Primary mitochondrial myopathy: Clinical features and outcome measures in 118 cases from Italy

Objective: To determine whether a set of functional tests, clinical scales, patient-reported questionnaires, and specific biomarkers can be considered reliable outcome measures in patients with primary mitochondrial myopathy (PMM), we analyzed a cohort of Italian patients. Methods: Baseline data were collected from 118 patients with PMM, followed by centers of the Italian network for mitochondrial diseases. We used the 6-Minute Walk Test (6MWT), Timed Up-and-Go Test (x3) (3TUG), Five-Times Sit-To-Stand Test (5XSST), Timed Water Swallow Test (TWST), and Test of Masticating and Swallowing Solids (TOMASS) as functional outcome measures; the Fatigue Severity Scale and West Haven-Yale Multidimensional Pain Inventory as patient-reported outcome measures; and FGF21, GDF15, lactate, and creatine kinase (CK) as biomarkers. Results: A total of 118 PMM cases were included. Functional outcome measures (6MWT, 3TUG, 5XSST, TWST, and TOMASS) and biomarkers significantly differed from healthy reference values and controls. Moreover, functional measures correlated with patients' perceived fatigue and pain severity. Patients with either mitochondrial or nuclear DNA point mutations performed worse in functional measures than patients harboring single deletion, even if the latter had an earlier age at onset but similar disease duration. Both the biomarkers FGF21 and GDF15 were significantly higher in the patients compared with a matched control population; however, there was no relation with severity of disease. Conclusions: We characterized a large cohort of PMM by evaluating baseline mitochondrial biomarkers and functional scales that represent potential outcome measures to monitor the efficacy of treatment in clinical trials; these outcome measures will be further reinvestigated longitudinally to define the natural history of PMM

Distribution of Exonic Variants in Glycogen Synthesis and Catabolism Genes in Late Onset Pompe Disease (LOPD)

Pompe disease (PD) is a monogenic autosomal recessive disorder caused by biallelic pathogenic variants of the GAA gene encoding lysosomal alpha-glucosidase; its loss causes glycogen storage in lysosomes, mainly in the muscular tissue. The genotype-phenotype correlation has been extensively discussed, and caution is recommended when interpreting the clinical significance of any mutation in a single patient. As there is no evidence that environmental factors can modulate the phenotype, the observed clinical variability in PD suggests that genetic variants other than pathogenic GAA mutations influence the mechanisms of muscle damage/repair and the overall clinical picture. Genes encoding proteins involved in glycogen synthesis and catabolism may represent excellent candidates as phenotypic modifiers of PD. The genes analyzed for glycogen synthesis included UGP2, glycogenin (GYG1-muscle, GYG2, and other tissues), glycogen synthase (GYS1-muscle and GYS2-liver), GBE1, EPM2A, NHLRC1, GSK3A, and GSK3B. The only enzyme involved in glycogen catabolism in lysosomes is alpha-glucosidase, which is encoded by GAA, while two cytoplasmic enzymes, phosphorylase (PYGB-brain, PGL-liver, and PYGM-muscle) and glycogen debranching (AGL) are needed to obtain glucose 1-phosphate or free glucose. Here, we report the potentially relevant variants in genes related to glycogen synthesis and catabolism, identified by whole exome sequencing in a group of 30 patients with late-onset Pompe disease (LOPD). In our exploratory analysis, we observed a reduced number of variants in the genes expressed in muscles versus the genes expressed in other tissues, but we did not find a single variant that strongly affected the phenotype. From our work, it also appears that the current clinical scores used in LOPD do not describe muscle impairment with enough qualitative/quantitative details to correlate it with genes that, even with a slightly reduced function due to genetic variants, impact the phenotype

Lipomatosis Incidence and Characteristics in an Italian Cohort of Mitochondrial Patients.

