High Oxidative Phosphorylation Research Articles

Abstract 6035: Glutaminase inhibitors block metabolic switch in prostate cancer and prevents anti-androgen treatment resistance

Abstract Background: All prostate cancers first manifest as androgen dependent tumors and most patients are initially treated with surgery or radiation. About 30% of these patients return to clinic with recurrent prostate cancer that initially responds to androgen ablation therapy (ADT). Unfortunately, almost all of them fail ADT and develop castrate resistant disease (CRPC) that responds poorly to most cancer therapies and progress to often lethal metastatic CRPC (mCRPC). There are accumulating evidence that at a certain state of progression of hormone sensitive prostate cancer (HSPC) to CRPC, prostate cancer cells adopt to androgen signaling axis targeted therapy (ASI) by switching their metabolism from glycolysis to mitochondrial oxidative phosphorylation (OXPHOS). High OXPHOS activity induces mitochondrial dysfunction, autophagy, cancer invasion, and metastasis. A mitochondrial enzyme glutaminase (GLS) produces L-glutamate from L-glutamine. L-glutamate is utilized in mitochondrial metabolism for energy production through TCA cycle. GLS inhibitors CB-839 and IACS-6274 are being clinically developed for treatment of various human malignancies. We have shown that growth of prostate cancer patient derived xenografts (PDX) is markedly inhibited by a combination treatment of a clinically used ASI enzalutamide (ENZA) followed by a GLS inhibitor. Methods: We followed the growth of one HSPC and two CRPC PDX tumors in mice and collected tumor tissues and circulating human tumor cells (CTC) from mouse blood samples at sacrifice. We used super resolution STED confocal microscopy to image mitochondria in the tissue samples and in the CTCs, desorption ionization mass spectrometry (DESI-MS) imaging of multiple metabolites in the tissue samples as well as hyperpolarized MRI (HP-MRI) of the xenografted tumors to follow the changes in cellular central carbon metabolism in vivo. Results: We observed that growth of PDXs developing ENZA resistance is markedly inhibited when ENZA is followed by adding GLS inhibitors with a concomitant reduction in OXPHOS activity. In addition, the ex vivo and in vivo methods introduced to monitor tumor metabolic activities can be easily translated into clinic to monitor the metabolic status of the tumors for early detection of resistance development and as a pharmacodynamic marker for metabolic inhibitor therapy. Conclusion: GLS inhibitors added to ASI could be effective against CRPC and mCRPC developing resistance to ASI treatment. Citation Format: Hirak Subhra Basu, Meredith Spradlin, Tian Weihua, Jose Enriquez, Pratip Bhattacharya, Livia Eberlin. Glutaminase inhibitors block metabolic switch in prostate cancer and prevents anti-androgen treatment resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6035.

Discovery of a Novel, Potent, Orally Active, and Safe Inhibitor Targeting Human Mitochondrial RNA Polymerase.

High oxidative phosphorylation (OXPHOS) happens in some tumors, which depends on OXPHOS for energy supply, particularly in slow-cycling tumor cells. Therefore, targeting human mitochondrial RNA polymerase (POLRMT) to inhibit mitochondrial gene expression emerges as a potential therapeutic strategy to eradicate tumor cells. In this work, exploration and optimization of the first-in-class POLRMT inhibitor IMT1B and its SAR led to the identification of a novel compound D26, which exerted a strong antiproliferative effect on several cancer cells and decreased mitochondrial-related genes expression. In addition, mechanism studies demonstrated that D26 arrested cell cycle at the G1 phase and had no effect on apoptosis, depolarized mitochondria, or reactive oxidative stress generation in A2780 cells. Importantly, D26 exhibited more potent anticancer activity than the lead IMT1B in A2780 xenograft nude mice and had no observable toxic effect. All results suggest that D26 deserves to be further investigated as a potent and safe antitumor candidate.

Disruption of mitochondrial oxidative phosphorylation by chidamide eradicates leukemic cells in AML

Nowadays, the oxidative phosphorylation (OXPHOS) correlatedwith leukemogenesis and treatment response is extensive. Thus, exploration of novel approaches in disrupting OXPHOS in AML is urgently needed. Bioinformatical analysis of TCGA AML dataset was performed to identify the molecular signaling of OXPHOS. The OXPHOS level was measured through a Seahorse XFe96 cell metabolic analyzer. Flow cytometry was applied to measure mitochondrial status. Real-time qPCR and western blot were used to analyze the expression of mitochondrial or inflammatory factors. MLL-AF9-induced leukemic mice were conducted to measure the anti-leukemia effect of chidamide. Here, we reported that AML patients with high OXPHOS level were in a poor prognosis, which was associated with high expression of HDAC1/3 (TCGA). Inhibition of HDAC1/3 by chidamide inhibited cell proliferation and induced apoptotic cell death in AML cells. Intriguingly, chidamide could disrupt mitochondrial OXPHOS as assessed by inducing mitochondrial superoxide and reducing oxygen consumption rate, as well as decreasing mitochondrial ATP production. We also observed that chidamide augmented HK1 expression, while glycolysis inhibitor 2-DG could reduce the elevation of HK1 and improve the sensitivity of AML cells exposed to chidamide. Furthermore, HDAC3 was correlated with hyperinflammatory status, while chidamide could downregulate the inflammatory signaling in AML. Notably, chidamide eradicated leukemic cells in vivo and prolonged the survival time of MLL-AF9-induced AML mice. Chidamide disrupted mitochondrial OXPHOS, promoted cell apoptosis and reduced inflammation in AML cells. These findings exhibited a novel mechanism that targeting OXPHOS would be a novel strategy for AML treatment.

