Higher Mitochondrial Mass Research Articles

The role of alternative autophagy in cell viability and response to paclitaxel treatment in v-Ha-ras-transformed NIH 3T3 cells

In confluent v-Ha-ras-transformed NIH 3T3 fibroblasts (Ras-NIH 3T3), LC3 downregulation may precede a decrease in canonical autophagy, thus contributing to cell survival. Herein, we aimed to investigate the role of alternative autophagy in the viability of long-term cultures of Ras-NIH 3T3 cells and their parental NIH 3T3 cells. As cell confluence increased with the culture period, the level of alternative autophagy, as assessed through Lamp2-Rab9 co-localization, gradually decreased in both cell lines. However, Ras-NIH 3T3 cells maintained higher levels of alternative autophagy than the parental cells did. Rab9 knockdown minimally affected NIH 3T3 cells while drastically reducing the viability of Ras-NIH 3T3 cells, which suggested that alternative autophagy plays a critical role in Ras-NIH 3T3 cells. In contrast, reactive oxygen species (ROS) production in Ras-NIH 3T3 cells was higher than that in NIH 3T3 cells during long-term culture. Moreover, NIH 3T3 cells exhibited a continual decrease in mitochondrial mass, whereas Ras-NIH 3T3 cells maintained high mitochondrial mass. Immunofluorescence analysis of mitochondrial membrane marker proteins and mitochondrial membrane potential (MMP) also demonstrated a temporal pattern of changes similar to those of mitochondrial mass. This finding could be attributed to the relatively higher level of alternative autophagy in Ras-NIH 3T3 cells facilitating the removal of damaged mitochondria. Paclitaxel treatment in Ras-NIH 3T3 cells induced an increase in canonical autophagy rates along with suppression of alternative autophagy. Ras-NIH 3T3 cells showed high sensitivity to paclitaxel at the early stage of culture, but as cell confluence increased, resistance to paclitaxel increased, showing a similar level of cell viability to the vehicle control group. The study findings suggest that alternative autophagy is more important than canonical autophagy for maintaining cell survival in response to an unfavorable environment, such as during high cell confluence and exposure to anticancer agents.

Mitochondrial disturbance related to increased caspase-1 of CD4+T cells in HIV-1 infection

BackgroundIn HIV-1 infection, more than 95% of CD4+T cells die of caspase-1 mediated pyroptosis. What governs the increased susceptibility of CD4+T cells to pyroptosis is poorly understood.MethodsBlood samples were obtained from 31 untreated HIV-infected patients (UNT), 29 antiretroviral therapy treated HIV-infected patients (ART), and 21 healthy control donors (HD). Plasma levels of IL-18 and IL-1β, caspase-1 expression, mitochondrial mass (MM) and mitochondrial fusion/fisson genes of CD4+T subsets were measured.ResultsA significantly higher IL-18 level in plasma and MM level of CD4+T cells were found in HIV-infected patients (UNT and ART) compared to HD, and the MMhigh phenotype was manifested, related to increased caspase-1 expression. Moreover, the increased MM was more pronounced in the early differentiated and inactivated CD4+T cells. However, higher MM was not intrinsically linked to T cell differentiation disorder or excessive activation of the CD4+T cells. Mechanistically, the increased MM was significantly correlated with an elevated level of expression of the mitochondrial fusion gene mitofusin1.ConclusionAn increase in MM was associated with heightened sensitivity of CD4+T cells to pyroptosis, even in early differentiated and inactivated CD4+T cells, in patients with HIV-1 infection, regardless of whether patients were on antiretroviral therapy or not. These new revelations have uncovered a previously unappreciated challenge to immune reconstitution with antiretroviral therapy.

The DPP4 Dependent Transcriptome Identifies Pathways to Improve Mouse Hematopoietic Cell Function

Hematopoietic cell transplantation (HCT) is a curative treatment for hematologic disorders wherein healthy donor hematopoietic stem (HSCs) and progenitor cells (HPCs) collected from bone marrow (BM), mobilized peripheral blood (PB) or umbilical cord blood (CB) reconstitute an ablated hematopoietic system. There is a strong need to enhance functional competency of HSCs/HPCs for HCT to prevent complications like graft versus host disease (GVHD), relapse, and graft failure. Dipeptidyl peptidase 4 (DPP4) is a protein that cleaves N-terminal dipeptides from hematopoietic factors, impacting their function. Inhibition of DPP4 improves HSC/HPC competency in mouse models of HCT and clinically speeds neutrophil recovery after CB transplantation and reduces incidences of GVHD after PB transplantation. Thus, DPP4 inhibition is a viable component of a multi-pronged strategy to improve engraftment and prevent GVHD. While DPP4 inhibition improved early neutrophil recovery in clinical trials, the effects were modest, required high dose treatment, and effects on platelet recovery were unclear. Examining mechanisms by which DPP4 inhibition drives HSC/HPC functional competency may yield insight into targets that can enhance DPP4 driven improvements to HCT. We harvested BM from mice treated with the DPP4 inhibitor Diprotin A (DPA) and sorted immunophenotypically defined HSCs, multipotent progenitors (MPPs), common myeloid progenitors (CMPs), and granulocyte-macrophage progenitors (GMPs) for transcriptomic analysis. Linear modeling to examine differential gene expression controlling for cell type revealed that DPP4 inhibition gave strong enrichment of gene programs associated with mitochondrial function. Using a mitochondrial stress test and the Seahorse metabolic flux analyzer, we found that lineage depleted mouse BM cells treated ex vivo with DPA had lower basal oxygen consumption rates and spare respiratory capacity. These seemingly paradoxical data are consistent with reports that highly functional HSCs have higher mitochondrial mass but exhibit less mitochondrial activity. Thus, DPP4 inhibition may preserve high levels of functional competency by reducing cellular dependence on oxidative phosphorylation. To examine DPP4 inhibition effects on individual subpopulations of HSCs/HPCs, we modeled each cell type separately for differential gene expression. We found that gene programs associated with neutrophil development were significantly enriched in HSCs and MPPs from mice treated with DPA, including genes Elane, Lcn2, Prtn3 and Ctsg/h, concomitant with downregulation of platelet development programs in HSCs and GMPs from mice treated with DPA, including genes Gp6/9, P2ry12 and Clec1b. This suggests that DPP4 drives improved engraftment in part by priming HSCs/HPCs for neutrophil recovery but does not enhance platelet development. We then sought to target a platelet activating pathway in combination with DPP4 inhibition to improve HCT. Recipient mice were treated on days -2, -1, and 0 with 75 mg/kg DPA and day 0 with 50 µg/kg and days 1, 3, 5, and 7 with 25 µg/kg recombinant human Thrombopoietin (TPO), a platelet activating agonist. On day -1 mice were lethally irradiated and transplanted via tail vein with 2.5x10 5 whole BM donor cells on day 0. Complete blood counts 2 weeks following transplantation showed that mice treated with DPA alone exhibited worse platelet recovery, in line with our transcriptomic data. However, mice treated with the combination of DPA and TPO exhibited significantly better platelet recovery than vehicle controls and DPA alone. Further, neutrophil recovery trended towards improved recovery in the combination treatment compared to the single agent treatments. Flow cytometry of the PB revealed no differences in BM homing or in donor PB chimerism one month following HCT in the combination treatment compared to single agents (though all treatments showed improved homing over vehicle controls), suggesting the primary effects were on early recovery and not on initial or long-term engraftment. These data elucidate the DPP4 dependent transcriptome in the hematopoietic compartment, providing molecular insights into genes that drive enhanced functional competency of HSCs/HPCs. We also demonstrate that the DPP4 dependent transcriptome can reveal gene programs that can be targeted in combination to provide additive improvement on transplantation recovery.

