Biallelic NFATC1 mutations cause an inborn error of immunity with impaired CD8+ T-cell function and perturbed glycolysis.

The nuclear factor of activated T cells (NFAT) family of transcription factors plays central roles in adaptive immunity in murine models; however, their contribution to human immune homeostasis remains poorly defined. In a multigenerational pedigree, we identified 3 patients who carry germ line biallelic missense variants in NFATC1, presenting with recurrent infections, hypogammaglobulinemia, and decreased antibody responses. The compound heterozygous NFATC1 variants identified in these patients caused decreased stability and reduced the binding of DNA and interacting proteins. We observed defects in early activation and proliferation of T and B cells from these patients, amenable to rescue upon genetic reconstitution. Stimulation induced early T-cell activation and proliferation responses were delayed but not lost, reaching that of healthy controls at day 7, indicative of an adaptive capacity of the cells. Assessment of the metabolic capacity of patient T cells revealed that NFATc1 dysfunction rendered T cells unable to engage in glycolysis after stimulation, although oxidative metabolic processes were intact. We hypothesized that NFATc1-mutant T cells could compensate for the energy deficit due to defective glycolysis by using enhanced lipid metabolism as an adaptation, leading to a delayed, but not lost, activation responses. Indeed, we observed increased 13C-labeled palmitate incorporation into citrate, indicating higher fatty acid oxidation, and we demonstrated that metformin and rosiglitazone improved patient T-cell effector functions. Collectively, enabled by our molecular dissection of the consequences of loss-of-function NFATC1 mutations and extending the role of NFATc1 in human immunity beyond receptor signaling, we provide evidence of metabolic plasticity in the context of impaired glycolysis observed in patient T cells, alleviating delayed effector responses.

Protocols for Understanding the Redox Behavior of Copper-Containing Systems.

Suitability of single-reference density functional theory (DFT) methods for the calculation of redox potentials of copper-containing macrocycle complexes was confirmed by the use of T 1 diagnostics along with a verification of negligible spin contamination or wave function instability. When examining the effect of improvement in the cc-pVnZ basis set series on calculated redox potentials, the results readily converged at the cc-pVTZ level. The all-electron Def2-TZVPP basis set is shown to be a suitable choice of a basis set for the calculation of redox potentials when utilizing a cc-pVTZ geometry. The best-performing model chemistries are determined to be the M06/polarizable continuum model (PCM); therefore, a scheme for redox potential calculations of copper macrocycles using either M06/cc-pVTZ with PCM solvation is proposed to reliably reproduce experimental trends.

Multi-omics & pathway analysis identify potential roles for tumor N-acetyl aspartate accumulation in murine models of castration-resistant prostate cancer.

Castration-resistant prostate cancer (CRPC) is incurable and remains a significant worldwide challenge (Oakes and Papa, 2015). Matched untargeted multi-level omic datasets may reveal biological changes driving CRPC, identifying novel biomarkers and/or therapeutic targets. Untargeted RNA sequencing, proteomics, and metabolomics were performed on xenografts derived from three independent sets of hormone naive and matched CRPC human cell line models of local, lymph node, and bone metastasis grown as murine orthografts. Collectively, we tested the feasibility of muti-omics analysis on models of CRPC in revealing pathways of interest for future validation investigation. Untargeted metabolomics revealed NAA and NAAG commonly accumulating in CRPC across three independent models and proteomics showed upregulation of related enzymes, namely N-acetylated alpha-linked acidic dipeptidases (FOLH1/NAALADL2). Based on pathway analysis integrating multiple omic levels, we hypothesize that increased NAA in CRPC may be due to upregulation of NAAG hydrolysis via NAALADLases providing a pool of acetyl Co-A for upregulated sphingolipid metabolism and a pool of glutamate and aspartate for nucleotide synthesis during tumor growth.

THEM6-mediated reprogramming of lipid metabolism supports treatment resistance in prostate cancer.