Astrocyte heterogeneity within white matter tracts and a unique subpopulation of optic nerve head astrocytes.

Much of what we know about astrocyte form and function is derived from the study of gray matter protoplasmic astrocytes, whereas white matter fibrous astrocytes remain relatively unexplored. Here, we used the ribotag approach to isolate ribosome-associated mRNA and investigated the transcriptome of uninjured fibrous astrocytes from three regions: unmyelinated optic nerve head, myelinated optic nerve proper, and corpus callosum. Astrocytes from each region were transcriptionally distinct and we identified region-specific astrocyte genes and pathways. Energy metabolism, particularly oxidative phosphorylation and mitochondrial protein translation emerged as key differentiators of astrocyte populations. Optic nerve astrocytes expressed higher levels of neuroinflammatory pathways than corpus callosum astrocytes and we further identified CARTPT as a new marker of optic nerve head astrocytes. These previously uncharacterized transcriptional profiles of white matter astrocyte types reveal their functional diversity and a greater heterogeneity than previously appreciated.

Gene signature predicting recurrence in oral squamous cell carcinoma is characterized by increased oxidative phosphorylation.

Although numerous studies have used systemic approaches to identify prognostic predictors in oral squamous cell carcinoma (OSCC), the effectiveness of these approaches has not been assessed clinically. Further, the mechanism underlying malignant behaviors in OSCC is poorly characterized. This study aimed to develop and verify accurate prognostic predictors for OSCC patients and assess the associated biology. We identified an OSCC-recurrence-related gene signature (ORGS) using a Cox regression analysis. Functional enrichment analysis was used to identify enriched pathways and biological processes to reveal the underlying mechanism of OSCC malignant behavior. The ORGS successfully divided OSCC patients into low- and high-risk groups with significantly different overall survivals. Pathway analysis revealed oxidative phosphorylation (OXPHOS) as a signaling pathway associated with the ORGS in OSCC. Interestingly, high OXPHOS status was strongly associated with poor overall survival in OSCC patients. Mediator complex subunit 30 (MED30) was a predicted upstream regulator of OXPHOS, and knockdown of MED30 reduced histone acetylation. We identified that the ORGS was strongly correlated with OXPHOS regulatory processes, suggesting OXPHOS as a key mechanism leading to poor prognosis in OSCC.

Multi-omics analysis identifies osteosarcoma subtypes with distinct prognosis indicating stratified treatment

Osteosarcoma (OS) is a primary malignant bone tumor that most commonly affects children, adolescents, and young adults. Here, we comprehensively analyze genomic, epigenomic and transcriptomic data from 121 OS patients. Somatic mutations are diverse within the cohort, and only TP53 is significantly mutated. Through unsupervised integrative clustering of the multi-omics data, we classify OS into four subtypes with distinct molecular features and clinical prognosis: (1) Immune activated (S-IA), (2) Immune suppressed (S-IS), (3) Homologous recombination deficiency dominant (S-HRD), and (4) MYC driven (S-MD). MYC amplification with HR proficiency tumors is identified with a high oxidative phosphorylation signature resulting in resistance to neoadjuvant chemotherapy. Potential therapeutic targets are identified for each subtype, including platinum-based chemotherapy, immune checkpoint inhibitors, anti-VEGFR, anti-MYC and PARPi-based synthetic lethal strategies. Our comprehensive integrated characterization provides a valuable resource that deepens our understanding of the disease, and may guide future clinical strategies for the precision treatment of OS.

