Hidden secrets of the cancer genome: unlocking the impact of non-coding mutations in gene regulatory elements.

Discoveries in the field of genomics have revealed that non-coding genomic regions are not merely "junk DNA", but rather comprise critical elements involved in gene expression. These gene regulatory elements (GREs) include enhancers, insulators, silencers, and gene promoters. Notably, new evidence shows how mutations within these regions substantially influence gene expression programs, especially in the context of cancer. Advances in high-throughput sequencing technologies have accelerated the identification of somatic and germline single nucleotide mutations in non-coding genomic regions. This review provides an overview of somatic and germline non-coding single nucleotide alterations affecting transcription factor binding sites in GREs, specifically involved in cancer biology. It also summarizes the technologies available for exploring GREs and the challenges associated with studying and characterizing non-coding single nucleotide mutations. Understanding the role of GRE alterations in cancer is essential for improving diagnostic and prognostic capabilities in the precision medicine era, leading to enhanced patient-centered clinical outcomes.

Long non-coding intergenic RNA, LINC00273 induces cancer metastasis and stemness via miRNA sponging in triple negative breast cancer.

LncRNAs and miRNAs, being the master regulators of gene expression, are crucial functional mediators in cancer. Our study unveils the critical regulatory role of the metastatic long non-coding RNA LINC00273 as the master regulator of oncogenes involved in cancer metastasis, stemness, and chemoresistance via its miRNA sponging mechanism. M2 (a salt of bis-Schiff base) mediated G quadruplex (G4) stabilization at the LINC00273 gene promoter remarkably inhibits LINC00273 transcription. Therefore, low-level LINC00273 transcripts are unable to efficiently sponge the miRNAs, which subsequently become available to bind and downregulate their target oncogenes. We have observed significantly different global transcriptomic scenarios in LINC00273 upregulated and downregulated circumstances in MDA-MB-231 triple-negative breast cancer model. Additionally, we have found the G4 sequence in the LINC00273 RNA to play a critical role in miRNA sequestration. miRNAs (miR-6789-5p, miR200b, miR-125b-5p, miR-4268, miR3978) have base pairing complementarity within the G4 region of LINC00273 RNA and the 3'-UTR (untranslated region) of MAPK12, TGF-ß1, and SIX-1 transcripts. We have reported TGF-ß1, SIX-1, and MAPK12 to be the direct downstream targets of LINC00273. The correlation between abnormal expression of lncRNA LINC00273 and TNBC aggressiveness strongly evidenced in our study shall accelerate the development of lncRNA-based anti-metastatic therapeutics.

Air Quality and Health Impacts of Onshore Oil and Gas Flaring and Venting Activities Estimated Using Refined Satellite-Based Emissions.

Emissions from flaring and venting (FV) in oil and gas (O&G) production are difficult to quantify due to their intermittent activities and lack of adequate monitoring and reporting. Given their potentially significant contribution to total emissions from the O&G sector in the United States, we estimate emissions from FV using Visible Infrared Imaging Radiometer Suite satellite observations and state/local reported data on flared gas volume. These refined estimates are higher than those reported in the National Emission Inventory: by up to 15 times for fine particulate matter (PM2.5), two times for sulfur dioxides, and 22% higher for nitrogen oxides (NOx). Annual average contributions of FV to ozone (O3), NO2, and PM2.5 in the conterminous U.S. (CONUS) are less than 0.15%, but significant contributions of up to 60% are found in O&G fields with FV. FV contributions are higher in winter than in summer months for O3 and PM2.5; an inverse behavior is found for NO2. Nitrate aerosol contributions to PM2.5 are highest in the Denver basin whereas in the Permian and Bakken basins, sulfate and elemental carbon aerosols are the major contributors. Over four simulated months in 2016 for the entire CONUS, FV contributes 210 additional instances of exceedances to the daily maximum 8-hr average O3 and has negligible contributions to exceedance of NO2 and PM2.5, given the current form of the national ambient air quality standards. FV emissions are found to cause over $7.4 billion in health damages, 710 premature deaths, and 73,000 asthma exacerbations among children annually.

