Malattie da mutazioni dei tRNA mitocondriali: implementazione di strategie terapeutiche e modelli cellulari innovativi

INTRODUZIONE E SCOPO DELLO STUDIO Le malattie mitocondriali sono patologie multisistemiche associate a mutazioni del DNA mitocondriale (mtDNA) e/o nucleare. La maggior parte delle mutazioni patogene a carico del mtDNA sono localizzate su geni dei tRNA (mt-tRNA) e causano quadri patologici complessi con coinvolgimento di singoli organi (es. cardiomiopatie) o di organi multipli (miopatie, encefalopatie). Ad oggi non è stata identificata alcuna terapia efficace per queste malattie e ciò appare riconducibile a due ordini di problemi; da un lato la mancanza di modelli animali in grado di riprodurre le mutazioni del mtDNA e dall’altro la difficoltà di veicolare molecole potenzialmente terapeutiche nei mitocondri. Studi condotti nel nostro laboratorio hanno recentemente dimostrato come il peptide β32-33 derivato dalla porzione carbossi-terminale (Cterm) della leucil-tRNA sintetasi mitocondriale (LeuRS) sia in grado di migliorare il fenotipo patologico associato alle mutazioni a carico di diversi mt-tRNA. L’analisi in vitro delle interazioni tra il peptide e i tRNA sintetici suggerisce che l’effetto terapeutico non sia correlabile all’azione catalitica ma a un’attività chaperonica che comporta una stabilizzazione del tRNA. Questo dato è in linea con il fatto che il peptide derivi dalla porzione non catalitica dell’intero enzima e trova conferma nell’effetto di rescue anche nei confronti di mutazioni su tRNA non cognati. I cibridi transmitocondriali (cibridi) ad oggi rappresentano il modello cellulare più utilizzato nello studio delle malattie mitocondriali; tuttavia derivando da cloni neoplastici non sono in grado di ricapitolare la tessuto-specificità del danno da mutazioni del mtDNA. La tecnologia delle cellule staminali pluripotenti indotte (iPSCs) ha aperto a nuove strategie rivoluzionando il campo dei modelli cellulari utilizzati nella ricerca biomedica. Lo scopo del mio dottorato è stato quello di avvicinare all’applicazione terapeutica la molecola peptidica, da noi precedentemente identificata, con il seguente approccio: i.) Sviluppo di strategie per veicolare le molecole in studio alla matrice mitocondriale. ii.) Analisi dell’effetto tessuto-specifico delle mutazioni dei mt-tRNA ed individuazione di eventuali meccanismi terapeutici dotati di specificità tissutale. MATERIALI E METODI Per testare le molecole oggetto della prima parte dello studio, abbiamo utilizzato cibridi che derivano dalla fusione di cellule di osteosarcoma private del proprio mtDNA con fibroblasti enucleati provenienti da soggetti di controllo (mtDNA wild-type) e da pazienti portatori della mutazione m.3243A>G a carico del tRNALeu(UUR) che causa la sindrome MELAS (Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like episodes; cibridi MELAS). Abbiamo quindi valutato il fenotipo patologico (vitalità cellulare e consumo di ossigeno) dei cibridi mutanti prima e dopo il trattamento con il peptide inserito in diversi costrutti disegnati per facilitarne l’accesso alla matrice mitocondriale. Il secondo obiettivo di questo progetto è stato lo sviluppo di modelli cellulari adeguati allo studio dell’effetto tessuto-specifico delle mutazioni dei mt-tRNA. Nel corso del mio dottorato abbiamo sviluppato cellule staminali pluripotenti indotte (iPSCs) a partire da fibroblasti MELAS con percentuali variabili di eteroplasmia (percentuale di molecole di mtDNA mutato) e di soggetti di controllo (WT), differenziando