L-asparaginase anti-tumor activity in pancreatic cancer is dependent on its glutaminase activity and resistance is mediated by glutamine synthetase.

l-Asparaginase is a cornerstone of acute lymphoblastic leukemia (ALL) therapy since lymphoblasts lack asparagine synthetase (ASNS) and rely on extracellular asparagine availability for survival. Resistance mechanisms are associated with increased ASNS expression in ALL. However, the association between ASNS and l-Asparaginase efficacy in solid tumors remains unclear, thus limiting clinical development. Interestingly, l-Asparaginase also has a glutaminase co-activity that is crucial in pancreatic cancer where KRAS mutations activate glutamine metabolism. By developing l-Asparaginase-resistant pancreatic cancer cells and using OMICS approaches, we identified glutamine synthetase (GS) as a marker of resistance to l-Asparaginase. GS is the only enzyme able to synthesize glutamine, and its expression also correlates with l-Asparaginase efficacy in 27 human cell lines from 11 cancer indications. Finally, we further demonstrated that GS inhibition prevents cancer cell adaptation to l-Asparaginase-induced glutamine starvation. These findings could pave the way to the development of promising drug combinations to overcome l-Asparaginase resistance.

Innovative approach in Pompe disease therapy: Induction of immune tolerance by antigen-encapsulated red blood cells.

Pompe disease is a glycogen storage disease caused by acid α-glucosidase enzyme deficiency. Currently, the unique treatment is lifelong enzyme replacement therapy ERT with frequent intravenous administration of the recombinant analog alglucosidase-α (AGA), which ultimately generates a sustained humoral response resulting in treatment discontinuation. Our aim is to use the tolerogenic properties of antigen-encapsulated red blood cells (RBCs) to abolish the humoral response against AGA and to restore tolerance to replacement therapy. To demonstrate that our approach could prevent the AGA-induced immune response, mice were intravenously injected three times with AGA encapsulated into RBCs before being sensitized to AGA with several adjuvant molecules. Control animals received injections of free AGA instead of the encapsulated molecule. One-week after treatment with AGA-loaded RBCs, a strong decrease in specific humoral response was observed despite three stimulations with AGA and adjuvant molecules. Furthermore, this specific immunomodulation was maintained for at least two months without affecting the overall immune response. AGA-loaded RBCs represent a promising strategy to induce or restore tolerance in Pompe disease patients who develop hypersensitivity reactions following repeated AGA administrations.

Red blood cells as innovative antigen carrier to induce specific immune tolerance.

The route of administration, the dose of antigen as well as the type of antigen-presenting cells (APCs) targeted are important factors to induce immune tolerance. Despite encouraging results obtained in animal models, intravenous injection of soluble antigen is unsuccessful in human clinical trials on autoimmune disease due to inefficient antigen delivery. To improve antigen delivery, we used mouse red blood cells (RBCs) as antigen vehicles to specifically target APCs which are responsible for removal of senescent RBCs after phagocytosis. In this study, we demonstrated that antigen-delivery by RBCs induced a strong decrease in the humoral response compared with the ovalbumin (OVA) free form in mice. In addition, OVA-loaded RBC treated with [bis(sulphosuccinimidyl)] suberate (BS3), a chemical compound known to enhance RBC phagocytosis, induced an inhibition of antigen-specific T cell responses and an increase in the percentage of regulatory T cells. The state of tolerance induced is long lasting, antigen-specific and sufficiently robust to withstand immunization with antigen mixed with cholera toxin adjuvant. This RBC strategy, which does not abolish the immune system, constitutes an attractive approach for induction of tolerance compared to systemic immunosuppressant therapies already in use.

Tumor growth control using red blood cells as the antigen delivery system and poly(I:C).

The goal of most current vaccines in tumor immunology is to induce an efficient immune response against the tumor cells. The use of red blood cells (RBCs) for the delivery of tumor-associated antigen to antigen-presenting cells is an innovative approach for cancer immunotherapy. The induction of antigen-specific immune responses after administration of antigen-loaded RBCs has been demonstrated previously in mice. In this paper, we show the utility of this delivery system for cancer immunotherapy in 2 tumor mouse models, using the E.G7-OVA and the B16F10 tumor cells. The non-self-antigen, ovalbumin, loaded in RBCs and the self-tumor antigen, tyrosinase-related protein 2, loaded in RBCs were tested in the E.G7-OVA and the B16F10 tumor models, respectively. We showed that not only protein but also peptide could be efficiently entrapped in RBCs by a controlled lysis/resealing process. In both antigen models, the administration of a small quantity of antigen loaded in RBCs combined with polyinosinic-polycytidylic acid induced an antigen-specific T-cell response and the control of tumor growth in mice, whereas the injection of the same quantity of free antigen did not. The intensity of the T-cell response was dependent on the concentrations of antigen entrapped and the treatment performed on the RBC membrane (antibody coating and heat treatment) to improve antigen delivery. In summary, these results support the use of RBCs as an antigen delivery system for a powerful cancer immunotherapy approach.

Differential muscular myosin heavy chain expression of the pectoral and pelvic girdles during early growth in the king penguin (Aptenodytes patagonicus) chick.

Continuous growth, associated with a steady parental food supply, is a general pattern in offspring development. So that young chicks can acquire their locomotor independence, this period is usually marked by a fast maturation of muscles, during which different myosin heavy chain (MyHC) isoforms are expressed. However, parental food provisioning may fluctuate seasonally, and offspring therefore face a challenge to ensure the necessary maturation of their tissues when energy is limited. To address this trade-off we investigated muscle maturation in both the pectoral and pelvic girdles of king penguin chicks. This species has an exceptionally long rearing period (1 year), which is prolonged when parental food provisioning is drastically reduced during the sub-Antarctic winter. Approximately 1 month post hatching, chicks acquire a functional pedestrian locomotion, which uses pelvic muscles, whereas swimming, which uses the pectoral muscles, only occurs 1 year later. We therefore tested the hypothesis that the MyHC content of the leg muscles reaches a mature state before those of the pectoral muscles. We found that leg muscle MyHC composition changed with the progressive acquisition of pedestrian locomotion, whereas pectoral muscle fibres reached their mature MyHC profile as early as hatching. Contrary to our predictions, the acquisition of the adult profile in pectoral muscles could be related to an early maturation of the contractile muscular proteins, presumably associated with early thermoregulatory capacities of chicks, necessary for survival in their cold environment. This differential maturation appears to reconcile both the locomotor and environmental constraints of king penguin chicks during growth.