LINE-1 expression in cancer correlates with p53 mutation, copy number alteration, and S phase checkpoint.

Retrotransposons are genomic DNA sequences that copy themselves to new genomic locations via RNA intermediates; LINE-1 is the only active and autonomous retrotransposon in the human genome. The mobility of LINE-1 is largely repressed in somatic tissues but is derepressed in many cancers, where LINE-1 retrotransposition is correlated with p53 mutation and copy number alteration (CNA). In cell lines, inducing LINE-1 expression can cause double-strand breaks (DSBs) and replication stress. Reanalyzing multiomic data from breast, ovarian, endometrial, and colon cancers, we confirmed correlations between LINE-1 expression, p53 mutation status, and CNA. We observed a consistent correlation between LINE-1 expression and the abundance of DNA replication complex components, indicating that LINE-1 may also induce replication stress in human tumors. In endometrial cancer, high-quality phosphoproteomic data allowed us to identify the DSB-induced ATM-MRN-SMC S phase checkpoint pathway as the primary DNA damage response (DDR) pathway associated with LINE-1 expression. Induction of LINE-1 expression in an in vitro model led to increased phosphorylation of MRN complex member RAD50, suggesting that LINE-1 directly activates this pathway.

Nivolumab with or without ipilimumab in patients with recurrent or metastatic cervical cancer (CheckMate 358): a phase 1-2, open-label, multicohort trial.

BACKGROUND: In preliminary findings from the recurrent or metastatic cervical cancer cohort of CheckMate 358, nivolumab showed durable anti-tumour responses, and the combination of nivolumab plus ipilimumab showed promising clinical activity. Here, we report long-term outcomes from this cohort. METHODS: CheckMate 358 was a phase 1-2, open-label, multicohort trial. The metastatic cervical cancer cohort enrolled patients from 30 hospitals and cancer centres across ten countries. Female patients aged 18 years or older with a histologically confirmed diagnosis of squamous cell carcinoma of the cervix with recurrent or metastatic disease, an Eastern Cooperative Oncology Group performance status of 0 or 1, and up to two previous systemic therapies were enrolled into the nivolumab 240 mg every 2 weeks group, the randomised groups (nivolumab 3 mg/kg every 2 weeks plus ipilimumab 1 mg/kg every 6 weeks [NIVO3 plus IPI1] or nivolumab 1 mg/kg every 3 weeks plus ipilimumab 3 mg/kg every 3 weeks for four cycles then nivolumab 240 mg every 2 weeks [NIVO1 plus IPI3]), or the NIVO1 plus IPI3 expansion group. All doses were given intravenously. Patients were randomly assigned (1:1) to NIVO3 plus IPI1 or NIVO1 plus IPI3 via an interactive voice response system. Treatment continued until disease progression, unacceptable toxicity, or consent withdrawal, or for up to 24 months. The primary endpoint was investigator-assessed objective response rate. Anti-tumour activity and safety were analysed in all treated patients. This study is registered with ClinicalTrials.gov (NCT02488759) and is now completed. FINDINGS: Between October, 2015, and March, 2020, 193 patients were recruited in the recurrent or metastatic cervical cancer cohort of CheckMate 358, of whom 176 were treated. 19 patients received nivolumab monotherapy, 45 received NIVO3 plus IPI1, and 112 received NIVO1 plus IPI3 (45 in the randomised group and 67 in the expansion group). Median follow-up times were 19·9 months (IQR 8·2-44·8) with nivolumab, 12·6 months (7·8-37·1) with NIVO3 plus IPI1, and 16·7 months (7·2-27·5) with pooled NIVO1 plus IPI3. Objective response rates were 26% (95% CI 9-51; five of 19 patients) with nivolumab, 31% (18-47; 14 of 45 patients) with NIVO3 plus IPI1, 40% (26-56; 18 of 45 patients) with randomised NIVO1 plus IPI3, and 38% (29-48; 43 of 112 patients) with pooled NIVO1 plus IPI3. The most common grade 3-4 treatment-related adverse events were diarrhoea, hepatic cytolysis, hyponatraemia, pneumonitis, and syncope (one [5%] patient each; nivolumab group), diarrhoea, increased gamma-glutamyl transferase, increased lipase, and vomiting (two [4%] patients each; NIVO3 plus IPI1 group), and increased lipase (nine [8%] patients) and anaemia (seven [6%] patients; pooled NIVO1 plus IPI3 group). Serious treatment-related adverse events were reported in three (16%) patients in the nivolumab group, 12 (27%) patients in the NIVO3 plus IPI1 group, and 47 (42%) patients in the pooled NIVO1 plus IPI3 group. There was one treatment-related death due to immune-mediated colitis in the NIVO1 plus IPI3 group. INTERPRETATION: Nivolumab monotherapy and nivolumab plus ipilimumab combination therapy showed promise in the CheckMate 358 study as potential treatment options for recurrent or metastatic cervical cancer. Future randomised controlled trials of nivolumab plus ipilimumab or other dual immunotherapy regimens are warranted to confirm treatment benefit in this patient population. FUNDING: Bristol Myers Squibb and Ono Pharmaceutical.

