Ghrelin is an acyl-peptide gastric hormone acting on the pituitary and hypothalamus to stimulate growth hormone (GH) release, adiposity, and appetite. Ghrelin endocrine activities are entirely dependent on its acylation and are mediated by GH secretagogue (GHS) receptor (GHSR)-1a, a G protein–coupled receptor mostly expressed in the pituitary and hypothalamus, previously identified as the receptor for a group of synthetic molecules featuring GH secretagogue (GHS) activity. Des-acyl ghrelin, which is far more abundant than ghrelin, does not bind GHSR-1a, is devoid of any endocrine activity, and its function is currently unknown. Ghrelin, which is expressed in heart, albeit at a much lower level than in the stomach, also exerts a cardio protective effect through an unknown mechanism, independent of GH release. Here we show that both ghrelin and des-acyl ghrelin inhibit apoptosis of primary adult and H9c2 cardiomyocytes and endothelial cells in vitro through activation of extracellular signal–regulated kinase-1/2 and Akt serine kinases. In addition, ghrelin and des-acyl ghrelin recognize common high affinity binding sites on H9c2 cardiomyocytes, which do not express GHSR-1a. Finally, both MK-0677 and hexarelin, a nonpeptidyl and a peptidyl synthetic GHS, respectively, recognize the common ghrelin and des-acyl ghrelin binding sites, inhibit cell death, and activate MAPK and Akt. These findings provide the first evidence that, independent of its acylation, ghrelin gene product may act as a survival factor directly on the cardiovascular system through binding to a novel, yet to be identified receptor, which is distinct from GHSR-1a.

Ghrelin is an acylated peptidyl gastric hormone acting on the pituitary and hypothalamus to stimulate appetite, adiposity, and growth hormone release, through activation of growth hormone secretagogue receptor (GHSR)-1a receptor. Moreover, ghrelin features several activities such as inhibition of apoptosis, regulation of differentiation, and stimulation or inhibition of proliferation of several cell types. Ghrelin acylation is absolutely required for both GHSR-1a binding and its central endocrine activities. However, the unacylated ghrelin form, des-acyl ghrelin, which does not bind GHSR-1a and is devoid of any endocrine activity, is far more abundant than ghrelin in plasma, and it shares with ghrelin some of its cellular activities. Inhere we show that both ghrelin and des-acyl ghrelin stimulate proliferating C2C12 skeletal myoblasts to differentiate and to fuse into multinucleated myotubes in vitro through activation of p38. Consistently, both ghrelin and des-acyl ghrelin inhibit C2C12 proliferation in growth medium. Moreover, the ectopic expression of ghrelin in C2C12 enhances differentiation and fusion of these myoblasts in differentiation medium. Finally, we show that C2C12 cells do not express GHSR-1a, but they do contain a common high-affinity binding site recognized by both acylated and des-acylated ghrelin, suggesting that the described activities on C2C12 are likely mediated by this novel, yet unidentified receptor for both ghrelin forms.

Immunotherapy with chimeric antigen receptor (CAR)–engineered T cells showed exceptional successes in patients with refractory B cell malignancies. However, first-in-human studies in solid tumors revealed unique hurdles contributing to poor demonstration of efficacy. Understanding the determinants of tumor recognition by CAR T cells should translate into the design of strategies that can overcome resistance. Here, we show that multiple carcinomas express extracellular N-glycans, whose abundance negatively correlates with CAR T cell killing. By knocking out mannoside acetyl-glucosaminyltransferase 5 ( MGAT5 ) in pancreatic adenocarcinoma (PAC), we showed that N-glycans protect tumors from CAR T cell killing by interfering with proper immunological synapse formation and reducing transcriptional activation, cytokine production, and cytotoxicity. To overcome this barrier, we exploited the high metabolic demand of tumors to safely inhibit N-glycans synthesis with the glucose/mannose analog 2-deoxy- d -glucose (2DG). Treatment with 2DG disrupts the N-glycan cover on tumor cells and results in enhanced CAR T cell activity in different xenograft mouse models of PAC. Moreover, 2DG treatment interferes with the PD-1–PD-L1 axis and results in a reduced exhaustion profile of tumor-infiltrating CAR T cells in vivo. The combined 2DG and CAR T cell therapy was successful against multiple carcinomas besides PAC, including those arising from the lung, ovary, and bladder, and with different clinically relevant CAR specificities, such as CD44v6 and CEA. Overall, our results indicate that tumor N-glycosylation regulates the quality and magnitude of CAR T cell responses, paving the way for the rational design of improved therapies against solid malignancies.