Lipomas have often been associated with mtDNA mutations and were mainly observed in patients with mutation in mitochondrial tRNAlysine which is also the most frequent mutation associated with MERRF. Up to date, no systematic studies have been developed in order to assess the incidence of lipomas in large cohorts of mitochondrial patients.The aim of this study is to analyze the incidence and characteristics of lipomas among an Italian cohort of patients with mitochondrial diseases. A retrospective, database-based study (Nation-wide Italian Collaborative Network of Mitochondrial Diseases) of patients with lipomas was performed. A total of 22 (1.7%) patients with lipomas have been identified among the 1,300 mitochondrial patients, enrolled in the Italian database. In about 18% multiple systemic lipomatosis (MSL) was the only clinical manifestation; 54% of patients showed a classical MERRF syndrome. Myopathy, alone or in association with other symptoms, was found in 27% of patients. Lactate was elevated in all the 12 patients in which was measured. Muscle biopsy was available in 18/22 patients: in all of them mitochondrial abnormalities were present. Eighty six percent had mutations in mtDNA coding for tRNA lysine. In most of patients, lipomas were localized along the cervical-cranial-thoracic region. In 68% of the patients were distributed symmetrically. Only two patients had lipomas in a single anatomical site (1 in right arm and 1 in gluteus maximum). MSL is often overlooked by clinicians in patients with mitochondrial diseases where the clinical picture could be dominated by a severe multi-systemic involvement. Our data confirmed that MSL is a rare sign of mitochondrial disease with a strong association between multiple lipomas and lysine tRNA mutations. MSL could be considered, even if rare, a red flag for mitochondrial disorders, even in patients with an apparently isolated MSL

Drug Repurposing in Pediatric Brain Tumors: Posterior Fossa Ependymoma and Diffuse Midline Glioma under the Looking Glass

Tumors of the Central Nervous System (CNS) represent the leading cause of cancer-related deaths in children. Current treatment options are not curative for most malignant histologies, and intense preclinical and clinical research is needed to develop more effective therapeutic interventions against these tumors, most of which meet the FDA definition for orphan diseases. Increased attention is being paid to the repositioning of already-approved drugs for new anticancer indications as a fast-tracking strategy for identifying new and more effective therapies. Two pediatric CNS tumors, posterior fossa ependymoma (EPN-PF) type A and diffuse midline glioma (DMG) H3K27-altered, share loss of H3K27 trimethylation as a common epigenetic hallmark and display early onset and poor prognosis. These features suggest a potentially common druggable vulnerability. Successful treatment of these CNS tumors raises several challenges due to the location of tumors, chemoresistance, drug blood-brain barrier penetration, and the likelihood of adverse side effects. Recently, increasing evidence demonstrates intense interactions between tumor cell subpopulations and supportive tumor microenvironments (TMEs) including nerve, metabolic, and inflammatory TMEs. These findings suggest the use of drugs, and/or multi-drug combinations, that attack both tumor cells and the TME simultaneously. In this work, we present an overview of the existing evidence concerning the most preclinically validated noncancer drugs with antineoplastic activity. These drugs belong to four pharmacotherapeutic classes: antiparasitic, neuroactive, metabolic, and anti-inflammatory. Preclinical evidence and undergoing clinical trials in patients with brain tumors, with special emphasis on pediatric EPN-PF and DMG, are summarized and critically discussed

Cell-of-Origin and Genetic, Epigenetic, and Microenvironmental Factors Contribute to the Intra-Tumoral Heterogeneity of Pediatric Intracranial Ependymoma

