MAP4K3 inhibits Sirtuin-1 to repress LKB1-AMPK to promote amino acid dependent activation of mTORC1

mTORC1 is the key rheostat controlling cellular metabolic state. Of the various inputs to mTORC1, the most potent effector of intracellular nutrient status is amino acid supply. Despite an established role for MAP4K3 in promoting mTORC1 activation in the presence of amino acids, the signaling pathway by which MAP4K3 controls mTORC1 activation remains unknown. Here we examined the process of MAP4K3 activation of mTORC1 and found that MAP4K3 represses the LKB1-AMPK pathway to prevent TSC1/2 complex inactivation of Rheb. When we sought the regulatory link between MAP4K3 and LKB1 inhibition, we discovered that MAP4K3 physically interacts with the master nutrient regulatory factor Sirtuin-1 and phosphorylates Sirtuin-1 to repress LKB1 activation. Our results reveal the existence of a novel signaling pathway linking amino acid satiety with MAP4K3-dependent suppression of SIRT1 to inactivate the repressive LKB1-AMPK pathway and thereby potently activate the mTORC1 complex to dictate the metabolic disposition of the cell.

Novel human neutralizing mAbs specific for Spike-RBD of SARS-CoV-2

Among the therapies against the pandemic SARS-CoV-2 virus, monoclonal Antibodies (mAbs) targeting the Spike glycoprotein represent good candidates to interfere in the Spike/ACE2 interaction, preventing virus cell entry. Since anti-spike mAbs, used individually, might be unable to block the virus entry in the case of resistant mutations, we designed an innovative strategy for the isolation of multiple novel human scFvs specific for the binding domain (RBD) of Spike. By panning a large phage display antibody library on immobilized RBD, we obtained specific binders by eluting with ACE2 in order to identify those scFvs recognizing the epitope of Spike interacting with its receptor. We converted the novel scFvs into full size IgG4, differently from the previously isolated IgG1 mAbs, to avoid unwanted potential side effects of IgG1 potent effector functions on immune system. The novel antibodies specifically bind to RBD in a nanomolar range and interfere in the interaction of Spike with ACE2 receptor, either used as purified protein or when expressed on cells in its native conformation. Furthermore, some of them have neutralizing activity for virus infection in cell cultures by using two different SARS-CoV-2 isolates including the highly contagious VOC 202012/01 variant and could become useful therapeutic tools to fight against the SARS-CoV-2 virus.

Hypoxia-Driven HIF-1α Activation Reprograms Pre-Activated NK Cells towards Highly Potent Effector Phenotypes via ERK/STAT3 Pathways

NK cells are the predominant innate lymphocyte subsets specialized to kill malignant tumor cells. In patients with advanced cancer, hypoxic stress shapes NK cells toward tumor-resistant and immunosuppressive phenotypes, hence a strategy to restore NK function is critical for successful tumor immunotherapy. Here, we present evidence that pre-activation and subsequent HIF-1α-dependent metabolic shift of NK cells from oxidative phosphorylation into glycolysis are keys to overcome hypoxia-mediated impairment in NK cell survival, proliferation, and tumor cytotoxicity. Specifically, exposing NK cells to 7–9 days of normoxic culture followed by a pO2 of 1.5% hypoxia led to a highly potent effector phenotype via HIF-1α stabilization and upregulation of its target genes, BNIP3, PDK1, VEGF, PKM2, and LDHA. RNA sequencing and network analyses revealed that concomitant reduction of p21/p53 apoptotic pathways along with upregulation of cell cycle-promoting genes, CCNE1, CDC6, CDC20, and downregulation of cell cycle-arrest genes, CDKN1A, GADD45A, and MDM2 were accountable for superior expansion of NK cells via ERK/STAT3 activation. Furthermore, HIF-1α-dependent upregulation of the NKp44 receptor in hypoxia-exposed NK cells resulted in increased killing against K562, CEM, and A375 tumor targets both in-vitro and in-vivo tumor clearance assays. Therefore, hypoxic exposure on pre-activated proliferating NK cells triggered HIF-1α-dependent pathways to initiate coordinated regulation of cell cycle, apoptosis, and cytotoxicity at the global gene transcription level. Our results uncover a previously unidentified role of HIF-1α-mediated metabolic reprogramming that can reverse impaired NK effector phenotypes to generate requisite numbers of functionally robust NK cells for adoptive cellular therapy for clinical evaluation.

