Riboflavin Deficiency-Implications for General Human Health and Inborn Errors of Metabolism.

As an essential vitamin, the role of riboflavin in human diet and health is increasingly being highlighted. Insufficient dietary intake of riboflavin is often reported in nutritional surveys and population studies, even in non-developing countries with abundant sources of riboflavin-rich dietary products. A latent subclinical riboflavin deficiency can result in a significant clinical phenotype when combined with inborn genetic disturbances or environmental and physiological factors like infections, exercise, diet, aging and pregnancy. Riboflavin, and more importantly its derivatives, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), play a crucial role in essential cellular processes including mitochondrial energy metabolism, stress responses, vitamin and cofactor biogenesis, where they function as cofactors to ensure the catalytic activity and folding/stability of flavoenzymes. Numerous inborn errors of flavin metabolism and flavoenzyme function have been described, and supplementation with riboflavin has in many cases been shown to be lifesaving or to mitigate symptoms. This review discusses the environmental, physiological and genetic factors that affect cellular riboflavin status. We describe the crucial role of riboflavin for general human health, and the clear benefits of riboflavin treatment in patients with inborn errors of metabolism.

CLPB variants associated with autosomal-recessive mitochondrial disorder with cataract, neutropenia, epilepsy, and methylglutaconic aciduria.

3-methylglutaconic aciduria (3-MGA-uria) is a nonspecific finding associated with mitochondrial dysfunction, including defects of oxidative phosphorylation. 3-MGA-uria is classified into five groups, of which one, type IV, is genetically heterogeneous. Here we report five children with a form of type IV 3-MGA-uria characterized by cataracts, severe psychomotor regression during febrile episodes, epilepsy, neutropenia with frequent infections, and death in early childhood. Four of the individuals were of Greenlandic descent, and one was North American, of Northern European and Asian descent. Through a combination of homozygosity mapping in the Greenlandic individuals and exome sequencing in the North American, we identified biallelic variants in the caseinolytic peptidase B homolog (CLPB). The causative variants included one missense variant, c.803C>T (p.Thr268Met), and two nonsense variants, c.961A>T (p.Lys321*) and c.1249C>T (p.Arg417*). The level of CLPB protein was markedly decreased in fibroblasts and liver of affected individuals. CLPB is proposed to function as a mitochondrial chaperone involved in disaggregation of misfolded proteins, resulting from stress such as heat denaturation.

Metformin targets brown adipose tissue in vivo and reduces oxygen consumption in vitro.

AIMS: To test the hypothesis that brown adipose tissue (BAT) is a metformin target tissue by investigating in vivo uptake of [11 C]-metformin tracer in mice and studying in vitro effects of metformin on cultured human brown adipocytes. MATERIALS AND METHODS: Tissue-specific uptake of metformin was assessed in mice by PET/CT imaging after injection of [11 C]-metformin. Human brown adipose tissue was obtained from elective neck surgery and metformin transporter expression levels in human and murine BAT were determined by qPCR. Oxygen consumption in metformin-treated human brown adipocyte cell models was assessed by Seahorse XF technology. RESULTS: Injected [11 C]-metformin showed avid uptake in the murine interscapular BAT depot. Metformin exposure in BAT was similar to hepatic exposure. Non-specific inhibition of the organic cation transporter (OCT) protein by cimetidine administration eliminated BAT exposure to metformin, demonstrating OCT-mediated uptake. Gene expression profiles of OCTs in BAT revealed ample OCT3 expression in both human and mouse BAT. Incubation of a human brown adipocyte cell models with metformin reduced cellular oxygen consumption in a dose-dependent manner. CONCLUSION: These results support BAT as a putative metformin target.

Enhanced genome editing in mammalian cells with a modified dual-fluorescent surrogate system.

