Preclinical assessment of antigen-specific chimeric antigen receptor regulatory T cells for use in solid organ transplantation.

A primary goal in transplantation medicine is the induction of a tolerogenic environment for prevention of transplant rejection without the need for long-term pharmacological immunosuppression. Generation of alloantigen-specific regulatory T cells (Tregs) by transduction with chimeric antigen receptors (CARs) is a promising strategy to achieve this goal. This publication reports the preclinical characterization of Tregs (TR101) transduced with a human leukocyte antigen (HLA)-A*02 CAR lentiviral vector (TX200) designated to induce immunosuppression of allograft-specific effector T cells in HLA-A*02-negative recipients of HLA-A*02-positive transplants. In vitro results demonstrated specificity, immunosuppressive function, and safety of TX200-TR101. In NOD scid gamma (NSG) mice, TX200-TR101 prevented graft-versus-host disease (GvHD) in a xenogeneic GvHD model and TX200-TR101 Tregs localized to human HLA-A*02-positive skin transplants in a transplant model. TX200-TR101 persisted over the entire duration of a 3-month study in humanized HLA-A*02 NSG mice and remained stable, without switching to a proinflammatory phenotype. Concomitant tacrolimus did not impair TX200-TR101 Treg survival or their ability to inhibit peripheral blood mononuclear cell (PBMC) engraftment. These data demonstrate that TX200-TR101 is specific, stable, efficacious, and safe in preclinical models, and provide the basis for a first-in-human study.

GAPDH Overexpression in the T Cell Lineage Promotes Angioimmunoblastic T Cell Lymphoma through an NF-κB-Dependent Mechanism.

GAPDH is emerging as a key player in T cell development and function. To investigate the role of GAPDH in T cells, we generated a transgenic mouse model overexpressing GAPDH in the T cell lineage. Aged mice developed a peripheral Tfh-like lymphoma that recapitulated key molecular, pathological, and immunophenotypic features of human angioimmunoblastic T cell lymphoma (AITL). GAPDH induced non-canonical NF-κB pathway activation in mouse T cells, which was strongly activated in human AITL. We developed a NIK inhibitor to reveal that targeting the NF-κB pathway prolonged AITL-bearing mouse survival alone and in combination with anti-PD-1. These findings suggest the therapeutic potential of targeting NF-κB signaling in AITL and provide a model for future AITL therapeutic investigations.

GAPDH Expression Predicts the Response to R-CHOP, the Tumor Metabolic Status, and the Response of DLBCL Patients to Metabolic Inhibitors.

Diffuse large B cell lymphoma (DLBCL) is a heterogeneous disease treated with anti-CD20-based immuno-chemotherapy (R-CHOP). We identified that low levels of GAPDH predict a poor response to R-CHOP treatment. Importantly, we demonstrated that GAPDHlow lymphomas use OxPhos metabolism and rely on mTORC1 signaling and glutaminolysis. Consistently, disruptors of OxPhos metabolism (phenformin) or glutaminolysis (L-asparaginase) induce cytotoxic responses in GAPDHlow B cells and improve GAPDHlow B cell-lymphoma-bearing mice survival, while they are low or not efficient on GAPDHhigh B cell lymphomas. Ultimately, we selected four GAPDHlow DLBCL patients, who were refractory to all anti-CD20-based therapies, and targeted DLBCL metabolism using L-asparaginase (K), mTOR inhibitor (T), and metformin (M) (called KTM therapy). Three out of the four patients presented a complete response upon one cycle of KTM. These findings establish that the GAPDH expression level predicts DLBCL patients' response to R-CHOP treatment and their sensitivity to specific metabolic inhibitors.

The oncogenic tyrosine kinase Lyn impairs the pro-apoptotic function of Bim.

