SETD2 Loss and ATR Inhibition Synergize to Promote cGAS Signaling and Immunotherapy Response in Renal Cell Carcinoma.

PURPOSE: Immune checkpoint blockade (ICB) demonstrates durable clinical benefits in a minority of patients with renal cell carcinoma (RCC). We aimed to identify the molecular features that determine the response and develop approaches to enhance it. EXPERIMENTAL DESIGN: We investigated the effects of SET domain-containing protein 2 (SETD2) loss on the DNA damage response pathway, the cytosolic DNA-sensing pathway, the tumor immune microenvironment, and the response to ataxia telangiectasia and rad3-related (ATR) and checkpoint inhibition in RCC. RESULTS: ATR inhibition activated the cyclic GMP-AMP synthase (cGAS)-interferon regulatory factor 3 (IRF3)-dependent cytosolic DNA-sensing pathway, resulting in the concurrent expression of inflammatory cytokines and immune checkpoints. Among the common RCC genotypes, SETD2 loss is associated with preferential ATR activation and sensitizes cells to ATR inhibition. SETD2 knockdown promoted the cytosolic DNA-sensing pathway in response to ATR inhibition. Treatment with the ATR inhibitor VE822 concurrently upregulated immune cell infiltration and immune checkpoint expression in Setd2 knockdown Renca tumors, providing a rationale for ATR inhibition plus ICB combination therapy. Setd2-deficient Renca tumors demonstrated greater vulnerability to ICB monotherapy or combination therapy with VE822 than Setd2-proficient tumors. Moreover, SETD2 mutations were associated with a higher response rate and prolonged overall survival in patients with ICB-treated RCC but not in patients with non-ICB-treated RCC. CONCLUSIONS: SETD2 loss and ATR inhibition synergize to promote cGAS signaling and enhance immune cell infiltration, providing a mechanistic rationale for the combination of ATR and checkpoint inhibition in patients with RCC with SETD2 mutations.

VHL-P138R and VHL-L163R Novel Variants: Mechanisms of VHL Pathogenicity Involving HIF-Dependent and HIF-Independent Actions.

The von Hippel-Lindau (VHL) disease is an autosomal dominant cancer syndrome caused by mutations in the VHL tumor suppressor gene. VHL protein (pVHL) forms a complex (VBC) with Elongins B-C, Cullin2, and Rbx1. Although other functions have been discovered, the most described function of pVHL is to recognize and target hypoxia-inducible factor (HIF) for degradation. This work comprises the functional characterization of two novel variants of the VHL gene (P138R and L163R) that have been described in our center in patients with VHL disease by in vitro, in vivo, and in silico approaches. In vitro, we found that these variants have a significantly shorter half-life compared to wild-type VHL but still form a functional VBC complex. Altered fibronectin deposition was evidenced for both variants using immunofluorescence. In vivo studies revealed that both variants failed to suppress tumor growth. By means of molecular dynamics simulations, we inspected in silico the nature of the changes introduced by each variant in the VBC complex. We have demonstrated the pathogenicity of P138R and L163R novel variants, involving HIF-dependent and HIF-independent mechanisms. These results provide the basis for future studies regarding the impact of structural alterations on posttranslational modifications that drive pVHL's fate and functions.

Clinical Features and Multiplatform Molecular Analysis Assist in Understanding Patient Response to Anti-PD-1/PD-L1 in Renal Cell Carcinoma.

Predicting response to ICI therapy among patients with renal cell carcinoma (RCC) has been uniquely challenging. We analyzed patient characteristics and clinical correlates from a retrospective single-site cohort of advanced RCC patients receiving anti-PD-1/PD-L1 monotherapy (N = 97), as well as molecular parameters in a subset of patients, including multiplexed immunofluorescence (mIF), whole exome sequencing (WES), T cell receptor (TCR) sequencing, and RNA sequencing (RNA-seq). Clinical factors such as the development of immune-related adverse events (odds ratio (OR) = 2.50, 95% confidence interval (CI) = 1.05-5.91) and immunological prognostic parameters, including a higher percentage of circulating lymphocytes (23.4% vs. 17.4%, p = 0.0015) and a lower percentage of circulating neutrophils (61.8% vs. 68.5%, p = 0.0045), correlated with response. Previously identified gene expression signatures representing pathways of angiogenesis, myeloid inflammation, T effector presence, and clear cell signatures also correlated with response. High PD-L1 expression (>10% cells) as well as low TCR diversity (≤644 clonotypes) were associated with improved progression-free survival (PFS). We corroborate previously published findings and provide preliminary evidence of T cell clonality impacting the outcome of RCC patients. To further biomarker development in RCC, future studies will benefit from integrated analysis of multiple molecular platforms and prospective validation.