Despite the clinical benefit of androgen-deprivation therapy (ADT), the majority of patients with advanced prostate cancer (PCa) ultimately develop lethal castration-resistant prostate cancer (CRPC). In this study, we identified thioesterase superfamily member 6 (THEM6) as a marker of ADT resistance in PCa. THEM6 deletion reduces in vivo tumour growth and restores castration sensitivity in orthograft models of CRPC. Mechanistically, we show that the ER membrane-associated protein THEM6 regulates intracellular levels of ether lipids and is essential to trigger the induction of the ER stress response (UPR). Consequently, THEM6 loss in CRPC cells significantly alters ER function, reducing de novo sterol biosynthesis and preventing lipid-mediated activation of ATF4. Finally, we demonstrate that high THEM6 expression is associated with poor survival and correlates with high levels of UPR activation in PCa patients. Altogether, our results highlight THEM6 as a novel driver of therapy resistance in PCa as well as a promising target for the treatment of CRPC.

Germline biallelic mutation affecting the transcription factor Helios causes pleiotropic defects of immunity.

Helios, a member of the Ikaros family of transcription factors, is predominantly expressed in developing thymocytes, activated T cells, and regulatory T cells (Tregs). Studies in mice have emphasized its role in maintenance of Treg immunosuppressive functions by stabilizing Foxp3 expression and silencing the Il2 locus. However, its contribution to human immune homeostasis and the precise mechanisms by which Helios regulates other T cell subsets remain unresolved. Here, we investigated a patient with recurrent respiratory infections and hypogammaglobulinemia and identified a germline homozygous missense mutation in IKZF2 encoding Helios (p.Ile325Val). We found that HeliosI325V retains DNA binding and dimerization properties but loses interaction with several partners, including epigenetic remodelers. Whereas patient Tregs showed increased IL-2 production, patient conventional T cells had decreased accessibility of the IL2 locus and consequently reduced IL-2 production. Reduced chromatin accessibility was not exclusive to the IL2 locus but involved a variety of genes associated with T cell activation. Single-cell RNA sequencing of peripheral blood mononuclear cells revealed gene expression signatures indicative of a shift toward a proinflammatory, effector-like status in patient CD8+ T cells. Moreover, patient CD4+ T cells exhibited a pronounced defect in proliferation with delayed expression of surface checkpoint inhibitors, suggesting an impaired onset of the T cell activation program. Collectively, we identified a previously uncharacterized, germline-encoded inborn error of immunity and uncovered a cell-specific defect in Helios-dependent epigenetic regulation. Binding of Helios with specific partners mediates this regulation, which is ultimately necessary for the transcriptional programs that enable T cell homeostasis in health and disease.

Landscape of Bone Marrow Metastasis in Human Neuroblastoma Unraveled by Transcriptomics and Deep Multiplex Imaging.

While the bone marrow attracts tumor cells in many solid cancers leading to poor outcome in affected patients, comprehensive analyses of bone marrow metastases have not been performed on a single-cell level. We here set out to capture tumor heterogeneity and unravel microenvironmental changes in neuroblastoma, a solid cancer with bone marrow involvement. To this end, we employed a multi-omics data mining approach to define a multiplex imaging panel and developed DeepFLEX, a pipeline for subsequent multiplex image analysis, whereby we constructed a single-cell atlas of over 35,000 disseminated tumor cells (DTCs) and cells of their microenvironment in the metastatic bone marrow niche. Further, we independently profiled the transcriptome of a cohort of 38 patients with and without bone marrow metastasis. Our results revealed vast diversity among DTCs and suggest that FAIM2 can act as a complementary marker to capture DTC heterogeneity. Importantly, we demonstrate that malignant bone marrow infiltration is associated with an inflammatory response and at the same time the presence of immuno-suppressive cell types, most prominently an immature neutrophil/granulocytic myeloid-derived suppressor-like cell type. The presented findings indicate that metastatic tumor cells shape the bone marrow microenvironment, warranting deeper investigations of spatio-temporal dynamics at the single-cell level and their clinical relevance.

18F-Fluciclovine PET metabolic imaging reveals prostate cancer tumour heterogeneity associated with disease resistance to androgen deprivation therapy.

