Mutations in INTS11, the catalytic subunit of the Integrator complex essential for RNA processing and transcriptional termination, have been linked to neurodevelopmental disorders (NDDs), yet the underlying mechanisms remain poorly understood. To address this gap, we developed and characterized a novel ints11 loss-of-function zebrafish model using CRISPR/Cas9 and morpholino-based approaches, which recapitulates key phenotypic traits observed in human patients, including motor and behavioral deficits. ints11 deficiency led to marked impairments in locomotor activity and visual motor response, consistent with the neurological manifestations reported in INTS11-mutated patients. These behavioral abnormalities were paralleled by significant dysregulation of neurodevelopmental gene expression, including decreased expression of islet1, map2, gfap, and mag, and upregulation of the progenitor marker nestin, indicating defective neuronal differentiation and glial maturation. Interestingly, the observed phenotypes are rescued not only by mRNA-mediated re-expression of ints11, but also through pharmacological administration with brain-derived neurotrophic factor (BDNF) and the GM1 ganglioside-derived oligosaccharide (OligoGM1). These findings highlight neurotrophic signaling as a potential compensatory axis counteracting RNA-processing defects. In conclusion, our work establishes the first in vivo zebrafish model of INTS11-associated neurodevelopmental dysfunction, uncovering conserved molecular mechanisms that link Integrator complex activity, neurotrophic support, and neuronal maturation and providing a valuable platform for dissecting disease mechanisms and evaluating therapeutic strategies targeting RNA processing pathways and neurotrophic support in NDDs.

OBJECTIVES:Primary ovarian insufficiency (POI) is a fertility disorder with a well-established genetic component, but many cases still remain idiopathic. Approximately 1.5-12% of patients with POI can carry a variant in the BMP15 gene, depending on the population and the diagnostic criteria. We hypothesize that genetic variations within pathways downstream of BMP15 activity in ovarian granulosa cells (GCs) may contribute to unexplained cases of POI. The main goal of this study is to identify novel variants associated with POI in genes induced by BMP15 in GCs. STUDY DESIGN:Primary cultures of human GCs were stimulated with recombinant human BMP15. Microarray analysis profiled the BMP15-induced transcriptome in GCs. Validation was achieved by qPCR and immunoblot. Further, target exome sequencing of the differentially expressed genes was performed on 64 women with early POI onset in search of novel variants. MAIN OUTCOME MEASURES:Transcriptome profiling of human GCs stimulated with BMP15 and target exome sequencing in women with early onset of POI. RESULTS:Transcriptome analysis revealed significant upregulation of 19 genes (p < 0.05). Ontology analysis of these genes converged towards two main pathways: TGF-beta signaling and regulation of stem cell pluripotency. Target exome sequencing identified six novel rare variants in five BMP15-induced genes (SAMD11, SMAD6, ID1, USP35, GPCR137C) in 9 of the 64 women with early POI (14%). CONCLUSIONS:BMP15 action in human ovarian GCs defines TGF-beta signaling and pluripotency fate in ovarian follicles. In addition, this study uncovers new potential candidate genes for the pathogenesis of POI.

Deficiency of adenosine deaminase 2 (DADA2) is an inborn error of immunity caused by loss-of-function mutations in the adenosine deaminase 2 (ADA2) gene. Clinical manifestations of DADA2 include vasculopathy and immuno-hematological abnormalities, culminating in bone marrow failure. A major gap exists in our knowledge of the regulatory functions of ADA2 during inflammation and hematopoiesis, mainly due to the absence of an ADA2 orthologue in rodents. Exploring these mechanisms is essential for understanding disease pathology and developing new treatments. Zebrafish possess two ADA2 orthologues, cecr1a and cecr1b, with the latter showing functional conservation with human ADA2. We establish a cecr1b-loss-of-function zebrafish model that recapitulates the immuno-hematological and vascular manifestations observed in humans. Loss of Cecr1b disrupts hematopoietic stem cell specification, resulting in defective hematopoiesis. This defect is caused by induced inflammation in the vascular endothelium. Blocking inflammation, pharmacological modulation of the A2r pathway, or the administration of the recombinant human ADA2 corrects these defects, providing insights into the mechanistic link between ADA2 deficiency, inflammation and immuno-hematological abnormalities. Our findings open up potential therapeutic avenues for DADA2 patients.