Senescence and Mitochondrial Transfer Ability of Bone Marrow Mesenchymal Stromal Cells Influence Patterns of Alloreactivity after Allogeneic Hematopoietic Stem Cell Transplantation

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains the only curative treatment for acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). However post-transplant disease recurrence and alloreactive complications (chiefly graft versus host diseases - GvHD) are still associated with severe morbidity and mortality. Proliferative advantage of transformed cells as well as immune and microenvironmental dysregulations contribute both to promoting relapse and escape from graft versus leukemia (GvL) effect. Bone marrow (BM) mesenchymal stromal cells (MSC) are well known for their role in hematopoiesis and immune modulation. MSCs have been shown able to transfer mitochondria (MT) to different cell populations, including immune effectors, in order to (re)establish a healthy oxidative metabolism within the recipient cell. Here we hypothesize that these mechanisms are highly active in BM microenvironment after allo-HSCT and may contribute to alloreactivity, modifying the immune environment. On this basis we stipulate that those intercellular exchanges are deeply impacted by MSCs' senescence, that could be deployed as a useful parameter to predict post-transplant outcomes. To that end, we conducted a translational research, based on BM specimens prospectively collected at our Institution (NCT03357172, NCT03964922), involving patients undergoing allo-HSCT for high-risk MDS and AML and receiving fludarabine/busulfan-based conditioning regimens, anti-lymphoglobuline and cyclosporin/mycophenolate mofetil. MSC phenotype and functionality at the pre-transplant stage, MT capacity, immunomodulation towards T cells and phenotype were studied by multiparametric flow cytometry. Cell metabolism, in terms of oxygen consumption and extracellular acidification rate, was characterized with Seahorse XF technology. Percentages of senescent MSCs were determined with SA β-galactosidase test. First, we compared phenotypic and metabolic characteristics of MSC from MDS/AML (N=29) patients with those retrieved from a cohort of healthy controls (N=8). We observed that patient MSCs were characterized by higher expression of CD157 and CD146 and lower expression of CD200 as compared to healthy MSCs. Pre-transplant senescence quantification highlighted a certain degree of heterogeneity within the MSC compartment in leukemic patients (median = 37.42% sd=16.30 for AML patient and m=11.74% sd=6.4 for HC). In an attempt to link clinical phenotypes to this heterogeneity, we observed that patients with high degree of MSC senescence in pretransplant samples (mean= 51.6% sd=16.6 cells/sample), developed more frequently acute GvHD, as compared to those with low degree of pre-transplant MSC senescence (mean= 23.8% sd=14.3) who experienced instead a higher incidence of disease relapse (p=0,004). Assuming that the link between senescence and alloreactivity could rely to different metabolic states and MT capabilities in MSCs, we analyzed these biological features in our samples. Indeed, we observed that low degree of MSC senescence was associated with higher MT to T cells, in particular to CD4+ cells, shifting their metabolism and phenotype toward an immunosuppressive state The characterization of cellular metabolism showed in highly senescent MSCs the presence of markers of mitochondrial dysfunction with high oxidative power, but reduced coupling efficiency of respiratory chain. This metabolic profile involved a high mitochondrial mass characterized by a high maximal respiration capacity compared to HC MSCs. The in vitro modulation of MSC senescence with nicotinamide administration restored healthy mitochondrial metabolism and improved coupling efficacy of oxidative phosphorylation, possibly impacting on the immunomodulation capabilities of this microenvironment component. Here for the first time, we provide the evidence that MSC senescence plays an pivotal role in defining an altered immune state in transplanted AML possibly shifting the degree of alloreactivity from a high GvH/GvL response (high GvHD and low relapse) to a high immunosuppressive state (low GvHD and high relapse). These findings pave the way for implementing MSC senescence as a biomarker able to predict post-transplant outcomes and show new biological rationales to integrate donor-derived MSC products to better adjust GvH responses.

