bims-midysc 2025-01-19 papers

bims-midysc

Biomed News

on Mitochondria dysfunction in cancer

Issue of 2025–01–19
sixteen papers selected by
Papachristodoulou Lab

Mitochondrial uncouplers inhibit oncogenic E2F1 activity and prostate cancer growth.
NADH-bound AIF activates the mitochondrial CHCHD4/MIA40 chaperone by a substrate-mimicry mechanism.
Using HBmito Crimson to Observe Mitochondrial Cristae Through STED Microscopy.
Sex-dependent adaptations in heart mitochondria from transgenic mice overexpressing cytochrome b5 reductase-3.
Mitochondrial chaperonin DNAJC15 promotes vulnerability to ferroptosis of chemoresistant ovarian cancer cells.
Differential Mitochondrial Redox Responses to the Inhibition of NAD+ Salvage Pathway of Triple Negative Breast Cancer Cells.
Mitochondrial genome variability and metabolic alterations reveal new biomarkers of resistance in testicular germ cell tumors.
Imaging flow cytometry reveals divergent mitochondrial phenotypes in mitochondrial disease patients.
DYRK1A-TGF-β signaling axis determines sensitivity to OXPHOS inhibition in hepatocellular carcinoma.
Somatic mutation as an explanation for epigenetic aging.
A review of the pathogenesis of mitochondria in breast cancer and progress of targeting mitochondria for breast cancer treatment.
Quantitative analyses of single mitochondrial components reveal an early role of small-mitochondrial-networks in priming neoplasticity.
Rewriting nuclear epigenetic scripts in mitochondrial diseases as a strategy for heteroplasmy control.
DNA replication stress underpins the vulnerability to oxidative phosphorylation inhibition in colorectal cancer.
Protocol for mitochondrial variant enrichment from single-cell RNA sequencing using MAESTER.
Disease stage-specific role of the mitochondrial pyruvate carrier suppresses differentiation in temozolomide and radiation-treated glioblastoma.

Cell Rep Med. 2024 Dec 30. pii: S2666-3791(24)00661-X. [Epub ahead of print] 101890

Mitochondrial uncouplers inhibit oncogenic E2F1 activity and prostate cancer growth.

Ohuod Hawsawi, Weinan Xue, Tingting Du, Mengqi Guo, Xiaolin Yu, Mingyi Zhang, Paul S Hoffman, Roni Bollag, Jun Li, Jia Zhou, Hongbo Wang, Junran Zhang, Zheng Fu, Xiaoguang Chen, Chunhong Yan.

  Mitochondrial uncouplers dissipate proton gradients and deplete ATP production from oxidative phosphorylation (OXPHOS). While the growth of prostate cancer depends on OXPHOS-generated ATP, the oncogenic pathway mediated by the transcription factor E2F1 is crucial for the progression of this deadly disease. Here, we report that mitochondrial uncouplers, including tizoxanide (TIZ), the active metabolite of the Food and Drug Administration (FDA)-approved anthelmintic nitazoxanide (NTZ), inhibit E2F1-mediated expression of genes involved in cell cycle progression, DNA synthesis, and lipid synthesis. Consequently, NTZ/TIZ induces S-phase kinase-associated protein 2 (SKP2)-mediated G1 arrest while impeding DNA synthesis, lipogenesis, and the growth of prostate cancer cells. The anti-cancer activity of TIZ correlates with its OXPHOS-uncoupling activity. NTZ/TIZ appears to inhibit ATP production, thereby activating the AMP-activated kinase (AMPK)-p38 pathway, leading to cyclin D1 degradation, Rb dephosphorylation, and subsequent E2F1 inhibition. Our results thus connect OXPHOS uncoupling to the inhibition of an essential oncogenic pathway, supporting repositioning NTZ and other mitochondrial uncouplers for prostate cancer therapy.

Keywords:  AMPK; E2F1; SKP2; cyclin D1; mitochondrial uncoupler; nitazoxanide; oxidative phosphorylation; p38; prostate cancer; tizoxanide

DOI:  https://doi.org/10.1016/j.xcrm.2024.101890
EMBO J. 2025 Jan 13.