le colonie ottenute in cellule neuronali staminali, che mantengono i diversi livelli di eteroplasmia. RISULTATI E DISCUSSIONE I. Sviluppo di strategie per veicolare le molecole in studio alla matrice mitocondriale Abbiamo disegnato e testato i seguenti 5 costrutti che hanno mostrato una co-localizzazione mitocondriale in assenza di tossicità cellulare: 1. FRFK-β32_33-FITC (Fβ): composto da un piccolo oligomero sintetico in grado di penetrare le membrane cellulari e mitocondriali (FRFK) e dalla sequenza β32_33; 2. FRFK-scramble-FITC (FS): composto da FRFK e dalla sequenza β32_33 variata nell’ordine dei residui amminoacidici; 3. FRFK-β32_33 (noKKR)-FITC (FA): un composto in cui i tre residui carichi positivamente (K-K-R) della sequenza β32_33 vengono sostituiti da residui neutri di alanina (A); 4. β32_33-FITC (β): composto dalla sola sequenza β32_33; 5. FRFK-FITC (F): composto dalla sola presequenza FRFK da utilizzare come controllo negativo. Abbiamo successivamente valutato l’effetto dei peptidi sulla vitalità cellulare e sul consumo di ossigeno dei cibridi dimostrando come esclusivamente il costrutto Fβ fosse in grado di migliorare il fenotipo dei cibridi MELAS. Un dato interessante è che né FS né FA siano in grado di migliorare il difetto biochimico dei cibridi; questo risultato permette di ipotizzare come la funzione del peptide β32_33 dipenda sia dall’ordine dei residui amminoacidici che dal numero delle cariche positive. Stante la prospettiva futura di veicolare nelle cellule le molecole terapeutiche oggetto del nostro studio, abbiamo parallelamente iniziato la ricerca di un ipotetico carrier, focalizzando la nostra attenzione sulla ferritina umana. La molecola di ferritina è stata coniugata al frammento β32_33 sia in presenza che in assenza di una sequenza di escape lisosomiale (RLA), non mostrando tuttavia in nessuna di queste condizioni una localizzazione mitocondriale. Per favorirne la localizzazione mitocondriale la ferritina è stata quindi fusa a una molecola di targeting mitocondriale MTS (Cox8A) che, tuttavia, ad oggi non siamo riusciti ancora ad esprimere nei ceppi di E. Coli. II. Analisi dell’effetto tessuto-specifico delle mutazioni dei mt-tRNA ed individuazione di eventuali meccanismi terapeutici dotati di specificità tissutale Per la produzione di iPSCs abbiamo selezionato fibroblasti di pazienti con sindrome MELAS con una percentuale di eteroplasmia pari a circa il 50% ottenendo, dopo 30 giorni, colonie mature che mostravano positività al marcatore di staminalità TRA 1-60. Dopo l’espansione abbiamo ottenuto 12 colonie di cui 7 costituite da cellule staminali pluripotenti, identificate attraverso pCR. Dopo averne verificato i livelli di eteroplasmia (compresi tra il 7% e il 77%), per gli studi successivi di differenziamento abbiamo deciso di utilizzare la colonia con la percentuale maggiore (77%, iMS-4) e quella con la percentuale minore (7%, iMS-1) da utilizzare come controllo isogenico. Tali colonie sono state inoculate in topi immunodepressi portando allo sviluppo di teratomi. Una volta caratterizzate nel dettaglio le colonie, abbiamo iniziato il differenziamento delle iPSCs in neuroni ottenendo inizialmente cloni di cellule staminali neurali (NSCs, dato confermato dalla positività per diversi markers neurali). Ad oggi è in corso il differenziamento di tali cellule in neuroni. Gli studi successivi saranno incentrati sull’analisi dettagliata del fenotipo di questi nuovi modelli cellulari e sullo studio dell’effetto tessuto-specifico dei nostri costrutti

