Tumor-Infiltrating Lymphocyte Therapy or Ipilimumab in Advanced Melanoma.

Background: Immune checkpoint inhibitors and targeted therapies have dramatically improved outcomes in patients with advanced melanoma, but approximately half these patients will not have a durable benefit. Phase 1-2 trials of adoptive cell therapy with tumor-infiltrating lymphocytes (TILs) have shown promising responses, but data from phase 3 trials are lacking to determine the role of TILs in treating advanced melanoma.

Methods: In this phase 3, multicenter, open-label trial, we randomly assigned patients with unresectable stage IIIC or IV melanoma in a 1:1 ratio to receive TIL or anti-cytotoxic T-lymphocyte antigen 4 therapy (ipilimumab at 3 mg per kilogram of body weight).

MART-1 TCR gene-modified peripheral blood T cells for the treatment of metastatic melanoma: a phase I/IIa clinical trial.

Background: Adoptive cell therapy with peripheral blood T cells expressing transgenic T-cell receptors (TCRs) is an innovative therapeutic approach for solid malignancies. We investigated the safety and feasibility of adoptive transfer of autologous T cells expressing melanoma antigen recognized by T cells 1 (MART-1)-specific TCR, cultured to have less differentiated phenotypes, in patients with metastatic melanoma.

Materials And Methods: In this phase I/IIa trial, peripheral blood T cells from HLA-A2∗02:01-positive patients with unresectable stage IIIC/IV melanoma expressing MART-1 were selected and stimulated with anti-CD3/CD28 beads, transduced with a modified MART-1-specific 1D3 TCR (1D3HMCys) and expanded in interleukin (IL)-7 and IL-15.

Regulation of intestinal LDLR by the LXR-IDOL axis.

Background And Aims: Cholesterol metabolism is tightly regulated by transcriptional and post-transcriptional mechanisms. Accordingly, dysregulation of cholesterol metabolism is a major risk factor for the development of coronary artery disease and associated complications. In recent years, it has become apparent that next to the liver, the intestine plays a key role in systemic cholesterol metabolism by governing cholesterol absorption, secretion, and incorporation into lipoprotein particles.

The Iminosugar AMP-DNM Improves Satiety and Activates Brown Adipose Tissue Through GLP1.

Obesity is taking on worldwide epidemic proportions, yet effective pharmacological agents with long-term efficacy remain unavailable. Previously, we designed the iminosugar N-adamantine-methyloxypentyl-deoxynojirimycin (AMP-DNM), which potently improves glucose homeostasis by lowering excessive glycosphingolipids. Here we show that AMP-DNM promotes satiety and activates brown adipose tissue (BAT) in obese rodents.

Differential use of E2 ubiquitin conjugating enzymes for regulated degradation of the rate-limiting enzymes HMGCR and SQLE in cholesterol biosynthesis.

Background And Aims: Cholesterol is an essential lipid for cellular function and membrane integrity, and hence its cellular levels and distribution must be tightly regulated. Biosynthesis of cholesterol is ramped when its cellular levels are low. Herein, the ER-resident and rate-limiting enzymes 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) and squalene monooxygenase (SQLE) play a prominent role.

Inactivation of the E3 Ubiquitin Ligase IDOL Attenuates Diet-Induced Obesity and Metabolic Dysfunction in Mice.

Objective- The E3 ubiquitin ligase IDOL (inducible degrader of the LDLR [LDL (low-density lipoprotein) receptor]) is a post-transcriptional regulator of LDLR abundance. Model systems and human genetics support a role for IDOL in regulating circulating LDL levels. Whether IDOL plays a broader metabolic role and affects development of metabolic syndrome-associated comorbidities is unknown.

HEPES activates a MiT/TFE-dependent lysosomal-autophagic gene network in cultured cells: A call for caution.

In recent years, the lysosome has emerged as a highly dynamic, transcriptionally regulated organelle that is integral to nutrient-sensing and metabolic rewiring. This is coordinated by a lysosome-to-nucleus signaling nexus in which MTORC1 controls the subcellular distribution of the microphthalmia-transcription factor E (MiT/TFE) family of "master lysosomal regulators". Yet, despite the importance of the lysosome in cellular metabolism, the impact of traditional in vitro culture media on lysosomal dynamics and/or MiT/TFE localization has not been fully appreciated.

Haploid Mammalian Genetic Screen Identifies UBXD8 as a Key Determinant of HMGCR Degradation and Cholesterol Biosynthesis.