NADH-bound AIF activates the mitochondrial CHCHD4/MIA40 chaperone by a substrate-mimicry mechanism.

Chris A Brosey, Runze Shen, John A Tainer.

  Mitochondrial metabolism requires the chaperoned import of disulfide-stabilized proteins via CHCHD4/MIA40 and its enigmatic interaction with oxidoreductase Apoptosis-inducing factor (AIF). By crystallizing human CHCHD4's AIF-interaction domain with an activated AIF dimer, we uncover how NADH allosterically configures AIF to anchor CHCHD4's β-hairpin and histidine-helix motifs to the inner mitochondrial membrane. The structure further reveals a similarity between the AIF-interaction domain and recognition sequences of CHCHD4 substrates. NMR and X-ray scattering (SAXS) solution measurements, mutational analyses, and biochemistry show that the substrate-mimicking AIF-interaction domain shields CHCHD4's redox-sensitive active site. Disrupting this shield critically activates CHCHD4 substrate affinity and chaperone activity. Regulatory-domain sequestration by NADH-activated AIF directly stimulates chaperone binding and folding, revealing how AIF mediates CHCHD4 mitochondrial import. These results establish AIF as an integral component of the metazoan disulfide relay and point to NADH-activated dimeric AIF as an organizational import center for CHCHD4 and its substrates. Importantly, AIF regulation of CHCHD4 directly links AIF's cellular NAD(H) sensing to CHCHD4 chaperone function, suggesting a mechanism to balance tissue-specific oxidative phosphorylation (OXPHOS) capacity with NADH availability.

Keywords:  Apoptosis-inducing Factor (AIF); CHCHD4/MIA40; OXPHOS; Small-angle X-ray Scattering (SAXS); X-ray Crystallography

DOI:  https://doi.org/10.1038/s44318-024-00360-6
Bio Protoc. 2025 Jan 05. 15(1): e5150

Using HBmito Crimson to Observe Mitochondrial Cristae Through STED Microscopy.

Xichuan Ge, Wei Ren, Chunyan Shan, Peng Xi, Baoxiang Gao.

  Mitochondrial cristae, formed by folding the mitochondrial inner membrane (IM), are essential for cellular energy supply. However, the observation of the IM is challenging due to the limitations in spatiotemporal resolution offered by conventional microscopy and the absence of suitable in vitro probes speciﬁcally targeting the IM. Here, we describe a detailed imaging protocol for the mitochondrial inner membrane using the Si-rhodamine dye HBmito Crimson, which has excellent photophysical properties, to label live cells for imaging via stimulated emission depletion (STED) microscopy. This allows for STED imaging over more than 500 frames (approximately one hour), with a spatial resolution of 40 nm, enabling the observation of cristae dynamics during various mitochondrial processes. The protocol includes detailed steps for cell staining, image acquisition, image processing, and resolution analysis. Utilizing the superior resolution of STED microscopy, the structure and complex dynamic changes of cristae can be visualized. Key features • The protocol is designed to visualize mitochondrial cristae in living cells using STED microscopy. • The protocol enables nanoscale observation of dynamic mitochondrial cristae. • Real-time observation of mitochondrial morphological changes, fusion, and fission events.

Keywords:  Fluorescence labeling; Live cell imaging; Low-saturation power; Mitochondria cristae; Super-resolution imaging

DOI:  https://doi.org/10.21769/BioProtoc.5150
Mitochondrion. 2025 Jan 09. pii: S1567-7249(25)00001-7. [Epub ahead of print]81 102004

Sex-dependent adaptations in heart mitochondria from transgenic mice overexpressing cytochrome b5 reductase-3.

Luz Marina Sánchez-Mendoza, José A González-Reyes, Sandra Rodríguez-López, Miguel Calvo-Rubio, Pilar Calero-Rodríguez, Rafael de Cabo, M Isabel Burón, José M Villalba.

