Multiplexed serum biomarkers to discriminate nonviable and ectopic pregnancy.

OBJECTIVE: To evaluate combinations of candidate biomarkers to develop a multiplexed prediction model for identifying the viability and location of an early pregnancy. In this study, we assessed 24 biomarkers with multiple machine learning-based methodologies to assess if multiplexed biomarkers may improve the diagnosis of normal and abnormal early pregnancies. DESIGN: A nested case-control design evaluated the predictive ability and discrimination of biomarkers in patients at risk of early pregnancy failure in the first trimester to classify viability and location. SETTING: Three university hospitals. PATIENTS: A total of 218 individuals with pain and/or bleeding in early pregnancy: 75 had an ongoing intrauterine gestation; 68 had ectopic pregnancies (EPs); and 75 had miscarriages. INTERVENTIONS: Serum levels of 24 biomarkers were assessed in the same patients. Multiple machine learning-based methodologies to evaluate combinations of these top candidates to develop a multiplexed prediction model for the identification of a nonviable pregnancy (ongoing intrauterine pregnancy vs. miscarriage or EP) and an EP (EP vs. ongoing intrauterine pregnancy or miscarriage). MAIN OUTCOME MEASURES: The predicted classification using each model was compared with the actual diagnosis, and sensitivity, specificity, positive predictive value, negative predictive value, conclusive classification, and accuracy were calculated. RESULTS: Models using classification regression tree analysis using 3 (pregnancy-specific beta-1-glycoprotein 3 [PSG3], chorionic gonadotropin-alpha subunit, and pregnancy-associated plasma protein-A) biomarkers were able to predict a maximum sensitivity of 93.3% and a maximum specificity of 98.6%. The model with the highest accuracy was 97.4% (with 70.2% receiving classification). Models using an overlapping group of 3 (soluble fms-like tyrosine kinase-1, PSG3, and tissue factor pathway inhibitor 2) biomarkers achieved a maximum sensitivity of 98.5% and a maximum specificity of 95.3%. The model with the highest accuracy was 94.4% (with 65.6% receiving classification). When the models were used simultaneously, the conclusive classification increased to 72.7% with an accuracy of 95.9%. The predictive ability of the biomarkers in the random forest produced similar test characteristics when using 11 predictive biomarkers. CONCLUSION: We have demonstrated a pool of biomarkers from divergent biological pathways that can be used to classify individuals with potential early pregnancy loss. The biomarkers choriogonadotropin alpha, pregnancy-associated plasma protein-A, and PSG3 can be used to predict viability, and soluble fms-like tyrosine kinase-1, tissue factor pathway inhibitor 2, and PSG3 can be used to predict pregnancy location.

TXNRD1 drives the innate immune response in senescent cells with implications for age-associated inflammation.

Sterile inflammation, also known as 'inflammaging', is a hallmark of tissue aging. Cellular senescence contributes to tissue aging, in part, through the secretion of proinflammatory factors collectively known as the senescence-associated secretory phenotype (SASP). The genetic variability of thioredoxin reductase 1 (TXNRD1) is associated with aging and age-associated phenotypes such as late-life survival, activity of daily living and physical performance in old age. TXNRD1's role in regulating tissue aging has been attributed to its enzymatic role in cellular redox regulation. Here, we show that TXNRD1 drives the SASP and inflammaging through the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) innate immune response pathway independently of its enzymatic activity. TXNRD1 localizes to cytoplasmic chromatin fragments and interacts with cGAS in a senescence-status-dependent manner, which is necessary for the SASP. TXNRD1 enhances the enzymatic activity of cGAS. TXNRD1 is required for both the tumor-promoting and immune surveillance functions of senescent cells, which are mediated by the SASP in vivo in mouse models. Treatment of aged mice with a TXNRD1 inhibitor that disrupts its interaction with cGAS, but not with an inhibitor of its enzymatic activity alone, downregulated markers of inflammaging in several tissues. In summary, our results show that TXNRD1 promotes the SASP through the innate immune response, with implications for inflammaging. This suggests that the TXNRD1-cGAS interaction is a relevant target for selectively suppressing inflammaging.