Profibrogenic macrophage-targeted delivery of mitochondrial protector via exosome formula for alleviating pulmonary fibrosis

Pulmonary fibrosis (PF) is a devastating lung disease with limited treatment options. During this pathological process, the profibrogenic macrophage subpopulation plays a crucial role, making the characterization of this subpopulation fundamentally important. The present study revealed a positive correlation between pulmonary macrophages with higher mitochondrial mass (Mømitohigh) and fibrosis. Among the Mømitohigh subpopulation of CD206+ M2, characterized by higher expression of dynamin 1-like (Drp1), as determined by flow cytometry and RNA-seq analysis, a therapeutic intervention was developed using an exosome-based formula composed of pathfinder and therapeutics. A pathfinder exosome called “exosomeMMP19 (ExoMMP19)”, was constructed to display matrix metalloproteinase-19 (MMP19) on the surface to locally break down the excessive extracellular matrix (ECM) in the fibrotic lung. A therapeutic exosome called “exosome therapeutics (ExoTx)”, was engineered to display D-mannose on the surface while encapsulating siDrp1 inside. Prior delivery of ExoMMP19 degraded excessive ECM and thus paved the way for ExoTx to be delivered into Mømitohigh, where ExoTx inhibited mitochondrial fission and alleviated PF. This study has not only identified Mømitohigh as profibrotic macrophages but it has also provided a potent strategy to reverse PF via a combination of formulated exosomes.

Bypassing mitochondrial defects rescues Huntington's phenotypes in Drosophila

Huntington's disease (HD) is a fatal neurodegenerative disease with limited treatment options. Human and animal studies have suggested that metabolic and mitochondrial dysfunctions contribute to HD pathogenesis. Here, we use high-resolution respirometry to uncover defective mitochondrial oxidative phosphorylation and electron transfer capacity when a mutant huntingtin fragment is targeted to neurons or muscles in Drosophila and find that enhancing mitochondrial function can ameliorate these defects. In particular, we find that co-expression of parkin, an E3 ubiquitin ligase critical for mitochondrial dynamics and homeostasis, produces significant enhancement of mitochondrial respiration when expressed either in neurons or muscles, resulting in significant rescue of neurodegeneration, viability and longevity in HD model flies. Targeting mutant HTT to muscles results in larger mitochondria and higher mitochondrial mass, while co-expression of parkin increases mitochondrial fission and decreases mass. Furthermore, directly addressing HD-mediated defects in the fly's mitochondrial electron transport system, by rerouting electrons to either bypass mitochondrial complex I or complexes III-IV, significantly increases mitochondrial respiration and results in a striking rescue of all phenotypes arising from neuronal mutant huntingtin expression. These observations suggest that bypassing impaired mitochondrial respiratory complexes in HD may have therapeutic potential for the treatment of this devastating disorder.

Abstract 4783: ADO is essential for redox homeostasis in liver cancer

Abstract 2-Aminoethanethiol dioxygenase (ADO) is a thiol dioxygenase that plays a direct role in both metabolism and protein stability. Oxidation of the metabolite cysteamine produces hypotaurine, while oxidation of N-terminal cysteines targets protein substrates for N-degron pathway-mediated degradation. Despite these known functions, the (patho)physiological roles of ADO remain obscure. By analyzing TCGA datasets we discovered that high ADO expression is associated with poor outcome for patients with hepatocellular carcinoma (HCC) (HR 1.2, p<0.001). HCC is linked to viral hepatitis, alcohol, metabolic syndrome, non-alcoholic fatty liver disease and chronic inflammation. It is the third leading cause of cancer-related deaths worldwide and is significantly more common in males than females. With poor outcomes, there is a need to better understand HCC biology and vulnerabilities. To assess the functional roles of ADO, we created ADO knockout mice as well as two liver cancer cell line models where ADO was depleted by siRNA, doxycycline-inducible shRNA, or CRISPR/Cas9. While male ADO knockout mice were viable, fertile and healthy, depletion of ADO in liver cancer models significantly reduced cancer cell proliferation and colony formation. ADO was also essential for supporting xenograft growth when implanted subcutaneously in immune-compromised mice. Taken together, this suggested that ADO depletion represents a cancer-specific vulnerability. Comprehensive metabolomic, transcriptomic and proteomic characterization of HCC cells isogenic for ADO demonstrated that loss of ADO resulted in dysregulation of glutathione, ascorbate, polyamine and proline metabolism. Consistent with this, ADO depleted cells had high levels of reactive oxygen species (ROS) measured by CellROX flowcytometry, and an increased ratio of oxidized to reduced glutathione, indicative of oxidative stress. Since mitochondria represent a substantial source of ROS, we assessed mitochondrial mass and function using MitotrackerTM flow cytometry and Seahorse stress tests, respectively. ADO depleted cancer cells had higher mitochondrial mass and higher maximal respiration rates compared to control cells. Finally, exogenously supplied antioxidants could rescue the survival of HCC cells with ADO depletion, demonstrating that the observed loss of viability is due to oxidative stress. This work shows that ADO is essential for redox homeostasis in liver cancer models and suggests that interfering with ADO function may represent a novel targeting strategy for HCC. Citation Format: Sandy C-E Lee, Andrea H. Pyo, Helia Mohammadi, Ji Zhang, Anna Dvorkin-Gheva, Lucie Malbeteau, Stephen Chung, Shahbaz Khan, Thomas Kislinger, Julie A. Reisz, Courtney Jones, Marianne Koritzinsky. ADO is essential for redox homeostasis in liver cancer. [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 4783.