Intra-tumoral heterogeneity (ITH) is a complex multifaceted phenomenon that posits major challenges for the clinical management of cancer patients. Genetic, epigenetic, and microenvironmental factors are concurrent drivers of diversity among the distinct populations of cancer cells. ITH may also be installed by cancer stem cells (CSCs), that foster unidirectional hierarchy of cellular phenotypes or, alternatively, shift dynamically between distinct cellular states. Ependymoma (EPN), a molecularly heterogeneous group of tumors, shows a specific spatiotemporal distribution that suggests a link between ependymomagenesis and alterations of the biological processes involved in embryonic brain development. In children, EPN most often arises intra-cranially and is associated with an adverse outcome. Emerging evidence shows that EPN displays large intra-patient heterogeneity. In this review, after touching on EPN inter-tumoral heterogeneity, we focus on the sources of ITH in pediatric intra-cranial EPN in the framework of the CSC paradigm. We also examine how single-cell technology has shed new light on the complexity and developmental origins of EPN and the potential impact that this understanding may have on the therapeutic strategies against this deadly pediatric malignancy

UPTAKE AND STORAGE OF M-IODOBENZYLGUANIDINE ARE FREQUENT NEURONAL FUNCTIONS OF HUMAN NEUROBLASTOMA CELL-LINES

The mechanisms of the uptake and release of m-iodobenzylguanidine (MIGB) have been studied in 5 neuroblastoma (NB) cell lines and in 4 clonal NB sublines with a homogeneous phenotype. A specific uptake system for MIBG was found in 8 of 9 NB cell lines or subpopulations. The uptake was characterized by temperature dependency, high affinity, saturability, sodium dependency, and imipramine sensitivity. The majority of NB cell lines that possessed a specific uptake system for MIBG were also able to efficiently store the incorporated drug. However, 3 NB cell lines were identified without the ability to retain high levels of MIBG, despite the presence of a specific uptake system. We also report that a clonal subline, SH-EP1, which has a nonneuroblastic phenotype, failed both MIBG uptake and retention. Conversely, the parental cell line, SK-N-SH, and the neuroblastic subline SH-SY5Y possessed both a specific uptake system and the ability to store MIBG. In addition, the induction of neuronal differentiation with retinoic acid increased the velocity of uptake and the storage efficiency for MIBG in the clonal subline SH-SY5Y. We conclude that MIBG uptake and storage should be considered to be frequent but independent neuronal functions of human NB cells

Chemoresistant tumor cell lines display altered epidermal growth factor receptor and HER3 signaling and enhanced sensitivity to Gefitinib.

Deregulated signaling through the epidermal growth factor receptor (EGFR) is involved in chemoresistance. To identify the molecular determinants of sensitivity to the EGFR inhibitor gefitinib (Iressa, ZD1839) in chemoresistance, we compared the response of matched chemosensitive and chemoresistant glioma and ovarian cancer cell lines. We found that chemoresistant cell lines were 2- to 3-fold more sensitive to gefitinib growth-inhibitory effects, because of decreased proliferation rather than survival. Sensitivity to gefitinib correlated with overexpression and constitutive phosphorylation of HER2 and HER3, but not EGFR, altered HER ligand expression, and enhanced activation of EGF-triggered EGFR pathway. No activating mutations were found in EGFR. Gefitinib fully inhibited EGF-induced and constitutive Akt activation only in chemoresistant cells. In parallel, gefitinib downregulated constitutively phosphorylated HER2 and HER3, and activated GSK3beta with a concomitant degradation of cyclin D1. Ectopically overexpressed HER2 on its own was insufficient to sensitize chemonaive cells to gefitinib. pHER3 coimmunoprecipitated with p85-PI3K in chemoresistant cells and gefitinib dissociated these complexes. siRNA-mediated inhibition of HER3 decreased constitutive activation of Akt and sensitivity to gefitinib in chemoresistant cells. Our study indicates that in chemoresistant cells gefitinib inhibits both an enhanced EGF-triggered pathway and a constitutive HER3-mediated Akt activation, indicating that inhibition of HER3 together with that of EGFR could be relevant in chemorefractory tumors. Furthermore, in combination experiments gefitinib enhanced the effects of coadministered drugs more in chemoresistant than chemosensitive ovarian cancer cells. Combined treatment might be therapeutically beneficial in chemoresistant tumors from ovary and likely from other tissues