BACKGROUND: Prostate cancer is highly prevalent worldwide. Androgen deprivation therapy (ADT) remains the treatment of choice for incurable prostate cancer, but majority of patients develop disease recurrence following ADT. There is therefore an urgent need for early detection of treatment resistance. METHODS: Isogenic androgen-responsive (CWR22Res) and castration-resistant (22Rv1) human prostate cancer cells were implanted into the anterior lobes of the prostate in CD-1 Nu mice to generate prostate orthografts. Castrated mice bearing CWR22Res and 22Rv1 orthografts mimic clinical prostate cancer following acute and chronic ADT, respectively. 18F-Fluciclovine (1-amino-3-fluorocyclobutane-1-carboxylic acid) with a radiochemical purity of > 99% was produced on a FASTlab synthesiser. Ki67 staining in endpoint orthografts was studied. Western blot, quantitative RT-PCR and next-generation sequencing transcriptomic analyses were performed to assess the expression levels of amino acid transporters (including LAT1 and ASCT2, which have been implicated for Fluciclovine uptake). Longitudinal metabolic imaging with 18F-Fluciclovine-based positron emission tomography (PET) was performed to study tumour response following acute and chronic ADT. RESULTS: Both immunohistochemistry analysis of endpoint prostate tumours and longitudinal 18F-Fluciclovine imaging revealed tumour heterogeneity, particularly following ADT, with in vivo 18F-Fluciclovine uptake correlating to viable cancer cells in both androgen-proficient and castrated environment. Highlighting tumour subpopulation following ADT, both SUVpeak and coefficient of variation (CoV) values of 18F-Fluciclovine uptake are consistent with tumour heterogeneity revealed by immunohistochemistry. We studied the expression of amino acid transporters (AATs) for 18F-Fluciclovine, namely LAT1 (SLC7A5 and SLC3A2) and ASCT2 (SLC1A5). SLC7A5 and SLC3A2 were expressed at relatively high levels in 22Rv1 castration-resistant orthografts following chronic ADT (modelling clinical castration-resistant disease), while SLC1A5 was preferentially expression in CWR22Res tumours following acute ADT. Additional AATs such as SLC43A2 (LAT4) were shown to be upregulated following chronic ADT by transcriptomic analysis; their role in Fluciclovine uptake warrants investigation. CONCLUSION: We studied in vivo 18F-Fluciclovine uptake in human prostate cancer orthograft models following acute and chronic ADT. 18F-Fluciclovine uptakes highlight tumour heterogeneity that may explain castration resistance and can be exploited as a clinical biomarker.

In vivo CRISPR/Cas9 knockout screen: TCEAL1 silencing enhances docetaxel efficacy in prostate cancer.

Docetaxel chemotherapy in metastatic prostate cancer offers only a modest survival benefit because of emerging resistance. To identify candidate therapeutic gene targets, we applied a murine prostate cancer orthograft model that recapitulates clinical invasive prostate cancer in a genome-wide CRISPR/Cas9 screen under docetaxel treatment pressure. We identified 17 candidate genes whose suppression may enhance the efficacy of docetaxel, with transcription elongation factor A-like 1 (Tceal1) as the top candidate. TCEAL1 function is not fully characterised; it may modulate transcription in a promoter dependent fashion. Suppressed TCEAL1 expression in multiple human prostate cancer cell lines enhanced therapeutic response to docetaxel. Based on gene set enrichment analysis from transcriptomic data and flow cytometry, we confirmed that loss of TCEAL1 in combination with docetaxel leads to an altered cell cycle profile compared with docetaxel alone, with increased subG1 cell death and increased polyploidy. Here, we report the first in vivo genome-wide treatment sensitisation CRISPR screen in prostate cancer, and present proof of concept data on TCEAL1 as a candidate for a combinational strategy with the use of docetaxel.

2,4-dienoyl-CoA reductase regulates lipid homeostasis in treatment-resistant prostate cancer.

Despite the clinical success of Androgen Receptor (AR)-targeted therapies, reactivation of AR signalling remains the main driver of castration-resistant prostate cancer (CRPC) progression. In this study, we perform a comprehensive unbiased characterisation of LNCaP cells chronically exposed to multiple AR inhibitors (ARI). Combined proteomics and metabolomics analyses implicate an acquired metabolic phenotype common in ARI-resistant cells and associated with perturbed glucose and lipid metabolism. To exploit this phenotype, we delineate a subset of proteins consistently associated with ARI resistance and highlight mitochondrial 2,4-dienoyl-CoA reductase (DECR1), an auxiliary enzyme of beta-oxidation, as a clinically relevant biomarker for CRPC. Mechanistically, DECR1 participates in redox homeostasis by controlling the balance between saturated and unsaturated phospholipids. DECR1 knockout induces ER stress and sensitises CRPC cells to ferroptosis. In vivo, DECR1 deletion impairs lipid metabolism and reduces CRPC tumour growth, emphasizing the importance of DECR1 in the development of treatment resistance.