Glioblastoma multiforme (GBM) is the most common and malignant brain tumor in adults. The invasiveness and the rapid progression that characterize GBM negatively impact patients' survival. Temozolomide (TMZ) is currently considered the first-choice chemotherapeutic agent. Unfortunately, over 50% of patients with GBM do not respond to TMZ treatment, and the mutation-prone nature of GBM enables the development of resistance mechanisms. Therefore, efforts have been devoted to the dissection of aberrant pathways involved in GBM insurgence and resistance in order to identify new therapeutic targets. Among them, sphingolipid signaling, Hedgehog (Hh) pathway, and the histone deacetylase 6 (HDAC6) activity are frequently dysregulated and may represent key targets to counteract GBM progression. Given the positive correlation between Hh/HDAC6/sphingolipid metabolism in GBM, we decided to perform a dual pharmacological inhibition of Hh and HDAC6 through cyclopamine and tubastatin A, respectively, in a human GMB cell line and zebrafish embryos. The combined administration of these compounds elicited a more significant reduction of GMB cell viability than did single treatments in vitro and in cells orthotopically transplanted in the zebrafish hindbrain ventricle. We demonstrated, for the first time, that the inhibition of these pathways induces lysosomal stress which results in an impaired fusion of lysosomes with autophagosomes and a block of sphingolipid degradation in GBM cell lines. This condition, which we also recapitulated in zebrafish embryos, suggests an impairment of lysosome-dependent processes involving autophagy and sphingolipid homeostasis and might be instrumental in the reduction of GBM progression.

Aberrant activation of the Hh pathway promotes cell proliferation and multi-drug resistance (MDR) in several cancers, including Acute Myeloid Leukemia (AML). Notably, only one Hh inhibitor, glasdegib, has been approved for AML treatment, and most patients eventually relapse, highlighting the urgent need to discover new therapeutic targets. Hh signal is transduced through the membrane of the primary cilium, a structure expressed by non-proliferating mammalian cells, whose stabilization depends on the activity of HDAC6. Here we describe a positive correlation between Hh, HDAC6, and MDR genes in a cohort of adult AML patients, human leukemic cell lines, and a zebrafish model of Hh overexpression. The hyper-activation of Hh or HDAC6 in zebrafish drove the increased proliferation of hematopoietic stem and progenitor cells (HSPCs). Interestingly, this phenotype was rescued by inhibition of HDAC6 but not of Hh. Also, in human leukemic cell lines, a reduction in vitality was obtained through HDAC6, but not Hh inhibition. Our data showed the presence of a cross-talk between Hh and HDAC6 mediated by stabilization of the primary cilium, which we detect for the first time in zebrafish HSPCs. Inhibition of HDAC6 activity alone or in combination therapy with the chemotherapeutic agent cytarabine, efficiently rescued the hematopoietic phenotype. Our results open the possibility to introduce HDAC6 as therapeutic target to reduce proliferation of leukemic blasts in AML patients.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Abstract Primary Ovarian Insufficiency (POI) is a female fertility disorder which affects 1% of women before 40 years of age and manifests with amenorrhea, elevation of serum gonadotrophins and low estrogens. POI has a strong genetic component with incomplete penetrance. Several candidate genes have been described so far, however, its etiopathogenesis is mostly unknown. In order to discover the POI-related causative mechanisms, microarray transcriptome analysis in human granulosa cells (hGCs) stimulated with recombinant human BMP15 (rhBMP15) and next generation sequencing analysis (NGS) on the identified differentially expressed genes in a selected group of patients with POI were conducted on NGS Illumina platform. In the present study, we obtained 19 differentially expressed genes upon rhBMP15 stimulation in hGCs. Results: showed that all identified genes were upregulated and associated to pluripotency, inhibition of apoptosis, cell proliferation, BMP signaling and apoptosis. Moreover, we identified nine POI patients bearing six rare variants in 5 of the BMP15-induced genes (SAMD11, SMAD6, ID1, USP35, GPCR137C). The BMP15-induced transcriptome analysis in hGCs contributed the understanding of BMP15 role as transcriptional regulator, through the activation of transcriptional repressors, by inducing pathways inhibiting the ovarian follicle maturation, thus possibly maintaining an undifferentiated state of hGCs. These findings lead to the identification of novel candidate genes for POI.

