Circular extrachromosomal DNA promotes tumor heterogeneity in high-risk medulloblastoma.

Circular extrachromosomal DNA (ecDNA) in patient tumors is an important driver of oncogenic gene expression, evolution of drug resistance and poor patient outcomes. Applying computational methods for the detection and reconstruction of ecDNA across a retrospective cohort of 481 medulloblastoma tumors from 465 patients, we identify circular ecDNA in 82 patients (18%). Patients with ecDNA-positive medulloblastoma were more than twice as likely to relapse and three times as likely to die within 5 years of diagnosis. A subset of tumors harbored multiple ecDNA lineages, each containing distinct amplified oncogenes. Multimodal sequencing, imaging and CRISPR inhibition experiments in medulloblastoma models reveal intratumoral heterogeneity of ecDNA copy number per cell and frequent putative 'enhancer rewiring' events on ecDNA. This study reveals the frequency and diversity of ecDNA in medulloblastoma, stratified into molecular subgroups, and suggests copy number heterogeneity and enhancer rewiring as oncogenic features of ecDNA.

Central Nervous System Distribution of Panobinostat in Preclinical Models to Guide Dosing for Pediatric Brain Tumors.

Achieving adequate exposure of the free therapeutic agent at the target is a critical determinant of efficacious chemotherapy. With this in mind, a major challenge in developing therapies for central nervous system (CNS) tumors is to overcome barriers to delivery, including the blood-brain barrier (BBB). Panobinostat is a nonselective pan-histone deacetylase inhibitor that is being tested in preclinical and clinical studies, including for the treatment of pediatric medulloblastoma, which has a propensity for leptomeningeal spread and diffuse midline glioma, which can infiltrate into supratentorial brain regions. In this study, we examined the rate, extent, and spatial heterogeneity of panobinostat CNS distribution in mice. Transporter-deficient mouse studies show that panobinostat is a dual substrate of P-glycoprotein (P-gp) and breast cancer resistant protein (Bcrp), which are major efflux transporters expressed at the BBB. The CNS delivery of panobinostat was moderately limited by P-gp and Bcrp, and the unbound tissue-to-plasma partition coefficient of panobinostat was 0.32 and 0.21 in the brain and spinal cord in wild-type mice. In addition, following intravenous administration, panobinostat demonstrated heterogeneous distribution among brain regions, indicating that its efficacy would be influenced by tumor location or the presence and extent of leptomeningeal spread. Simulation using a compartmental BBB model suggests inadequate exposure of free panobinostat in the brain following a recommended oral dosing regimen in patients. Therefore, alternative approaches to CNS delivery may be necessary to have adequate exposure of free panobinostat for the treatment of a broad range of pediatric brain tumors. SIGNIFICANCE STATEMENT: This study shows that the central nervous system (CNS) penetration of panobinostat is limited by P-gp and Bcrp, and its efficacy may be limited by inadequate distribution to the tumor. Panobinostat has heterogeneous distribution into various brain regions, indicating that its efficacy might depend on the anatomical location of the tumors. These distributional parameters in the mouse CNS can inform both preclinical and clinical trial study design and may guide treatment for these devastating brain tumors in children.

Honest, Open, Proud (HOP) for people with mental illness in Hong Kong: a randomized controlled trial.

BACKGROUND: Self-stigma among people with mental illness is negatively associated with personal and clinical recovery. Due to the concealable nature of mental illness, people with mental illness experience constant struggles between concealment and disclosure. Disclosure of mental health challenges can potentially minimize negative impacts of self-stigma and enhance self-esteem and sense of empowerment. Honest, Open, Proud (HOP) is a peer-led intervention that promotes autonomous and dignified decisions about disclosure. PURPOSE: This study examined the effectiveness of HOP on concealment motivation, empowerment, self-stigma, stigma stress, and recovery among people with lived experience of mental illness in Hong Kong. METHODOLOGY: A total of 162 participants with a mean age of 45.38 were recruited and randomized into intervention group and waitlist control group. Participants in the intervention group were invited to attend a 6-session HOP group intervention. RESULTS: Significant improvement in optimism score from the empowerment scale was found in the intervention group compared to the waitlist control group and the effect was sustained at 1-month follow-up. However, significant changes were not found in other outcome variables. CONCLUSION: Only improvement in optimism was observed in the current study. Future study needs to examine the effects of HOP with further modification to maximize the benefit for people with lived experience of mental illness in the local context.

