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.

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.

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.

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.

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.

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.

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.