Anticancer Drugs of Lysine Specific Histone Demethylase-1 (LSD1) Display Variable Inhibition on Nucleosome Substrates.

Lysine specific demethylase-1 (LSD1) serves as a regulator of transcription and represents a promising epigenetic target for anticancer treatment. LSD1 inhibitors are in clinical trials for the treatment of Ewing's sarcoma (EWS), acute myeloid leukemia, and small cell lung cancer, and the development of robust inhibitors requires accurate methods for probing demethylation, potency, and selectivity. Here, the inhibition kinetics on the H3K4me2 peptide and nucleosome substrates was examined, comparing the rates of demethylation in the presence of reversible [CC-90011 (PD) and SP-2577 (SD)] and irreversible [ORY-1001 (ID) and tranylcypromine (TCP)] inhibitors. Inhibitors were also subject to viability studies in three human cell lines and Western blot assays to monitor H3K4me2 nucleosome levels in EWS (TC-32) cells, enabling a correlation of drug potency, inhibition in vitro, and cell-based studies. For example, SP-2577, a drug in clinical trials for EWS, inhibits activity on small peptide substrates (Ki = 60 ± 20 nM) using an indirect coupled assay but does not inhibit demethylation on H3K4me2 peptides or nucleosomes using direct Western blot approaches. In addition, the drug has no effect on H3K4me2 levels in TC-32 cells. These data show that SP-2577 is not an LSD1 enzyme inhibitor, although the drug may function independent of demethylation due to its cytotoxic selectivity in TC-32 cells. Taken together, this work highlights the pitfalls of using coupled assays to ascribe a drug's mode of action, emphasizes the use of physiologically relevant substrates in epigenetic drug targeting strategies, and provides insight into the development of substrate-selective inhibitors of LSD1.

Piperacetazine Directly Binds to the PAX3::FOXO1 Fusion Protein and Inhibits Its Transcriptional Activity.

The tumor-specific chromosomal translocation product, PAX3::FOXO1, is an aberrant fusion protein that plays a key role for oncogenesis in the alveolar subtype of rhabdomyosarcoma (RMS). PAX3::FOXO1 represents a validated molecular target for alveolar RMS and successful inhibition of its oncogenic activity is likely to have significant clinical applications. Even though several PAX3::FOXO1 function-based screening studies have been successfully completed, a directly binding small-molecule inhibitor of PAX3::FOXO1 has not been reported. Therefore, we screened small-molecule libraries to identify compounds that were capable of directly binding to PAX3::FOXO1 protein using surface plasmon resonance technology. Compounds that directly bound to PAX3::FOXO1 were further evaluated in secondary transcriptional activation assays. We discovered that piperacetazine can directly bind to PAX3::FOXO1 protein and inhibit fusion protein-derived transcription in multiple alveolar RMS cell lines. Piperacetazine inhibited anchorage-independent growth of fusion-positive alveolar RMS cells but not embryonal RMS cells. On the basis of our findings, piperacetazine is a molecular scaffold upon which derivatives could be developed as specific inhibitors of PAX3::FOXO1. These novel inhibitors could potentially be evaluated in future clinical trials for recurrent or metastatic alveolar RMS as novel targeted therapy options. SIGNIFICANCE: RMS is a malignant soft-tissue tumor mainly affecting the pediatric population. A subgroup of RMS with worse prognosis harbors a unique chromosomal translocation creating an oncogenic fusion protein, PAX3::FOXO1. We identified piperacetazine as a direct inhibitor of PAX3::FOXO1, which may provide a scaffold for designing RMS-specific targeted therapy.

A longitudinal study of sibling bullying and mental health in autistic adolescents: The role of self-esteem.

Sibling bullying is associated with poor mental health in autistic adolescents. The reasons for this remain unknown. In the current study, we attempted to replicate the existing findings on the direct associations between sibling bullying and mental health in autistic adolescents and expand knowledge by focusing on the indirect associations through self-esteem. We made use of existing data from the Millennium Cohort Study, a nationally representative UK-based birth cohort study. We fitted a mediation model to longitudinal data from a sample of 416 autistic adolescents aged 11, 14, and 17 years old who had at least one sibling. We found that sibling bullying was prevalent in the lives of autistic adolescents, especially in those who were late-diagnosed, had a shared bedroom, and lived in a low-income household. Additionally, increased sibling bullying in early adolescence was a significant predictor of reduced self-esteem in mid-adolescence; in turn, reduced self-esteem predicted poorer mental health and wellbeing in late adolescence. Our findings indicate that sibling bullying in early adolescence may indirectly lead to poorer mental health and wellbeing in late adolescence through a reduction in self-esteem in mid-adolescence in autistic adolescents. We discuss the implications of these findings further.