Programmable DNA nucleases such as TALENs and CRISPR/Cas9 are emerging as powerful tools for genome editing. Dual-fluorescent surrogate systems have been demonstrated by several studies to recapitulate DNA nuclease activity and enrich for genetically edited cells. In this study, we created a single-strand annealing-directed, dual-fluorescent surrogate reporter system, referred to as C-Check. We opted for the Golden Gate Cloning strategy to simplify C-Check construction. To demonstrate the utility of the C-Check system, we used the C-Check in combination with TALENs or CRISPR/Cas9 in different scenarios of gene editing experiments. First, we disrupted the endogenous pIAPP gene (3.0 % efficiency) by C-Check-validated TALENs in primary porcine fibroblasts (PPFs). Next, we achieved gene-editing efficiencies of 9.0-20.3 and 4.9 % when performing single- and double-gene targeting (MAPT and SORL1), respectively, in PPFs using C-Check-validated CRISPR/Cas9 vectors. Third, fluorescent tagging of endogenous genes (MYH6 and COL2A1, up to 10.0 % frequency) was achieved in human fibroblasts with C-Check-validated CRISPR/Cas9 vectors. We further demonstrated that the C-Check system could be applied to enrich for IGF1R null HEK293T cells and CBX5 null MCF-7 cells with frequencies of nearly 100.0 and 86.9 %, respectively. Most importantly, we further showed that the C-Check system is compatible with multiplexing and for studying CRISPR/Cas9 sgRNA specificity. The C-Check system may serve as an alternative dual-fluorescent surrogate tool for measuring DNA nuclease activity and enrichment of gene-edited cells, and may thereby aid in streamlining programmable DNA nuclease-mediated genome editing and biological research.

Do lamin A and lamin C have unique roles?

The A-type lamins, lamin A and lamin C, generated from a single gene, LMNA, are major structural components of the nuclear lamina. The two alternative splice products have mostly been studied together because they have been considered to be interchangeable. However, several lines of evidence indicate that in spite of being generated from the same gene and having high similarities in their primary sequences, the two isoforms are not equivalent in different biological aspects in both health and disease. The key question is whether they have both overlapping and unique functions and whether they are distinctly regulated. Based on the so far available experimental evidence, lamin A appears to be the most regulated A-type isoform during development, aging, and disease which indicates that lamin A is implicated in many different biological aspects and may have a greater repertoire of specialized functions than lamin C. The aim of this review is to point out differences between the two major LMNA splice variants and the consequences of these differences on their functions. This may guide further research and be of prime importance for the understanding of the pathogenesis of LMNA mutations.

Molecular mechanisms of riboflavin responsiveness in patients with ETF-QO variations and multiple acyl-CoA dehydrogenation deficiency.

Riboflavin-responsive forms of multiple acyl-CoA dehydrogenation deficiency (RR-MADD) have been known for years, but with presumed defects in the formation of the flavin adenine dinucleotide (FAD) co-factor rather than genetic defects of electron transfer flavoprotein (ETF) or electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO). It was only recently established that a number of RR-MADD patients carry genetic defects in ETF-QO and that the well-documented clinical efficacy of riboflavin treatment may be based on a chaperone effect that can compensate for inherited folding defects of ETF-QO. In the present study, we investigate the molecular mechanisms and the genotype-phenotype relationships for the riboflavin responsiveness in MADD, using a human HEK-293 cell expression system. We studied the influence of riboflavin and temperature on the steady-state level and the activity of variant ETF-QO proteins identified in patients with RR-MADD, or non- and partially responsive MADD. Our results showed that variant ETF-QO proteins associated with non- and partially responsive MADD caused severe misfolding of ETF-QO variant proteins when cultured in media with supplemented concentrations of riboflavin. In contrast, variant ETF-QO proteins associated with RR-MADD caused milder folding defects when cultured at the same conditions. Decreased thermal stability of the variants showed that FAD does not completely correct the structural defects induced by the variation. This may cause leakage of electrons and increased reactive oxygen species, as reflected by increased amounts of cellular peroxide production in HEK-293 cells expressing the variant ETF-QO proteins. Finally, we found indications of prolonged association of variant ETF-QO protein with the Hsp60 chaperonin in the mitochondrial matrix, supporting indications of folding defects in the variant ETF-QO proteins.

Proteomic investigation of cultivated fibroblasts from patients with mitochondrial short-chain acyl-CoA dehydrogenase deficiency.

