Enhancing standard of care chemotherapy efficacy using DNA-dependent protein kinase (DNA-PK) inhibition in preclinical models of Ewing sarcoma.

Disruption of DNA damage repair via impaired homologous recombination is characteristic of Ewing sarcoma (EWS) cells. We hypothesize that this disruption results in increased reliance on non-homologous end joining (NHEJ) to repair DNA damage. In this study, we investigated if pharmacological inhibition of the enzyme responsible for NHEJ, the DNA-PK holoenzyme, alters the response of EWS cells to genotoxic standard of care chemotherapy. We used analyses of cell viability and proliferation to investigate the effects of clinical DNA-PK inhibitors (DNA-PKi) in combination with six therapeutic or experimental agents for EWS. We performed calculations of synergy using the Loewe Additivity Model. Immunoblotting evaluated treatment effects on DNA-PK, DNA damage, and apoptosis. Flow cytometric analyses evaluated effects on cell cycle and fate. We used orthotopic xenograft models to interrogate tolerability, drug mechanism, and efficacy in vivo. DNA-PKi demonstrated on-target activity, reducing phosphorylated DNA-PK levels in EWS cells. DNA-PKi sensitized EWS cell lines to agents that function as topoisomerase 2 (TOP2) poisons and enhanced the DNA damage induced by TOP2 poisons. Nanomolar concentrations of single agent TOP2 poisons induced G2M arrest and little apoptotic response, while adding DNA-PKi mediated apoptosis. In vivo, the combination of AZD-7648 and etoposide had limited tolerability but resulted in enhanced DNA damage, apoptosis, and EWS tumor shrinkage. The combination of DNA-PKi with standard of care TOP2 poisons in EWS models is synergistic, enhances DNA damage and cell death, and may form the basis of a promising future therapeutic strategy for EWS.

Endogenous EWSR1 Exists in Two Visual Modalities That Reflect Its Associations with Nucleic Acids and Concentration at Sites of Active Transcription.

EWSR1 is a member of the FET family of nucleic acid binding proteins that includes FUS and TAF15. Here, we report the systematic analysis of endogenous EWSR1's cellular organization in human cells. We demonstrate that EWSR1, which contains low complexity and nucleic acid binding domains, is present in cells in faster and slower-recovering fractions, indicative of a protein undergoing both rapid exchange and longer-term interactions. The employment of complementary high-resolution imaging approaches shows EWSR1 exists in two visual modalities, a distributed state which is present throughout the nucleoplasm, and a concentrated state consistent with the formation of foci. Both EWSR1 visual modalities localize with nascent RNA. EWSR1 foci concentrate in regions of euchromatin, adjacent to protein markers of transcriptional activation, and significantly colocalize with phosphorylated RNA polymerase II. Our results contribute to bridging the gap between our understanding of the biophysical and biochemical properties of FET proteins, including EWSR1, their functions as transcriptional regulators, and the participation of these proteins in tumorigenesis and neurodegenerative disease.

The plasma metabolome is associated with preservation of physiological function following lifelong aerobic exercise in mice.

Declines in physiological function with aging are strongly linked to age-related diseases. Lifelong voluntary aerobic exercise (LVAE) preserves physiological function with aging, possibly by increasing cellular quality control processes, but the circulating molecular transducers mediating these processes are incompletely understood. The plasma metabolome may predict biological aging and is impacted by a single bout of aerobic exercise. Here, we conducted an ancillary analysis using plasma samples, and physiological function data, from previously reported studies of LVAE in male C57BL/6N mice randomized to LVAE (wheel running) or sedentary (SED) (n = 8-9/group) to determine if LVAE alters the plasma metabolome and whether these changes correlated with preservation of physiological function with LVAE. Physical function (grip strength, coordination, and endurance) was assessed at 3 and 18 months of age; vascular endothelial function and the plasma metabolome were assessed at 19 months. Physical function was preserved (%decline; mean ± SEM) with LVAE vs SED (all p < 0.05)-grip strength, 0.4 ± 1.7% vs 12 ± 4.0%; coordination, 10 ± 4% vs 73 ± 10%; endurance, 1 ± 15% vs 61 ± 5%. Vascular endothelial function with LVAE (88.2 ± 2.0%) was higher than SED (79.1 ± 2.5%; p = 0.03) and similar to the young controls (91.4 ± 2.9%). Fifteen metabolites were different with LVAE compared to SED (FDR < 0.05) and correlated with the preservation of physiological function. Plasma spermidine, a polyamine that increases cellular quality control (e.g., autophagy), correlated with all assessed physiological indices. Autophagy (LC3A/B abundance) was higher in LVAE skeletal muscle compared to SED (p < 0.01) and inversely correlated with plasma spermidine (r = - 0.5297; p = 0.054). These findings provide novel insight into the circulating molecular transducers by which LVAE may preserve physiological function with aging.

