DHODH inhibition enhances the efficacy of immune checkpoint blockade by increasing cancer cell antigen presentation.

Pyrimidine nucleotide biosynthesis is a druggable metabolic dependency of cancer cells, and chemotherapy agents targeting pyrimidine metabolism are the backbone of treatment for many cancers. Dihydroorotate dehydrogenase (DHODH) is an essential enzyme in the de novo pyrimidine biosynthesis pathway that can be targeted by clinically approved inhibitors. However, despite robust preclinical anticancer efficacy, DHODH inhibitors have shown limited single-agent activity in phase 1 and 2 clinical trials. Therefore, novel combination therapy strategies are necessary to realize the potential of these drugs. To search for therapeutic vulnerabilities induced by DHODH inhibition, we examined gene expression changes in cancer cells treated with the potent and selective DHODH inhibitor brequinar (BQ). This revealed that BQ treatment causes upregulation of antigen presentation pathway genes and cell surface MHC class I expression. Mechanistic studies showed that this effect is 1) strictly dependent on pyrimidine nucleotide depletion, 2) independent of canonical antigen presentation pathway transcriptional regulators, and 3) mediated by RNA polymerase II elongation control by positive transcription elongation factor B (P-TEFb). Furthermore, BQ showed impressive single-agent efficacy in the immunocompetent B16F10 melanoma model, and combination treatment with BQ and dual immune checkpoint blockade (anti-CTLA-4 plus anti-PD-1) significantly prolonged mouse survival compared to either therapy alone. Our results have important implications for the clinical development of DHODH inhibitors and provide a rationale for combination therapy with BQ and immune checkpoint blockade.

What does high value care for musculoskeletal conditions mean and how do you apply it in practice? A consensus statement from a research network of physiotherapists in New South Wales, Australia.

OBJECTIVES: To develop a physiotherapist-led consensus statement on the definition and provision of high-value care for people with musculoskeletal conditions. DESIGN: We performed a three-stage study using Research And Development/University of California Los Angeles Appropriateness Method methodology. We reviewed evidence about current definitions through a rapid literature review and then performed a survey and interviews with network members to gather consensus. Consensus was finalised in a face-to-face meeting. SETTING: Australian primary care. PARTICIPANTS: Registered physiotherapists who are members of a practice-based research network (n=31). RESULTS: The rapid review revealed two definitions, four domains of high value care and seven themes of high-quality care. Online survey responses (n=26) and interviews (n=9) generated two additional high-quality care themes, a definition of low-value care, and 21 statements on the application of high value care. Consensus was reached for three working definitions (high value, high-quality and low value care), a final model of four high value care domains (high-quality care, patient values, cost-effectiveness, reducing waste), nine high-quality care themes and 15 statements on application. CONCLUSION: High value care for musculoskeletal conditions delivers most value for the patient, and the clinical benefits outweigh the costs to the individual or system providing the care. High-quality care is evidence based, effective and safe care that is patient-centred, consistent, accountable, timely, equitable and allows easy interaction with healthcare providers and healthcare systems.

Meaningful coproduction with clinicians: establishing a practice-based research network with physiotherapists in regional Australia.

BACKGROUND: The disconnect between research and clinical practice leads to research evidence that is often not useful for clinical practice. Practice-based research networks are collaborations between researchers and clinicians aimed at coproducing more useful research. Such networks are rare in the physiotherapy field. We aimed to describe (i) clinicians' motivations behind, and enablers to, participating in a network, (ii) the process of network establishment and (iii) research priorities for a practice-based network of physiotherapists in the Hunter Region of New South Wales (NSW), Australia that supports research coproduction. METHODS: We describe the methods and outcomes of the three steps we used to establish the network. Step 1 involved consultation with local opinion leaders and a formative evaluation to understand clinicians' motivations behind, and enablers to, participating in a network. Step 2 involved establishment activities to generate a founding membership group and codesign a governance model. Step 3 involved mapping clinical problems through a workshop guided by systems thinking theory with local stakeholders and prioritizing research areas. RESULTS: Through formative evaluation focus groups, we generated five key motivating themes and three key enablers for physiotherapists' involvement in the network. Establishment activities led to a founding membership group (n = 29, 67% from private practice clinics), a network vision and mission statement, and a joint governance group (9/13 [70%] are private practice clinicians). Our problem-mapping and prioritization process led to three clinically relevant priority research areas with the potential for significant change in practice and patient outcomes. CONCLUSIONS: Clinicians are motivated to break down traditional siloed research generation and collaborate with researchers to solve a wide array of issues with the delivery of care. Practice-based research networks have promise for both researchers and clinicians in the common goal of improving patient outcomes.