Objective: The cellular demand for cholesterol requires control of its biosynthesis by the mevalonate pathway. Regulation of HMGCR (3-hydroxy-3-methylglutaryl coenzyme A reductase), a rate-limiting enzyme in this pathway and the target of statins, is a key control point herein. Accordingly, HMGCR is subject to negative and positive regulation.

Stabilization of Glucocerebrosidase by Active Site Occupancy.

Glucocerebrosidase (GBA) is a lysosomal β-glucosidase that degrades glucosylceramide. Its deficiency results in Gaucher disease (GD). We examined the effects of active site occupancy of GBA on its structural stability.

Identification of the ER-resident E3 ubiquitin ligase RNF145 as a novel LXR-regulated gene.

Cellular cholesterol metabolism is subject to tight regulation to maintain adequate levels of this central lipid molecule. Herein, the sterol-responsive Liver X Receptors (LXRs) play an important role owing to their ability to reduce cellular cholesterol load. In this context, identifying the full set of LXR-regulated genes will contribute to our understanding of their role in cholesterol metabolism.

Investigations on therapeutic glucocerebrosidases through paired detection with fluorescent activity-based probes.

Deficiency of glucocerebrosidase (GBA) causes Gaucher disease (GD). In the common non-neuronopathic GD type I variant, glucosylceramide accumulates primarily in the lysosomes of visceral macrophages. Supplementing storage cells with lacking enzyme is accomplished via chronic intravenous administration of recombinant GBA containing mannose-terminated N-linked glycans, mediating the selective uptake by macrophages expressing mannose-binding lectin(s).

EEPD1 Is a Novel LXR Target Gene in Macrophages Which Regulates ABCA1 Abundance and Cholesterol Efflux.

Objective: The sterol-responsive nuclear receptors, liver X receptors α (LXRα, ) and β (LXRβ, ), are key determinants of cellular cholesterol homeostasis. LXRs are activated under conditions of high cellular sterol load and induce expression of the cholesterol efflux transporters and to promote efflux of excess cellular cholesterol. However, the full set of genes that contribute to LXR-stimulated cholesterol efflux is unknown, and their identification is the objective of this study.

A Specific Activity-Based Probe to Monitor Family GH59 Galactosylceramidase, the Enzyme Deficient in Krabbe Disease.

Galactosylceramidase (GALC) is the lysosomal β-galactosidase responsible for the hydrolysis of galactosylceramide. Inherited deficiency in GALC causes Krabbe disease, a devastating neurological disorder characterized by accumulation of galactosylceramide and its deacylated counterpart, the toxic sphingoid base galactosylsphingosine (psychosine). We report the design and application of a fluorescently tagged activity-based probe (ABP) for the sensitive and specific labeling of active GALC molecules from various species.

Endo-β-Glucosidase Tag Allows Dual Detection of Fusion Proteins by Fluorescent Mechanism-Based Probes and Activity Measurement.

β-Glucoside-configured cyclophellitols are activity-based probes (ABPs) that allow sensitive detection of β-glucosidases. Their applicability to detect proteins fused with β-glucosidase was investigated in the cellular context. The tag was Rhodococcus sp.

A PPARγ-Bnip3 Axis Couples Adipose Mitochondrial Fusion-Fission Balance to Systemic Insulin Sensitivity.

Aberrant mitochondrial fission plays a pivotal role in the pathogenesis of skeletal muscle insulin resistance. However, fusion-fission dynamics are physiologically regulated by inherent tissue-specific and nutrient-sensitive processes that may have distinct or even opposing effects with respect to insulin sensitivity. Based on a combination of mouse population genetics and functional in vitro assays, we describe here a regulatory circuit in which peroxisome proliferator-activated receptor γ (PPARγ), the adipocyte master regulator and receptor for the thiazolidinedione class of antidiabetic drugs, controls mitochondrial network fragmentation through transcriptional induction of Bnip3.

Lysosomal glycosphingolipid catabolism by acid ceramidase: formation of glycosphingoid bases during deficiency of glycosidases.

Glycosphingoid bases are elevated in inherited lysosomal storage disorders with deficient activity of glycosphingolipid catabolizing glycosidases. We investigated the molecular basis of the formation of glucosylsphingosine and globotriaosylsphingosine during deficiency of glucocerebrosidase (Gaucher disease) and α-galactosidase A (Fabry disease). Independent genetic and pharmacological evidence is presented pointing to an active role of acid ceramidase in both processes through deacylation of lysosomal glycosphingolipids.

