Structure-guided optimization of 3-hydroxybenzoisoxazole derivatives as inhibitors of Aldo-keto reductase 1C3 (AKR1C3) to target prostate cancer.

AKR1C3 is an enzyme that is overexpressed in several types of radiotherapy- and chemotherapy-resistant cancers. Despite AKR1C3 is a validated target for drug development, no inhibitor has been approved for clinical use. In this manuscript, we describe our study of a new series of potent AKR1C3-targeting 3-hydroxybenzoisoxazole based inhibitors that display high selectivity over the AKR1C2 isoform and low micromolar activity in inhibiting 22Rv1 prostate cancer cell proliferation. In silico studies suggested proper substituents to increase compound potency and provided with a mechanistic explanation that could clarify their different activity, later confirmed by X-ray crystallography. Both the in-silico studies and the crystallographic data highlight the importance of 90° rotation around the single bond of the biphenyl group, in ensuring that the inhibitor can adopt the optimal binding mode within the active pocket. The p-biphenyls that bear the meta-methoxy, and the ortho- and meta-trifluoromethyl substituents (in compounds 6a, 6e and 6f respectively) proved to be the best contributors to cellular potency as they provided the best IC50 values in series (2.3, 2.0 and 2.4 µM respectively) and showed no toxicity towards human MRC-5 cells. Co-treatment with scalar dilutions of either compound 6 or 6e and the clinically used drug abiraterone led to a significant reduction in cell proliferation, and thus confirmed that treatment with both CYP171A1-and AKR1C3-targeting compounds possess the potential to intervene in key steps in the steroidogenic pathway. Taken together, the novel compounds display desirable biochemical potency and cellular target inhibition as well as good in-vitro ADME properties, which highlight their potential for further preclinical studies.

Development and validation of a clinical decision support system based on PSA, microRNAs, and MRI for the detection of prostate cancer.

OBJECTIVES: The aims of this study are to develop and validate a clinical decision support system based on demographics, prostate-specific antigen (PSA), microRNA (miRNA), and MRI for the detection of prostate cancer (PCa) and clinical significant (cs) PCa, and to assess if this system performs better compared to MRI alone. METHODS: This retrospective, multicenter, observational study included 222 patients (mean age 66, range 46-75 years) who underwent prostate MRI, miRNA (let-7a-5p and miR-103a-3p) assessment, and biopsy. Monoparametric and multiparametric models including age, PSA, miRNA, and MRI outcome were trained on 65% of the data and then validated on the remaining 35% to predict both PCa (any Gleason grade [GG]) and csPCa (GG ≥ 2 vs GG = 1/negative). Accuracy, sensitivity, specificity, positive and negative predictive value (NPV), and area under the receiver operating characteristic curve were calculated. RESULTS: MRI outcome was the best predictor in the monoparametric model for both detection of PCa, with sensitivity of 90% (95%CI 73-98%) and NPV of 93% (95%CI 82-98%), and for csPCa identification, with sensitivity of 91% (95%CI 72-99%) and NPV of 95% (95%CI 84-99%). Sensitivity and NPV of PSA + miRNA for the detection of csPCa were not statistically different from the other models including MRI alone. CONCLUSION: MRI stand-alone yielded the best prediction models for both PCa and csPCa detection in biopsy-naïve patients. The use of miRNAs let-7a-5p and miR-103a-3p did not improve classification performances compared to MRI stand-alone results. CLINICAL RELEVANCE STATEMENT: The use of miRNA (let-7a-5p and miR-103a-3p), PSA, and MRI in a clinical decision support system (CDSS) does not improve MRI stand-alone performance in the detection of PCa and csPCa. KEY POINTS: • Clinical decision support systems including MRI improve the detection of both prostate cancer and clinically significant prostate cancer with respect to PSA test and/or microRNA. • The use of miRNAs let-7a-5p and miR-103a-3p did not significantly improve MRI stand-alone performance. • Results of this study were in line with previous works on MRI and microRNA.

Androgen receptor is a determinant of melanoma targeted drug resistance.