CD69 Marks Leukemic Regenerating Cells and Regulates Their Metabolism in AML

The treatment of relapsed or refractory (R/R) acute myeloid leukemia (AML) has been a challenge for several decades. Despite the widespread use of cytarabine-based chemotherapy and novel targeted agents, the 5-year overall survival (OS) of AML is still only about 30%, and patients mostly die from relapsed AML. Single-cell RNA sequencing (scRNA-seq) has been applied in the study of recurrence and metastasis of solid tumors, but there is still a lack of in-depth exploration of AML. Here, we investigated the transcriptome features of leukemia stem cells (LSCs) in R/R AML, explored their markers and mechanisms of relapse and chemoresistance, and provided potential therapeutic targets for the clinic. To explore the features of LSCs in R/R AML, we collected 13 bone marrow samples for scRNA-seq, including 8 samples from 4 AML patients at the time of initial diagnosis and their relapse after chemotherapy between 2017 and 2021 (median time to relapse was 333.5 days), 2 samples at initial diagnosis from AML patients with relapse-free survival for three years after chemotherapy, and 3 healthy donors (Figure.1A). Through clustering algorithm, activity analysis of relugons and copy number variation analysis comparing with normal bone marrow samples, we isolated AML cells from normal cells (Figure.1B). Changes of cell number of each cluster and leukemia stemness gene scores were used to define stemness-deficient leukemia cells (Bulk), chemotherapy-sensitive LSCs (S-LSC), and resistant LSCs (R-LSC) (Figure.1C-D). Compared with S-LSCs or Bulk, R-LSCs significantly upregulated the oxidative phosphorylation pathway (Figure.1E). Besides, we found that CD69 was significantly upgraded in R-LSCs, indicating that CD69 may represent a group of leukemic-regenerating cells (LRCs) associated with both leukemic stemness and chemoresistance (Figure.1F) . To investigate the impact of upregulated genes in R-LSC on the clinical prognosis of AML, we explored GEO database and found that the highly expressed genes in R-LSCs including CD69 were associated with a worse clinical prognosis (Figure.1G). TCGA database was used as a validation, and consistent results were obtained (Figure.1H). To explore the stemness, chemoresistance, and metabolic function of CD69+ LRCs, we sorted CD69- and CD69+ AML cells from patients. Through in vitro chemotherapy, we found that CD69+ AML cells were more resistant to chemotherapy than CD69- AML cells (Figure.2A). By clone formation assay, we found that CD69+ AML had better clonal proliferation ability (Figure.2B). To explore the stemness of CD69+ AML cells, we performed limiting dilution analysis (LDA) in vivo and found that the LSCs ratio of CD69+ AML was 109 times that of CD69- AML (p < 0.0001) (Figure.2C). Next, we explored the metabolic function of CD69+ AML cells and found that CD69+ AML cells had higher levels of pyruvate and ATP metabolism, while lactate metabolism levels were lower than those of CD69- AML cells (Figure.2D-F). After culturing with the ATP synthase inhibitor, the chemosensitivity of CD69+ AML cells was significantly improved (Figure.2G). The above evidence proves that CD69 marks a population of LRCs with high oxidative phosphorylation, stemness and chemoresistance in AML, which is consistent with the scRNA-seq results. Based on the above conclusions, we further explored the mechanism by which CD69+ LRCs maintain chemoresistance properties. By scRNA-seq analysis, we found that CD69+ LRCs had enhanced activity of STAT5 in the regulatory network (Figure.3A). We found the expression of phospho-STAT5A, MYC and BCL-2 proteins in CD69+ AML cells of patients was significantly higher than that in CD69- AML cells. To explore whether the function of CD69+ LRCs is affected by the JAK3-STAT5 pathway, we knocked down STAT5 in human AML cells. The protein expressions of MYC and BCL-2 were significantly reduced (Figure.3B). The chemosensitivity in vitro was significantly reduced in STAT5 knockdown (KD) (Figure.3C). To explore the effect of STAT5 KD on the metabolism of CD69+ LRCs, we detected metabolic index. We found that CD69+ LRCs with STAT5 KD produced decreased levels of pyruvate (Figure.3D). In conclusion, CD69 markers LRCs with high oxidative phosphorylation. CD69+ LRCs affect their metabolism and stemness through JAK3-STAT5 activation of downstream MYC and BCL-2. CD69-targeted and STAT5-targeted inhibitors may be candidate strategies for clinical R/R AML treatment. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

ACC1 Inhibition Enhances Treg Gvhd Treatment Efficacy through Reciprocal Fatty-Acid Synthesis and Oxidative Phosphorylation Regulation

Regulatory T-cells (Treg) play a critical role in preventing autoimmune and alloimmune reactions. Clinical trials demonstrated that Treg adoptive transfer significantly reduced but did not eliminate grades II-IV graft-vs-host disease (GVHD). We hypothesized that increasing fatty acid oxidation (FAO) would augment Treg potency, and ultimately enhance Treg-mediated GVHD reduction. Acetyl-CoA carboxylase-1 (ACC1) negatively regulates FAO by augmenting fatty acid synthesis (FAS), which results in downstream inhibition of CPT1a, the rate-limiting enzyme in FAO. ACC1 genetic deletion or inhibition increases T-cell FAO and skews naive CD4 T-cell differentiation from Th17 to Treg. However, the role of ACC1 in thymic Treg function and metabolic fitness is unknown. Here we show that a 2-hour treatment of murine Treg with ND630, a pharmacological inhibitor of ACC1 currently in clinical trials for hepatic steatosis, enhances Treg suppressive function. As compared to control Tregs, ND630 treatment also increased expression of suppressive molecules Lag3, TIGIT, and IL-10. Treatment of Tregs with either of two other ACC1 inhibitors (TOFA, sorafin A) resulted in similar augmentations in Treg function, disfavoring the likelihood of off-target effects. Additionally, ND630 treatment increased Treg oxygen consumption rate (OCR), a surrogate for mitochondrial oxidative phosphorylation (OXPHOS), a critical source of ATP for Treg. Similar to murine Treg, ND630 treatment of in vitro expanded human Treg enhanced their suppressive function and OCR/OXPHOS. IACS, an OXPHOS inhibitor, abrogated the positive ND630 effect on in vitro Treg suppressive function and OXPHOS. Given the augmentation in OXPHOS with ND630 treatment, we further evaluated ND630 effects on Treg mitochondria. Compared to controls, ND630 treatment increased mitochondrial mass and membrane potential, essential for energy storage during OXPHOS, as well as mitochondrial polarization, which is required for full Treg functionality. Microscopy analysis revealed enhanced mitochondrial fusion and elongation after ND630 or TOFA treatment (FigA). This mitochondrial structural change boosts OXPHOS, consistent with a direct effect of ACC1 inhibition on mitochondrial fitness. Since ACC1-driven FAS ultimately inhibits FAO, we next evaluated contributors to FAO. Expression of CPT1a, a critical enzyme for mitochondrial FAO, fatty acid binding protein 5, a key shuttle of fatty acids (FA), and exogenous FA uptake were all enhanced after ND630 treatment. Using an unbiased metabolomic screen, a metabolic signature was observed that was consistent with enhanced FAO. Together, these data suggest that ND630 increases mitochondrial fitness, and augments both FA uptake and FAO. To identify additional ND603-induced intracellular effects in Treg, activated murine Treg were subjected to RNAseq. Differential gene expression analysis indicated that 82 genes differed between DMSO and ND630 treated Treg. These included increased expression of genes associated with OXPHOS, as well as mTOR, consistent with high OXPHOS and suppressor cell function. Flow cytometry analysis of phosphorylated proteins showed reduced pFOXO3a and pAKT, both mTORC2 targets, but increased mTORC1 downstream phospho-proteins. An imbalance of mTORC1/2 is consistent to our previous studies reflective of higher Treg function and metabolic fitness (McDonald-Hyman C, JCI 2018). To exclude potential bystander effects of ND630 on Treg function, we utilized murine Treg with genetic deletion of ACC1 (ACC1KO). Supporting our studies with chemical ACC1 inhibitors, ACC1KO Treg were functionally and metabolically superior to wild-type Treg. Furthermore, as our goal is to use ACC1 inhibited or deleted Treg in treatment of GVHD, we tested ACC1KO Treg in treating established bronchiolitis obliterans (BOS) in a murine model of chronic GVHD. ACC1KO Treg were more potent than wild-type Treg in reversing BOS pathology (FigB), a finding consistent in each of 3 replicate transplants. In summary, ACC1 inhibition or deletion amplifies murine (and human) Treg potency resulting in superior metabolic fitness, and greater efficacy in treating established BOS in murine chronic GVHD. Since ND630 is already under investigation in patients, ACC1 inhibition with ND630 could be readily translatable for clinical trials with a goal of utilizing ACC1 inhibited Treg infusion to improve GVHD therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

