Mitochondrial protein import is regulated by p17/PERMIT to mediate lipid metabolism and cellular stress.

How lipid metabolism is regulated at the outer mitochondrial membrane (OMM) for transducing stress signaling remains largely unknown. We show here that this process is controlled by trafficking of ceramide synthase 1 (CerS1) from the endoplasmic reticulum (ER) to the OMM by a previously uncharacterized p17, which is now renamed protein that mediates ER-mitochondria trafficking (PERMIT). Data revealed that p17/PERMIT associates with newly translated CerS1 on the ER surface to mediate its trafficking to the OMM. Cellular stress induces Drp1 nitrosylation/activation, releasing p17/PERMIT to retrieve CerS1 for its OMM trafficking, resulting in mitochondrial ceramide generation, mitophagy and cell death. In vivo, CRISPR-Cas9-dependent genetic ablation of p17/PERMIT prevents acute stress-mediated CerS1 trafficking to OMM, attenuating mitophagy in p17/PERMIT-/- mice, compared to controls, in various metabolically active tissues, including brain, muscle, and pancreas. Thus, these data have implications in diseases associated with accumulation of damaged mitochondria such as cancer and/or neurodegeneration.

Pro-Survival Lipid Sphingosine-1-Phosphate Metabolically Programs T Cells to Limit Anti-tumor Activity.

Sphingosine 1-phosphate (S1P), a bioactive lysophospholipid generated by sphingosine kinase 1 (SphK1), regulates lymphocyte egress into circulation via S1P receptor 1 (S1PR1) signaling, and it controls the differentiation of regulatory T cells (Tregs) and T helper-17 cells. However, the mechanisms by which receptor-independent SphK1-mediated intracellular S1P levels modulate T cell functionality remains unknown. We show here that SphK1-deficient T cells maintain central memory phenotype and exhibit higher mitochondrial respiration and reduced differentiation to Tregs. Mechanistically, we discovered a direct correlation between SphK1-generated S1P and lipid transcription factor PPARγ (peroxisome proliferator-activated receptor gamma) activity, which in turn regulates lipolysis in T cells. Genetic and pharmacologic inhibition of SphK1 improved metabolic fitness and anti-tumor activity of T cells against murine melanoma. Further, inhibition of SphK1 and PD1 together led to improved control of melanoma. Overall, these data highlight the clinical potential of limiting SphK1/S1P signaling for enhancing anti-tumor-adoptive T cell therapy.

Thioredoxin-1 confines T cell alloresponse and pathogenicity in graft-versus-host disease.

Oxidative stress is elevated in the recipients of allogeneic hematopoietic transplantation (allo-HCT) and likely contributes to the development of graft-versus-host disease (GVHD). GVHD is characterized by activation, expansion, cytokine production and migration of alloreactive donor T cells, and remains a major cause of morbidity and mortality after allo-HCT. Hence, strategies to limit oxidative stress in GVHD are highly desirable. Thioredoxin1 (Trx1) counteracts oxidative stress by scavenging reactive oxygen species (ROS) and regulating other enzymes that metabolize H2O2. The present study sought to elucidate the role of Trx1 in the pathophysiology of GVHD. Using murine and xenograft models of allogeneic bone marrow transplantation (allo-BMT) and genetic (human Trx1-transgenic, Trx1-Tg) as well as pharmacologic (human recombinant Trx1, RTrx1) strategies; we found that Trx1-Tg donor T cells or administration of the recipients with RTrx1 significantly reduced GVHD severity. Mechanistically, we observed RTrx1 reduced ROS accumulation and cytokine production of mouse and human T cells in response to alloantigen stimulation in vitro. In allo-BMT settings, we found that Trx1-Tg or RTrx1 decreased downstream signaling molecules including NFκB activation and T-bet expression, and reduced proliferation, IFN-γ production and ROS accumulation in donor T cells within GVHD target organs. More importantly, administration of RTrx1 did not impair the graft-versus-leukemia (GVL) effect. Taken together, the current work provides a strong rationale and demonstrates feasibility to target the ROS pathway, which can be readily translated into clinic.

