The reliance of many cancers on aerobic glycolysis has stimulated efforts to develop lactate dehydrogenase (LDH) inhibitors. However, despite significant efforts, LDH inhibitors (LDHi) with ...sufficient specificity and in vivo activity to determine whether LDH is a feasible drug target are lacking. We describe an LDHi with potent, on-target, in vivo activity. Using hyperpolarized magnetic resonance spectroscopic imaging (HP-MRSI), we demonstrate in vivo LDH inhibition in two glycolytic cancer models, MIA PaCa-2 and HT29, and we correlate depth and duration of LDH inhibition with direct anti-tumor activity. HP-MRSI also reveals a metabolic rewiring that occurs in vivo within 30 min of LDH inhibition, wherein pyruvate in a tumor is redirected toward mitochondrial metabolism. Using HP-MRSI, we show that inhibition of mitochondrial complex 1 rapidly redirects tumor pyruvate toward lactate. Inhibition of both mitochondrial complex 1 and LDH suppresses metabolic plasticity, causing metabolic quiescence in vitro and tumor growth inhibition in vivo.
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•Specific LDH inhibition in vivo reduces growth rate of glycolytic tumors•Depth and duration of tumor LDH inhibition can be monitored in real time by HP-MRSI•LDH inhibition in vivo redirects pyruvate to support oxidative phosphorylation•Inhibiting mitochondrial complex 1 and LDH enhances durability of anti-tumor response
Oshima et al. use hyperpolarized magnetic resonance spectroscopy to dynamically monitor tumor glycolysis and oxidative phosphorylation. LDH inhibition slows tumor growth but rapidly redirects pyruvate to support mitochondrial metabolism. Inhibiting both mitochondrial complex 1 and LDH suppresses metabolic plasticity of glycolytic tumors in vivo, significantly prolonging tumor growth inhibition.
We report a novel mechanism of action of ONC201 as a mitochondria-targeting drug in cancer cells. ONC201 was originally identified as a small molecule that induces transcription of TNF-related ...apoptosis-inducing ligand (TRAIL) and subsequently kills cancer cells by activating TRAIL death receptors. In this study, we examined ONC201 toxicity on multiple human breast and endometrial cancer cell lines. ONC201 attenuated cell viability in all cancer cell lines tested. Unexpectedly, ONC201 toxicity was not dependent on either TRAIL receptors nor caspases. Time-lapse live cell imaging revealed that ONC201 induces cell membrane ballooning followed by rupture, distinct from the morphology of cells undergoing apoptosis. Further investigation found that ONC201 induces phosphorylation of AMP-dependent kinase and ATP loss. Cytotoxicity and ATP depletion were significantly enhanced in the absence of glucose, suggesting that ONC201 targets mitochondrial respiration. Further analysis indicated that ONC201 indirectly inhibits mitochondrial respiration. Confocal and electron microscopic analysis demonstrated that ONC201 triggers mitochondrial structural damage and functional impairment. Moreover, ONC201 decreased mitochondrial DNA (mtDNA). RNAseq analysis revealed that ONC201 suppresses expression of multiple mtDNA-encoded genes and nuclear-encoded mitochondrial genes involved in oxidative phosphorylation and other mitochondrial functions. Importantly, fumarate hydratase deficient cancer cells and multiple cancer cell lines with reduced amounts of mtDNA were resistant to ONC201. These results indicate that cells not dependent on mitochondrial respiration are ONC201-resistant. Our data demonstrate that ONC201 kills cancer cells by disrupting mitochondrial function and further suggests that cancer cells that are dependent on glycolysis will be resistant to ONC201.
Abstract
Increased conversion of pyruvate to lactate by Lactate Dehydrogenase A (LDHA) is a feature of many neoplasms. Therefore, LDHA inhibition is considered a promising approach toward developing ...a new therapeutic strategy against cancers expressing the Warburg phenotype. To develop this strategy for clinical use, a feasible and sensitive noninvasive imaging approach that can dynamically evaluate LDHA activity in vivo would be highly beneficial since in vitro metabolic profiling dose not always predict in vivo cancer metabolism.
Hyperpolarized (HP) 13C Magnetic Resonance Imaging (MRI) can be used to perform dynamic 13C metabolic flux analysis in vivo. In particular, use of 1-13Cpyruvate (13C-pyr) with 13C MRI permits real-time monitoring of intratumoral LDHA activity through dynamic observation of conversion of 13C-pyr to 1-13Clactate (13C-lac).
This study aimed to apply 13C MRI technology in support of a therapeutic strategy to explore efficacy of a newly developed and highly potent LDH inhibitor (LDHi) in a glycolytic tumor model using MiaPaCa2 xenografts in mice.