Short-chain acyl-CoA dehydrogenase (SCAD) deficiency is a rare inherited autosomal recessive disorder with not yet well established mechanisms of disease. In the present study, the mitochondrial proteome of five symptomatic patients homozygous for missense variations in the SCAD gene ACADS was investigated in an extensive large-scale proteomic study to map protein perturbations linked to the disease. Fibroblast cultures of patient cells homozygous for either c.319C>T/p.Arg107Cys (n=2) or c.1138C>T/p.Arg380Trp (n=3) in ACADS, and healthy controls (normal human dermal fibroblasts), were studied. The mitochondrial proteome derived from these cultures was analyzed by label free proteomics using high mass accuracy nanoliquid chromatography tandem mass spectrometry (nanoLC-MS/MS). More than 300 mitochondrial proteins were identified and quantified. Thirteen proteins had significant alteration in protein levels in patients carrying variation c.319C>T in ACADS compared to controls and they belonged to various pathways, such as the antioxidant system and amino acid metabolism. Twenty-two proteins were found significantly altered in patients carrying variation c.1138C>T which included proteins associated with fatty acid ß-oxidation, amino acid metabolism and protein quality control system. Three proteins were found significantly regulated in both patient groups: adenylate kinase 4 (AK4), nucleoside diphosphate kinase A (NME1) and aldehyde dehydrogenase family 4 member A1 (ALDH4A1). Proteins AK4 and NME1 deserve further investigation because of their involvement in energy reprogramming, cell survival and proliferation with relevance for SCAD deficiency and related metabolic disorders.

The LMNA mutation p.Arg321Ter associated with dilated cardiomyopathy leads to reduced expression and a skewed ratio of lamin A and lamin C proteins.

Dilated cardiomyopathy (DCM) is a disease of the heart muscle characterized by cardiac chamber enlargement and reduced systolic function of the left ventricle. Mutations in the LMNA gene represent the most frequent known genetic cause of DCM associated with disease of the conduction systems. The LMNA gene generates two major transcripts encoding the nuclear lamina major components lamin A and lamin C by alternative splicing. Both haploinsuffiency and dominant negative effects have been proposed as disease mechanism for premature termination codon (PTC) mutations in LMNA. These mechanisms however are still not clearly established. In this study, we used a representative LMNA nonsense mutation, p.Arg321Ter, to shed light on the molecular disease mechanisms. Cultured fibroblasts from three DCM patients carrying this mutation were analyzed. Quantitative reverse transcriptase PCR and sequencing of these PCR products indicated that transcripts from the mutant allele were degraded by the nonsense-mediated mRNA decay (NMD) mechanism. The fact that no truncated mutant protein was detectable in western blot (WB) analysis strengthens the notion that the mutant transcript is efficiently degraded. Furthermore, WB analysis showed that the expression of lamin C protein was reduced by the expected approximately 50%. Clearly decreased lamin A and lamin C levels were also observed by immunofluorescence microscopy analysis. However, results from both WB and nano-liquid chromatography/mass spectrometry demonstrated that the levels of lamin A protein were more reduced suggesting an effect on expression of lamin A from the wild type allele. PCR analysis of the ratio of lamin A to lamin C transcripts showed unchanged relative amounts of lamin A transcript suggesting that the effect on the wild type allele was operative at the protein level. Immunofluorescence microscopy analysis showed no abnormal nuclear morphology of patient fibroblast cells. Based on these data, we propose that heterozygosity for the nonsense mutation causes NMD degradation of the mutant transcripts blocking expression of the truncated mutant protein and an additional trans effect on lamin A protein levels expressed from the wild type allele. We discuss the possibility that skewing of the lamin A to lamin C ratio may contribute to ensuing processes that destabilize cardiomyocytes and trigger cardiomyopathy.

