Sphingolipids inhibit endosomal recycling of nutrient transporters by inactivating ARF6.

Endogenous sphingolipids (ceramide) and related synthetic molecules (FTY720, SH-BC-893) reduce nutrient access by decreasing cell surface expression of a subset of nutrient transporter proteins. Here, we report that these sphingolipids disrupt endocytic recycling by inactivating the small GTPase ARF6. Consistent with reported roles for ARF6 in maintaining the tubular recycling endosome, MICAL-L1-positive tubules were lost from sphingolipid-treated cells. We propose that ARF6 inactivation may occur downstream of PP2A activation since: (1) sphingolipids that fail to activate PP2A did not reduce ARF6-GTP levels; (2) a structurally unrelated PP2A activator disrupted tubular recycling endosome morphology and transporter localization; and (3) overexpression of a phosphomimetic mutant of the ARF6 GEF GRP1 prevented nutrient transporter loss. ARF6 inhibition alone was not toxic; however, the ARF6 inhibitors SecinH3 and NAV2729 dramatically enhanced the killing of cancer cells by SH-BC-893 without increasing toxicity to peripheral blood mononuclear cells, suggesting that ARF6 inactivation contributes to the anti-neoplastic actions of sphingolipids. Taken together, these studies provide mechanistic insight into how ceramide and sphingolipid-like molecules limit nutrient access and suppress tumor cell growth and survival

Multiorgan MRI findings after hospitalisation with COVID-19 in the UK (C-MORE): a prospective, multicentre, observational cohort study

Introduction: The multiorgan impact of moderate to severe coronavirus infections in the post-acute phase is still poorly understood. We aimed to evaluate the excess burden of multiorgan abnormalities after hospitalisation with COVID-19, evaluate their determinants, and explore associations with patient-related outcome measures. Methods: In a prospective, UK-wide, multicentre MRI follow-up study (C-MORE), adults (aged ≥18 years) discharged from hospital following COVID-19 who were included in Tier 2 of the Post-hospitalisation COVID-19 study (PHOSP-COVID) and contemporary controls with no evidence of previous COVID-19 (SARS-CoV-2 nucleocapsid antibody negative) underwent multiorgan MRI (lungs, heart, brain, liver, and kidneys) with quantitative and qualitative assessment of images and clinical adjudication when relevant. Individuals with end-stage renal failure or contraindications to MRI were excluded. Participants also underwent detailed recording of symptoms, and physiological and biochemical tests. The primary outcome was the excess burden of multiorgan abnormalities (two or more organs) relative to controls, with further adjustments for potential confounders. The C-MORE study is ongoing and is registered with ClinicalTrials.gov, NCT04510025. Findings: Of 2710 participants in Tier 2 of PHOSP-COVID, 531 were recruited across 13 UK-wide C-MORE sites. After exclusions, 259 C-MORE patients (mean age 57 years [SD 12]; 158 [61%] male and 101 [39%] female) who were discharged from hospital with PCR-confirmed or clinically diagnosed COVID-19 between March 1, 2020, and Nov 1, 2021, and 52 non-COVID-19 controls from the community (mean age 49 years [SD 14]; 30 [58%] male and 22 [42%] female) were included in the analysis. Patients were assessed at a median of 5·0 months (IQR 4·2–6·3) after hospital discharge. Compared with non-COVID-19 controls, patients were older, living with more obesity, and had more comorbidities. Multiorgan abnormalities on MRI were more frequent in patients than in controls (157 [61%] of 259 vs 14 [27%] of 52; p<0·0001) and independently associated with COVID-19 status (odds ratio [OR] 2·9 [95% CI 1·5–5·8]; padjusted=0·0023) after adjusting for relevant confounders. Compared with controls, patients were more likely to have MRI evidence of lung abnormalities (p=0·0001; parenchymal abnormalities), brain abnormalities (p<0·0001; more white matter hyperintensities and regional brain volume reduction), and kidney abnormalities (p=0·014; lower medullary T1 and loss of corticomedullary differentiation), whereas cardiac and liver MRI abnormalities were similar between patients and controls. Patients with multiorgan abnormalities were older (difference in mean age 7 years [95% CI 4–10]; mean age of 59·8 years [SD 11·7] with multiorgan abnormalities vs mean age of 52·8 years [11·9] without multiorgan abnormalities; p<0·0001), more likely to have three or more comorbidities (OR 2·47 [1·32–4·82]; padjusted=0·0059), and more likely to have a more severe acute infection (acute CRP >5mg/L, OR 3·55 [1·23–11·88]; padjusted=0·025) than those without multiorgan abnormalities. Presence of lung MRI abnormalities was associated with a two-fold higher risk of chest tightness, and multiorgan MRI abnormalities were associated with severe and very severe persistent physical and mental health impairment (PHOSP-COVID symptom clusters) after hospitalisation. Interpretation: After hospitalisation for COVID-19, people are at risk of multiorgan abnormalities in the medium term. Our findings emphasise the need for proactive multidisciplinary care pathways, with the potential for imaging to guide surveillance frequency and therapeutic stratification

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Targeting cancer metabolism at the plasma membrane by limiting amino acid access through SLC6A14.