Melanoma provides a primary benchmark for targeted drug therapy. Most melanomas with BRAFV600 mutations regress in response to BRAF/MEK inhibitors (BRAFi/MEKi). However, nearly all relapse within the first two years, and there is a connection between BRAFi/MEKi-resistance and poor response to immune checkpoint therapy. We reported that androgen receptor (AR) activity is required for melanoma cell proliferation and tumorigenesis. We show here that AR expression is markedly increased in BRAFi-resistant melanoma cells, and in sensitive cells soon after BRAFi exposure. Increased AR expression is sufficient to render melanoma cells BRAFi-resistant, eliciting transcriptional changes of BRAFi-resistant subpopulations, including elevated EGFR and SERPINE1 expression, of likely clinical significance. Inhibition of AR expression or activity blunts changes in gene expression and suppresses proliferation and tumorigenesis of BRAFi-resistant melanoma cells, promoting clusters of CD8+ T cells infiltration and cancer cells killing. Our findings point to targeting AR as possible co-therapeutical approach in melanoma treatment.

Mechanisms of antiviral activity of the new hDHODH inhibitor MEDS433 against respiratory syncytial virus replication.

Human respiratory syncytial virus (RSV) is an important cause of acute lower respiratory infections, for which no effective drugs are currently available. The development of new effective anti-RSV agents is therefore an urgent priority, and Host-Targeting Antivirals (HTAs) can be considered to target RSV infections. As a contribution to this antiviral avenue, we have characterized the molecular mechanisms of the anti-RSV activity of MEDS433, a new inhibitor of human dihydroorotate dehydrogenase (hDHODH), a key cellular enzyme of de novo pyrimidine biosynthesis. MEDS433 was found to exert a potent antiviral activity against RSV-A and RSV-B in the one-digit nanomolar range. Analysis of the RSV replication cycle in MEDS433-treated cells, revealed that the hDHODH inhibitor suppressed the synthesis of viral genome, consistently with its ability to specifically target hDHODH enzymatic activity. Then, the capability of MEDS433 to induce the expression of antiviral proteins encoded by Interferon-Stimulated Genes (ISGs) was identified as a second mechanism of its antiviral activity against RSV. Indeed, MEDS433 stimulated secretion of IFN-ß and IFN-λ1 that, in turn, induced the expression of some ISG antiviral proteins, such as IFI6, IFITM1 and IRF7. Singly expression of these ISG proteins reduced RSV-A replication, thus likely contributing to the overall anti-RSV activity of MEDS433. Lastly, MEDS433 proved to be effective against RSV-A replication even in a primary human small airway epithelial cell model. Taken as a whole, these observations provide new insights for further development of MEDS433, as a promising candidate to develop new strategies for treatment of RSV infections.

Plasma microRNA ratios associated with breast cancer detection in a nested case-control study from a mammography screening cohort.

Mammographic breast cancer screening is effective in reducing breast cancer mortality. Nevertheless, several limitations are known. Therefore, developing an alternative or complementary non-invasive tool capable of increasing the accuracy of the screening process is highly desirable. The objective of this study was to identify circulating microRNA (miRs) ratios associated with BC in women attending mammography screening. A nested case-control study was conducted within the ANDROMEDA cohort (women of age 46-67 attending BC screening). Pre-diagnostic plasma samples, information on life-styles and common BC risk factors were collected. Small-RNA sequencing was carried out on plasma samples from 65 cases and 66 controls. miR ratios associated with BC were selected by two-sample Wilcoxon test and lasso logistic regression. Subsequent assessment by RT-qPCR of the miRs contained in the selected miR ratios was carried out as a platform validation. To identify the most promising biomarkers, penalised logistic regression was further applied to candidate miR ratios alone, or in combination with non-molecular factors. Small-RNA sequencing yielded 20 candidate miR ratios associated with BC, which were further assessed by RT-qPCR. In the resulting model, penalised logistic regression selected seven miR ratios (miR-199a-3p_let-7a-5p, miR-26b-5p_miR-142-5p, let-7b-5p_miR-19b-3p, miR-101-3p_miR-19b-3p, miR-93-5p_miR-19b-3p, let-7a-5p_miR-22-3p and miR-21-5p_miR-23a-3p), together with body mass index (BMI), menopausal status (MS), the interaction term BMI * MS, life-style score and breast density. The ROC AUC of the model was 0.79 with a sensitivity and specificity of 71.9% and 76.6%, respectively. We identified biomarkers potentially useful for BC screening measured through a widespread and low-cost technique. This is the first study reporting circulating miRs for BC detection in a screening setting. Validation in a wider sample is warranted.Trial registration: The Andromeda prospective cohort study protocol was retrospectively registered on 27-11-2015 (NCT02618538).