Circulating exosomal microRNAs as novel potential detection biomarkers in pancreatic cancer.

Circulating exosomal microRNAs (ex-miRNAs) are reflective of the characteristics of the tumor and are valuable biomarkers in different types of tumor. In addition, miRNAs serve important roles in tumor progression and metastasis. The present study aimed to investigate the circulating ex-miRNA-21 and miRNA-210 as novel biomarkers for patients with pancreatic cancer (PC). For this purpose, serum ex-miRNAs were extracted from the serum of patients with PC (n=30) and chronic pancreatitis (CP) (n=10) using an RNA isolation kit. For exosome identification in serum, transmission electron micrographs were used to determine crystalline structure, western blotting was used to identify exosomal markers, and NanoSight was used for nanoparticle characterization. The relative expression levels of ex-miRNAs were quantified using quantitative PCR and compared between patients with PC and CP. The expression levels of both ex-miRNA-21 and miRNA-210 were significantly higher in patients with PC compared with patients with CP (both P<0.001). However, no significant difference in the relative serum levels of free miR-21 and miR-210 was observed between the 2 groups of patients (both P>0.05). ex-miRNA-21 and miRNA-210 were associated with tumor stage, as well as other factors. The diagnostic potential of ex-miRNA-21 and miRNA-210 levels was 83 and 85%, respectively. In addition, when ex-miRNA and serum carbohydrate antigen 19-9 expression levels were combined, the accuracy increased to 90%. The present study identified that serum ex-miRNAs, miRNA-21 and miRNA-210 may be of value as potential biomarkers and therapeutic targets for the diagnosis and treatment of PC.

Macrophage HIF-1α Is an Independent Prognostic Indicator in Kidney Cancer.

PURPOSE: Clear cell renal cell carcinoma (ccRCC) is frequently associated with inactivation of the von Hippel-Lindau tumor suppressor, resulting in activation of HIF-1α and HIF-2α. The current paradigm, established using mechanistic cell-based studies, supports a tumor promoting role for HIF-2α, and a tumor suppressor role for HIF-1α. However, few studies have comprehensively examined the clinical relevance of this paradigm. Furthermore, the hypoxia-associated factor (HAF), which regulates the HIFs, has not been comprehensively evaluated in ccRCC. EXPERIMENTAL DESIGN: To assess the involvement of HAF/HIFs in ccRCC, we analyzed their relationship to tumor grade/stage/outcome using tissue from 380 patients, and validated these associations using tissue from 72 additional patients and a further 57 patients treated with antiangiogenic therapy for associations with response. Further characterization was performed using single-cell mRNA sequencing (scRNA-seq), RNA-in situ hybridization (RNA-ISH), and IHC. RESULTS: HIF-1α was primarily expressed in tumor-associated macrophages (TAMs), whereas HIF-2α and HAF were expressed primarily in tumor cells. TAM-associated HIF-1α was significantly associated with high tumor grade and increased metastasis and was independently associated with decreased overall survival. Furthermore, elevated TAM HIF-1α was significantly associated with resistance to antiangiogenic therapy. In contrast, high HAF or HIF-2α were associated with low grade, decreased metastasis, and increased overall survival. scRNA-seq, RNA-ISH, and Western blotting confirmed the expression of HIF-1α in M2-polarized CD163-expressing TAMs. CONCLUSIONS: These findings highlight a potential role of TAM HIF-1α in ccRCC progression and support the reevaluation of HIF-1α as a therapeutic target and marker of disease progression.