In vitro analysis showed that the LDHi dose-dependently inhibited human LDH activity and suppressed in vitro cell growth in MiaPaCa2 cells. By ex vivo assay, LDH activity in MiaPaCa2 xenografts was significantly suppressed (82.2 ± 5.6 % decrease compared to vehicle controls) following a single intravenous(IV) injection of 50 mg/kg LDHi.
Next, 13C MRI imaging of HP 13C-pyr metabolism was performed before and after a single LDHi IV injection to assess inhibitor impact on intratumoral metabolic flux in vivo. 13C MR spectroscopy confirmed that LDHi suppressed intratumor LDHA activity dose- and time-dependently. The maximum effective dose was 50 mg/kg and inhibitor impact in the tumor persisted for 10-12 hrs after a single injection. In addition, lactate production in the tumor was suppressed 30 minutes after IV administration of LDHi, as was the 13C-lac to 13C-pyr ratio decreased by 83.3 ± 4.4 % compared to vehicle controls. Importantly, the close correlation of these data with the results of the ex vivo LDH activity assay, suggests that 13C MRI can reliably monitor in vivo on target effects of LDHi without need for tissue sampling. Based on the data using 13C MRI, we developed a therapeutic strategy for using LDHi in an efficacy study of MiaPaCa2 xenografts. Intermittent IV administration of LDHi significantly suppressed tumor growth in this glycolytic model.
In conclusion, intratumoral inhibition of LDHA in vivo upon IV administration of a novel LDHi was readily visualized by HP 13C MRI, confirming the utility of this noninvasive method. This methodology can be of great value in developing new therapeutic strategies using LDHi and perhaps other metabolic inhibitors to treat cancers characterized by the Warburg phenotype. Further, HP 13C MRI should allow for selection of those patients likely to respond to such treatments.
Citation Format: Nobu Oshima, Shun Kishimoto, Kristin Beebe, Dan Crooks, Michael Moses, Kazutoshi Yamamoto, Jeffry R. Brender, Anastasia Sowers, Ganesha Rai, Daniel Urban, Goria Benavides, Giuseppe Squadrito, Victor Darley-Usmar, Matthew Hall, James B. Mitchell, Murali C. Krishna, Leonard M. Neckers. Evaluation of a novel LDH inhibitor efficacy in vivo in a glycolytic cancer model using hyperpolarized 13C magnetic resonance imaging abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4104.
Abstract
The tumor suppressor p53 is mutated (mt-p53) in over 50% of human cancers causing gain-of-function oncogenic effects, including metabolic changes that reduce tumor responsiveness to ...radio/chemotherapy. Common hot-spot mutations within the DNA-binding domain can be categorized as conformational (R175H) or DNA binding (R248W). NSC59984 has been characterized as a small molecule that targets mt-p53 for degradation and restores wt-p53 signaling. Using esophageal adenocarcinoma cells and CRISPR generated isogenic cell lines bearing matching hot-spot p53 mutations, we aim to understand how the molecular features of mt-p53 affect drug efficiency and enable the development of targeted therapies to limit cancer cell growth. We found that NSC59984 covalently modifies p53 by Michael addition at cysteine residues 124 and 229, which promote interactions that would stabilize the protein/DNA complex leading to increased p53 transcriptional activity. In cells, the effects of NSC59984 were substantially greater in cells harboring the R248W mutation compared with the R175H mutation. Treatment with NSC59984 reduced proliferation and increased apoptosis via the intrinsic mitochondrial pathway. It also induced changes in OXPHOS, ATP level, mitochondrial membrane potential, glycolysis, and lactate production. Furthermore, treatment of cells with NSC59984 increased reactive oxygen species production and decreased glutathione levels; effects were enhanced by the addition of buthionine sulfoximine and inhibited by N-acetyl cysteine. NSC59984 treatment increased G6PD activity, total NADPH levels, and expression of TIGAR. Knockout of TIGAR partially removed the antiproliferative effects of the drug and reduced G6PD levels in the p53-R248W cells. Incorporation of 13C6 into cellular metabolites suggests that p53-regulated transcription of TIGAR increased utilization of the pentose phosphate pathway and inhibited glycolysis at the fructose-6-P fructose-1,6-bisphosphate junction, supported by an increase in Hexokinase 2 and a decrease of phosphofructokinase-1. Thermal proteome profiling identified TIGAR as an additional reaction target of NSC59984, suggesting increased involvement in modulating these metabolic effects. Combining currently available therapeutic metabolic inhibitors with NSC59984 enhanced the antiproliferative effects in cells harboring p53-R248W creating a therapeutic window when compared to the wt-p53 expressing cells. This suggests these combinations could be used in a clinically relevant setting. Overall, this work has identified a distinctive mode of action for p53 reactivation resulting in not only transcriptional activity, but also a unique effect on cellular energetics. This study shows evidence of variation in responsiveness of different mt-p53 forms and allows the development of specific therapeutics directed to individuals for patient-centered precision medicine. Importantly, we have shown that targeting p53 signaling has significant effects on the metabolic profiles of cancer cells rendering them more vulnerable to neoadjuvant therapy.