N-myc-Mediated Translation Control Is a Therapeutic Vulnerability in Medulloblastoma.

Deregulation of neuroblastoma-derived myc (N-myc) is a leading cause of malignant brain tumors in children. To target N-myc-driven medulloblastoma, most research has focused on identifying genomic alterations or on the analysis of the medulloblastoma transcriptome. Here, we have broadly characterized the translatome of medulloblastoma and shown that N-myc unexpectedly drives selective translation of transcripts that promote protein homeostasis. Cancer cells are constantly exposed to proteotoxic stress associated with alterations in protein production or folding. It remains poorly understood how cancers cope with proteotoxic stress to promote their growth. Here, our data revealed that N-myc regulates the expression of specific components (∼5%) of the protein folding machinery at the translational level through the major cap binding protein, eukaryotic initiation factor eIF4E. Reducing eIF4E levels in mouse models of medulloblastoma blocked tumorigenesis. Importantly, targeting Hsp70, a protein folding chaperone translationally regulated by N-myc, suppressed tumor growth in mouse and human medulloblastoma xenograft models. These findings reveal a previously hidden molecular program that promotes medulloblastoma formation and identify new therapies that may have impact in the clinic. SIGNIFICANCE: Translatome analysis in medulloblastoma shows that N-myc drives selective translation of transcripts that promote protein homeostasis and that represent new therapeutic vulnerabilities.

Integrated genome and tissue engineering enables screening of cancer vulnerabilities in physiologically relevant perfusable ex vivo cultures.

Genetic screens are powerful tools for both resolving biological function and identifying potential therapeutic targets, but require physiologically accurate systems to glean biologically useful information. Here, we enable genetic screens in physiologically relevant ex vivo cancer tissue models by integrating CRISPR-Cas-based genome engineering and biofabrication technologies. We first present a novel method for generating perfusable tissue constructs, and validate its functionality by using it to generate three-dimensional perfusable dense cultures of cancer cell lines and sustain otherwise ex vivo unculturable patient-derived xenografts. Using this system we enable large-scale CRISPR screens in perfused tissue cultures, as well as emulate a novel point-of-care diagnostics scenario of a clinically actionable CRISPR knockout (CRISPRko) screen of genes with FDA-approved drug treatments in ex vivo PDX cell cultures. Our results reveal differences across in vitro and in vivo cancer model systems, and highlight the utility of programmable tissue engineered models for screening therapeutically relevant cancer vulnerabilities.

Factor analysis of the neurobehavioral symptom inventory in veterans with PTSD and no history of mild TBI.

It is well established that long-term postconcussive symptoms following a mild traumatic brain injury (mTBI) are associated with underlying physical, emotional, and behavioral conditions. The Neurobehavioral Symptom Inventory (NSI) is a measure used to assess neurobehavioral symptoms that can occur following a mTBI and has demonstrated a 3- or 4-factor structure in veterans. The present study aimed to investigate the factor structure of veterans with PTSD without a history of mTBI. A confirmatory factor analysis (CFA) was conducted on a sample of 221 treatment-seeking veterans and service members with PTSD and without a history of mTBI. Results supported a 4-factor structure comprised of vestibular, somatic, cognitive, and affective domains in veterans with PTSD. Subsequent, correlational analyses between the four NSI factors and the four subscales of the Posttraumatic Stress Disorder Checklist - Fifth Edition (PCL-5) revealed high correspondence between the cognitive and affective factors of the NSI and the negative alterations in mood and cognitions and hyperarousal symptom subscales of PTSD. Collectively, findings demonstrated that the NSI functions similarly in veterans with PTSD with or without a history of mTBI. Findings suggest that neurobehavioral symptoms assessed by the NSI appear to be nonspecific and not explicitly associated with mTBI.

ChIPseqSpikeInFree: a ChIP-seq normalization approach to reveal global changes in histone modifications without spike-in.

