Expression and processing of mature human frataxin after gene therapy in mice.

Friedreich's ataxia is a degenerative and progressive multisystem disorder caused by mutations in the highly conserved frataxin (FXN) gene that results in FXN protein deficiency and mitochondrial dysfunction. While gene therapy approaches are promising, consistent induction of therapeutic FXN protein expression that is sub-toxic has proven challenging, and numerous therapeutic approaches are being tested in animal models. FXN (hFXN in humans, mFXN in mice) is proteolytically modified in mitochondria to produce mature FXN. However, unlike endogenous hFXN, endogenous mFXN is further processed into N-terminally truncated, extra-mitochondrial mFXN forms of unknown function. This study assessed mature exogenous hFXN expression levels in the heart and liver of C57Bl/6 mice 7-10 months after intravenous administration of a recombinant adeno-associated virus encoding hFXN (AAVrh.10hFXN) and examined the potential for hFXN truncation in mice. AAVrh.10hFXN induced dose-dependent expression of hFXN in the heart and liver. Interestingly, hFXN was processed into truncated forms, but found at lower levels than mature hFXN. However, the truncations were at different positions than mFXN. AAVrh.10hFXN induced mature hFXN expression in mouse heart and liver at levels that approximated endogenous mFXN levels. These results suggest that AAVrh.10hFXN can likely induce expression of therapeutic levels of mature hFXN in mice.

Ferroptosis and HMGB2 induced calreticulin translocation required for immunogenic cell death are controlled by the nuclear exporter XPO1.

Cisplatin and oxaliplatin cause the secretion of high mobility group box 1 (HMGB1) from cancer cells, which is necessary for initiation of immunogenic cell death (ICD). Calreticulin (CRT) translocation from the endoplasmic reticulum to the plasma membrane is also required; oxaliplatin induces this translocation but cisplatin does not. We have discovered that oxaliplatin causes the secretion of both HMGB1 and HMGB2 from the nucleus into the extracellular milieu. We previously showed that cisplatin mediated secretion of HMGB1 is controlled by the nuclear exporter XPO1 (chromosomal maintenance 1; CRM1). We now find that XPO1 regulates oxaliplatin mediated secretion of both HMGB1 and HMGB2. XPO1 inhibition causes nuclear accumulation of both proteins, inhibition of oxaliplatin-mediated ferroptosis of colon cancer cells, and inhibition of CRT translocation to the plasma membrane of lung and colon cancer cells. Incubation of cancer cells with cell targeted (CT)-HMGB2 confirmed that HMGB2 is responsible for translocation of CRT to the plasma membrane. CT-HMGB2 is three orders of magnitude more potent than oxaliplatin at inducing CRT translocation. Inhibition of HMGB1 and HMGB2 secretion and/or their activation of nuclear factor-kappa B (NF-kB) has potential utility for treating cardiovascular, and neurodegenerative diseases; whereas CT-HMGB2 could augment therapeutic approaches to cancer treatment.

Bile Acid Metabolism Mediates Cholesterol Homeostasis and Promotes Tumorigenesis in Clear Cell Renal Cell Carcinoma.

Clear cell renal cell carcinoma (ccRCC) incidence has risen steadily over the last decade. Elevated lipid uptake and storage is required for ccRCC cell viability. As stored cholesterol is the most abundant component in ccRCC intracellular lipid droplets, it may also play an important role in ccRCC cellular homeostasis. In support of this hypothesis, ccRCC cells acquire exogenous cholesterol through the high-density lipoprotein receptor SCARB1, inhibition or suppression of which induces apoptosis. Here, we showed that elevated expression of 3 beta-hydroxy steroid dehydrogenase type 7 (HSD3B7), which metabolizes cholesterol-derived oxysterols in the bile acid biosynthetic pathway, is also essential for ccRCC cell survival. Development of an HSD3B7 enzymatic assay and screening for small-molecule inhibitors uncovered the compound celastrol as a potent HSD3B7 inhibitor with low micromolar activity. Repressing HSD3B7 expression genetically or treating ccRCC cells with celastrol resulted in toxic oxysterol accumulation, impaired proliferation, and increased apoptosis in vitro and in vivo. These data demonstrate that bile acid synthesis regulates cholesterol homeostasis in ccRCC and identifies HSD3B7 as a plausible therapeutic target. SIGNIFICANCE: The bile acid biosynthetic enzyme HSD3B7 is essential for ccRCC cell survival and can be targeted to induce accumulation of cholesterol-derived oxysterols and apoptotic cell death.