NCAPH serves as a prognostic factor and promotes the tumor progression in glioma through PI3K/AKT signaling pathway.

Non-SMC (Structural Maintenance of Chromosomes) condensin I complex subunit H (NCAPH) has been shown to facilitate progression and predict adverse prognostic outcome in many cancer types. However, the function of NCAPH in gliomas is still unclear. Series of experiments were taken to uncover the function of NCAPH in glioma. The expression of NCAPH and potential mechanism regulating progression of glioma was verified by bioinformatics analysis. Lentiviral transfection was used for establishment of loss-of-function and gain-of-function cell lines. CCK-8 assay and Colony-formation assay were used to evaluate proliferation. Transwell assay and Cell wound healing assay were used to assess migration and invasion. Cell cycle and apoptosis were measured by flow cytometry. Protein and RNA were quantified by WB and RT-PCR, respectively. The nude mice model of glioma was used to evaluate the effect of NCAPH in vivo. The expression of NCAPH increased significantly in glioma tissues and correlated with WHO grade, IDH wild-type and non-1p/19q codeletion. Glioma patients with high expression of NCAPH had an undesirable prognosis. Functionally, upregulated NCAPH promotes the malignant hallmarks of glioma cells in vivo and in vitro. NCAPH correlated with DNA damage repair ability of glioma cells and facilitated the proliferation, invasion, and migration of glioma cells by promoting the PI3K/AKT signaling pathway. This study identifies the important pro-tumor role of NCAPH in glioma and suggests that NCAPH is a potential therapeutic target.

GPR65 contributes to constructing immunosuppressive microenvironment in glioma.

Glioma, especially glioblastoma patients, present highly heterogeneous and immunosuppressive microenvironment, leading to their poor response to treatment and survival. Targeting the tumor microenvironment is considered a promising therapeutic strategy. M2 macrophages are highly infiltrated in glioma tissue, even up to 50% of the total number of bulk tissue cells. Here, we identified GPR65 as the hub gene of the M2 macrophage-related module in glioma through WGCNA analysis. The expression and prognosis analysis suggested that GPR65 was positively correlated with the malignancy and poor prognosis of glioma, and the heterogeneity analysis found that GPR65 was highly expressed in the vascular proliferation area of glioma, which matched the spatial expression characteristics of M2 macrophages. We further verified that GPR65 was highly expressed in macrophages but not tumor cells in the glioma microenvironment by single-cell data analysis and immunofluorescence. Most importantly, we found that inhibition of GPR65 was sufficient to reduce macrophages' polarization response to glioma cell and break the malignant cooperation with glioma cells. Our study reports the expression characteristics and malignant behavior of GPR65 in the glioma microenvironment, which provides a new alternative target of treatment to glioma microenvironment.

TCAF2 drives glioma cellular migratory/invasion properties through STAT3 signaling.

Glioma is an intracranial tumor characterized by high mortality and recurrence rates. In the present study, the association of TRPM8 channel-associated factor 2 (TCAF2) in glioma was investigated using bioinformatics, showing significant relationships with age, WHO grade, IDH, and 1p/19q status, as well as being an independent predictor of prognosis. Immunohistochemistry of a glioma sample microarray showed markedly increased TCAF2 expression in glioblastoma relative to lower-grade glioma, with elevated expression predominating in the tumor center. Raised TCAF2 levels promote glioma cell migratory/invasion properties through the epithelial-to-mesenchymal transition-like (EMT-like) process, shown by Transwell and scratch assays and western blotting. It was further found that the effects of TCAF2 were mediated by the activation of STAT3. These results suggest that TCAF2 promotes glioma cell migration and invasion, rendering it a potential drug target in glioma therapy.