MOTIVATION: The traditional reads per million normalization method is inappropriate for the evaluation of ChIP-seq data when treatments or mutations have global effects. Changes in global levels of histone modifications can be detected with exogenous reference spike-in controls. However, most ChIP-seq studies overlook the normalization that must be corrected with spike-in. A method that retrospectively renormalizes datasets without spike-in is lacking. RESULTS: ChIPseqSpikeInFree is a novel ChIP-seq normalization method to effectively determine scaling factors for samples across various conditions and treatments, which does not rely on exogenous spike-in chromatin or peak detection to reveal global changes in histone modification occupancy. Application of ChIPseqSpikeInFree on five datasets demonstrates that this in silico approach reveals a similar magnitude of global changes as the spike-in method does. AVAILABILITY AND IMPLEMENTATION: St. Jude Cloud (https://pecan.stjude.cloud/permalink/spikefree) and St. Jude Github ( https://github.com/stjude/ChIPseqSpikeInFree). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Correction to: H3.3 K27M depletion increases differentiation and extends latency of diffuse intrinsic pontine glioma growth in vivo.

The original article can be found online.

H3.3 K27M depletion increases differentiation and extends latency of diffuse intrinsic pontine glioma growth in vivo.

Histone H3 K27M mutation is the defining molecular feature of the devastating pediatric brain tumor, diffuse intrinsic pontine glioma (DIPG). The prevalence of histone H3 K27M mutations indicates a critical role in DIPGs, but the contribution of the mutation to disease pathogenesis remains unclear. We show that knockdown of this mutation in DIPG xenografts restores K27M-dependent loss of H3K27me3 and delays tumor growth. Comparisons of matched DIPG xenografts with and without K27M knockdown allowed identification of mutation-specific effects on the transcriptome and epigenome. The resulting transcriptional changes recapitulate expression signatures from K27M primary DIPG tumors and are strongly enriched for genes associated with nervous system development. Integrated analysis of ChIP-seq and expression data showed that genes upregulated by the mutation are overrepresented in apparently bivalent promoters. Many of these targets are associated with more immature differentiation states. Expression profiles indicate K27M knockdown decreases proliferation and increases differentiation within lineages represented in DIPG. These data suggest that K27M-mediated loss of H3K27me3 directly regulates a subset of genes by releasing poised promoters, and contributes to tumor phenotype and growth by limiting differentiation. The delayed tumor growth associated with knockdown of H3 K27M provides evidence that this highly recurrent mutation is a relevant therapeutic target.

Sleeping Beauty Insertional Mutagenesis Reveals Important Genetic Drivers of Central Nervous System Embryonal Tumors.

Medulloblastoma and central nervous system primitive neuroectodermal tumors (CNS-PNET) are aggressive, poorly differentiated brain tumors with limited effective therapies. Using Sleeping Beauty (SB) transposon mutagenesis, we identified novel genetic drivers of medulloblastoma and CNS-PNET. Cross-species gene expression analyses classified SB-driven tumors into distinct medulloblastoma and CNS-PNET subgroups, indicating they resemble human Sonic hedgehog and group 3 and 4 medulloblastoma and CNS neuroblastoma with FOXR2 activation. This represents the first genetically induced mouse model of CNS-PNET and a rare model of group 3 and 4 medulloblastoma. We identified several putative proto-oncogenes including Arhgap36, Megf10, and Foxr2. Genetic manipulation of these genes demonstrated a robust impact on tumorigenesis in vitro and in vivo. We also determined that FOXR2 interacts with N-MYC, increases C-MYC protein stability, and activates FAK/SRC signaling. Altogether, our study identified several promising therapeutic targets in medulloblastoma and CNS-PNET. SIGNIFICANCE: A transposon-induced mouse model identifies several novel genetic drivers and potential therapeutic targets in medulloblastoma and CNS-PNET.

Histone H3.3 K27M Accelerates Spontaneous Brainstem Glioma and Drives Restricted Changes in Bivalent Gene Expression.