Evaluation of novel biomarkers for early pregnancy outcome prediction†.

OBJECTIVE: To assess performance and discriminatory capacity of commercially available enzyme-linked immunosorbent assays of biomarkers for predicting first trimester pregnancy outcome in a multi-center cohort. DESIGN: In a case-control study at three academic centers of women with pain and bleeding in early pregnancy, enzyme-linked immunosorbent assays of biomarkers were screened for assay performance. Performance was assessed via functional sensitivity, assay reportable range, recovery/linearity, and intra-assay precision (%Coefficient of Variation). Top candidates were analyzed for discriminatory capacity for viability and location among 210 women with tubal ectopic pregnancy, viable intrauterine pregnancy, or miscarriage. Assay discrimination was assessed by visual plots, area under the curve with 95% confidence intervals, and measures of central tendency with two-sample t-tests. RESULTS: Of 25 biomarkers evaluated, 22 demonstrated good or acceptable assay performance. Transgelin-2, oviductal glycoprotein, and integrin-linked kinase were rejected due to poor performance. The best biomarkers for discrimination of pregnancy location were pregnancy-specific beta-1-glycoprotein 9, pregnancy-specific beta-1-glycoprotein 1, insulin-like growth factor binding protein 1, kisspeptin (KISS1), pregnancy-specific beta-1-glycoprotein 3, and beta parvin (PARVB). The best biomarkers for discrimination of pregnancy viability were pregnancy-specific beta-1-glycoprotein 9, pregnancy-specific beta-1-glycoprotein 3, EH domain-containing protein 3, KISS1, WAP four-disulfide core domain protein 2 (HE4), quiescin sulfhydryl oxidase 2, and pregnancy-specific beta-1-glycoprotein 1. CONCLUSION: Performance of commercially available enzyme-linked immunosorbent assays was acceptable for a panel of novel biomarkers to predict early pregnancy outcome. Of these, six and seven candidates demonstrated good discriminatory capacity of pregnancy location and viability, respectively, when validated in a distinct external population. Four markers demonstrated good discrimination for both location and viability.

Identification and verification of plasma protein biomarkers that accurately identify an ectopic pregnancy.

BACKGROUND: Differentiating between a normal intrauterine pregnancy (IUP) and abnormal conditions including early pregnancy loss (EPL) or ectopic pregnancy (EP) is a major clinical challenge in early pregnancy. Currently, serial ß-human chorionic gonadotropin (ß-hCG) and progesterone are the most commonly used plasma biomarkers for evaluating pregnancy prognosis when ultrasound is inconclusive. However, neither biomarker can predict an EP with sufficient and reproducible accuracy. Hence, identification of new plasma biomarkers that can accurately diagnose EP would have great clinical value. METHODS: Plasma was collected from a discovery cohort of 48 consenting women having an IUP, EPL, or EP. Samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) followed by a label-free proteomics analysis to identify significant changes between pregnancy outcomes. A panel of 14 candidate biomarkers were then verified in an independent cohort of 74 women using absolute quantitation by targeted parallel reaction monitoring mass spectrometry (PRM-MS) which provided the capacity to distinguish between closely related protein isoforms. Logistic regression and Lasso feature selection were used to evaluate the performance of individual biomarkers and panels of multiple biomarkers to predict EP. RESULTS: A total of 1391 proteins were identified in an unbiased plasma proteome discovery. A number of significant changes (FDR ≤ 5%) were identified when comparing EP vs. non-EP (IUP + EPL). Next, 14 candidate biomarkers (ADAM12, CGA, CGB, ISM2, NOTUM, PAEP, PAPPA, PSG1, PSG2, PSG3, PSG9, PSG11, PSG6/9, and PSG8/1) were verified as being significantly different between EP and non-EP in an independent cohort (FDR ≤ 5%). Using logistic regression models, a risk score for EP was calculated for each subject, and four multiple biomarker logistic models were identified that performed similarly and had higher AUCs than models with single predictors. CONCLUSIONS: Overall, four multivariable logistic models were identified that had significantly better prediction of having EP than those logistic models with single biomarkers. Model 4 (NOTUM, PAEP, PAPPA, ADAM12) had the highest AUC (0.987) and accuracy (96%). However, because the models are statistically similar, all markers in the four models and other highly correlated markers should be considered in further validation studies.