The experiences and preferences of individuals living with a musculoskeletal disorder regarding prognosis: A qualitative study.

QUESTION(S): What are the experiences and preferences of individuals living with a musculoskeletal disorder regarding prognosis? DESIGN: Exploratory phenomenological study. PARTICIPANTS: Individuals aged 18-year or older currently experiencing a musculoskeletal disorder and receiving treatment from a physiotherapist. DATA ANALYSIS: Data were collected through semi-structured interviews and analysed using inductive coding and thematic analysis. RESULTS: Five themes were identified. First, participants described seeking a cause for their pain. This influenced their experience of prognosis as they perceived a diagnosis was required to inform their prognosis. Second, whilst participants wished to receive a prognosis from their physiotherapist, this was often not their experience. Third, participants perceived that physiotherapists have the potential to impact prognosis through exercise prescription, management of conditions, and improving function. Fourth, receiving a prognosis can have both a positive and negative impact on the individual. Positive impacts include planning for the future, motivation, knowledge acquisition, and instilling hope. However, receiving a prognosis can be disheartening if a patient's expectations are unmet. Finally, participants have several preferences regarding receiving a prognosis including when and how often the prognosis is discussed, what prognostic information is provided, how the prognosis is presented, and what prognosis based on. CONCLUSION: Individuals wish to receive a prognosis, although this is not always their experience. Individuals perceive that physiotherapists have the ability to provide a prognosis and impact their prognosis. Furthermore, receiving a prognosis has an impact on itself. To ensure patient-centred care, physiotherapists should explicitly discuss the prognosis with patients and consider their preferences when providing a prognosis.

Nucleotide metabolism: a pan-cancer metabolic dependency.

Metabolic alterations are a key hallmark of cancer cells, and the augmented synthesis and use of nucleotide triphosphates is a critical and universal metabolic dependency of cancer cells across different cancer types and genetic backgrounds. Many of the aggressive behaviours of cancer cells, including uncontrolled proliferation, chemotherapy resistance, immune evasion and metastasis, rely heavily on augmented nucleotide metabolism. Furthermore, most of the known oncogenic drivers upregulate nucleotide biosynthetic capacity, suggesting that this phenotype is a prerequisite for cancer initiation and progression. Despite the wealth of data demonstrating the efficacy of nucleotide synthesis inhibitors in preclinical cancer models and the well-established clinical use of these drugs in certain cancer settings, the full potential of these agents remains unrealized. In this Review, we discuss recent studies that have generated mechanistic insights into the diverse biological roles of hyperactive cancer cell nucleotide metabolism. We explore opportunities for combination therapies that are highlighted by these recent advances and detail key questions that remain to be answered, with the goal of informing urgently warranted future studies.

The perceptions of individuals with musculoskeletal disorders towards prognosis: An exploratory qualitative study.

QUESTION(S): How do individuals living with musculoskeletal disorders perceive the concept of prognosis? DESIGN: Exploratory phenomenological study. PARTICIPANTS: Individuals aged 18 years or older currently experiencing a musculoskeletal disorder. DATA ANALYSIS: Single semi-structured one-on-one interviews were conducted. Data was analysed using inductive coding and thematic analysis. RESULTS: Five themes were identified. First, participants defined prognosis as the likely outcome associated with their diagnosis. Their prognosis was often associated with outcomes related to pain, tissue health, and function. Second, participants perceived pain as having a negative impact on their prognosis by limiting their function and having a psychological impact. Third, participants held biomedical views in that tissue health was perceived as a cause for their pain and that tissue healing was essential for pain cessation. It was also difficult for participants to distinguish between pain related to tissue damage, and pain that was not. Fourth, participants use their ability to complete leisure and functional activities to determine the success of their recovery. Finally, participants perceived receiving individual prognoses for pain, tissue health, and function that may be simultaneously occurring as both important and beneficial. CONCLUSION: Overall, participants viewed receiving prognostic information as important and beneficial. When constructing their views on prognosis participants perceived that pain, tissue health, and functional ability could all impact upon prognosis, whilst having a prognosis of their own. Physiotherapists should consider conceptualising and discussing prognosis in terms of pain, tissue health, and function when managing musculoskeletal disorders.