HSP90-CDC37 functions as a chaperone for the oncogenic FGFR3-TACC3 fusion.

The FGFR3-TACC3 (F3-T3) fusion gene was discovered as an oncogenic molecule in glioblastoma and bladder cancers, and has subsequently been found in many cancer types. Notably, F3-T3 was found to be highly expressed in both untreated and matched recurrence glioblastoma under the concurrent radiotherapy and temozolomide (TMZ) treatment, suggesting that targeting F3-T3 is a valid strategy for treatment. Here, we show that the F3-T3 protein is a client of heat shock protein 90 (HSP90), forming a ternary complex with the cell division cycle 37 (CDC37). Deprivation of HSP90 or CDC37 disrupts the formation of the ternary complex, which destabilizes glycosylated F3-T3, and thereby suppresses F3-T3 oncogenic activity. Gliomas harboring F3-T3 are resistant to TMZ chemotherapy. HSP90 inhibitors sensitized F3-T3 glioma cells to TMZ via the inhibition of F3-T3 activation and potentiated TMZ-induced DNA damage. These results demonstrate that F3-T3 oncogenic function is dependent on the HSP90 chaperone system and suggests a new clinical option for targeting this genetic aberration in cancer.

Transcription factor profiling reveals molecular choreography and key regulators of human retrotransposon expression.

Transposable elements (TEs) represent a substantial fraction of many eukaryotic genomes, and transcriptional regulation of these factors is important to determine TE activities in human cells. However, due to the repetitive nature of TEs, identifying transcription factor (TF)-binding sites from ChIP-sequencing (ChIP-seq) datasets is challenging. Current algorithms are focused on subtle differences between TE copies and thus bias the analysis to relatively old and inactive TEs. Here we describe an approach termed "MapRRCon" (mapping repeat reads to a consensus) which allows us to identify proteins binding to TE DNA sequences by mapping ChIP-seq reads to the TE consensus sequence after whole-genome alignment. Although this method does not assign binding sites to individual insertions in the genome, it provides a landscape of interacting TFs by capturing factors that bind to TEs under various conditions. We applied this method to screen TFs' interaction with L1 in human cells/tissues using ENCODE ChIP-seq datasets and identified 178 of the 512 TFs tested as bound to L1 in at least one biological condition with most of them (138) localized to the promoter. Among these L1-binding factors, we focused on Myc and CTCF, as they play important roles in cancer progression and 3D chromatin structure formation. Furthermore, we explored the transcriptomes of The Cancer Genome Atlas breast and ovarian tumor samples in which a consistent anti-/correlation between L1 and Myc/CTCF expression was observed, suggesting that these two factors may play roles in regulating L1 transcription during the development of such tumors.

An Analysis of the Sensitivity of Proteogenomic Mapping of Somatic Mutations and Novel Splicing Events in Cancer.

Improvements in mass spectrometry (MS)-based peptide sequencing provide a new opportunity to determine whether polymorphisms, mutations, and splice variants identified in cancer cells are translated. Herein, we apply a proteogenomic data integration tool (QUILTS) to illustrate protein variant discovery using whole genome, whole transcriptome, and global proteome datasets generated from a pair of luminal and basal-like breast-cancer-patient-derived xenografts (PDX). The sensitivity of proteogenomic analysis for singe nucleotide variant (SNV) expression and novel splice junction (NSJ) detection was probed using multiple MS/MS sample process replicates defined here as an independent tandem MS experiment using identical sample material. Despite analysis of over 30 sample process replicates, only about 10% of SNVs (somatic and germline) detected by both DNA and RNA sequencing were observed as peptides. An even smaller proportion of peptides corresponding to NSJ observed by RNA sequencing were detected (<0.1%). Peptides mapping to DNA-detected SNVs without a detectable mRNA transcript were also observed, suggesting that transcriptome coverage was incomplete (∼80%). In contrast to germline variants, somatic variants were less likely to be detected at the peptide level in the basal-like tumor than in the luminal tumor, raising the possibility of differential translation or protein degradation effects. In conclusion, this large-scale proteogenomic integration allowed us to determine the degree to which mutations are translated and identify gaps in sequence coverage, thereby benchmarking current technology and progress toward whole cancer proteome and transcriptome analysis.