Can IDO activity predict primary resistance to anti-PD-1 treatment in NSCLC?

BACKGROUND: Immune checkpoint inhibitors have revolutionized the treatment paradigm of highly lethal malignancies like advanced non-small cell lung cancer (NSCLC), demonstrating long-term tumour control and extended patient survival. Unfortunately, only 25-30% of patients experience a durable benefit, while the vast majority demonstrate primary or acquired resistance. Recently, indoleamine 2,3-dioxygenase (IDO) activity has been proposed as a possible mechanism of resistance to anti-PD-1 treatment leading to an immunosuppressive microenvironment. METHODS: Pre-treatment serum concentrations of tryptophan (trp) and kynurenine (kyn) were measured by high-performance liquid chromatography tandem mass spectrometry in NSCLC patients treated with second-line nivolumab. The IDO activity was expressed with kyn/trp ratio. The associations between kyn/trp ratio and early progression, performance status (PS), age, sex, brain metastases, pleural effusion, progression free survival (PFS) and overall survival (OS) were analyzed using Spearman test and Mann-Whitney test. RESULTS: Twenty-six NSCLC patients were included in our study; 14 of them (54%) presented early progression (< 3 months) to nivolumab treatment. The median value of kyn/trp ratio was 0.06 µg/ml and the median value of quinolinic acid was 68.45 ng/ml. A significant correlation between early progression and higher kyn/trp ratio and quinolinic acid concentration was observed (p = 0.017 and p = 0.005, respectively). Patients presenting lower values of kyn/trp ratio and quinolinic acid levels showed longer PFS (median PFS not reached versus 3 months; HR: 0.3; p = 0.018) and OS (median OS not reached vs 3 months; HR: 0.18; p = 0.0005). CONCLUSION: IDO activity, expressed as kyn/trp ratio, is associated with response to immunotherapy; in particular, higher kyn/trp ratio could predict resistance to anti-PD-1 treatment. These preliminary results suggest the possibility of using anti-PD-1 plus IDO inhibitor in those patients with high level of kyn/trp ratio

Nonischemic left ventricular scar and cardiac sudden death in the young

Nonischemic Left Ventricular Scar (NLVS) is a pattern of myocardial injury characterized by midventricular and/or subepicardial gadolinium hyper enhancement at cardiac magnetic resonance, in absence of significant coronary artery disease. We aimed to evaluate the prevalence of NLVS in juvenile sudden cardiac death and to ascertain its aetiology at autopsy. We examined 281 consecutive cases of sudden death of subjects aged 1 to 35 years of age. NLVS was defined as a thin, grey rim of subepicardial and/or midmyocardial scar in the left ventricular free wall and/or the septum, in absence of significant stenosis of coronary arteries. NLVS was the most frequent finding (25%) in sudden deaths occurring during sports. Myocardial scar was localized most frequently within the left ventricular posterior wall, and affected the subepicardial myocardium, often extending to the midventricular layer. On histology it consisted of fibrous or fibro-adipose tissue. Right ventricular involvement was always present. Patchy lymphocytic infiltrates were frequent. Genetic and molecular analyses clarified the aetiology of NLVS in a subset of cases. ECG recordings were available in over half of subjects. The most frequent abnormality was the presence of low QRS voltages (< 0,5 mV) in limb leads. In serial ECG tracings, the decrease in QRS voltages appeared, in some way progressive. NLVS is the most frequent morphologic substrate of juvenile cardiac sudden death in sports. It can be suspected based on ECG findings. Autopsy study and clinical screening of family members are required to differentiate between Arrhythmogenic Right Ventricular Cardiomyopathy/Dysplasia and chronic acquired myocarditis

Degradation rate of 5-fluorouracil in metastatic colorectal cancer. A new predictive outcome biomarker?

BACKGROUND: 5-FU based chemotherapy is the most common first line regimen used for metastatic colorectal cancer (mCRC). Identification of predictive markers of response to chemotherapy is a challenging approach for drug selection. The present study analyzes the predictive role of 5-FU degradation rate (5-FUDR) and genetic polymorphisms (MTHFR, TSER, DPYD) on survival. MATERIALS AND METHODS: Genetic polymorphisms of MTHFR, TSER and DPYD, and the 5-FUDR of homogenous patients with mCRC were retrospectively studied. Genetic markers and the 5-FUDR were correlated with clinical outcome. RESULTS: 133 patients affected by mCRC, treated with fluoropyrimidine-based chemotherapy from 2009 to 2014, were evaluated. Patients were classified into three metabolic classes, according to normal distribution of 5-FUDR in more than 1000 patients, as previously published: poor-metabolizer (PM) with 5-FU-DR ≤ 0,85 ng/ml/106 cells/min (8 pts); normal metabolizer with 0,85 < 5-FU-DR < 2,2 ng/ml/106 cells/min (119 pts); ultra-rapid metabolizer (UM) with 5-FU-DR ≥ 2,2 ng/ml/106 cells/min (6 pts). PM and UM groups showed a longer PFS respect to normal metabolizer group (14.5 and 11 months respectively vs 8 months; p = 0.029). A higher G3-4 toxicity rate was observed in PM and UM, respect to normal metabolizer (50% in both PM and UM vs 18%; p = 0.019). No significant associations between genes polymorphisms and outcomes or toxicities were observed. CONCLUSION: 5-FUDR seems to be significantly involved in predicting survival of patients who underwent 5-FU based CHT for mCRC. Although our findings require confirmation in large prospective studies, they reinforce the concept that individual genetic variation may allow personalized selection of chemotherapy to optimize clinical outcomes