Microscopy-based phenotypic monitoring of MDA-MB-231 spheroids allows the evaluation of phenotype-directed therapy

Breast cancer (BC) is the most commonly diagnosed cancer among women. Prognosis has improved over the years, to a large extent, owing to personalized therapy informed by molecular profiling of hormone receptors. However, there is a need for new therapeutic approaches for a subgroup of BCs lacking molecular markers, the Triple Negative Breast Cancer (TNBC) subgroup. TNBC is the most aggressive type of BC, lacks an effective standard of care, shows high levels of resistance and relapse is often inevitable. High resistance to therapy has been hypothesized to be associated with high intratumoral phenotypic heterogeneity. To characterize and treat this phenotypic heterogeneity, we optimized a whole-mount staining and image analysis protocol for three-dimensions (3D) spheroids.Applying this protocol to TNBC spheroids located in the outer region of the spheroid the cells with selected phenotypes: dividing, migrating, and high mitochondrial mass phenotypes. To evaluate the relevance of phenotype-based targeting these cell populations were targeted with Paclitaxel, Trametinib, and Everolimus, respectively, in a dose-dependent manner. Single agents cannot specifically target all phenotypes at the same time. Therefore, we combined drugs that should target independent phenotype. With this rationale we observed that combining Trametinib and Everolimus achieves the highest cytotoxicity at lower doses from all the tested combinations. These findings suggest a rational approach to design treatments can be evaluated in spheroids prior to pre-clinical models and potentially reduce adverse effects.

Small molecule-mediated rapid maturation of human induced pluripotent stem cell-derived cardiomyocytes

BackgroundHuman induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPSC-CMs) do not display all hallmarks of mature primary cardiomyocytes, especially the ability to use fatty acids (FA) as an energy source, containing high mitochondrial mass, presenting binucleation and increased DNA content per nuclei (polyploidism), and synchronized electrical conduction. This immaturity represents a bottleneck to their application in (1) disease modelling—as most cardiac (genetic) diseases have a middle-age onset—and (2) clinically relevant models, where integration and functional coupling are key. So far, several methods have been reported to enhance iPSC-CM maturation; however, these protocols are laborious, costly, and not easily scalable. Therefore, we developed a simple, low-cost, and rapid protocol to promote cardiomyocyte maturation using two small molecule activators of the peroxisome proliferator-activated receptor β/δ and gamma coactivator 1-alpha (PPAR/PGC-1α) pathway: asiatic acid (AA) and GW501516 (GW).Methods and ResultsMonolayers of iPSC-CMs were incubated with AA or GW every other day for ten days resulting in increased expression of FA metabolism-related genes and markers for mitochondrial activity. AA-treated iPSC-CMs responsiveness to the mitochondrial respiratory chain inhibitors increased and exhibited higher flexibility in substrate utilization. Additionally, structural maturity improved after treatment as demonstrated by an increase in mRNA expression of sarcomeric-related genes and higher nuclear polyploidy in AA-treated samples. Furthermore, treatment led to increased ion channel gene expression and protein levels.ConclusionsCollectively, we developed a fast, easy, and economical method to induce iPSC-CMs maturation via PPAR/PGC-1α activation. Treatment with AA or GW led to increased metabolic, structural, functional, and electrophysiological maturation, evaluated using a multiparametric quality assessment.

Generation of mature compact ventricular cardiomyocytes from human pluripotent stem cells

Abstract Introduction Compact cardiomyocytes that make up the ventricular wall of the adult heart represent an important therapeutic target population for modeling and treating cardiovascular diseases. Although human pluripotent stem cell (hPSC) represents a promising source to generate target cells in vitro, there has been no optimal protocols to generate mature compact cardiomyocytes derived from hPSCs. Purpose To establish the protocol to generate mature compact ventricular cardiomyocytes derived from hPSCs. Methods By mimicking the early ventricular development and postnatal maturation process observed in the heart in vivo, we searched for the specific factors to induce compact lineage and promote their maturation level. Results We established a differentiation strategy that promotes the specification, proliferation and maturation of compact ventricular cardiomyocytes from hPSCs. The treatment of Wnt signaling activator, CHIR, and IGF2 significantly increased the expression level of a compact marker, MYCN (p=0.006), and suppressed the trabecular markers, NPPA (p=0.0117) and BMP10 (p=0.0032). Those factors induced a proliferation of compact cardiomyocytes by two folds (p<0.0001). Additionally, we identified that the combination of PPARα agonist, dexamethasone, T3 hormone, and palmitate in the low glucose containing media following the compact specification period significantly induced the fatty acid transporter, CD36, in the compact cardiomyocytes (p<0.0001). The CD36-positive compact cardiomyocytes generated under these conditions displayed the ability to use fatty acids as an energy source, a high mitochondrial mass, well-defined sarcomere structures and enhanced contraction force. These mature compact cardiomyocytes showed metabolic changes indicative of those associated with heart failure when challenged in vitro with pathological stimuli and were found to generate grafts consisting of more mature cells than those derived from immature cardiomyocytes following transplantation into infarcted rat hearts. Conclusions We successfully generated the mature compact cardiomyocytes derived from hPSCs. These findings highlight the power of recapitulating key aspects of embryonic and postnatal development for generating therapeutically relevant cell types from hPSCs. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Canadian Institutes of Health Research (CIHR)

Abstract 125: The role of oxidative stress on the polyaneuploid cancer cell state in metastatic cancer