Correction: BRF1 accelerates prostate tumourigenesis and perturbs immune infiltration.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

BRF1 accelerates prostate tumourigenesis and perturbs immune infiltration.

BRF1 is a rate-limiting factor for RNA Polymerase III-mediated transcription and is elevated in numerous cancers. Here, we report that elevated levels of BRF1 associate with poor prognosis in human prostate cancer. In vitro studies in human prostate cancer cell lines demonstrated that transient overexpression of BRF1 increased cell proliferation whereas the transient downregulation of BRF1 reduced proliferation and mediated cell cycle arrest. Consistent with our clinical observations, BRF1 overexpression in a Pten-deficient mouse (PtenΔ/Δ BRF1Tg) prostate cancer model accelerated prostate carcinogenesis and shortened survival. In PtenΔ/Δ BRF1Tg tumours, immune and inflammatory processes were altered, with reduced tumoral infiltration of neutrophils and CD4 positive T cells, which can be explained by decreased levels of complement factor D (CFD) and C7 components of the complement cascade, an innate immune pathway that influences the adaptive immune response. We tested if the secretome was involved in BRF1-driven tumorigenesis. Unbiased proteomic analysis on BRF1-overexpresing PC3 cells confirmed reduced levels of CFD in the secretome, implicating the complement system in prostate carcinogenesis. We further identify that expression of C7 significantly correlates with expression of CD4 and has the potential to alter clinical outcome in human prostate cancer, where low levels of C7 associate with poorer prognosis.

Activation of ß-Catenin Cooperates with Loss of Pten to Drive AR-Independent Castration-Resistant Prostate Cancer.

Inhibition of the androgen receptor (AR) is the main strategy to treat advanced prostate cancers. AR-independent treatment-resistant prostate cancer is a major unresolved clinical problem. Patients with prostate cancer with alterations in canonical WNT pathway genes, which lead to ß-catenin activation, are refractory to AR-targeted therapies. Here, using clinically relevant murine prostate cancer models, we investigated the significance of ß-catenin activation in prostate cancer progression and treatment resistance. ß-Catenin activation, independent of the cell of origin, cooperated with Pten loss to drive AR-independent castration-resistant prostate cancer. Prostate tumors with ß-catenin activation relied on the noncanonical WNT ligand WNT5a for sustained growth. WNT5a repressed AR expression and maintained the expression of c-Myc, an oncogenic effector of ß-catenin activation, by mediating nuclear localization of NFκBp65 and ß-catenin. Overall, WNT/ß-catenin and AR signaling are reciprocally inhibited. Therefore, inhibiting WNT/ß-catenin signaling by limiting WNT secretion in concert with AR inhibition may be useful for treating prostate cancers with alterations in WNT pathway genes. SIGNIFICANCE: Targeting of both AR and WNT/ß-catenin signaling may be required to treat prostate cancers that exhibit alterations of the WNT pathway.

Sprouty2 loss-induced IL6 drives castration-resistant prostate cancer through scavenger receptor B1.

Metastatic castration-resistant prostate cancer (mCRPC) is a lethal form of treatment-resistant prostate cancer and poses significant therapeutic challenges. Deregulated receptor tyrosine kinase (RTK) signalling mediated by loss of tumour suppressor Sprouty2 (SPRY2) is associated with treatment resistance. Using pre-clinical human and murine mCRPC models, we show that SPRY2 deficiency leads to an androgen self-sufficient form of CRPC Mechanistically, HER2-IL6 signalling axis enhances the expression of androgen biosynthetic enzyme HSD3B1 and increases SRB1-mediated cholesterol uptake in SPRY2-deficient tumours. Systemically, IL6 elevated the levels of circulating cholesterol by inducing host adipose lipolysis and hepatic cholesterol biosynthesis. SPRY2-deficient CRPC is dependent on cholesterol bioavailability and SRB1-mediated tumoral cholesterol uptake for androgen biosynthesis. Importantly, treatment with ITX5061, a clinically safe SRB1 antagonist, decreased treatment resistance. Our results indicate that cholesterol transport blockade may be effective against SPRY2-deficient CRPC.

Metformin selectively targets redox control of complex I energy transduction.

