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Relationship between CNA level and TMB in (A) MSK pan-cancer non-ICI treated cohort and (B) other three ICI-treated cohorts (MSK NSCLC, SKCM, and Dana-Farber ICI cohort). For each cancer type and each cohort, a plot is shown containing the CNA level versus tumor mutation burden. The spearman correlation coefficient and P value are shown.

Contradictory characteristics of elevated mutational burden and a “cold” tumor microenvironment (TME) coexist in liver kinase B1 (LKB1)–mutant non–small cell lung cancers (NSCLC). The molecular basis underlying this paradox and strategies tailored to these historically difficult to treat cancers are lacking. Here, by mapping the single-cell transcriptomic landscape of genetically engineered mouse models with Kras versus Kras/Lkb1-driven lung tumors, we detected impaired tumor-intrinsic IFNγ signaling in Kras/Lkb1-driven tumors that explains the inert immune context. Mechanistic analysis showed that mutant LKB1 led to deficiency in the DNA damage repair process and abnormally activated PARP1. Hyperactivated PARP1 attenuated the IFNγ pathway by physically interacting with and enhancing the poly(ADP-ribosyl)ation of STAT1, compromising its phosphorylation and activation. Abrogation of the PARP1-driven program triggered synthetic lethality in NSCLC on the basis of the LKB1 mutation–mediated DNA repair defect, while also restoring phosphorylated STAT1 to favor an immunologically “hot” TME. Accordingly, PARP1 inhibition restored the disrupted IFNγ signaling and thus mounted an adaptive immune response to synergize with PD-1 blockade in multiple LKB1-deficient murine tumor models. Overall, this study reveals an unexplored interplay between the DNA repair process and adaptive immune response, providing a molecular basis for dual PARP1 and PD-1 inhibition in treating LKB1-mutant NSCLC.Significance:Targeting PARP exerts dual effects to overcome LKB1 loss–driven immunotherapy resistance through triggering DNA damage and adaptive immunity, providing a rationale for dual PARP and PD-1 inhibition in treating LKB1-mutant lung cancers.

Demographic and clinicopathologic characteristics of three cohorts treated with immune checkpoint inhibitors.

Univariate and Multivariable Cox regression analysis of predictive factors in two cohorts treated with immune checkpoint inhibitors.

Sequences for qPCR primers.

Sensitivity analysis of overall survival (OS) or progression-free survival (PFS) in three cohorts treated with immune checkpoint inhibitor (ICI) by altering cut-point (median, tertile, quartile) of TMB and CNA. (A) Univariate analysis of treatment survival in three ICI treated cohorts with different cut-points (median, tertile, quartile) of TMB and CNA. (B-D) Multivariate analysis of treatment survival in three ICI treated cohorts with the inclusion of alternate cut-points of CNA and TMB, and illustrated in three groups stratified by the cut-points of CNA (B: {greater than or equal to}median vs

PARP1 inhibition restores phosphorylated STAT1 from poly(ADP-ribosyl)ation and triggers synthetic lethality in LKB1-deficient cells. (a) A549 lung cancer cells were transfected with lentivirus expressing the indicated genes (LV-Ctrl, LV-LKB1-WT, and LV-LKB1-Mut) followed by treatment with olaparib (5μM) for 24 hours. The expression of pSTAT1 and PARP1 was analyzed by western blotting. (b-d) LLC1-shLkb1 and LLC1-shCtrl cells were pre-treated with olaparib (5μmol/L) for 6 hours and then stimulated with IFNγ for another 24 hours. Total pSTAT1 and STAT1 were quantified using western blot (b) and cell-surface PD-L1 was measured by flow cytometry (c). Q-PCR analysis was used to detect the expression of CXCL9 and PD-L1 (d). (e, f) The three groups of A549 cells were treated with IFNγ or IFNγ/olaparib and then refreshed the culture medium. The ratio of CD8+ T (effector cells) to A549 cells (target cell) ratio was 5:1. After 6 hours of incubation, QT-PCR analysis was used to detect the expression of indicated molecules of CD8+ T cells. (g) CD8+ T cells were treated with PBS or olaparib (5μmol/L) for 24 hours, and then the cells were collected for quantification of GZMB, PRF1, TNF, and IFNγ. CD8a was selected as the reference gene. (h) LLC-LV-Ctrl cells were treated with IFNγ/anti-PD-1 antibodies (20 μg/mL) in the absence or presence of olaparib (5 µM) or BMDCs (Tumor cells: DCs= 1:1) for 24 hours. Flow cytometry assay was used for apoptosis analysis of tumor cells. The right graph showed the quantification of tumor cell apoptosis. The data are presented as the mean ± SD of triplicate experiments. Statistical significance was determined by Student's t-test; ns, not significant; ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05.

Study flow: The flowchart of our study design and specific cohort selected.

The outcome-based cutpoint of TMB and CNA as continuous variables in three ICI treated cohort analyzed by X-tile software. Kaplan-Meier curves of survival according to the cutpoints of TMB and CNA calculated by X-tile software in (A) NSCLC ICI, (B) SKCM ICI, and (C) Dana-Farber ICI cohorts; (D) The summary of subpopulation cutpoints of TMB and CNA in the above three cohorts. Hi, high; Lo, low.