Receptor-interacting Ser/Thr kinase 1 (RIPK1) and myosin IIA-dependent ceramidosomes form membrane pores that mediate blebbing and necroptosis.

Formation of membrane pores/channels regulates various cellular processes, such as necroptosis or stem cell niche signaling. However, the roles of membrane lipids in the formation of pores and their biological functions are largely unknown. Here, using the cellular stress model evoked by the sphingolipid analog drug FTY720, we show that formation of ceramide-enriched membrane pores, referred to here as ceramidosomes, is initiated by a receptor-interacting Ser/Thr kinase 1 (RIPK1)-ceramide complex transported to the plasma membrane by nonmuscle myosin IIA-dependent trafficking in human lung cancer cells. Molecular modeling/simulation coupled with site-directed mutagenesis revealed that Asp147 or Asn169 of RIPK1 are key for ceramide binding and that Arg258 or Leu293 residues are involved in the myosin IIA interaction, leading to ceramidosome formation and necroptosis. Moreover, generation of ceramidosomes independently of any external drug/stress stimuli was also detected in the plasma membrane of germ line stem cells in ovaries during the early stages of oogenesis in Drosophila melanogaster Inhibition of ceramidosome formation via myosin IIA silencing limited germ line stem cell signaling and abrogated oogenesis. In conclusion, our findings indicate that the RIPK1-ceramide complex forms large membrane pores we named ceramidosomes. They further suggest that, in addition to their roles in stress-mediated necroptosis, these ceramide-enriched pores also regulate membrane integrity and signaling and might also play a role in D. melanogaster ovary development.

CD38-NAD+Axis Regulates Immunotherapeutic Anti-Tumor T Cell Response.

Heightened effector function and prolonged persistence, the key attributes of Th1 and Th17 cells, respectively, are key features of potent anti-tumor T cells. Here, we established ex vivo culture conditions to generate hybrid Th1/17 cells, which persisted long-term in vivo while maintaining their effector function. Using transcriptomics and metabolic profiling approaches, we showed that the enhanced anti-tumor property of Th1/17 cells was dependent on the increased NAD+-dependent activity of the histone deacetylase Sirt1. Pharmacological or genetic inhibition of Sirt1 activity impaired the anti-tumor potential of Th1/17 cells. Importantly, T cells with reduced surface expression of the NADase CD38 exhibited intrinsically higher NAD+, enhanced oxidative phosphorylation, higher glutaminolysis, and altered mitochondrial dynamics that vastly improved tumor control. Lastly, blocking CD38 expression improved tumor control even when using Th0 anti-tumor T cells. Thus, strategies targeting the CD38-NAD+ axis could increase the efficacy of anti-tumor adoptive T cell therapy.

S. pombe Uba1-Ubc15 Structure Reveals a Novel Regulatory Mechanism of Ubiquitin E2 Activity.

Ubiquitin (Ub) E1 initiates the Ub conjugation cascade by activating and transferring Ub to tens of different E2s. How Ub E1 cooperates with E2s that differ substantially in their predicted E1-interacting residues is unknown. Here, we report the structure of S. pombe Uba1 in complex with Ubc15, a Ub E2 with intrinsically low E1-E2 Ub thioester transfer activity. The structure reveals a distinct Ubc15 binding mode that substantially alters the network of interactions at the E1-E2 interface compared to the only other available Ub E1-E2 structure. Structure-function analysis reveals that the intrinsically low activity of Ubc15 largely results from the presence of an acidic residue at its N-terminal region. Notably, Ub E2 N termini are serine/threonine rich in many other Ub E2s, leading us to hypothesize that phosphorylation of these sites may serve as a novel negative regulatory mechanism of Ub E2 activity, which we demonstrate biochemically and in cell-based assays.