Xeno-free cultured mesenchymal stromal cells release extracellular vesicles with a "therapeutic" miRNA cargo ameliorating cartilage inflammation in vitro.

Rationale: Mesenchymal stromal cells (MSCs)-derived extracellular vesicles (EVs) emerged as an innovative strategy for the treatment of chronic disorders such as osteoarthritis (OA). Biological activity of EVs is generally driven by their cargo, which might be influenced by microenvironment. Therefore, pre-conditioning strategies, including modifications in culture conditions or oxygen tension could directly impact on MSCs paracrine activity. In this study we selected an appropriate preconditioning system to induce cells to perform the most suitable therapeutic response by EV-encapsulated bioactive factors. Methods: A xeno-free supplement (XFS) was used for isolation and expansion of MSCs and compared to conventional fetal bovine serum (FBS) culture. Bone Marrow-derived MSCs (BMSCs) were pre-conditioned under normoxia (20% O2) or under hypoxia (1% O2) and EVs production was evaluated. Anti-OA activity was evaluated by using an in vitro inflammatory model. miRNA content was also explored, to select putative miRNA that could be involved in a biological function. Results: Modulation of IL-6, IL-8, COX-2 and PGE2 was evaluated on hACs simultaneously treated with IL-1α and BMSC-derived EVs. FBS-sEVs exerted a blunt inhibitory effect, while a strong anti-inflammatory outcome was achieved by XFS-sEVs. Interestingly, in both cases hypoxia pre-conditioning allowed to increase EVs effectiveness. Analysis of miRNA content showed the upregulation in XFS-hBMSC-derived EVs of miRNA known to have a chondroprotective role, such as let-7b-5p, miR-17, miR-145, miR-21-5p, miR-214-3p, miR-30b-5p, miR-30c-5p. Activated pathways and target genes were investigated in silico and upregulated miRNAs functionally validated in target cells. MiR-145 and miR-214 were found to protect chondrocytes from IL-1α-induced inflammation and to reduce production of pro-inflammatory cytokines. Conclusions: XFS medium was found to be suitable for isolation and expansion of MSCs, secreting EVs with a therapeutic cargo. The application of cells cultured exclusively in XFS overcomes issues of safety associated with serum-containing media and makes ready-to-use clinical therapies more accessible.

SOMAscan Proteomics Identifies Novel Plasma Proteins in Amyotrophic Lateral Sclerosis Patients.

Amyotrophic lateral sclerosis (ALS) is a complex disease characterized by the interplay of genetic and environmental factors for which, despite decades of intense research, diagnosis remains rather delayed, and most therapeutic options fail. Therefore, unravelling other potential pathogenetic mechanisms and searching for reliable markers are high priorities. In the present study, we employ the SOMAscan assay, an aptamer-based proteomic technology, to determine the circulating proteomic profile of ALS patients. The expression levels of ~1300 proteins were assessed in plasma, and 42 proteins with statistically significant differential expression between ALS patients and healthy controls were identified. Among these, four were upregulated proteins, Thymus- and activation-regulated chemokine, metalloproteinase inhibitor 3 and nidogen 1 and 2 were selected and validated by enzyme-linked immunosorbent assays in an overlapping cohort of patients. Following statistical analyses, different expression patterns of these proteins were observed in the familial and sporadic ALS patients. The proteins identified in this study might provide insight into ALS pathogenesis and represent potential candidates to develop novel targeted therapies.