Ex-TFRs: A Missing Piece of the SLE Puzzle?

Systemic lupus erythematosus (SLE) is a chronic multi-organ autoimmune disease involving the production of a wide range of autoantibodies and complement activation. The production of these high-affinity autoantibodies requires T cell/B cell collaboration as well as germinal center (GC) formation. T follicular regulatory cells (TFRs) are functional specialized T regulatory cells (Tregs) that safeguard against both self-reactive T and B cells. However, recent evidence suggests that TFRs are not always stable and can lose Foxp3 expression to become pathogenic “ex-TFRs” that gain potent effector functions. In this review, we summarize the literature on intrinsic and extrinsic mechanisms of regulation of TFR stability and discuss the potential role of TFR reprogramming in autoantibody production and SLE pathogenesis.

Structural Basis for Epitopes in the gp120 Cluster A Region that Invokes Potent Effector Cell Activity

While a number of therapeutic options to control the progression of human immunodeficiency virus (HIV-1) now exist, a broadly effective preventive vaccine is still not available. Through detailed structural analysis of antibodies able to induce potent effector cell activity, a number of Env epitopes have been identified which have the potential to be considered vaccine candidates. These antibodies mainly target the gp120 Cluster A region which is only exposed upon viral binding to the target cell with epitopes becoming available for antibody binding during viral entry and fusion and, therefore, after the effective window for neutralizing antibody activity. This review will discuss recent advances in the structural characterization of these important targets with a special focus on epitopes that are involved in Fc-mediated effector function without direct viral neutralizing activities.

The nuclear export inhibitor aminoratjadone is a potent effector in extracellular-targeted drug conjugates

Ratjadone derivatives have been successfully introduced as suitable payloads with new mode of action for targeted drug conjugates.

The negative effect of GH/IGF-1 excess on NAD- and NADP-dependent blood lymphocytes dehydrogenases activity in acromegaly

Background. Acromegaly is a rare serious condition characterized by chronic hypersecretion of growth hormone (GH) from a pituitary adenoma and induces the synthesis of insulin-like growth factor I (IGF-1). The idea of the crucial GH importance not only in the control of cell proliferation and differentiation, but, also, in the regulation of immune cells metabolism allows to think that chronic excess GH/IGF-I in acromegaly is the potent effector distortion of the immune response mechanisms. Aim. To study the NAD(P)-dependent dehydrogenases level in blood lymphocytes and their interaction with GH/IGF-1 concentration in patients with active acromegaly.Methods. The level of NAD(P)-dependent dehydrogenases in blood lymphocytes was studied in a group of 88 patients with active acromegaly, mean age 51.0±12.5 years. The NAD(P)-dependent dehydrogenases activity was determined by biochemiluminescence method. The concentrations of GH and IGF-1 were measured by ELISA.Results. Studying the activity of mitochondrial NAD(P)-dependent dehydrogenases found a decrease in all NAD-dependent oxidoreductase: NADIDH, NADGDH, and MDH (P<0.01), which allows to state the low level flow in the tricarboxylic acid cycle. In active acromegaly were revealed the decreasing activity of all studied oxidoreductases: glucose-6-phosphate dehydrogenase (P<0.01), NAD–lactate dehydrogenase (LDH) (P<0.001), NADH–LDH (P<0.001), NAD–malate dehydrogenase (MDH) (P<0.001), NADH–MDH (P<0.001), NADP–MDH (P<0.001), NAD–glutamate dehydrogenases (GDH) and NADH–GDH (P<0.001), NADP–GDH and NADPH–GDH (P<0.001), NAD–isocitrate dehydrogenases (IDH) and NADP–IDH (P<0.01 and P<0.001 respectively), and, also, glutathione reductase (P<0.001). Our data observed that decreasing activity of NADP–GDH positively correlated with the basal GH level (r=+0.23, P=0.04) and NADP–MDH activity with IGF-1 level (r=+0.30, P=0.008). The low NADH–MDH activity negatively correlated to the basal GH concentration (r=−0.23, P=0.04).Conclusion. The chronic excess of GH/IGF-1 causes a significant depletion of metabolic lymphocytes reserves and may play an important role in several systems malignancies of acromegaly patients. This pathway continues to attract interest as a potentially useful target for therapeutic design of acromegaly.