Evaluating the sensitivity of mortality attributable to pollution to modeling Choices: A case study for Colorado.

We evaluated the sensitivity of estimated PM2.5 and NO2 health impacts to varying key input parameters and assumptions including: 1) the spatial scale at which impacts are estimated, 2) using either a single concentration-response function (CRF) or using racial/ethnic group specific CRFs from the same epidemiologic study, 3) assigning exposure to residents based on home, instead of home and work locations for the state of Colorado. We found that the spatial scale of the analysis influences the magnitude of NO2, but not PM2.5, attributable deaths. Using county-level predictions instead of 1 km2 predictions of NO2 resulted in a lower estimate of mortality attributable to NO2 by âˆ¼ 50 % for all of Colorado for each year between 2000 and 2020. Using an all-population CRF instead of racial/ethnic group specific CRFs results in a 130 % higher estimate of annual mortality attributable for the white population and a 40 % and 80 % lower estimate of mortality attributable to PM2.5 for Black and Hispanic residents, respectively. Using racial/ethnic group specific CRFs did not result in a different estimation of NO2 attributable mortality for white residents, but led to âˆ¼ 50 % lower estimates of mortality for Black residents, and 290 % lower estimate for Hispanic residents. Using NO2 based on home instead of home and workplace locations results in a smaller estimate of annual mortality attributable to NO2 for all of Colorado by 2 % each year and 0.3 % for PM2.5. Our results should be interpreted as an exercise to make methodological recommendations for future health impact assessments of pollution.

Switchable tetraplex elements in the heterogeneous nuclear ribonucleoprotein K promoter: micro-environment dictated structural transitions of G/C rich elements.

We have elucidated the hnRNP K promoter as a hotspot for tetraplex-based molecular switches receptive to micro-environmental stimuli. We have characterised the structural features of four tetraplex-forming loci and identified them as binding sites of transcription factors. These segments form either G-quadruplex or i-motif structures, the structural dynamicity of which has been studied in depth via several biophysical techniques. The tetraplexes display high dynamicity and are influenced by both pH and KCl concentrations in vitro. The loci complementary to these sequences form additional non-canonical secondary structures. In the cellular context, the most eminent observation of this study is the binding of hnRNP K to the i-motif forming sequences in its own promoter. We are the first to report a probable transcriptional autoregulatory function of hnRNP K in coordination with higher-order DNA structures. Herein, we also report the positive interaction of the endogenous tetraplexes with Sp1, a well-known transcriptional regulator. Treatment with tetraplex-specific small molecule ligands further uncovered G-quadruplexes' functioning as repressors and i-motifs as activators in this context. Together, our findings strongly indicate the critical regulatory role of the identified tetraplex elements in the hnRNP K promoter.Communicated by Ramaswamy H. Sarma.

Phase I LITESPARK-001 study of belzutifan for advanced solid tumors: Extended 41-month follow-up in the clear cell renal cell carcinoma cohort.

BACKGROUND: Accumulation of the HIF-2α transcription factor is an oncogenic event implicated in the tumorigenesis of clear cell renal cell carcinoma (ccRCC). In the phase I LITESPARK-001 study, the first-in-class HIF-2α inhibitor belzutifan demonstrated antitumor activity and an acceptable safety profile for pretreated patients with advanced ccRCC. Updated data with additional follow-up of > 40 months are presented. METHODS: LITESPARK-001 is an ongoing open-label study with a 3 + 3 dose-escalation design followed by an expansion phase. Patients with ccRCC enrolled at 7 sites received belzutifan 120 mg orally once daily until disease progression, unacceptable toxicity, or patient withdrawal. The data cutoff date was July 15, 2021. The primary end point was identifying the maximum tolerated dose and/or the recommended phase II dose. Secondary end points included objective response rate (ORR) and duration of response (DOR) per RECIST v1.1 by investigator assessment and safety. RESULTS: Median follow-up was 41.2 months (range, 38.2-47.7). Patients received a median of 3 (range, 1-9) prior systemic therapies. Of 55 patients, 14 (25 %) achieved an objective response. Median DOR was not reached (range, 3.1 + to 38.0 + months). Adverse events (AEs) attributed to study treatment by investigator assessment were reported in 53 patients (96 %). 22 patients (40 %) had grade 3 treatment-related AEs; the most common were anemia (n = 13; 24 %) and hypoxia (n = 7; 13 %). No grade 4 or 5 treatment-related AEs occurred. CONCLUSION: After a median follow-up of 41.2 months, belzutifan monotherapy demonstrated durable antitumor activity in patients with advanced ccRCC and acceptable safety. CLINICALTRIALS: gov. NCT02974738.