Quantification of human mature frataxin protein expression in nonhuman primate hearts after gene therapy.

Deficiency in human mature frataxin (hFXN-M) protein is responsible for the devastating neurodegenerative and cardiodegenerative disease of Friedreich's ataxia (FRDA). It results primarily through epigenetic silencing of the FXN gene by GAA triplet repeats on intron 1 of both alleles. GAA repeat lengths are most commonly between 600 and 1200 but can reach 1700. A subset of approximately 3% of FRDA patients have GAA repeats on one allele and a mutation on the other. FRDA patients die most commonly in their 30s from heart disease. Therefore, increasing expression of heart hFXN-M using gene therapy offers a way to prevent early mortality in FRDA. We used rhesus macaque monkeys to test the pharmacology of an adeno-associated virus (AAV)hu68.CB7.hFXN therapy. The advantage of using non-human primates for hFXN-M gene therapy studies is that hFXN-M and monkey FXN-M (mFXN-M) are 98.5% identical, which limits potential immunologic side-effects. However, this presented a formidable bioanalytical challenge in quantification of proteins with almost identical sequences. This could be overcome by the development of a species-specific quantitative mass spectrometry-based method, which has revealed for the first time, robust transgene-specific human protein expression in monkey heart tissue. The dose response is non-linear resulting in a ten-fold increase in monkey heart hFXN-M protein expression with only a three-fold increase in dose of the vector.

Chimeric kinase ALK induces expression of NAMPT and selectively depends on this metabolic enzyme to sustain its own oncogenic function.

As we show in this study, NAMPT, the key rate-limiting enzyme in the salvage pathway, one of the three known pathways involved in NAD synthesis, is selectively over-expressed in anaplastic T-cell lymphoma carrying oncogenic kinase NPM1::ALK (ALK + ALCL). NPM1::ALK induces expression of the NAMPT-encoding gene with STAT3 acting as transcriptional activator of the gene. Inhibition of NAMPT affects ALK + ALCL cells expression of numerous genes, many from the cell-signaling, metabolic, and apoptotic pathways. NAMPT inhibition also functionally impairs the key metabolic and signaling pathways, strikingly including enzymatic activity and, hence, oncogenic function of NPM1::ALK itself. Consequently, NAMPT inhibition induces cell death in vitro and suppresses ALK + ALCL tumor growth in vivo. These results indicate that NAMPT is a novel therapeutic target in ALK + ALCL and, possibly, other similar malignancies. Targeting metabolic pathways selectively activated by oncogenic kinases to which malignant cells become "addicted" may become a novel therapeutic approach to cancer, alternative or, more likely, complementary to direct inhibition of the kinase enzymatic domain. This potential therapy to simultaneously inhibit and metabolically "starve" oncogenic kinases may not only lead to higher response rates but also delay, or even prevent, development of drug resistance, frequently seen when kinase inhibitors are used as single agents.

The androgen receptor.

