Mitochondrial respiratory function is preserved under cysteine starvation via glutathione catabolism in NSCLC.

Cysteine metabolism occurs across cellular compartments to support diverse biological functions and prevent the induction of ferroptosis. Though the disruption of cytosolic cysteine metabolism is implicated in this form of cell death, it is unknown whether the substantial cysteine metabolism resident within the mitochondria is similarly pertinent to ferroptosis. Here, we show that despite the rapid depletion of intracellular cysteine upon loss of extracellular cystine, cysteine-dependent synthesis of Fe-S clusters persists in the mitochondria of lung cancer cells. This promotes a retention of respiratory function and a maintenance of the mitochondrial redox state. Under these limiting conditions, we find that glutathione catabolism by CHAC1 supports the mitochondrial cysteine pool to sustain the function of the Fe-S proteins critical to oxidative metabolism. We find that disrupting Fe-S cluster synthesis under cysteine restriction protects against the induction of ferroptosis, suggesting that the preservation of mitochondrial function is antagonistic to survival under starved conditions. Overall, our findings implicate mitochondrial cysteine metabolism in the induction of ferroptosis and reveal a mechanism of mitochondrial resilience in response to nutrient stress.

Cys-regulation: oxidized CHK1 controls cross-compartment circuit of chemoresistance.

In a recent study published in Cell, Zhang et al. integrate genome-wide CRISPRi screening with cysteine chemoproteomics to identify functionally relevant oxidation events associated with the cellular response to chemotherapy. This work uncovered checkpoint kinase 1 (CHK1) as a nuclear reactive oxygen species (ROS) sensor that mediates chemoresistance through the suppression of mitochondrial protein synthesis.

Glutathione supports lipid abundance in vivo.

Cells rely on antioxidants to survive. The most abundant antioxidant is glutathione (GSH). The synthesis of GSH is non-redundantly controlled by the glutamate-cysteine ligase catalytic subunit (GCLC). GSH imbalance is implicated in many diseases, but the requirement for GSH in adult tissues is unclear. To interrogate this, we developed a series of in vivo models to induce Gclc deletion in adult animals. We find that GSH is essential to lipid abundance in vivo. GSH levels are reported to be highest in liver tissue, which is also a hub for lipid production. While the loss of GSH did not cause liver failure, it decreased lipogenic enzyme expression, circulating triglyceride levels, and fat stores. Mechanistically, we found that GSH promotes lipid abundance by repressing NRF2, a transcription factor induced by oxidative stress. These studies identify GSH as a fulcrum in the liver's balance of redox buffering and triglyceride production.

NADK-mediated de novo NADP(H) synthesis is a metabolic adaptation essential for breast cancer metastasis.

Metabolic reprogramming and metabolic plasticity allow cancer cells to fine-tune their metabolism and adapt to the ever-changing environments of the metastatic cascade, for which lipid metabolism and oxidative stress are of particular importance. NADPH is a central co-factor for both lipid and redox homeostasis, suggesting that cancer cells may require larger pools of NADPH to efficiently metastasize. NADPH is recycled through reduction of NADP+ by several enzymatic systems in cells; however, de novo NADP+ is synthesized only through one known enzymatic reaction, catalyzed by NAD+ kinase (NADK). Here, we show that NADK is upregulated in metastatic breast cancer cells enabling de novo production of NADP(H) and the expansion of the NADP(H) pools thereby increasing the ability of these cells to adapt to the challenges of the metastatic cascade and efficiently metastasize. Mechanistically, we found that metastatic signals lead to a histone H3.3 variant-mediated epigenetic regulation of the NADK promoter, resulting in increased NADK levels in cells with metastatic ability. Together, our work presents a previously uncharacterized role for NADK and de novo NADP(H) production as a contributor to breast cancer progression and suggests that NADK constitutes an important and much needed therapeutic target for metastatic breast cancers.

Exercise and Airway Clearance Techniques in Cystic Fibrosis.