Regulation of CTR2 mRNA by the nonsense-mediated mRNA decay pathway.

The nonsense-mediated mRNA decay (NMD) pathway was originally identified as a pathway that degrades mRNAs with premature termination codons; however, NMD is now known to regulate natural mRNAs as well. Natural mRNAs are degraded by NMD due to the presence of specific NMD targeting features. An atypically long 3'-UTR is one of the features that has been shown to induce the rapid degradation of mRNAs by NMD in Saccharomyces cerevisiae and other organisms. S. cerevisiae CTR2 mRNAs have long 3'-UTRs and are sensitive to NMD, although the extent by which these long 3'-UTRs target the CTR2 mRNAs to the pathway is unknown. Here, we investigated the sequence elements that induce NMD of the CTR2 mRNAs and determined that the long CTR2 3'-UTR is sufficient to target an NMD-insensitive mRNA to the pathway. We also found that, although the CTR2 3'-UTR contributes to NMD-induced degradation, CTR2 mRNAs contain additional NMD-inducing features that function cooperatively with the atypically long 3'-UTR to trigger mRNA degradation. Lengthening the CTR2 ORF abrogates NMD and renders the mRNAs immune to the NMD pathway. Moreover, we found that transcription of CTR2 driven by the GPD promoter, which is not identical to the CTR2 promoter, affects degradation of the transcripts by NMD.

Physiological basis of copper tolerance of Saccharomyces cerevisiae nonsense-mediated mRNA decay mutants.

The eukaryotic nonsense-mediated mRNA decay pathway (NMD) is a specialized pathway that contributes to the recognition and rapid degradation of mRNA with premature termination codons. In addition to mRNAs containing premature termination codons, NMD degrades non-nonsense-containing, natural mRNAs. Approximately 5-10% of the total Saccharomyces cerevisiae transcriptome is affected when NMD is inactivated. The regulation of natural mRNAs by NMD has physiological consequences. However, the physiological outcomes associated with the degradation of specific natural mRNAs by NMD are not fully understood. Here, we examined the physiological consequences resulting from the NMD-mediated regulation of an mRNA involved in copper homeostasis, in an attempt to understand why nmd mutant strains are more tolerant of toxic copper levels than wild-type yeast strains. We found that wild-type (UPF1) and upf1Δ mutants accumulate similar amounts of total copper when grown in medium containing elevated levels of copper; however, the copper levels in the cytoplasm of wild-type yeast cells were higher than in the upf1Δ mutant. Copper tolerance by the upf1Δ mutant is dependent on the presence of CTR2. Deletion of CTR2 resulted in similar cytoplasmic copper levels in wild-type and upf1Δ mutant strains, regardless of the environmental copper levels. This suggests that CTR2 plays a role in regulating the level of copper in the cytoplasm. We also found that the upf1Δ mutant contained elevated copper levels in the vacuole relative to wild-type yeast cells, after both strains were exposed to elevated copper levels.

PDPN contributes to constructing immunosuppressive microenvironment in IDH wildtype glioma.

The tumor immunosuppressive microenvironment (IME) significantly affects tumor occurrence, progression, and prognosis, but the underlying molecular mechanisms remain to make known. We investigated the prognostic significance of PDPN and its role in IME in glioma. Weighted gene co-expression network analysis (WGCNA) found PDPN closely related to IDH wildtype status and higher immune score. Correlation analysis suggested PDPN was highly positively relevant to immune checkpoints expression and immune checkpoints block responding status. Correlation analysis together with verification in vitro suggested PDPN highly positively relevant tumor-associated neutrophils (TANs) and tumor-associated macrophages (TAMs). Least absolute shrinkage and selection operator (LASSO) regression employed to develop the prediction model with TANs and TAMs markers showed that high risk scores predicted worse prognosis. We highlight that PDPN overexpression is an independent prognostic indicator, and promotes macrophage M2 polarization and neutrophil degranulation, ultimately devotes to the formation of an immunosuppressive tumor microenvironment. Our findings contribute to re-recognizing the role of PDPN in IDH wildtype gliomas and implicate promising target therapy combined with immunotherapy for this highly malignant tumor.