Different forms of sudden cardiac death have been described, including a recently identified form of genetic arrhythmogenic disorder, named “Triadin KnockOut Syndrome” (TKOS). TKOS is associated with recessive mutations in the TRDN gene, encoding for TRIADIN, but the pathogenic mechanism underlying the malignant phenotype has yet to be completely defined. Moreover, patients with TKOS are often refractory to conventional treatment, substantiating the need to identify new therapeutic strategies in order to prevent or treat cardiac events. The zebrafish (Danio rerio) heart is highly comparable to the human heart in terms of functions, signal pathways and ion channels, representing a good model to study cardiac disorders. In this work, we generated the first zebrafish model for trdn loss-of-function, by means of trdn morpholino injections, and characterized its phenotype. Although we did not observe any gross cardiac morphological defect between trdn loss-of-function embryos and controls, we found altered cardiac rhythm that was recovered by the administration of arrhythmic drugs. Our model will provide a suitable platform to study the effect of TRDN mutations and to perform drug screening to identify new pharmacological strategies for patients carrying TRDN mutations.

Duchenne muscular dystrophy (DMD) causes progressive skeletal muscle degeneration and currently there are few therapeutic options. The identification of new drug targets and their validation in model systems of DMD could be a promising approach to make progress in finding new treatments for this lethal disease. Histone deacetylases (HDACs) play key roles in myogenesis and the therapeutic approach targeting HDACs in DMD is in an advanced phase of clinical trial. Here, we show that the expression of HDAC8, one of the members of the HDAC family, is increased in DMD patients and dystrophic zebrafish. The selective inhibition of HDAC8 with the PCI-34051 inhibitor rescues skeletal muscle defects, similarly to the treatment with the pan-HDAC inhibitor Givinostat. Through acetylation profile of zebrafish with HDAC8 dysregulation, we identified new HDAC8 targets involved in cytoskeleton organization such as tubulin that, when acetylated, is a marker of stable microtubules. Our work provides evidence of HDAC8 overexpression in DMD patients and zebrafish and supports its specific inhibition as a new valuable therapeutic approach in the treatment of this pathology.

Primary ovarian insufficiency (POI) is one of the major causes of female infertility associated with the premature loss of ovarian function in about 3.7% of women before the age of 40. This disorder is highly heterogeneous and can manifest with a wide range of clinical phenotypes, ranging from ovarian dysgenesis and primary amenorrhea to post-pubertal secondary amenorrhea, with elevated serum gonadotropins and hypoestrogenism. The ovarian defect still remains idiopathic in some cases; however, a strong genetic component has been demonstrated by the next-generation sequencing (NGS) approach of familiar and sporadic POI cases. As recent evidence suggested an oligogenic architecture for POI, we developed a target NGS panel with 295 genes including known candidates and novel genetic determinants potentially involved in POI pathogenesis. Sixty-four patients with early onset POI (range: 10–25 years) of our cohort have been screened with 90% of target coverage at 50×. Here, we report 48 analyzed patients with at least one genetic variant (75%) in the selected candidate genes. In particular, we found the following: 11/64 patients (17%) with two variants, 9/64 (14%) with three variants, 9/64 (14%) with four variants, 3/64 (5%) with five variants, and 2/64 (3%) with six variants. The most severe phenotypes were associated with either the major number of variations or a worse prediction in pathogenicity of variants. Bioinformatic gene ontology analysis identified the following major pathways likely affected by gene variants: 1) cell cycle, meiosis, and DNA repair; 2) extracellular matrix remodeling; 3) reproduction; 4) cell metabolism; 5) cell proliferation; 6) calcium homeostasis; 7) NOTCH signaling; 8) signal transduction; 9) WNT signaling; 10) cell death; and 11) ubiquitin modifications. Consistently, the identified pathways have been described in other studies dissecting the mechanisms of folliculogenesis in animal models of altered fertility. In conclusion, our results contribute to define POI as an oligogenic disease and suggest novel candidates to be investigated in patients with POI.