PBRM1 loss defines a nonimmunogenic tumor phenotype associated with checkpoint inhibitor resistance in renal carcinoma.

A non-immunogenic tumor microenvironment (TME) is a significant barrier to immune checkpoint blockade (ICB) response. The impact of Polybromo-1 (PBRM1) on TME and response to ICB in renal cell carcinoma (RCC) remains to be resolved. Here we show that PBRM1/Pbrm1 deficiency reduces the binding of brahma-related gene 1 (BRG1) to the IFNγ receptor 2 (Ifngr2) promoter, decreasing STAT1 phosphorylation and the subsequent expression of IFNγ target genes. An analysis of 3 independent patient cohorts and of murine pre-clinical models reveals that PBRM1 loss is associated with a less immunogenic TME and upregulated angiogenesis. Pbrm1 deficient Renca subcutaneous tumors in mice are more resistance to ICB, and a retrospective analysis of the IMmotion150 RCC study also suggests that PBRM1 mutation reduces benefit from ICB. Our study sheds light on the influence of PBRM1 mutations on IFNγ-STAT1 signaling and TME, and can inform additional preclinical and clinical studies in RCC.

[Characteristics of Leontopodium leontopodioides leaf stochiometry with altitude and their relationship with soil nutrients in Qilian Mountains, Northwest China]. / ç¥è¿žå±±ä¸åŒæµ·æ‹”ç«ç»’è‰å¶ç‰‡ç”Ÿæ€åŒ–å­¦è®¡é‡ç‰¹å¾åŠå ¶ä¸ŽåœŸå£¤å »åˆ†çš„å ³ç³».

Foliar stoichiometry provides information on the biotic and abiotic changes of environment. We examined the stoichiometric characteristics of plant leaves at different altitudes to understand how plants adapt to environmental changes. Foliar stoichiometry of Leontopodium leontopodioides at various altitudes (2400, 2600, 2800, 3000 and 3200 m) were analyzed in the Qilian Mountains of China. Across the altitude gradient, mean value of leaf carbon content (LC), nitrogen content (LN), and phosphorous content (LP) of L. leontopodioides was 401.27, 23.99 and 1.22 g·kg-1, respectively. The mean value of LC:LN, LC:LP and LN:LP was 16.8, 352.5 and 20.7, respectively. LC, LC:LN, LC:LP and LN:LP initially increased with increases in altitude, rea-ching the maximum at 2600 m, then decreased, reaching the minimum at 3000 m, and finally increased again. LP exhibited the opposite trend. LN demonstrated an initial decrease with altitude, reaching the minimum at 2800 m, followed by an increase at higher altitudes. LC did not correlate with LN, but was significantly negatively correlated with LP. LN was significantly positively correlated with LP. There was no correlation between LN and any other stoichiometry ratios. LP showed a significantly negative correlation with other stoichiometry ratios. LC:LN, LC:LP, and LN:LP were positively correlated with each other. Both soil total nitrogen and total phosphorus affected LC and LN, whereas LP was significantly negatively correlated with soil total phosphorus. The results suggested that the growth of L. leontopodioides in the study region was mainly limited by P availability.

Reproducibility of CRISPR-Cas9 methods for generation of conditional mouse alleles: a multi-center evaluation.

BACKGROUND: CRISPR-Cas9 gene-editing technology has facilitated the generation of knockout mice, providing an alternative to cumbersome and time-consuming traditional embryonic stem cell-based methods. An earlier study reported up to 16% efficiency in generating conditional knockout (cKO or floxed) alleles by microinjection of 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides as donors (referred herein as "two-donor floxing" method). RESULTS: We re-evaluate the two-donor method from a consortium of 20 laboratories across the world. The dataset constitutes 56 genetic loci, 17,887 zygotes, and 1718 live-born mice, of which only 15 (0.87%) mice contain cKO alleles. We subject the dataset to statistical analyses and a machine learning algorithm, which reveals that none of the factors analyzed was predictive for the success of this method. We test some of the newer methods that use one-donor DNA on 18 loci for which the two-donor approach failed to produce cKO alleles. We find that the one-donor methods are 10- to 20-fold more efficient than the two-donor approach. CONCLUSION: We propose that the two-donor method lacks efficiency because it relies on two simultaneous recombination events in cis, an outcome that is dwarfed by pervasive accompanying undesired editing events. The methods that use one-donor DNA are fairly efficient as they rely on only one recombination event, and the probability of correct insertion of the donor cassette without unanticipated mutational events is much higher. Therefore, one-donor methods offer higher efficiencies for the routine generation of cKO animal models.