Immune Checkpoint Blockade Outcome in Small-Cell Lung Cancer and Its Relationship With Retinoblastoma Mutation Status and Function.

PURPOSE: Immune checkpoint blockade (ICB) in conjunction with chemotherapy is approved for the treatment of extensive-stage small-cell lung cancer (SCLC). Although specific genomic abnormalities such as KEAP1 and STK11 gene mutations are associated with resistance to ICB in non-SCLC, no genomic abnormality has been found in association with resistance to ICB in SCLC. MATERIALS AND METHODS: We first analyzed a retrospective cohort of 42 patients with SCLC treated with single-agent ICB or ICB combination (data set A). We then validated our results in a large prospective clinical trial of 460 patients (CheckMate 032, data set B). DNA and RNA sequencing were performed. RESULTS: In data set A, patients treated with ICB with RB1 wild-type (WT) had a median overall survival (OS) of 23.1 months (95% CI, 9 to 37.5), whereas the RB1 mutant OS was 5 months (95% CI, 2.5 to 26; P = .04). Differentially expressed gene analysis between RB1 mutant and RB1 WT samples indicated the enrichment of downregulated immune-related genes and an immune exclusion phenotype among RB1 mutant but not in the RB1 WT tumor samples. We then assessed results from 460 patients enrolled in CheckMate 032, a trial of nivolumab (NIVO) or NIVO + ipilimumab only in SCLC. In this large cohort, RB1 WT patients had significantly improved outcome with NIVO therapy compared with mutant patients (hazard ratio, 1.41; 95% CI, 1.02 to 2.01; P = .041). High RB1 loss-of-function (LOF) signature scores significantly associated with neuroendocrine subtypes (ASCL1 and NeuroD1). However, neuroendocrine subtypes did not associate with OS. Remarkably, patients with lower RB1 LOF scores had longer OS following treatment with NIVO. CONCLUSION: SCLC patients with RB1 WT status or lower RB1 LOF signature scores by transcriptomics have better outcomes with ICB monotherapy.

A Novel Prognostic Signature Based on Glioma Essential Ferroptosis-Related Genes Predicts Clinical Outcomes and Indicates Treatment in Glioma.

Background: Ferroptosis is a form of programmed cell death (PCD) that has been implicated in cancer progression, although the specific mechanism is not known. Here, we used the latest DepMap release CRISPR data to identify the essential ferroptosis-related genes (FRGs) in glioma and their role in patient outcomes. Methods: RNA-seq and clinical information on glioma cases were obtained from the Chinese Glioma Genome Atlas (CGGA) and The Cancer Genome Atlas (TCGA). FRGs were obtained from the FerrDb database. CRISPR-screened essential genes (CSEGs) in glioma cell lines were downloaded from the DepMap portal. A series of bioinformatic and machine learning approaches were combined to establish FRG signatures to predict overall survival (OS) in glioma patients. In addition, pathways analysis was used to identify the functional roles of FRGs. Somatic mutation, immune cell infiltration, and immune checkpoint gene expression were analyzed within the risk subgroups. Finally, compounds for reversing high-risk gene signatures were predicted using the GDSC and L1000 datasets. Results: Seven FRGs (ISCU, NFS1, MTOR, EIF2S1, HSPA5, AURKA, RPL8) were included in the model and the model was found to have good prognostic value (p < 0.001) in both training and validation groups. The risk score was found to be an independent prognostic factor and the model had good efficacy. Subgroup analysis using clinical parameters demonstrated the general applicability of the model. The nomogram indicated that the model could effectively predict 12-, 36-, and 60-months OS and progression-free interval (PFI). The results showed the presence of more aggressive phenotypes (lower numbers of IDH mutations, higher numbers of EGFR and PTEN mutations, greater infiltration of immune suppressive cells, and higher expression of immune checkpoint inhibitors) in the high-risk group. The signaling pathways enriched closely related to the cell cycle and DNA damage repair. Drug predictions showed that patients with higher risk scores may benefit from treatment with RTK pathway inhibitors, including compounds that inhibit RTKs directly or indirectly by targeting downstream PI3K or MAPK pathways. Conclusion: In summary, the proposed cancer essential FRG signature predicts survival and treatment response in glioma.