Mutated desmoglein-2 proteins are incorporated into desmosomes and exhibit dominant-negative effects in arrhythmogenic right ventricular cardiomyopathy.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a hereditary cardiac condition associated with ventricular arrhythmias, heart failure, and sudden death. The most frequent ARVC genes encode desmosomal proteins of which mutations in desmoglein-2 (DSG2), account for 10%-20% of cases. This study aimed to investigate how DSG2 mutations contribute to the pathogenesis of ARVC. Initial mutation analysis of DSG2 in 71 probands identified the first family reported with recessively inherited ARVC due to a missense mutation. In addition, three recognized DSG2 mutations were identified in 12 families. These results and further mutation analyses of four additional desmosomal genes indicated that ARVC caused by DSG2 mutations is often transmitted by recessive or digenic inheritance. Because desmosomal proteins are also expressed in skin tissue, keratinocytes served as a cell model to investigate DSG2 protein expression by Western blotting, 2D-PAGE, and liquid chromatography-mass spectrometry. The results showed that heterozygous mutation carriers expressed both mutated and wild-type DSG2 proteins. These findings were consistent with the results obtained by immunohistochemistry of endomyocardial biopsies and epidermal tissue of mutation carriers, which indicated a normal cellular distribution of DSG2. The results suggested a dominant-negative effect of the mutated DSG2 proteins because they were incorporated into the desmosomes.

Late onset motoneuron disorder caused by mitochondrial Hsp60 chaperone deficiency in mice.

Cells rely on efficient protein quality control systems (PQCs) to maintain proper activity of mitochondrial proteins. As part of this system, the mitochondrial chaperone Hsp60 assists folding of matrix proteins and it is an essential protein in all organisms. Mutations in Hspd1, the gene encoding Hsp60, are associated with two human inherited diseases of the nervous system, a dominantly inherited form of spastic paraplegia (SPG13) and an autosomal recessively inherited white matter disorder termed MitCHAP60 disease. Although the connection between mitochondrial failure and neurodegeneration is well known in many neurodegenerative disorders, such as Huntington's disease, Parkinson's disease, and hereditary spastic paraplegia, the molecular basis of the neurodegeneration associated with these diseases is still ill-defined. Here, we investigate mice heterozygous for a knockout allele of the Hspd1 gene encoding Hsp60. Our results demonstrate that Hspd1 haploinsufficiency is sufficient to cause a late onset and slowly progressive deficit in motor functions in mice. We furthermore emphasize the crucial role of the Hsp60 chaperone in mitochondrial function by showing that the motor phenotype is associated with morphological changes of mitochondria, deficient ATP synthesis, and in particular, a defect in the assembly of the respiratory chain complex III in neuronal tissues. In the current study, we propose that our heterozygous Hsp60 mouse model is a valuable model system for the investigation of the link between mitochondrial dysfunction and neurodegeneration.

Advancing Clinical Trial Design for Non-Muscle Invasive Bladder Cancer.

BACKGROUND: Despite recent drug development for non-muscle invasive bladder cancer (NMIBC), few therapies have been approved by the US Food and Drug Administration (FDA), and there remains an unmet clinical need. Bacillus Calmette-Guerin (BCG) supply issues underscore the importance of developing safe and effective drugs for NMIBC. OBJECTIVE: On November 18-19, 2021, the FDA held a public virtual workshop to discuss NMIBC research needs and potential trial designs for future development of effective therapies. METHODS: Representatives from various disciplines including urologists, oncologists, pathologists, statisticians, basic and translational scientists, and the patient advocacy community participated. The workshop format included invited lectures, panel discussions, and opportunity for audience discussion and comment. RESULTS: In a pre-workshop survey, 92% of urologists surveyed considered the development of alternatives to BCG as a high drug development priority for BCG-naïve high-risk patients. Key topics discussed included definitions of disease states; trial design for BCG-naïve NMIBC, BCG-unresponsive carcinoma in situ, and BCG-unresponsive papillary carcinoma; strengths and limitations of single-arm trial designs; assessing patient-reported outcomes; and considerations for assessing avoidance of cystectomy as an efficacy measure. CONCLUSIONS: The workshop discussed several important opportunities for trial design refinement in NMIBC. FDA encourages sponsors to meet with the appropriate review division to discuss trial design proposals for NMIBC early in drug development.

Cofactors and metabolites as potential stabilizers of mitochondrial acyl-CoA dehydrogenases.

