shed light not only on the effects of these pollutants on fundamental ecosystem processes, such as litter decomposition, but also on the degradation of these pollutants by soil microbial community. This allows to understand the effect of atmospheric PAH contamination on soil PAH content via litterfall. At this aim, we studied mass and PAH dynamics of Quercus ilex leaf litters collected from urban, industrial and remote sites, incubated in mesocosms under controlled conditions for 361d. The results highlighted a litter decomposition rate of leaves sampled in urban>industrial>remote sites; the faster decomposition of litter of the urban site is also related to the low C/N ratio of the leaves. The PAHs showed concentrations at the beginning of the incubation of 887, 650 and 143 ng g(-1)d.w., respectively in leaf litters from urban, industrial and remote sites. The PAHs in litter decreased along the time, with the same trend observed for mass litter, showing the highest decrease at 361 d for the urban leaf litter. Anyway, PAH dynamics in all the litters exhibited two phases of loss, separated by a PAH increase observed at 246 d and mainly linked to benzo[e]pyrene.

Polycyclic aromatic hydrocarbon contamination in an urban area assessed by Quercus ilex leaves and soil.

We investigated the PAH contamination of Naples urban area, densely populated and with high traffic flow, by analyses of environmental matrices: soil and Quercus ilex leaves. Being some PAHs demonstrated to have hazardous effects on human health, the accumulation of carcinogenic and toxic PAHs (expressed as B(a)Peq) was evaluated in the leaves and soil. The main sources of the PAHs were discriminated by the diagnostic ratios in the two matrices. The urban area appeared heavily contaminated by PAHs, showing in soil and leaves total PAH concentrations also fivefold higher than those from the remote area. The soil mainly accumulated heavy PAHs, whereas leaves the lightest ones. Median values of carcinogenic PAH concentrations were higher in soil (440 ng g(-1) d.w.) and leaves (340 ng g(-1) d.w.) from the urban than the remote area (60 and 70 ng g(-1) d.w., respectively, for soil and leaves). Also, median B(a)Peq concentrations were higher both in soil and leaves from the urban (137 and 63 ng g(-1) d.w., respectively) than those from the remote area (19 and 49 ng g(-1) d.w., respectively). Different from the soils, the diagnostic ratios found for the leaves discerned PAH sources in the remote and urban areas, highlighting a great contribution of vehicular traffic emission as main PAH source in the urban area.

Improved biomonitoring of airborne contaminants by combined use of holm oak leaves and epiphytic moss.

Concentrations of 12 elements (Ca, Cd, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, Pb and Zn) and 16 EPA-listed PAHs were detected in Quercus ilex leaves and the epiphytic moss Leptodon smithii collected at urban, periurban and extraurban holm oak stands, in two Italian Regions (Campania and Tuscany). Levels of environmental contaminants were generally higher in leaves and moss from urban areas than periurban and extraurban ones and samples from Campania had the highest PAH content. The epiphytic moss accumulated higher concentrations of trace elements than leaves and the latter showed a higher accumulation capability for PAHs, especially for those with low molecular weight. The different bioaccumulation in leaves and moss were explained in terms of their distinctive morphological and ecophysiological characteristics. The combined approach seems a promising tool for the monitoring of a wide range of pollutants in Mediterranean urban and extraurban environments.

Suitability of two types of organic wastes for the growth of sclerophyllous shrubs on limestone debris: a mesocosm trial.