Differential angiogenesis of bone and muscle endothelium in aging and inflammatory processes.

Different tissues have different endothelial features, however, the implications of this heterogeneity in pathological responses are not clear yet. "Inflamm-aging" has been hypothesized as a possible trigger of diseases, including osteoarthritis (OA) and sarcopenia, often present in the same patient. To highlight a possible contribution of organ-specific endothelial cells (ECs), we compare ECs derived from bone and skeletal muscle of the same OA patients. OA bone ECs show a pro-inflammatory signature and higher angiogenic sprouting as compared to muscle ECs, in control conditions and stimulated with TNFα. Furthermore, growth of muscle but not bone ECs decreases with increasing patient age and systemic inflammation. Overall, our data demonstrate that inflammatory conditions in OA patients differently affect bone and muscle ECs, suggesting that inflammatory processes increase angiogenesis in subchondral bone while associated systemic low-grade inflammation impairs angiogenesis in muscle, possibly highlighting a vascular trigger linking OA and sarcopenia.

Fluoropyrimidine­induced cardiotoxicity in colorectal cancer patients: a prospective observational trial (CHECKPOINT).

Fluoropyrimidines (FP) are the backbone chemotherapy in colorectal cancer (CRC) treatment; however, their use is associated with cardiotoxicity, which is underreported. In the present study, it was aimed to prospectively determine the incidence rates and related risk factors of FP­induced cardiotoxicity (FIC) in CRC patients and at identifying predictive biomarkers. A total of 129 consecutive previously untreated CRC patients underwent active cardiological monitoring, including 5­items simplified questionnaire on symptoms, electrocardiogram (ECG) and plasma sample collection during FP chemotherapy. FIC was defined as the presence of ECG alterations and/or the arising of at least one symptom of chest pain, dyspnoea, palpitations or syncope. The primary objective was the evaluation of FIC incidence. Secondary objectives were the correlation of FIC with well­known cardiological risk factors and the identification of circulating biomarkers (serum levels of troponin I, pro hormone BNP; miRNA analysis) as predictors of FIC. A total of 20 out of 129 (15.5%) patients experienced FIC. The most common symptoms were dyspnoea (60%) and chest pain (40%), while only 15% of patients presented ECG alterations, including one acute myocardial infarction. Retreatment with FP was attempted in 90% of patients with a favourable outcome. Despite 48% of patients having cardiological comorbidities, an increased FIC was not observed in this subgroup. Only the subgroup of females with the habit of alcohol consumption showed an increased risk of FIC. None of the circulating biomarkers evaluated demonstrated a clinical utility as FIC predictors. FIC can be an unexpected, life­threatening adverse event that can limit the subsequent treatment choices in patients with CRC. In this prospective study, well­known cardiological comorbidities were not related to higher FIC risk and circulating biomarkers predictive of toxicity could not be found. With careful monitoring, mainly based on symptoms, almost all patients completed the FP treatment.

Identification of a minimum number of genes to predict triple-negative breast cancer subgroups from gene expression profiles.