Exercise and airway clearance techniques (ACTs) have been a cornerstone of treatment for people with cystic fibrosis (pwCF) for many decades. Exercise may confer both respiratory and nonrespiratory benefits for pwCF, with greater exercise capacity associated with improved survival. A wide variety of exercise interventions for pwCF have been investigated. ACTs may assist in reducing respiratory symptoms for pwCF and are currently recommended to be performed daily, with the types of ACTs used varying globally. While recommended components of care, both exercise and ACTs are time-intensive and maintaining adherence to the recommendations over the longer term can be challenging. It has been proposed that with advances in the therapeutic options for pwCF, a rationalization of the therapeutic regimen may be possible. We summarize the current evidence for the use of exercise and ACTs by pwCF, discuss the implications of the introduction of cystic fibrosis transmembrane conductance regulator modulators on both exercise and ACTs, and highlight areas for further research.

Web-based physical activity promotion in young people with CF: a randomised controlled trial.

BACKGROUND: Physical activity levels are known to decline following hospitalisation for people with cystic fibrosis (pwCF). However, optimal physical activity promotion strategies are unclear. This study investigated the effect of a web-based application (ActivOnline) in promoting physical activity in young pwCF. METHODS: Multicentre randomised controlled trial with assessor blinding and qualitative evaluation. People with CF (12-35 years) admitted to hospital for a respiratory cause were eligible and randomised to the 12-week ActivOnline intervention (AO) or usual care (UC). The primary outcome was change in device-based time spent in moderate-to-vigorous physical activity (MVPA) from baseline to post-intervention. Follow-up was at 6 months from hospital discharge when qualitative evaluation was undertaken. RESULTS: 107 participants were randomised to AO (n=52) or UC (n=55). Sixty-three participants (59%) contributed to the intention-to-treat analysis. Mean (SD) age was 21 (6) years (n=46, <18 years). At baseline, physical activity levels were high in both groups (AO 102 (52) vs UC 127 (73) min/day). There was no statistically significant difference in MVPA between groups at either timepoint (post-intervention mean difference (95% CI) -14 mins (-45 to 16)). Uptake of the intervention was low with only 40% (n=21) of participants accessing the web application. CONCLUSION: A web-based application, including individualised goal setting, real-time feedback and motivation for behavioural change, was no better than usual care at promoting physical activity in young pwCF following hospital discharge. High levels of baseline physical activity levels in both groups, and limited engagement with the intervention, suggest alternative strategies may be necessary to identify and support young pwCF who would benefit from enhanced physical activity. TRIAL REGISTRATION NUMBER: ACTRN12617001009303, 13 July 13 2017.

Imaging the master regulator of the antioxidant response in non-small cell lung cancer with positron emission tomography.

Mutations in the NRF2-KEAP1 pathway are common in non-small cell lung cancer (NSCLC) and confer broad-spectrum therapeutic resistance, leading to poor outcomes. The cystine/glutamate antiporter, system xc-, is one of the >200 cytoprotective proteins controlled by NRF2, which can be non-invasively imaged by (S)-4-(3-18F-fluoropropyl)-l-glutamate ([18F]FSPG) positron emission tomography (PET). Through genetic and pharmacologic manipulation, we show that [18F]FSPG provides a sensitive and specific marker of NRF2 activation in advanced preclinical models of NSCLC. We validate imaging readouts with metabolomic measurements of system xc- activity and their coupling to intracellular glutathione concentration. A redox gene signature was measured in patients from the TRACERx 421 cohort, suggesting an opportunity for patient stratification prior to imaging. Furthermore, we reveal that system xc- is a metabolic vulnerability that can be therapeutically targeted for sustained tumour growth suppression in aggressive NSCLC. Our results establish [18F]FSPG as predictive marker of therapy resistance in NSCLC and provide the basis for the clinical evaluation of both imaging and therapeutic agents that target this important antioxidant pathway.