The Androgen Receptor (AR) is a ligand (androgen) activated transcription factor and a member of the nuclear receptor (NR) superfamily. It is required for male sex hormone function. AR-FL (full-length) has the domain structure of NRs, an N-terminal domain (NTD) required for transactivation, a DNA-binding domain (DBD), a nuclear localization signal (NLS) and a ligand-binding domain (LBD). Paradoxes exist in that endogenous ligands testosterone (T) and 5α-dihydrotestosterone (DHT) have differential effects on male sexual development while binding to the same receptor and transcriptional specificity is achieved even though the androgen response elements (AREs) are identical to those seen for the progesterone, glucocorticoid and mineralocorticoid receptors. A high resolution 3-dimensional structure of AR-FL by either cryo-EM or X-ray crystallography has remained elusive largely due to the intrinsic disorder of the NTD. AR function is regulated by post-translational modification leading to a large number of proteoforms. The interaction of these proteoforms in multiprotein complexes with co-activators and co-repressors driven by interdomain coupling mediates the AR transcriptional output. The AR is a drug target for selective androgen receptor modulators (SARMS) that either have anabolic or androgenic effects. Protstate cancer is treated with androgen deprivation therapy or by the use of AR antagonists that bind to the LBD. Drug resistance occurs due to adaptive AR upregulation and the appearance of splice variants that lack the LBD and become constitutively active. Bipolar T treatment and NTD-antagonists could surmount these resistance mechanisms, respectively. These recent advances in AR signaling are described.

Cisplatin Dependent Secretion of Immunomodulatory High Mobility Group Box 1 (HMGB1) Protein from Lung Cancer Cells.

High mobility group box 1 (HMGB1) is secreted from activated immune cells, necrotic cells, and certain cancers. Previous studies have reported that different patterns of post-translational modification, particularly acetylation and oxidation, mediate HMGB1 release and confer distinct extracellular HMGB1 signaling activity. Here we report that cisplatin but not carboplatin induces secretion of HMGB1 from human A549 non-small cell lung cancer (NSCLC) cells. Cisplatin-mediated HMGB1 secretion was dose-dependent and was regulated by nuclear exportin 1 (XPO1) also known as chromosomal maintenance 1 (CRM1) rather than adenosine diphosphate (ADP)-ribosylation, acetylation, or oxidation. HMGB1, as well as lysine acetylation and cysteine disulfide oxidation of secreted HMGB1, were monitored by sensitive and specific assays using immunoprecipitation, stable isotope dilution, differential alkylation, and nano liquid chromatography parallel reaction monitoring/high-resolution mass spectrometry (nano-LC-PRM/HRMS). A major fraction of the HMGB1 secreted by low-dose cisplatin treatment of A549 NSCLC cells was found to be in the fully reduced form. In contrast, mainly oxidized forms of HMGB1 were secreted by dimethyl sulfoxide (DMSO)-mediated apoptosis. These findings suggest that inhibition of XPO1 could potentiate the anti-tumor activity of cisplatin by increasing the nuclear accumulation of HMGB1 protein, an inhibitor of cisplatin DNA-adduct repair. Furthermore, low-dose cisplatin therapy could modulate the immune response in NSCLC through the established chemokine activity of extracellular reduced HMGB1. This could potentially enhance the efficacy of subsequent immunotherapy treatment.

Quantification of human mature frataxin protein expression in nonhuman primate hearts after gene therapy.

Deficiency in human mature frataxin (hFXN-M) protein is responsible for the devastating neurodegenerative and cardiodegenerative disease of Friedreich's ataxia (FRDA). It results primarily by epigenetic silencing the FXN gene due to up to 1400 GAA triplet repeats in intron 1 of both alleles of the gene; a subset of approximately 3% of FRDA patients have a mutation on one allele. FRDA patients die most commonly in their 30s from heart disease. Therefore, increasing expression of heart hFXN-M using gene therapy offers a way to prevent early mortality in FRDA. We used rhesus macaque monkeys to test the pharmacology of an adeno-associated virus (AAV)hu68.CB7.hFXN therapy. The advantage of using non-human primates for hFXN-M gene therapy studies is that hFXN-M and monkey FXN-M (mFXN-M) are 98.5% identical, which limits potential immunologic side-effects. However, this presented a formidable bioanalytical challenge in quantification of proteins with almost identical sequences. This was overcome by development of a species-specific quantitative mass spectrometry-based method, which revealed for the first time, robust transgene-specific human protein expression in monkey heart tissue. The dose response was non-linear resulting in a ten-fold increase in monkey heart hFXN-M protein expression with only a three-fold increase in dose of the vector.