TMEM158 promotes the proliferation and migration of glioma cells via STAT3 signaling in glioblastomas.

Glioblastoma is the most common primary intracranial malignant tumor in adults and has high morbidity and high mortality. TMEM158 has been reported to promote the progression of solid tumors. However, its potential role in glioma is still unclear. Here, we found that TMEM158 expression in human glioma cells in the tumor core was significantly higher than that in noncancerous cells at the tumor edge using bioinformatics analysis. Cancer cells in patients with primary GBMs harbored significantly higher expression of TMEM158 than those in patients with WHO grade II or III gliomas. Interestingly, regardless of tumor grading, human glioma samples that were IDH1-wild-type (IDH1-WT) exhibited higher expression of TMEM158 than those with IDH1-mutant (IDH1-Mut). We also illustrated that TMEM158 mRNA expression was correlated with poor overall survival in glioma patients. Furthermore, we demonstrated that silencing TMEM158 inhibited the proliferation of glioma cells and that TMEM158 overexpression promoted the migration and invasion of glioma cells by stimulating the EMT process. We found that the underlying mechanism involves STAT3 activation mediating TMEM158-driven glioma progression. In vivo results further confirmed the inhibitory effect of the TMEM158 downregulation on glioma growth. Collectively, these findings further our understanding of the oncogenic function of TMEM158 in gliomas, which represents a potential therapeutic target, especially for GBMs.

Nivolumab plus rucaparib for metastatic castration-resistant prostate cancer: results from the phase 2 CheckMate 9KD trial.

BACKGROUND: CheckMate 9KD (NCT03338790) is a non-randomized, multicohort, phase 2 trial of nivolumab plus other anticancer treatments for metastatic castration-resistant prostate cancer (mCRPC). We report results from cohorts A1 and A2 of CheckMate 9KD, specifically evaluating nivolumab plus rucaparib. METHODS: CheckMate 9KD enrolled adult patients with histologically confirmed mCRPC, ongoing androgen deprivation therapy, and an Eastern Cooperative Oncology Group performance status of 0-1. Cohort A1 included patients with postchemotherapy mCRPC (1-2 prior taxane-based regimens) and ≤2 prior novel hormonal therapies (eg, abiraterone, enzalutamide, apalutamide); cohort A2 included patients with chemotherapy-naïve mCRPC and prior novel hormonal therapy. Patients received nivolumab 480 mg every 4 weeks plus rucaparib 600 mg two times per day (nivolumab dosing ≤2 years). Coprimary endpoints were objective response rate (ORR) per Prostate Cancer Clinical Trials Working Group 3 and prostate-specific antigen response rate (PSA50-RR; ≥50% PSA reduction) in all-treated patients and patients with homologous recombination deficiency (HRD)-positive tumors, determined before enrollment. Secondary endpoints included radiographic progression-free survival (rPFS), overall survival (OS), and safety. RESULTS: Outcomes (95% CI) among all-treated, HRD-positive, and BRCA1/2-positive populations for cohort A1 were confirmed ORR: 10.3% (3.9-21.2) (n=58), 17.2% (5.8-35.8) (n=29), and 33.3% (7.5-70.1) (n=9); confirmed PSA50-RR: 11.9% (5.9-20.8) (n=84), 18.2% (8.2-32.7) (n=44), and 41.7% (15.2-72.3) (n=12); median rPFS: 4.9 (3.7-5.7) (n=88), 5.8 (3.7-8.4) (n=45), and 5.6 (2.8-15.7) (n=12) months; and median OS: 13.9 (10.4-15.8) (n=88), 15.4 (11.4-18.2) (n=45), and 15.2 (3.0-not estimable) (n=12) months. For cohort A2 they were confirmed ORR: 15.4% (5.9-30.5) (n=39), 25.0% (8.7-49.1) (n=20), and 33.3% (7.5-70.1) (n=9); confirmed PSA50-RR: 27.3% (17.0-39.6) (n=66), 41.9 (24.5-60.9) (n=31), and 84.6% (54.6-98.1) (n=13); median rPFS: 8.1 (5.6-10.9) (n=71), 10.9 (6.7-12.0) (n=34), and 10.9 (5.6-12.0) (n=15) months; and median OS: 20.2 (14.1-22.8) (n=71), 22.7 (14.1-not estimable) (n=34), and 20.2 (11.1-not estimable) (n=15) months. In cohorts A1 and A2, respectively, the most common any-grade and grade 3-4 treatment-related adverse events (TRAEs) were nausea (40.9% and 40.8%) and anemia (20.5% and 14.1%). Discontinuation rates due to TRAEs were 27.3% and 23.9%, respectively. CONCLUSIONS: Nivolumab plus rucaparib is active in patients with HRD-positive postchemotherapy or chemotherapy-naïve mCRPC, particularly those harboring BRCA1/2 mutations. Safety was as expected, with no new signals identified. Whether the addition of nivolumab incrementally improves outcomes versus rucaparib alone cannot be determined from this trial. TRIAL REGISTRATION NUMBER: NCT03338790.