Survival of people with cystic fibrosis in Australia.

Survival statistics, estimated using data from national cystic fibrosis (CF) registries, inform the CF community and monitor disease progression. This study aimed to estimate survival among people with CF in Australia and to identify factors associated with survival. This population-based cohort study used prospectively collected data from 23 Australian CF centres participating in the Australian CF Data Registry (ACFDR) from 2005-2020. Period survival analysis was used to calculate median age of survival estimates for each 5-year window from 2005-2009 until 2016-2020. The overall median survival was estimated using the Kaplan-Meier method. Between 2005-2020 the ACFDR followed 4,601 people with CF, noting 516 (11.2%) deaths including 195 following lung transplantation. Out of the total sample, more than half (52.5%) were male and 395 (8.6%) had undergone lung transplantation. Two thirds of people with CF (66.1%) were diagnosed before six weeks of age or by newborn/prenatal screening. The overall median age of survival was estimated as 54.0 years (95% CI: 51.0-57.04). Estimated median survival increased from 48.9 years (95% CI: 44.7-53.5) for people with CF born in 2005-2009, to 56.3 years (95% CI: 51.2-60.4) for those born in 2016-2020. Factors independently associated with reduced survival include receiving a lung transplant, having low FEV1pp and BMI. Median survival estimates are increasing in CF in Australia. This likely reflects multiple factors, including newborn screening, improvement in diagnosis, refinements in CF management and centre-based multidisciplinary care.

Coordinated Transcriptional and Catabolic Programs Support Iron-Dependent Adaptation to RAS-MAPK Pathway Inhibition in Pancreatic Cancer.

The mechanisms underlying metabolic adaptation of pancreatic ductal adenocarcinoma (PDA) cells to pharmacologic inhibition of RAS-MAPK signaling are largely unknown. Using transcriptome and chromatin immunoprecipitation profiling of PDA cells treated with the MEK inhibitor (MEKi) trametinib, we identify transcriptional antagonism between c-MYC and the master transcription factors for lysosome gene expression, the MiT/TFE proteins. Under baseline conditions, c-MYC and MiT/TFE factors compete for binding to lysosome gene promoters to fine-tune gene expression. Treatment of PDA cells or patient organoids with MEKi leads to c-MYC downregulation and increased MiT/TFE-dependent lysosome biogenesis. Quantitative proteomics of immunopurified lysosomes uncovered reliance on ferritinophagy, the selective degradation of the iron storage complex ferritin, in MEKi-treated cells. Ferritinophagy promotes mitochondrial iron-sulfur cluster protein synthesis and enhanced mitochondrial respiration. Accordingly, suppressing iron utilization sensitizes PDA cells to MEKi, highlighting a critical and targetable reliance on lysosome-dependent iron supply during adaptation to KRAS-MAPK inhibition. SIGNIFICANCE: Reduced c-MYC levels following MAPK pathway suppression facilitate the upregulation of autophagy and lysosome biogenesis. Increased autophagy-lysosome activity is required for increased ferritinophagy-mediated iron supply, which supports mitochondrial respiration under therapy stress. Disruption of ferritinophagy synergizes with KRAS-MAPK inhibition and blocks PDA growth, thus highlighting a key targetable metabolic dependency. See related commentary by Jain and Amaravadi, p. 2023. See related article by Santana-Codina et al., p. 2180. This article is highlighted in the In This Issue feature, p. 2007.

A CRISPR screen identifies redox vulnerabilities for KEAP1/NRF2 mutant non-small cell lung cancer.