ALS-linked CCNF variant disrupts motor neuron ubiquitin homeostasis.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative disorders that share pathological features, including the aberrant accumulation of ubiquitinated protein inclusions within motor neurons. Previously, we have shown that the sequestration of ubiquitin (Ub) into inclusions disrupts Ub homeostasis in cells expressing ALS-associated variants superoxide dismutase 1 (SOD1), fused in sarcoma (FUS) and TAR DNA-binding protein 43 (TDP-43). Here, we investigated whether an ALS/FTD-linked pathogenic variant in the CCNF gene, encoding the E3 Ub ligase Cyclin F (CCNF), also perturbs Ub homeostasis. The presence of a pathogenic CCNF variant was shown to cause ubiquitin-proteasome system (UPS) dysfunction in induced pluripotent stem cell-derived motor neurons harboring the CCNF  S621G mutation. The expression of the CCNFS621G variant was associated with an increased abundance of ubiquitinated proteins and significant changes in the ubiquitination of key UPS components. To further investigate the mechanisms responsible for this UPS dysfunction, we overexpressed CCNF in NSC-34 cells and found that the overexpression of both wild-type (WT) and the pathogenic variant of CCNF (CCNFS621G) altered free Ub levels. Furthermore, double mutants designed to decrease the ability of CCNF to form an active E3 Ub ligase complex significantly improved UPS function in cells expressing both CCNFWT and the CCNFS621G variant and were associated with increased levels of free monomeric Ub. Collectively, these results suggest that alterations to the ligase activity of the CCNF complex and the subsequent disruption to Ub homeostasis play an important role in the pathogenesis of CCNF-associated ALS/FTD.

Expression and processing of mature human frataxin after gene therapy in mice.

Friedreich's ataxia is a degenerative and progressive multisystem disorder caused by mutations in the highly conserved frataxin (FXN) gene that results in FXN protein deficiency and mitochondrial dysfunction. While gene therapy approaches are promising, consistent induction of therapeutic FXN protein expression that is sub-toxic has proven challenging, and numerous therapeutic approaches are being tested in animal models. FXN (hFXN in humans, mFXN in mice) is proteolytically modified in mitochondria to produce mature FXN. However, unlike endogenous hFXN, endogenous mFXN is further processed into N-terminally truncated, extra-mitochondrial mFXN forms of unknown function. This study assessed mature exogenous hFXN expression levels in the heart and liver of C57Bl/6 mice 7-10 months after intravenous administration of a recombinant adeno-associated virus encoding hFXN (AAVrh.10hFXN) and examined the potential for hFXN truncation in mice. AAVrh.10hFXN induced dose-dependent expression of hFXN in the heart and liver. Interestingly, hFXN was processed into truncated forms, but found at lower levels than mature hFXN. However, the truncations were at different positions than mFXN. AAVrh.10hFXN induced mature hFXN expression in mouse heart and liver at levels that approximated endogenous mFXN levels. These results demonstrate that AAVrh.10hFXN may induce expression of therapeutic levels of mature hFXN in mice.

Ruxolitinib and exemestane for estrogen receptor positive, aromatase inhibitor resistant advanced breast cancer.