Intermittent supplementation with fisetin improves arterial function in old mice by decreasing cellular senescence.

Cellular senescence and the senescence-associated secretory phenotype (SASP) contribute to age-related arterial dysfunction, in part, by promoting oxidative stress and inflammation, which reduce the bioavailability of the vasodilatory molecule nitric oxide (NO). In the present study, we assessed the efficacy of fisetin, a natural compound, as a senolytic to reduce vascular cell senescence and SASP factors and improve arterial function in old mice. We found that fisetin decreased cellular senescence in human endothelial cell culture. In old mice, vascular cell senescence and SASP-related inflammation were lower 1 week after the final dose of oral intermittent (1 week on-2 weeks off-1 weeks on dosing) fisetin supplementation. Old fisetin-supplemented mice had higher endothelial function. Leveraging old p16-3MR mice, a transgenic model allowing genetic clearance of p16INK4A -positive senescent cells, we found that ex vivo removal of senescent cells from arteries isolated from vehicle- but not fisetin-treated mice increased endothelium-dependent dilation, demonstrating that fisetin improved endothelial function through senolysis. Enhanced endothelial function with fisetin was mediated by increased NO bioavailability and reduced cellular- and mitochondrial-related oxidative stress. Arterial stiffness was lower in fisetin-treated mice. Ex vivo genetic senolysis in aorta rings from p16-3MR mice did not further reduce mechanical wall stiffness in fisetin-treated mice, demonstrating lower arterial stiffness after fisetin was due to senolysis. Lower arterial stiffness with fisetin was accompanied by favorable arterial wall remodeling. The findings from this study identify fisetin as promising therapy for clinical translation to target excess cell senescence to treat age-related arterial dysfunction.

Electronic hookah (waterpipe) vaping reduces vascular endothelial function: the role of nicotine.

Vaping has risen substantially in recent years, particularly among young adults. Electronic (e-) hookahs are a newer category of vaping devices touted as safer tobacco alternatives. Although e-hookah vaping acutely reduces endothelial function, the role of nicotine and the mechanisms by which it may impair endothelial function remain understudied. In a randomized crossover study, we investigated the acute effects of vaping e-hookah, with and without nicotine, as compared with sham on endothelial function assessed by brachial artery flow-mediated dilation (FMD), among 18 overtly healthy young adults. To determine the role of changes in circulating factors in plasma on endothelial cell function, human umbilical vein endothelial cells (HUVECs) were cultured with participants' plasma, and acetylcholine-stimulated nitric oxide (NO) production and basal reactive oxygen species (ROS) bioactivity were assessed. Plasma nicotine was measured before and after the sessions. E-hookah vaping with nicotine, which acutely increased heart rate (HR) by 8 ± 3 beats/min and mean arterial pressure (MAP) by 7 ± 2 mmHg (means ± SE; P < 0.05), decreased endothelial-dependent FMD by 1.57 ± 0.19%Δ (P = 0.001), indicating impairment in endothelial function. Vaping e-hookah without nicotine, which mildly increased hemodynamics (HR, 2 ± 2 beats/min and MAP 1 ± 1 mmHg; P = ns), did not significantly impair endothelial function. No changes were observed after sham vaping. HUVECs cultured with participants' plasma after versus before e-hookah vaping with nicotine, but not without nicotine or sham vaping, exhibited reductions in endothelial cell NO bioavailability and increases in ROS bioactivity (P < 0.05). Plasma nicotine concentrations increased after vaping e-hookah with nicotine (6.7 ± 1.8 ng/mL; P = 0.002), whereas no changes were observed after vaping e-hookah without nicotine or sham (P = ns). Acute e-hookah vaping induces endothelial dysfunction by impairing NO bioavailability associated with increased ROS production, and these effects are attributable to nicotine, not to nonnicotine constituents, present in the flavored e-liquid.NEW & NOTEWORTHY Despite safety claims heavily advertised by the hookah tobacco industry, acute e-hookah vaping induces in vivo endothelial dysfunction by impairing ex vivo NO bioavailability associated with increased ROS production. These effects are attributable to nicotine, not to nonnicotine constituents, present in the flavored e-liquid.