Regulation of EBNA1 protein stability and DNA replication activity by PLOD1 lysine hydroxylase.

Epstein-Barr virus (EBV) is a ubiquitous human γ-herpesvirus that is causally associated with various malignancies and autoimmune disease. Epstein-Barr Nuclear Antigen 1 (EBNA1) is the viral-encoded DNA binding protein required for viral episome maintenance and DNA replication during latent infection in proliferating cells. EBNA1 is known to be a highly stable protein, but the mechanisms regulating protein stability and how this may be linked to EBNA1 function is not fully understood. Proteomic analysis of EBNA1 revealed interaction with Procollagen Lysine-2 Oxoglutarate 5 Dioxygenase (PLOD) family of proteins. Depletion of PLOD1 by shRNA or inhibition with small molecule inhibitors 2,-2' dipyridyl resulted in the loss of EBNA1 protein levels, along with a selective growth inhibition of EBV-positive lymphoid cells. PLOD1 depletion also caused a loss of EBV episomes from latently infected cells and inhibited oriP-dependent DNA replication. Mass spectrometry identified EBNA1 peptides with lysine hydroxylation at K460 or K461. Mutation of K460, but not K461 abrogates EBNA1-driven DNA replication of oriP, but did not significantly affect EBNA1 DNA binding. Mutations in both K460 and K461 perturbed interactions with PLOD1, as well as decreased EBNA1 protein stability. These findings suggest that PLOD1 is a novel interaction partner of EBNA1 that regulates EBNA1 protein stability and function in viral plasmid replication, episome maintenance and host cell survival.

Parkin ubiquitination of Kindlin-2 enables mitochondria-associated metastasis suppression.

Mitochondria are signaling organelles implicated in cancer, but the mechanisms are elusive. Here, we show that Parkin, an E3 ubiquitination (Ub) ligase altered in Parkinson's disease, forms a complex with the regulator of cell motility, Kindlin-2 (K2), at mitochondria of tumor cells. In turn, Parkin ubiquitinates Lys581 and Lys582 using Lys48 linkages, resulting in proteasomal degradation of K2 and shortened half-life from ∼5 h to ∼1.5 h. Loss of K2 inhibits focal adhesion turnover and ß1 integrin activation, impairs membrane lamellipodia size and frequency, and inhibits mitochondrial dynamics, altogether suppressing tumor cell-extracellular matrix interactions, migration, and invasion. Conversely, Parkin does not affect tumor cell proliferation, cell cycle transitions, or apoptosis. Expression of a Parkin Ub-resistant K2 Lys581Ala/Lys582Ala double mutant is sufficient to restore membrane lamellipodia dynamics, correct mitochondrial fusion/fission, and preserve single-cell migration and invasion. In a 3D model of mammary gland developmental morphogenesis, impaired K2 Ub drives multiple oncogenic traits of EMT, increased cell proliferation, reduced apoptosis, and disrupted basal-apical polarity. Therefore, deregulated K2 is a potent oncogene, and its Ub by Parkin enables mitochondria-associated metastasis suppression.

Lysosomal lipid peroxidation regulates tumor immunity.