Atypical cellular neurothekeoma (ACN) of the elderly: case report and brief review of the literature

Atypical cellular neurothekeoma (ACN) is an aggressive and rare variant of cellular neurothekeoma. Only few cases have been reported in the literature and the biological behavior seems to be uncertain. We describe the case of an ACN presenting on the scalp of an elderly man, emphasizing the cytologic features of malignancy. In addition, we provide a brief overview of the literature and discuss the differential diagnosis with other entities, and the possible diagnostic pitfalls

Neuromuscular magnetic stimulation counteracts muscle decline in ALS patients: results of a randomized, double-blind, controlled study

The aim of the study was to verify whether neuromuscular magnetic stimulation (NMMS) improves muscle function in spinal-onset amyotrophic lateral sclerosis (ALS) patients. Twenty-two ALS patients were randomized in two groups to receive, daily for two weeks, NMMS in right or left arm (referred to as real-NMMS, rNMMS), and sham NMMS (sNMMS) in the opposite arm. All the patients underwent a median nerve conduction (compound muscle action potential, CMAP) study and a clinical examination that included a handgrip strength test and an evaluation of upper limb muscle strength by means of the Medical Research Council Muscle Scale (MRC). Muscle biopsy was then performed bilaterally on the flexor carpi radialis muscle to monitor morpho-functional parameters and molecular changes. Patients and physicians who performed examinations were blinded to the side of real intervention. The primary outcome was the change in the muscle strength in upper arms. The secondary outcomes were the change from baseline in the CMAP amplitudes, in the nicotinic ACh currents, in the expression levels of a selected panel of genes involved in muscle growth and atrophy, and in histomorphometric parameters of ALS muscle fibers. The Repeated Measures (RM) ANOVA with a Greenhouse-Geisser correction (sphericity not assumed) showed a significant effect [F(3, 63) = 5.907, p < 0.01] of rNMMS on MRC scale at the flexor carpi radialis muscle, thus demonstrating that the rNMMS significantly improves muscle strength in flexor muscles in the forearm. Secondary outcomes showed that the improvement observed in rNMMS-treated muscles was associated to counteracting muscle atrophy, down-modulating the proteolysis, and increasing the efficacy of nicotinic ACh receptors (AChRs). We did not observe any significant difference in pre- and post-stimulation CMAP amplitudes, evoked by median nerve stimulation. This suggests that the improvement in muscle strength observed in the stimulated arm is unlikely related to reinnervation. The real and sham treatments were well tolerated without evident side effects. Although promising, this is a proof of concept study, without an immediate clinical translation, that requires further clinical validation

The role of opioids in cancer response to immunotherapy

BACKGROUND: The response to immunotherapy can be impaired by several factors including external intervention such as drug interactions with immune system. We aimed to examine the immunomodulatory action of opioids, since immune cells express opioid receptors able to negatively influence their activities.METHODS: This observational, multicenter, retrospective study, recruited patients with different metastatic solid tumors, who have received immunotherapy between September 2014 and September 2019. Immunotherapy was administered according to the standard schedule approved for each primary tumor and line of treatment. The concomitant intake of antibiotics, antifungals, corticosteroids and opioids were evaluated in all included patients. The relationship between tumor response to immunotherapy and the oncological outcomes were evaluated. A multivariate Cox-proportional hazard model was used to identify independent prognostic factors for survival.RESULTS: One hundred ninety-three patients were recruited. Overall, progression-free survival (PFS) and overall survival (OS) were significantly shorter in those patients taking opioids than in those who didn't (median PFS, 3months vs. 19months, HR 1.70, 95% CI 1.37-2.09, p&lt;0.0001; median OS, 4months vs. 35months, HR 1.60, 95% CI 1.26-2.02, p&lt;0.0001). In addition, PFS and OS were significantly impaired in those patients taking corticosteroids, antibiotics or antifungals, in those patients with an ECOG PS≥1 and in patients with a high tumor burden. Using the multivariate analyses, opioids and ECOG PS were independent prognostic factors for PFS, whereas only ECOG PS resulted to be an independent prognostic factor for OS, with trend toward significance for opioids as well as tumor burden.DISCUSSION: Our study suggests that the concomitant administration of drugs as well as some clinical features could negatively predict the outcomes of cancer patients receiving immunotherapy. In particular, opioids use during immunotherapy is associated with early progression, potentially representing a predictive factor for PFS and negatively influencing OS as well.CONCLUSIONS: A possible negative drug interaction able to impair the immune response to anti-PD-1/PD-L1 agents has been highlighted. Our findings suggest the need to further explore the impact of opioids on immune system modulation and their role in restoring the response to immunotherapy treatment, thereby improving patients' outcomes