Circulating IL-6, an activator of JAK/STAT signaling, is associated with poor prognosis and aromatase inhibitor (AI) resistance in hormone-receptor positive (HR+) breast cancer. Here we report the results of a phase 2 single-arm Simon 2-stage trial combining Ruxolitinib, an oral selective inhibitor of JAK1/2, with exemestane, a steroidal AI, in patients with HR+ metastatic breast cancer (MBC) after progression on non-steroidal AI (NSAI). Safety and efficacy were primary objectives, and analysis of inflammatory markers as predictors of response was a key secondary objective. Twenty-five subjects enrolled. The combination of ruxolitinib and exemestane was safe, though anemia requiring transfusion in 5/15 (33%) at the 25 mg dose in stage 1 led to a reduction to 15 mg twice daily in stage 2 (with no additional transfusions). Clinical benefit rate (CBR) in the overall study population was 24% (95% CI 9.4-45.1); 6/25 patients demonstrated stable disease for ≥6 months. Median progression-free survival was 2.8 months (95% CI 2.6-3.9). Exploratory biomarkers revealed high levels of systemic inflammation and 60% harbored a high-risk IL-6 genotype. Pharmacodynamics demonstrated modest on-target inhibition of phosphorylated-STAT3 by ruxolitinib at a tolerable dose. Thus, ruxolitinib combined with exemestane at a tolerable dose was safe but minimally active in AI-resistant tumors of patients with high levels of systemic inflammation. These findings highlight the need for more potent and specific therapies targeting inflammation in MBC.

Characterization and Quantification of Oxidized High Mobility Group Box 1 Proteoforms Secreted from Hepatocytes by Toxic Levels of Acetaminophen.

The high mobility group box 1 (HMGB1), which is released during acute acetaminophen (APAP) overdose, is thought to mediate a subsequent immune response, particularly hepatic infiltration of macrophages. The redox behavior of HMGB1 and the proteoforms of HMGB1 present in oxidative environments has been the subject of a number of confusing and contradictory studies. Therefore, a stable isotope dilution two-dimensional nanoultrahigh-performance liquid chromatography parallel reaction monitoring/high-resolution mass spectrometry method was developed in order to characterize and quantify oxidative modifications to the cysteine (Cys) residues (Cys-23, Cys-45, and Cys-106) that are present in HMGB1. Disulfide linkages were determined using carbamidoethyl derivatization before and after reduction as well as by direct analysis of disulfide cross-linked peptides. A stable isotope labeled form of HMGB1 was used as an internal standard to correct for sample to sample differences in immunoaffinity precipitation, derivatization, and electrospray ionization. Four discrete HMGB1 proteoforms were found to be released from a hepatocarcinoma cell model of APAP overdose after 24 h. Fully reduced HMGB1 with all three Cys-residues in their free thiol state accounted for 18% of the secreted HMGB1. The proteoform with disulfide between Cys-23 and Cys-45 accounted for 24% of the HMGB1. No evidence was obtained for a disulfide cross-link between Cys-106 and the other two Cys-residues. However, 45% of the HMGB1 formed a cross-link with unidentified intracellular proteins via an intermolecular disulfide bond, and 12% was present as the terminally oxidized cysteic acid. Surprisingly, there was no evidence for the formation of HMGB1 disulfides with GSH or other low molecular weight thiols. Secreted plasma HMGB1 Cys-23/Cys45 disulfide proteoform together with the Cys-106/protein disulfide proteoforms could potentially serve as early biomarkers of hepatoxicity after APAP overdose as well as biomarkers of drug-induced liver injury.

Association between DNA methylation variability and self-reported exposure to heavy metals.

Individuals encounter varying environmental exposures throughout their lifetimes. Some exposures such as smoking are readily observed and have high personal recall; others are more indirect or sporadic and might only be inferred from long occupational histories or lifestyles. We evaluated the utility of using lifetime-long self-reported exposures for identifying differential methylation in an amyotrophic lateral sclerosis cases-control cohort of 855 individuals. Individuals submitted paper-based surveys on exposure and occupational histories as well as whole blood samples. Genome-wide DNA methylation levels were quantified using the Illumina Infinium Human Methylation450 array. We analyzed 15 environmental exposures using the OSCA software linear and MOA models, where we regressed exposures individually by methylation adjusted for batch effects and disease status as well as predicted scores for age, sex, cell count, and smoking status. We also regressed on the first principal components on clustered environmental exposures to detect DNA methylation changes associated with a more generalised definition of environmental exposure. Five DNA methylation probes across three environmental exposures (cadmium, mercury and metalwork) were significantly associated using the MOA models and seven through the linear models, with one additionally across a principal component representing chemical exposures. Methylome-wide significance for four of these markers was driven by extreme hyper/hypo-methylation in small numbers of individuals. The results indicate the potential for using self-reported exposure histories in detecting DNA methylation changes in response to the environment, but also highlight the confounded nature of environmental exposure in cohort studies.