Sphingosine kinase/sphingosine 1-phosphate signaling in cancer therapeutics and drug resistance.

In this chapter, roles of bioactive sphingolipids, specifically sphingosine kinase 1 (SK1) and 2 (SK2) and their product-sphingosine 1-phosphate (S1P)-will be reviewed with respect to regulation of cancer growth, metastasis, chemotherapeutics, and drug resistance. Sphingolipids are known to be key bioeffector molecules that regulate cancer proliferation, angiogenesis, and cell death. Sphingolipid molecules such as ceramide and S1P have been shown to control cancer cell death and proliferation, respectively. Roles of S1P have been described with respect to their intracellular and extracellular pro-survival and drug resistance functions mostly through S1P receptor (S1PR1-5) engagement. Identification of novel intracellular SK/S1P targets has broadened the existing complex regulatory roles of bioactive sphingolipids in cancer pathogenesis and therapeutics. Thus, deciphering the biochemical and molecular regulation of SK/S1P/S1PR signaling could permit development of novel therapeutic interventions to improve cancer therapy and/or overcome drug resistance.

Ceramide targets autophagosomes to mitochondria and induces lethal mitophagy.

Mechanisms by which autophagy promotes cell survival or death are unclear. We provide evidence that C(18)-pyridinium ceramide treatment or endogenous C(18)-ceramide generation by ceramide synthase 1 (CerS1) expression mediates autophagic cell death, independent of apoptosis in human cancer cells. C(18)-ceramide-induced lethal autophagy was regulated via microtubule-associated protein 1 light chain 3 ß-lipidation, forming LC3B-II, and selective targeting of mitochondria by LC3B-II-containing autophagolysosomes (mitophagy) through direct interaction between ceramide and LC3B-II upon Drp1-dependent mitochondrial fission, leading to inhibition of mitochondrial function and oxygen consumption. Accordingly, expression of mutant LC3B with impaired ceramide binding, as predicted by molecular modeling, prevented CerS1-mediated mitochondrial targeting, recovering oxygen consumption. Moreover, knockdown of CerS1 abrogated sodium selenite-induced mitophagy, and stable LC3B knockdown protected against CerS1- and C(18)-ceramide-dependent mitophagy and blocked tumor suppression in vivo. Thus, these data suggest a new receptor function of ceramide for anchoring LC3B-II autophagolysosomes to mitochondrial membranes, defining a key mechanism for the induction of lethal mitophagy.

Communication between host organism and cancer cells is transduced by systemic sphingosine kinase 1/sphingosine 1-phosphate signalling to regulate tumour metastasis.

Mechanisms by which cancer cells communicate with the host organism to regulate lung colonization/metastasis are unclear. We show that this communication occurs via sphingosine 1-phosphate (S1P) generated systemically by sphingosine kinase 1 (SK1), rather than via tumour-derived S1P. Modulation of systemic, but not tumour SK1, prevented S1P elevation, and inhibited TRAMP-induced prostate cancer growth in TRAMP(+/+) SK1(-/-) mice, or lung metastasis of multiple cancer cells in SK1(-/-) animals. Genetic loss of SK1 activated a master metastasis suppressor, Brms1 (breast carcinoma metastasis suppressor 1), via modulation of S1P receptor 2 (S1PR2) in cancer cells. Alterations of S1PR2 using pharmacologic and genetic tools enhanced Brms1. Moreover, Brms1 in S1PR2(-/-) MEFs was modulated by serum S1P alterations. Accordingly, ectopic Brms1 in MB49 bladder cancer cells suppressed lung metastasis, and stable knockdown of Brms1 prevented this process. Importantly, inhibition of systemic S1P signalling using a novel anti-S1P monoclonal antibody (mAb), Sphingomab, attenuated lung metastasis, which was prevented by Brms1 knockdown in MB49 cells. Thus, these data suggest that systemic SK1/S1P regulates metastatic potential via regulation of tumour S1PR2/Brms1 axis.