STUDY QUESTION:Can a targeted whole exome sequencing (WES) on a cohort of women showing a primary ovarian insufficiency (POI) phenotype at a young age, combined with a study of copy number variations, identify variants in candidate genes confirming their deleterious effect on ovarian function?SUMMARY ANSWER:This integrated approach has proved effective in identifying novel candidate genes unveiling mechanisms involved in POI pathogenesis.WHAT IS KNOWN ALREADY:POI, a condition occurring in 1% of women under 40 years of age, affects women's fertility leading to a premature loss of ovarian reserve. The genetic causes of POI are highly heterogeneous and several determinants contributing to its prominent oligogenic inheritance pattern still need to be elucidated.STUDY DESIGN, SIZE, DURATION:WES screening for pathogenic variants of 41 Italian women with non-syndromic primary and early secondary amenorrhoea occurring before age 25 was replicated on another 60 POI patients, including 35 French and 25 American women, to reveal statistically significant shared variants.PARTICIPANTS/MATERIALS, SETTING, METHODS:The Italian POI patients' DNA were processed by targeted WES including 542 RefSeq genes expressed or functioning during distinct reproductive or ovarian processes (e.g. DNA repair, meiosis, oocyte maturation, folliculogenesis and menopause). Extremely rare variants were filtered and selected by means of a Fisher Exact test using several publicly available datasets. A case-control Burden test was applied to highlight the most significant genes using two ad-hoc control female cohorts. To support the obtained data, the identified genes were screened on a novel cohort of 60 Caucasian POI patients and the same case-control analysis was carried out. Comparative analysis of the human identified genes was performed on mouse and Drosophila melanogaster by analysing the orthologous genes in their ovarian phenotype, and two of the selected genes were fruit fly modelled to explore their role in fertility.MAIN RESULTS AND THE ROLE OF CHANCE:The filtering steps applied to search for extremely rare pathogenic variants in the Italian cohort revealed 64 validated single-nucleotide variants/Indels in 59 genes in 30 out of 41 screened women. Burden test analysis highlighted 13 ovarian genes as being the most enriched and significant. To validate these findings, filtering steps and Burden analysis on the second cohort of Caucasian patients yielded 11 significantly enriched genes. Among them, AFP, DMRT3, MOV10, FYN and MYC were significant in both patient cohorts and hence were considered strong candidates for POI. Mouse and Drosophila comparative analysis evaluated a conserved role through the evolution of several candidates, and functional studies using a Drosophila model, when applicable, supported the conserved role of the MOV10 armitage and DMRT3 dmrt93B orthologues in female fertility.LARGE SCALE DATA:The datasets for the Italian cohort generated during the current study are publicly available at ClinVar database (http://www.ncbi.nlm.nih.gov/clinvar/): accession numbers SCV001364312 to SCV001364375.LIMITATIONS, REASONS FOR CAUTION:This is a targeted WES analysis hunting variants in candidate genes previously identified by different genomic approaches. For most of the investigated sporadic cases, we could not track the parental inheritance, due to unavailability of the parents' DNA samples; in addition, we might have overlooked additional rare variants in novel candidate POI genes extracted from the exome data. On the contrary, we might have considered some inherited variants whose clinical significance is uncertain and might not be causative for the patients' phenotype. Additionally, as regards the Drosophila model, it will be extremely important in the future to have more mutants or RNAi strains available for each candidate gene in order to validate their role in POI pathogenesis.WIDER IMPLICATIONS OF THE FINDINGS:The genomic, statistical, comparative and functional approaches integrated in our study convincingly support the extremely heterogeneous oligogenic nature of POI, and confirm the maintenance across the evolution of some key genes safeguarding fertility and successful reproduction. Two principal classes of genes were identified: (i) genes primarily involved in meiosis, namely in synaptonemal complex formation, asymmetric division and oocyte maturation and (ii) genes safeguarding cell maintenance (piRNA and DNA repair pathways).STUDY FUNDING/COMPETING INTEREST(S):This work was supported by Italian Ministry of Health grants 'Ricerca Corrente' (08C621_2016 and 08C924_2019) provided to IRCCS Istituto Auxologico Italiano, and by 'Piano Sostegno alla Ricerca' (PSR2020_FINELLI_LINEA_B) provided by the University of Milan; M.P.B. was supported by Telethon-Italy (grant number GG14181). There are no conflicts of interest.