Dual Inhibitor AEE788 Reduces Tumor Growth in Preclinical Models of Medulloblastoma1

Medulloblastoma is the most frequent malignant pediatric brain tumor with a dismal prognosis in 30% of cases. We examined the activity of AEE788, a dual inhibitor of human epidermal receptor (HER) 1/2 and vascular endothelial growth factor receptor (VEGFR) 1/2, in medulloblastoma preclinical models. Established lines (Daoy and D283), chemoresistant (DaoyPt), and ectopically HER2-overexpressing (DaoyHER2) cells expressed diverse levels of total and activated AEE788 target receptors. In vitro, AEE788 inhibited cell proliferation (IC50 from 1.7 to 3.8 µM) and prevented epidermal growth factor- and neuregulin-induced HER1, HER2, and HER3 activation. Inhibition of Akt paralleled that of HER receptors. In vivo, AEE788 growth inhibited Daoy, DaoyPt, and DaoyHER2 xenografts by 51%, 45%, and 72%, respectively. Immunohistochemical analysis of mock- and HER2-transfected xenografts revealed that the latter showed, along with high HER2 expression, high VEGFR2 staining in tumor and endothelial cells and increased expression of the endothelial marker CD31. AEE788 reduced the activation of target receptors and angiogenesis. In 21 primary medulloblastoma, HER2 expression significantly correlated (P < .01) with VEGFR2 (r = 0.56) and VEGF (r = 0.61). In conclusion, AEE788 shows similar growth-suppressive activities in chemosensitive and chemoresistant medulloblastoma cells in vitro and in vivo. Ectopic HER2 overexpression sensitizes cells to AEE788 in vivo, but not in vitro, possibly through host-mediated processes. Together with the experimental data, the finding that HER2 positively correlates with VEGFR2 and VEGF in human medulloblastoma specimens indicates HER2-overexpressing medulloblastoma as the subset that most likely might benefit from AEE788 treatment

Antitumor effect in medulloblastoma cells by gefitinib: Ectopic HER2 overexpression enhances gefitinib effects in vivo

The effects of the epidermal growth factor receptor (EGFR) inhibitor gefitinib on cell growth and signaling were evaluated in three medulloblastoma (MB) cell lines (D283, D341, Daoy), one supratentorial primitive neuroectodermal tumor cell line (PFSK), and four MB primary cultures. Cell lines showed diverse expression of EGFR and human epidermal receptor 2 (HER2), with high levels of constitutively activated HER2 in the HER2-overexpressing D341 and D283 cells. Gefitinib sensitivity varied across lines and was not related to expression of HER receptors or receptor baseline activation. Gefitinib induced G0/G1 arrest in all lines, whereas apoptosis was dose-dependently induced only in D283 and D341 cells. The molecular response to gefitinib was investigated in Daoy and D341 lines, which showed a higher (half-maximal inhibitory concentration [IC50], 3.8 μM) and lower (IC50, 6.6 μM) sensitivity to the agent, respectively. Gefitinib inhibited constitutive and EGF-triggered EGFR phosphorylation in both lines but was ineffective in constitutive activation of HER2 in D341 cells. Phosphorylated AKT inhibition paralleled that of phosphorylated EGFR, suggesting the presence of an autocrine gefitinib-sensitive EGFR/AKT pathway. On the whole, EGF-dependent signaling was less responsive to ligand stimulation and gefitinib inhibition in D341 cells, which correlated with the lower sensitivity to gefitinib’s antiproliferative effect of this line. In vivo, the growth of D341 and Daoy xenografts treated with gefitinib at 150 mg/kg per day was inhibited by approximately 50%. Ectopically overexpressed HER2 in Daoy cells significantly increased sensitivity to gefitinib’s antitumor effects in vivo (tumor volume inhibition = 78%). Our data indicate that gefitinib might be a molecularly targeted agent for the treatment of MB