Abstract Metastatic cancer is responsible for 90% of cancer deaths and is currently incurable due to resistance to all systemic anticancer therapies. Our lab hypothesizes that this resistance is driven by a subpopulation of cells in the tumor that enter the polyaneuploid cancer cell (PACC) state by undergoing a polyaneuploid transition (PAT). Cells in the PACC state are found in cell culture, xenograft, and genetically engineered mouse models, as well as in clinical samples. We have shown that cancer cells are induced to undergo a PAT upon exposure to external stress, such as chemotherapeutic treatments that include platinum-based therapy, etoposide, and taxane drugs. In addition, cells that enter the PACC state are resistant to that stress. One mechanism of therapy-induced cancer cell death is due to the resultant oxidative stress. We hypothesize that this oxidative stress drives a subset of cancer cells to undergo the PAT, thus enabling them to persist following treatment. To test the effects of oxidative stress on the PACC state, we treated cancer cells with cisplatin to induce the PAT and measured mitochondrial function and levels of reactive oxygen species (ROS). Using fluorescence microscopy with live-cell mitochondrial and ROS dyes, we showed that cells in the PACC state have higher mitochondrial mass, exhibit two to three times more mitochondrial activity per total mitochondria, and have over a five-fold increase in intracellular ROS levels per unit area when compared to typical cancer cell populations that they arise from. Moreover, PACCs exhibit a three-fold increase in their oxygen consumption rate and have higher expression of genes related to mitochondrial respiration and antioxidant pathways when compared to cells that did not undergo the PAT. Taken together, this suggests that cells in the PACC state may have a higher tolerance for stress since they can withstand higher amounts of ROS that result from both therapy and mitochondrial activity without exhibiting cell death. Our goal is to investigate how oxidative stress affects PACC formation and identify mechanisms of how PACCs manage stress to ultimately target these cells. If we can prevent PACC formation or find a method to eliminate cells in the PACC state, we can attack and resolve the issue of therapeutic resistance and incurability of metastatic cancer. Citation Format: Melvin Li, Sarah R. Amend, Kenneth J. Pienta. The role of oxidative stress on the polyaneuploid cancer cell state in metastatic 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 125.

Abstract 2329: Variable HPV E6 levels among oropharyngeal cancers govern therapy response via the PGC1/ERR axis

Abstract BACKGROUND: Elevated mitochondrial antioxidant defenses contribute to progression and therapy resistance of some poor prognosis cancer types. By contrast, HPV+ oropharyngeal squamous cell carcinomas (OPSCCs) are generally sensitive to oxidative damage from radiation plus cisplatin, which are often curative. It is unknown whether the subset of HPV+ OPSCCs with poor outcomes exploit antioxidant capacity provided by high mitochondrial mass to evade therapy. It is also unclear whether the wide range of HPV oncoprotein expression among them contributes to diversity in mitochondrial mass and treatment outcomes. AIMS: (1) evaluate HPV+ OPSCCs for associations between mitochondrial mass and clinical outcome (2) elucidate relationships among HPV oncoprotein levels, mitochondrial mass, and therapy response (3) delineate mechanisms by which variable HPV oncoprotein levels govern therapy response. METHODS/RESULTS: High expression of genes involved in oxidative metabolism was associated with decreased survival in three patient cohorts. In PDX and cell line models, cisplatin resistance positively correlated with mitochondrial mass (MTCO1/B2M), oxidative metabolism (basal OCR), and antioxidant capacity (NADPH/NADP+). Among the HPV oncoproteins, only full length E6 (fl-E6) was linked to these features, showing negative correlation with mitochondrial mass in the cell lines, PDXs, and patient tumors. Fl-E6 levels also positively correlated with cisplatin response in the PDXs and cell lines. To test whether fl-E6 can mediate such effects, lentiviral fl-E6 expression in HPV+ cancer cell lines with low endogenous fl-E6 was used to increase levels to the high end of the range seen in human tumors. Doing so reduced MTCO1/B2M, basal OCR, and NADPH/NADP+ while sensitizing to radiation and cisplatin in vitro and in vivo. The same effects on mitochondrial biogenesis were maintained in nTERT/E7 keratinocytes. Using luciferase reporter assays, fl-E6 was shown to repress the PGC-1/ERR axis for mitochondrial biogenesis by attenuating p53-dependent PGC1α promoter activity. The prediction of PGC1/ERR axis activation in tumors with low fl-E6 was confirmed in PDXs and patient cohorts, where high ERRα was negatively prognostic. CONCLUSIONS: These results reveal a novel ability of E6-mediated p53 downregulation to contribute to treatment sensitivity by depleting mitochondrial antioxidant capacity. They also provide evidence that differing fl-E6 levels across HPV+ cancers variably repress the PGC1/ERR pathway, leading to diversity in mitochondrial mass that impacts therapy response. Therapy-refractory features may prove identifiable using expression-based biomarkers in the PGC1/ERR axis and be mitigated through targetable nodes in this pathway. In addition, HPV+ OPSCCs may find competitive advantage in down-regulating fl-E6 upon acquiring alternate drivers to compensate decrease in its oncogenic functions. Citation Format: Malay K. Sannigrahi, Pavithra Rajagopalan, Ling Lai, Xinyi Liu, Varun Sahu, Hiroshi Nakagawa, Robert Brody, Iain M. Morgan, Bradford E. Windle, Xiaowei Wang, Phyllis A. Gimotty, Daniel P. Kelly, Elizabeth A. White, Devraj Basu. Variable HPV E6 levels among oropharyngeal cancers govern therapy response via the PGC1/ERR axis [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 2329.

Small molecule-mediated rapid maturation of human induced pluripotent stem cell derived cardiomyocytes