VHL substrate transcription factor ZHX2 as an oncogenic driver in clear cell renal cell carcinoma.

Inactivation of the von Hippel-Lindau (VHL) E3 ubiquitin ligase protein is a hallmark of clear cell renal cell carcinoma (ccRCC). Identifying how pathways affected by VHL loss contribute to ccRCC remains challenging. We used a genome-wide in vitro expression strategy to identify proteins that bind VHL when hydroxylated. Zinc fingers and homeoboxes 2 (ZHX2) was found as a VHL target, and its hydroxylation allowed VHL to regulate its protein stability. Tumor cells from ccRCC patients with VHL loss-of-function mutations usually had increased abundance and nuclear localization of ZHX2. Functionally, depletion of ZHX2 inhibited VHL-deficient ccRCC cell growth in vitro and in vivo. Mechanistically, integrated chromatin immunoprecipitation sequencing and microarray analysis showed that ZHX2 promoted nuclear factor κB activation. These studies reveal ZHX2 as a potential therapeutic target for ccRCC.

miR-200c inhibits TGF-ß-induced-EMT to restore trastuzumab sensitivity by targeting ZEB1 and ZEB2 in gastric cancer.

Gastric cancer is the fifth most common malignancy in the world, with Eastern Asia as one of areas with the highest incidence rates. Trastuzumab, a HER2-targeting antibody, combined with chemotherapy has been successfully employed for the gastric cancer patients with HER2 overexpression/amplification. However, trastuzumab resistance is a major problem in clinical practice. Here we observed that the trastuzumab-resistant gastric cancer cell line NCI-N87/TR expressed high levels of epithelial-mesenchymal transition factors and demonstrated increased migration and invasion capability compared with NCI-N87 cells. Downregulated E-cadherin and increased N-cadherin, TGF-ß, ZEB1, ZEB2, TWIST1, and Snail were detected in NCI-N87/TR cells. We also found that miR-200c was downregulated in NCI-N87/TR cells compared with parental cells NCI-87 by qRT-PCR. Treatment with TGF-ß downregulated the expression of miR-200c and upregulated ZEB2, and significantly decreased the trastuzumab sensitivity of NCI-N87 cells. miR-200c restored trastuzumab sensitivity and inhibited migration and invasion through suppressing ZEB1 and ZEB2. In summary, TGF-ß/ZEB2 axis plays an encouraging role in trastuzumab resistance of gastric cancer, while miR-200c overexpression downregulates ZEB1/ZEB2 and resensitizes drugs resistance. Our findings might provide a potential therapeutic strategy for trastuzumab resistance of gastric cancer.

HNF1B Loss Exacerbates the Development of Chromophobe Renal Cell Carcinomas.

Chromophobe renal cell carcinoma (ChRCC) is characterized by major changes in chromosomal copy number (CN). No model is available to precisely elucidate the molecular drivers of this tumor type. HNF1B is a master regulator of gene expression. Here, we report that the transcription factor HNF1B is downregulated in the majority of ChRCC and that the magnitude of HNF1B loss is unique to ChRCC. We also observed a strong correlation between reduced HNF1B expression and aneuploidy in ChRCC patients. In murine embryonic fibroblasts or ACHN cells, HNF1B deficiency reduced expression of the spindle checkpoint proteins MAD2L1 and BUB1B, and the cell-cycle checkpoint proteins RB1 and p27. Furthermore, it altered the chromatin accessibility of Mad2l1, Bub1b, and Rb1 genes and triggered aneuploidy development. Analysis of The Cancer Genome Atlas database revealed TP53 mutations in 33% of ChRCC where HNF1B expression was repressed. In clinical specimens, combining HNF1B loss with TP53 mutation produced an association with poor patient prognosis. In cells, combining HNF1B loss and TP53 mutation increased cell proliferation and aneuploidy. Our results show how HNF1B loss leads to abnormal mitotic protein regulation and induction of aneuploidy. We propose that coordinate loss of HNF1B and TP53 may enhance cellular survival and confer an aggressive phenotype in ChRCC. Cancer Res; 77(19); 5313-26. ©2017 AACR.