  Cytochrome b5 reductase 3 (CYB5R3) overexpression upregulates mitochondrial biogenesis, function, and abundance in skeletal muscle and kidneys, and mimics some of the salutary effects of calorie restriction, with the most striking effects being observed in females. We aimed to investigate the mitochondrial adaptations prompted by CYB5R3 overexpression in the heart, an organ surprisingly overlooked in studies focused on this long-lived transgenic model despite the critical role played by CYB5R3 in supporting cardiomyocytes mitochondrial respiration. Given that CYB5R3 effects have been found to be sex-dependent, we focused our research on both males and females. CYB5R3 was efficiently overexpressed in cardiac tissue from transgenic mice, without any difference between sexes. The abundance of electron transport chain complexes markers and cytochrome c was higher in males than in females. CYB5R3 overexpression downregulated the levels of complexes markers in males but not females, without decreasing oxygen consumption capacity. CYB5R3 increased the size and abundance of cardiomyocytes mitochondria, and reduced thickness and preserved the length of mitochondria-endoplasmic reticulum contact sites in heart from males but not females. Metabolic changes were also highlighted in transgenic mice, with an upregulation of fatty acid oxidation markers, particularly in males. Our results support that CYB5R3 overexpression upregulates markers consistent with enhanced mitochondrial function in the heart, producing most of these actions in males, with illustrates the complexity of the CYB5R3-overexpressing transgenic model.

Keywords:  CYB5R3; Cardiomyocyte; Heart; Mitochondria; Sexual dimorphism

DOI:  https://doi.org/10.1016/j.mito.2025.102004
Open Biol. 2025 Jan;15(1): 240151

Mitochondrial chaperonin DNAJC15 promotes vulnerability to ferroptosis of chemoresistant ovarian cancer cells.

Stefano Miglietta, Manuela Sollazzo, Iacopo Gherardi, Sara Milioni, Beatrice Cavina, Lorena Marchio, Monica De Luise, Camelia Alexandra Coada, Marco Fiorillo, Anna Myriam Perrone, Ivana Kurelac, Giuseppe Gasparre, Luisa Iommarini, Anna Maria Ghelli, Anna Maria Porcelli.

  DNAJC15 is a mitochondrial TIMM23-related co-chaperonin known for its role in regulating oxidative phosphorylation efficiency, oxidative stress response and lipid metabolism. Recently, it has been proposed that the loss of DNAJC15 correlates with cisplatin (CDDP)-resistance onset in ovarian cancer (OC), suggesting this protein as a potential prognostic factor during OC progression. However, the molecular mechanisms through which DNAJC15 contributes to CDDP response remains poorly investigated. Here, we show that high levels of DNAJC15 are associated with accumulation of lipid droplets, decreased tumorigenic features and increased sensitivity to CDDP in OC cells. When overexpressed, DNAJC15 induced a phenotype displaying increased lipid peroxidation and subsequent ferroptosis induction. To prove a role for DNAJC15-induced ferroptosis in promoting sensitivity to CDDP, we reduced lipid peroxidation upon Ferrostatin 1 treatment, which decreased cells' vulnerability to ferroptosis ultimately recovering their CDDP-resistant phenotype. In conclusion, our study uncovers the role of DNAJC15 in modulating ferroptosis activation and in the onset of CDDP resistance in OC cells.

Keywords:  DNAJC15; cisplatin resistance; ferroptosis; mitochondria; ovarian cancer

DOI:  https://doi.org/10.1098/rsob.240151
Cancers (Basel). 2024 Dec 24. pii: 7. [Epub ahead of print]17(1):

Differential Mitochondrial Redox Responses to the Inhibition of NAD+ Salvage Pathway of Triple Negative Breast Cancer Cells.

Jack Kollmar, Junmei Xu, Diego Gonzalves, Joseph A Baur, Lin Z Li, Julia Tchou, He N Xu.

