GC-MS analysis of 4-hydroxyproline: elevated proline hydroxylation in metformin-associated lactic acidosis and metformin-treated Becker muscular dystrophy patients.

Metformin (N,N-dimethylbiguanide), an inhibitor of gluconeogenesis and insulin sensitizer, is widely used for the treatment of type 2 diabetes. In some patients with renal insufficiency, metformin can accumulate and cause lactic acidosis, known as metformin-associated lactic acidosis (MALA, defined as lactate ≥ 5 mM, pH < 7.35, and metformin concentration > 38.7 µM). Here, we report on the post-translational modification (PTM) of proline (Pro) to 4-hydroxyproline (OH-Pro) in metformin-associated lactic acidosis and in metformin-treated patients with Becker muscular dystrophy (BMD). Pro and OH-Pro were measured simultaneously by gas chromatography-mass spectrometry before, during, and after renal replacement therapy in a patient admitted to the intensive care unit (ICU) because of MALA. At admission to the ICU, plasma metformin concentration was 175 µM, with a corresponding lactate concentration of 20 mM and a blood pH of 7.1. Throughout ICU admission, the Pro concentration was lower compared to healthy controls. Renal excretion of OH-Pro was initially high and decreased over time. Moreover, during the first 12 h of ICU admission, OH-Pro seems to be renally secreted while thereafter, it was reabsorbed. Our results suggest that MALA is associated with hyper-hydroxyprolinuria due to elevated PTM of Pro to OH-Pro by prolyl-hydroxylase and/or inhibition of OH-Pro metabolism in the kidneys. In BMD patients, metformin, at the therapeutic dose of 3 × 500 mg per day for 6 weeks, increased the urinary excretion of OH-Pro suggesting elevation of Pro hydroxylation to OH-Pro. Our study suggests that metformin induces specifically the expression/activity of prolyl-hydroxylase in metformin intoxication and BMD.

The regulation of plasma gelsolin by DNA methylation in ovarian cancer chemo-resistance.

BACKGROUND: Ovarian cancer (OVCA) is the most lethal gynecologic cancer and chemoresistance remains a major hurdle to successful therapy and survival of OVCA patients. Plasma gelsolin (pGSN) is highly expressed in chemoresistant OVCA compared with their chemosensitive counterparts, although the mechanism underlying the differential expression is not known. Also, its overexpression significantly correlates with shortened survival of OVCA patients. In this study, we investigated the methylation role of Ten eleven translocation isoform-1 (TET1) in the regulation of differential pGSN expression and chemosensitivity in OVCA cells. METHODS: Chemosensitive and resistant OVCA cell lines of different histological subtypes were used in this study to measure pGSN and TET1 mRNA abundance (qPCR) as well as protein contents (Western blotting). To investigate the role of DNA methylation specifically in pGSN regulation and pGSN-induced chemoresistance, DNMTs and TETs were pharmacologically inhibited in sensitive and resistant OVCA cells using specific inhibitors. DNA methylation was quantified using EpiTYPER MassARRAY system. Gain-and-loss-of-function assays were used to investigate the relationship between TET1 and pGSN in OVCA chemoresponsiveness. RESULTS: We observed differential protein and mRNA expressions of pGSN and TET1 between sensitive and resistant OVCA cells and cisplatin reduced their expression in sensitive but not in resistant cells. We observed hypomethylation at pGSN promoter upstream region in resistant cells compared to sensitive cells. Pharmacological inhibition of DNMTs increased pGSN protein levels in sensitive OVCA cells and decreased their responsiveness to cisplatin, however we did not observe any difference in methylation level at pGSN promoter region. TETs inhibition resulted in hypermethylation at multiple CpG sites and decreased pGSN protein level in resistant OVCA cells which was also associated with enhanced response to cisplatin, findings that suggested the methylation role of TETs in the regulation of pGSN expression in OVCA cells. Further, we found that TET1 is inversely related to pGSN but positively related to chemoresponsiveness of OVCA cells. CONCLUSION: Our findings broaden our knowledge about the epigenetic regulation of pGSN in OVCA chemoresistance and reveal a novel potential target to re-sensitize resistant OVCA cells. This may provide a future therapeutic strategy to improve the overall OVCA patient survival.