State-of-the-Science Data and Methods Need to Guide Place-Based Efforts to Reduce Air Pollution Inequity.

BACKGROUND: Recently enacted environmental justice policies in the United States at the state and federal level emphasize addressing place-based inequities, including persistent disparities in air pollution exposure and associated health impacts. Advances in air quality measurement, models, and analytic methods have demonstrated the importance of finer-scale data and analysis in accurately quantifying the extent of inequity in intraurban pollution exposure, although the necessary degree of spatial resolution remains a complex and context-dependent question. OBJECTIVE: The objectives of this commentary were to a) discuss ways to maximize and evaluate the effectiveness of efforts to reduce air pollution disparities, and b) argue that environmental regulators must employ improved methods to project, measure, and track the distributional impacts of new policies at finer geographic and temporal scales. DISCUSSION: The historic federal investments from the Inflation Reduction Act, the Infrastructure Investment and Jobs Act, and the Biden Administration's commitment to Justice40 present an unprecedented opportunity to advance climate and energy policies that deliver real reductions in pollution-related health inequities. In our opinion, scientists, advocates, policymakers, and implementing agencies must work together to harness critical advances in air quality measurements, models, and analytic methods to ensure success. https://doi.org/10.1289/EHP13063.

Ethnoracial Disparities in Nitrogen Dioxide Pollution in the United States: Comparing Data Sets from Satellites, Models, and Monitors.

In the United States (U.S.), studies on nitrogen dioxide (NO2) trends and pollution-attributable health effects have historically used measurements from in situ monitors, which have limited geographical coverage and leave 66% of urban areas unmonitored. Novel tools, including remotely sensed NO2 measurements and estimates of NO2 estimates from land-use regression and photochemical models, can aid in assessing NO2 exposure gradients, leveraging their complete spatial coverage. Using these data sets, we find that Black, Hispanic, Asian, and multiracial populations experience NO2 levels 15-50% higher than the national average in 2019, whereas the non-Hispanic White population is consistently exposed to levels that are 5-15% lower than the national average. By contrast, the in situ monitoring network indicates more moderate ethnoracial NO2 disparities and different rankings of the least- to most-exposed ethnoracial population subgroup. Validating these spatially complete data sets against in situ observations reveals similar performance, indicating that all these data sets can be used to understand spatial variations in NO2. Integrating in situ monitoring, satellite data, statistical models, and photochemical models can provide a semiobservational record, complete geospatial coverage, and increasingly high spatial resolution, enhancing future efforts to characterize, map, and track exposure and inequality for highly spatially heterogeneous pollutants like NO2.

Unraveling the structural aspects of the G-quadruplex in SMO promoter and elucidating its contribution in transcriptional regulation.

We located a 25 nt G-rich sequence in the promoter region of SMO oncogene. We performed an array of biophysical and biochemical assays and confirmed the formation of a parallel G quadruplex (SMO1-GQ) by the identified sequence. SMO1-GQ is highly conserved in primates. For a comprehensive characterization of the SMO quadruplex structure, we have performed spectroscopic and in silico analysis with established GQ binder small molecules TMPyP4 and BRACO-19. We observed comparatively higher stable interaction of BRACO-19 with SMO1-GQ. Structure-based, rational drug design against SMO1-GQ to target SMO oncogene requires a detailed molecular anatomy of the G-quadruplex. We structurally characterised the SMO1-GQ using DMS footprinting assay and molecular modelling, docking, and MD simulation to identify the probable atomic regions that interact with either of the small molecules. We further investigated SMO1-GQ in vivo by performing chromatin immunoprecipitation (ChIP) assay. ChIP data revealed that this gene element functions as a scaffold for a number of transcription factors: specificity protein (Sp1), nucleolin (NCL), non-metastatic cell 2 (NM23-H2), cellular nucleic acid binding protein (CNBP), and heterogeneous nuclear ribonucleoprotein K (hnRNPK) which reflects the SMO1-P1 G-quadruplex to be the master regulator of SMO1 transcriptional activity. The strong binding interaction detected between SMO1-GQ and BRACO-19 contemplates the potential of the G quadruplex as a promising anti-cancer druggable target to downregulate SMO1 oncogene driven cancers.Communicated by Ramaswamy H. Sarma.