Impaired mitochondrial biogenesis is a common feature to myocardial hypertrophy and end-stage ischemic heart failure

Mitochondrial (mt) DNA depletion and oxidative mtDNA damage have been implicated in the process of pathological cardiac remodeling. Whether these features are present in the early phase of maladaptive cardiac remodeling, that is, during compensated cardiac hypertrophy, is still unknown. We compared the morphologic and molecular features of mt biogenesis and markers of oxidative stress in human heart from adult subjects with compensated hypertrophic cardiomyopathy and heart failure. We have shown that mtDNA depletion is a constant feature of both conditions. A quantitative loss of mtDNA content was associated with significant down-regulation of selected modulators of mt biogenesis and decreased expression of proteins involved in mtDNA maintenance. Interestingly, mtDNA depletion characterized also the end-stage phase of cardiomyopathies due to a primary mtDNA defect. Oxidative stress damage was detected only in failing myocardium

Novel compound mutations in the mitochondrial translation elongation factor (TSFM) gene cause severe cardiomyopathy with myocardial fibro-adipose replacement

Primary mitochondrial dysfunction is an under-appreciated cause of cardiomyopathy, especially when cardiac symptoms are the unique or prevalent manifestation of disease. Here, we report an unusual presentation of mitochondrial cardiomyopathy, with dilated phenotype and pathologic evidence of biventricular fibro-adipose replacement, in a 33-year old woman who underwent cardiac transplant. Whole exome sequencing revealed two novel compound heterozygous variants in the TSFM gene, coding for the mitochondrial translation elongation factor EF-Ts. This protein participates in the elongation step of mitochondrial translation by binding and stabilizing the translation elongation factor Tu (EF-Tu). Bioinformatics analysis predicted a destabilization of the EF-Ts variants complex with EF-Tu, in agreement with the dramatic steady-state level reduction of both proteins in the clinically affected myocardium, which demonstrated a combined respiratory chain enzyme deficiency. In patient fibroblasts, the decrease of EF-Ts was paralleled by up-regulation of EF-Tu and induction of genes involved in mitochondrial biogenesis, along with increased expression of respiratory chain subunits and normal oxygen consumption rate. Our report extends the current picture of morphologic phenotypes associated with mitochondrial cardiomyopathies and confirms the heart as a main target of TSFM dysfunction. The compensatory response detected in patient fibroblasts might explain the tissue-specific expression of TSFM-associated disease

Involvement of substance P (SP) and its related NK1 receptor in primary Sjögren’s syndrome (pSS) pathogenesis

Primary Sjogren's Syndrome (pSS) is a systemic autoimmune disease that primarily attacks the lacrimal and salivary glands, resulting in impaired secretory function characterized by xerostomia and xerophthalmia. Patients with pSS have been shown to have impaired salivary gland innervation and altered circulating levels of neuropeptides thought to be a cause of decreased salivation, including substance P (SP). Using Western blot analysis and immunofluorescence studies, we examined the expression levels of SP and its preferred G protein-coupled TK Receptor 1 (NK1R) and apoptosis markers in biopsies of the minor salivary gland (MSG) from pSS patients compared with patients with idiopathic sicca syndrome. We confirmed a quantitative decrease in the amount of SP in the MSG of pSS patients and demonstrated a significant increase in NK1R levels compared with sicca subjects, indicating the involvement of SP fibers and NK1R in the impaired salivary secretion observed in pSS patients. Moreover, the increase in apoptosis (PARP-1 cleavage) in pSS patients was shown to be related to JNK phosphorylation. Since there is no satisfactory therapy for the treatment of secretory hypofunction in pSS patients, the SP pathway may be a new potential diagnostic tool or therapeutic target