  Background/Objectives: Cancer cells rely on metabolic reprogramming that is supported by altered mitochondrial redox status and an increased demand for NAD+. Over expression of Nampt, the rate-limiting enzyme of the NAD+ biosynthesis salvage pathway, is common in breast cancer cells, and more so in triple negative breast cancer (TNBC) cells. Targeting the salvage pathway has been pursued for cancer therapy. However, TNBC cells have heterogeneous responses to Nampt inhibition, which contributes to the diverse outcomes. There is a lack of imaging biomarkers to differentiate among TNBC cells under metabolic stress and identify which are responsive. We aimed to characterize and differentiate among a panel of TNBC cell lines under NAD-deficient stress and identify which subtypes are more dependent on the NAD salvage pathway. Methods: Optical redox imaging (ORI), a label-free live cell imaging microscopy technique was utilized to acquire intrinsic fluorescence intensities of NADH and FAD-containing flavoproteins (Fp) thus the mitochondrial redox ratio Fp/(NADH + Fp) in a panel of TNBC cell lines. Various fluorescence probes were then added to the cultures to image the mitochondrial ROS, mitochondrial membrane potential, mitochondrial mass, and cell number. Results: Various TNBC subtypes are sensitive to Nampt inhibition in a dose- and time-dependent manner, they have differential mitochondrial redox responses; furthermore, the mitochondrial redox indices linearly correlated with mitochondrial ROS induced by various doses of a Nampt inhibitor. Moreover, the changes in the redox indices correlated with growth inhibition. Additionally, the redox state was found fully recovered after removing the Nampt inhibitor. Conclusions: This study supports the utility of ORI in rapid metabolic phenotyping of TNBC cells under NAD-deficient stress to identify responsive cells and biomarkers of treatment response, facilitating combination therapy strategies.

Keywords:  FAD-containing flavoproteins; FK866; GMX1778; NAD+ biosynthesis; Nampt; TNBC; label-free optical redox/metabolic imaging; mitochondrial redox state; optical redox ratio

DOI:  https://doi.org/10.3390/cancers17010007
Cancer Drug Resist. 2024 ;7 54

Mitochondrial genome variability and metabolic alterations reveal new biomarkers of resistance in testicular germ cell tumors.

Pavlina Kabelikova, Danica Ivovic, Zuzana Sumbalova, Miloslav Karhanek, Lucia Tatayova, Martina Skopkova, Michal Cagalinec, Vladimira Bruderova, Jan Roska, Dana Jurkovicova.

  Aim: Mutations in the mitochondrial (mt) genome contribute to metabolic dysfunction and their accumulation relates to disease progression and resistance development in cancer cells. This study explores the mutational status of the mt genome of cisplatin-resistant vs. -sensitive testicular germ cell tumor (TGCT) cells and explores its association with their respiration parameters, expression of respiratory genes, and preferences for metabolic pathways to reveal new markers of therapy resistance in TGCTs. Methods: Using Illumina sequencing with Twist Enrichment Panel, the mutations of mt genomes of sensitive 2102EP, H12.1, NTERA-2, T-cam and resistant 2102EP Cis, H12.1 ODM, 1411HP, 1777NRpmet, NTERA-2 Cis and T-cam Cis cell lines were identified. The mt respiration of the cells was assessed using high-resolution respirometry method (O2k-respirometer Oroboros) and the differential expression profiles of mt respiratory genes were determined using RT-qPCR. Associated preferences for metabolic pathways were compared using Glycolysis/OXPHOS assay. Results: In resistant TGCT cells, new mutations in mt genes MT-ND1-6, MT-RNR, MT-CO1-3, MT-ATP6, and MT-CYB were recognized. The respiratory rates of the 1777NRpmet cell line were the highest, while those of the 1411HP line the lowest; rates of the control and all other TGCT cell lines fell between these two lines. The statistically significant differences in gene expression of the respiratory genes were recorded only in NTERA-2 Cis and T-cam Cis cell lines. Sensitive cell lines NTERA-2 and 2102EP preferred oxidative phosphorylation (OXPHOS), while glycolysis was typical for resistant NTERA-2 Cis, 2102EP Cis and 1411HP cell lines. Metastatic 1777NRpmet cells seem to utilize both. An isogenic pair of cell lines H12.1 and H12.1ODM showed the opposite dependence, sensitive H12.1 preferring glycolysis, while resistant H12.1ODM OXPHOS. Conclusion: In summary, our study identified new mutations in mt genes of resistant TGCT cell lines that are associated with different mt respiration parameters, gene expression patterns and preferences for metabolic pathways, providing potential novel molecular biomarkers that distinguish the resistant TGCT phenotype or specify its histological classification.

Keywords:  OXPHOS; Testicular cancer; chemoresistance; glycolysis; mitochondria; mtDNA mutation

DOI:  https://doi.org/10.20517/cdr.2024.141
iScience. 2025 Jan 17. 28(1): 111496

Imaging flow cytometry reveals divergent mitochondrial phenotypes in mitochondrial disease patients.