Using evolutionary constraint to define novel candidate driver genes in medulloblastoma.

Current knowledge of cancer genomics remains biased against noncoding mutations. To systematically search for regulatory noncoding mutations, we assessed mutations in conserved positions in the genome under the assumption that these are more likely to be functional than mutations in positions with low conservation. To this end, we use whole-genome sequencing data from the International Cancer Genome Consortium and combined it with evolutionary constraint inferred from 240 mammals, to identify genes enriched in noncoding constraint mutations (NCCMs), mutations likely to be regulatory in nature. We compare medulloblastoma (MB), which is malignant, to pilocytic astrocytoma (PA), a primarily benign tumor, and find highly different NCCM frequencies between the two, in agreement with the fact that malignant cancers tend to have more mutations. In PA, a high NCCM frequency only affects the BRAF locus, which is the most commonly mutated gene in PA. In contrast, in MB, >500 genes have high levels of NCCMs. Intriguingly, several loci with NCCMs in MB are associated with different ages of onset, such as the HOXB cluster in young MB patients. In adult patients, NCCMs occurred in, e.g., the WASF-2/AHDC1/FGR locus. One of these NCCMs led to increased expression of the SRC kinase FGR and augmented responsiveness of MB cells to dasatinib, a SRC kinase inhibitor. Our analysis thus points to different molecular pathways in different patient groups. These newly identified putative candidate driver mutations may aid in patient stratification in MB and could be valuable for future selection of personalized treatment options.

Noncoding RNA as an influential epigenetic modulator with promising roles in cancer therapeutics.

The epigenetic landscape has an important role in cellular homeostasis and its deregulation leads to cancer. Noncoding (nc)RNA networks function as major regulators of cellular epigenetic hallmarks via regulation of vital processes, such as histone modification and DNA methylation. They are integral intracellular components affecting multiple oncogenic pathways. Thus, it is important to elucidate the effects of ncRNA networks on epigenetic programming that lead to the initiation and progression of cancer. In this review, we summarize the effects of epigenetic modification influenced by ncRNA networks and crosstalk between diverse classes of ncRNA, which could aid the development of patient-specific cancer therapeutics targeting ncRNAs, thereby altering cellular epigenetics.

Belzutifan plus cabozantinib for patients with advanced clear cell renal cell carcinoma previously treated with immunotherapy: an open-label, single-arm, phase 2 study.