C/EBPα Confers Dependence to Fatty Acid Biosynthetic Pathways and Vulnerability to Lipid Oxidative Stress in FLT3-Mutant Leukemia

Despite therapeutic breakthroughs, drug resistance and relapse remain the major barrier for the treatment of patients with acute myeloid leukemia (AML). Therefore, understanding and targeting therapy-resistant AML cells responsible for relapse ("persisters") represent an urgent need in the development of new treatments. In this context, we demonstrated that the hyperactivation of mitochondrial oxidative metabolism plays a crucial role in drug resistance of AML. This high oxidative phosphorylation (OxPHOS) phenotype associated with enhanced mitochondrial BCL2 dependency and ROS detoxification is the consequence of a mitochondrial adaptation driven by inflammatory stress responses in a cAMP-PKA-ATF4-dependent manner. Targeting elevated oxidative phosphorylation (OxPHOS) activity with direct and indirect mitochondrial inhibitors sensitizes resistant cells to chemotherapy cytarabine (AraC) in AML. Furthermore, we have recently shown that the mitochondrial OxPHOS state supports IDH mutant cell proliferation and chemoresistance in a C/EBPa-dependent manner. Importantly, multi-omics analyses uncovered a coordinated activation of CEBPA and Fms-like tyrosine kinase 3 (FLT3) to control lipid metabolism in FLT3-mutant AML cells from primary AML patient specimens, cell lines, and PDX models. Mechanistically, C/EBPα regulated FASN-SCD axis to promote uptake, biosynthesis and desaturation of fatty acids (FA). We further demonstrated that FLT3 or C/EBPα inactivation decreased mono-unsaturated FAs incorporation to membrane phospholipids through SCD downregulation in leukemic cells. Consequently, SCD inhibition enhanced susceptibility to lipid redox stress and accumulation of poly-unsaturated FA increased lipid peroxidation and vulnerability to redox stress. Accordingly, this C/EBPα-dependent adaptation of FA homeostasis was exploited by combining FLT3 and glutathione peroxidase 4 (GPX4) inhibition to trigger lipid oxidative stress, enhancing ferroptotic death of FLT3-mutant AML cells ex vivo and in vivo. Altogether, this study reveals a non-canonical C/EBPα function in lipid homeostasis and adaptation to redox stress, and a previously unreported vulnerability of FLT3-mutant AML with promising therapeutic application. Altogether, our works highlight the role of metabolic adaptations to support the plasticity and drug resistance in AML.

Lactate dehydrogenases promote glioblastoma growth and invasion via a metabolic symbiosis

Lactate is a central metabolite in brain physiology but also contributes to tumor development. Glioblastoma (GB) is the most common and malignant primary brain tumor in adults, recognized by angiogenic and invasive growth, in addition to its altered metabolism. We show herein that lactate fuels GB anaplerosis by replenishing the tricarboxylic acid (TCA) cycle in absence of glucose. Lactate dehydrogenases (LDHA and LDHB), which we found spatially expressed in GB tissues, catalyze the interconversion of pyruvate and lactate. However, ablation of both LDH isoforms, but not only one, led to a reduction in tumor growth and an increase in mouse survival. Comparative transcriptomics and metabolomics revealed metabolic rewiring involving high oxidative phosphorylation (OXPHOS) in the LDHA/B KO group which sensitized tumors to cranial irradiation, thus improving mouse survival. When mice were treated with the antiepileptic drug stiripentol, which targets LDH activity, tumor growth decreased. Our findings unveil the complex metabolic network in which both LDHA and LDHB are integrated and show that the combined inhibition of LDHA and LDHB strongly sensitizes GB to therapy.