NEK1 and STMN2 short tandem repeat lengths are not associated with Australian amyotrophic lateral sclerosis risk.

Sporadic amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with a complex genetic architecture. The lengths of two short tandem repeats (STRs), at the NEK1 and STMN2 loci, were recently associated with ALS risk in cohorts of European descent. The STMN2 STR was proposed to be predictive of clinical features including the age of onset and disease duration in bulbar onset cases. We sought to investigate NEK1 and STMN2 STR lengths in a cohort of Australian sporadic ALS cases (n = 608) and neurologically healthy controls (n = 4689) of European ancestry. ExpansionHunter was used to determine NEK1 and STMN2 STR length genotypes from whole-genome sequencing data followed by PCR validation of predicted lengths. No significant association was identified between sporadic ALS risk and the length of either STR. Further, neither NEK1 nor STMN2 STR lengths were indicative of the age of onset or disease duration. We report that the NEK1 and STMN2 STRs were not associated with ALS risk or clinical features in this Australian sporadic ALS cohort.

Bcl-xL Enforces a Slow-Cycling State Necessary for Survival in the Nutrient-Deprived Microenvironment of Pancreatic Cancer.

Solid tumors possess heterogeneous metabolic microenvironments where oxygen and nutrient availability are plentiful (fertile regions) or scarce (arid regions). While cancer cells residing in fertile regions proliferate rapidly, most cancer cells in vivo reside in arid regions and exhibit a slow-cycling state that renders them chemoresistant. Here, we developed an in vitro system enabling systematic comparison between these populations via transcriptome analysis, metabolomic profiling, and whole-genome CRISPR screening. Metabolic deprivation led to pronounced transcriptional and metabolic reprogramming, resulting in decreased anabolic activities and distinct vulnerabilities. Reductions in anabolic, energy-consuming activities, particularly cell proliferation, were not simply byproducts of the metabolic challenge, but rather essential adaptations. Mechanistically, Bcl-xL played a central role in the adaptation to nutrient and oxygen deprivation. In this setting, Bcl-xL protected quiescent cells from the lethal effects of cell-cycle entry in the absence of adequate nutrients. Moreover, inhibition of Bcl-xL combined with traditional chemotherapy had a synergistic antitumor effect that targeted cycling cells. Bcl-xL expression was strongly associated with poor patient survival despite being confined to the slow-cycling fraction of human pancreatic cancer cells. These findings provide a rationale for combining traditional cancer therapies that target rapidly cycling cells with those that target quiescent, chemoresistant cells associated with nutrient and oxygen deprivation. SIGNIFICANCE: The majority of pancreatic cancer cells inhabit nutrient- and oxygen-poor tumor regions and require Bcl-xL for their survival, providing a compelling antitumor metabolic strategy.

Glutamine deprivation triggers NAGK-dependent hexosamine salvage.

Tumors frequently exhibit aberrant glycosylation, which can impact cancer progression and therapeutic responses. The hexosamine biosynthesis pathway (HBP) produces uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), a major substrate for glycosylation in the cell. Prior studies have identified the HBP as a promising therapeutic target in pancreatic ductal adenocarcinoma (PDA). The HBP requires both glucose and glutamine for its initiation. The PDA tumor microenvironment is nutrient poor, however, prompting us to investigate how nutrient limitation impacts hexosamine synthesis. Here, we identify that glutamine limitation in PDA cells suppresses de novo hexosamine synthesis but results in increased free GlcNAc abundance. GlcNAc salvage via N-acetylglucosamine kinase (NAGK) is engaged to feed UDP-GlcNAc pools. NAGK expression is elevated in human PDA, and NAGK deletion from PDA cells impairs tumor growth in mice. Together, these data identify an important role for NAGK-dependent hexosamine salvage in supporting PDA tumor growth.

