Interplay between α-thalassemia and ß-hemoglobinopathies: Translating genotype-phenotype relationships into therapies.

α-Thalassemia represents one of the most important genetic modulators of ß-hemoglobinopathies. During this last decade, the ongoing interest in characterizing genotype-phenotype relationships has yielded incredible insights into α-globin gene regulation and its impact on ß-hemoglobinopathies. In this review, we provide a holistic update on α-globin gene expression stemming from DNA to RNA to protein, as well as epigenetic mechanisms that can impact gene expression and potentially influence phenotypic outcomes. Here, we highlight defined α-globin targeted strategies and rationalize the use of distinct molecular targets based on the restoration of balanced α/ß-like globin chain synthesis. Considering the therapies that either increase ß-globin synthesis or reactivate γ-globin gene expression, the modulation of α-globin chains as a disease modifier for ß-hemoglobinopathies still remains largely uncharted in clinical studies.

Pediatric glioma histone H3.3 K27M/G34R mutations drive abnormalities in PML nuclear bodies.

BACKGROUND: Point mutations in histone variant H3.3 (H3.3K27M, H3.3G34R) and the H3.3-specific ATRX/DAXX chaperone complex are frequent events in pediatric gliomas. These H3.3 point mutations affect many chromatin modifications but the exact oncogenic mechanisms are currently unclear. Histone H3.3 is known to localize to nuclear compartments known as promyelocytic leukemia (PML) nuclear bodies, which are frequently mutated and confirmed as oncogenic drivers in acute promyelocytic leukemia. RESULTS: We find that the pediatric glioma-associated H3.3 point mutations disrupt the formation of PML nuclear bodies and this prevents differentiation down glial lineages. Similar to leukemias driven by PML mutations, H3.3-mutated glioma cells are sensitive to drugs that target PML bodies. We also find that point mutations in IDH1/2-which are common events in adult gliomas and myeloid leukemias-also disrupt the formation of PML bodies. CONCLUSIONS: We identify PML as a contributor to oncogenesis in a subset of gliomas and show that targeting PML bodies is effective in treating these H3.3-mutated pediatric gliomas.

Histone H3.3 phosphorylation promotes heterochromatin formation by inhibiting H3K9/K36 histone demethylase.

Histone H3.3 is an H3 variant which differs from the canonical H3.1/2 at four residues, including a serine residue at position 31 which is evolutionarily conserved. The H3.3 S31 residue is phosphorylated (H3.3 S31Ph) at heterochromatin regions including telomeres and pericentric repeats. However, the role of H3.3 S31Ph in these regions remains unknown. In this study, we find that H3.3 S31Ph regulates heterochromatin accessibility at telomeres during replication through regulation of H3K9/K36 histone demethylase KDM4B. In mouse embryonic stem (ES) cells, substitution of S31 with an alanine residue (H3.3 A31 -phosphorylation null mutant) results in increased KDM4B activity that removes H3K9me3 from telomeres. In contrast, substitution with a glutamic acid (H3.3 E31, mimics S31 phosphorylation) inhibits KDM4B, leading to increased H3K9me3 and DNA damage at telomeres. H3.3 E31 expression also increases damage at other heterochromatin regions including the pericentric heterochromatin and Y chromosome-specific satellite DNA repeats. We propose that H3.3 S31Ph regulation of KDM4B is required to control heterochromatin accessibility of repetitive DNA and preserve chromatin integrity.

Chromatin mutations in pediatric high grade gliomas.

Pediatric high grade gliomas (HGG) are lethal tumors which are currently untreatable. A number of recent studies have provided much needed insights into the mutations and mechanisms which drive oncogenesis in pediatric HGGs. It is now clear that mutations in chromatin proteins, particularly H3.3 and its associated chaperone complex (ATRX), are a hallmark feature of pediatric HGGs. We review the current literature on the normal roles of the ATRX/H3.3 complex and how these functions are disrupted by oncogenic mutations. We discuss the current clinical trials and pre-clinical models that target chromatin and DNA, and how these agents fit into the ATRX/H3.3 mutation model. As chromatin mutations are a relatively new discovery in pediatric HGGs, developing clear mechanistic insights are a key step to improving therapies for these tumors.

Coordinated ß-globin expression and α2-globin reduction in a multiplex lentiviral gene therapy vector for ß-thalassemia.