MXRA5 Is a Novel Immune-Related Biomarker That Predicts Poor Prognosis in Glioma.

BACKGROUND: Glioma is the most common primary intracranial tumor and is associated with poor prognosis. Identifying effective biomarkers for glioma is particularly important. MXRA5, a secreted glycoprotein, is involved in cell adhesion and extracellular matrix remodeling and has been reported to be expressed in many cancers. However, the role and mechanism of action of MXRA5 in gliomas remain unclear. This study was aimed at investigating the role of MXRA5 at the transcriptome level and its clinical prognostic value. METHODS: In this study, RNA microarray data of 301 glioma patients from the Chinese Glioma Genome Atlas (CGGA) were collected as a training cohort and RNA-seq data of 702 glioma samples from The Cancer Genome Atlas (TCGA) were used for validation. We analyzed the clinical and molecular characteristics as well as the prognostic value of MXRA5 in glioma. In addition, the expression level of MXRA was evaluated in 28 glioma tissue samples. RESULTS: We found that MXRA5 expression was significantly upregulated in high-grade gliomas and IDH wild-type gliomas compared to controls. Receiver operating characteristic (ROC) analysis showed that MXRA5 is a potential marker of the mesenchymal subtype of glioblastoma multiforme (GBM). We found that MXRA5 expression is highly correlated with immune checkpoint molecule expression levels and tumor-associated macrophage infiltration. High MXRA5 expression could be used as an independent indicator of poor prognosis in glioma patients. CONCLUSION: Our study suggests that MXRA5 expression is associated with the clinicopathologic features and poor prognosis of gliomas. MXRA5 may play an important role in the immunosuppressive microenvironment of glioma. As a secreted glycoprotein, MXRA5 is a potential circulating biomarker for glioma, deserving further investigation.

ACT001 reduces the expression of PD-L1 by inhibiting the phosphorylation of STAT3 in glioblastoma.

ACT001, which is derived from an ancient anti-inflammatory drug, has been shown to cross the blood-brain barrier in preclinical studies and has demonstrated anti-glioblastoma (GBM) activity in clinical trials. However, its pharmacological potential for anti-GBM immune response modulation remains unclear. The chemical structure of ACT001 indicates that it may bind to STAT3 and thus modulate antitumor immune response. Methods: Bioinformatics and immunohistochemistry (IHC) were used to assess STAT3 and PD-L1 expression in gliomas. Western blotting, RT-PCR and immunofluorescence were used to detect PD-L1 and p-STAT3 expression in glioma cells exposed to ACT001. Chromatin immunoprecipitation, an ACT001-Biotin probe, and a dual-luciferase reporter assay were used to detect direct modulation. The in vivo efficacy of ACT001 was evaluated in GL261 murine glioma model. Survival analyses were conducted using the log-rank (Mantel-Cox) test. Results: Bioinformatic analysis of 1,837 samples from 4 public glioma datasets showed that STAT3 mRNA expression was correlated with the degree of malignancy and therapeutic resistance and that STAT3 mRNA expression was related to immunosuppression, leukocyte infiltration, and PD-L1 expression. IHC staining of 53 tissue samples confirmed that relatively high phosphorylated STAT3 and PD-L1 protein expression was associated with a relatively advanced World Health Organization (WHO) glioma grade. Next, we confirmed that ACT001 treatment reduced PD-L1 expression and STAT3 phosphorylation. An ACT001-biotin probe was used to verify that ACT001 bound to STAT3. We also demonstrated that STAT3 bound to the PD-L1 promoter. The inhibition of PD-L1 expression and STAT3 phosphorylation by ACT001 could be rescued by STAT3 overexpression. Additionally, ACT001 inhibited GBM growth and decreased PD-L1 expression in vivo. The expression of the M2 markers CD206 and CD163 was decreased, while that of the antitumor immune markers iNOS and IFNγ was increased by ACT001 in vivo. Conclusion: Our results demonstrate that STAT3 plays a key role in immunosuppression of glioma and is inhibited by ACT001. ACT001 inhibits PD-L1 transcription and modulates anti-tumor immune response in glioma bearing mice. These findings will help us to understand the mechanism of ACT001 in GBM therapy.