Rapidly proliferating cancer cells increase flux through anabolic pathways to build the mass necessary to support cell division. Imported amino acids and glucose lie at the apex of the anabolic pyramid. Consistent with this, elevated expression of nutrient transporter proteins is characteristic of aggressive and highly malignant cancers. Because tumour cells are more dependent than their normal neighbours on accelerated nutrient import, these up-regulated transporters could be excellent targets for selective anti-cancer therapies. A study by Babu et al. in a recent issue of the Biochemical Journal definitively shows that SLC6A14 (where SLC is solute carrier) is one such cancer-specific amino acid transporter. Although mice completely lacking SLC6A14 are viable and exhibit normal mammary gland development, these animals are highly resistant to mammary tumour initiation and progression driven by potent oncogenes. Because SLC6A14 is essential for tumour growth yet dispensable for normal development and tissue maintenance, small molecules that block amino acid import through this transporter could be effective and selective anti-cancer agents, particularly as components of rational drug combinations

Design, Synthesis, and Anti-leukemic Activity of Stereochemically Defined Constrained Analogs of FTY720 (Gilenya).

FTY720 functions as an immunosuppressant due to its effect on sphingosine-1-phosphate receptors. At doses well above those needed for immunosuppression, FTY720 also has anti-neoplastic actions. Our published work suggests that at least some of FTY720's anti-cancer activity is independent of its effects on S1P receptors and due instead to its ability to induce nutrient transporter down-regulation. Compounds that trigger nutrient transporter loss but lack FTY720's S1P receptor-related, dose-limiting toxicity have the potential to be effective and selective anti-tumor agents. In this study, a series of enantiomerically pure and stereochemically diverse O-substituted benzyl ethers of pyrrolidines was generated and tested for the ability to kill human leukemia cells. The stereochemistry of the hydroxymethyl was found to be a key determinant of compound activity. Moreover, phosphorylation of this group was not required for anti-leukemic activity

In search of constrained FTY720 and phytosphingosine analogs as dual acting anticancer agents targeting metabolic and epigenetic pathways.

A series of compounds containing pyrrolidine and pyrrolizidine cores with appended hydrophobic substituents were prepared as constrained analogs of FTY720 and phytosphingosine. The effect of these compounds on the viability of cancer cells, on downregulation of the nutrient transport systems, and on their ability to cause vacuolation was studied. An attempt to inhibit HDACs with some phosphate esters of our analogs was thwarted by our failure to reproduce the reported inhibitory action of FTY720-phosphate

Dynamic Phosphoproteomics Uncovers Signaling Pathways Modulated by Anti-oncogenic Sphingolipid Analogs.

The anti-neoplastic sphingolipid analog SH-BC-893 starves cancer cells to death by down-regulating cell surface nutrient transporters and blocking lysosomal trafficking events. These effects are mediated by the activation of protein phosphatase 2A (PP2A). To identify putative PP2A substrates, we used quantitative phosphoproteomics to profile the temporal changes in protein phosphorylation in FL5.12 cells following incubation with SH-BC-893 or the specific PP2A inhibitor LB-100. These analyses enabled the profiling of more than 15,000 phosphorylation sites, of which 958 sites on 644 proteins were dynamically regulated. We identified 114 putative PP2A substrates including several nutrient transporter proteins, GTPase regulators (e.g. Agap2, Git1), and proteins associated with actin cytoskeletal remodeling (e.g. Vim, Pxn). To identify SH-BC-893-induced cell signaling events that disrupt lysosomal trafficking, we compared phosphorylation profiles in cells treated with SH-BC-893 or C2-ceramide, a non-vacuolating sphingolipid that does not impair lysosomal fusion. These analyses combined with functional assays uncovered the differential regulation of Akt and Gsk3b by SH-BC-893 (vacuolating) and C2-ceramide (non-vacuolating). Dynamic phosphoproteomics of cells treated with compounds affecting PP2A activity thus enabled the correlation of cell signaling with phenotypes to rationalize their mode of action

Design, Synthesis, and Antileukemic Activity of Stereochemically Defined Constrained Analogues of FTY720 (Gilenya)

FTY720 functions as an immunosuppressant due to its effect on sphingosine-1-phosphate receptors. At doses well above those needed for immunosuppression, FTY720 also has antineoplastic actions. Our published work suggests that at least some of FTY720’s anticancer activity is independent of its effects on S1P receptors and due instead to its ability to induce nutrient transporter down-regulation. Compounds that trigger nutrient transporter loss but lack FTY720’s S1P receptor-related, dose-limiting toxicity have the potential to be effective and selective antitumor agents. In this study, a series of enantiomerically pure and stereochemically diverse <i>O</i>-substituted benzyl ethers of pyrrolidines was generated and tested for the ability to kill human leukemia cells. The stereochemistry of the hydroxymethyl was found to be a key determinant of compound activity. Moreover, phosphorylation of this group was not required for antileukemic activity