Protein misfolding is a hallmark of a number of metabolic diseases, in which fatty acid oxidation defects are included. The latter result from genetic deficiencies in transport proteins and enzymes of the mitochondrial ß-oxidation, and milder disease conditions frequently result from conformational destabilization and decreased enzymatic function of the affected proteins. Small molecules which have the ability to raise the functional levels of the affected protein above a certain disease threshold are thus valuable tools for effective drug design. In this work we have investigated the effect of mitochondrial cofactors and metabolites as potential stabilizers in two ß-oxidation acyl-CoA dehydrogenases: short chain acyl-CoA dehydrogenase and the medium chain acyl-CoA dehydrogenase as well as glutaryl-CoA dehydrogenase, which is involved in lysine and tryptophan metabolism. We found that near physiological concentrations (low micromolar) of FAD resulted in a spectacular enhancement of the thermal stabilities of these enzymes and prevented enzymatic activity loss during a 1h incubation at 40°C. A clear effect of the respective substrate, which was additive to that of the FAD effect, was also observed for short- and medium-chain acyl-CoA dehydrogenase but not for glutaryl-CoA dehydrogenase. In conclusion, riboflavin may be beneficial during feverish crises in patients with short- and medium-chain acyl-CoA dehydrogenase as well as in glutaryl-CoA dehydrogenase deficiencies, and treatment with substrate analogs to butyryl- and octanoyl-CoAs could theoretically enhance enzyme activity for some enzyme proteins with inherited folding difficulties.

MCAD deficiency in Denmark.

Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is the most common defect of fatty acid oxidation. Many countries have introduced newborn screening for MCADD, because characteristic acylcarnitines can easily be identified in filter paper blood spot samples by tandem mass spectrometry (MS/MS), because MCADD is a frequent disease, and because of the success of early treatment initiated before clinical symptoms have emerged. In Denmark we have screened 519,350 newborns for MCADD by MS/MS and identified 58 affected babies. The diagnosis of MCADD was confirmed in all 58 newborns by mutation analysis. This gives an incidence of MCADD detected by newborn screening in Denmark of 1/8954. In sharp contrast to this we found that the incidence of clinically presenting MCADD in Denmark in the 10 year period preceding introduction of MS/MS-based screening was only 1 in 39,691. This means that four times more newborns with MCADD are detected by screening than what is expected based on the number of children presenting clinically in an unscreened population. The mutation spectrum in the newborns detected by screening is different from that observed in clinically presenting patients with a much lower proportion of newborns being homozygous for the prevalent disease-causing c.985A>G mutation. A significant number of the newborns have genotypes with mutations that have not been observed in patients detected clinically. Some of these mutations, like c.199T>C and c.127G>A, are always associated with a milder biochemical phenotype and may cause a milder form of MCADD with a relatively low risk of disease manifestation, thereby explaining part of the discrepancy between the frequency of clinically manifested MCADD and the frequency of MCADD determined by screening. In addition, our data suggest that some of this discrepancy can be explained by a reduced penetrance of the c.985A>G mutation, with perhaps only 50% of c.985A>G homozygotes presenting with disease manifestations. Interestingly, we also report that the observed number of newborns identified by screening who are homozygous for the c.985A>G mutation is twice that predicted from the estimated carrier frequency. We therefore redetermined the carrier frequency in a new sample of 1946 blood spots using a new assay, but this only confirmed that the c.985A>G carrier frequency in Denmark is approximately 1/105. We conclude that MCADD is much more frequent than expected, has a reduced penetrance and that rapid genotyping using the initial blood spot sample is important for correct diagnosis and counseling.

Heterozygosity for an in-frame deletion causes glutaryl-CoA dehydrogenase deficiency in a patient detected by newborn screening: investigation of the effect of the mutant allele.