The redox regulator NRF2 is hyperactivated in a large percentage of non-small cell lung cancer (NSCLC) cases, which is associated with chemotherapy and radiation resistance. To identify redox vulnerabilities for KEAP1/NRF2 mutant NSCLC, we conducted a CRISPR-Cas9-based negative selection screen for antioxidant enzyme genes whose loss sensitized cells to sub-lethal concentrations of the superoxide (O2•-) -generating drug ß-Lapachone. While our screen identified expected hits in the pentose phosphate pathway, the thioredoxin-dependent antioxidant system, and glutathione reductase, we also identified the mitochondrial superoxide dismutase 2 (SOD2) as one of the top hits. Surprisingly, ß-Lapachone did not generate mitochondrial O2•- but rather SOD2 loss enhanced the efficacy of ß-Lapachone due to loss of iron-sulfur protein function, loss of mitochondrial ATP maintenance and deficient NADPH production. Importantly, inhibition of mitochondrial electron transport activity sensitized cells to ß-Lapachone, demonstrating that these effects may be translated to increase ROS sensitivity therapeutically.

Development of a device to measure adherence and pressure characteristics of positive expiratory pressure therapies used by adults with cystic fibrosis.

INTRODUCTION: Positive expiratory pressure (PEP) and oscillating positive expiratory pressure (OscPEP) therapies are often used by people with cystic fibrosis (CF) to facilitate airway clearance. However, suboptimal adherence and poor technique may reduce their effectiveness. OBJECTIVE: To develop a device (PEPtrac) to accurately measure and provide preliminary clinical data of adherence and technique characteristics when airway clearance is performed using PEP/OscPEP devices. METHODS: This study comprised two distinct phases: 1) a benchtop validation study; and 2) clinical study. Benchtop study: Accuracy of PEPtrac was measured by comparing it to video analysis for five different PEP/OscPEP devices. Clinical study: Clinical data were then collected for 18 adults with CF using one of three PEP/OscPEP devices (PariPEP S®, Acapella DH® or Aerobika®) unsupervised. RESULTS: There was 100% agreement between PEPtrac and video analysis data. Clinical data revealed significant variability in expiratory duration and pressure properties between the three PEP/OscPEP devices and between participants. For example, expiratory duration with PariPEP S® (mean [SD] = 4.8 [1.2] sec) was longer (p < .001) than Acapella DH® (3.7 [0.8] sec) and Aerobika® (2.9 [1.1] sec) and Aerobika® had a higher oscillation amplitude than Acapella DH® (6.4 [1.7] vs 5.3 [1.5] cmH2O, p < .001). DISCUSSION: Accurate measurement of PEP/OscPEP adherence and technique using a device such as PEPtrac was possible. Further research is required to investigate the clinical importance of the variability in technique seen in our clinical data.

The Golgi: Keeping It Unapologetically Basic.

Tumor cells maintain a reverse pH gradient relative to normal cells, conferring cell-intrinsic and cell-extrinsic benefits that sustain tumor growth. In this issue of Cancer Discovery, Galenkamp and colleagues reveal that NHE7 mediates acidification of the trans-Golgi network in pancreatic ductal adenocarcinoma, which is critical for the maintenance of cytosolic pH and consequently tumor growth.See related article by Galenkamp et al., p. 822.

Ketone Bodies Attenuate Wasting in Models of Atrophy.