BACKGROUND: Few treatment options are available for patients with advanced renal cell carcinoma who have received previous anti-PD-1-based or anti-PD-L1-based immunotherapy. Combining belzutifan, an HIF-2α inhibitor, with cabozantinib, a multitargeted tyrosine-kinase inhibitor of VEGFR, c-MET, and AXL, might provide more antitumoural effects than either agent alone. We aimed to investigate the antitumour activity and safety of belzutifan plus cabozantinib in patients with advanced clear cell renal cell carcinoma that was previously treated with immunotherapy. METHODS: This open-label, single-arm, phase 2 study was conducted at ten hospitals and cancer centres in the USA. Patients were enrolled into two cohorts. Patients in cohort 1 had treatment-naive disease (results will be reported separately). In cohort 2, eligible patients were aged 18 years or older with locally advanced or metastatic clear cell renal cell carcinoma, measurable disease according to Response Evaluation Criteria in Solid Tumours version 1.1, an Eastern Cooperative Oncology Group performance status score of 0 or 1, and had previously received immunotherapy and up to two systemic treatment regimens. Patients were given belzutifan 120 mg orally once daily and cabozantinib 60 mg orally once daily until disease progression, unacceptable toxicity, or patient withdrawal. The primary endpoint was confirmed objective response assessed by the investigator. Antitumour activity and safety were assessed in all patients who received at least one dose of study treatment. This trial is registered with ClinicalTrials.gov, NCT03634540, and is ongoing. FINDINGS: Between Sept 27, 2018, and July 14, 2020, 117 patients were screened for eligibility, 52 (44%) of whom were enrolled in cohort 2 and received at least one dose of study treatment. Median age was 63·0 years (IQR 57·5-68·5), 38 (73%) of 52 patients were male, 14 (27%) were female, 48 (92%) were White, two (4%) were Black or African American, and two were Asian (4%). As of data cutoff (Feb 1, 2022), median follow-up was 24·6 months (IQR 22·1-32·2). 16 (30·8% [95% CI 18·7-45·1]) of 52 patients had a confirmed objective response, including one (2%) who had a complete response and 15 (29%) who had partial responses. The most common grade 3-4 treatment-related adverse event was hypertension (14 [27%] of 52 patients). Serious treatment-related adverse events occurred in 15 (29%) patients. One death was considered treatment related by the investigator (respiratory failure). INTERPRETATION: Belzutifan plus cabozantinib has promising antitumour activity in patients with pretreated clear cell renal cell carcinoma and our findings provide rationale for further randomised trials with belzutifan in combination with a VEGFR tyrosine-kinase inhibitor. FUNDING: Merck Sharp & Dohme (a subsidiary of Merck & Co) and the National Cancer Institute.

Leveraging base-pair mammalian constraint to understand genetic variation and human disease.

Thousands of genomic regions have been associated with heritable human diseases, but attempts to elucidate biological mechanisms are impeded by an inability to discern which genomic positions are functionally important. Evolutionary constraint is a powerful predictor of function, agnostic to cell type or disease mechanism. Single-base phyloP scores from 240 mammals identified 3.3% of the human genome as significantly constrained and likely functional. We compared phyloP scores to genome annotation, association studies, copy-number variation, clinical genetics findings, and cancer data. Constrained positions are enriched for variants that explain common disease heritability more than other functional annotations. Our results improve variant annotation but also highlight that the regulatory landscape of the human genome still needs to be further explored and linked to disease.

Leveraging Base Pair Mammalian Constraint to Understand Genetic Variation and Human Disease.

Although thousands of genomic regions have been associated with heritable human diseases, attempts to elucidate biological mechanisms are impeded by a general inability to discern which genomic positions are functionally important. Evolutionary constraint is a powerful predictor of function that is agnostic to cell type or disease mechanism. Here, single base phyloP scores from the whole genome alignment of 240 placental mammals identified 3.5% of the human genome as significantly constrained, and likely functional. We compared these scores to large-scale genome annotation, genome-wide association studies (GWAS), copy number variation, clinical genetics findings, and cancer data sets. Evolutionarily constrained positions are enriched for variants explaining common disease heritability (more than any other functional annotation). Our results improve variant annotation but also highlight that the regulatory landscape of the human genome still needs to be further explored and linked to disease.

Identification and characterization of a flexile G-quadruplex in the distal promoter region of stemness gene REX1.

