Deleting the mitochondrial respiration negative regulator MCJ enhances the efficacy of CD8(+) T cell adoptive therapies in pre-clinical studies.

Mitochondrial respiration is essential for the survival and function of T cells used in adoptive cellular therapies. However, strategies that specifically enhance mitochondrial respiration to promote T cell function remain limited. Here, we investigate methylation-controlled J protein (MCJ), an endogenous negative regulator of mitochondrial complex I expressed in CD8 cells, as a target for improving the efficacy of adoptive T cell therapies. We demonstrate that MCJ inhibits mitochondrial respiration in murine CD8+ CAR-T cells and that deletion of MCJ increases their in vitro and in vivo efficacy against murine B cell leukaemia. Similarly, MCJ deletion in ovalbumin (OVA)-specific CD8+ T cells also increases their efficacy against established OVA-expressing melanoma tumors in vivo. Furthermore, we show for the first time that MCJ is expressed in human CD8 cells and that the level of MCJ expression correlates with the functional activity of CD8+ CAR-T cells. Silencing MCJ expression in human CD8 CAR-T cells increases their mitochondrial metabolism and enhances their anti-tumor activity. Thus, targeting MCJ may represent a potential therapeutic strategy to increase mitochondrial metabolism and improve the efficacy of adoptive T cell therapies.

Wu MH ，Valenca-Pereira F ，Cendali F ，Giddings EL ，Pham-Danis C ，Yarnell MC ，Novak AJ ，Brunetti TM ，Thompson SB ，Henao-Mejia J ，Flavell RA ，D'Alessandro A ，Kohler ME ，Rincon M ... -  《Nature Communications》

Tumor growth suppression in adoptive T cell therapy via IFN-γ targeting of tumor vascular endothelial cells.

Rationale: In adoptive T cell therapy (ACT), the direct cytotoxic effects of CD8 T cells on tumor cells, including the release of interferon-gamma (IFN-γ), are considered the primary mechanism for tumor eradication. Cancer antigen escape diminishes the T cell responses, thereby limiting the therapeutic success. The impacts of IFN-γ targeting non-tumor cells in ACT, on the other hand, remains under-investigated. We hypothesized that IFN-γ action on non-tumor cells, particularly tumor vascular endothelial cells within the physiological tumor microenvironment, could influence therapeutic efficacy. Methods: ACT was performed against ovalbumin (OVA)- or OVA-peptide SIINFEKL-expressing syngeneic mouse tumors, MCA-205-OVA-GFP fibrosarcoma or MOC2-SIINFEKL oral squamous cell carcinoma, using ex vivo-activated OT-1 CD8 T cells expressing the T cell receptor against OVA. Efficacy was examined in wild-type mice, mice deficient for IFN-γ receptor 1 (IFN-γR1KO), and bone marrow chimeras lacking IFN-γR1 expression in endothelial cells. To exclude direct IFN-γ action against tumor cells, IFN-γR1KO-MCA-205-OVA-GFP tumors were used. IFN-γ production, STAT1 induction in its targets, and subsequent changes, especially in vasculatures in the tumor, were examined. Results: ACT suppressed the growth of MCA-205-OVA-GFP and MOC2-SIINFEKL tumors in wild-type mice but failed in IFNγR1KO mice. Furthermore, in the bone marrow chimeras lacking endothelial cell IFN-γR1, ACT efficacy was lost, thus implicating a vital role of IFN-γ action on the endothelium. IFN-γR1KO-MCA-205-OVA-GFP tumor growth was successfully suppressed by ACT in wild-type mice, suggesting that IFN-γ targeting of tumor cells may not be essential for ACT efficacy. OT-1 CD8 T cells interacted with endothelial cells or localized in proximity to the vessels on Day 1.5 after transfer, as observed by intravital microscopy. The OT-1 T cells found in tumors were limited in number but produced high levels of IFN-γ on Day 1.5, while their number peaked on Day 5.5 with negligible IFN-γ production. Together with IFN-γ production by endogenous lymphocytes, IFN-γ levels in the whole tumor peaked on Day 1.5, inducing IFN-γ/STAT1 signaling in endothelial cells. Early targeting of tumor vascular endothelial cells by IFN-γ led to endothelial regression, reduced perfusion, and tumor hypoxia/necrosis (Day 4.5-7). Conclusions: These findings highlight the critical role of T cell-derived IFN-γ action on endothelial cells early in ACT, emphasizing its dynamic influence on the tumor microenvironment, and offering insights into addressing antigen escape.