BACKGROUND: Cancer Anorexia Cachexia Syndrome (CACS) is a distinct atrophy disease negatively influencing multiple aspects of clinical care and patient quality of life. Although it directly causes 20% of all cancer-related deaths, there are currently no model systems that encompass the entire multifaceted syndrome, nor are there any effective therapeutic treatments. METHODS: A novel model of systemic metastasis was evaluated for the comprehensive CACS (metastasis, skeletal muscle and adipose tissue wasting, inflammation, anorexia, anemia, elevated protein breakdown, hypoalbuminemia, and metabolic derangement) in both males and females. Ex vivo skeletal muscle analysis was utilized to determine ubiquitin proteasome degradation pathway activation. A novel ketone diester (R/S 1,3-Butanediol Acetoacetate Diester) was assessed in multifaceted catabolic environments to determine anti-atrophy efficacy. RESULTS: Here, we show that the VM-M3 mouse model of systemic metastasis demonstrates a novel, immunocompetent, logistically feasible, repeatable phenotype with progressive tumor growth, spontaneous metastatic spread, and the full multifaceted CACS with sex dimorphisms across tissue wasting. We also demonstrate that the ubiquitin proteasome degradation pathway was significantly upregulated in association with reduced insulin-like growth factor-1/insulin and increased FOXO3a activation, but not tumor necrosis factor-α-induced nuclear factor-kappa B activation, driving skeletal muscle atrophy. Additionally, we show that R/S 1,3-Butanediol Acetoacetate Diester administration shifted systemic metabolism, attenuated tumor burden indices, reduced atrophy/catabolism and mitigated comorbid symptoms in both CACS and cancer-independent atrophy environments. CONCLUSIONS: Our findings suggest the ketone diester attenuates multifactorial CACS skeletal muscle atrophy and inflammation-induced catabolism, demonstrating anti-catabolic effects of ketone bodies in multifactorial atrophy.

Nicotinamide nucleotide transhydrogenase regulates mitochondrial metabolism in NSCLC through maintenance of Fe-S protein function.

Human lung tumors exhibit robust and complex mitochondrial metabolism, likely precipitated by the highly oxygenated nature of pulmonary tissue. As ROS generation is a byproduct of this metabolism, reducing power in the form of nicotinamide adenine dinucleotide phosphate (NADPH) is required to mitigate oxidative stress in response to this heightened mitochondrial activity. Nicotinamide nucleotide transhydrogenase (NNT) is known to sustain mitochondrial antioxidant capacity through the generation of NADPH; however, its function in non-small cell lung cancer (NSCLC) has not been established. We found that NNT expression significantly enhances tumor formation and aggressiveness in mouse models of lung tumor initiation and progression. We further show that NNT loss elicits mitochondrial dysfunction independent of substantial increases in oxidative stress, but rather marked by the diminished activities of proteins dependent on resident iron-sulfur clusters. These defects were associated with both NADPH availability and ROS accumulation, suggesting that NNT serves a specific role in mitigating the oxidation of these critical protein cofactors.

Exercise as a substitute for traditional airway clearance in cystic fibrosis: a systematic review.

BACKGROUND: Exercise and traditional airway clearance techniques (ACTs) are both routinely recommended for people with cystic fibrosis (CF), with some people using exercise as a substitute for traditional ACTs. The effectiveness of this is unclear. We systematically reviewed the evidence for using exercise as a substitute for traditional ACTs in people with CF. METHODS: A systematic database and literature search were undertaken of studies comparing exercise to rest or traditional ACTs. Primary outcomes were respiratory function, respiratory exacerbations and health-related quality of life. Secondary outcomes included mucociliary clearance (MCC), sputum weight and ease of expectoration. Data are mean difference (95% CI). RESULTS: A total of 12 studies (15 reports) were included, all of short duration (single session to 2 weeks). In crossover trials, exercise did not improve forced expiratory volume in one second in comparison to rest, but peak expiratory flow was increased during treadmill exercise (mean difference (MD) range 1.00-1.16 L/s) and cycle ergometry (1.19 (0.96 to 1.42) L/s). Treadmill exercise improved MCC (2.6 (1.6 to 3.6)%) and ease of expectoration (MD range 1.3-1.8 cm) compared with rest. No consistent differences in respiratory function were evident when exercise was compared with traditional ACTs (four crossover studies). There was no significant difference in MCC or sputum weight in studies where forced expirations were included in the exercise intervention. CONCLUSIONS: Exercise improves ease of expectoration and sputum clearance compared with rest. Exercise, incorporating forced expirations, may have similar effects to traditional ACTs over the short term. There are no data comparing exercise to traditional ACTs over the longer term. PROSPERO REGISTRATION NUMBER: CRD42018102780.

A web-based intervention to promote physical activity in adolescents and young adults with cystic fibrosis: protocol for a randomized controlled trial.