Citation Format: Kate Brown, Lisa Jenkins, Dan Crooks, Deborah Surman, Sharlyn Mazur, Yuan Xu, Bhargav Arimilli, Ye Yang, Andrew Lane, Stewart Durell, Teresa Fan, David Schrump, Marston Marston, Taylor Ripley, Ettore Appella, Gaelyn Lyons, Andrew Perciaccante, Jerry Dinan, Marco Robello, Herman Nikolayevskiy, Robert O’Connor, Daniel Appella. Targeting mutant p53-R248W reactivates WT p53 function and alters the onco-metabolic profile abstract. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A103.
Abstract
ONC201 is a small molecule originally identified as a TRAIL (TNF-related apoptosis-inducing ligand) inducing compound currently being tested in phase1/2 clinical trials in multiple cancer ...types. Two recent studies reported that ONC201 also induces an atypical stress response mediated in part by ATF4 and CHOP. In this study, we tested ONC201 toxicity in breast cancer cell lines. ONC201 was obtained from Oncoceutics, Inc. Cell viability was tested with MTS assay and CytoTox-Glo assay. ATP level was measured with CellTiter-Glo 2.0 assay. RNA-seq and Western blotting were performed to investigate change of gene expression. Mitochondrial respiration was monitored by Seahorse XF analyzer. Live cell imaging was performed to examine the mode of cell death. Confocal and electron microscopy analysis were performed to study mitochondrial morphology. Mitochondrial DNA (mtDNA) copy number was analyzed by Quantitative PCR (qPCR). Mitochondrial defective (rho0) cell lines were generated by ethidium bromide treatment. We tested the effects of ONC201 on 18 human breast cancer cell lines that represent ER+, HER2-amplified, basal A triple-negative (TNBC), and basal B TNBC breast cancer. ONC201 reduced cell viability in breast cancer cell lines in all subtypes tested with IC50s ranging from 0.8-5 μM, similar to what has been reported for other cancer cell types. Unexpectedly, ONC201 toxicity was not dependent on TRAIL receptors or caspases. Live cell imaging revealed ONC201 induces cell membrane ballooning followed by rupture. By contrast, GST-TRAIL induced TRAIL-receptor dependent caspase mediated death and classic apoptosis morphology. These results suggested that ONC201 kills breast cancer cells via a caspase-independent, TRAIL-receptor-independent mechanism. Western blots confimed that ONC201 induces ATF4 and CHOP, consistent with reported observations. ONC201 also induced phosphorylation of AMP-dependent kinase (AMPK) and depletion of cellular ATP in multiple breast cancer cell lines. Cytotoxicity and ATP depletion induced by ONC201 were significantly enhanced in nonglucose (galactose) medium compared with glucose-containing medium, suggesting that ONC201 targets mitochondrial respiration. Supplementing glucose to cells grown in galactose medium rescued ONC201-dependent ATP depletion, cytotoxicity, and induction of p-AMPK, ATF4, and CHOP. Seahorse XF analyzer indicated that ONC201 inhibits mitochondrial respiration via an indirect mechanism. Confocal imaging revealed that ONC201 induces mitochondrial fission and reduces membrane potential. Electron microscopic analysis revealed that ONC201 induces mitochondrial structural damages, such as mitochondrial swelling, matrix lysis, and fragmentation. Confocal imaging and qPCR revealed that ONC201 decreases mtDNA. Importantly, ONC201 induced mitochondrial damage and mtDNA depletion even in ONC201-resistant cells, implying that cells that are not dependent on mitochondrial respiration are ONC201 resistant. Indeed, multiple rho0 cell lines were ONC201-resistant. RNAseq analysis confirmed that ONC201 inhibits expression of multiple mtDNA-encoded genes and nuclear encoded mitochondrial genes involved in oxidative phosphorylation and other mitochondrial functions, and Western blot reinforced those findings. In summary, our data demonstrate that ONC201 kills breast cancer cells by targeting mitochondria.