Lin Q ，Olkowski CP ，Choyke PL ，Sato N ... -  《Theranostics》

Icariin promoted ferroptosis by activating mitochondrial dysfunction to inhibit colorectal cancer and synergistically enhanced the efficacy of PD-1 inhibitors.

A controlled type of cell death called ferroptosis is linked to increased reactive oxygen species (ROS), lipid peroxidation, and iron buildup. Furthermore, evidence indicates that ferroptosis may act as an immunogenic form of cell death with potential physiological functions in tumors and immunosuppression. Inducing ferroptosis in tumor cells may have the potential to complement cancer immunotherapy strategies. The development of colorectal cancer (CRC) and the poor efficacy of immunotherapy are associated with the crosstalk of cellular ferroptosis. Currently, Icariin (ICA), the main bioactive component extracted from Epimedium, has been shown to inhibit a variety of cancers. However, the specific role and potential mechanism of ICA in regulating ferroptosis in CRC remains unclear. The aim of this investigation was to clarify the mechanism underlying the anti-CRC cancer properties of ICA and how it induces ferroptosis to enhance immunotherapy. To evaluate cell viability, the Cell Counting Kit-8 (CCK-8) test was utilized. The transwell test and the wound healing assay were used to assess cell migration. A subcutaneous graft tumor model was constructed with C57BL/6 mice using MC38 colorectal cancer cell lines. The inhibitory effect of ICA on CRC, ferroptosis level and immunomodulatory effects were detected by serum biochemical assay, cytokine assay, hematoxylin-eosin (H&E) staining, immunofluorescence staining, CyTOF mass spectrometry flow screening and Western blotting. Western blotting, proteomics, molecular docking and microscale thermophoresis (MST) were used to forecast and confirm ICA's binding and interaction with HMGA2, STAT3, and HIF-1α. Moreover, the levels of lipid peroxidation and ferroptosis were assessed through the use of the C11-BODIPY fluorescent probe, the FerroOrange fluorescent probe, the iron level, the malondialdehyde (MDA) and reduced glutathione (GSH) assay kit, and Western blotting analysis. To assess alterations in mitochondrial structure and membrane potential, transmission electron microscopy (TEM) and JC-1 immunofluorescence were employed. It was demonstrated in the current study that ICA treatment inhibits CRC and enhances anti-PD-1 therapy efficacy by inciting ferroptosis. As shown in vitro, ICA inhibits CRC cell proliferation, migration, and apoptosis. As demonstrated in vivo, ICA has a dose-dependent tumor suppressor effect when combined with anti-PD-1, it can significantly inhibit tumor growth, increase the expression of serum TNF-α, IFN-γ, and granzyme B, and promote CD69+CD8+ T, CD69+CD8+Tem, CD69+CD8+Teff, TCRβ+CD8+ T, TCRβ+CD8+ T, TCRβ+CD8+Tem, TCRβ+CD8+Teff. The inhibitory effect of ICA on CRC was associated with the binding of HMGA2, STAT3, and HIF-1α proteins, which inhibited CRC by increasing the levels of reactive oxygen species (ROS) and malondialdehyde (MDA), promoting the accumulation of iron (Fe2+), depletion of reduced glutathione (GSH), inhibiting SLC7A11 and GPX4 expressions, thereby inducing ferroptosis in CRC. As a consequence of ICA-induced ferroptosis, mitochondria are dysfunctional, with increased ROS production, membrane potential depolarization (MMP), and ATP production reduced. This process can be efficiently reversed by the mitochondria-targeted antioxidant Mito-Q. It is noteworthy that the ferroptosis inhibitor liproxstatin-1 (lip-1), anti-CD8, and anti-IFN-γ exhibited a significant inhibitory effect on the level of ferroptosis and antitumor capacity of ICA combined with anti-PD-1. This finding suggests that the antitumor immunopotentiating effect of ICA on anti-PD-1 is dependent on the secretion of IFN-γ-induced ferroptosis of CRC cells by the CD8+ T cell. Our study represents the inaugural demonstration of the mechanism whereby ICA exerts anti-CRC effects and synergistically enhances the efficacy of anti-PD-1, inducing mitochondrial damage and leading to ferroptosis. ICA promotes ferroptosis of CRC cells by inducing mitochondrial dysfunction, and ICA combined with anti-PD-1 significantly promotes CD69, TCRβ signalling, activates effector CD8+ T cells to secrete IFN-γ, and achieves immunopotentiation by promoting ferroptosis of CRC cells, thus inhibiting CRC development. This study is built upon existing research into the pharmacodynamic mechanisms of ICA in the context of CRC, and offers a novel therapeutic approach in addressing the issue of CRC immunotherapy potentiation.