BACKGROUND: Regular participation in physical activity by people with cystic fibrosis (CF) promotes positive clinical and health outcomes including reduced rate of decline in lung function, fewer hospitalizations and greater wellbeing. However adherence to exercise and activity programs is low, in part due to the substantial daily therapy burden for young people with CF. Strict infection control requirements limit the role of group exercise programs that are commonly used in other clinical groups. Investigation of methods to promote physical activity in this group has been limited. The Active Online Physical Activity in Cystic fibrosis Trial (ActionPACT) is an assessor-blinded, multi-centre, randomized controlled trial designed to compare the efficacy of a novel web-based program (ActivOnline) compared to usual care in promoting physical activity participation in adolescents and young adults with CF. METHODS: Adolescents and young adults with CF will be recruited on discharge from hospital for a respiratory exacerbation. Participants randomized to the intervention group will have access to a web-based physical activity platform for the 12-week intervention period. ActivOnline allows users to track their physical activity, set goals, and self-monitor progress. All participants in both groups will be provided with standardised information regarding general physical activity recommendations for adolescents and young adults. Outcomes will be assessed by a blinded assessor at baseline, after completion of the intervention, and at 3-months followup. Healthcare utilization will be assessed at 12 months from intervention completion. The primary outcome is change in moderate-to-vigorous physical activity participation measured objectively by accelerometry. Secondary outcomes include aerobic fitness, health-related quality of life, anxiety and depression and sleep quality. DISCUSSION: This trial will establish whether a web-based application can improve physical activity participation more effectively than usual care in the period following hospitalization for a respiratory exacerbation. The web-based application under investigation can be made readily and widely available to all individuals with CF, to support physical activity and exercise participation at a time and location of the user's choosing, regardless of microbiological status. TRIAL REGISTRATION: Clinical trial registered on July 13, 2017 with the Australian and New Zealand Clinical Trials Register at (ACTRN12617001009303).

Sulfur metabolism and its contribution to malignancy.

Metabolic dysregulation is an appreciated hallmark of cancer and a target for therapeutic intervention. Cellular metabolism involves a series of oxidation/reduction (redox) reactions that yield the energy and biomass required for tumor growth. Cells require diverse molecular species with constituent sulfur atoms to facilitate these processes. For humans, this sulfur is derived from the dietary consumption of the proteinogenic amino acids cysteine and methionine, as only lower organisms (e.g., bacteria, fungi, and plants) can synthesize them de novo. In addition to providing the sulfur required to sustain redox chemistry, the metabolism of these sulfur-containing amino acids yield intermediate metabolites that constitute the cellular antioxidant system, mediate inter- and intracellular signaling, and facilitate the epigenetic regulation of gene expression, all of which contribute to tumorigenesis.

Exercise as a therapeutic intervention for people with cystic fibrosis.

INTRODUCTION: The complex multisystem nature of cystic fibrosis (CF) commonly results in reduced exercise tolerance, which is independently associated with poor clinical outcomes. Exercise is routinely recommended as part of the therapeutic regimen in CF to improve both respiratory and non-respiratory impairments. Areas covered: This article summarises the most recent evidence regarding the use of exercise as a therapeutic intervention in CF and discusses some of the practical considerations for exercise prescription in this setting. Clinical trials in progress and future research priorities are outlined. Expert opinion: On the balance of available evidence, exercise is likely to assist in improving physical fitness and health-related quality of life (HRQOL) and may be associated with a slower rate of decline in respiratory function in CF. Limitations to current studies include small sample sizes, study durations insufficient to achieve a training effect and difficulty distinguishing the effects of exercise training from that of other interventions implemented as part of a package of care. Larger, multi-centred trials are required to clarify the role of exercise in CF in improving physical fitness, respiratory function, HRQOL, as a substitute for traditional airway clearance techniques and in the management of common CF-related comorbidities.