Lysosomal inhibition elicited by palmitoyl-protein thioesterase 1 (PPT1) inhibitors such as DC661 can produce cell death, but the mechanism for this is not completely understood. Programmed cell death pathways (autophagy, apoptosis, necroptosis, ferroptosis, and pyroptosis) were not required to achieve the cytotoxic effect of DC661. Inhibition of cathepsins, or iron or calcium chelation, did not rescue DC661-induced cytotoxicity. PPT1 inhibition induced lysosomal lipid peroxidation (LLP), which led to lysosomal membrane permeabilization and cell death that could be reversed by the antioxidant N-acetylcysteine (NAC) but not by other lipid peroxidation antioxidants. The lysosomal cysteine transporter MFSD12 was required for intralysosomal transport of NAC and rescue of LLP. PPT1 inhibition produced cell-intrinsic immunogenicity with surface expression of calreticulin that could only be reversed with NAC. DC661-treated cells primed naive T cells and enhanced T cell-mediated toxicity. Mice vaccinated with DC661-treated cells engendered adaptive immunity and tumor rejection in "immune hot" tumors but not in "immune cold" tumors. These findings demonstrate that LLP drives lysosomal cell death, a unique immunogenic form of cell death, pointing the way to rational combinations of immunotherapy and lysosomal inhibition that can be tested in clinical trials.

Targeting UGCG Overcomes Resistance to Lysosomal Autophagy Inhibition.

Lysosomal autophagy inhibition (LAI) with hydroxychloroquine or DC661 can enhance cancer therapy, but tumor regrowth is common. To elucidate LAI resistance, proteomics and immunoblotting demonstrated that LAI induced lipid metabolism enzymes in multiple cancer cell lines. Lipidomics showed that LAI increased cholesterol, sphingolipids, and glycosphingolipids. These changes were associated with striking levels of GM1+ membrane microdomains (GMM) in plasma membranes and lysosomes. Inhibition of cholesterol/sphingolipid metabolism proteins enhanced LAI cytotoxicity. Targeting UDP-glucose ceramide glucosyltransferase (UGCG) synergistically augmented LAI cytotoxicity. Although UGCG inhibition decreased LAI-induced GMM and augmented cell death, UGCG overexpression led to LAI resistance. Melanoma patients with high UGCG expression had significantly shorter disease-specific survival. The FDA-approved UGCG inhibitor eliglustat combined with LAI significantly inhibited tumor growth and improved survival in syngeneic tumors and a therapy-resistant patient-derived xenograft. These findings nominate UGCG as a new cancer target, and clinical trials testing UGCG inhibition in combination with LAI are warranted. SIGNIFICANCE: We discovered UGCG-dependent lipid remodeling drives resistance to LAI. Targeting UGCG with a drug approved for a lysosomal storage disorder enhanced LAI antitumor activity without toxicity. LAI and UGCG inhibition could be tested clinically in multiple cancers. This article is highlighted in the In This Issue feature, p. 247.

Peroxynitrite in the tumor microenvironment changes the profile of antigens allowing escape from cancer immunotherapy.

Cancer immunotherapy often depends on recognition of peptide epitopes by cytotoxic T lymphocytes (CTLs). The tumor microenvironment (TME) is enriched for peroxynitrite (PNT), a potent oxidant produced by infiltrating myeloid cells and some tumor cells. We demonstrate that PNT alters the profile of MHC class I bound peptides presented on tumor cells. Only CTLs specific for PNT-resistant peptides have a strong antitumor effect in vivo, whereas CTLs specific for PNT-sensitive peptides are not effective. Therapeutic targeting of PNT in mice reduces resistance of tumor cells to CTLs. Melanoma patients with low PNT activity in their tumors demonstrate a better clinical response to immunotherapy than patients with high PNT activity. Our data suggest that intratumoral PNT activity should be considered for the design of neoantigen-based therapy and also may be an important immunotherapeutic target.

Circadian lncRNA ADIRF-AS1 binds PBAF and regulates renal clear cell tumorigenesis.