Autophagy degrades hypoxia inducible factors.

Hypoxia inducible factors are subjected to degradation by the ubiquitin-proteasome system (UPS), macroautophagy, and chaperone-mediated autophagy. The E3 ligases, ubiquitination, autophagy receptor proteins, and oxygen are determinants that direct hypoxia-inducible factors to different degradation pathways.

Resistance to Antiangiogenic Therapy Is Associated with an Immunosuppressive Tumor Microenvironment in Metastatic Renal Cell Carcinoma.

Renal cell carcinoma (RCC) is an immunogenic and proangiogenic cancer, and antiangiogenic therapy is the current mainstay of treatment. Patients with RCC develop innate or adaptive resistance to antiangiogenic therapy. There is a need to identify biomarkers that predict therapeutic resistance and guide combination therapy. We assessed the interaction between antiangiogenic therapy and the tumor immune microenvironment and determined their impact on clinical outcome. We found that antiangiogenic therapy-treated RCC primary tumors showed increased infiltration of CD4(+) and CD8(+) T lymphocytes, which was inversely related to patient overall survival and progression-free survival. Furthermore, specimens from patients treated with antiangiogenic therapy showed higher infiltration of CD4(+)FOXP3(+) regulatory T cells and enhanced expression of checkpoint ligand programed death-ligand 1 (PD-L1). Both immunosuppressive features were correlated with T-lymphocyte infiltration and were negatively related to patient survival. Treatment of RCC cell lines and RCC xenografts in immunodeficient mice with sunitinib also increased tumor PD-L1 expression. Results from this study indicate that antiangiogenic treatment may both positively and negatively regulate the tumor immune microenvironment. These findings generate hypotheses on resistance mechanisms to antiangiogenic therapy and will guide the development of combination therapy with PD-1/PD-L1-blocking agents.

The impact of FGFR1 and FRS2α expression on sorafenib treatment in metastatic renal cell carcinoma.

BACKGROUND: Angiogenesis plays a role in tumor growth and is partly mediated by factors in both the fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) pathways. Durable clinical responses with VEGF tyrosine kinase inhibitors (TKIs) may be limited by intrinsic tumor resistance. We hypothesized that FGF signaling may impact clinical responses to sorafenib. METHODS: Nephrectomy material was available from 40 patients with metastatic renal cell carcinoma (RCC) enrolled in a phase II clinical trial of sorafenib ± interferon (ClinicalTrials.gov Identifier NCT00126594). Fibroblast growth factor receptor 1 (FGFR1) and fibroblast growth factor receptor substrate 2 alpha (FRS2α) expression was assessed by in situ hybridization and immunofluorescence, respectively. The relationship between fibroblast growth factor pathway marker levels and progression-free survival (PFS) was analyzed using Kaplan-Meier and Cox proportional hazards regression methods. RESULTS: Univariate analysis indicated that more intense FGFR1 staining was associated with shorter PFS (log-rank P = 0.0452), but FRS2α staining was not significantly associated with PFS (log-rank P = 0.2610). Multivariate Cox proportional hazards regression models were constructed for FGFR1 and FRS2α individually, adjusting for baseline Eastern Cooperative Oncology Group performance status, treatment arm and anemia status. When adjusted for each of these variables, the highest intensity level of FGFR1 (level 3 or 4) had increased progression risk relative to the lowest intensity level of FGFR1 (level 1) (P = 0.0115). The highest intensity level of FRS2α (level 3 or 4) had increased progression risk relative to the lowest intensity level of FRS2α (level 1) (P = 0.0126). CONCLUSIONS: Increased expression of FGFR1 and FRS2α was associated with decreased PFS among patients with metastatic RCC treated with sorafenib. The results suggest that FGF pathway activation may impact intrinsic resistance to VEGF receptor inhibition.