LKB1 deficiency impairs IFNγ pathway by hindering STAT1 phosphorylation. (a) Western blotting for detection of LKB1 and pAMPK expression in K and KL lung tumors. (b) UMAP of sub-clustered CD45+ immune cells from lung tumor samples of K and KL, colored by cell types. (c) The proportion of each immune cell type in K vs KL. (d) UMAP of sub-clustered cells from lung tumor samples of KP and KPL mice, colored by their 10 major cell types. (e) Gene set enrichment analysis (GSEA) showing downregulated response to interferon-gamma in KPL compared with KP. (f) A549 lung cancer cells were transfected with lentivirus expressing the indicated genes (LV-Ctrl, LV-LKB1-WT, and LV-LKB1-Mut) followed by stimulation with IFNγ. The expression of LKB1 and pAMPK was analyzed by western blotting. (g) Expression of pSTAT1 was measured by immunofluorescent staining in the three groups of A549 cells treated with IFNγ for 24 hours. Nuclei were visualized with DAPI (blue). Scale bar = 100 μm. (h) Immunoblot of the indicated IFNγ-induced proteins in different lung cancer cells. (i) Decreased IFNγ signaling downstream molecules, PD-L1, and chemokines, in KPL vs KP malignant cells. (j) PD-L1 transcription and chemokine signature score in tumor cells of K vs KL. (k) Western blot illustrating LKB1 expression in LLC1 and CMT-167 cells respectively. (l, m) Lewis lung cancer cells (LLC1) were transfected with shRNA specific for Lkb1 (shLkb1) or noneffective scrambled shRNA (shCtrl) followed by treatment with IFNγ for 24 hours. Western blotting for the detection of pSTAT1, STAT1, LKB1, and pAMPK expression was performed. (n) Flow cytometry analysis of cell surface PD-L1 of tumor cells from mice bearing LLC1-shLkb1 and LLC1-shCtrl tumors. (o) Tumors from mice bearing LLC1-shLkb1 and LLC1-shCtrl derived tumors were analyzed by Q-PCR for detecting CXCL9, CXCL10, CXCL11 and CCL5. (p) Representative IHC staining of CD8+ T cells and quantification of infiltrated CD8+ T cells in LLC-LV-Lkb1 tumors treated with indicated treatment (n=3 mice each group). Scale bar: 100 µm. n = 5 fields from independent tumors. Error bars represent mean ± SD. Statistical analyses were performed using unpaired t-tests. (****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05). (q) LLC1/LV-Ctrl or LLC1/LV-Lkb1 tumors were administered with different treatments, anti-PD-1 monotherapy or combination treatment (n=7 mice/group). Tumor size was monitored. Results are presented as mean ± SEM; * P < 0.05; two-way ANOVA.

Olaparib-derived dual effects sensitize LKB1-deficient tumors to anti-PD-1 immunotherapy in preclinical models. (a) Mice-bearing orthotopic lung tumors were treated with different therapies. Bioluminescent images were shown. (b) Immunofluorescence staining of PD-L1 and CD8+T cells in resected lung tumors of mice bearing orthotopic tumors (n= 6 fields from independent tumors). Scale bar, 100 μm. (c). Quantification of tumor-infiltrating CD8+ T cells in immunofluorescence staining. (d, e) IHC statistical analysis of PAR and PD-L1 expression in resected lung tumors of mice bearing orthotopic tumors (n=5 fields from independent tumors). (f) Quantification of tumor-infiltrating CD8+ T cells in IHC staining in resected lung tumors of mice bearing orthotopic tumors (n= 5 fields from independent tumors). Data are represented as mean ± SD; Student's t-tests; * P < 0.05; ** P < 0.01; ***P

Relationship between HRD mutation and TMB in (A) MSK NSCLC ICI cohort and (B) Dana-Farber ICI cohort. For each cohort, a plot is shown containing the HRD mutation status versus the tumor mutation burden. The P value is shown.

Olaparib promoted STING activation in LKB1-mutant/loss NSCLC cells and LLC-derived tumors. (a) Cells were pre-treated with olaparib (5 μM) for 6 hours and then stimulated with IFNγ for 24 hours. The cell lysates were then subjected to western blot analysis for key proteins in the STING signaling pathway. (b) Representative IHC staining of STING and pSTING in LLC-LV-Lkb1 tumors and LLC-LV-Ctrl tumors following different treatments. Scale bar: 200 μm. (c) IHC statistical analysis of STING and pSTING expression in LLC-LV-Ctrl/Lkb1 tumors following indicated treatment. n=5 fields from independent tumors. Data are represented as mean ± SD; Student's t-tests; ***P

LKB1-interacting proteins identified by LC-MS/MS analysis.

Supplementary figure legends

Anti-PD-1/olaparib treatment promotes IFNγ secretion by CD8+ T cells in the tumor microenvironment. Mice-bearing orthotopic lung tumors were treated with different therapies. Representative IHC stainings of IFNγ expression in tumor tissues were shown. Scale bar, 50 μm.The right graph showed the IHC statistical analysis of IFNγ expression in tumor tissues. n = 5 fields from independent tumors. Data are represented as mean ± SD; Student's t-tests; **P

Mechanistic link between LKB1 and PARP1. (a) A bioinformatics tool (Pepsite) was utilized to predict the possibility of PARP1 docking on the di-Arg ER retention motif of LKB1 (PRR). (b) Localization of PARP1 (green) relative to LKB1 (red) in LKB1-WT A549 cells and H1299 cells. Scale bar = 20 μm. (c) Lysates from H1299 cells were subjected to IP with anti-PARP1 and anti-LKB1 antibodies and western blot analysis with the indicated antibodies. (d) IHC statistical analysis of PAR expression in LLC-shCtrl and LLC-shLkb1 tumors. n=5 fields from independent tumors. Data are represented as mean ± SD; Student's t-tests; **, P < 0.01.