Design, synthesis and biological evaluation of Nucleosidic CD99 inhibitors that selectively reduce Ewing sarcoma viability.

Ewing Sarcoma (ES) is a cancer of bone and soft tissues affecting mostly children and young adults. Aggressive progression and poor prognosis of this malignancy call for novel and targeted treatments. CD99 is a transmembrane protein that is abundantly expressed on ES cells and is a diagnostic marker for the disease. ES cells are selectively sensitive to CD99 inhibition compared to most normal cells and other tumors. Therefore, CD99 is a good molecular target for ES treatment. Clofarabine and cladribine are two FDA approved drugs that are administered for their inhibitory acts on DNA synthesis to treat relapsed or refractory acute lymphoblastic and myeloid leukemia. They have also been shown to directly bind to CD99 and inhibit ES growth through a distinct mechanism. In the current study, we designed, synthesized and tested new ES specific derivatives of both drugs that would continue to target CD99 but with expected reduction in cellular membrane permeability and rendered unsuitable for inhibiting DNA synthesis. By using commercially available clofarabine and cladribine purine nucleoside analogs, we modified the primary alcohol moiety at the deoxyribose C-5' terminal site to suppress phosphorylation and thus inhibition of subsequent DNA synthesis pathways. In addition, we incorporated a variety of polar groups in the ribose and purine rings to reduce membrane permeability and investigated the effects of configurational changes in the sugar moiety. Among 26 new derivatives, we identified two compounds, BK50164 and BK60106, that cause cell death specifically in ES primarily due to inhibition of CD99 but not via inhibition of DNA synthesis. These findings provide a road map for the future development selective CD99 inhibitors for targeted treatment of ES.

Play-based interventions for mental health: A systematic review and meta-analysis focused on children and adolescents with autism spectrum disorder and developmental language disorder.

Background and aims: Play-based interventions are used ubiquitously with children with social, communication, and language needs but the impact of these interventions on the mental health of this group of children is unknown. Despite their pre-existing challenges, the mental health of children with developmental language disorder (DLD) and autism spectrum disorder (ASD) should be given equal consideration to the other more salient features of their condition. To this aim, a systematic literature review with meta-analysis was undertaken to assess the impact of play-based interventions on mental health outcomes from studies of children with DLD and ASD, as well as to identify the characteristics of research in this field. Methods: The study used full systematic review design reported to the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines (PRISMA prisma-statement.org) with pre-specified inclusion criteria and explicit, transparent and replicable methods at each stage of the review. The study selection process involved a rigorous systematic search of seven academic databases, double screening of abstracts, and full-text screening to identify studies using randomised controlled trial (RCT) and quasi-experimental (QE) designs to assess mental health outcomes from interventions supporting children with DLD and ASD. For reliability, data extraction of included studies, as well as risk of bias assessments were conducted by two study authors. Qualitative data were synthesised narratively and quantified data were used in the metaanalytic calculation. Main contribution: A total of 2,882 papers were identified from the literature search which were double screened at the abstract (n = 1,785) and full-text (n = 366) levels resulting in 10 papers meeting the criteria for inclusion in the review. There were 8 RCTs and 2 QEs using 7 named play-based interventions with ASD participants only. Meta-analysis of 5 studies addressing positive mental health outcomes (e.g. positive affect and emotional functioning) found a significant overall intervention effect (Cohen's d = 1.60 (95% CI [0.37, 2.82], p = 0.01); meta-analysis of 6 studies addressing negative mental health outcomes (e.g., negative affect, internalising and externalising problems) found a non-significant overall intervention effect (Cohen's d = 0.04 -0.17 (95% CI [-0.04, 0.51], p = 0.88). Conclusions: A key observation is the diversity of study characteristics relating to study sample size, duration of interventions, study settings, background of interventionists, and variability of specific mental health outcomes. Play-based interventions appear to have a beneficial effect on positive, but not negative, mental health in children with ASD. There are no high quality studies investigating the efficacy of such interventions in children with DLD. Implications: This review provides good evidence of the need for further research into how commonly used play-based interventions designed to support the social, communication, and language needs of young people may impact the mental health of children with ASD or DLD.

Parent-mediated play-based interventions to improve social communication and language skills of preschool autistic children: A systematic review and meta-analysis protocol.