Many guanide-containing drugs are antihyperglycaemic but most exhibit toxicity, to the extent that only the biguanide metformin has enjoyed sustained clinical use. Here, we have isolated unique mitochondrial redox control properties of metformin that are likely to account for this difference. In primary hepatocytes and H4IIE hepatoma cells we found that antihyperglycaemic diguanides DG5-DG10 and the biguanide phenformin were up to 1000-fold more potent than metformin on cell signalling responses, gluconeogenic promoter expression and hepatocyte glucose production. Each drug inhibited cellular oxygen consumption similarly but there were marked differences in other respects. All diguanides and phenformin but not metformin inhibited NADH oxidation in submitochondrial particles, indicative of complex I inhibition, which also corresponded closely with dehydrogenase activity in living cells measured by WST-1. Consistent with these findings, in isolated mitochondria, DG8 but not metformin caused the NADH/NAD+ couple to become more reduced over time and mitochondrial deterioration ensued, suggesting direct inhibition of complex I and mitochondrial toxicity of DG8. In contrast, metformin exerted a selective oxidation of the mitochondrial NADH/NAD+ couple, without triggering mitochondrial deterioration. Together, our results suggest that metformin suppresses energy transduction by selectively inducing a state in complex I where redox and proton transfer domains are no longer efficiently coupled.

General Force-Field Parametrization Scheme for Molecular Dynamics Simulations of Conjugated Materials in Solution.

We describe a general scheme to obtain force-field parameters for classical molecular dynamics simulations of conjugated polymers. We identify a computationally inexpensive methodology for calculation of accurate intermonomer dihedral potentials and partial charges. Our findings indicate that the use of a two-step methodology of geometry optimization and single-point energy calculations using DFT methods produces potentials which compare favorably to high level theory calculation. We also report the effects of varying the conjugated backbone length and alkyl side-chain lengths on the dihedral profiles and partial charge distributions and determine the existence of converged lengths above which convergence is achieved in the force-field parameter sets. We thus determine which calculations are required for accurate parametrization and the scope of a given parameter set for variations to a given molecule. We perform simulations of long oligomers of dioctylfluorene and hexylthiophene in explicit solvent and find peristence lengths and end-length distributions consistent with experimental values.

Sleeping Beauty screen reveals Pparg activation in metastatic prostate cancer.

Prostate cancer (CaP) is the most common adult male cancer in the developed world. The paucity of biomarkers to predict prostate tumor biology makes it important to identify key pathways that confer poor prognosis and guide potential targeted therapy. Using a murine forward mutagenesis screen in a Pten-null background, we identified peroxisome proliferator-activated receptor gamma (Pparg), encoding a ligand-activated transcription factor, as a promoter of metastatic CaP through activation of lipid signaling pathways, including up-regulation of lipid synthesis enzymes [fatty acid synthase (FASN), acetyl-CoA carboxylase (ACC), ATP citrate lyase (ACLY)]. Importantly, inhibition of PPARG suppressed tumor growth in vivo, with down-regulation of the lipid synthesis program. We show that elevated levels of PPARG strongly correlate with elevation of FASN in human CaP and that high levels of PPARG/FASN and PI3K/pAKT pathway activation confer a poor prognosis. These data suggest that CaP patients could be stratified in terms of PPARG/FASN and PTEN levels to identify patients with aggressive CaP who may respond favorably to PPARG/FASN inhibition.

Biomolecular mode of action of metformin in relation to its copper binding properties.

Metformin (Metf), the most commonly used type 2 diabetes drug, is known to affect the cellular housekeeping of copper. Recently, we discovered that the structurally closely related propanediimidamide (PDI) shows a cellular behavior different from that of Metf. Here we investigate the binding of these compounds to copper, to compare their binding strength. Furthermore, we take a closer look at the electronic properties of these compounds and their copper complexes such as molecular orbital interactions and electrostatic potential surfaces. Our results clearly show that the copper binding energies cannot alone be the cause of the biochemical differentiation between Metf and PDI. We conclude that other factors such as pKa values and hydrophilicity of the compounds play a crucial role in their cellular activity. Metf in contrast to PDI can occur as an anion in aqueous medium at moderate pH, forming much stronger complexes particularly with Cu(II) ions, suggesting that biguanides but not PDI may induce easy oxidation of Cu(I) ions extracted from proteins. The higher hydrophobicity and the lack of planarity of PDI may further differentiate it from biguanides in terms of their molecular recognition characteristics. These different properties could hold the key to metformin's mitochondrial activity because they suggest that the drug could act at least in part as a pro-oxidant of accessible protein-bound Cu(I) ions.