A patient with suspected glutaric aciduria type 1 (GA-1) was detected by newborn screening. GA-1 is known as an autosomal recessively inherited disease due to defects in the gene coding for glutaryl-CoA dehydrogenase (GCDH), a mitochondrial enzyme involved in the catabolism of the amino acids hydroxylysine, lysine and tryptophan. DNA and cDNA sequencing revealed a 18 bp deletion (c.553_570del18) resulting in deletion of six amino acids (p.Gly185_Ser190del) in one allele and no sequence changes in the other allele. Confirmatory biochemical analysis of blood, urine and cultured fibroblasts from the proband were consistent with a mild biochemical GA-1 phenotype. Recombinant expression of the mutant variant in E. coli showed that the GCDH-(p.Gly185_Ser190del) protein displayed severely decreased assembly into tetramers and enzyme activity. To discover a potential dominant negative effect of the mutant protein, we engineered a prokaryotic expression system in which expression of a wild type and a mutant GCDH allele is controlled by separately inducible promoters. These cells displayed decreased levels of GCDH tetramer and enzyme activity when expressing both the wild type and the mutant GCDH variant protein compared to the situation when only the wild type allele was expressed. Further experiments suggest that the major impact of the GCDH-(p.Gly185_Ser190del) protein in heterozygous cells consists of hampering the assembly of wild type GCDH into tetramers. Our experimental data are consistent with the hypothesis that heterozygosity for this mutation confers a dominant negative effect resulting in a GCDH enzyme activity that is significantly lower than the expected 50%.

Clinical characteristics and symptom duration among outpatients with COVID-19.

BACKGROUND: Approximately 80% of people with COVID-19 do not require hospitalization. Studies examining the outpatient experience have not tracked symptoms to resolution leading to unknown expected symptom duration. Our objectives were to (1) determine symptom duration among patients with COVID-19 who do not require hospitalization and (2) identify potential risk factors associated with prolonged symptom duration. DESIGN: This is a retrospective cohort study conducted across an academic healthcare system including adult patients with laboratory-confirmed SARS-CoV-2 infection between March 18th and April 28th, 2020 who were not hospitalized. Symptom duration encompassed time from patient-reported symptom onset as documented in the chart until documented symptom resolution. We calculated the median symptom duration and tested if demographics, comorbidities, or reported symptoms were associated with symptom duration. KEY RESULTS: Of 294 patients meeting inclusion criteria, 178 (60.5%) had documented symptom resolution. The median [interquartile range (IQR)] symptom duration for included patients was 15 (8-24) days. No associations were found between comorbidities and symptom duration. Factors associated with prolonged symptom duration were presence vs lack of lower respiratory symptoms [median (IQR) 16.5 (10.75-33.5) vs 14.5 (7-21.75) days respectively, P < .001] and neurologic symptoms [median (IQR) 17 (9-28) vs 9.5 (4-17) days, P < .001] at disease onset. CONCLUSIONS: The median symptom duration in outpatients is 15 days and over 25% of patients have symptoms longer than 21 days.

The psychiatric risk gene BRD1 modulates mitochondrial bioenergetics by transcriptional regulation.

Bromodomain containing 1 (BRD1) encodes an epigenetic regulator that controls the expression of genetic networks linked to mental illness. BRD1 is essential for normal brain development and its role in psychopathology has been demonstrated in genetic and preclinical studies. However, the neurobiology that bridges its molecular and neuropathological effects remains poorly explored. Here, using publicly available datasets, we find that BRD1 targets nuclear genes encoding mitochondrial proteins in cell lines and that modulation of BRD1 expression, irrespective of whether it is downregulation or upregulation of one or the other existing BRD1 isoforms (BRD1-L and BRD1-S), leads to distinct shifts in the expression profile of these genes. We further show that the expression of nuclear genes encoding mitochondrial proteins is negatively correlated with the expression of BRD1 mRNA during human brain development. In accordance, we identify the key gate-keeper of mitochondrial metabolism, Peroxisome proliferator-activated receptor (PPAR) among BRD1's co-transcription factors and provide evidence that BRD1 acts as a co-repressor of PPAR-mediated transcription. Lastly, when using quantitative PCR, mitochondria-targeted fluorescent probes, and the Seahorse XFe96 Analyzer, we demonstrate that modulation of BRD1 expression in cell lines alters mitochondrial physiology (mtDNA content and mitochondrial mass), metabolism (reducing power), and bioenergetics (among others, basal, maximal, and spare respiration) in an expression level- and isoform-dependent manner. Collectively, our data suggest that BRD1 is a transcriptional regulator of nuclear-encoded mitochondrial proteins and that disruption of BRD1's genomic actions alters mitochondrial functions. This may be the mechanism underlying the cellular and atrophic changes of neurons previously associated with BRD1 deficiency and suggests that mitochondrial dysfunction may be a possible link between genetic variation in BRD1 and psychopathology in humans.