Sphingosine kinase-1 and sphingosine 1-phosphate receptor 2 mediate Bcr-Abl1 stability and drug resistance by modulation of protein phosphatase 2A.

The mechanisms by which sphingosine kinase-1 (SK-1)/sphingosine 1-phosphate (S1P) activation contributes to imatinib resistance in chronic myeloid leukemia (CML) are unknown. We show herein that increased SK-1/S1P enhances Bcr-Abl1 protein stability, through inhibition of its proteasomal degradation in imatinib-resistant K562/IMA-3 and LAMA-4/IMA human CML cells. In fact, Bcr-Abl1 stability was enhanced by ectopic SK-1 expression. Conversely, siRNA-mediated SK-1 knockdown in K562/IMA-3 cells, or its genetic loss in SK-1(-/-) MEFs, significantly reduced Bcr-Abl1 stability. Regulation of Bcr-Abl1 by SK-1/S1P was dependent on S1P receptor 2 (S1P2) signaling, which prevented Bcr-Abl1 dephosphorylation, and degradation via inhibition of PP2A. Molecular or pharmacologic interference with SK-1/S1P2 restored PP2A-dependent Bcr-Abl1 dephosphorylation, and enhanced imatinib- or nilotinib-induced growth inhibition in primary CD34(+) mononuclear cells obtained from chronic phase and blast crisis CML patients, K562/IMA-3 or LAMA4/IMA cells, and 32Dcl3 murine progenitor cells, expressing the wild-type or mutant (Y253H or T315I) Bcr-Abl1 in situ. Accordingly, impaired SK-1/S1P2 signaling enhanced the growth-inhibitory effects of nilotinib against 32D/T315I-Bcr-Abl1-derived mouse allografts. Since SK-1/S1P/S1P2 signaling regulates Bcr-Abl1 stability via modulation of PP2A, inhibition of SK-1/S1P2 axis represents a novel approach to target wild-type- or mutant-Bcr-Abl1 thereby overcoming drug resistance.

Sphingolipids and cancer: ceramide and sphingosine-1-phosphate in the regulation of cell death and drug resistance.

Sphingolipids have emerged as bioeffector molecules, controlling various aspects of cell growth and proliferation in cancer, which is becoming the deadliest disease in the world. These lipid molecules have also been implicated in the mechanism of action of cancer chemotherapeutics. Ceramide, the central molecule of sphingolipid metabolism, generally mediates antiproliferative responses, such as cell growth inhibition, apoptosis induction, senescence modulation, endoplasmic reticulum stress responses and/or autophagy. Interestingly, recent studies suggest de novo-generated ceramides may have distinct and opposing roles in the promotion/suppression of tumors, and that these activities are based on their fatty acid chain lengths, subcellular localization and/or direct downstream targets. For example, in head and neck cancer cells, ceramide synthase 6/C(16)-ceramide addiction was revealed, and this was associated with increased tumor growth, whereas downregulation of its synthesis resulted in ER stress-induced apoptosis. By contrast, ceramide synthase 1-generated C(18)-ceramide has been shown to suppress tumor growth in various cancer models, both in situ and in vivo. In addition, ceramide metabolism to generate sphingosine-1-phosphate (S1P) by sphingosine kinases 1 and 2 mediates, with or without the involvement of G-protein-coupled S1P receptor signaling, prosurvival, angiogenesis, metastasis and/or resistance to drug-induced apoptosis. Importantly, recent findings regarding the mechanisms by which sphingolipid metabolism and signaling regulate tumor growth and progression, such as identifying direct intracellular protein targets of sphingolipids, have been key for the development of new chemotherapeutic strategies. Thus, in this article, we will present conclusions of recent studies that describe opposing roles of de novo-generated ceramides by ceramide synthases and/or S1P in the regulation of cancer pathogenesis, as well as the development of sphingolipid-based cancer therapeutics and drug resistance.