Purpose:Although tumor mutation burden (TMB) has been well known to predict the response to immune checkpoint inhibitors (ICI), lack of randomized clinical trial data has restricted its clinical application. This study aimed to explore the significance and feasibility of biomarker combination based on TMB and copy-number alteration (CNA) for the prognosis of each tumor and prediction for ICI therapy in metastatic pan-cancer milieu.Experimental Design:Non-ICI–treated MSK pan-cancer cohort was used for prognosis analysis. Three independent immunotherapy cohorts, including non–small cell lung cancer (n = 240), skin cutaneous melanoma (n = 174), and mixed cancer (Dana-Farber, n = 98) patients from previous studies, were analyzed for efficacy of ICI therapy.Results:TMB and CNA showed optimized combination for the prognosis of most metastatic cancer types, and patients with TMBlowCNAlow showed better survival. In the predictive analysis, both TMB and CNA were independent predictive factors for ICI therapy. Remarkably, when TMB and CNA were jointly analyzed, those with TMBhighCNAlow showed favorable responses to ICI therapy. Meanwhile, TMBhighCNAlow as a new biomarker showed better prediction for ICI efficacy compared with either TMB-high or CNA-low alone. Furthermore, analysis of the non-ICI–treated MSK pan-cancer cohort supported that the joint stratification of TMB and CNA can be used to categorize tumors into distinct sensitivity to ICI therapy across pan-tumors.Conclusions:The combination of TMB and CNA can jointly stratify multiple metastatic tumors into groups with different prognosis and heterogeneous clinical responses to ICI treatment. Patients with TMBhighCNAlow cancer can be an optimal subgroup for ICI therapy.

Kaplan-Meier curves of overall survival (OS) and progression-free survival (PFS) according to TMB and CNA in patients receiving immune checkpoint inhibitor treatment. (A) Kaplan-Meier curves of OS based on TMB or CNA classification in SKCM ICI cohort treated with anti-CTLA-4. (B) Kaplan-Meier curves of PFS based on TMB or CNA classification in MSK NSCLC ICI cohort treated with anti-PD1/L1. Kaplan-Meier curves of (C) OS and (D) PFS based on TMB or CNA classification in Dana-Farber ICI cohort treated with anti-PD1/L1. We calculated p values using the log-rank test.

Multivariate analysis of clinical and genetic features associated with overall survival in 8 cancer types of MSK pan-cancer cohort.

Overexpression of mutant PARP1 promotes activation of the IFNγ signaling pathway. (a, b) A549 lung cancer cells and LLC1 lung cancer cells were transfected with the catalytic mutant Flag-PARP1 (E988) followed by stimulation with IFNγ. The lysates were then subjected to western blot analysis for indicated proteins.

Liver kinase B1 (LKB1) mutation is prevalent and a driver of resistance to immune checkpoint blockade (ICB) therapy for lung adenocarcinoma. Here leveraging single cell RNA sequencing data, we demonstrate that trafficking and adhesion process of activated T cells are defected in genetically engineered Kras-driven mouse model with Lkb1 conditional knockout. LKB1 mutant cancer cells result in marked suppression of intercellular adhesion molecule-1 (ICAM1). Ectopic expression of Icam1 in Lkb1-deficient tumor increases homing and activation of adoptively transferred SIINFEKL-specific CD8+ T cells, reactivates tumor-effector cell interactions and re-sensitises tumors to ICB. Further discovery proves that CDK4/6 inhibitors upregulate ICAM1 transcription by inhibiting phosphorylation of retinoblastoma protein RB in LKB1 deficient cancer cells. Finally, a tailored combination strategy using CDK4/6 inhibitors and anti-PD-1 antibodies promotes ICAM1-triggered immune response in multiple Lkb1-deficient murine models. Our findings renovate that ICAM1 on tumor cells orchestrates anti-tumor immune response, especially for adaptive immunity.