Irena J J Muffels, Richard Rodenburg, Hanneke L D Willemen, Désirée van Haaften-Visser, Hans Waterham, Niels Eijkelkamp, Sabine A Fuchs, Peter M van Hasselt.

  Traditional classification by clinical phenotype or oxidative phosphorylation (OXPHOS) complex deficiencies often fails to clarify complex genotype-phenotype correlations in mitochondrial disease. A multimodal functional assessment may better reveal underlying disease patterns. Using imaging flow cytometry (IFC), we evaluated mitochondrial fragmentation, swelling, membrane potential, reactive oxygen species (ROS) production, and mitochondrial mass in fibroblasts from 31 mitochondrial disease patients. Significant changes were observed in 97% of patients, forming two overarching groups with distinct responses to mitochondrial pathology. One group displayed low-to-normal membrane potential, indicating a hypometabolic state, while the other showed elevated membrane potential and swelling, suggesting a hypermetabolic state. Literature analysis linked these clusters to complex I stability defects (hypometabolic) and proton pumping activity (hypermetabolic). Thus, our IFC-based platform offers a novel approach to identify disease-specific patterns through functional responses, supporting improved diagnostic and therapeutic strategies.

Keywords:  Biological sciences; Genetics; Health sciences; Human genetics; Medicine; Natural sciences

DOI:  https://doi.org/10.1016/j.isci.2024.111496
Dev Cell. 2025 Jan 07. pii: S1534-5807(24)00775-5. [Epub ahead of print]

DYRK1A-TGF-β signaling axis determines sensitivity to OXPHOS inhibition in hepatocellular carcinoma.

Ying Cao, Ruolan Qian, Ruilian Yao, Quan Zheng, Chen Yang, Xupeng Yang, Shuyi Ji, Linmen Zhang, Shujie Zhan, Yiping Wang, Tianshi Wang, Hui Wang, Chun-Ming Wong, Shengxian Yuan, Christopher Heeschen, Qiang Gao, René Bernards, Wenxin Qin, Cun Wang.

  Intervening in mitochondrial oxidative phosphorylation (OXPHOS) has emerged as a potential therapeutic strategy for certain types of cancers. Employing kinome-based CRISPR screen, we find that knockout of dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) synergizes with OXPHOS inhibitor IACS-010759 in liver cancer cells. Targeting DYRK1A combined with OXPHOS inhibitors activates TGF-β signaling, which is crucial for OXPHOS-inhibition-triggered cell death. Mechanistically, upregulation of glutamine transporter solute carrier family 1 member 5 (SLC1A5) transcription compensates for the increased glutamine requirement upon OXPHOS inhibition. DYRK1A directly phosphorylates SMAD3 Thr132, thereby suppressing the negative impact of TGF-β signaling on transcription of SLC1A5, leading to intrinsic resistance of liver cancer cells to OXPHOS inhibition. Moreover, we demonstrate the therapeutic efficacy of IACS-010759 in combination with DYRK1A inhibition in multiple liver cancer models, including xenografts, patient-derived xenografts, and spontaneous tumor model. Our study elucidates how the DYRK1A-TGF-β signaling axis controls the response of tumor cells to OXPHOS inhibition and provides valuable insights into targeting OXPHOS for liver cancer therapy.

Keywords:  DYRK1A; IACS-010759; TGF-β signaling; hepatocellular carcinoma; oxidative phosphorylation

DOI:  https://doi.org/10.1016/j.devcel.2024.12.035
Nat Aging. 2025 Jan 13.

Somatic mutation as an explanation for epigenetic aging.

Zane Koch, Adam Li, Daniel S Evans, Steven Cummings, Trey Ideker.