BACKGROUND: Triple-negative breast cancer (TNBC) is a very heterogeneous disease. Several gene expression and mutation profiling approaches were used to classify it, and all converged to the identification of distinct molecular subtypes, with some overlapping across different approaches. However, a standardised tool to routinely classify TNBC in the clinics and guide personalised treatment is lacking. We aimed at defining a specific gene signature for each of the six TNBC subtypes proposed by Lehman et al. in 2011 (basal-like 1 (BL1); basal-like 2 (BL2); mesenchymal (M); immunomodulatory (IM); mesenchymal stem-like (MSL); and luminal androgen receptor (LAR)), to be able to accurately predict them. METHODS: Lehman's TNBCtype subtyping tool was applied to RNA-sequencing data from 482 TNBC (GSE164458), and a minimal subtype-specific gene signature was defined by combining two class comparison techniques with seven attribute selection methods. Several machine learning algorithms for subtype prediction were used, and the best classifier was applied on microarray data from 72 Italian TNBC and on the TNBC subset of the BRCA-TCGA data set. RESULTS: We identified two signatures with the 120 and 81 top up- and downregulated genes that define the six TNBC subtypes, with prediction accuracy ranging from 88.6 to 89.4%, and even improving after removal of the least important genes. Network analysis was used to identify highly interconnected genes within each subgroup. Two druggable matrix metalloproteinases were found in the BL1 and BL2 subsets, and several druggable targets were complementary to androgen receptor or aromatase in the LAR subset. Several secondary drug-target interactions were found among the upregulated genes in the M, IM and MSL subsets. CONCLUSIONS: Our study took full advantage of available TNBC data sets to stratify samples and genes into distinct subtypes, according to gene expression profiles. The development of a data mining approach to acquire a large amount of information from several data sets has allowed us to identify a well-determined minimal number of genes that may help in the recognition of TNBC subtypes. These genes, most of which have been previously found to be associated with breast cancer, have the potential to become novel diagnostic markers and/or therapeutic targets for specific TNBC subsets.

A Circulating Risk Score, Based on Combined Expression of Exo-miR-130a-3p and Fibrinopeptide A, as Predictive Biomarker of Relapse in Resectable Non-Small Cell Lung Cancer Patients.

To date, the 5-year overall survival rate of 60% for early-stage non-small cell lung cancer (NSCLC) is still unsatisfactory. Therefore, reliable prognostic factors are needed. Growing evidence shows that cancer progression may depend on an interconnection between cancer cells and the surrounding tumor microenvironment; hence, circulating molecules may represent promising markers of cancer recurrence. In order to identify a prognostic score, we performed in-depth high-throughput analyses of plasma circulating markers, including exosomal microRNAs (Exo-miR) and peptides, in 67 radically resected NSCLCs. The miRnome profile selected the Exo-miR-130a-3p as the most overexpressed in relapsed patients. Peptidome analysis identified four progressively more degraded forms of fibrinopeptide A (FpA), which were depleted in progressing patients. Notably, stepwise Cox regression analysis selected Exo-miR-130a-3p and the greatest FpA (2-16) to build a score predictive of recurrence, where high-risk patients had 18 months of median disease-free survival. Moreover, in vitro transfections showed that higher levels of miR-130a-3p lead to a deregulation of pathways involved in metastasis and angiogenesis, including the coagulation process and metalloprotease increase which might be linked to FpA reduction. In conclusion, by integrating circulating markers, the identified risk score may help clinicians predict early-stage NSCLC patients who are more likely to relapse after primary surgery.

Ki-67 proliferation index to further stratify invasive breast cancer molecular subtypes: Northern African comparative cohort-study with external TCGA-BRCA and METABRIC validation.

Introduction: breast cancer (BC) is a malignancy with very high incidence and mortality in Africa, especially in Western Africa, where more than 25 thousand deaths are registered every year. Not all BC have the same prognosis, and being able to personalize treatment and predict aggressiveness is of crucial importance. The purpose of our study is to explore further subdivisions associated with prognosis, beyond breast cancer molecular classification that is routinely established in pathology departments. Methods: we conducted a 5-year retrospective cohort study on 1266 invasive BC of Moroccan patients, collected at the Pathology Department of Ibn-Rochd University Hospital in Casablanca, and followed at King Mohammed VI National Centre for the Treatment of Cancers. We elaborated an Estimation-Maximization Clustering, based on the main BC biomarkers: Ki-67, HER2, estrogen and progesterone receptors, evaluated by immunohistochemistry. Two independent datasets (TCGA-BRCA and Metabric) were also analyzed to assess the external reproducibility of the results. Results: each molecular subgroup could be partitioned into two further subdivisions: Cluster1, with average Ki-67 of 16.26% (±11.9) across all molecular subgroups and higher frequency within luminal BC, and Cluster2, with average Ki-67 of 68.8%(±18) across all molecular subgroups and higher frequency in HER2 as well as in triple-negative BC. Overall survival of the two Clusters was significantly different, with 5-year rates of 52 and 37 months for Custer1 and Cluster2, respectively (p=0.000001). Moreover, mortality rates within the same molecular subgroup, especially in luminal B HER2-, varied remarkably depending on Cluster membership (6% for C1 and 18% for C2 after 1 year of follow-up). Two different algorithms to evaluate the prognostic importance, variable selection using random forests (VSURF) and Minimal depth, ranked the subdivision proposed as one of the 4 most influential features being able to predict patient survival better than several histoprognostic features, both in the Moroccan and in the external datasets. Conclusion: our results highlight a new refinement of the BC molecular classification and provide a simple and improved way to classify tumors that could be applied in low to middle-income countries. This is the first study of its kind addressed in an African context.