Background Immune checkpoint inhibitor (ICI) therapy combined with conventional therapies is being broadly applied in non-small cell lung cancer (NSCLC) patients. However, the risk of interstitial pneumonitis (IP) following a combined regimen is incompletely characterized. Methods A total of 46,127 NSCLC patients were extracted for disproportionality analyses of IP from the Food and Drug Administration’s Adverse Event Reporting System (FAERS) database. A total of 1108 NSCLC patients who received ICI treatment at Nanfang Hospital of Southern Medical University were collected and utilized for real-world validation. Results Of the 46,127 patients with NSCLC, 3830 cases (8.3%; 95% confidence interval [CI], 8.05–8.56) developed IP. Multivariable logistic regression analyses revealed that the adjusted ROR of ICI combined with radiation (RT) was the highest (121.69; 95% CI, 83.60–184.96; P < 0.0001) among all therapies, while that of ICI combined with chemotherapy (CHEMO) or targeted therapy (TARGET) was 0.90 (95% CI, 0.78–1.04; P = 0.160) and 1.49 (95% CI, 0.95–2.23; P = 0.065), respectively, using ICI monotherapy as reference. Furthermore, analyses from our validation cohort of 1108 cases showed that the adjusted odds ratio of ICI combined with RT was the highest (12.25; 95% CI, 3.34–50.22; P < 0.01) among all the therapies, while that of ICI combined with CHEMO or TARGET was 2.32 (95% CI, 0.89–7.92; P = 0.12) and 0.66 (95% CI, 0.03–4.55; P = 0.71), respectively, using ICI monotherapy as reference. Conclusions Compared with ICI monotherapy, ICI combined with RT, rather than with CHEMO or TARGET, is associated with a higher risk of IP in NSCLC patients. Hence, patients receiving these treatments should be carefully monitored for IP.

Contradictory characteristics of elevated mutational burden and a “cold” tumor microenvironment (TME) coexist in liver kinase B1 (LKB1)–mutant non–small cell lung cancers (NSCLC). The molecular basis underlying this paradox and strategies tailored to these historically difficult to treat cancers are lacking. Here, by mapping the single-cell transcriptomic landscape of genetically engineered mouse models with Kras versus Kras/Lkb1-driven lung tumors, we detected impaired tumor-intrinsic IFNγ signaling in Kras/Lkb1-driven tumors that explains the inert immune context. Mechanistic analysis showed that mutant LKB1 led to deficiency in the DNA damage repair process and abnormally activated PARP1. Hyperactivated PARP1 attenuated the IFNγ pathway by physically interacting with and enhancing the poly(ADP-ribosyl)ation of STAT1, compromising its phosphorylation and activation. Abrogation of the PARP1-driven program triggered synthetic lethality in NSCLC on the basis of the LKB1 mutation–mediated DNA repair defect, while also restoring phosphorylated STAT1 to favor an immunologically “hot” TME. Accordingly, PARP1 inhibition restored the disrupted IFNγ signaling and thus mounted an adaptive immune response to synergize with PD-1 blockade in multiple LKB1-deficient murine tumor models. Overall, this study reveals an unexplored interplay between the DNA repair process and adaptive immune response, providing a molecular basis for dual PARP1 and PD-1 inhibition in treating LKB1-mutant NSCLC.Significance:Targeting PARP exerts dual effects to overcome LKB1 loss–driven immunotherapy resistance through triggering DNA damage and adaptive immunity, providing a rationale for dual PARP and PD-1 inhibition in treating LKB1-mutant lung cancers.

Background Organ-specific metastatic context has not been incorporated into the clinical practice of guiding programmed death-(ligand) 1 [PD-(L)1] blockade, due to a lack of understanding of its predictive versus prognostic value. We aim at delineating and then incorporating both the predictive and prognostic effects of the metastatic-organ landscape to dissect PD-(L)1 blockade efficacy in non-small cell lung cancer (NSCLC). Methods A total of 2062 NSCLC patients from a double-arm randomized trial (OAK), two immunotherapy trials (FIR, BIRCH), and a real-world cohort (NFyy) were included. The metastatic organs were stratified into two categories based on their treatment-dependent predictive significance versus treatment-independent prognosis. A metastasis-based scoring system (METscore) was developed and validated for guiding PD-(L)1 blockade in clinical trials and real-world practice. Results Patients harboring various organ-specific metastases presented significantly different responses to immunotherapy, and those with brain and adrenal gland metastases survived longer than others [overall survival (OS), p = 0.0105; progression-free survival (PFS), p = 0.0167]. In contrast, survival outcomes were similar in chemotherapy-treated patients regardless of metastatic sites (OS, p = 0.3742; PFS, p = 0.8242). Intriguingly, the immunotherapeutic predictive significance of the metastatic-organ landscape was specifically presented in PD-L1-positive populations (PD-L1 > 1%). Among them, a paradoxical coexistence of a favorable predictive effect coupled with an unfavorable prognostic effect was observed in metastases to adrenal glands, brain, and liver (category I organs), whereas metastases to bone, pleura, pleural effusion, and mediastinum yielded consistent unfavorable predictive and prognostic effects (category II organs). METscore was capable of integrating both predictive and prognostic effects of the entire landscape and dissected OS outcome of NSCLC patients received PD-(L)1 blockade (p < 0.0001) but not chemotherapy (p = 0.0805) in the OAK training cohort. Meanwhile, general performance of METscore was first validated in FIR (p = 0.0350) and BIRCH (p < 0.0001), and then in the real-world NFyy cohort (p = 0.0181). Notably, METscore was also applicable to patients received PD-(L)1 blockade as first-line treatment both in the clinical trials (OS, p = 0.0087; PFS, p = 0.0290) and in the real-world practice (OS, p = 0.0182; PFS, p = 0.0045). Conclusions Organ-specific metastatic landscape served as a potential predictor of immunotherapy, and METscore might enable noninvasive forecast of PD-(L)1 blockade efficacy using baseline radiologic assessments in advanced NSCLC.