This study investigated whether overlaying organic wastes directly on limestone debris allowed the growth of sclerophyllous shrubs; the aim was to explore the feasibility of rehabilitation of sites destroyed by quarrying activity. In an open air mesocosm experiment two types of organic material were compared: compost from municipal wastes (C) and a mixture of compost and poultry manure added with wheat husk (C-PW). Mesocosms were pots (1m diameter, 60cm height) containing limestone debris covered by the organic material. Seven mesocosms with C and seven mesocosms with C-PW were planted with sclerophyllous shrubs (Laurus nobilis L., Phillyrea angustifolia L. and Quercus ilex L.). The substrates were characterised in terms of chemical and physical parameters, microbial activity and biomass, and total and active fungal biomass. Shrub photosynthetic performance and growth were evaluated. Over the whole experimental period, organic matter mineralization was higher in C-PW. Microbial biomass and respiration were higher in C-PW than in C but after one year no statistically significant difference between the two substrates occurred. Fungal mycelium was a minor fraction of the microbial community in both types of substrates and decreased dramatically after setting up the mesocosms. The metabolic quotient was higher in C suggesting more stressful conditions as compared to C-PW. Both substrates allowed shrub growth; however photosynthetic rates and the increase of plant size were higher on C-PW than on C. The results demonstrated that, as compared to only compost, the mixture of compost and poultry manure added with wheat husk is a substrate more suitable to both microbial processes and plant growth. Therefore a plan to revegetate quarries based on the use of organic wastes as a substrate for sclerophyllous shrubs could be feasible and, what is more, helpful to mitigate the environmental impact of organic wastes disposal.

Che-1 activates XIAP expression in response to DNA damage.

X-linked inhibitor of apoptosis protein (XIAP) is a member of the inhibitor of apoptosis proteins family that selectively binds and inhibits caspase-3, -7 and -9. As such, XIAP is an extremely potent suppressor of apoptosis and an attractive target for cancer treatment. Che-1 is an antiapoptotic agent involved in the control of gene transcription and cell proliferation. Recently, we showed that the checkpoint kinases ATM/ATR and checkpoint kinase 2 physically and functionally interact with Che-1 and promote its phosphorylation and accumulation in response to DNA damage. These Che-1 modifications induce transcription of p53, and Che-1 depletion strongly sensitizes tumor cells to anticancer drugs. Here we show that Che-1 activates XIAP expression in response to DNA damage. This effect is mediated by Che-1 phosphorylation and requires NF-kappaB. Notably, we found that XIAP expression is necessary for antiapoptotic activity of Che-1 and that in vivo downregulation of Che-1 by small interference RNA strongly enhanced the cytotoxicity of anticancer drugs.

Leaf accumulation of trace elements and polycyclic aromatic hydrocarbons (PAHs) in Quercus ilex L.

Quercus ilex L. leaves were collected four times in one year at six urban sites and one remote area in order to determine trace element and PAH accumulation through concomitant analyses of unwashed and water-washed leaves. Both unwashed and washed leaves showed the highest amounts of trace elements and PAHs in the urban area. Unwashed leaves showed greater differences between urban and remote areas and among the urban sites than washed leaves for trace element and PAH concentrations. Water-washing resulted in a significant (P<0.001) decrease in leaf concentrations of Cr, Cu, Fe, Pb, V and Zn. By contrast, Cd and total PAH concentrations showed no differences between unwashed and washed leaves.

Assessment of nutritional status and trace element contamination of holm oak woodlands through analyses of leaves and surrounding soils.

The nutritional status and trace element contamination of holm oak woodlands in Vesuvius National Park were assessed by analyses of Quercus ilex L. leaves and surrounding soils. The concentrations of Cd, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, Pb, V and Zn were measured in 1-year-old leaves, and in the soils at 0-5 and 15-20 cm depths. The potentially available concentrations were also measured for the soils. The leaf element concentrations were similar to the Q. ilex chemical fingerprint, thus indicating a good nutritional status and the absence of short-term trace element depositions. Total K and V were more abundant in the deep soil layers than in the surface ones, whereas Cd and Pb showed higher values in the surface soils. This suggests that long-term soil accumulations of Cd and Pb are due to atmospheric input. The soil availabilities of Cd, Pb and Zn were high, and Cr availability was very low. A correlation between the available concentrations in the deep soil layers and leaf concentrations was found only for Zn.