DNA methylation marks have recently been used to build models known as epigenetic clocks, which predict calendar age. As methylation of cytosine promotes C-to-T mutations, we hypothesized that the methylation changes observed with age should reflect the accrual of somatic mutations, and the two should yield analogous aging estimates. In an analysis of multimodal data from 9,331 human individuals, we found that CpG mutations indeed coincide with changes in methylation, not only at the mutated site but with pervasive remodeling of the methylome out to ±10 kilobases. This one-to-many mapping allows mutation-based predictions of age that agree with epigenetic clocks, including which individuals are aging more rapidly or slowly than expected. Moreover, genomic loci where mutations accumulate with age also tend to have methylation patterns that are especially predictive of age. These results suggest a close coupling between the accumulation of sporadic somatic mutations and the widespread changes in methylation observed over the course of life.

DOI: https://doi.org/10.1038/s43587-024-00794-x
J Transl Med. 2025 Jan 15. 23(1): 70

A review of the pathogenesis of mitochondria in breast cancer and progress of targeting mitochondria for breast cancer treatment.

Aoling Huang, Haochen Xue, Ting Xie, Lingyan Xiang, Zhengzhuo Chen, Aolong Ma, Honglin Yan, Jingping Yuan.

  With breast cancer being the most common tumor among women in the world today, it is also the leading cause of cancer-related deaths. Standard treatments include chemotherapy, surgery, endocrine therapy, and targeted therapy. However, the heterogeneity, drug resistance, and poor prognosis of breast cancer highlight an urgent need for further exploration of its underlying mechanisms. Mitochondria, highly dynamic intracellular organelles, play a pivotal role in maintaining cellular energy metabolism. Altered mitochondrial function plays a critical role in various diseases, and recent studies have elucidated its pathophysiological mechanisms in breast carcinogenesis. This review explores the role of mitochondrial dysfunction in breast cancer pathogenesis and assesses potential mitochondria-targeted therapies.

Keywords:  Apoptosis; Breast cancer; Mitochondria; Mitochondrial dynamics; Mitochondrial metabolism

DOI:  https://doi.org/10.1186/s12967-025-06077-2
bioRxiv. 2025 Jan 02. pii: 2024.12.26.630414. [Epub ahead of print]

Quantitative analyses of single mitochondrial components reveal an early role of small-mitochondrial-networks in priming neoplasticity.

Mayank Saini, Swati Agarwala, Biratal Wagle, Brian Spurlock, Bharti Golchha, Danitra Parker, Kasturi Mitra.

Quantitative understanding of mitochondrial heterogeneity is necessary for elucidating the precise role of these multifaceted organelles in tumor cell development. We demonstrate an early mechanistic role of mitochondria in initiating neoplasticity by performing quantitative analyses of structure-function of single mitochondrial components coupled with single cell transcriptomics. We demonstrate that the large Hyperfused-Mitochondrial-Networks (HMNs) of keratinocytes promptly get converted to the heterogenous Small-Mitochondrial-Networks (SMNs) as the stem cell enriching dose of the model carcinogen, TCDD, depolarizes mitochondria. This happens by physical reorganization of the HMN nodes and edges, which enriches redox tuned SMNs with distinct network complexity. This leads to establishment of transcriptomic interaction between the upregulated redox relevant mtDNA genes and the lineage specific stemness gene, KRT15, prior to cell cycle exit. The SMN enrichment and related transcriptomic connections are sustained in the neoplastic cell population. Consistently, carcinogenic dose incapable of causing pronounced neoplastic stem cell enrichment fails to establish specific enrichment of SMNs and its linked mtDNA-KRT15(stemness) transcriptomic interaction prior to cell cycle exit. The mtDNA-KRT15 modulation is confirmed in cSCC tumors, while highlighting patient heterogeneity. Therefore, we propose that early enrichment of redox-tuned SMNs primes neoplastic transformation by establishing mtDNA-stemness transcriptomic interaction prior to cell cycle exit towards specifying quiescent neoplastic stem cells. Our data implies that redox-tuned SMNs, created by mitochondrial fission, would be sustained by tuning the balance of mitochondrial fission-fusion during neoplastic transformation. The proposed early role of mitochondria in cancer etiology is potentially relevant for designing precision strategies for cancer prevention and therapy.
Significance Statement: The challenges of understanding the complex role of the multifaceted and heterogenous cellular organelles, mitochondria, can be potentially overcome with their quantitative analyses. We use a combinatorial approach of quantitative analyses of single-mitochondrial-components and scRNA-seq to elucidate a mechanism of mitochondrial priming of cancer initiation by a model carcinogen. We propose that conversion of large Hyperfused-Mitochondrial-Networks (HMNs) to Small-Mitochondrial-Networks (SMNs) primes non-transformed keratinocytes towards their neoplastic transformation. Mechanistically, the SMNs, enriched by modulation of the physical nodes and edges of mitochondrial networks, tunes mitochondrial redox balance to establish transcriptomic interactions towards specifying a state of stemness. Further probing of our fundamental findings in the light of cancer heterogeneity may facilitate refinement of the various proposed mitochondria based targeted cancer therapies.