LINE-1 ORF2p expression is nearly imperceptible in human cancers.

BACKGROUND: Long interspersed element-1 (LINE-1, L1) is the major driver of mobile DNA activity in modern humans. When expressed, LINE-1 loci produce bicistronic transcripts encoding two proteins essential for retrotransposition, ORF1p and ORF2p. Many types of human cancers are characterized by L1 promoter hypomethylation, L1 transcription, L1 ORF1p protein expression, and somatic L1 retrotransposition. ORF2p encodes the endonuclease and reverse transcriptase activities required for L1 retrotransposition. Its expression is poorly characterized in human tissues and cell lines. RESULTS: We report mass spectrometry-based tumor proteome profiling studies wherein ORF2p eludes detection. To test whether ORF2p could be detected with specific reagents, we developed and validated five rabbit monoclonal antibodies with immunoreactivity for specific epitopes on the protein. These reagents readily detect ectopic ORF2p expressed from bicistronic L1 constructs. However, endogenous ORF2p is not detected in human tumor samples or cell lines by western blot, immunoprecipitation, or immunohistochemistry despite high levels of ORF1p expression. Moreover, we report endogenous ORF1p-associated interactomes, affinity isolated from colorectal cancers, wherein we similarly fail to detect ORF2p. These samples include primary tumors harboring hundreds of somatically acquired L1 insertions. The new data are available via ProteomeXchange with identifier PXD013743. CONCLUSIONS: Although somatic retrotransposition provides unequivocal genetic evidence for the expression of ORF2p in human cancers, we are unable to directly measure its presence using several standard methods. Experimental systems have previously indicated an unequal stoichiometry between ORF1p and ORF2p, but in vivo, the expression of these two proteins may be more strikingly uncoupled. These findings are consistent with observations that ORF2p is not tolerable for cell growth.

Comprehensive Scanning Mutagenesis of Human Retrotransposon LINE-1 Identifies Motifs Essential for Function.

Long Interspersed Nuclear Element-1 (LINE-1, L1) is the only autonomous active transposable element in the human genome. The L1-encoded proteins ORF1p and ORF2p enable the element to jump from one locus to another via a "copy-and-paste" mechanism. ORF1p is an RNA-binding protein, and ORF2p has endonuclease and reverse transcriptase activities. The huge number of truncated L1 remnants in the human genome suggests that the host has likely evolved mechanisms to prevent full L1 replication, and thereby decrease the proliferation of active elements and reduce the mutagenic potential of L1. In turn, L1 appears to have a minimized length to increase the probability of successful full-length replication. This streamlining would be expected to lead to high information density. Here, we describe the construction and initial characterization of a library of 538 consecutive trialanine substitutions that scan along ORF1p and ORF2p to identify functionally important regions. In accordance with the streamlining hypothesis, retrotransposition was overall very sensitive to mutations in ORF1p and ORF2p; only 16% of trialanine mutants retained near-wild-type (WT) activity. All ORF1p mutants formed near-WT levels of mRNA transcripts and 75% formed near-WT levels of protein. Two ORF1p mutants presented a unique nucleolar-relocalization phenotype. Regions of ORF2p that are sensitive to mutagenesis but lack phylogenetic conservation were also identified. We provide comprehensive information on the regions most critical to retrotransposition. This resource will guide future studies of intermolecular interactions that form with RNA, proteins, and target DNA throughout the L1 life cycle.