A primary challenge in lentiviral gene therapy of ß-hemoglobinopathies is to maintain low vector copy numbers to avoid genotoxicity while being reliably therapeutic for all genotypes. We designed a high-titer lentiviral vector, LVß-shα2, that allows coordinated expression of the therapeutic ßA-T87Q-globin gene and of an intron-embedded miR-30-based short hairpin RNA (shRNA) selectively targeting the α2-globin mRNA. Our approach was guided by the knowledge that moderate reduction of α-globin chain synthesis ameliorates disease severity in ß-thalassemia. We demonstrate that LVß-shα2 reduces α2-globin mRNA expression in erythroid cells while keeping α1-globin mRNA levels unchanged and ßA-T87Q-globin gene expression identical to the parent vector. Compared with the first ßA-T87Q-globin lentiviral vector that has received conditional marketing authorization, BB305, LVß-shα2 shows 1.7-fold greater potency to improve α/ß ratios. It may thus result in greater therapeutic efficacy and reliability for the most severe types of ß-thalassemia and provide an improved benefit/risk ratio regardless of the ß-thalassemia genotype.

Inhibition of a K9/K36 demethylase by an H3.3 point mutation found in paediatric glioblastoma.

An array of oncogenic histone point mutations have been identified across a number of different cancer studies. It has been suggested that some of these mutant histones can exert their effects by inhibiting epigenetic writers. Here, we report that the H3.3 G34R (glycine to arginine) substitution mutation, found in paediatric gliomas, causes widespread changes in H3K9me3 and H3K36me3 by interfering with the KDM4 family of K9/K36 demethylases. Expression of a targeted single-copy of H3.3 G34R at endogenous levels induced chromatin alterations that were comparable to a KDM4 A/B/C triple-knockout. We find that H3.3 G34R preferentially binds KDM4 while simultaneously inhibiting its enzymatic activity, demonstrating that histone mutations can act through inhibition of epigenetic erasers. These results suggest that histone point mutations can exert their effects through interactions with a range of epigenetic readers, writers and erasers.

Ribosomal DNA copy loss and repeat instability in ATRX-mutated cancers.

ATRX (alpha thalassemia/mental retardation X-linked) complexes with DAXX to deposit histone variant H3.3 into repetitive heterochromatin. Recent genome sequencing studies in cancers have revealed mutations in ATRX and their association with ALT (alternative lengthening of telomeres) activation. Here we report depletion of ATRX in mouse ES cells leads to selective loss in ribosomal RNA gene (rDNA) copy number. Supporting this, ATRX-mutated human ALT-positive tumors also show a substantially lower rDNA copy than ALT-negative tumors. Further investigation shows that the rDNA copy loss and repeat instability are caused by a disruption in H3.3 deposition and thus a failure in heterochromatin formation at rDNA repeats in the absence of ATRX. We also find that ATRX-depleted cells are reduced in ribosomal RNA transcription output and show increased sensitivity to RNA polymerase I (Pol I) transcription inhibitor CX5461. In addition, human ALT-positive cancer cell lines are also more sensitive to CX5461 treatment. Our study provides insights into the contribution of ATRX loss of function to tumorigenesis through the loss of rDNA stability and suggests the therapeutic potential of targeting Pol I transcription in ALT cancers.

Reliable sonographic features for nodal thyroglobulin to diagnose recurrent lymph node metastasis from papillary thyroid carcinoma.

OBJECTIVES: Ultrasound-guided aspiration cytology (US-FNAC) was previously used to diagnose lymph node metastasis of papillary thyroid carcinoma (PTC). Combined US-FNAC with nodal thyroglobulin (LN-FNA-Tg) significantly improved the diagnostic rate. However, diagnostic accuracy depends on proper node selection. Therefore, it is crucial to choose the nodes with reliable sonographic features to guide clinician for confirmation. DESIGN AND SETTING: Retrospective cohort study was carried out in one medical centre from 2011 to 2014. PARTICIPANTS: A total of 148 patients with PTC, being treated by total thyroidectomy and radioiodine, were assessed for potential nodal metastases by ultrasound. MAIN OUTCOME MEASURES: Lymph nodes with cystic content, peripheral hypervascularity, calcification, hyperechoic content, the absence of hilum and Solbiati index < 2 indicated risk of malignancy. US-FNAC and LN-FNA-Tg were both performed. Positive nodal metastasis was further confirmed by dissection. Risk impact of these sonographic features on LN-FNA-Tg to diagnose nodal metastasis was tested by logistic regression analysis based on the significance in both univariate and multivariate models. RESULTS: Overall, 49 lymph nodes were documented as recurrent nodal metastasis. LN-FNA-Tg greater than serum thyroglobulin and higher than 1 ng/mL achieved 100% of diagnostic rate for recurrent nodal metastasis. The malignant sonographic features that significantly cohered with positive LN-FNA-Tg were cystic and hyperechoic content and lack hilum, in sequence. CONCLUSIONS: LN-FNA-Tg is an excellent tool to quantitatively diagnose nodal metastasis. To achieve ideal diagnosis, the most reliable sonographic features were cystic content, hyperechoic content and the absence of hilum in lymph nodes, but not calcification or Solbiati index < 2.