Glucosylated cholesterol in mammalian cells and tissues: formation and degradation by multiple cellular β-glucosidases.

The membrane lipid glucosylceramide (GlcCer) is continuously formed and degraded. Cells express two GlcCer-degrading β-glucosidases, glucocerebrosidase (GBA) and GBA2, located in and outside the lysosome, respectively. Here we demonstrate that through transglucosylation both GBA and GBA2 are able to catalyze in vitro the transfer of glucosyl-moieties from GlcCer to cholesterol, and vice versa.

Deubiquitylase Inhibition Reveals Liver X Receptor-independent Transcriptional Regulation of the E3 Ubiquitin Ligase IDOL and Lipoprotein Uptake.

Cholesterol metabolism is subject to complex transcriptional and nontranscriptional regulation. Herein, the role of ubiquitylation is emerging as an important post-translational modification that regulates cholesterol synthesis and uptake. Similar to other post-translational modifications, ubiquitylation is reversible in a process dependent on activity of deubiquitylating enzymes (DUBs).

Identification of the (Pro)renin Receptor as a Novel Regulator of Low-Density Lipoprotein Metabolism.

Rationale: The (pro)renin receptor ([P]RR) interacts with (pro)renin at concentrations that are >1000× higher than observed under (patho)physiological conditions. Recent studies have identified renin-angiotensin system-independent functions for (P)RR related to its association with the vacuolar H(+)-ATPase.

Objective: To uncover renin-angiotensin system-independent functions of the (P)RR.

and comparative and competitive activity-based protein profiling of GH29 α-l-fucosidases.

GH29 α-l-fucosidases catalyze the hydrolysis of α-l-fucosidic linkages. Deficiency in human lysosomal α-l-fucosidase (FUCA1) leads to the recessively inherited disorder, fucosidosis. Herein we describe the development of fucopyranose-configured cyclophellitol aziridines as activity-based probes (ABPs) for selective and labeling of GH29 α-l-fucosidases from bacteria, mice and man.

LIM-only protein FHL2 is a positive regulator of liver X receptors in smooth muscle cells involved in lipid homeostasis.

The LIM-only protein FHL2 is expressed in smooth muscle cells (SMCs) and inhibits SMC-rich-lesion formation. To further elucidate the role of FHL2 in SMCs, we compared the transcriptomes of SMCs derived from wild-type (WT) and FHL2 knockout (KO) mice. This revealed that in addition to the previously recognized involvement of FHL2 in SMC proliferation, the cholesterol synthesis and liver X receptor (LXR) pathways are altered in the absence of FHL2.

Identification and development of biphenyl substituted iminosugars as improved dual glucosylceramide synthase/neutral glucosylceramidase inhibitors.

This work details the evaluation of a number of N-alkylated deoxynojirimycin derivatives on their merits as dual glucosylceramide synthase/neutral glucosylceramidase inhibitors. Building on our previous work, we synthesized a series of D-gluco and L-ido-configured iminosugars N-modified with a variety of hydrophobic functional groups. We found that iminosugars featuring N-pentyloxymethylaryl substituents are considerably more potent inhibitors of glucosylceramide synthase than their aliphatic counterparts.

Potent and selective activity-based probes for GH27 human retaining α-galactosidases.

Lysosomal degradation of glycosphingolipids is mediated by the consecutive action of several glycosidases. Malfunctioning of one of these hydrolases can lead to a lysosomal storage disorder such as Fabry disease, which is caused by a deficiency in α-galactosidase A. Herein we describe the development of potent and selective activity-based probes that target retaining α-galactosidases.

Design and synthesis of 4'-O-alkyl-chitobiosyl-4-methylumbelliferone as human chitinase fluorogenic substrates.

The synthesis of three fluorogenic chitobiosyl derivatives, modified at the non-reducing 4'-OH with, either a methyl, an isopropyl or a cyclohexylmethyl substituent, is described. The 4'-capped 4-methylumbelliferyl chitobiosides are hydrolysed by the human chitinase CHIT1 following Michaelis-Menten kinetics and in contrast to unmodified chitobiosyl-4-methylumbelliferone do not undergo transglycosylation. The compounds are also relatively poor hexosaminidase substrates and thus provide useful alternatives to 4'-deoxychitobiosyl-4-methylumbelliferone, previously reported by us as fluorogenic substrate to monitor CHIT1 activity as a marker for Gaucher disease state.