Time-efficient, high-resistance inspiratory muscle strength training increases cerebrovascular reactivity in midlife and older adults.

Aging is associated with increased risk for cognitive decline and dementia due in part to increases in systolic blood pressure (SBP) and cerebrovascular dysfunction. High-resistance inspiratory muscle strength training (IMST) is a time-efficient, intensive respiratory training protocol (30 resisted inspirations/day) that lowers SBP and improves peripheral vascular function in midlife/older adults with above-normal SBP. However, whether, and by what mechanisms, IMST can improve cerebrovascular function is unknown. We hypothesized that IMST would increase cerebrovascular reactivity to hypercapnia (CVR to CO2), which would coincide with changes to the plasma milieu that improve brain endothelial cell function and enhance cognitive performance (NIH Toolbox). We conducted a 6-wk double-blind, randomized, controlled clinical trial investigating high-resistance IMST [75% maximal inspiratory pressure (PImax); 6×/wk; 4 females, 5 males] vs. low-resistance sham training (15% PImax; 6×/wk; 2 females, 5 males) in midlife/older adults (age 50-79 yr) with initial above-normal SBP. Human brain endothelial cells (HBECs) were exposed to participant plasma and assessed for acetylcholine-stimulated nitric oxide (NO) production. CVR to CO2 increased after high-resistance IMST (pre: 1.38 ± 0.66 cm/s/mmHg; post: 2.31 ± 1.02 cm/s/mmHg, P = 0.020). Acetylcholine-stimulated NO production increased in HBECs exposed to plasma from after vs. before the IMST intervention [pre: 1.49 ± 0.33; post: 1.73 ± 0.35 arbitrary units (AU); P < 0.001]. Episodic memory increased modestly after the IMST intervention (pre: 95 ± 13; post: 103 ± 17 AU; P = 0.045). Cerebrovascular and cognitive function were unchanged in the sham control group. High-resistance IMST may be a promising strategy to improve cerebrovascular and cognitive function in midlife/older adults with above-normal SBP, a population at risk for future cognitive decline and dementia.NEW & NOTEWORTHY Midlife/older adults with above-normal blood pressure are at increased risk of developing cognitive decline and dementia. Our findings suggest that high-resistance inspiratory muscle strength training (IMST), a novel, time-efficient (5-10 min/day) form of physical training, may increase cerebrovascular reactivity to CO2 and episodic memory in midlife/older adults with initial above-normal blood pressure.

NAD+-boosting compounds enhance nitric oxide production and prevent oxidative stress in endothelial cells exposed to plasma from patients with COVID-19.