A comparative and ontogenetic examination of mitochondrial function in Antarctic notothenioid species

Notothenioidei fishes have evolved under stable cold temperatures; however, ocean conditions are changing globally, with polar regions poised to experience the greatest changes in environmental factors, such as warming. These stressors have the potential to dramatically affect energetic demands, and the persistence of the notothenioids will be dependent on metabolic capacity, or the ability to match energy supply with energy demand, to restore homeostasis in the face of changing climate conditions. In this study we examined aerobic metabolic capacity in three species, Trematomus bernacchii, T. pennellii and T. newnesi, and between two life stages, juvenile and adult, by assessing mitochondrial function of permeabilized cardiac fibers. Respiratory capacity differed among the adult notothenioids in this study, with greater oxidative phosphorylation (OXPHOS) respiration in the pelagic T. newnesi than the benthic T. bernacchii and T. pennellii. The variation in mitochondrial respiratory capacity was likely driven by differences in the mitochondrial content, as measured by citrate synthase activity, which was the highest in T. newnesi. In addition to high OXPHOS, T. newnesi exhibited lower LEAK respiration, resulting in greater mitochondrial efficiency than either T. bernacchii or T. pennellii. Life stage largely had an effect on mitochondrial efficiency and excess complex IV capacity, but there were little differences in OXPHOS respiration and electron transfer capacity, pointing to a lack of significant differences in the metabolic capacity between juveniles and adults. Overall, these results demonstrate species-specific differences in cardiac metabolic capacity, which may influence the acclimation potential of notothenioid fishes to changing environmental conditions.

Early posttraumatic CSF1R inhibition via PLX3397 leads to time- and sex-dependent effects on inflammation and neuronal maintenance after traumatic brain injury in mice

BackgroundThere is a need for early therapeutic interventions after traumatic brain injury (TBI) to prevent neurodegeneration. Microglia/macrophage (M/M) depletion and repopulation after treatment with colony stimulating factor 1 receptor (CSF1R) inhibitors reduces neurodegeneration. The present study investigates short- and long-term consequences after CSF1R inhibition during the early phase after TBI. MethodsSex-matched mice were subjected to TBI and CSF1R inhibition by PLX3397 for 5 days and sacrificed at 5 or 30 days post injury (dpi). Neurological deficits were monitored and brain tissues were examined for histo- and molecular pathological markers. RNAseq was performed with 30 dpi TBI samples. ResultsAt 5 dpi, CSF1R inhibition attenuated the TBI-induced perilesional M/M increase and associated gene expressions by up to 50%. M/M attenuation did not affect structural brain damage at this time-point, impaired hematoma clearance, and had no effect on IL-1β expression. At 30 dpi, following drug discontinuation at 5 dpi and M/M repopulation, CSF1R inhibition attenuated brain tissue loss regardless of sex, as well as hippocampal atrophy and thalamic neuronal loss in male mice. Selected gene markers of brain inflammation and apoptosis were reduced in males but increased in females after early CSF1R inhibition as compared to corresponding TBI vehicle groups. Neurological outcome in behaving mice was almost not affected. RNAseq and gene set enrichment analysis (GSEA) of injured brains at 30 dpi revealed more genes associated with dendritic spines and synapse function after early CSF1R inhibition as compared to vehicle, suggesting improved neuronal maintenance and recovery. In TBI vehicle mice, GSEA showed high oxidative phosphorylation, oxidoreductase activity and ribosomal biogenesis suggesting oxidative stress and increased abundance of metabolically highly active cells. More genes associated with immune processes and phagocytosis in PLX3397 treated females vs males, suggesting sex-specific differences in response to early CSF1R inhibition after TBI. ConclusionsM/M attenuation after CSF1R inhibition via PLX3397 during the early phase of TBI reduces long-term brain tissue loss, improves neuronal maintenance and fosters synapse recovery. Overall effects were not sex-specific but there is evidence that male mice benefit more than female mice.