DOI: https://doi.org/10.1101/2024.12.26.630414
bioRxiv. 2024 Dec 30. pii: 2024.12.30.630791. [Epub ahead of print]

Rewriting nuclear epigenetic scripts in mitochondrial diseases as a strategy for heteroplasmy control.

María J Pérez, Rocío B Colombo, Sebastián M Real, María T Branham, Sergio R Laurito, Carlos T Moraes, Lía Mayorga.

Mitochondrial diseases, caused by mutations in either nuclear or mitochondrial DNA (mtDNA), currently have limited treatment options. For mtDNA mutations, reducing mutant-to-wild-type mtDNA ratio (heteroplasmy shift) is a promising therapeutic option, though current approaches face significant challenges. Previous research has shown that severe mitochondrial dysfunction triggers an adaptive nuclear epigenetic response, characterized by changes in DNA methylation, which does not occur or is less important when mitochondrial impairment is subtle. Building on this, we hypothesized that targeting nuclear DNA methylation could selectively compromise cells with high levels of mutant mtDNA, favor ones with lower mutant load and thereby reduce overall heteroplasmy. Using cybrid models harboring two disease-causing mtDNA mutations-m.13513G>A and m.8344A>G-at varying heteroplasmy levels, we discovered that both the mutation type and load distinctly shape the nuclear DNA methylome. We found this methylation pattern to be critical for the survival of high-heteroplasmy cells but not for the low-heteroplasmy ones. Consequently, by disrupting this epigenetic programming with FDA approved DNA methylation inhibitors we managed to selectively impact high-heteroplasmy cybrids and reduce heteroplasmy. These findings were validated in both cultured cells and an in vivo xenograft model. Our study reveals a previously unrecognized role for nuclear DNA methylation in regulating cell survival in the context of mitochondrial heteroplasmy. This insight not only advances our understanding of mitochondrial-nuclear interactions but also introduces epigenetic modulation as a possible therapeutic avenue for mitochondrial diseases.

DOI: https://doi.org/10.1101/2024.12.30.630791
Cell Death Dis. 2025 Jan 14. 16(1): 16

DNA replication stress underpins the vulnerability to oxidative phosphorylation inhibition in colorectal cancer.

Xiao Hong Zhao, Man Man Han, Qian Qian Yan, Yi Meng Yue, Kaihong Ye, Yuan Yuan Zhang, Liu Teng, Liang Xu, Xiao-Jing Shi, Ting La, Yu Chen Feng, Ran Xu, Vinod K Narayana, David P De Souza, Lake-Ee Quek, Jeff Holst, Tao Liu, Mark A Baker, Rick F Thorne, Xu Dong Zhang, Lei Jin.

Mitochondrial oxidative phosphorylation (OXPHOS) is a therapeutic vulnerability in glycolysis-deficient cancers. Here we show that inhibiting OXPHOS similarly suppresses the proliferation and tumorigenicity of glycolytically competent colorectal cancer (CRC) cells in vitro and in patient-derived CRC xenografts. While the increased glycolytic activity rapidly replenished the ATP pool, it did not restore the reduced production of aspartate upon OXPHOS inhibition. This shortage in aspartate, in turn, caused nucleotide deficiencies, leading to S phase cell cycle arrest, replication fork stalling, and enrichment of the p53 pathway, manifestations of replication stress. The addition of purine nucleobases adenine and guanine along with the pyrimidine nucleoside uridine restored replication fork progression and cell proliferation, whereas the supplementation of exogenous aspartate recovered the nucleotide pool, demonstrating a causal role of the aspartate shortage in OXPHOS inhibition-induced nucleotide deficiencies and consequently replication stress and reductions in proliferation. Moreover, we demonstrate that glutamic-oxaloacetic transaminase 1 (GOT1) is critical for maintaining the minimum aspartate pool when OXPHOS is inhibited, as knockdown of GOT1 further reduced aspartate levels and rendered CRC cells more sensitive to OXPHOS inhibition both in vitro and in vivo. These results propose GOT1 targeting as a potential avenue to sensitize cancer cells to OXPHOS inhibitors, thus lowering the necessary doses to efficiently inhibit cancer growth while alleviating their adverse effects.