Inside tumors, cancer cells often have to compete with each other for food and other resources they need to survive. This is a key factor driving the growth and progression of cancer. One of the resources cells need is a molecule called UDP-GlcNAc, which they use to modify many proteins so they can work properly. Because cancer cells grow quickly, they likely need much more UDP-GlcNAc than healthy cells. Many tumors, including those derived from pancreatic cancers, have very poor blood supplies, so their cells cannot get the nutrients and other resources they need to grow from the bloodstream. This means that tumor cells have to find new ways to use what they already have. One example of this is developing alternative ways to obtain UDP-GlcNAc. Cells require a nutrient called glutamine to produce UDP-GlcNAc. Limiting the supply of glutamine to cells allows researchers to study how cells are producing UDP-GlcNAc in the lab. Campbell et al. used this approach to study how pancreatic cancer cells obtain UDP-GlcNAc when their access to glutamine is limited. They used a technique called isotope tracing, which allows researchers to track how a specific chemical is processed inside the cell, and what it turns into. The results showed that the pancreatic cancer cells do not make new UDP-GlcNAc but use a protein called NAGK to salvage GlcNAc (another precursor of UDP-GlcNAc), which may be obtained from cellular proteins. Cancer cells that lacked NAGK formed smaller tumors, suggesting that the cells grow more slowly because they cannot recycle UDP-GlcNAc fast enough. Pancreatic cancer is one of the most common causes of cancer deaths and is notable for being difficult to detect and treat. Campbell et al. have identified one of the changes that allows pancreatic cancers to survive and grow quickly. Next steps will include examining the role of NAGK in healthy cells and testing whether it could be targeted for cancer treatment.

Primary saturation of α, ß-unsaturated carbonyl containing fatty acids does not abolish electrophilicity.

Metabolism of polyunsaturated fatty acids results in the formation of hydroxylated fatty acids that can be further oxidized by dehydrogenases, often resulting in the formation of electrophilic, α,ß-unsaturated ketone containing fatty acids. As electrophiles are associated with redox signaling, we sought to investigate the metabolism of the oxo-fatty acid products in relation to their double bond architecture. Using an untargeted liquid chromatography mass spectrometry approach, we identified mono- and di-saturated products of the arachidonic acid-derived 11-oxoeicosatetraenoic acid (11-oxoETE) and mono-saturated metabolites of 15-oxoETE and docosahexaenoic acid-derived 17-oxodocosahexaenoinc acid (17-oxoDHA) in both human A549 lung carcinoma and umbilical vein endothelial cells. Notably, mono-saturated oxo-fatty acids maintained their electrophilicity as determined by nucleophilic conjugation to glutathione while a second saturation of 11-oxoETE resulted in a loss of electrophilicity. These results would suggest that prostaglandin reductase 1 (PTGR1), known only for its reduction of the α,ß-unsaturated double bond, was not responsible for the saturation of oxo-fatty acids at alternative double bonds. Surprisingly, knockdown of PTGR1 expression by shRNA confirmed its participation in the formation of 15-oxoETE and 17-oxoDHA mono-saturated metabolites. Furthermore, overexpression of PTGR1 in A549 cells increased the rate and total amount of oxo-fatty acid saturation. These findings will further facilitate the study of electrophilic fatty acid metabolism and signaling in the context of inflammatory diseases and cancer where they have been shown to have anti-inflammatory and anti-proliferative signaling properties.

Cholesterol Auxotrophy as a Targetable Vulnerability in Clear Cell Renal Cell Carcinoma.