S251: DISRUPTION OF SUCCINATE RECEPTOR SIGNALING PROMOTES MYELOPROLIFERATION AND ACUTE MYELOID LEUKAEMIA

Background: Unlike healthy haematopoietic stem cells (HSC), leukaemia stem cells (LSC) have high oxidative phosphorylation and hence targeting the tricarboxylic acid (TCA) cycle seems a promising therapeutic strategy in acute myeloid leukemia (AML) treatment. Accumulation of the TCA cycle oncometabolite succinate is associated with tumourigenesis, but its potential involvement in HSC function and leukaemia is unkown. Succinate stabilizes the oxygen-sensing transcription factor hypoxia-inducible factor (HIF)-1-α and activates interleukin (IL)-1β production in macrophages. Succinate may also be exported and has cell extrinsic effects through its receptor, succinate receptor-1 (SUCNR1), which may be pro- or anti-inflammatory. Aims: To investigate the in vivo role and molecular mechanisms of succinate and its receptor in normal and malignant haematopoiesis. Methods: To study the effect of succinate receptor on haematopoiesis, we characterized the immunophenotype of Sucnr1-KO mice. To dissect the role of Sucnr1 on healthy haematopoiesis via cell-autonomous effects, we transplanted fl/Sucnr1/fl control or Mx1-Cre fl/Sucnr1/fl bone marrow (BM) cells into wild-type (WT) C57BL/6J mice, which were later injected with pIpC. To study the role of Sucnr1 through non-cell autonomous effects, we transplanted WT BM into Nes-CreERT2fl/Sucnr1/fl recipients or fl/Sucnr1/fl controls, which were induced with tamoxifen. We characterized the effects of Sucnr1 deletion at the molecular level by bulk and single-cell RNA-sequencing (scRNA-seq), and aimed to uncover the molecular mechanisms mediating the effects of Sucnr1 ablation using in vivo tageting with drugs. To study the effect of succinate on leukaemic progression, we used two different mouse models, i.e. Mx1-Cre NrasG12D and MLL-AF9 knock-in (KI). In human AML, we quantified SUCNR1 expression in peripheral blood mononuclear cells (PBMC) from AML patients versus healthy volunteers, and treated xenograft recipients of CD34+ AML cells with succinate or vehicle. Results:In vivo deletion of Sucnr1 induced myeloid bias and expansion of the haematopoietic stem and progenitor cell (HSPC) compartment. In vivo deletion of Sucnr1 from Nestin+ stromal cells promoted expansion of long-term HSC (LT-HSC) versus intact recipients. Conversely, in vivo deletion of Sucnr1 from the haematopoietic system induced expansion of multipotent progenitors (MPP) versus intact haematopoietic cells. RNA-seq in HSPC from Sucnr1-KO versus WT mice showed changes in genes associated with myeloid output, and pinpointed related pathways potentially involved. scRNA-seq confirmed expansion of LT-HSC in Sucnr1-KO versus WT mice. In vivo treatments targeted signalling pathways at least partially responsible for the haematopoietic abnormalities in Sucnr1-KO. We found higher content of succinate in the BM extracellular fluid of leukaemic Mx1-Cre NrasG12D. Succinate injection to xenograft recipients of CD34+ AML cells increased leukaemic output versus vehicle, promoted myeloid bias in Mx1-Cre NrasG12D mice and reduced survival in mice transplanted with BM of MLL-AF9 leukaemic donors. PBMC from AML patients expressed lower level of SUCNR1 versus healthy donors and BM nucleated cells from Mx1-Cre NrasG12D also expressed less Sucnr1 than their healthy counterparts. Summary/Conclusion: We show a novel role for succinate and its receptor in the haematopoietic system and pinpoint this pathway as a novel means of communication within the BM HSC microenvironment, whose disruption may be involved in myeloid malignancy. These findings may help design new therapeutic strategies for AML patients.

Deregulation and epigenetic modification of BCL2-family genes cause resistance to venetoclax in hematologic malignancies

AbstractThe BCL2 inhibitor venetoclax has been approved to treat different hematological malignancies. Because there is no common genetic alteration causing resistance to venetoclax in chronic lymphocytic leukemia (CLL) and B-cell lymphoma, we asked if epigenetic events might be involved in venetoclax resistance. Therefore, we employed whole-exome sequencing, methylated DNA immunoprecipitation sequencing, and genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 screening to investigate venetoclax resistance in aggressive lymphoma and high-risk CLL patients. We identified a regulatory CpG island within the PUMA promoter that is methylated upon venetoclax treatment, mediating PUMA downregulation on transcript and protein level. PUMA expression and sensitivity toward venetoclax can be restored by inhibition of methyltransferases. We can demonstrate that loss of PUMA results in metabolic reprogramming with higher oxidative phosphorylation and adenosine triphosphate production, resembling the metabolic phenotype that is seen upon venetoclax resistance. Although PUMA loss is specific for acquired venetoclax resistance but not for acquired MCL1 resistance and is not seen in CLL patients after chemotherapy-resistance, BAX is essential for sensitivity toward both venetoclax and MCL1 inhibition. As we found loss of BAX in Richter’s syndrome patients after venetoclax failure, we defined BAX-mediated apoptosis to be critical for drug resistance but not for disease progression of CLL into aggressive diffuse large B-cell lymphoma in vivo. A compound screen revealed TRAIL-mediated apoptosis as a target to overcome BAX deficiency. Furthermore, antibody or CAR T cells eliminated venetoclax resistant lymphoma cells, paving a clinically applicable way to overcome venetoclax resistance.

Abstract 6384: Morphological and functional plasticity of mitochondria promotes chemotherapy resistance in triple negative breast cancer

Abstract Mitochondrial metabolism plays a key role in triple negative breast cancer (TNBC) aggressiveness. As TNBC has limited targeted therapy options, chemotherapies remain the mainstay treatment. Nearly 50% of TNBC patients harbor substantial residual cancer following chemotherapy, leading to high rates of recurrence. Using longitudinal biopsies from orthotopic patient-derived xenograft (PDX) models and TNBC patients, we found residual tumors following chemotherapy transitioned to a unique metabolic state characterized by high mitochondrial oxidative phosphorylation (oxphos). This state was transient, with tumors reverting to their baseline glycolysis-high phenotype when they were allowed to regrow in the absence of treatment. Using genomic sequencing and cellular barcode-mediated clonal tracking, we found this mechanism of chemoresistance arose in the absence of clonal selection, suggesting chemotherapy induced plastic (i.e., non-genomic) programs enabling cell survival following treatment. Blocking oxphos with an inhibitor of electron transport chain Complex I (IACS010759; PMID:29892070) was significantly more efficacious against residual than pre-treated tumors (PMID:30996079), providing evidence that dynamic metabolic phenotypes represent targetable therapeutic vulnerabilities for TNBC. Using longitudinal samples collected from PDX models undergoing treatments with anthracyclines, platinums, and/or taxanes, we visualized and quantified mitochondrial structure in two- and three-dimensions by electron microscopy. These studies revealed extensive alteration of mitochondrial structure and number in residual tumor cells, and these changes reverted when residual tumors were allowed to regrow in the absence of treatment. We then administered chemotherapeutics to human TNBC cells, revealing that DNA-damaging chemotherapeutics increased mitochondrial elongation, but microtubule poisons increased mitochondrial fragmentation. These findings suggested chemotherapeutics may alter the dynamics of mitochondrial fission and fusion in TNBC cells. These structural changes were accompanied by increased or decreased oxphos rates, glucose-driven TCA cycle flux, and mitochondrial content, respectively. Driving mitochondrial fusion by genetic or pharmacologic inhibition of the mitochondrial fission factor Drp1 increased oxphos and chemoresistance, whereas driving mitochondrial fission by genetic or pharmacologic inhibition of the mitochondrial fusion protein Opa1 decreased oxphos and chemoresistance. These findings provide evidence that modulating mitochondrial fission and fusion may be a promising strategy to overcome metabolic states contributing to chemoresistance in TNBC. Our ongoing investigations are aimed at rational targeted therapies and scheduling approaches to overcome chemoresistance in in vivo models of TNBC. Citation Format: Lily Baek, Junegoo Lee, Mariah J. Berner, Katherine E. Pendleton, Emily B. Goff, Karen Wang, James P. Barrish, Bora Lim, Philip J. Lorenzi, Weston Porter, Michael T. Lewis, Gloria V. Echeverria. Morphological and functional plasticity of mitochondria promotes chemotherapy resistance in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6384.