Multiplex epigenome editing of MECP2 to rescue Rett syndrome neurons.

Rett syndrome (RTT) is an X-linked neurodevelopmental disorder caused by loss-of-function heterozygous mutations of methyl CpG-binding protein 2 (MECP2) on the X chromosome in young females. Reactivation of the silent wild-type MECP2 allele from the inactive X chromosome (Xi) represents a promising therapeutic opportunity for female patients with RTT. Here, we applied a multiplex epigenome editing approach to reactivate MECP2 from Xi in RTT human embryonic stem cells (hESCs) and derived neurons. Demethylation of the MECP2 promoter by dCas9-Tet1 with target single-guide RNA reactivated MECP2 from Xi in RTT hESCs without detectable off-target effects at the transcriptional level. Neurons derived from methylation-edited RTT hESCs maintained MECP2 reactivation and reversed the smaller soma size and electrophysiological abnormalities, two hallmarks of RTT. In RTT neurons, insulation of the methylation-edited MECP2 locus by dCpf1-CTCF (a catalytically dead Cpf1 fused with CCCTC-binding factor) with target CRISPR RNA enhanced MECP2 reactivation and rescued RTT-related neuronal defects, providing a proof-of-concept study for epigenome editing to treat RTT and potentially other dominant X-linked diseases.

Long-term outcomes of osilodrostat in Cushing's disease: LINC 3 study extension.

Objective: To investigate the long-term efficacy and tolerability of osilodrostat, a potent oral 11ß-hydroxylase inhibitor, for treating Cushing's disease (CD). Design/methods: A total of 137 adults with CD and mean 24-h urinary free cortisol (mUFC) > 1.5 × upper limit of normal (ULN) received osilodrostat (starting dose 2 mg bid; maximum 30 mg bid) during the prospective, Phase III, 48-week LINC 3 (NCT02180217) core study. Patients benefiting from osilodrostat at week 48 could enter the optional extension (ending when all patients had received ≥ 72 weeks of treatment or discontinued). Efficacy and safety were assessed for all enrolled patients from the core study baseline. Results: Median osilodrostat exposure from the core study baseline to study end was 130 weeks (range 1-245) and median average dose was 7.4 mg/day (range 0.8-46.6). The reduction in mean mUFC achieved during the core was maintained during the extension and remained ≤ ULN. Of 106 patients, 86 (81%) patients who entered the extension had mUFC ≤ ULN at week 72. Improvements in cardiovascular/metabolic-related parameters, physical manifestations of hypercortisolism (fat pads, central obesity, rubor, striae, and hirsutism in females), and quality of life in the core study were also maintained or improved further during the extension. No new safety signals were reported; 15/137 (10.9%) and 12/106 (11.3%) patients discontinued for adverse events during the core and extension, respectively. Mean testosterone in females decreased towards baseline levels during the extension. Conclusions: Data from this large, multicentre trial show that long-term treatment with osilodrostat sustains cortisol normalisation alongside clinical benefits in most patients with CD and is well tolerated.

TET1 mutations as a predictive biomarker for immune checkpoint inhibitors in colon adenocarcinoma.

BACKGROUND: The ten-eleven translocation 1 (TET1), which is essential for active DNA demethylation, plays a multifaceted role in the pathogenesis of colorectal cancer. The study has demonstrated the association of TET1 mutations with a high response to immune checkpoint inhibitors (ICIs) in diverse cancers. However, the relationship between TET1 mutations and the response to ICIs in colon cancer is still lacking. METHODS: The prognosis, predictive markers, immune characteristics, mutation number of DNA damage repair (DDR) pathways, pathway enrichment, and drug sensitivity conditions were all compared between TET1-mutated and wild-type patients with colon adenocarcinoma (COAD). RESULTS: The overall survival of patients with TET1 mutations in the ICI-treated cohort was significantly longer than those without (p = 0.0059). Compared with the wild-type patients, TET1-mutated patients had higher tumor mutational burden and neoantigen load, enhanced abundance of tumor-infiltrating immune cells, increased expression of immune-related genes, and mutation number of DDR pathways. Additionally, the patients with TET1 mutations were found to be more sensitive to lapatinib and 5-fluorouracil. CONCLUSION: These findings suggest that TET1 mutations may serve as a potential biomarker for the response to ICIs in COAD patients.