A genome-wide CRISPR-Cas9 knockout screen identifies novel PARP inhibitor resistance genes in prostate cancer.

DNA repair gene mutations are frequent in castration-resistant prostate cancer (CRPC), suggesting eligibility for poly(ADP-ribose) polymerase inhibitor (PARPi) treatment. However, therapy resistance is a major clinical challenge and genes contributing to PARPi resistance are poorly understood. Using a genome-wide CRISPR-Cas9 knockout screen, this study aimed at identifying genes involved in PARPi resistance in CRPC. Based on the screen, we identified PARP1, and six novel candidates associated with olaparib resistance upon knockout. For validation, we generated multiple knockout populations/clones per gene in C4 and/or LNCaP CRPC cells, which confirmed that loss of PARP1, ARH3, YWHAE, or UBR5 caused olaparib resistance. PARP1 or ARH3 knockout caused cross-resistance to other PARPis (veliparib and niraparib). Furthermore, PARP1 or ARH3 knockout led to reduced autophagy, while pharmacological induction of autophagy partially reverted their PARPi resistant phenotype. Tumor RNA sequencing of 126 prostate cancer patients identified low ARH3 expression as an independent predictor of recurrence. Our results advance the understanding of PARPi response by identifying four novel genes that contribute to PARPi sensitivity in CRPC and suggest a new model of PARPi resistance through decreased autophagy.

Paediatric Strategy Forum for medicinal product development of chimeric antigen receptor T-cells in children and adolescents with cancer: ACCELERATE in collaboration with the European Medicines Agency with participation of the Food and Drug Administration.

The seventh multi-stakeholder Paediatric Strategy Forum focused on chimeric antigen receptor (CAR) T-cells for children and adolescents with cancer. The development of CAR T-cells for patients with haematological malignancies, especially B-cell precursor acute lymphoblastic leukaemia (BCP-ALL), has been spectacular. However, currently, there are scientific, clinical and logistical challenges for use of CAR T-cells in BCP-ALL and other paediatric malignancies, particularly in acute myeloid leukaemia (AML), lymphomas and solid tumours. The aims of the Forum were to summarise the current landscape of CAR T-cell therapy development in paediatrics, too identify current challenges and future directions, with consideration of other immune effector modalities and ascertain the best strategies to accelerate their development and availability to children. Although the effect is of limited duration in about half of the patients, anti-CD19 CAR T-cells produce high response rates in relapsed/refractory BCP-ALL and this has highlighted previously unknown mechanisms of relapse. CAR T-cell treatment as first- or second-line therapy could also potentially benefit patients whose disease has high-risk features associated with relapse and failure of conventional therapies. Identifying patients with very early and early relapse in whom CAR T-cell therapy may replace haematopoietic stem cell transplantation and be definitive therapy versus those in whom it provides a more effective bridge to haematopoietic stem cell transplantation is a very high priority. Development of approaches to improve persistence, either by improving T cell fitness or using more humanised/fully humanised products and co-targeting of multiple antigens to prevent antigen escape, could potentially further optimise therapy. Many differences exist between paediatric B-cell non-Hodgkin lymphomas (B-NHL) and BCP-ALL. In view of the very small patient numbers with relapsed lymphoma, careful prioritisation is needed to evaluate CAR T-cells in children with Burkitt lymphoma, primary mediastinal B cell lymphoma and other NHL subtypes. Combination trials of alternative targets to CD19 (CD20 or CD22) should also be explored as a priority to improve efficacy in this population. Development of CD30 CAR T-cell immunotherapy strategies in patients with relapsed/refractory Hodgkin lymphoma will likely be most efficiently accomplished by joint paediatric and adult trials. CAR T-cell approaches are early in development for AML and T-ALL, given the unique challenges of successful immunotherapy actualisation in these diseases. At this time, CD33 and CD123 appear to be the most universal targets in AML and CD7 in T-ALL. The results of ongoing or planned first-in-human studies are required to facilitate further understanding. There are promising early results in solid tumours, particularly with GD2 targeting cell therapies in neuroblastoma and central nervous system gliomas that represent significant unmet clinical needs. Further understanding of biology is critical to success. The comparative benefits of autologous versus allogeneic CAR T-cells, T-cells engineered with T cell receptors T-cells engineered with T cell receptor fusion constructs, CAR Natural Killer (NK)-cell products, bispecific T-cell engager antibodies and antibody-drug conjugates require evaluation in paediatric malignancies. Early and proactive academia and multi-company engagement are mandatory to advance cellular immunotherapies in paediatric oncology. Regulatory advice should be sought very early in the design and preparation of clinical trials of innovative medicines, for which regulatory approval may ultimately be sought. Aligning strategic, scientific, regulatory, health technology and funding requirements from the inception of a clinical trial is especially important as these are very expensive therapies. The model for drug development for cell therapy in paediatric oncology could also involve a 'later stage handoff' to industry after early development in academic hands. Finally, and very importantly, strategies must evolve to ensure appropriate ease of access for children who need and could potentially benefit from these therapies.