SARS-CoV-2, the virus that causes coronavirus disease 2019 (COVID-19), induces vascular endothelial dysfunction, but the mechanisms are unknown. We tested the hypothesis that the "circulating milieu" (plasma) of patients with COVID-19 would cause endothelial cell dysfunction (characterized by lower nitric oxide (NO) production), which would be linked to greater reactive oxygen species (ROS) bioactivity and depletion of the critical metabolic co-substrate, nicotinamide adenine dinucleotide (NAD+). We also investigated if treatment with NAD+-boosting compounds would prevent COVID-19-induced reductions in endothelial cell NO bioavailability and oxidative stress. Human aortic endothelial cells (HAECs) were exposed to plasma from men and women (age 18-85 years) who were hospitalized and tested positive (n = 34; 20 M) or negative (n = 13; 10 M) for COVID-19. HAECs exposed to plasma from patients with COVID-19 also were co-incubated with NAD+ precursors nicotinamide riboside (NR) or nicotinamide mononucleotide (NMN). Acetylcholine-stimulated NO production was 27% lower and ROS bioactivity was 54% higher in HAECs exposed to plasma from patients with COVID-19 (both p < 0.001 vs. control); these responses were independent of age and sex. NAD+ concentrations were 30% lower in HAECs exposed to plasma from patients with COVID-19 (p = 0.001 vs. control). Co-incubation with NR abolished COVID-19-induced reductions in NO production and oxidative stress (both p > 0.05 vs. control). Co-treatment with NMN produced similar results. Our findings suggest the circulating milieu of patients with COVID-19 promotes endothelial cell dysfunction, characterized by lower NO bioavailability, greater ROS bioactivity, and NAD+ depletion. Supplementation with NAD+ precursors may exert a protective effect against COVID-19-evoked endothelial cell dysfunction and oxidative stress.

Cellular Senescence Contributes to Large Elastic Artery Stiffening and Endothelial Dysfunction With Aging: Amelioration With Senolytic Treatment.

BACKGROUND: Here, we assessed the role of cellular senescence and the senescence associated secretory phenotype (SASP) in age-related aortic stiffening and endothelial dysfunction. METHODS: We studied young (6-8 mo) and old (27-29 mo) p16-3MR mice, which allows for genetic-based clearance of senescent cells with ganciclovir (GCV). We also treated old C57BL/6N mice with the senolytic ABT-263. RESULTS: In old mice, GCV reduced aortic stiffness assessed by aortic pulse wave velocity (PWV; 477±10 vs. 382±7 cm/s, P<0.05) to young levels (old-GCV vs. young-vehicle, P=0.35); ABT-263 also reduced aortic PWV in old mice (446±9 to 356±11 cm/s, P<0.05). Aortic adventitial collagen was reduced by GCV (P<0.05) and ABT-263 (P=0.12) in old mice. To show an effect of the circulating SASP, we demonstrated that plasma exposure from Old-vehicle p16-3MR mice, but not from Old-GCV mice, induced aortic stiffening assessed ex vivo (elastic modulus; P<0.05). Plasma proteomics implicated glycolysis in circulating SASP-mediated aortic stiffening. In old p16-3MR mice, GCV increased endothelial function assessed via peak carotid artery endothelium-dependent dilation (EDD; Old-GCV, 94±1% vs. Old-vehicle, 84±2%, P<0.05) to young levels (Old-GCV vs. young-vehicle, P=0.98), and EDD was higher in old C57BL/6N mice treated with ABT-263 vs. vehicle (96±1% vs. 82±3%, P<0.05). Improvements in endothelial function were mediated by increased nitric oxide (NO) bioavailability (P<0.05) and reduced oxidative stress (P<0.05). Circulating SASP factors related to NO signaling were associated with greater NO-mediated EDD following senescent cell clearance. CONCLUSIONS: Cellular senescence and the SASP contribute to vascular aging and senolytics hold promise for improving age-related vascular function.

EWSR1's visual modalities are defined by its association with nucleic acids and RNA polymerase II.

We report systematic analysis of endogenous EWSR1's cellular organization. We demonstrate that EWSR1, which contains low complexity and nucleic acid binding domains, is present in cells in faster and slower-recovering fractions, indicative of a protein undergoing both rapid exchange and longer-term interactions. The employment of complementary high-resolution imaging approaches shows EWSR1 exists in in two visual modalities, a distributed state which is present throughout the nucleoplasm, and a concentrated state consistent with the formation of foci. Both EWSR1 visual modalities localize with nascent RNA. EWSR1 foci concentrate in regions of euchromatin, adjacent to protein markers of transcriptional activation, and significantly colocalize with phosphorylated RNA polymerase II. Interestingly, EWSR1 and FUS, another FET protein, exhibit distinct spatial organizations. Our results contribute to bridging the gap between our understanding of the biophysical and biochemical properties of FET proteins, including EWSR1, their functions as transcriptional regulators, and the participation of these proteins in tumorigenesis and neurodegenerative disease.