Abstract Funding Acknowledgements Type of funding sources: Other. Main funding source(s): Gravitation Program “Materials Driven Regeneration” by the Netherlands Organization for Scientific Research (RegmedXB #024.003.013) and the Marie Skłodowska-Curie Actions (Grant agreement RESCUE #801540). The EU-funded project BRAV3 (H2020, ID:874827) Background Human-induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPSC-CMs) do not display all hallmarks of mature human primary cardiomyocytes: the ability to use fatty acids as an energy source, high mitochondrial mass, increased nuclei polyploidism, synchronized electrical conduction, and forceful contractions. Instead, their phenotype is similar to immature cardiomyocytes in the late fetal stage. This immaturity represents a bottleneck to their application in 1) disease modeling – as most cardiac (genetic) diseases have a middle-age onset – and 2) clinical use, where integration and functional coupling are key. So far, the mainly used methods to enhance iPSC-CM maturation include prolonged time-in-culture, 3D culture, cyclic mechanical stretch, and electrical stimulation with specialized media. However, these protocols are laborious, costly, and not easily scalable. Methods In this study, we developed a simple, low cost, and rapid protocol using two peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A/PGC-1α) activating small molecules: Asiatic Acid (AA) and GW501516 (GW) to promote cardiomyocyte maturity by inducing a metabolic switch to fatty acid utilization and increased mitochondrial biogenesis. Results Monolayers of iPSC-CMs were incubated with AA and GW every other day for 10 days resulting in increased expression of fatty acid-metabolism-related genes (5 and 10-fold increase in CPT1B gene expression, respectively), mitochondria biogenesis (protein expression of ATP5A) and fusion (50 and 100-fold increase in OPA1 gene expression, respectively). In addition, AA treated iPSC-CMs responded in the seahorse mitochondria stress test more rapidly to an artificial increase in mitochondrial activity and showed a higher flexibility in substrate utilization in the seahorse stress test. A more mature electrophysiological functionality was shown by increased ion channel gene expression (KCNA4, SCN5A, GJA1, CACNA1C, and SCN1B) and enhanced synchronous contraction in treated samples. Moreover, maturation was further shown by increased sarcomeric gene expression (5 and 7-fold increase in TNNI3 in AA and GW respectively) and nuclear polyploidism (>4N fold 2.16 and 1.48-fold increase in AA and GW respectively). Conclusions Collectively, these findings show that AA and GW trigger a metabolic switch and induce extensive maturation of iPSC-CMs, providing a rapid and cost-effective method to obtain iPSC-CMs that more closely resemble their adult counterparts.

Automated quantitative high-throughput multiplex immunofluorescence pipeline to evaluate OXPHOS defects in formalin-fixed human prostate tissue

Advances in multiplex immunofluorescence (mIF) and digital image analysis has enabled simultaneous assessment of protein defects in electron transport chain components. However, current manual methodology is time consuming and labour intensive. Therefore, we developed an automated high-throughput mIF workflow for quantitative single-cell level assessment of formalin fixed paraffin embedded tissue (FFPE), leveraging tyramide signal amplification on a Ventana Ultra platform coupled with automated multispectral imaging on a Vectra 3 platform. Utilising this protocol, we assessed the mitochondrial oxidative phosphorylation (OXPHOS) protein alterations in a cohort of benign and malignant prostate samples. Mitochondrial OXPHOS plays a critical role in cell metabolism, and OXPHOS perturbation is implicated in carcinogenesis. Marked inter-patient, intra-patient and spatial cellular heterogeneity in OXPHOS protein abundance was observed. We noted frequent Complex IV loss in benign prostate tissue and Complex I loss in age matched prostate cancer tissues. Malignant regions within prostate cancer samples more frequently contained cells with low Complex I & IV and high mitochondrial mass in comparison to benign–adjacent regions. This methodology can now be applied more widely to study the frequency and distribution of OXPHOS alterations in formalin-fixed tissues, and their impact on long-term clinical outcomes.

Docosahexaenoic acid supplementation represses the early immune response against murine cytomegalovirus but enhances NK cell effector function

BackgroundDocosahexaenoic acid (DHA) supplementation is beneficial for several chronic diseases; however, its effect on immune regulation is still debated. Given the prevalence of cytomegalovirus (CMV) infection and because natural killer (NK) cells are a component of innate immunity critical for controlling CMV infection, the current study explored the effect of a DHA-enriched diet on susceptibility to murine (M) CMV infection and the NK cell effector response to MCMV infection.ResultsMale C57BL/6 mice fed a control or DHA-enriched diet for 3 weeks were infected with MCMV and sacrificed at the indicated time points postinfection. Compared with control mice, DHA-fed mice had higher liver and spleen viral loads at day 7 postinfection, but final MCMV clearance was not affected. The total numbers of NK cells and their terminal mature cell subset (KLRG1+ and Ly49H+ NK cells) were reduced compared with those in control mice at day 7 postinfection but not day 21. DHA feeding resulted in higher IFN-γ and granzyme B expression in splenic NK cells at day 7 postinfection. A mechanistic analysis showed that the splenic NK cells of DHA-fed mice had enhanced glucose uptake, increased CD71 and CD98 expression, and higher mitochondrial mass than control mice. In addition, DHA-fed mice showed reductions in the total numbers and activation levels of CD4+ and CD8+ T cells.ConclusionsThese results suggest that DHA supplementation represses the early response to CMV infection but preserves NK cell effector functions by improving mitochondrial activity, which may play critical roles in subsequent MCMV clearance.

Molecular Hydrogen Enhances Proliferation of Cancer Cells That Exhibit Potent Mitochondrial Unfolded Protein Response.

Molecular hydrogen ameliorates pathological states in a variety of human diseases, animal models, and cell models, but the effects of hydrogen on cancer have been rarely reported. In addition, the molecular mechanisms underlying the effects of hydrogen remain mostly unelucidated. We found that hydrogen enhances proliferation of four out of seven human cancer cell lines (the responders). The proliferation-promoting effects were not correlated with basal levels of cellular reactive oxygen species. Expression profiling of the seven cells showed that the responders have higher gene expression of mitochondrial electron transport chain (ETC) molecules than the non-responders. In addition, the responders have higher mitochondrial mass, higher mitochondrial superoxide, higher mitochondrial membrane potential, and higher mitochondrial spare respiratory capacity than the non-responders. In the responders, hydrogen provoked mitochondrial unfolded protein response (mtUPR). Suppression of cell proliferation by rotenone, an inhibitor of mitochondrial ETC complex I, was rescued by hydrogen in the responders. Hydrogen triggers mtUPR and induces cell proliferation in cancer cells that have high basal and spare mitochondrial ETC activities.