We identify ADIRF-AS1 circadian long non-coding RNA (lncRNA). Deletion of ADIRF-AS1 in U2OS cells alters rhythmicity of clock-controlled genes and expression of extracellular matrix genes. ADIRF-AS1 interacts with all components of the PBAF (PBRM1/BRG1) complex in U2OS cells. Because PBRM1 is a tumor suppressor mutated in over 40% of clear cell renal carcinoma (ccRCC) cases, we evaluate ADIRF-AS1 in ccRCC cells. Reducing ADIRF-AS1 expression in ccRCC cells decreases expression of some PBAF-suppressed genes. Expression of these genes is partially rescued by PBRM1 loss, consistent with ADIRF-AS1 acting in part to modulate PBAF. ADIRF-AS1 expression correlates with survival in human ccRCC, particularly in PBRM1 wild-type, but not mutant, tumors. Loss of ADIRF-AS1 eliminates in vivo tumorigenesis, partially rescued by concurrent loss of PBRM1 only when co-injected with Matrigel, suggesting a PBRM1-independent function of ADIRF-AS1. Our findings suggest that ADIRF-AS1 functions partly through PBAF to regulate specific genes as a BMAL1-CLOCK-regulated, oncogenic lncRNA.

HRS phosphorylation drives immunosuppressive exosome secretion and restricts CD8+ T-cell infiltration into tumors.

The lack of tumor infiltration by CD8+ T cells is associated with poor patient response to anti-PD-1 therapy. Understanding how tumor infiltration is regulated is key to improving treatment efficacy. Here, we report that phosphorylation of HRS, a pivotal component of the ESCRT complex involved in exosome biogenesis, restricts tumor infiltration of cytolytic CD8+ T cells. Following ERK-mediated phosphorylation, HRS interacts with and mediates the selective loading of PD-L1 to exosomes, which inhibits the migration of CD8+ T cells into tumors. In tissue samples from patients with melanoma, CD8+ T cells are excluded from the regions where tumor cells contain high levels of phosphorylated HRS. In murine tumor models, overexpression of phosphorylated HRS increases resistance to anti-PD-1 treatment, whereas inhibition of HRS phosphorylation enhances treatment efficacy. Our study reveals a mechanism by which phosphorylation of HRS in tumor cells regulates anti-tumor immunity by inducing PD-L1+ immunosuppressive exosomes, and suggests HRS phosphorylation blockade as a potential strategy to improve the efficacy of cancer immunotherapy.

Stromal changes in the aged lung induce an emergence from melanoma dormancy.

Disseminated cancer cells from primary tumours can seed in distal tissues, but may take several years to form overt metastases, a phenomenon that is termed tumour dormancy. Despite its importance in metastasis and residual disease, few studies have been able to successfully characterize dormancy within melanoma. Here we show that the aged lung microenvironment facilitates a permissive niche for efficient outgrowth of dormant disseminated cancer cells-in contrast to the aged skin, in which age-related changes suppress melanoma growth but drive dissemination. These microenvironmental complexities can be explained by the phenotype switching model, which argues that melanoma cells switch between a proliferative cell state and a slower-cycling, invasive state1-3. It was previously shown that dermal fibroblasts promote phenotype switching in melanoma during ageing4-8. We now identify WNT5A as an activator of dormancy in melanoma disseminated cancer cells within the lung, which initially enables the efficient dissemination and seeding of melanoma cells in metastatic niches. Age-induced reprogramming of lung fibroblasts increases their secretion of the soluble WNT antagonist sFRP1, which inhibits WNT5A in melanoma cells and thereby enables efficient metastatic outgrowth. We also identify the tyrosine kinase receptors AXL and MER as promoting a dormancy-to-reactivation axis within melanoma cells. Overall, we find that age-induced changes in distal metastatic microenvironments promote the efficient reactivation of dormant melanoma cells in the lung.

Protein Posttranslational Signatures Identified in COVID-19 Patient Plasma.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a highly contagious virus of the coronavirus family that causes coronavirus disease-19 (COVID-19) in humans and a number of animal species. COVID-19 has rapidly propagated in the world in the past 2 years, causing a global pandemic. Here, we performed proteomic analysis of plasma samples from COVID-19 patients compared to healthy control donors in an exploratory study to gain insights into protein-level changes in the patients caused by SARS-CoV-2 infection and to identify potential proteomic and posttranslational signatures of this disease. Our results suggest a global change in protein processing and regulation that occurs in response to SARS-CoV-2, and the existence of a posttranslational COVID-19 signature that includes an elevation in threonine phosphorylation, a change in glycosylation, and a decrease in arginylation, an emerging posttranslational modification not previously implicated in infectious disease. This study provides a resource for COVID-19 researchers and, longer term, and will inform our understanding of this disease and its treatment.