Early years interventions have shown to be effective in improving the social communication and language skills of autistic children. Therefore, various play-based interventions have been developed to support those developmental areas of autistic children. Although researchers have previously reported the overall effectiveness of different types of play-based interventions on the social communication and language skills of autistic children, no previous systematic reviews have yet evaluated the effectiveness of parent-mediated play-based interventions in preschool autistic children. The overarching aims of the study will be to (i) report the key characteristics and (ii) synthesise the results of studies evaluating parent-mediated play-based interventions targeting the social communication and language skills of preschool autistic children using experimental designs. A comprehensive search for and screening of the relevant studies published between 2000 and 2021 will be undertaken. To be included, studies will have to (i) use either a randomised control trial or quasi-experimental design, (ii) focus on preschool autistic children aged six years old or younger, (iii) deliver a play-based intervention in non-educational settings, and (iv) include at least one parent as the mediator of the intervention. Data extraction of all included studies will be undertaken using a specially devised template and they will also be assessed for risk of bias using an adapted form from the Cochrane Risk of Bias tool. The overall characteristics of the included studies will be reported and a narrative synthesis of the results of the included studies will be undertaken. A meta-analysis may be performed (if justified) to report the pooled effect size of the parent-mediated play-based interventions on the social communication and language skills of preschool autistic children. Trial registration: The current study protocol was pre-registered with the international prospective register of systematic reviews (PROSPERO: CRD42022302220).

Targeting mitochondrial DNA polymerase gamma for selective inhibition of MLH1 deficient colon cancer growth.

Synthetic lethality in DNA repair pathways is an important strategy for the selective treatment of cancer cells without harming healthy cells and developing cancer-specific drugs. The synthetic lethal interaction between the mismatch repair (MMR) protein, MutL homolog 1 (MLH1), and the mitochondrial base excision repair protein, DNA polymerase γ (Pol γ) was used in this study for the selective treatment of MLH1 deficient cancers. Germline mutations in the MLH1 gene and aberrant MLH1 promoter methylation result in an increased risk of developing many cancers, including nonpolyposis colorectal and endometrial cancers. Because the inhibition of Pol γ in MLH1 deficient cancer cells provides the synthetic lethal selectivity, we conducted a comprehensive small molecule screening from various databases and chemical drug library molecules for novel Pol γ inhibitors that selectively kill MLH1 deficient cancer cells. We characterized these Pol γ inhibitor molecules in vitro and in vivo, and identified 3,3'-[(1,1'-Biphenyl)-4',4'-diyl)bis(azo)]bis[4-amino-1-naphthalenesulfonic acid] (congo red; CR; Zinc 03830554) as a high-affinity binder to the Pol γ protein and potent inhibitor of the Pol γ strand displacement and one-nucleotide incorporation DNA synthesis activities in vitro and in vivo. CR reduced the cell proliferation of MLH1 deficient HCT116 human colon cancer cells and suppressed HCT116 xenograft tumor growth whereas it did not affect the MLH1 proficient cell proliferation and xenograft tumor growth. CR caused mitochondrial dysfunction and cell death by inhibiting Pol γ activity and oxidative mtDNA damage repair, increasing the production of reactive oxygen species and oxidative mtDNA damage in MLH1 deficient cells. This study suggests that the Pol γ inhibitor, CR may be further evaluated for the MLH1 deficient cancers' therapy.

How is COVID-19 Affecting the Mental Health of Children with Special Educational Needs and Disabilities and Their Families?

Parents of children with Special Educational Needs and Disabilities in the UK (n = 241) were asked to describe the impact of COVID-19 on their own mental health and that of their child. An inductive content analysis of the data was undertaken. Both parents and children appear to be experiencing loss, worry and changes in mood and behaviour as a result of the rapid social changes that have occurred. Some parents reported feeling overwhelmed and described the impact of child understanding and awareness. Finally, a minority of parents reported that COVID-19 has had little impact on mental health in their family, or has even led to improvements. Implications for how to support these families in the immediate future are discussed.

Long noncoding RNA (lincRNA), a new paradigm in gene expression control.

Long intergenic non-coding RNAs (lincRNAs) are defined as RNA transcripts that are longer than 200 nucleotides. By definition, these RNAs must not have open reading frames that encode proteins. Many of these transcripts are encoded by RNA polymerase II, are spliced, and are poly-adenylated. This final fact indicates that there is a trove of information about lincRNAs in databases such as the Gene Expression Omnibus (GEO), which is a repository for RNAseq and microarray data. Recent experiments indicate that there are upwards of 15,000 lincRNAs encoded by the human genome. The term "intergenic" refers to the identification of these transcripts from regions of the genome that do not contain protein-encoding genes. These regions coincide with what was once labeled as the "junk DNA" portions of our genomes, which, upon careful examination by whole genome RNA sequencing experiments, clearly encode RNA transcripts. LincRNAs also contain promoter- or enhancer-associated RNAs that are gene proximal and can be either in the sense or antisense orientation, relative to the protein-coding gene with which they are associated. In this review, we describe the functions of lincRNAs playing roles in biological processes such as gene expression control, scaffold formation, and epigenetic control.

Screening of new antileukemic agents from essential oils of algae extracts and computational modeling of their interactions with intracellular signaling nodes.