Diffuse intrinsic pontine gliomas (DIPGs) are incurable childhood brainstem tumors with frequent histone H3 K27M mutations and recurrent alterations in PDGFRA and TP53. We generated genetically engineered inducible mice and showed that H3.3 K27M enhanced neural stem cell self-renewal while preserving regional identity. Neonatal induction of H3.3 K27M cooperated with activating platelet-derived growth factor receptor α (PDGFRα) mutant and Trp53 loss to accelerate development of diffuse brainstem gliomas that recapitulated human DIPG gene expression signatures and showed global changes in H3K27 posttranslational modifications, but relatively restricted gene expression changes. Genes upregulated in H3.3 K27M tumors were enriched for those associated with neural development where H3K27me3 loss released the poised state of apparently bivalent promoters, whereas downregulated genes were enriched for those encoding homeodomain transcription factors.

Engineering Inducible Knock-In Mice to Model Oncogenic Brain Tumor Mutations from Endogenous Loci.

To maximize the physiological relevance of in vivo brain tumor mouse models designed to study the downstream effects of oncogenic mutations, it is important to express the mutated genes at appropriate levels, in relevant cell types, and in the proper developmental context. For recurrent mutations found in the heterozygous state in tumors, expression of the mutation from the endogenous locus is a more physiologically relevant recapitulation of the brain tumor genome. Here, we describe an approach to generate knock-in mice with an inducible mutation recombined into the endogenous locus. In these engineered mice, the mutated allele is designed for expression controlled by the endogenous promoter and regulatory elements after Cre recombinase-mediated deletion of a loxP-STOP-loxP cassette inserted upstream of the translational start site. To preserve the structure of the endogenous locus, mutations or additional elements may need to be inserted at a considerable distance from the loxP-STOP-loxP cassette. We used recombineering to build a construct with two selectable markers and multiple genetic alterations that can be introduced into the endogenous allele in cis with a single ES cell targeting.

The Impact of a Peer Navigator Program in Addressing the Health Needs of Latinos With Serious Mental Illness.

OBJECTIVE: The impact of a peer navigator program (PNP) on efforts to address the health needs of Latinos with serious mental illness was examined in a randomized controlled trial. METHODS: Latinos with a serious mental illness (N=110) were randomly assigned to the PNP (integrated care with a peer navigator [PN]) or to a treatment-as-usual control group (integrated care without a PN) for one year. Data on service engagement (scheduled and received appointments) were assessed weekly, and self-reports of recovery, empowerment, and quality of life were collected at baseline and at four, eight, and 12 months. RESULTS: Findings from group × trial analyses of covariance (ANCOVAs) found main and interaction effects for scheduled and achieved appointments, showing better engagement for the PNP group compared with the control group over the course of the study. Significant interactions were found for recovery, empowerment, and quality of life, showing greater improvement for the PNP group compared with the control group over year 1 of the study (multivariate ANCOVA; F=3.27, df=9 and 98, p<.01). CONCLUSIONS: In-the-field navigation by peers seems to enhance service engagement, recovery, and quality of life. Whether these results occurred because navigators helped overcome barriers to treatment-regardless of whether they were peers per se-needs to be examined in future research.

PTEN Signaling in the Postnatal Perivascular Progenitor Niche Drives Medulloblastoma Formation.

Loss of the tumor suppressor gene PTEN exerts diverse outcomes on cancer in different developmental contexts. To gain insight into the effect of its loss on outcomes in the brain, we conditionally inactivated the murine Pten gene in neonatal neural stem/progenitor cells. Pten inactivation created an abnormal perivascular proliferative niche in the cerebellum that persisted in adult animals but did not progress to malignancy. Proliferating cells showed undifferentiated morphology and expressed the progenitor marker Nestin but not Math1, a marker of committed granule neuron progenitors. Codeletion of Pten and Trp53 resulted in fully penetrant medulloblastoma originating from the perivascular niche, which exhibited abnormal blood vessel networks and advanced neuronal differentiation of tumor cells. EdU pulse-chase experiments demonstrated a perivascular cancer stem cell population in Pten/Trp53 double mutant medulloblastomas. Genetic analyses revealed recurrent somatic inactivations of the tumor suppressor gene Ptch1 and a recapitulation of the sonic hedgehog subgroup of human medulloblastomas. Overall, our results showed that PTEN acts to prevent the proliferation of a progenitor niche in postnatal cerebellum predisposed to oncogenic induction of medulloblastoma. Cancer Res; 77(1); 123-33. ©2016 AACR.