Phosphorylation of Ser/Thr residues is a well-established modulating mechanism of the pro-apoptotic function of the BH3-only protein Bim. However, nothing is known about the putative tyrosine phosphorylation of this Bcl-2 family member and its potential impact on Bim function and subsequent Bax/Bak-mediated cytochrome c release and apoptosis. As we have previously shown that the tyrosine kinase Lyn could behave as an anti-apoptotic molecule, we investigated whether this Src family member could directly regulate the pro-apoptotic function of Bim. In the present study, we show that Bim is phosphorylated onto tyrosine residues 92 and 161 by Lyn, which results in an inhibition of its pro-apoptotic function. Mechanistically, we show that Lyn-dependent tyrosine phosphorylation of Bim increases its interaction with anti-apoptotic members such as Bcl-xL, therefore limiting mitochondrial outer membrane permeabilization and subsequent apoptosis. Collectively, our data uncover one molecular mechanism through which the oncogenic tyrosine kinase Lyn negatively regulates the mitochondrial apoptotic pathway, which may contribute to the transformation and/or the chemotherapeutic resistance of cancer cells.

Parkin-Independent Mitophagy Controls Chemotherapeutic Response in Cancer Cells.

Mitophagy is an evolutionarily conserved process that selectively targets impaired mitochondria for degradation. Defects in mitophagy are often associated with diverse pathologies, including cancer. Because the main known regulators of mitophagy are frequently inactivated in cancer cells, the mechanisms that regulate mitophagy in cancer cells are not fully understood. Here, we identified an E3 ubiquitin ligase (ARIH1/HHARI) that triggers mitophagy in cancer cells in a PINK1-dependent manner. We found that ARIH1/HHARI polyubiquitinates damaged mitochondria, leading to their removal via autophagy. Importantly, ARIH1 is widely expressed in cancer cells, notably in breast and lung adenocarcinomas; ARIH1 expression protects against chemotherapy-induced death. These data challenge the view that the main regulators of mitophagy are tumor suppressors, arguing instead that ARIH1-mediated mitophagy promotes therapeutic resistance.

Mitochondrial permeabilization engages NF-κB-dependent anti-tumour activity under caspase deficiency.

Apoptosis represents a key anti-cancer therapeutic effector mechanism. During apoptosis, mitochondrial outer membrane permeabilization (MOMP) typically kills cells even in the absence of caspase activity. Caspase activity can also have a variety of unwanted consequences that include DNA damage. We therefore investigated whether MOMP-induced caspase-independent cell death (CICD) might be a better way to kill cancer cells. We find that cells undergoing CICD display potent pro-inflammatory effects relative to apoptosis. Underlying this, MOMP was found to stimulate NF-κB activity through the downregulation of inhibitor of apoptosis proteins. Strikingly, engagement of CICD displays potent anti-tumorigenic effects, often promoting complete tumour regression in a manner dependent on intact immunity. Our data demonstrate that by activating NF-κB, MOMP can exert additional signalling functions besides triggering cell death. Moreover, they support a rationale for engaging caspase-independent cell death in cell-killing anti-cancer therapies.

Low carbohydrate diet prevents Mcl-1-mediated resistance to BH3-mimetics.

Overexpression of Mcl-1 is implicated in resistance of several cancers to chemotherapeutic treatment, therefore identifying a safe way to decrease its expression in tumor cells represents a central goal. We investigated if a modulation of the diet could impact on Mcl-1 expression using a Myc-driven lymphoma model. We established that a partial reduction of caloric intake by 25% represents an efficient way to decrease Mcl-1 expression in tumor cells. Furthermore, using isocaloric custom diets, we observed that carbohydrates (CHO) are the main regulators of Mcl-1 expression within the food. Indeed, feeding lymphoma-bearing mice with a diet having 25% less carbohydrates was sufficient to decrease Mcl-1 expression by 50% in lymphoma cells. We showed that a low CHO diet resulted in AMPK activation and mTOR inhibition leading to eukaryotic elongation factor 2 (eEF2) inhibition, blocking protein translation elongation. Strikingly, a low CHO diet was sufficient to sensitize Myc-driven lymphoma-bearing mice to ABT-737-induced cell death in vivo. Thus reducing carbohydrate intake may represent a safe way to decrease Mcl-1 expression and to sensitize tumor cells to anti-cancer therapeutics.