ENT1 blockade by CNX-774 overcomes resistance to DHODH inhibition in pancreatic cancer.

Inhibitors of dihydroorotate dehydrogenase (DHODH), a key enzyme for de novo synthesis of pyrimidine nucleotides, have failed in clinical trials for various cancers despite robust efficacy in preclinical animal models. To probe for druggable mediators of DHODH inhibitor resistance, we performed a combination screen with a small molecule library against pancreatic cancer cell lines that are highly resistant to the DHODH inhibitor brequinar (BQ). The screen revealed that CNX-774, a preclinical Bruton tyrosine kinase (BTK) inhibitor, sensitizes resistant cell lines to BQ. Mechanistic studies showed that this effect is independent of BTK and instead results from inhibition of equilibrative nucleoside transporter 1 (ENT1) by CNX-774. We show that ENT1 mediates BQ resistance by taking up extracellular uridine, which is salvaged to generate pyrimidine nucleotides in a DHODH-independent manner. In BQ-resistant cell lines, BQ monotherapy slowed proliferation and caused modest pyrimidine nucleotide depletion, whereas combination treatment with BQ and CNX-774 led to profound cell viability loss and pyrimidine starvation. We also identify N-acetylneuraminic acid accumulation as a potential marker of the therapeutic efficacy of DHODH inhibitors. In an aggressive, immunocompetent pancreatic cancer mouse model, combined targeting of DHODH and ENT1 dramatically suppressed tumor growth and prolonged mouse survival. Overall, our study defines CNX-774 as a previously uncharacterized ENT1 inhibitor and provides strong proof of concept support for dual targeting of DHODH and ENT1 in pancreatic cancer.

Metabolic Rewiring by Loss of Sirt5 Promotes Kras-Induced Pancreatic Cancer Progression.

BACKGROUND & AIMS: SIRT5 plays pleiotropic roles via post-translational modifications, serving as a tumor suppressor, or an oncogene, in different tumors. However, the role SIRT5 plays in the initiation and progression of pancreatic ductal adenocarcinoma (PDAC) remains unknown. METHODS: Published datasets and tissue arrays with SIRT5 staining were used to investigate the clinical relevance of SIRT5 in PDAC. Furthermore, to define the role of SIRT5 in the carcinogenesis of PDAC, we generated autochthonous mouse models with conditional Sirt5 knockout. Moreover, to examine the mechanistic role of SIRT5 in PDAC carcinogenesis, SIRT5 was knocked down in PDAC cell lines and organoids, followed by metabolomics and proteomics studies. A novel SIRT5 activator was used for therapeutic studies in organoids and patient-derived xenografts. RESULTS: SIRT5 expression negatively regulated tumor cell proliferation and correlated with a favorable prognosis in patients with PDAC. Genetic ablation of Sirt5 in PDAC mouse models promoted acinar-to-ductal metaplasia, precursor lesions, and pancreatic tumorigenesis, resulting in poor survival. Mechanistically, SIRT5 loss enhanced glutamine and glutathione metabolism via acetylation-mediated activation of GOT1. A selective SIRT5 activator, MC3138, phenocopied the effects of SIRT5 overexpression and exhibited antitumor effects on human PDAC cells. MC3138 also diminished nucleotide pools, sensitizing human PDAC cell lines, organoids, and patient-derived xenografts to gemcitabine. CONCLUSIONS: Collectively, we identify SIRT5 as a key tumor suppressor in PDAC, whose loss promotes tumorigenesis through increased noncanonic use of glutamine via GOT1, and that SIRT5 activation is a novel therapeutic strategy to target PDAC.

SIRT1-NOX4 signaling axis regulates cancer cachexia.