Key ingredients to contact-based stigma change: a cross-validation.

OBJECTIVE: Contact-based antistigma programs seemingly have a larger and more sustained impact than educational strategies. Previous qualitative research of advocates with lived experiences yielded 32 key ingredients of contact-based programs comprising 5 categories. This study sought an independent sample's feedback of the 32 ingredients. METHODS: One hundred advocates with lived experience of mental health conditions who have led, coordinated, and/or delivered antistigma presentations completed an online survey to rank importance of key ingredients. RESULTS: Analysis of rank distributions showed most important ingredients in program categories: (a) design: face-to-face presentations, audience discussion; (b) target: specific group identified (e.g., employers), assessment completed with targets to derive stigma change goals relevant to needs; (c) staff: presenters are people with lived experience; (d) message: message includes on the-way-up stories; and (e) evaluation/follow-up: post-presentation follow-up actions discussed with targets. CONCLUSIONS AND IMPLICATIONS FOR PRACTICE: Future research hopes to yield a fidelity measure for contact-based programs.

Production of homozygous transgenic rainbow trout with enhanced disease resistance.

Previous studies conducted in our laboratory showed that transgenic medaka expressing cecropin B transgenes exhibited resistant characteristic to fish bacterial pathogens, Pseudomonas fluorescens and Vibrio anguillarum. To confirm whether antimicrobial peptide gene will also exhibit anti-bacterial and anti-viral characteristics in aquaculture important fish species, we produced transgenic rainbow trout expressing cecropin P1 or a synthetic cecropin B analog, CF-17, transgene by sperm-mediated gene transfer method. About 30 % of fish recovered from electroporation were shown to carry the transgene as determined by polymerase chain reaction (PCR) amplification assay. Positive P1 transgenic fish were crossed to non-transgenic fish to establish F1 transgenic founder families, and subsequently generating F2, and F3 progeny. Expression of cecropin P1 and CF-17 transgenes was detected in transgenic fish by reverse transcription (RT)-PCR analysis. The distribution of body sizes among F1 transgenic fish were not significantly different from those of non-transgenic fish. Results of challenge studies revealed that many families of F2 and F3 transgenic fish exhibited resistance to infection by Aeromonas salmonicida and infectious hematopoietic necrosis virus (IHNV). All-male homozygous cecropin P1 transgenic families were produced by androgenesis from sperm of F3 heterozygous transgenic fish in one generation. The resistant characteristic to A. salmonicida was confirmed in progeny derived from the outcross of all-male fish to non-transgenic females. Results of our current studies confirmed the possibility of producing disease-resistant homozygous rainbow trout strains by transgenesis of cecropin P1 or CF-17 gene and followed by androgenesis.

The California schedule of key ingredients for contact-based antistigma programs.

OBJECTIVE: A major public health priority has been to eliminate stigma's egregious effects on life opportunities for people with mental illnesses. Research shows contact-based antistigma programs are among the most effective. Such findings call for clarity to define the components of consumer-directed antistigma programs. This article represents community-based participatory research (CBPR) and the first step of a mixed methods design to describe the active ingredients comprising these kinds of programs. METHOD: CBPR investigators developed an interview guide and subsequently facilitated four focus groups to identify key components. Participants included antistigma experts with lived experience. Using grounded theory, two independent raters identified 641 discrete themes. Two additional coders then sorted themes into constructs representing key ingredients of contact-based programs. Coders agreed upon 198 constructs and then grouped them into a hierarchical model of key ingredients in consumer-directed stigma change. RESULTS: Five criteria represent indicators of successful consumer-directed programs: (a) program design-factors necessary for trained presenters to facilitate programs; (b) targeting-tailored presentation congruent with target's goal; (c) staffing-facilitators and leadership are diverse people with lived experience; (d) messaging-presentation discusses struggles and recovery from mental health challenges; and (e) follow-up and evaluation-assessment of sustained audience change. CONCLUSIONS AND IMPLICATIONS FOR PRACTICE: This study identified key ingredients of consumer-directed antistigma programs. Part two of the mixed methods design, a quantitative cross-validation study, will yield a sound fidelity measure.