Chronic mitochondria antioxidant treatment in older adults alters the circulating milieu to improve endothelial cell function and mitochondrial oxidative stress.

Excessive reactive oxygen species production by mitochondria (mtROS) is a key contributor to age-related vascular endothelial dysfunction. We recently showed in a crossover design, placebo-controlled clinical trial in older adults that 6 wk of treatment with the mitochondria-targeted antioxidant (MitoQ) improved endothelial function, as measured by nitric oxide (NO)-mediated endothelium-dependent dilation (EDD), by lowering mtROS and was associated with reduced circulating levels of oxidized low-density lipoprotein (oxLDL). Here, we conducted an ancillary analysis using plasma samples from our clinical trial to determine if MitoQ treatment-mediated changes in the "circulating milieu" (plasma) contribute to improvements in endothelial function and the mechanisms involved. With the use of an ex vivo model of endothelial function, acetylcholine-stimulated NO production was quantified in human aortic endothelial cells (HAECs) exposed to plasma collected after chronic MitoQ and placebo supplementation in 19 older adults (67 ± 1 yr; 11 females). We also assessed the influence of plasma on endothelial cell (EC) mtROS bioactivity and the role of lower circulating oxLDL in plasma-mediated changes. NO production was ∼25% higher (P = 0.0002) and mtROS bioactivity was ∼25% lower (P = 0.003) in HAECs exposed to plasma collected from subjects after MitoQ treatment versus placebo. Improvements in NO production ex vivo and NO-mediated EDD in vivo with MitoQ were correlated (r = 0.4683; P = 0.0431). Increasing oxLDL in plasma collected after MitoQ to placebo levels abolished MitoQ treatment effects on NO production and mtROS bioactivity, whereas inhibition of endogenous oxLDL binding to its lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1) prevented these effects. These findings provide novel insight into the mechanisms by which MitoQ treatment improves endothelial function in older adults.NEW & NOTEWORTHY Chronic supplementation with a mitochondria-targeted antioxidant (MitoQ) improves vascular endothelial function in older adults, but the mechanisms of action are incompletely understood. Here, we show that MitoQ supplementation leads to changes in the circulating milieu (plasma), including reductions in oxidized low-density lipoprotein that enhance nitric oxide production and reduce mitochondrial oxidative stress in endothelial cells. These findings provide new information regarding the mechanisms by which MitoQ improves age-related endothelial dysfunction.

Female C57BL/6N mice are a viable model of aortic aging in women.

The aorta stiffens with aging in both men and women, which predicts cardiovascular mortality. Aortic wall structural and extracellular matrix (ECM) remodeling, induced in part by chronic low-grade inflammation, contribute to aortic stiffening. Male mice are an established model of aortic aging. However, there is little information regarding whether female mice are an appropriate model of aortic aging in women, which we aimed to elucidate in the present study. We assessed two strains of mice and found that in C57BL/6N mice, in vivo aortic stiffness (pulse wave velocity, PWV) was higher with aging in both sexes, whereas in B6D2F1 mice, PWV was higher in old versus young male mice, but not in old versus young female mice. Because the age-related stiffening that occurs in men and women was reflected in male and female C57BL/6N mice, we examined the mechanisms of stiffening in this strain. In both sexes, aortic modulus of elasticity (pin myography) was lower in old mice, occurred in conjunction with and was related to higher plasma levels of the elastin-degrading enzyme matrix metalloproteinase-9 (MMP-9), and was accompanied by higher numbers of aortic elastin breaks and higher abundance of adventitial collagen-1. Plasma levels of the inflammatory cytokines interferon-γ, interleukin 6, and monocyte chemoattractant protein-1 were higher in both sexes of old mice. In conclusion, female C57BL/6N mice exhibit aortic stiffening, reduced modulus of elasticity and structural/ECM remodeling, and associated increases in MMP-9 and systemic inflammation with aging, and thus are an appropriate model of aortic aging in women.NEW & NOTEWORTHY Our study demonstrates that with aging, female C57BL/6N mice exhibit higher in vivo aortic stiffness, reduced modulus of elasticity, aortic wall structural and extracellular matrix remodeling, and elevations in systemic inflammation. These changes are largely reflective of those that occur with aging in women. Thus, female C57BL/6N mice are a viable model of human aortic aging and the utility of these animals should be considered in future biomedical investigations.