Meta-analysis of diagnostic cell-free circulating microRNAs for breast cancer detection.

BACKGROUND: Breast cancer (BC) is the most frequently diagnosed cancer among women. Numerous studies explored cell-free circulating microRNAs as diagnostic biomarkers of BC. As inconsistent and rarely intersecting microRNA panels have been reported thus far, we aim to evaluate the overall diagnostic performance as well as the sources of heterogeneity between studies. METHODS: Based on the search of three online search engines performed up to March 21st 2022, 56 eligible publications that investigated diagnostic circulating microRNAs by utilizing Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) were obtained. Primary studies' potential for bias was evaluated with the revised tool for the quality assessment of diagnostic accuracy studies (QUADAS-2). A bivariate generalized linear mixed-effects model was applied to obtain pooled sensitivity and specificity. A novel methodology was utilized in which the sample and study models' characteristics were analysed to determine the potential preference of studies for sensitivity or specificity. RESULTS: Pooled sensitivity and specificity of 0.85 [0.81-0.88] and 0.83 [0.79-0.87] were obtained, respectively. Subgroup analysis showed a significantly better performance of multiple (sensitivity: 0.90 [0.86-0.93]; specificity: 0.86 [0.80-0.90]) vs single (sensitivity: 0.82 [0.77-0.86], specificity: 0.83 [0.78-0.87]) microRNA panels and a comparable pooled diagnostic performance between studies using serum (sensitivity: 0.87 [0.81-0.91]; specificity: 0.83 [0.78-0.87]) and plasma (sensitivity: 0.83 [0.77-0.87]; specificity: 0.85 [0.78-0.91]) as specimen type. In addition, based on bivariate and univariate analyses, miRNA(s) based on endogenous normalizers tend to have a higher diagnostic performance than miRNA(s) based on exogenous ones. Moreover, a slight tendency of studies to prefer specificity over sensitivity was observed. CONCLUSIONS: In this study the diagnostic ability of circulating microRNAs to diagnose BC was reaffirmed. Nonetheless, some subgroup analyses showed between-study heterogeneity. Finally, lack of standardization and of result reproducibility remain the biggest issues regarding the diagnostic application of circulating cell-free microRNAs.

Comprehensive Gene Expression Analysis to Identify Differences and Similarities between Sex- and Stage-Stratified Melanoma Samples.

Overall lower incidence and better prognosis are observed in female melanoma patients compared to males. As sex and stage differences in the context of melanoma gene expression are understudied, we aim to highlight them through statistical analysis of melanoma gene expression datasets. Data from seven online datasets, including normal skin, commonly acquired nevi, and melanomas, were collected and analyzed. Sex/stage-related differences were assessed using statistical analyses on survival, gene expression, and its variability. Significantly better overall survival in females was observed in stage I, II but not in stage III. Gene expression variability was significantly different between stages and sexes. Specifically, we observed a significantly lower variability in genes expressed in normal skin and nevi in females compared to males, as well as in female stage I, II melanomas. However, in stage III, variability was lower in males. Similarly, class comparison showed that the gene expression differences between sexes are most notable in non-melanoma followed by early-stage-melanoma samples. Sexual dimorphism is an important aspect to consider for a holistic understanding of early-stage melanomas, not only from the tumor characteristics but also from the gene expression points of view.