Clear cell renal cell carcinoma (ccRCC) is characterized by large intracellular lipid droplets containing free and esterified cholesterol; however, the functional significance of cholesterol accumulation in ccRCC cells is unknown. We demonstrate that, surprisingly, genes encoding cholesterol biosynthetic enzymes are repressed in ccRCC, suggesting a dependency on exogenous cholesterol. Mendelian randomization analyses based on 31,000 individuals indicate a causal link between elevated circulating high-density lipoprotein (HDL) cholesterol and ccRCC risk. Depriving ccRCC cells of either cholesterol or HDL compromises proliferation and survival in vitro and tumor growth in vivo; in contrast, elevated dietary cholesterol promotes tumor growth. Scavenger Receptor B1 (SCARB1) is uniquely required for cholesterol import, and inhibiting SCARB1 is sufficient to cause ccRCC cell-cycle arrest, apoptosis, elevated intracellular reactive oxygen species levels, and decreased PI3K/AKT signaling. Collectively, we reveal a cholesterol dependency in ccRCC and implicate SCARB1 as a novel therapeutic target for treating kidney cancer. SIGNIFICANCE: We demonstrate that ccRCC cells are auxotrophic for exogenous cholesterol to maintain PI3K/AKT signaling pathway and ROS homeostasis. Blocking cholesterol import through the HDL transporter SCARB1 compromises ccRCC cell survival and tumor growth, suggesting a novel pharmacologic target for this disease. This article is highlighted in the In This Issue feature, p. 2945.

Splicing factor proline and glutamine rich intron retention, reduced expression and aggregate formation are pathological features of amyotrophic lateral sclerosis.

AIM: Splicing factor proline and glutamine rich (SFPQ) is an RNA-DNA binding protein that is dysregulated in Alzheimer's disease and frontotemporal dementia. Dysregulation of SFPQ, specifically increased intron retention and nuclear depletion, has been linked to several genetic subtypes of amyotrophic lateral sclerosis (ALS), suggesting that SFPQ pathology may be a common feature of this heterogeneous disease. Our study aimed to investigate this hypothesis by providing the first comprehensive assessment of SFPQ pathology in large ALS case-control cohorts. METHODS: We examined SFPQ at the RNA, protein and DNA levels. SFPQ RNA expression and intron retention were examined using RNA-sequencing and quantitative PCR. SFPQ protein expression was assessed by immunoblotting and immunofluorescent staining. At the DNA level, SFPQ was examined for genetic variation novel to ALS patients. RESULTS: At the RNA level, retention of SFPQ intron nine was significantly increased in ALS patients' motor cortex. In addition, SFPQ RNA expression was significantly reduced in the central nervous system, but not blood, of patients. At the protein level, neither nuclear depletion nor reduced expression of SFPQ was found to be a consistent feature of spinal motor neurons. However, SFPQ-positive ubiquitinated protein aggregates were observed in patients' spinal motor neurons. At the DNA level, our genetic screen identified two novel and two rare SFPQ sequence variants not previously reported in the literature. CONCLUSIONS: Our findings confirm dysregulation of SFPQ as a pathological feature of the central nervous system of ALS patients and indicate that investigation of the functional consequences of this pathology will provide insight into ALS biology.

Efficient Labeling of Native Human IgG by Proximity-Based Sortase-Mediated Isopeptide Ligation.

Antibody-drug conjugates (ADCs) have demonstrated great therapeutic potential due to their ability to target the delivery of potent cytotoxins. However, the heterogeneous nature of conventional drug conjugation strategies can affect the safety, efficacy, and stability of ADCs. Site-specific conjugations can resolve these issues, but often require genetic modification of Immunoglobulin G (IgG), which can impact yield or cost of production, or require undesirable chemical linkages. Here, we describe a near-traceless conjugation method that enables the efficient modification of native IgG, without the need for genetic engineering or glycan modification. This method utilizes engineered variants of sortase A to catalyze noncanonical isopeptide ligation. Sortase A was fused to an antibody-binding domain to improve ligation efficiency. Antibody labeling is limited to five lysine residues on the heavy chain and one on the light chain of human IgG1. The ADCs exhibit conserved antigen and Fc-receptor interactions, as well as potent cytolytic activity.