Targeting Residual Chemotherapy-Resistant Acute Myeloid Leukemia Cells By a Novel Oxphos Inhibitor IACS010759

Acute myeloid leukemia (AML) is initiated and maintained by a relatively rare leukemia stem cells (LSCs) that are capable of self-renewing, proliferation and differentiation into leukemia blasts. Recent data showed that LSCs (Lagadinou et al. Cell Stem Cell 2013) and residual cytarabine (Ara-C)-resistant AML cells (Farge et al. Cancer Discovery 2017) are highly dependent on mitochondrial function for survival. This unique metabolic biology makes LSCs and AML cells resistant to chemotherapy vulnerable to oxidative phosphorylation (OXPHOS) inhibition. We have identified a novel potent inhibitor, IACS-010759, which blocks mitochondrial complex I, suppresses OXPHOS and selectively inhibits the growth of AML cells (Molina et al. AACR 2016). In this study, we aimed to determine the effects of OXPHOS inhibition with IACS-010759 on residual AML cells and AML LSCs surviving standard chemotherapy (Doxorubicin/Ara-C, DA) in patient-derived xenograft (PDX) AML models.Consistent with Farge et al study, OCI-AML3 cells treated with DA in vitro produced elevated levels of reactive oxygen species, displayed increased mitochondrial membrane potential and higher mitochondrial mass (data not shown), indicating reliance on mitochondrial metabolism. Further, Ara-C facilitated statistically significant increase in the basal and maximal oxygen consumption rates (OCR) (36%±8%, p=0.03; 36%±3%, p=0.003, respectively) compared to control. In turn, OXPHOS inhibition with IACS-010759 at 30 nM fully inhibited basal and Ara-C-induced OCR (Fig. 1A). These findings indicate that chemotherapy fosters mitochondrial respiration in AML, which could be abrogated by OXPHOS inhibitor.To examine the efficacy of IACS-010759 on residual leukemia cells surviving chemotherapy in vivo, we first established a chemosensitive PDX AML model of minimal residual disease. Treatment of mice inoculated with a human AML PDX harboring FLT3-ITD mutation with DA (D: 1.5 mg/kg; A: 50 mg/kg) reduced leukemia burden (PB, 0.8 ± 0.6% vs 45.8 ± 8.2% in vehicle-treated mice, p=0.001). The residual AML cells in DA-treated mice expanded and caused rapidly progressing leukemia (78.2 ± 6.2% vs 95.3 ± 1.0% in vehicle-treated mice, p=0.047) on week 6 post DA. Daily oral treatment of mice with IACS-010759 (7.5 mg/kg) as a single agent reduced leukemia burden, and caused further reduction when administered post completion of DA (Fig. 1B,C). The CyTOF mass cytometry, which enables measurement of up to 40 parameters per cell, demonstrated reduced frequency of CD34+CD38lowCD123+ AML LSCs and resurgence of CD11c+ differentiated cells in both IACS and IACS/DA groups (Fig. 1D). On the contrary, chemotherapy alone failed to significantly reduce fractions of LSCs or induce differentiation (Fig. 1D). Proliferation measured by Ki67 was greatly reduced by IACS/DA combination in all populations (1.4 ± 0.3% vs 5.5 ± 0.4% in vehicle group, p<0.01). The expression of hypoxia-inducible factor 1α was downregulated, consistent with the decreased oxygen consumption by IACS-010759 (not shown).We next investigated the efficacy of IACS-010759 in a chemoresistant PDX model inoculated with chemorefractory MLL-rearranged AML. CyTOF analysis demonstrated that DA chemotherapy failed to reduce the primitive leukemia stem/progenitor populations, which comprised 84.7% of total cells in DA versus 71.2% in vehicle groups, while the combination of IACS-010759 and DA efficiently reduced this population down to 30.2%. Additionally, the combination increased the proportion of mature cells characterized by CD34-CD64+CD11b+ (43.1% versus 12.8% in vehicle), which remained low in the DA-only group (5.6%). DA/OXPHOS inhibitor combination significantly extended survival, compared to the vehicle or single-agent treatment arms (Fig. 1E).In conclusion, AML cells surviving chemotherapy depend on OXPHOS for survival, and OXPHOS inhibition with complex I inhibitor IACS-010759 reduces AML LSC pool, induces differentiation and extends survival in AML PDX models in vivo . Our data advocate for combining IACS-010759 with standard chemotherapy, upon identification of a recommended Phase II dose in an ongoing clinical trial of IACS-010759 in AML (NCT02882321). [Display omitted] DisclosuresBattula:United Therapeutics Corporation: Research Funding. Andreeff:Daiichi Sankyo: Consultancy. Kantarjian:ARIAD: Research Funding; Delta-Fly Pharma: Research Funding; Amgen: Research Funding; Pfizer: Research Funding; Novartis: Research Funding; Bristol-Meyers Squibb: Research Funding. Daver:Karyopharm: Consultancy, Research Funding; Incyte Corporation: Honoraria, Research Funding; Bristol-Myers Squibb Company: Consultancy, Research Funding; Daiichi-Sankyo: Research Funding; Sunesis Pharmaceuticals, Inc.: Consultancy, Research Funding; Jazz: Consultancy; Otsuka America Pharmaceutical, Inc.: Consultancy; Pfizer Inc.: Consultancy, Research Funding; Novartis Pharmaceuticals Corporation: Consultancy; Kiromic: Research Funding; Immunogen: Research Funding.