Mutational hotspots in electron transfer flavoprotein underlie defective folding and function in multiple acyl-CoA dehydrogenase deficiency.

We have carried out an extensive in silico analysis on 18 disease associated missense mutations found in electron transfer flavoprotein (ETF), and found that mutations fall essentially in two groups, one in which mutations affect protein folding and assembly, and another one in which mutations impair catalytic activity and disrupt interactions with partner dehydrogenases. We have further experimentally analyzed three of these mutations, ETFß-p.Cys42Arg, ETFß-p.Asp128Asn and ETFß-p.Arg191Cys, which have been found in homozygous form in patients and which typify different scenarios in respect to the clinical phenotypes. The ETFß-p.Cys42Arg mutation, related to a severe form of multiple acyl-CoA dehydrogenase deficiency (MADD), affects directly the AMP binding site and intersubunit contacts and impairs correct protein folding. The two other variations, ETFß-p.Asp128Asn and ETFß-p.Arg191Cys, are both associated with mild MADD, but these mutations have a different impact on ETF. Although none affects the overall α/ß fold topology as shown by far-UV CD, analysis of the purified proteins shows that both have substantially decreased enzymatic activity and conformational stability. Altogether, this study combines in silico analysis of mutations with experimental data and has allowed establishing structural hotspots within the ETF fold that are useful to provide a rationale for the prediction of effects of mutations in ETF.

Mitochondrial hsp60 chaperonopathy causes an autosomal-recessive neurodegenerative disorder linked to brain hypomyelination and leukodystrophy.

Hypomyelinating leukodystrophies (HMLs) are disorders involving aberrant myelin formation. The prototype of primary HMLs is the X-linked Pelizaeus-Merzbacher disease (PMD) caused by mutations in PLP1. Recently, homozygous mutations in GJA12 encoding connexin 47 were found in patients with autosomal-recessive Pelizaeus-Merzbacher-like disease (PMLD). However, many patients of both genders with PMLD carry neither PLP1 nor GJA12 mutations. We report a consanguineous Israeli Bedouin kindred with clinical and radiological findings compatible with PMLD, in which linkage to PLP1 and GJA12 was excluded. Using homozygosity mapping and mutation analysis, we have identified a homozygous missense mutation (D29G) not previously described in HSPD1, encoding the mitochondrial heat-shock protein 60 (Hsp60) in all affected individuals. The D29G mutation completely segregates with the disease-associated phenotype. The pathogenic effect of D29G on Hsp60-chaperonin activity was verified by an in vivo E. coli complementation assay, which demonstrated compromised ability of the D29G-Hsp60 mutant protein to support E. coli survival, especially at high temperatures. The disorder, which we have termed MitCHAP-60 disease, can be distinguished from spastic paraplegia 13 (SPG13), another Hsp60-associated autosomal-dominant neurodegenerative disorder, by its autosomal-recessive inheritance pattern, as well as by its early-onset, profound cerebral involvement and lethality. Our findings suggest that Hsp60 defects can cause neurodegenerative pathologies of varying severity, not previously suspected on the basis of the SPG13 phenotype. These findings should help to clarify the important role of Hsp60 in myelinogenesis and neurodegeneration.