We have identified a parallel G-quadruplex (R1WT) in the distal promoter region (-821 base-pairs upstream of the TSS) of the pluripotent gene REX1. Through biophysical and biochemical approach, we have characterized the G-quadruplex (GQ) as a potential molecular switch that may control REX1 promoter activity to determine the transcriptional fate. Small- molecule interactive study of the monomeric form of R1WT (characterized as R1mut2) with TMPyP4 and BRACO-19 revealed GQ destabilization upon interaction with TMPyP4 and stabilization upon interaction with BRACO-19. This distinctive drug interactivity suggests the in cellulo R1WT to be a promising drug target. The endogenous existence of R1WT was confirmed by BG4 antibody derived chromatin immunoprecipitation experiment. Here in, we also report the endogenous interaction of GQ specific transcription factors (TFs) with R1WT region in the human chromatin of cancer cell. The wild-type G-quadruplex was found to interact with four important transcription factors, (i) specificity protein (Sp1) (ii) non-metastatic cell 2 (NM23-H2): a diphosphatase (iii) cellular nucleic acid binding protein (CNBP) and (iv) heterogenous nuclear ribonucleoprotein K (hnRNPK) in the REX1 promoter. In contrast, nucleolin protein (NCL) binding was found to be low to the said G-quadruplex. The flexibility of R1WT between folded and unfolded states, obtained from experimental and computational analysis strongly suggests R1WT to be an important gene regulatory element in the genome. It controls promoter DNA relaxation with the coordinated interaction of transcription factors, the deregulation of which seeds stemness characteristic in cancer cells for further metastatic progression.

Association between daily ambient temperature and drug overdose in Tokyo: a time-series study.

BACKGROUND: Previous studies have reported that high ambient temperature is associated with increased risk of suicide; however, the association has not been extensively investigated with drug overdose which is the most common method of unsuccessful suicidal behavior in Japan. Therefore, this study aims to examine the short-term association between daily mean temperature and the incidence of self-harm attempts by drug overdose in Tokyo, Japan. METHODS: We collected the emergency ambulance dispatch data and daily meteorological data in Tokyo from 2010 to 2014. A quasi-Poisson regression model incorporating a distributed lag non-linear function was applied to estimate the non-linear and delayed association between temperature and drug overdose, adjusting for relative humidity, seasonal and long-term trends, and days of the week. Sex, age and location-specific associations of ambient temperature with drug overdose was also estimated. RESULTS: 12,937 drug overdose cases were recorded during the study period, 73.9% of which were female. We observed a non-linear association between temperature and drug overdose, with the highest risk observed at 21 °C. The highest relative risk (RR) was 1.30 (95% Confidence Interval (CI): 1.10-1.67) compared with the risk at the first percentile of daily mean temperature (2.9 °C) over 0-4 days lag period. In subgroup analyses, the RR of a drug overdose at 21 °C was 1.36 (95% CI: 1.02-1.81) for females and 1.07 (95% CI: 0.66-1.75) for males. Also, we observed that the risk was highest among those aged ≥65 years (RR = 2.54; 95% CI: 0.94-6.90), followed by those aged 15-34 years (RR = 1.25; 95% CI: 0.89-1.77) and those aged 35-64 years (RR = 1.15; 95% CI: 0.78-1.68). There was no evidence for the difference in RRs between urban (23 special wards) and sub-urban areas in Tokyo. CONCLUSIONS: An increase in daily mean temperature was associated with increased drug overdose risk. This study indicated the positive non-linear association between temperature and incomplete attempts by drug overdose. The findings of this study may add further evidence of the association of temperature on suicidal behavior and suggests increasing more research and investigation of other modifying factors.

Insights from Application of a Hierarchical Spatio-Temporal Model to an Intensive Urban Black Carbon Monitoring Dataset.

Existing regulatory pollutant monitoring networks rely on a small number of centrally located measurement sites that are purposefully sited away from major emission sources. While informative of general air quality trends regionally, these networks often do not fully capture the local variability of air pollution exposure within a community. Recent technological advancements have reduced the cost of sensors, allowing air quality monitoring campaigns with high spatial resolution. The 100×100 black carbon (BC) monitoring network deployed 100 low-cost BC sensors across the 15 km2 West Oakland, CA community for 100 days in the summer of 2017, producing a nearly continuous site-specific time series of BC concentrations which we aggregated to one-hour averages. Leveraging this dataset, we employed a hierarchical spatio-temporal model to accurately predict local spatio-temporal concentration patterns throughout West Oakland, at locations without monitors (average cross-validated hourly temporal R 2=0.60). Using our model, we identified spatially varying temporal pollution patterns associated with small-scale geographic features and proximity to local sources. In a sub-sampling analysis, we demonstrated that fine scale predictions of nearly comparable accuracy can be obtained with our modeling approach by using ~30% of the 100×100 BC network supplemented by a shorter-term high-density campaign.