Polygenic risk for schizophrenia and neurocognitive performance in patients with schizophrenia.

Both neurocognitive deficits and schizophrenia are highly heritable. Genetic overlap between neurocognitive deficits and schizophrenia has been observed in both the general population and in the clinical samples. This study aimed to examine if the polygenic architecture of susceptibility to schizophrenia modified neurocognitive performance in schizophrenia patients. Schizophrenia polygenic risk scores (PRSs) were first derived from the Psychiatric Genomics Consortium (PGC) on schizophrenia, and then the scores were calculated in our independent sample of 1130 schizophrenia trios, who had PsychChip data and were part of the Schizophrenia Families from Taiwan project. Pseudocontrols generated from the nontransmitted parental alleles of the parents in these trios were compared with alleles in schizophrenia patients in assessing the replicability of PGC-derived susceptibility variants. Schizophrenia PRS at the P-value threshold (PT) of 0.1 explained 0.2% in the variance of disease status in this Han-Taiwanese samples, and the score itself had a P-value 0.05 for the association test with the disorder. Each patient underwent neurocognitive evaluation on sustained attention using the continuous performance test and executive function using the Wisconsin Card Sorting Test. We applied a structural equation model to construct the neurocognitive latent variable estimated from multiple measured indices in these 2 tests, and then tested the association between the PRS and the neurocognitive latent variable. Higher schizophrenia PRS generated at the PT of 0.1 was significantly associated with poorer neurocognitive performance with explained variance 0.5%. Our findings indicated that schizophrenia susceptibility variants modify the neurocognitive performance in schizophrenia patients.

Inhibition of cyclooxygenase-2-mediated matriptase activation contributes to the suppression of prostate cancer cell motility and metastasis.

Chronic inflammation plays an important role in cancer development and progression. Cyclooxygenases-2 (COX-2) is a key enzyme in generating prostaglandins causing inflammation, is often found to be overexpressed in prostate cancer (PCa) and is correlated with PCa cell invasion and metastasis. We aim to investigate the molecular mechanism of how COX-2 promotes PCa cell invasion and metastasis and to evaluate the effect of COX-2 inhibitors in a selected model of PCa progression. Our results showed that the expression of COX-2 and Interleukin 1ß (IL-1ß) was upregulated in highly invasive PCa cells and was correlated with the activated levels of membrane-anchored serine protease matriptase. The expression levels of COX-2 were increased and were correlated with matriptase levels in PCa specimens. Moreover, results showed that COX-2 overexpression or a COX-2 product Prostaglandin E2 (PGE2) caused an increase in matriptase activation and PCa cell invasion, whereas COX-2 silencing antagonized matriptase activation and cell invasion. In addition, the inhibition of COX-2-mediated matriptase activation by Celebrex and sulindac sulfide suppressed the androgen-independent and COX2-overexpressing PCa PC-3 cell invasion, tumor growth and lung metastasis in an orthotopic xenograft model. Our results indicate that COX-2/matriptase signaling contributes to the invasion, tumor growth and metastasis of COX-2-overexpressing and androgen-independent PCa cells.

Maintaining memory of silencing at imprinted differentially methylated regions.

Imprinted genes are an exceptional cluster of genes which are expressed in a parent-of-origin dependent fashion. This allele-specific expression is dependent on differential DNA methylation which is established in the parental germlines in a sex-specific manner. The DNA methylation imprint is accompanied by heterochromatin modifications which must be continuously maintained through development. This review summarises the factors which are important for protecting the epigenetic modifications at imprinted differentially methylated regions (DMRs), including PGC7, ZFP57 and the ATRX/Daxx/H3.3 complex. We discuss how these factors maintain heterochromatin silencing, not only at imprinted DMRs, but also other heterochromatic regions in the genome.

New players in heterochromatin silencing: histone variant H3.3 and the ATRX/DAXX chaperone.

A number of studies have demonstrated that various components of the ATRX/DAXX/Histone H3.3 complex are important for heterochromatin silencing at multiple genomic regions. We provide an overview of the individual components (ATRX, DAXX and/or H3.3) tested in each study and propose a model where the ATRX/DAXX chaperone complex deposits H3.3 to maintain the H3K9me3 modification at heterochromatin throughout the genome.

Compromised Telomeric Heterochromatin Promotes ALTernative Lengthening of Telomeres.

Alternative lengthening of telomeres (ALT) is an enigmatic process that allows certain cancers to maintain telomeres in the absence of telomerase. ALT cancers are frequently defective for ATRX/DAXX, a chaperone complex that deposits histone variant H3.3 at telomeres. We propose that mutations in alpha thalassemia-mental retardation syndrome X-linked (ATRX)/death-domain associated protein (DAXX) prime ALT activation by disrupting telomeric heterochromatin.