The ubiquity and potential toxicity of phthalates make their exposure on humans a matter of great concern. Measuring the targeted phthalate metabolites using liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a commonly used approach for human biomonitoring of phthalates. However, the targeted LC-MS/MS methods have been unable to meet the desire to comprehensively and rapidly screen and identify for as many known and potentially unknown phthalate metabolites in human samples. Instead, non-targeted screening methods, allowing the rapid detection and identification of unknown compounds in a wide variety of environmental and human samples are complementary to the targeted methods. In this study, 23 phthalate metabolites were analyzed using liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-QTOF-MS) for development of a non-targeted screening method. The fragmentation patterns of the 23 phthalate metabolites were investigated using collision-induced dissociation experiments at 10, 20, and 40 eV. And three common specific product ions at m/z 121.0297, m/z 147.0088, and m/z 165.0193 corresponding to C7H5O2-, C8H3O3-, and C8H5O4-, respectively were observed in their mass spectra. Based on the three common product ions, a new strategy was proposed for non-targeted screening of the new phthalate metabolites as exposure biomarkers for human biomonitoring. It can greatly reduce the time spent on data processing for potential precursor ions. Additionally, the correlation of the retention time and logKow of the phthalate metabolites was explored, which provides a useful tool to reduce the number of identified candidates in the non-targeted screening.

Long non-coding RNA LincIN has been reported to be overexpressed and to be involved in the metastasis of breast cancer. However, the expression and role of LincIN in esophageal squamous cell carcinoma (ESCC) remain unsolved. In the present study, LincIN expression was examined in ESCC by RT-qPCR, and the roles of LincIN in ESCC were determined using cell growth, migration and invasion assays. In addition, the effects of LincIN on nuclear factor 90 (NF90) and microRNA/miR (miR)-7 were examined by RNA immunoprecipitation assay, RT-qPCR, dual-luciferase reporter assay and western blot analysis. The results revealed that LincIN expression was significantly increased in ESCC tissues and cell lines. The increased expression of LincIN was positively associated with invasion depth, lymph node metastasis, TNM stage and a poor prognosis. Functional assays revealed that the overexpression of LincIN promoted ESCC cell growth, migration and invasion. Mechanistic analysis revealed that LincIN physically bound to NF90, enhanced the binding between NF90 and primary miR-7 (pri-miR-7), and further enhanced the inhibitory effects of NF90 on miR-7 biogenesis. Therefore, LincIN downregulated miR-7 expression in ESCC. The expression of miR-7 inversely correlated with that of LincIN in ESCC tissues. By downregulating miR-7, LincIN increased the expression of HOXB13, a target of miR-7. The overexpression of miR-7 or the depletion of HOXB13 both attenuated the tumor-promoting roles of LincIN in ESCC cell growth, migration and invasion. On the whole, the findings of the present study suggest that LincIN is overexpressed and plays an oncogenic role in ESCC via the regulation of the NF90/miR-7/HOXB13 axis. Thus, LincIN may prove to be a promising prognostic biomarker and therapeutic target for ESCC.

Objectives: Clinical benefits of immune-checkpoint blockade (ICB) versus standard chemotherapy have been established in unselected non-small cell lung cancer (NSCLC). However, the response to ICB therapy among patients is heterogeneous in clinical practice. Materials and Methods: We retrospectively assessed the predicitive effect of the primary and metastatic lesion spectrum (baseline sum of the longest diameters [SLD], number of metastatic sites and specific organ metastases) on the efficacy of atezolizumab over docetaxel in OAK and POPLAR trial cohorts. A decision model, termed DSO (Diameter-Site-Organ), based on the spectrum was developed and validated for guiding ICB. Results: Higher SLD (>38 mm) and more metastatic sites (≥2) were characterized with pronounced overall survival (OS) benefits from atezolizumab versus docetaxel. Specifically, adrenal gland and brain metastases were identified as favorable predictors of atezolizumab treatment. The DSO model was developed in the discovery cohort to integrate the directive effect of the primary and metastatic lesion spectrum. Remarkably, a general pattern of enhanced efficacy of atezolizumab versus docetaxel was observed along with the increase of the DSO score. For patients with DSO score > 0, atezolizumab yielded a significantly prolonged OS than docetaxel, whereas OS was generally similar between two treatments in patients with DSO score ≤ 0. Equivalent findings were also seen in the internal and external validation cohorts. Conclusions: The response to anti-PD-L1 therapy among patients varied with the primary and metastatic lesion spectrum. The DSO-based system might provide promising medication guidance for ICB treatment in NSCLC patients.