Osilodrostat oral tablets for adults with Cushing's disease.

INTRODUCTION: Endogenous Cushing's syndrome (CS) is a rare, multi-systemic condition resulting from chronic glucocorticoid excess sustained by a pituitary adenoma (Cushing's disease, CD), an adrenal adenoma or, less frequently, a neuroendocrine tumor. The optimal first-line option is surgery, but when it is contraindicated/refused, or in case of severe, life-threatening disease, medical treatment is a first-line choice. Osilodrostat (LCI699, Isturisa®) is a new, orally active adrenal steroidogenesis inhibitor currently approved by the FDA and EMA for the treatment of endogenous CS. AREAS COVERED: We illustrate the pharmacologic profile of osilodrostat and summarize the efficacy and safety of osilodrostat from the first phase I studies to the most recent evidence. EXPERT OPINION: Osilodrostat acts as a potent, reversible inhibitor of 11ß-hydroxylase (CYP11B1) and 18-hydroxylase (or aldosterone synthase, CYP11B2), counteracting both gluco- and mineralocorticoid production. According to the results of the LINC1, LINC2, and LINC3 studies and the preliminary findings of LINC4, osilodrostat offers an excellent efficacy in controlling hypercortisolism with a good tolerability. The non-negligible risk of adrenal insufficiency/steroid withdrawal symptoms, hypokalemia, and hyperandrogenism disorders, and the possibility, albeit rare, of pituitary tumor enlargement, require further confirmation and careful monitoring.

Newly diagnosed autoimmune Addison's disease in a patient with COVID-19 with autoimmune disseminated encephalomyelitis.

A man in his 20s with a history of acute disseminated encephalomyelitis (ADEM) was brought into the emergency department (ED) after his family found him at home collapsed on the floor unresponsive with a blood glucose of 28 mg/dL at the field. In the ED, the patient was tachycardic, tachypnoeic and hypotensive, requiring pressors and intubation at 9 hours and 12 hours after arrival, respectively. Laboratory results revealed a positive COVID-19 test, serum sodium of 125 mmol/L and persistent hypoglycaemia. The patient was given a high dose of dexamethasone for COVID-19 treatment 1 hour before pressors were started. He was then continued on a stress dose of intravenous hydrocortisone with rapid clinical improvement leading to his extubation, and discontinuation of vasopressors and glucose on day 2 of admission. The patient received his last dose of intravenous hydrocortisone on day 4 in the early afternoon with the plan to order adrenal testing the following morning prior to discharge. On day 5, the aldosterone <3.0 ng/dL, adrenocorticotropic hormone (ACTH) level >1250 pg/mL, and ACTH stimulation test showed cortisol levels of 3 and 3 µg/dL at 30 and 60 min, respectively. The anti-21-hydroxylase antibody was positive. The patient was discharged on hydrocortisone and fludrocortisone. The patient's symptoms, elevated ACTH, low cortisol and presence of 21-hydroxylase antibodies are consistent with autoimmune Addison's disease. This is the first case reporting autoimmune Addison's disease in a patient with COVID-19 with a history of ADEM. The case highlights the importance of considering adrenal insufficiency as a diagnostic differential in haemodynamically unstable patients with COVID-19.

Potential therapeutics using tumor-secreted lactate in nonsmall cell lung cancer.

Targeted-therapy failure in treating nonsmall cell lung cancer (NSCLC) frequently occurs because of the emergence of drug resistance and genetic mutations. The same mutations also result in aerobic glycolysis, which further antagonizes outcomes by localized increases in lactate, an immune suppressor. Recent evidence indicates that enzymatic lowering of lactate can promote an oncolytic immune microenvironment within the tumour. Here, we review factors relating to lactate expression in NSCLC and the utility of lactate oxidase (LOX) for governing therapeutic delivery, its role in lactate oxidation and turnover, and relationships between lactate depletion and immune cell populations. The lactate-rich characteristic of NSCLC provides an exploitable property to potentially improve NSCLC outcomes and design new therapeutic strategies to integrate with conventional therapies.