Approximately one third of cancer patients die due to complexities related to cachexia. However, the mechanisms of cachexia and the potential therapeutic interventions remain poorly studied. We observed a significant positive correlation between SIRT1 expression and muscle fiber cross-sectional area in pancreatic cancer patients. Rescuing Sirt1 expression by exogenous expression or pharmacological agents reverted cancer cell-induced myotube wasting in culture conditions and mouse models. RNA-seq and follow-up analyses showed cancer cell-mediated SIRT1 loss induced NF-κB signaling in cachectic muscles that enhanced the expression of FOXO transcription factors and NADPH oxidase 4 (Nox4), a key regulator of reactive oxygen species production. Additionally, we observed a negative correlation between NOX4 expression and skeletal muscle fiber cross-sectional area in pancreatic cancer patients. Knocking out Nox4 in skeletal muscles or pharmacological blockade of Nox4 activity abrogated tumor-induced cachexia in mice. Thus, we conclude that targeting the Sirt1-Nox4 axis in muscles is an effective therapeutic intervention for mitigating pancreatic cancer-induced cachexia.

Hydrogen peroxide yields mechanistic insights into human mRNA capping enzyme function.

Capping of nascent RNA polymerase II (Pol II) transcripts is required for gene expression and the first two steps are catalyzed by separate 5' triphosphatase and guanylyltransferase activities of the human capping enzyme (HCE). The cap is added co-transcriptionally, but how the two activities are coordinated is unclear. Our previous in vitro work has suggested that an unidentified factor modulates the minimum length at which nascent transcripts can be capped. Using the same well-established in vitro system with hydrogen peroxide as a capping inhibitor, we show that this unidentified factor targets the guanylyltransferase activity of HCE. We also uncover the mechanism of HCE inhibition by hydrogen peroxide, and by using mass spectrometry demonstrate that the active site cysteine residue of the HCE triphosphatase domain becomes oxidized. Using recombinant proteins for the two separated HCE domains, we provide evidence that the triphosphatase normally acts on transcripts shorter than can be acted upon by the guanylyltransferase. Our further characterization of the capping reaction dependence on transcript length and its interaction with the unidentified modulator of capping raises the interesting possibility that the capping reaction could be regulated.

Oxidative stress rapidly stabilizes promoter-proximal paused Pol II across the human genome.

Oxidative stress has pervasive effects on cells but how they respond transcriptionally upon the initial insult is incompletely understood. We developed a nuclear walk-on assay that semi-globally quantifies nascent transcripts in promoter-proximal paused RNA polymerase II (Pol II). Using this assay in conjunction with ChIP-Seq, in vitro transcription, and a chromatin retention assay, we show that within a minute, hydrogen peroxide causes accumulation of Pol II near promoters and enhancers that can best be explained by a rapid decrease in termination. Some of the accumulated polymerases slowly move or 'creep' downstream. This second effect is correlated with and probably results from loss of NELF association and function. Notably, both effects were independent of DNA damage and ADP-ribosylation. Our results demonstrate the unexpected speed at which a global transcriptional response can occur. The findings provide strong support for the residence time of paused Pol II elongation complexes being much shorter than estimated from previous studies.

Comparison of energy expenditure and heart rate responses between three commercial group fitness classes.

OBJECTIVES: The objectives of this study were to compare the energy expenditure and heart rate responses between three commercial group fitness classes (group resistance exercise [PUMP]; indoor stationary cycling [RIDE]; and step aerobics [STEP]). DESIGN: One-Way Repeated Measures incorporating a Latin Square Design for class randomisation. METHODS: Ten participants (5 males and 5 females) completed each group fitness class in random order with energy expenditure and heart rate determined using an Actiheart monitor. RESULTS: STEP and RIDE produced significantly (p<0.05) higher average heart rates (HRavg) (85.8±5.1% and 86.4±4.3% of HRmax, respectively) compared to PUMP (73.7±7% of HRmax). HRpeak was also significantly (p<0.05) higher in STEP and RIDE (97.1±4.7% and 95.6±4.5% of HRmax, respectively) when compared with the PUMP class (90±5.9% of HRmax). Total energy expenditure (TEE), both absolute and relative, were significantly (p<0.05) higher for STEP (2101.7±560.2kJ and 0.59±0.07kJkg-1min-1) and RIDE (1880.4±420kJ and 0.58±0.03kJkg-1min-1) when compared with the PUMP class (1385.1±504kJ and 0.36±0.07kJkg-1min-1). CONCLUSIONS: These data suggest that overall exercise intensity and energy expenditure was highly comparable between RIDE and STEP, which suggests these group fitness classes are more effective for developing cardiovascular fitness and assisting with weight management compared with group resistance exercise classes when performed on a regular basis.