Background Dysregulation of long non-coding RNAs (lncRNAs) is responsible for cancer initiation and development, positioning lncRNAs as not only biomarkers but also promising therapeutic targets for cancer treatment. A growing number of lncRNAs have been reported in hepatocellular carcinoma (HCC), but their functional and mechanistic roles remain unclear. Methods Gene Set Enrichment Analysis was used to investigate the molecular mechanism of UPK1A antisense RNA 1 (UPK1A-AS1). Cell Counting Kit-8 assays, EdU assays, flow cytometry, western blotting, and xenograft assays were used to confirm the role of UPK1A-AS1 in the proliferation of HCC cells in vitro and in vivo. Bioinformatics analyses and quantitative polymerase chain reaction (qRT-PCR) were performed to explore the interplay between UPK1A-AS1 and enhancer of zeste homologue 2 (EZH2). RNA immunoprecipitation (RIP), RNA pull-down assays, western blotting, and qRT-PCR were conducted to confirm the interaction between UPK1A-AS1 and EZH2. The interaction between UPK1A-AS1 and miR-138-5p was examined by luciferase reporter and RIP assays. Finally, the expression level and prognosis value of UPK1A-AS1 in HCC were analyzed using RNA sequencing data from The Cancer Genome Atlas datasets. Results We showed that UPK1A-AS1, a newly identified lncRNA, promoted cellular proliferation and tumor growth by accelerating cell cycle progression. Cell cycle-related genes, including CCND1, CDK2, CDK4, CCNB1, and CCNB2, were significantly upregulated in HCC cells overexpressing UPK1A-AS1. Furthermore, overexpression of UPK1A-AS1 could protect HCC cells from cis-platinum toxicity. Mechanistically, UPK1A-AS1 interacted with EZH2 to mediate its nuclear translocation and reinforce its binding to SUZ12, leading to increased H27K3 trimethylation. Targeting EZH2 with specific small interfering RNA impaired the UPK1A-AS1-mediated upregulation of proliferation and cell cycle progression-related genes. Moreover, miR-138-5p was identified as a direct target of UPK1A-AS1. Additionally, UPK1A-AS1 was significantly upregulated in HCC, and the upregulation of UPK1A-AS1 predicted poor prognosis for patients with HCC. Conclusions Our study revealed that UPK1A-AS1 promotes HCC development by accelerating cell cycle progression through interaction with EZH2 and sponging of miR-138-5p, suggesting that UPK1A-AS1 possesses substantial potential as a novel biomarker for HCC prognosis and therapy.

Background: Dysregulations of lncRNA are responsible for cancer initiation and development, positioning lncRNAs as not only biomarkers but also promising therapeutic targets for cancer treatment. Growing number of lncRNAs have been reported in HCC but their functional and mechanistic roles remain unclear. Methods: Gene Set Enrichment Analysis was used to investigate the molecular mechanism of lncRNA UPK1A antisense RNA 1 (UPK1A-AS1). CCK-8 assay, EdU assay, flow cytometry, western blot, and xenograft assay were used to confirm the role of UPK1A-AS1 in the proliferation of HCC cells both in vitro and in vivo. Bioinformatics analysis and qRT-PCR were performed to explore the interplay between UPK1A-AS1 and Enhancer of Zeste Homologue 2 (EZH2). RNA immunoprecipitation (RIP), RNA-pull down assay, western blot, qRT-PCR, and were conducted to confirm the interaction between UPK1A-AS1 and EZH2. The interaction between UPK1A-AS1 and miR-138-5p was examined by luciferase reporter and RIP assays. Finally, the expression level and prognosis value of UPK1A-AS1 in HCC were analyzed using RNA-seq data from TCGA datasets.Results: We showed that UPK1A-AS1, a newly identified lncRNA, promoted cellular proliferation and tumor growth by accelerating cell cycle progression. Cell cycle related genes including CCND1, CDK2, CDK4, CCNB1 and CCNB2 were significantly upregulated in HCC cells with UPK1A-AS1 overexpression. Furthermore, overexpression of UPK1A-AS1 could protect HCC cells from cis-platinum toxicity. Mechanistically, UPK1A-AS1 interacted with EZH2 to mediate its nuclear translocation and reinforce its binding to SUZ12, leading to the increasing trimethylation of H27K3. Targeting EZH2 with specific siRNA impaired UPK1A-AS1-mediated upregulation of proliferation and cell cycle progression related genes. Moreover, miR-138-5p was identified as a direct target of UPK1A-AS1. Additionally, UPK1A-AS1 was significantly upregulated in HCC, and upregulation of UPK1A-AS1 predicted poor prognosis for patients with HCC. Conclusions: Our study reveals that UPK1A-AS1 promotes HCC development by accelerating cell cycle progression via interacting with EZH2 and sponging miR-138-5p, suggesting that UPK1A-AS1 possesses substantial potential as a novel biomarker for HCC prognosis and therapy.

Background: Clinical benefits of immune-checkpoint blockade (ICB) versus standard chemotherapy have been established in unselected non-small cell lung cancer (NSCLC). However, the response to ICB therapy among patients is still heterogeneous in clinical practice. Methods: We retrospectively assessed the impact of baseline sum of the longest diameters (SLD), number of metastatic sites and specific organ metastases on the efficacy of atezolizumab versus docetaxel in the pooled population from OAK and POPLAR studies. An assistant decision model based on the machine-learning method, incorporating these indicators, termed DSO (Diameter-Site-Organ), was developed and validated in OAK and POPLAR cohorts. Findings: Higher SLD (> 38mm) and more metastatic sites (≥ 2) were characterized with pronounced OS benefits from atezolizumab versus docetaxel. Specifically, brain and adrenal gland metastases were identified as favorable predictors of atezolizumab treatment. The DSO model for guiding second-line treatment was developed based on SLD and metastatic sites and organs in the discovery cohort. Remarkably, a general pattern of enhanced efficacy of atezolizumab versus docetaxel was observed along with the increase of the DSO score. For patients with DSO score > 0, atezolizumab yielded a prolonged OS than docetaxel (hazard ratio [HR] 0.67, 95% CI 0.54-0.84; P = 0.0003), whereas OS was generally similar between two treatments in patients with DSO score ≤ 0 (HR 1.46, 95% CI 0.89-2.39; P = 0.1346). Equivalent findings were seen in the internal and external validation cohorts. Interpretation: Our study for the first time revealed that patients with higher tumor burden were more suitable for ICB compared with standard chemotherapy. More importantly, the integrative DSO decision model might provide promising medication guidance for second-line ICB treatment in unselected NSCLC patients. Funding: This study was supported by the National Natural Science Foundation for YoungScientists of China (Grant No. 81802863 and 81902353), the Natural Science Foundation of Guangdong Province (Grant No. 2018030310285), and the Outstanding Youths Development Scheme of Nanfang Hospital, Southern Medical University (Grant No. 2017J003). Conflict of Interest: The authors have no actual or potential conflicts of interest to declare. Ethical Approval: The study was approved by theInstitutional Ethical Review Boards of Nanfang Hospital. All patients enrolled in OAK and POPLAR provided signed informed consent in accordance with the protocols of the corresponding studies.