Haoyue W ，Kexiang S ，Shan TW ，Jiamin G ，Luyun Y ，Junkai W ，Wanli D ... -  《-》

XCL1-secreting CEA CAR-T cells enhance endogenous CD8(+) T cell responses to tumor neoantigens to confer a long-term antitumor immunity.

Therapeutic efficacy of carcinoembryonic antigen (CEA)-specific chimeric antigen receptor (CAR) T cells against colorectal cancer (CRC) remains limited due to the unique characteristics and distinct microenvironments of tumor tissues. We modified CEA-specific CAR-T cells, aiming to stimulate endogenous CD8+ T cell responses against neoantigens that were derived from CEA-positive tumors destroyed by the CAR T cells. In a conventional CEA CAR (reg-CAR), we modified it to express lymphotactin XCL1 and interleukin (IL)-7 genes, constructing a modified 7XCL1-CAR. By generating the CEA-specific 7XCL1-CAR T cells, we assessed their antitumor efficacy against CRC cells with varying levels of CEA expression, both in cell-cultures and in two strains of tumor-bearing syngeneic mice. Following retroviral transduction, 7XCL1-CAR T cells and reg-CAR T cells exhibited similar positive proportions of CEA-CAR and CD4:CD8 ratios. In co-culture system with CEA-negative CT26 cells, no differences in cytotoxicity were observed between 7XCL1-CAR and reg-CAR T cells. However, in co-culture with CT26.CEAhigh and CT26.CEAint cells, 7XCL1-CAR T cells displayed higher cytotoxicity than that reg-CAR T cells after 60 hours. On interaction with CT26.CEA-positive cells, 7XCL1-CAR T cells secreted higher levels of XCL1 and IL-7, effectively recruited the most potent cross-presenting cDC1s (type-I conventional dendritic cells), and sustained the antitumor activity of CAR-T cells. In treating mice that carried tumors derived from universally CEA-positive cells, 7XCL1-CAR T cells exhibited no difference compared with reg-CAR T cells. However, in treating mice with tumors containing both CEA-positive and CEA-negative cells, 7XCL1-CAR T cells displayed greater inhibition than that of reg-CAR-T cells. After treatment of 7XCL1-CAR T cells, tumor-bearing mice exhibited enhanced infiltration of cDC1s, maintained CAR-T activity, and generation of endogenous neoantigen-specific T cells. Consequently, 7XCL1-CAR T cell-treated mice demonstrated resistance to challenge with CEA-negative CT26 cells. Treatment with CEA-specific, XCL1-secreting CAR-T cells for CEA-positive tumors promoted the generation of CD8+ T cells against tumor neoantigens, mediating a long-term antitumor immunity against heterogeneous CRCs.

Li XN ，Wang F ，Chen K ，Wu Z ，Zhang R ，Xiao C ，Zhao F ，Wang D ，Zhao H ，Ran Y ，Qu C ... -  《Journal for ImmunoTherapy of Cancer》

Identification of a novel toxicophore in anti-cancer chemotherapeutics that targets mitochondrial respiratory complex I.

Disruption of mitochondrial function selectively targets tumour cells that are dependent on oxidative phosphorylation. However, due to their high energy demands, cardiac cells are disproportionately targeted by mitochondrial toxins resulting in a loss of cardiac function. An analysis of the effects of mubritinib on cardiac cells showed that this drug did not inhibit HER2 as reported, but directly inhibits mitochondrial respiratory complex I, reducing cardiac-cell beat rate, with prolonged exposure resulting in cell death. We used a library of chemical variants of mubritinib and showed that modifying the 1H-1,2,3-triazole altered complex I inhibition, identifying the heterocyclic 1,3-nitrogen motif as the toxicophore. The same toxicophore is present in a second anti-cancer therapeutic carboxyamidotriazole (CAI) and we demonstrate that CAI also functions through complex I inhibition, mediated by the toxicophore. Complex I inhibition is directly linked to anti-cancer cell activity, with toxicophore modification ablating the desired effects of these compounds on cancer cell proliferation and apoptosis.

Stephenson ZA ，Harvey RF ，Pryde KR ，Mistry S ，Hardy RE ，Serreli R ，Chung I ，Allen TE ，Stoneley M ，MacFarlane M ，Fischer PM ，Hirst J ，Kellam B ，Willis AE ... -  《eLife》