1212-P: Adipose Tissue Mitochondrial Function in Humans with Varying Liver Histology

Adipose dysfunction may drive the development of insulin resistance, type 2 diabetes (T2D) and nonalcoholic (NA) fatty liver disease (NAFLD), comprising NA fatty liver and steatohepatitis (NAFL and NASH). Possible underlying mechanism comprise impaired lipid storage or mitochondrial oxidation favoring increased fatty acid flux to other tissues. Thus, we examined subcutaneous (SAT) and visceral adipose tissue (VAT) mitochondrial capacity in humans with different degrees of insulin resistance and histologically proven NAFLD. Obese people without NAFL (OBE-CON, n=20, 38±8 years, body mass index 53±6 kg/m2, 10% T2D), with NAFL (OBE-NAFL, n=20, 40±8 years, 51±5 kg/m2, 25% T2D) or NASH (OBE-NASH, n=20, 42±10 years, 51±6 kg/m2, 40% T2D) underwent metabolic phenotyping and tissue biopsies. O2 flux rates from different substrates were measured with high resolution respirometry in SAT and VAT. In VAT, maximal uncoupled respiration was lower in OBE-NAFL (least square means (LSM): 0.46 pmol*mg wet weight–1*s–1 [95% confidence interval 0.21;0.71], p&amp;lt;0.05) and OBE-NASH (LSM: 0.51 [0.26;0.76], p&amp;lt;0.001) compared to OBE-CON. Similar differences were seen for state 3 respiration related to complex I (glutamate) or combined complex I and II activity (succinate). In Pearson correlation analysis, combined complex I and II activity in VAT positively correlated with whole-body insulin sensitivity (M value; r= 0.29, p&amp;lt;0.05). VAT mitochondrial content, as assessed from mitochondrial DNA copy number, was not different between the groups. Protein expression of electron transport chain complexes was similar in VAT and SAT of all groups, except for complex IV that tended to be lower in VAT in OBE-NASH than in OBE-CON (p=0.065). Of note, in SAT, oxidative capacity was comparable between groups despite a higher mitochondrial mass in OBE-NASH than in OBE-CON (p=0.045). In conclusion, oxidative capacity is downregulated in VAT, but not in SAT of obese humans with NAFLD, which may be related to the degree of insulin resistance. Disclosure K. Pafili: None. M. Schlensak: None. M. Roden: Advisory Panel; Self; Allergan plc, Bristol-Myers Squibb Company, Novo Nordisk A/S, Research Support; Self; Boehringer Ingelheim International GmbH, Danone Nutricia, Sanofi-Aventis Deutschland GmbH. S. Kahl: None. D. Pesta: None. K. Strassburger: None. L. Mastrototaro: None. J. Puetzer-furmanczak: None. B. Dewidar: None. T. Sarabhai: None. I. Esposito: None.

Generation of mature compact ventricular cardiomyocytes from human pluripotent stem cells

Compact cardiomyocytes that make up the ventricular wall of the adult heart represent an important therapeutic target population for modeling and treating cardiovascular diseases. Here, we established a differentiation strategy that promotes the specification, proliferation and maturation of compact ventricular cardiomyocytes from human pluripotent stem cells (hPSCs). The cardiomyocytes generated under these conditions display the ability to use fatty acids as an energy source, a high mitochondrial mass, well-defined sarcomere structures and enhanced contraction force. These ventricular cells undergo metabolic changes indicative of those associated with heart failure when challenged in vitro with pathological stimuli and were found to generate grafts consisting of more mature cells than those derived from immature cardiomyocytes following transplantation into infarcted rat hearts. hPSC-derived atrial cardiomyocytes also responded to the maturation cues identified in this study, indicating that the approach is broadly applicable to different subtypes of the heart. Collectively, these findings highlight the power of recapitulating key aspects of embryonic and postnatal development for generating therapeutically relevant cell types from hPSCs.

Mitochondrial oxidative metabolism contributes to a cancer stem cell phenotype in cholangiocarcinoma

Little is known about the metabolic regulation of cancer stem cells (CSCs) in cholangiocarcinoma (CCA). We analyzed whether mitochondrial-dependent metabolism and related signaling pathways contribute to stemness in CCA. The stem-like subset was enriched by sphere culture (SPH) in human intrahepatic CCA cells (HUCCT1 and CCLP1) and compared to cells cultured in monolayer. Extracellular flux analysis was examined by Seahorse technology and high-resolution respirometry. In patients with CCA, expression of factors related to mitochondrial metabolism was analyzed for possible correlation with clinical parameters. Metabolic analyses revealed a more efficient respiratory phenotype in CCA-SPH than in monolayers, due to mitochondrial oxidative phosphorylation. CCA-SPH showed high mitochondrial membrane potential and elevated mitochondrial mass, and over-expressed peroxisome proliferator-activated receptor gamma coactivator (PGC)-1α, a master regulator of mitochondrial biogenesis. Targeting mitochondrial complex I in CCA-SPH using metformin, or PGC-1α silencing or pharmacologic inhibition (SR-18292), impaired spherogenicity and expression of markers related to the CSC phenotype, pluripotency, and epithelial-mesenchymal transition. In mice with tumor xenografts generated by injection of CCA-SPH, administration of metformin or SR-18292 significantly reduced tumor growth and determined a phenotype more similar to tumors originated from cells grown in monolayer. In patients with CCA, expression of PGC-1α correlated with expression of mitochondrial complex II and of stem-like genes. Patients with higher PGC-1α expression by immunostaining had lower overall and progression-free survival, increased angioinvasion and faster recurrence. In GSEA analysis, patients with CCA and high levels of mitochondrial complex II had shorter overall survival and time to recurrence. The CCA stem-subset has a more efficient respiratory phenotype and depends on mitochondrial oxidative metabolism and PGC-1α to maintain CSC features. The growth of many cancers is sustained by a specific type of cells with more embryonic characteristics, termed 'cancer stem cells'. These cells have been described in cholangiocarcinoma, a type of liver cancer with poor prognosis and limited therapeutic approaches. We demonstrate that cancer stem cells in cholangiocarcinoma have different metabolic features, and use mitochondria, an organelle located within the cells, as the major source of energy. We also identify PGC-1α, a molecule which regulates the biology of mitochondria, as a possible new target to be explored for developing new treatments for cholangiocarcinoma.