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Myc oncoproteins exert tumorigenic effects by regulating expression of target oncogenes. Histone H3 lysine 79 (H3K79) methylation at Myc-responsive elements of target gene promoters is a strict prerequisite for Myc-induced transcriptional activation, and DOT1L is the only known histone methyltransferase that catalyzes H3K79 methylation. Here, we show that N-Myc upregulates DOT1L mRNA and protein expression by binding to the DOT1L gene promoter. shRNA-mediated depletion of DOT1L reduced mRNA and protein expression of N-Myc target genes ODC1 and E2F2. DOT1L bound to the Myc Box II domain of N-Myc protein, and knockdown of DOT1L reduced histone H3K79 methylation and N-Myc protein binding at the ODC1 and E2F2 gene promoters and reduced neuroblastoma cell proliferation. Treatment with the small-molecule DOT1L inhibitor SGC0946 reduced H3K79 methylation and proliferation of MYCN gene–amplified neuroblastoma cells. In mice xenografts of neuroblastoma cells stably expressing doxycycline-inducible DOT1L shRNA, ablating DOT1L expression with doxycycline significantly reduced ODC1 and E2F2 expression, reduced tumor progression, and improved overall survival. In addition, high levels of DOT1L gene expression in human neuroblastoma tissues correlated with high levels of MYCN, ODC1, and E2F2 gene expression and independently correlated with poor patient survival. Taken together, our results identify DOT1L as a novel cofactor in N-Myc–mediated transcriptional activation of target genes and neuroblastoma oncogenesis. Furthermore, they characterize DOT1L inhibitors as novel anticancer agents against MYCN-amplified neuroblastoma. Cancer Res; 77(9); 2522–33. ©2017 AACR.

Supplementary Figure S1. PA2G4 is a MYCN transactivation target gene. A, Quantification of protein expression using anti-PA2G4 and anti-MYCN antibodies against whole cell protein lysates from BE(2)-C and Kelly cells, following MYCN siRNA knockdown for 48 hours. B, mRNA expression of PA2G4 and MYCN in SH-EP MYCN3 overexpression cells treated with 1µg/ml doxycycline for 24-96 hr. C, The effect of doxycycline-induced MYCN overexpression in SHEP-TRE-MYCN cells on c-MYC and PA2G4 protein expression. D, Chromatin immunoprecipitation (ChIP) assay in Kelly cells using an anti-MYCN antibody, and real-time PCR analysis with primers identifying the MYCN DNA binding sites in the PA2G4 gene promoter (500bP upstream of transcription start site [TSS]) or Intron 1a & 1b regions of the PA2G4 gene, with and without MYCN siRNA knockdown. ChIP and real-time PCR analysis using primers against a region 1200bp upstream of TSS was used as a negative control for MYCN chromatin binding. ChIP and real-time PCR analysis using primers against the ornithine decarboxylase (ODC1) gene promoter region was used as a positive control for MYCN chromatin binding. E, Immunoblot analysis of PA2G4, MYCN and MYC protein levels in a panel of human MYCN amplified and non-amplified neuroblastoma, and normal fibroblast, cell lines using antibodies recognising PA2G4, MYCN and MYC.

Supplementary Figure S1. F, Confocal microscopy of SHEP Tet21N cells using anti-PA2G4 (red) and anti-MYCN (green) antibodies. G, Immunoblot analysis of cytoplasmic and nuclear fractions from BE(2)-C and Kelly human neuroblastoma cell lines with antibodies recognizing PA2G4 and MYCN, or GAPDH and topoisomerase as loading controls. H, Confocal microscopy of neuroblastoma (BE(2)-C and Kelly) cells using anti-PA2G4 (red) and anti-MYCN (green) antibodies. Alexafluor 555 anti-rabbit (to detect PA2G4) and Alexafluor 488 anti-mouse (to detect MYCN) were used as the secondary antibodies. I, Real-time PCR mRNA expression of MYCN and PA2G4 in ganglia from homozygote TH-MYCN+/+ mice, compared to wild type littermate control mice, obtained at different postnatal age (weeks).

MYCN is a major driver for the childhood cancer, neuroblastoma, however, there are no inhibitors of this target. Enhanced MYCN protein stability is a key component of MYCN oncogenesis and is maintained by multiple feedforward expression loops involving MYCN transactivation target genes. Here, we reveal the oncogenic role of a novel MYCN target and binding protein, proliferation-associated 2AG4 (PA2G4). Chromatin immunoprecipitation studies demonstrated that MYCN occupies the PA2G4 gene promoter, stimulating transcription. Direct binding of PA2G4 to MYCN protein blocked proteolysis of MYCN and enhanced colony formation in a MYCN-dependent manner. Using molecular modeling, surface plasmon resonance, and mutagenesis studies, we mapped the MYCN–PA2G4 interaction site to a 14 amino acid MYCN sequence and a surface crevice of PA2G4. Competitive chemical inhibition of the MYCN–PA2G4 protein–protein interface had potent inhibitory effects on neuroblastoma tumorigenesis in vivo. Treated tumors showed reduced levels of both MYCN and PA2G4. Our findings demonstrate a critical role for PA2G4 as a cofactor in MYCN-driven neuroblastoma and highlight competitive inhibition of the PA2G4-MYCN protein binding as a novel therapeutic strategy in the disease.Significance:Competitive chemical inhibition of the PA2G4–MYCN protein interface provides a basis for drug design of small molecules targeting MYC and MYCN-binding partners in malignancies driven by MYC family oncoproteins.