Review: In vivo models for defining molecular subtypes of the primitive neuroectodermal tumor genome: current challenges and solutions.

Primitive neuroectodermal tumors (PNET) of the brain include medulloblastoma (MB) and central nervous system primitive neuroectodermal tumor (CNS PNET) subtypes, which share histological features yet differ at the genomic level and in clinical outcome. Delineation of the genetic anomalies between PNET subtypes is a current challenge for establishing effective targeted therapeutic strategies against these aggressive tumors. Current efforts have demonstrated that specific molecular pathways drive a subset of MB and CNS PNET, but the genetic basis for the deadliest forms of these tumors remains poorly understood and anecdotal. This is in part due to an overall lack of biologically relevant in vivo and in vitro model systems capable of direct comparison and identification of the genetic origins among PNET subtypes. Forward genetic, random mutagenesis in mice is an effective phenotype-driven method to model the genetic origins of human disease including cancer. We have applied this method to PNET by developing a single Sleeping Beauty transposon insertional mutagenesis mouse model that recapitulates the morphological similarities and genetic heterogeneity of MB and CNS PNET capable of identifying genetic drivers important for genesis of PNET. Importantly, this model has allowed new PNET phenotypes to be observed and is designed to reveal biologically relevant candidate oncogenes and tumor suppressor genes for MB and CNS PNET molecular subgroups in mice and humans. The ultimate goal of the approach we have taken is to uncover new understanding of the genetic basis for MB and CNS PNET development, how they are distinguished from each other, and offer potential targets for therapeutic testing to improve patient clinical outcome.

The lineage-specific gene ponzr1 is essential for zebrafish pronephric and pharyngeal arch development.

The Homeobox (Hox) and Paired box (Pax) gene families are key determinants of animal body plans and organ structure. In particular, they function within regulatory networks that control organogenesis. How these conserved genes elicit differences in organ form and function in response to evolutionary pressures is incompletely understood. We molecularly and functionally characterized one member of an evolutionarily dynamic gene family, plac8 onzin related protein 1 (ponzr1), in the zebrafish. ponzr1 mRNA is expressed early in the developing kidney and pharyngeal arches. Using ponzr1-targeting morpholinos, we show that ponzr1 is required for formation of the glomerulus. Loss of ponzr1 results in a nonfunctional glomerulus but retention of a functional pronephros, an arrangement similar to the aglomerular kidneys found in a subset of marine fish. ponzr1 is integrated into the pax2a pathway, with ponzr1 expression requiring pax2a gene function, and proper pax2a expression requiring normal ponzr1 expression. In addition to pronephric function, ponzr1 is required for pharyngeal arch formation. We functionally demonstrate that ponzr1 can act as a transcription factor or co-factor, providing the first molecular mode of action for this newly described gene family. Together, this work provides experimental evidence of an additional mechanism that incorporates evolutionarily dynamic, lineage-specific gene families into conserved regulatory gene networks to create functional organ diversity.

Sino-American employer perspective about behavioral-driven health conditions: predictive analyses.

OBJECTIVE: A large-scale quantitative study was conducted by stratified representative samples from Chicago (prototype of the United States; N = 293), Beijing (prototype of urbanized China; N = 302), and Hong Kong (prototype of East-meets-West culture; N = 284) to explore factors that might lead to their stigmatizing attitudes towards hiring individuals with (mental illness, alcohol abuse, drug abuse, and HIV/AIDS) and without (bone cancer) behavioral-driven health conditions. METHODS: Consented employers completed the Employer Survey pertaining to their attitudes towards specific health conditions, previous hiring experiences, resources, assets of applicants, and hiring concerns. RESULTS: The findings suggested that employers in Hong Kong and Beijing were more willing to hire individuals with alcohol abuse, whereas employers in Chicago were more willing to hire those with HIV/AIDS or bone cancer. Logistic regression suggested that the type of health conditions, assets of applicants, and perceived level of dangerousness of applicants were significant predictors that contributed to employers' hiring preference. CONCLUSION: Employers express different hiring preference towards individuals with or without behavioral-driven health conditions. Their hiring preference towards specific type of health conditions is discussed.