Ghost mitochondria drive metastasis through adaptive GCN2/Akt therapeutic vulnerability.

Cancer metabolism, including in mitochondria, is a disease hallmark and therapeutic target, but its regulation is poorly understood. Here, we show that many human tumors have heterogeneous and often reduced levels of Mic60, or Mitofilin, an essential scaffold of mitochondrial structure. Despite a catastrophic collapse of mitochondrial integrity, loss of bioenergetics, and oxidative damage, tumors with Mic60 depletion slow down cell proliferation, evade cell death, and activate a nuclear gene expression program of innate immunity and cytokine/chemokine signaling. In turn, this induces epithelial-mesenchymal transition (EMT), activates tumor cell movements through exaggerated mitochondrial dynamics, and promotes metastatic dissemination in vivo. In a small-molecule drug screen, compensatory activation of stress response (GCN2) and survival (Akt) signaling maintains the viability of Mic60-low tumors and provides a selective therapeutic vulnerability. These data demonstrate that acutely damaged, "ghost" mitochondria drive tumor progression and expose an actionable therapeutic target in metastasis-prone cancers.

Proteome-defined changes in cellular pathways for decidua and trophoblast tissues associated with location and viability of early-stage pregnancy.

BACKGROUND: In early pregnancy, differentiating between a normal intrauterine pregnancy (IUP) and abnormal gestations including early pregnancy loss (EPL) or ectopic pregnancy (EP) is a major clinical challenge when ultrasound is not yet diagnostic. Clinical treatments for these outcomes are drastically different making early, accurate diagnosis imperative. Hence, a greater understanding of the biological mechanisms involved in these early pregnancy complications could lead to new molecular diagnostics. METHODS: Trophoblast and endometrial tissue was collected from consenting women having an IUP (n = 4), EPL (n = 4), or EP (n = 2). Samples were analyzed by LC-MS/MS followed by a label-free proteomics analysis in an exploratory study. For each tissue type, pairwise comparisons of different pregnancy outcomes (EPL vs. IUP and EP vs. IUP) were performed, and protein changes having a fold change ≥ 3 and a Student's t-test p-value ≤ 0.05 were defined as significant. Pathway and network classification tools were used to group significantly changing proteins based on their functional similarities. RESULTS: A total of 4792 and 4757 proteins were identified in decidua and trophoblast proteomes. For decidua, 125 protein levels (2.6% of the proteome) were significantly different between EP and IUP, whereas EPL and IUP decidua were more similar with only 68 (1.4%) differences. For trophoblasts, there were 66 (1.4%) differences between EPL and IUP. However, the largest group of 344 differences (7.2%) was observed between EP and IUP trophoblasts. In both tissues, proteins associated with ECM remodeling, cell adhesion and metabolic pathways showed decreases in EP specimens compared with IUP and EPL. In trophoblasts, EP showed elevation of inflammatory and immune response pathways. CONCLUSIONS: Overall, differences between an EP and IUP are greater than the changes observed when comparing ongoing IUP and nonviable intrauterine pregnancies (EPL) in both decidua and trophoblast proteomes. Furthermore, differences between EP and IUP were much higher in the trophoblast than in the decidua. This observation is true for the total number of protein changes as well as the extent of changes in upstream regulators and related pathways. This suggests that biomarkers and mechanisms of trophoblast function may be the best predictors of early pregnancy location and viability.

Pyruvate dehydrogenase inactivation causes glycolytic phenotype in BAP1 mutant uveal melanoma.