TRF2 cooperates with CTCF for controlling the oncomiR-193b-3p in colorectal cancer.

The Telomeric Repeat binding Factor 2 (TRF2), a key protein involved in telomere integrity, is over-expressed in several human cancers and promotes tumor formation and progression. Recently, TRF2 has been also found outside telomeres where it can affect gene expression. Here we provide evidence that TRF2 is able to modulate the expression of microRNAs (miRNAs), small non-coding RNAs altered in human tumors. Among the miRNAs regulated by TRF2, we focused on miR-193b-3p, an oncomiRNA that positively correlates with TRF2 expression in human colorectal cancer patients from The Cancer Genome Atlas dataset. At the mechanistic level, the control of miR-193b-3p expression requires the cooperative activity between TRF2 and the chromatin organization factor CTCF. We found that CTCF physically interacts with TRF2, thus driving the proper positioning of TRF2 on a binding site located upstream the miR-193b-3p host-gene. The binding of TRF2 on the identified region is necessary for promoting the expression of miR-193b3p which, in turn, inhibits the translation of the onco-suppressive methyltransferase SUV39H1 and promotes tumor cell proliferation. The translational relevance of the oncogenic properties of miR-193b-3p was confirmed in patients, in whom the association between TRF2 and miR-193b-3p has a prognostic value.

PGC1α/ß Expression Predicts Therapeutic Response to Oxidative Phosphorylation Inhibition in Ovarian Cancer.

Ovarian cancer is the deadliest gynecologic cancer, and novel therapeutic options are crucial to improve overall survival. Here we provide evidence that impairment of oxidative phosphorylation (OXPHOS) can help control ovarian cancer progression, and this benefit correlates with expression of the two mitochondrial master regulators PGC1α and PGC1ß. In orthotopic patient-derived ovarian cancer xenografts (OC-PDX), concomitant high expression of PGC1α and PGC1ß (PGC1α/ß) fostered a unique transcriptional signature, leading to increased mitochondrial abundance, enhanced tricarboxylic acid cycling, and elevated cellular respiration that ultimately conferred vulnerability to OXPHOS inhibition. Treatment with the respiratory chain complex I inhibitor IACS-010759 caused mitochondrial swelling and ATP depletion that consequently delayed malignant progression and prolonged the lifespan of high PGC1α/ß-expressing OC-PDX-bearing mice. Conversely, low PGC1α/ß OC-PDXs were not affected by IACS-010759, thus pinpointing a selective antitumor effect of OXPHOS inhibition. The clinical relevance of these findings was substantiated by analysis of ovarian cancer patient datasets, which showed that 25% of all cases displayed high PGC1α/ß expression along with an activated mitochondrial gene program. This study endorses the use of OXPHOS inhibitors to manage ovarian cancer and identifies the high expression of both PGC1α and ß as biomarkers to refine the selection of patients likely to benefit most from this therapy. SIGNIFICANCE: OXPHOS inhibition in ovarian cancer can exploit the metabolic vulnerabilities conferred by high PGC1α/ß expression and offers an effective approach to manage patients on the basis of PGC1α/ß expression.

The DNA-PK Inhibitor AZD7648 Sensitizes Patient-Derived Ovarian Cancer Xenografts to Pegylated Liposomal Doxorubicin and Olaparib Preventing Abdominal Metastases.