Microalgae are very rich in bioactive compounds, minerals, polysaccharides, poly-unsaturated fatty acids and vitamins, and these rich constituents make microalgae an important resource for the discovery of new bioactive compounds with applications in biotechnology. In this study, we studied the antileukemic activity of several chosen microalgae species at the molecular level and assessed their potential for drug development. Here we identified Stichococcus bacillaris, Phaeodactylum tricornutum, Microcystis aeruginosa and Nannochloropsis oculata microalgae extracts with possible antileukemic agent potentials. Specifically we studied the effects of these extracts on intracellular signal nodes and apoptotic pathways. We characterized the composition of essential oils of these fifteen different algae extracts using gas chromatography-mass spectrometry (GC-MS). Finally, to identify potential molecular targets causing the phenotypic changes in leukemic cell lines, we docked a selected group of these essential oils to several key intracellular proteins. According to results of rank score algorithm, five of these essential oils analyzed might be considered as in silico plausible candidates to be used as antileukemic agents.

PATZ1 Is a DNA Damage-Responsive Transcription Factor That Inhibits p53 Function.

Insults to cellular health cause p53 protein accumulation, and loss of p53 function leads to tumorigenesis. Thus, p53 has to be tightly controlled. Here we report that the BTB/POZ domain transcription factor PATZ1 (MAZR), previously known for its transcriptional suppressor functions in T lymphocytes, is a crucial regulator of p53. The novel role of PATZ1 as an inhibitor of the p53 protein marks its gene as a proto-oncogene. PATZ1-deficient cells have reduced proliferative capacity, which we assessed by transcriptome sequencing (RNA-Seq) and real-time cell growth rate analysis. PATZ1 modifies the expression of p53 target genes associated with cell proliferation gene ontology terms. Moreover, PATZ1 regulates several genes involved in cellular adhesion and morphogenesis. Significantly, treatment with the DNA damage-inducing drug doxorubicin results in the loss of the PATZ1 transcription factor as p53 accumulates. We find that PATZ1 binds to p53 and inhibits p53-dependent transcription activation. We examine the mechanism of this functional inhibitory interaction and demonstrate that PATZ1 excludes p53 from DNA binding. This study documents PATZ1 as a novel player in the p53 pathway.

CD81 interacts with the T cell receptor to suppress signaling.

CD81 (TAPA-1) is a ubiquitously expressed tetraspanin protein identified as a component of the B lymphocyte receptor (BCR) and as a receptor for the Hepatitis C Virus. In an effort to identify trans-membrane proteins that interact with the T-cell antigen receptor (TCR), we performed a membrane yeast two hybrid screen and identified CD81 as an interactor of the CD3delta subunit of the TCR. We found that in the absence of CD81, in thymocytes from knockout mice, TCR engagement resulted in stronger signals. These results were recapitulated in T cell lines that express low levels of CD81 through shRNA mediated silencing. Increased signaling did not result from alterations in the levels of TCR on the surface of T lymphocytes. Although CD81 is not essential for normal T lymphocyte development, it plays an important role in regulating TCR and possibly pre-TCR signal transduction by controlling the strength of signaling. CD81 dependent alterations in thymocyte signaling are evident in increased CD5 expression on CD81 deficient double positive (DP) thymocytes. We conclude that CD81 interacts with the T cell receptor to suppress signaling.

CD8 lineage-specific regulation of interleukin-7 receptor expression by the transcriptional repressor Gfi1.

Interleukin-7 receptor α (IL-7Rα) is essential for T cell survival and differentiation. Glucocorticoids are potent enhancers of IL-7Rα expression with diverse roles in T cell biology. Here we identify the transcriptional repressor, growth factor independent-1 (Gfi1), as a novel intermediary in glucocorticoid-induced IL-7Rα up-regulation. We found Gfi1 to be a major inhibitory target of dexamethasone by microarray expression profiling of 3B4.15 T-hybridoma cells. Concordantly, retroviral transduction of Gfi1 significantly blunted IL-7Rα up-regulation by dexamethasone. To further assess the role of Gfi1 in vivo, we generated bacterial artificial chromosome (BAC) transgenic mice, in which a modified Il7r locus expresses GFP to report Il7r gene transcription. By introducing this BAC reporter transgene into either Gfi1-deficient or Gfi1-transgenic mice, we document in vivo that IL-7Rα transcription is up-regulated in the absence of Gfi1 and down-regulated when Gfi1 is overexpressed. Strikingly, the in vivo regulatory role of Gfi1 was specific for CD8(+), and not CD4(+) T cells or immature thymocytes. These results identify Gfi1 as a specific transcriptional repressor of the Il7r gene in CD8 T lymphocytes in vivo.