Ammonia-induced oxidative stress triggered proinflammatory response and apoptosis in pig lungs

Ammonia, a common toxic gas, is not only one of the main causes of haze, but also can enter respiratory tract and directly affect the health of humans and animals. Pig was used as an animal model for exploring the molecular mechanism and dose effect of ammonia toxicity to lung. In this study, the apoptosis of type II alveolar epithelial cells was observed in high ammonia exposure group using transmission electron microscopy. Gene and protein expression analysis using transcriptome sequencing and western blot showed that low ammonia exposure induced T-cell-involved proinflammatory response, but high ammonia exposure repressed the expression of DNA repair-related genes and affected ion transport. Moreover, high ammonia exposure significantly increased 8-hydroxy-2-deoxyguanosine (8-OHdG) level, meaning DNA oxidative damage occurred. In addition, both low and high ammonia exposure caused oxidative stress in pig lungs. Integrated analysis of transcriptome and metabolome revealed that the up-regulation of LDHB and ND2 took part in high ammonia exposure-affected pyruvate metabolism and oxidative phosphorylation progress, respectively. Inclusion, oxidative stress mediated ammonia-induced proinflammatory response and apoptosis of porcine lungs. These findings may provide new insights for understanding the ammonia toxicity to workers in livestock farms and chemical fertilizer plants.

Diphenyleneiodonium Triggers Cell Death of Acute Myeloid Leukemia Cells by Blocking the Mitochondrial Respiratory Chain, and Synergizes with Cytarabine.

Simple SummaryAcute myeloid leukemia (AML) is an aggressive heterogeneous cancer of the blood, of which 70% of cases develop relapse. Relapse is mainly due to chemoresistant leukemic cells (LCs) that are characterized by high mitochondrial oxidative phosphorylation (OxPhos) status, i.e., cells that are dependent on the mitochondrial respiratory chain (MRC) function. The aim of our study was to determine whether diphenyleneiodonium (DPI)—known as a potent inhibitor of flavoproteins—could be used to target AML cells. Herein, we demonstrate that DPI disrupts the mitochondrial function of AML cell lines. Interestingly, we found that cells with high OxPhos are more sensitive to the apoptotic effects of DPI. Moreover, we showed that DPI sensitizes AML cell lines to cytarabine (Ara-C) treatment, suggesting that MRC inhibitors could be employed to target LCs that are resistant to this chemotherapeutic agent.Acute myeloid leukemia (AML) is characterized by the accumulation of undifferentiated blast cells in the bone marrow and blood. In most cases of AML, relapse frequently occurs due to resistance to chemotherapy. Compelling research results indicate that drug resistance in cancer cells is highly dependent on the intracellular levels of reactive oxygen species (ROS). Modulating ROS levels is therefore a valuable strategy to overcome the chemotherapy resistance of leukemic cells. In this study, we evaluated the efficiency of diphenyleneiodonium (DPI)—a well-known inhibitor of ROS production—in targeting AML cells. Results showed that although inhibiting cytoplasmic ROS production, DPI also triggered an increase in the mitochondrial ROS levels, caused by the disruption of the mitochondrial respiratory chain. We also demonstrated that DPI blocks mitochondrial oxidative phosphorylation (OxPhos) in a dose-dependent manner, and that AML cells with high OxPhos status are highly sensitive to treatment with DPI, which synergizes with the chemotherapeutic agent cytarabine (Ara-C). Thus, our results suggest that targeting mitochondrial function with DPI might be exploited to target AML cells with high OxPhos status.