Purpose: Although tumor mutation burden (TMB) has been well known to predict the response to immune checkpoint inhibitors (ICI), lack of randomized clinical trial data has restricted its clinical application. This study aimed to explore the significance and feasibility of biomarker combination based on TMB and copy-number alteration (CNA) for the prognosis of each tumor and prediction for ICI therapy in metastatic pan-cancer milieu. Experimental Design: Non-ICI–treated MSK pan-cancer cohort was used for prognosis analysis. Three independent immunotherapy cohorts, including non–small cell lung cancer (n = 240), skin cutaneous melanoma (n = 174), and mixed cancer (Dana-Farber, n = 98) patients from previous studies, were analyzed for efficacy of ICI therapy. Results: TMB and CNA showed optimized combination for the prognosis of most metastatic cancer types, and patients with TMBlowCNAlow showed better survival. In the predictive analysis, both TMB and CNA were independent predictive factors for ICI therapy. Remarkably, when TMB and CNA were jointly analyzed, those with TMBhighCNAlow showed favorable responses to ICI therapy. Meanwhile, TMBhighCNAlow as a new biomarker showed better prediction for ICI efficacy compared with either TMB-high or CNA-low alone. Furthermore, analysis of the non-ICI–treated MSK pan-cancer cohort supported that the joint stratification of TMB and CNA can be used to categorize tumors into distinct sensitivity to ICI therapy across pan-tumors. Conclusions: The combination of TMB and CNA can jointly stratify multiple metastatic tumors into groups with different prognosis and heterogeneous clinical responses to ICI treatment. Patients with TMBhighCNAlow cancer can be an optimal subgroup for ICI therapy.

Over the last decade, advances related to high-resolution mass spectrometry (HRMS) have led to improved capabilities for non-targeted chemical analyses. Important applications for these capabilities include identifying unknown xenobiotics and discovering emerging contaminants in human samples as exposure biomarkers. Despite technological advances, identifying unknown compounds by non-targeted analyses remains challenging due in part to the lack of MS spectral libraries and inherent sample complexity resulting in the generation of large amounts of MS data. While high resolution can separate nominally isobaric compounds in a mass spectrum, isomers cannot be distinguished. Much work also remains to develop models to predict both mass spectra and retention times for the unexplored regions of chemical space. In this review, we focus on recent advances and applications of non-targeted analyses using liquid chromatography - high-resolution mass spectrometry (LC-HRMS) in human biomonitoring, including sample preparation, molecular formula assignments, and prediction models for retention times and mass fragmentations, to enable and improve identifications of unknown chemicals. The purpose of this review is to improve our understanding of the applicability and limitations in both the analytical methods and data analysis aspects of non-targeted analysis in human exposure studies. We also discuss the challenges and prospects in this field for future research on sample preparation, identification confidence and accuracy, data processing tools, MS spectra comparability, liquid chromatographic retention time (RT) prediction algorithms, and quantitative capabilities.

Asian honeybee venom is widely used in traditional oriental medicine. Melittin is the main component of Asian honeybee venom. In the present study, an ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QqTOF-MS) method was used for accurate qualitative and quantitative analyses of melittin in Asian honeybee venom. The results showed that the dynamic linear range of melittin was from 0.094 to 20 &mu, g/mL, and the limit of quantification was 0.3125 &mu, g/mL. The spiking recovery of melittin in honeybee venom ranged from 84.88% to 93.05%. Eighteen Asian honeybee venom samples in eighteen batches were collected from two different zones of China, and their melittin contents were measured. The contents of melittin in Asian honeybee venom samples was 33.9&ndash, 46.23% of dry weight. This method proved a useful tool for the rapid evaluation of the authenticity and quality of Asian honeybee venom in terms of the melittin contents, and will contribute to a broader understanding of Asian honeybee venom.