Sympathetic Neural Overdrive, Aortic Stiffening, Endothelial Dysfunction, and Impaired Exercise Capacity in Severe COVID-19 Survivors: A Mid-Term Study of Cardiovascular Sequelae.

BACKGROUND: COVID-19 has become a dramatic health problem during this century. In addition to high mortality rate, COVID-19 survivors are at increased risk for cardiovascular diseases 1-year after infection. Explanations for these manifestations are still unclear but can involve a constellation of biological alterations. We hypothesized that COVID-19 survivors compared with controls exhibit sympathetic overdrive, vascular dysfunction, cardiac morpho-functional changes, impaired exercise capacity, and increased oxidative stress. METHODS: Nineteen severe COVID-19 survivors and 19 well-matched controls completed the study. Muscle sympathetic nerve activity (microneurography), brachial artery flow-mediated dilation and blood flow (Doppler-Ultrasound), carotid-femoral pulse wave velocity (Complior), cardiac morpho-functional parameters (echocardiography), peak oxygen uptake (cardiopulmonary exercise testing), and oxidative stress were measured ~3 months after hospital discharge. Complementary experiments were conducted on human umbilical vein endothelial cells cultured with plasma samples from subjects. RESULTS: Muscle sympathetic nerve activity and carotid-femoral pulse wave velocity were greater and brachial artery flow-mediated dilation, brachial artery blood flow, E/e' ratio, and peak oxygen uptake were lower in COVID-19 survivors than in controls. COVID-19 survivors had lower circulating antioxidant markers compared with controls, but there were no differences in plasma-treated human umbilical vein endothelial cells nitric oxide production and reactive oxygen species bioactivity. Diminished peak oxygen uptake was associated with sympathetic overdrive, vascular dysfunction, and reduced diastolic function in COVID-19 survivors. CONCLUSIONS: Our study revealed that COVID-19 survivors have sympathetic overactivation, vascular dysfunction, cardiac morpho-functional changes, and reduced exercise capacity. These findings indicate the need for further investigation to determine whether these manifestations are persistent longer-term and their impact on the cardiovascular health of COVID-19 survivors.

Promoting healthy cardiovascular aging: emerging topics.

The development of age-related cardiovascular (CV) dysfunction increases the risk of CV disease as well as other chronic age-associated disorders, including chronic kidney disease, and Alzheimer's disease and related dementias. Major manifestations of age-associated CV dysfunction that increase disease risk are vascular dysfunction, primarily vascular endothelial dysfunction and arterial stiffening, and elevated systolic blood pressure. Declines in nitric oxide bioavailability secondary to increased oxidative stress and inflammation are established mechanisms of CV dysfunction with aging. Moreover, fundamental mechanisms of aging, termed the "hallmarks of aging" extend to the CV system and, as such, may be considered "hallmarks of CV aging". These mechanisms represent viable therapeutic targets for treating CV dysfunction with aging. Healthy lifestyle behaviors, such as regular aerobic exercise and certain dietary patterns, are considered "first-line" strategies to prevent and/or treat age-associated CV dysfunction. Despite the well-established benefits of these strategies, many older adults do not meet the recommended guidelines for exercise or consume a healthy diet. Therefore, it is important to establish alternative and/or complementary evidence-based approaches to prevent or reverse age-related CV dysfunction. Targeting fundamental mechanisms of CV aging with interventions such as time-efficient exercise training, food-derived molecules, termed nutraceuticals, or select synthetic pharmacological agents represents a promising approach. In the present review, we will highlight emerging topics in the field of healthy CV aging with a specific focus on how exercise, nutrition/dietary patterns, nutraceuticals and select synthetic pharmacological compounds may promote healthy CV aging, in part, by targeting the hallmarks of CV aging.