Abstract PR009: Investigating dynamics of the mitochondrial network in triple negative breast cancer chemotherapy resistance

Abstract Triple negative breast cancer (TNBC) is an aggressive disease with extremely limited targeted therapeutic options. Thus, standard cytotoxic chemotherapies (typically sequential and/or combined anthracyclines, taxanes, and/or platinums) remain a mainstay treatment for this subtype of breast cancer. Nearly 50% of TNBC patients harbor substantial residual cancer burden following standard chemotherapy treatment, leading to high rates of recurrence and death (PMID:28135148). Using longitudinal biopsies from orthotopic patient-derived xenograft (PDX) models and TNBC patients, we found that residual tumors following chemotherapy transitioned to a unique metabolic state characterized by high mitochondrial oxidative phosphorylation (oxphos). This metabolic state was transient, with tumors reverting to their baseline glycolysis-high phenotype when they were allowed to regrow in the absence of treatment. Using genomic sequencing and cellular barcode-mediated clonal tracking, we found this mechanism of chemoresistance arose in the absence of clonal selection, suggesting chemotherapy induced plastic (i.e., non-genomic) programs enabling cell survival following treatment. Oxphos inhibition using a specific inhibitor of electron transport chain Complex I (IACS010759; PMID:29892070) was significantly more efficacious against residual, rather than treatment-naive tumors (PMID:30996079), providing evidence that dynamic metabolic phenotypes represent targetable therapeutic vulnerabilities for TNBC. Mechanisms controlling mitochondrial bioenergetics are numerous but their roles in driving metabolic phenotypes of chemoresistant TNBC are not understood. We find that residual tumor cells following chemotherapy treatment have altered mitochondrial network morphology. Mitochondrial fission and fusion are well established regulators of mitochondrial morphology, yet their functional impacts on bioenergetic output of mitochondria is highly context dependent. Our analyses of mitochondrial structure in TNBC cells and PDX tumors reveal that while DNA-damaging chemotherapeutics (anthracyclines, platinums) increased mitochondrial elongation, microtubule-stabilizing chemotherapeutics (taxanes) increased mitochondrial fragmentation. This was accompanied by increased or decreased oxphos rates and mitochondrial content, respectively. Interestingly, these structural changes were reverted in PDX tumors that were allowed to regrow in the absence of treatment, suggesting mitochondrial adaptations are plastic in vivo. Using pharmacologic agents in TNBC cells, we found that mitochondrial fusion increased oxphos and chemoresistance, whereas mitochondrial fission decreased oxphos and chemoresistance. Further, knockdown of OPA1 or MFN2, mediators of mitochondrial inner and outer membrane fusion, respectively, diminished mitochondrial fusion, oxphos, and chemoresistance. Based on these findings, we hypothesize therapeutic targeting of mediators of mitochondrial network adaptability may be a promising strategy to overcome plastic metabolic states indued by chemotherapeutics in TNBC. Citation Format: Lily Baek, Mariah Berner, Junegoo Lee, Katherine Pendleton, Karen Wang, Emily B. Goff, James P. Barrish, Bora Lim, Michael T. Lewis, Philip L. Lorenzi, Weston Porter, Gloria V. Echeverria. Investigating dynamics of the mitochondrial network in triple negative breast cancer chemotherapy resistance [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr PR009.

Live isolation of naïve ESCs via distinct glucose metabolism and stored glycogen

Naïve and primed pluripotent stem cells recapitulate the peri- and post-implantation development, respectively. Thus, investigation of distinct traits between each pluripotent stem cell type would shed light on early embryonic processes. Herein, by screening a fluorescent probe library, we found that intracellular glycogen led to specific reactivity to CDg4, a glycogen fluorescence sensor, in both human and mouse naïve embryonic stem cells (ESCs). The requirement of constant inhibition of Gsk3β as well as high oxidative phosphorylation (OxPHOS) in naïve compared to primed ESCs was closely associated to high level of intracellular glycogen in naïve ESCs. Both capacity of OxPHOS and stored glycogen, rescued naïve ESCs by transient inhibition of glycolysis, which selectively eliminated primed ESCs. Additionally, naïve ESCs with active OxPHOS were enriched from a mixture with primed ESCs by high reactivity to ATP-Red1, a mitochondrial ATP fluorescence probe. These results indicate the active OxPHOS and high intracellular glycogen as a novel “biomarker” delineating metabolic remodeling during the transition of naïve pluripotency.

Kidney VISTA prevents IFN-γ/IL-9 axis-mediated tubulointerstitial fibrosis after acute glomerular injury.

Severe glomerular injury ultimately leads to tubulointerstitial fibrosis that determines patient outcome, but the immunological molecules connecting these processes remain undetermined. The present study addressed whether V-domain Ig suppressor of T cell activation (VISTA), constitutively expressed in kidney macrophages, plays a protective role in tubulointerstitial fibrotic transformation after acute antibody-mediated glomerulonephritis. After acute glomerular injury using nephrotoxic serum, tubules in the VISTA-deficient (Vsir–/–) kidney suffered more damage than those in WT kidneys. When interstitial immune cells were examined, the contact frequency of macrophages with infiltrated T cells increased and the immunometabolic features of T cells changed to showing high oxidative phosphorylation and fatty acid metabolism and overproduction of IFN-γ. The Vsir–/– parenchymal tissue cells responded to this altered milieu of interstitial immune cells as more IL-9 was produced, which augmented tubulointerstitial fibrosis. Blocking antibodies against IFN-γ and IL-9 protected the above pathological process in VISTA-depleted conditions. In human samples with acute glomerular injury (e.g., antineutrophil cytoplasmic autoantibody vasculitis), high VISTA expression in tubulointerstitial immune cells was associated with low tubulointerstitial fibrosis and good prognosis. Therefore, VISTA is a sentinel protein expressed in kidney macrophages that prevents tubulointerstitial fibrosis via the IFN-γ/IL-9 axis after acute antibody-mediated glomerular injury.