Citation Format: Yoshimi Endo Greer, Samuel Gilbert, Celia Islam, Ashley Ubaldini, Christina Stuelten, Natalie Porat-Shliom, Roberto Weigert, Yun Ji, Luca Gattinoni, Ferri Soheilian, Kunio Nagashima, Xiantao Wang, Markus Hafner, Jyoti Shetty, Bao Tran, Vishal Koparde, Parthav Jailwala, Maggie Cam, Dan Crooks, W. Marston Linehan, Donna Voeller, William Reinhold, Vinodh Rajapakse, Yves Pommier, Stanley Lipkowitz. ONC201 kills breast cancer cells by targeting mitochondria abstract. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B023.
ObjectiveTo assess medium-term organ impairment in symptomatic individuals following recovery from acute SARS-CoV-2 infection.DesignBaseline findings from a prospective, observational cohort ...study.SettingCommunity-based individuals from two UK centres between 1 April and 14 September 2020.ParticipantsIndividuals ≥18 years with persistent symptoms following recovery from acute SARS-CoV-2 infection and age-matched healthy controls.InterventionAssessment of symptoms by standardised questionnaires (EQ-5D-5L, Dyspnoea-12) and organ-specific metrics by biochemical assessment and quantitative MRI.Main outcome measuresSevere post-COVID-19 syndrome defined as ongoing respiratory symptoms and/or moderate functional impairment in activities of daily living; single-organ and multiorgan impairment (heart, lungs, kidneys, liver, pancreas, spleen) by consensus definitions at baseline investigation.Results201 individuals (mean age 45, range 21–71 years, 71% female, 88% white, 32% healthcare workers) completed the baseline assessment (median of 141 days following SARS-CoV-2 infection, IQR 110–162). The study population was at low risk of COVID-19 mortality (obesity 20%, hypertension 7%, type 2 diabetes 2%, heart disease 5%), with only 19% hospitalised with COVID-19. 42% of individuals had 10 or more symptoms and 60% had severe post-COVID-19 syndrome. Fatigue (98%), muscle aches (87%), breathlessness (88%) and headaches (83%) were most frequently reported. Mild organ impairment was present in the heart (26%), lungs (11%), kidneys (4%), liver (28%), pancreas (40%) and spleen (4%), with single-organ and multiorgan impairment in 70% and 29%, respectively. Hospitalisation was associated with older age (p=0.001), non-white ethnicity (p=0.016), increased liver volume (p<0.0001), pancreatic inflammation (p<0.01), and fat accumulation in the liver (p<0.05) and pancreas (p<0.01). Severe post-COVID-19 syndrome was associated with radiological evidence of cardiac damage (myocarditis) (p<0.05).ConclusionsIn individuals at low risk of COVID-19 mortality with ongoing symptoms, 70% have impairment in one or more organs 4 months after initial COVID-19 symptoms, with implications for healthcare and public health, which have assumed low risk in young people with no comorbidities.Trial registration numberNCT04369807; Pre-results.
As mortality rates from COVID-19 disease fall, the high prevalence of long-term sequelae (Long COVID) is becoming increasingly widespread, challenging healthcare systems globally. Traditional ...pathways of care for Long Term Conditions (LTCs) have tended to be managed by disease-specific specialties, an approach that has been ineffective in delivering care for patients with multi-morbidity. The multi-system nature of Long COVID and its impact on physical and psychological health demands a more effective model of holistic, integrated care. The evolution of integrated care systems (ICSs) in the UK presents an important opportunity to explore areas of mutual benefit to LTC, multi-morbidity and Long COVID care. There may be benefits in comparing and contrasting ICPs for Long COVID with ICPs for other LTCs.
This study aims to evaluate health services requirements for ICPs for Long COVID and their applicability to other LTCs including multi-morbidity and the overlap with medically not yet explained symptoms (MNYES). The study will follow a Delphi design and involve an expert panel of stakeholders including people with lived experience, as well as clinicians with expertise in Long COVID and other LTCs. Study processes will include expert panel and moderator panel meetings, surveys, and interviews. The Delphi process is part of the overall STIMULATE-ICP programme, aimed at improving integrated care for people with Long COVID.
Ethical approval for this Delphi study has been obtained (Research Governance Board of the University of York) as have approvals for the other STIMULATE-ICP studies. Study outcomes are likely to inform policy for ICPs across LTCs. Results will be disseminated through scientific publication, conference presentation and communications with patients and stakeholders involved in care of other LTCs and Long COVID.
Researchregistry: https://www.researchregistry.com/browse-the-registry#home/registrationdetails/6246bfeeeaaed6001f08dadc/.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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