HNRNPH1 destabilizes the G-quadruplex structures formed by G-rich RNA sequences that regulate the alternative splicing of an oncogenic fusion transcript.

In the presence of physiological monovalent cations, thousands of RNA G-rich sequences can form parallel G-quadruplexes (G4s) unless RNA-binding proteins inhibit, destabilize, or resolve the formation of such secondary RNA structures. Here, we have used a disease-relevant model system to investigate the biophysical properties of the RNA-binding protein HNRNPH1's interaction with G-rich sequences. We demonstrate the importance of two EWSR1-exon 8 G-rich regions in mediating the exclusion of this exon from the oncogenic EWS-FLI1 transcripts expressed in a subset of Ewing sarcomas, using complementary analysis of tumor data, long-read sequencing, and minigene studies. We determined that HNRNPH1 binds the EWSR1-exon 8 G-rich sequences with low nM affinities irrespective of whether in a non-G4 or G4 state but exhibits different kinetics depending on RNA structure. Specifically, HNRNPH1 associates and dissociates from G4-folded RNA faster than the identical sequences in a non-G4 state. Importantly, we demonstrate using gel shift and spectroscopic assays that HNRNPH1, particularly the qRRM1-qRRM2 domains, destabilizes the G4s formed by the EWSR1-exon 8 G-rich sequences in a non-catalytic fashion. Our results indicate that HNRNPH1's binding of G-rich sequences favors the accumulation of RNA in a non-G4 state and that this contributes to its regulation of RNA processing.

Spatial Stable Isotopic Labeling by Amino Acids in Cell Culture: Pulse-Chase Labeling of Three-Dimensional Multicellular Spheroids for Global Proteome Analysis.

Three-dimensional cell cultures, or spheroids, are important model systems for cancer research because they recapitulate chemical and phenotypic aspects of in vivo tumors. Spheroids develop radially symmetric chemical gradients, resulting in distinct cellular populations. Stable isotopic labeling by amino acids in cell culture (SILAC) is a well-established approach to quantify protein expression and has previously been used in a pulse-chase format to evaluate temporal changes. In this article, we demonstrate that distinct isotopic signatures can be introduced into discrete spatial cellular populations, effectively tracking proteins to original locations in the spheroid, using a platform that we refer to as spatial SILAC. Spheroid populations were grown with light, medium, and heavy isotopic media, and the concentric shells of cells were harvested by serial trypsinization. Proteins were quantitatively analyzed by ultraperformance liquid chromatography-tandem mass spectrometry. The isotopic signatures correlated with the spatial location and the isotope position do not significantly impact the proteome of each individual layer. Spatial SILAC can be used to examine the proteomic changes in the different layers of the spheroid and to identify protein biomarkers throughout the structure. We show that SILAC labels can be discretely pulsed to discrete positions, without altering the spheroid's proteome, promising future combined pharmacodynamic and pharmacokinetic studies.

Cancer biology functional genomics: From small RNAs to big dreams.

The year 2021 marks the 20th anniversary of the first publications reporting the discovery of the gene silencing mechanism, RNA interference (RNAi) in mammalian cells. Along with the many studies that delineated the proteins and substrates that form the RNAi pathway, this finding changed our understanding of the posttranscriptional regulation of mammalian gene expression. Furthermore, the development of methods that exploited the RNAi pathway began the technological revolution that eventually enabled the interrogation of mammalian gene function-from a single gene to the whole genome-in only a few days. The needs of the cancer research community have driven much of this progress. In this perspective, we highlight milestones in the development and application of RNAi-based methods to study carcinogenesis. We discuss how RNAi-based functional genetic analysis of exemplar tumor suppressors and oncogenes furthered our understanding of cancer initiation and progression and explore how such studies formed the basis of genome-wide scale efforts to identify cancer or cancer-type specific vulnerabilities, including studies conducted in vivo. Furthermore, we examine how RNAi technologies have revealed new cancer-relevant molecular targets and the implications for cancer of the first RNAi-based drugs. Finally, we discuss the future of functional genetic analysis, highlighting the increasing availability of complementary approaches to analyze cancer gene function.

ColoType: a forty gene signature for consensus molecular subtyping of colorectal cancer tumors using whole-genome assay or targeted RNA-sequencing.