The downregulation of miR-200c/141 promotes ZEB1/2 expression and gastric cancer progression.

Gastric cancer is the fourth most common malignancy in the world. Although microRNA-200 (miR-200) family members are thought to play roles in tumorigenesis, their functions in carcinogenesis are tumor specific, and the underlying mechanism of action still remains elusive. Few studies to date have addressed the dysregulation and function of miR-200 family members in gastric cancer progression. Here, we report that the miR-200 family members, miR-200c and miR-141, were significantly downregulated in gastric cancer specimens and gastric cancer cell lines. Importantly, on clinical samples, the expression of miR-200c and miR-141 was inversely correlated with TNM stage, tumor invasion depth (T), tumor embolus and disease-free survival. Wound-healing assay results showed that co-transfected miR-200c/141 could inhibit the migration and invasion capability of the gastric cell line SGC-7901. We also found that miR-200c and miR-141 directly targeted zinc finger E-box-binding homeobox 1/2 (ZEB1/2) and upregulated E-cadherin expression. In specimens from gastric cancer patients, reduced expression of miR-200c/141 was associated with increased expression of ZEB1 and/or ZEB2. In addition, the downregulation of miR-200c and miR-141 was found to be due to a highly methylated CpG island located upstream of their genomic sequence and/or upregulated TGF-ß signaling. Treatment with the chemotherapeutic agent decitabine, a known DNA methyltransferase inhibitor, increased miR-200c/141 expression and ameliorated decreased expression of miR-200c/141 induced by TGF-ß in SGC-7901 cells. Our study revealed that miR-200c/141 was downregulated by CpG island methylation and TGF-ß signaling, which decreased ZEB1/2 expression and increased E-cadherin expression to inhibit migration and invasion of gastric cancer cells and provides powerful evidence for the application of decitabine in gastric cancer treatment.

[Spatial heterogeneity of community structure of Picea crassifolia forest in Qilian Mountains, China].

We selected the grid of 5 m x 5 m in a dynamic monitoring plot (340 m x 300 m) as the sampling unites and chose 5 structural characteristics (density, average crown breadth, coverage, conspicuousness and average height) to study the spatial heterogeneity of community structure of Picea crassifolia forest in Dayekou Basin of Qilian Mountains by the fractal geometry and geostatistics methods. The results showed that the order of spatial variation in these characteristics was: density > average crown breadth > conspicuousness > coverage > average height, with the variation coefficient ranging from 43.7% to 79.6%. Moran's I index indicated that the structural variables had different degrees of spatial autocorrelation, and the order of autocorrelation was density > average height> coverage > average crown breadth > conspicuousness, with the range of -0.047-0.382. The exponential semivariation model well fitted the spatial variability in different structural features, and the range was 24.6-68.1 m. The variables displayed moderate spatial autocorrelation except for coverage, while the other variables had strong spatial autocorrelation, and the fractal dimension of the variables was close to 2, indicating a low spatial dependence among variables. The variables presented a superposing characteristic of zonal and patchy structures except for density and coverage, while the other variables presented strong patchiness property. Density and coverage had a certain spatial dependence on average crown breadth, conspicuousness and average height. Density and coverage for the spatial heterogeneity of community structural of P. crassifolia forests were 10 m and 0.5 hm2, respectively.

Dysregulation of HIF2α and autophagy in renal cell carcinoma.

Our recent study shows that autophagy collaborates with proteasomes to degrade endothelial PAS domain-containing protein 1 (EPAS1, also known as HIF2α) in a manner dependent on Von Hippel-Lindau (VHL) and sequestosome 1 (SQSTM1/p62). The genetic dysregulation of autophagy is a common feature of different subtypes of renal cell carcinoma (RCC).