Transcriptional changes normally occur during development but also underlie differences between healthy and pathological conditions. Transcription factors or chromatin modifiers are involved in orchestrating gene activity, such as the cohesin genes and their regulator NIPBL. In our previous studies, using a zebrafish model for nipblb knockdown, we described the effect of nipblb loss-of-function in specific contexts, such as central nervous system development and hematopoiesis. However, the genome-wide transcriptional impact of nipblb loss-of-function in zebrafish embryos at diverse developmental stages remains under investigation. By RNA-seq analyses in zebrafish embryos at 24 h post-fertilization, we examined genome-wide effects of nipblb knockdown on transcriptional programs. Differential gene expression analysis revealed that nipblb loss-of-function has an impact on gene expression at 24 h post fertilization, mainly resulting in gene inactivation. A similar transcriptional effect has also been reported in other organisms, supporting the use of zebrafish as a model to understand the role of Nipbl in gene regulation during early vertebrate development. Moreover, we unraveled a connection between nipblb-dependent differential expression and gene expression patterns of hematological cell populations and AML subtypes, enforcing our previous evidence on the involvement of NIPBL-related transcriptional dysregulation in hematological malignancies.

The transcription factor RUNX1, a pivotal regulator of HSCs and haematopoiesis, is a frequent target of chromosomal translocations, point mutations or altered gene/protein dosage. These modifications lead or contribute to the development of myelodysplasia, leukaemia or platelet disorders. A better understanding of how regulatory elements contribute to fine-tune the RUNX1 expression in haematopoietic tissues could improve our knowledge of the mechanisms responsible for normal haematopoiesis and malignancy insurgence. The cohesin RAD21 was reported to be a regulator of RUNX1 expression in the human myeloid HL60 cell line and during primitive haematopoiesis in zebrafish. In our study, we demonstrate that another cohesin, NIPBL, exerts positive regulation of RUNX1 in three different contexts in which RUNX1 displays important functions: in megakaryocytes derived from healthy donors, in bone marrow samples obtained from adult patients with acute myeloid leukaemia and during zebrafish haematopoiesis. In this model, we demonstrate that alterations in the zebrafish orthologue nipblb reduce runx1 expression with consequent defects in its erythroid and myeloid targets such as gata1a and spi1b in an opposite way to rad21. Thus, also in the absence of RUNX1 translocation or mutations, additional factors such as defects in the expression of NIPBL might induce haematological diseases.

Histone deacetylase 8 (HDAC8), a class I HDAC that modifies non-histone proteins such as p53, is highly expressed in different hematological neoplasms including a subtype of acute myeloid leukemia (AML) bearing inversion of chromosome 16 [inv(16)]. To investigate HDAC8 contribution to hematopoietic stem cell maintenance and myeloid leukemic transformation, we generated a zebrafish model with Hdac8 overexpression and observed an increase in hematopoietic stem/progenitor cells, a phenotype that could be reverted using a specific HDAC8 inhibitor, PCI-34051 (PCI). In addition, we demonstrated that AML cell lines respond differently to PCI treatment: HDAC8 inhibition elicits cytotoxic effect with cell cycle arrest followed by apoptosis in THP-1 cells, and cytostatic effect in HL60 cells that lack p53. A combination of cytarabine, a standard anti-AML chemotherapeutic, with PCI resulted in a synergistic effect in all the cell lines tested. We, then, searched for a mechanism behind cell cycle arrest caused by HDAC8 inhibition in the absence of functional p53 and demonstrated an involvement of the canonical WNT signaling in zebrafish and in cell lines. Together, we provide the evidence for the role of HDAC8 in hematopoietic stem cell differentiation in zebrafish and AML cell lines, suggesting HDAC8 inhibition as a therapeutic target in hematological malignancies. Accordingly, we demonstrated the utility of a highly specific HDAC8 inhibition as a therapeutic strategy in combination with standard chemotherapy.

The genes of the cohesin complex exert different functions, ranging from the adhesion of sister chromatids during the cell cycle, DNA repair, gene expression and chromatin architecture remodeling. In recent years, the improvement of DNA sequencing technologies allows the identification of cohesin mutations in different tumors such as acute myeloid leukemia (AML), acute megakaryoblastic leukemia (AMKL), and myelodysplastic syndromes (MDS). However, the role of cohesin dysfunction in cancer insurgence remains elusive. In this regard, cells harboring cohesin mutations do not show any increase in aneuploidy that might explain their oncogenic activity, nor cohesin mutations are sufficient to induce myeloid neoplasms as they have to co-occur with other causative mutations such as NPM1, FLT3-ITD, and DNMT3A. Several works, also using animal models for cohesin haploinsufficiency, correlate cohesin activity with dysregulated expression of genes involved in myeloid development and differentiation. These evidences support the involvement of cohesin mutations in myeloid neoplasms.