DOI: https://doi.org/10.1038/s41419-025-07334-4
STAR Protoc. 2025 Jan 15. pii: S2666-1667(24)00729-9. [Epub ahead of print]6(1): 103564

Protocol for mitochondrial variant enrichment from single-cell RNA sequencing using MAESTER.

Jonathan D Good, Ksenia R Safina, Tyler E Miller, Peter van Galen.

  Single-cell RNA sequencing (scRNA-seq) enables detailed characterization of cell states but often lacks insights into tissue clonal structures. Here, we present a protocol to probe cell states and clonal information simultaneously by enriching mitochondrial DNA (mtDNA) variants from 3'-barcoded full-length cDNA. We describe steps for input library preparation, mtDNA enrichment, PCR product cleanup, and paired-end sequencing. We then detail computational steps for running maegatk, variant calling, and data integration to illuminate cell states and clonal dynamics in primary human tissues. For complete details on the use and execution of this protocol, please refer to Miller et al.1.

Keywords:  Genetics; RNA-seq; Sequence analysis; Single Cell

DOI:  https://doi.org/10.1016/j.xpro.2024.103564
Neuro Oncol. 2025 Jan 11. pii: noaf008. [Epub ahead of print]

Disease stage-specific role of the mitochondrial pyruvate carrier suppresses differentiation in temozolomide and radiation-treated glioblastoma.

Emma Martell, Helgi Kuzmychova, Harshal Senthil, Ujala Chawla, Esha Kaul, Akaljot Grewal, Versha Banerji, Christopher M Anderson, Chitra Venugopal, Donald Miller, Tamra E Werbowetski-Ogilvie, Sheila K Singh, Tanveer Sharif.

   BACKGROUND: The mitochondrial pyruvate carrier (MPC), a central metabolic conduit linking glycolysis and mitochondrial metabolism, is instrumental in energy production. However, the role of the MPC in cancer is controversial. In particular, the importance of the MPC in glioblastoma (GBM) disease progression following standard temozolomide (TMZ) and radiation therapy (RT) remains unexplored.
METHODS: Leveraging in vitro and in vivo patient-derived models of TMZ-RT treatment in GBM, we characterize the temporal dynamics of MPC abundance and downstream metabolic consequences using state-of-the-art molecular, metabolic, and functional assays.
RESULTS: Our findings unveil a disease stage-specific role for the MPC, where in post-treatment GBM, but not therapy-naïve tumors, the MPC acts as a central metabolic regulator that suppresses differentiation. Temporal profiling reveals a dynamic metabolic rewiring where a steady increase in MPC abundance favors a shift towards enhanced mitochondrial metabolic activity across patient GBM samples. Intriguingly, while overall mitochondrial metabolism is increased, acetyl-CoA production is reduced in post-treatment GBM cells, hindering histone acetylation and silencing neural differentiation genes in an MPC-dependent manner. Finally, the therapeutic translations of these findings are highlighted by the successful pre-clinical patient-derived orthotopic xenograft (PDOX) trials utilizing a blood-brain-barrier (BBB) permeable MPC inhibitor, MSDC-0160, which augments standard TMZ-RT therapy to mitigate disease relapse and prolong animal survival.
CONCLUSION: Our findings demonstrate the critical role of the MPC in mediating GBM aggressiveness and molecular evolution following standard TMZ-RT treatment, illuminating a therapeutically-relevant metabolic vulnerability to potentially improve survival outcomes for GBM patients.

Keywords:  Glioblastoma; differentiation; metabolism; mitochondrial pyruvate carrier; tumor recurrence

DOI:  https://doi.org/10.1093/neuonc/noaf008