Effective therapeutic options are still lacking for uveal melanoma (UM) patients who develop metastasis. Metastatic traits of UM are linked to BRCA1-associated protein 1 (BAP1) mutations. Cell metabolism is re-programmed in UM with BAP1 mutant UM, but the underlying mechanisms and opportunities for therapeutic intervention remain unclear. BAP1 mutant UM tumors have an elevated glycolytic gene expression signature, with increased expression of pyruvate dehydrogenase (PDH) complex and PDH kinase (PDHK1). Furthermore, BAP1 mutant UM cells showed higher levels of phosphorylated PDHK1 and PDH that was associated with an upregulated glycolytic profile compared to BAP1 wild-type UM cells. Suppressing PDHK1-PDH phosphorylation decreased glycolytic capacity and cell growth, and induced cell cycle arrest of BAP1 mutant UM cells. Our results suggest that PDHK1-PDH phosphorylation is a causative factor of glycolytic phenotypes found in BAP1 mutant UM and propose a therapeutic opportunity for BAP1 mutant UM patients.

Neural Crest-Like Stem Cell Transcriptome Analysis Identifies LPAR1 in Melanoma Progression and Therapy Resistance.

Metastatic melanoma is challenging to clinically address. Although standard-of-care targeted therapy has high response rates in patients with BRAF-mutant melanoma, therapy relapse occurs in most cases. Intrinsically resistant melanoma cells drive therapy resistance and display molecular and biologic properties akin to neural crest-like stem cells (NCLSC) including high invasiveness, plasticity, and self-renewal capacity. The shared transcriptional programs and vulnerabilities between NCLSCs and cancer cells remains poorly understood. Here, we identify a developmental LPAR1-axis critical for NCLSC viability and melanoma cell survival. LPAR1 activity increased during progression and following acquisition of therapeutic resistance. Notably, genetic inhibition of LPAR1 potentiated BRAFi ± MEKi efficacy and ablated melanoma migration and invasion. Our data define LPAR1 as a new therapeutic target in melanoma and highlights the promise of dissecting stem cell-like pathways hijacked by tumor cells. SIGNIFICANCE: This study identifies an LPAR1-axis critical for melanoma invasion and intrinsic/acquired therapy resistance.

A cancer ubiquitome landscape identifies metabolic reprogramming as target of Parkin tumor suppression.

Changes in metabolism that affect mitochondrial and glycolytic networks are hallmarks of cancer, but their impact in disease is still elusive. Using global proteomics and ubiquitome screens, we now show that Parkin, an E3 ubiquitin ligase and key effector of mitophagy altered in Parkinson's disease, shuts off mitochondrial dynamics and inhibits the non-oxidative phase of the pentose phosphate pathway. This blocks tumor cell movements, creates metabolic and oxidative stress, and inhibits primary and metastatic tumor growth. Uniformly down-regulated in cancer patients, Parkin tumor suppression requires its E3 ligase function, is reversed by antioxidants, and is independent of mitophagy. These data demonstrate that cancer metabolic networks are potent oncogenes directly targeted by endogenous tumor suppression.

Targeting glutamine dependence through GLS1 inhibition suppresses ARID1A-inactivated clear cell ovarian carcinoma.

Alterations in components of the SWI/SNF chromatin-remodeling complex occur in ~20% of all human cancers. For example, ARID1A is mutated in up to 62% of clear cell ovarian carcinoma (OCCC), a disease currently lacking effective therapies. Here we show that ARID1A mutation creates a dependence on glutamine metabolism. SWI/SNF represses glutaminase (GLS1) and ARID1A inactivation upregulates GLS1. ARID1A inactivation increases glutamine utilization and metabolism through the tricarboxylic acid cycle to support aspartate synthesis. Indeed, glutaminase inhibitor CB-839 suppresses the growth of ARID1A mutant, but not wildtype, OCCCs in both orthotopic and patient-derived xenografts. In addition, glutaminase inhibitor CB-839 synergizes with immune checkpoint blockade anti-PDL1 antibody in a genetic OCCC mouse model driven by conditional Arid1a inactivation. Our data indicate that pharmacological inhibition of glutaminase alone or in combination with immune checkpoint blockade represents an effective therapeutic strategy for cancers involving alterations in the SWI/SNF complex such as ARID1A mutations.