Ovarian cancer is the deadliest gynecologic cancer, with a 5-year survival rate of 30%, when the disease has spread throughout the peritoneal cavity. We investigated the efficacy to delay disease progression by the DNA-dependent protein kinase (DNA-PK) inhibitor AZD7648, administered in combination with two of the therapeutic options for patient management: either pegylated liposomal doxorubicin (PLD) or the PARP inhibitor olaparib. Patient-derived ovarian cancer xenografts (OC-PDX) were transplanted subcutaneously to evaluate the effect of treatment on tumor growth, or orthotopically in the peritoneal cavity to evaluate the effect on metastatic spread. AZD7648 was administered orally in combination with PLD (dosed intravenously) or with olaparib (orally). To prove the inhibition of DNA-PK in the tumors, we measured pDNA-PKcs, pRPA32, and γH2AX, biomarkers of DNA-PK activity. AZD7648 enhanced the therapeutic efficacy of PLD in all the OC-PDXs tested, regardless of their BRCA status or sensitivity to cisplatin or PLD. The treatment caused disease stabilization, which persisted despite therapy discontinuation for tumors growing subcutaneously, and significantly impaired the abdominal metastatic dissemination, prolonging the lifespan of mice implanted orthotopically. AZD7648 potentiated the efficacy of olaparib in BRCA-deficient OC-PDXs but did not sensitize BRCA-proficient OC-PDXs to olaparib, despite an equivalent inhibition of DNA-PK, suggesting the need of a preexisting olaparib activity to benefit from the addition of AZD7648. This work suggests that AZD7648, an inhibitor of DNA-PK, dosed in combination with PLD or olaparib is an exciting therapeutic option that could benefit patients with ovarian cancer and should be explored in clinical trials.

Two Novel Ceramide-Like Molecules and miR-5100 Levels as Biomarkers Improve Prediction of Prostate Cancer in Gray-Zone PSA.

Reliable liquid biopsy-based tools able to accurately discriminate prostate cancer (PCa) from benign prostatic hyperplasia (BPH), when PSA is within the "gray zone" (PSA 4-10), are still urgent. We analyzed plasma samples from a cohort of 102 consecutively recruited patients with PSA levels between 4 and 16 ng/ml, using the SANIST-Cloud Ion Mobility Metabolomic Mass Spectrometry platform, combined with the analysis of a panel of circulating microRNAs (miR). By coupling CIMS ion mobility technology with SANIST, we were able to reveal three new structures among the most differentially expressed metabolites in PCa vs. BPH. In particular, two were classified as polyunsaturated ceramide ester-like and one as polysaturated glycerol ester-like. Penalized logistic regression was applied to build a model to predict PCa, using six circulating miR, seven circulating metabolites, and demographic/clinical variables, as covariates. Four circulating metabolites, miR-5100, and age were selected by the model, and the corresponding prediction score gave an AUC of 0.76 (C.I. = 0.66-0.85). At a specified cut-off, no high-risk tumor was misclassified, and 22 out of 53 BPH were correctly identified, reducing by 40% the false positives of PSA. We developed and applied a novel, minimally invasive, liquid biopsy-based powerful tool to characterize novel metabolites and identified new potential non-invasive biomarkers to better predict PCa, when PSA is uninformative as a tool for precision medicine in genitourinary cancers.

EZH2-induced lysine K362 methylation enhances TMPRSS2-ERG oncogenic activity in prostate cancer.

The TMPRSS2-ERG gene fusion is the most frequent alteration observed in human prostate cancer. However, its role in disease progression is still unclear. In this study, we uncover an important mechanism promoting ERG oncogenic activity. We show that ERG is methylated by Enhancer of zest homolog 2 (EZH2) at a specific lysine residue (K362) located within the internal auto-inhibitory domain. Mechanistically, K362 methylation modifies intra-domain interactions, favors DNA binding and enhances ERG transcriptional activity. In a genetically engineered mouse model of ERG fusion-positive prostate cancer (Pb-Cre4 Pten flox/flox Rosa26-ERG, ERG/PTEN), ERG K362 methylation is associated with PTEN loss and progression to invasive adenocarcinomas. In both ERG positive VCaP cells and ERG/PTEN mice, PTEN loss results in AKT activation and EZH2 phosphorylation at serine 21 that favors ERG methylation. We find that ERG and EZH2 interact and co-occupy several sites in the genome forming trans-activating complexes. Consistently, ERG/EZH2 co-regulated target genes are deregulated preferentially in tumors with concomitant ERG gain and PTEN loss and in castration-resistant prostate cancers. Collectively, these findings identify ERG methylation as a post-translational modification sustaining disease progression in ERG-positive prostate cancers.