Self-Assembled Multiple-Enzyme Composites for Enhanced Synergistic Cancer Starving-Catalytic Therapy.

Inspired by the particularity of tumor microenvironments, including acidity and sensibility to reactive oxygen species (ROS), advanced and smart responsive nanomaterials have recently been developed. The present study synthesized tumor-targeted and pH-sensitive supramolecular micelles that self-assembled via host-guest recognition. The micelles consumed intratumoral glucose and lactate via loading with glucose oxidase (GOD) and lactate oxidase (LOD). Intratumoral glucose and lactate were converted into hydrogen peroxide (H2O2) and were sequentially reduced to highly toxic hydroxyl radicals (•OH) via the peroxidase (POD)-like activity of the loaded C-dot nanozymes. Tumor-killing effects were observed via cascade catalytic reactions. After an intravenous injection, the nanocomposite exhibited an excellent tumor-targeted ability with good biocompatibility, which demonstrated its effective antitumor effect. The nanocomposite effectively combined starvation and catalytic therapies and exerted a synergistic anticancer effect with minimal side effects and without external addition.

Improving Cancer Immunotherapy by Targeting the Hypoxic Tumor Microenvironment: New Opportunities and Challenges.

Initially believed to be a disease of deregulated cellular and genetic expression, cancer is now also considered a disease of the tumor microenvironment. Over the past two decades, significant and rapid progress has been made to understand the complexity of the tumor microenvironment and its contribution to shaping the response to various anti-cancer therapies, including immunotherapy. Nevertheless, it has become clear that the tumor microenvironment is one of the main hallmarks of cancer. Therefore, a major challenge is to identify key druggable factors and pathways in the tumor microenvironment that can be manipulated to improve the efficacy of current cancer therapies. Among the different tumor microenvironmental factors, this review will focus on hypoxia as a key process that evolved in the tumor microenvironment. We will briefly describe our current understanding of the molecular mechanisms by which hypoxia negatively affects tumor immunity and shapes the anti-tumor immune response. We believe that such understanding will provide insight into the therapeutic value of targeting hypoxia and assist in the design of innovative combination approaches to improve the efficacy of current cancer therapies, including immunotherapy.

Anti-TNF-alpha therapy as a clinical intervention for periprosthetic osteolysis.

Aseptic loosening of total joint arthroplastics due to periprosthetic osteolysis is a frequent cause of implant failure. The absence of clinical interventions to arrest or prevent this complication limits the use of total joint replacement especially in younger patients. Here we review recent studies implicating tumor necrosis factor (TNF)-alpha in periprosthetic osteolysis and the rationale for clinical studies of anti-TNF therapy and other interventions for periprosthetic loosening.

Depletion of patients' plasma tryptophan using tryptophan side-chain oxidase columns.

The use of the enzyme tryptophan side-chain oxidase, isolated from Pseudomonas XA, was explored in 3 patients with refractory acute lymphocytic leukemia. Patients were given either a low-tryptophan diet or tryptophan-free hyperalimentation, prior to and during therapy. Their plasma, separated by pheresis, was continuously passed through a tryptophan depletion column containing the immobilized tryptophan side-chain oxidase. Up to 4 plasma volumes were passed through the column daily, 5 days per week for 2-3 weeks, and plasma tryptophan levels, both free and total, were measured by high-performance liquid chromatography. Pre- and postcolumn plasma samples were collected throughout the pheresis procedure. All postcolumn plasma samples had unmeasurable tryptophan levels throughout the treatment period, whereas precolumn samples were always measurable. Generally, tryptophan levels of plasma isolated from peripheral blood decreased after therapy, but rebounded by the next day. The enzyme depletion column reduces circulating plasma tryptophan levels, and its use is well tolerated by patients. However, further development of this method will require study of the effects of diet and of the duration, interval, and frequency of use of this column on therapeutic efficacy. Problems include difficulties with extended diet compliance and apparently intensive mobilization of tryptophan from body stores, which may preclude the clinical application of this enzyme depletion column.