BackgroundRadioimmunotherapy has a promising antitumor effect in hepatocellular carcinoma (HCC), depending on the regulatory effect of radiotherapy on tumor immune microenvironment. Ionizing radiation (IR)-induced DNA damage repair (DDR) pathway activation leads to the inhibition of immune microenvironment, thus impairing the antitumor effect of radioimmunotherapy. However, it is unclear whether inhibition of the DDR pathway can enhance the effect of radioimmunotherapy. In this study, we aim to explore the role of DDR inhibitor AZD6738 on the combination of radiotherapy and immune checkpoint inhibitors (ICIs) in HCC.MethodsC57BL/6 mouse subcutaneous tumor model was used to evaluate the ability of different treatment regimens in tumor growth control and tumor recurrence inhibition. Effects of each treatment regimen on the alterations of immunophenotypes including the quantification, activation, proliferating ability, exhaustion marker expression, and memory status were assessed by flow cytometry.ResultsAZD6738 further increased radiotherapy-stimulated CD8+T cell infiltration and activation and reverted the immunosuppressive effect of radiation on the number of Tregs in mice xenografts. Moreover, compared with radioimmunotherapy (radiotherapy plus anti-PD-L1 (Programmed death ligand 1)), the addition of AZD6738 boosted the infiltration, increased cell proliferation, enhanced interferon (IFN)-γ production ability of TIL (tumor-infiltrating lymphocyte) CD8+T cells, and caused a decreasing trend in the number of TIL Tregs and exhausted T cells in mice xenografts. Thus, the tumor immune microenvironment was significantly improved. Meanwhile, triple therapy (AZD6738 plus radiotherapy plus anti-PD-L1) also induced a better immunophenotype than radioimmunotherapy in mice spleens. As a consequence, triple therapy displayed greater benefit in antitumor efficacy and mice survival than radioimmunotherapy. Mechanism study revealed that the synergistic antitumor effect of AZD6738 with radioimmunotherapy relied on the activation of cyclic GMP–AMP synthase /stimulator of interferon genes (cGAS/STING) signaling pathway. Furthermore, triple therapy led to stronger immunologic memory and lasting antitumor immunity than radioimmunotherapy, thus preventing tumor recurrence in mouse models.ConclusionsOur findings indicate that AZD6738 might be a potential synergistic treatment for radioimmunotherapy to control the proliferation of HCC cells, prolong survival, and prevent tumor recurrence in patients with HCC by improving the immune microenvironment.

The fragmentation of patterns of moxifloxacin, 2-N-methylated moxifloxacin (analog 1), and 1-cyclopropyl-6,7-difluoro-8-methoxy-4-oxo-3-quinolinecarboxylic acid (analog 2) were investigated by electrospray ionization quadrupole time-of-flight tandem mass spectrometry in the positive-ion mode. Many unusual ions were detected in the tandem mass spectra of moxifloxacin. Although the structures of moxifloxacin and analog 1 were similar, the relative abundances of products varied greatly. Comparison of the relative abundances of the product ions that lost CO2 or H2O and complementary product ions resulting from sequential four-membered hydrogen rearrangement showed that the differences were related to the protonation sites. The loss of HF, probably though the formation of an ion/neutral complex, is of scientific interest. The identities of the major product ions were confirmed by deuterium-labeling experiments that demonstrated an unusual loss of a deuterium atom. The major differences in fragmentation patterns were compared to previous reports in the literature.

A series of six C-3 alkyl-substituted indole derivatives were investigated using electrospray, ionization quadrupole time-of-flight mass spectrometry (ESI-QTOF-MS). Anomalous [M-H](+) ions, were detected. Three possible pathways forming [M-H](+) were proposed. MS/MS spectra and radical, trapping experiment support direct loss of hydride anion. Notably, the positions losing the hydride, anion were different for these compounds, and were tentatively confirmed by D-labeled and MS/MS, spectra. Both steric hindrance and electronic effect might contribute the difference. (c) 2014 Elsevier B.V. All rights reserved.

Paeoniflorin and albiflorin were investigated by electrospray quadrupole time-of-flight mass spectrometry (ESI-QTOF-MS) in positive-ion mode, when ultrapure water was selected as solvent. In full-scan mode, the ions [M+H2O+Ca](2+), [M+2H(2)O+Ca](2+), [2M+Ca](2+), [3M+Ca](2+), and [4M+Ca](2+) as the primary ions were detected. Low-energy collision-induced experiments for the precursor[M+H2O+Ca](2+) and [2M+Ca](2+) ions provided many interesting productions. Most product ions are formed by combinations of a paeoniflorin (or albiflorin) molecule, benzoic acid anion, aglycone moiety, glucosyl group, H2O, or Ca2+ ion. Strong noncovalent bond formed by Ca2+ and O atom of neutral molecules and proper coordination number might contribute to producing these complex ions. A labeling experiment involving HID exchange of paeoniflorin indicates the H2O molecule did not come from solvent. Although the fragmentation patterns of the isomers of paeoniflorin and albiflorin are similar, they can be completely distinguished by the MS/MS spectra of either [M+H2O+Ca](2+) or [2M+Ca](2+). (C) 2013 Elsevier B.V. All rights reserved.

Liquid chromatography-Mass Spectrometry (LC-MS) has been widely used in natural product analysis. Global detection and identification of nontargeted components are desirable in natural product research, for example, in quality control of Chinese herbal medicine. Nontargeted components analysis continues to expand to exciting life science application domains such as metabonomics. With this background, the present review summarizes recent developments in the analysis of minor unknown natural products using LC-MS and mainly focuses on the determination of the molecular formulae, selection of precursor ions, and characteristic fragmentation patterns of the known compounds. This review consists of three parts. Firstly, the methods used to determine unique molecular formula of unknown compounds such as accurate mass measurements, MS