Colorectal cancer (CRC) tumors can be partitioned into four biologically distinct consensus molecular subtypes (CMS1-4) using gene expression. Evidence is accumulating that tumors in different subtypes are likely to respond differently to treatments. However, to date, there is no clinical diagnostic test for CMS subtyping. In this study, we used novel methodology in a multi-cohort training domain (n = 1,214) to develop the ColoType scores and classifier to predict CMS1-4 based on expression of 40 genes. In three validation cohorts (n = 1,744, in total) representing three distinct gene-expression measurement technologies, ColoType predicted gold-standard CMS subtypes with accuracies 0.90, 0.91, 0.88, respectively. To accommodate for potential intratumoral heterogeneity and tumors of mixed subtypes, ColoType was designed to report continuous scores measuring the prevalence of each of CMS1-4 in a tumor, in addition to specifying the most prevalent subtype. For analysis of clinical specimens, ColoType was also implemented with targeted RNA-sequencing (Illumina AmpliSeq). In a series of formalin-fixed, paraffin-embedded CRC samples (n = 49), ColoType by targeted RNA-sequencing agreed with subtypes predicted by two independent methods with accuracies 0.92, 0.82, respectively. With further validation, ColoType by targeted RNA-sequencing, may enable clinical application of CMS subtyping with widely-available and cost-effective technology.

Capillary Zone Electrophoresis-Tandem Mass Spectrometry for Large-Scale Phosphoproteomics with the Production of over 11,000 Phosphopeptides from the Colon Carcinoma HCT116 Cell Line.

Phosphoproteomics requires better separation of phosphopeptides to boost the coverage of the phosphoproteome. We argue that an alternative separation method that produces orthogonal phosphopeptide separation to the widely used LC needs to be considered. Capillary zone electrophoresis (CZE) is one important alternative because CZE and LC are orthogonal for phosphopeptide separation and because the migration time of peptides in CZE can be accurately predicted. In this work, we coupled strong cation exchange (SCX)-reversed-phase LC (RPLC) to CZE-MS/MS for large-scale phosphoproteomics of the colon carcinoma HCT116 cell line. The CZE-MS/MS-based platform identified 11,555 phosphopeptides. The phosphopeptide data set is at least 100% larger than that from previous CZE-MS/MS studies and will be a valuable resource for building a model for predicting the migration time of phosphopeptides in CZE. Phosphopeptides migrate significantly slower than corresponding unphosphopeptides under acidic conditions of CZE separations and in a normal polarity. According to our modeling data, phosphorylation decreases peptide's charge roughly by one charge unit, resulting in dramatic decrease in electrophoretic mobility. Preliminary investigations demonstrate that electrophoretic mobility of phosphopeptides containing one phosphoryl group can be predicted with the same accuracy as for nonmodified peptides ( R2 ≈ 0.99). The CZE-MS/MS and LC-MS/MS were complementary in large-scale phosphopeptide identifications and produced different phosphosite motifs from the HCT116 cell line. The data highlight the value of CZE-MS/MS for phosphoproteomics as a complementary separation approach for not only improving the phosphoproteome coverage but also providing more insight into the phosphosite motifs.

Combined Short-Term Glucose Starvation and Chemotherapy in 3D Colorectal Cancer Cell Culture Decreases 14-3-3 Family Protein Expression and Phenotypic Response to Therapy.

Short-term glucose starvation prior to chemotherapy has the potential to preferentially weaken cancer cells, making them more likely to succumb to treatment, while protecting normal cells. In this study, we used 3D cell cultures of colorectal cancer and assessed the effects of short-term glucose starvation and chemotherapy compared to treatment of either individually. We evaluated both phenotypic changes and protein expression levels. Our findings indicate that the combined treatment results in more significant phenotypic responses, including decreased cell viability and clonogenicity. These phenotypic responses can be explained by the decreased expression of LDHA and 14-3-3 family proteins, which were found only in the combined treatment groups. This study indicates that short-term glucose starvation has the potential to increase the efficacy of current cancer treatment regimes. Graphical Abstract ᅟ.