Supplementary Figure S2. PA2G4 increases MYCN protein stability. A and B, Representative immunoblots from cycloheximide (CHX) chase assays measuring the half-life of MYCN protein after PA2G4 knockdown (A) or overexpression (B) in BE(2)-C and Kelly cells for 48 hours. Cells were then treated with 100 µg/µl CHX for up to 60 minutes followed by immunoblotting. C, Co-IP of total protein from BE(2)-C cells using IgG, anti-MYCN, anti-PA2G4 antibodies, followed by immunoblotting with anti-MYCN, anti-PA2G4, anti-AURKA, anti-Fbxw7 or anti-vinculin antibodies.

Supplementary Figure S4. Characterization of the PA2G4-MYCN protein-protein interface. A, BE(2)-C cells were transiently transfected with EV, wildtype PA2G4 or 6 different PA2G4 point mutants for 48 hours, then treated with 100 µg/µl Cycloheximide (CHX) for up to 60 minutes, followed by immunoblot analysis for MYCN protein half-life. B, Differential Scanning Fluorimetry (DSF) showed both the seven amino acid (DHKALST, aa248-254) and large peptide (GGDHKALSTGEDTL, aa246-259) MYCN oligopeptides, along with the MYCN oligopeptide shown not to bind via SPR (DHAALAT) changed the melting temperature of the PA2G4 protein, relative to baseline (i.e. 0mM), in a dose-response manner. Shown are the means of 3 independent experiments {plus minus} SEM. C, An example of the raw data for DHKALST, with the shift to the left correlating to an increase in concentration. D, Raw SPR data for PA2G4 triple mutant (R271A, R272A and S47A) and single mutants (S47A and R272A). Also, raw SPR data for MYCN oligopeptide mutants (DHAALST, DHAALAT and DHKALAT). For the mutants and triple mutations, no binding was observed, thus analysis could not be conducted and is not shown. E, Root Mead Squared Deviation (RMSD) of the peptide over the time of the simulation. After the first 100 frames the peptide is relatively stable. Analysis was only conducted after this time.

Supplementary Information

Supplementary Figure S6. PA2G4 increases neuroblastoma tumorigenicity. A, Representative images of athymic nude mice inoculated with neuroblastoma (SH-EP) cells stably transfected with either EV control or a PA2G4 expression vector at 10 weeks post-injection. B, Images of tumor formation after mice were culled 12 weeks post-injection. C, Real-time PCR analysis of PA2G4 mRNA expression level in tumors from mice injected with either SH-EP cells overexpressing PA2G4, or SH-EP EV control cells. β2-microglobulin was used as a reference gene for total RNA loading. D, Protein was extracted from SH-EP tumor xenografts overexpressing PA2G4 and control vectors, then analysed for PA2G4-Flag and MYCN expression by immunoblotting using anti-MYCN and anti-PA2G4 antibodies, using a Vinculin loading control. E, Immunoblots of three tumor samples from each siRNA-treated cohort showing the levels of PA2G4 and MYCN protein expression, using a Vinculin loading control. F, Real-time PCR analysis of PA2G4 and MYCN mRNA expression in tumor samples taken from tumour-bearing mice xenografted with BE(2)-C neuroblastoma cells, which had been treated with either nano-particle encapsulated siRNA control, PA2G4 siRNA or PA2G4-p48 siRNA. Data are shown as means and SD derived from 6 mice per group, P-values were calculated by t-test.

Supplementary Figure S5 F-H F), at 72 hours post-transfection of EV or a PA2G4 expression vector. G, Cell viability measured by the Alamar Blue assay and immunoblot analyses using an antibody identifying MYC-tagged PA2G4-p42 protein expression in Kelly and SH-SY5Y cells transfected with EV or the MYC-tagged PA2G4-p42 expression vector at 48 and 72 hours post-transfection. Vinculin was used as loading control. H, Colony formation in vitro by Kelly and SH-SY5Y cells following transfection with either EV or MYC-tagged PA2G4-p42.

Supplementary Figure S3 F-G F, Left panel: Histopathologic and immunohistochemical analyses of MYCN;GFP tumors treated with vehicle (left) or WS6 (right). Left Panel, Top to Bottom: Neuroblastoma tumour sections immunohistochemically stained for Haematoxylin & Eosin (H&E), Proliferating Cell Nuclear Antigen (pCNA), Neural Hu protein C (Hu-C), MYCN, PA2G4 and Tyrosine Hydroxylase. Scale bar, 50 μm. Right Panel: Histograms illustrating the staining intensity of cells of vehicle (left) or WS6 treated neuroblastoma tumours expressing either MYCN, PA2G4 or Tyrosine Hydroxylase as measured by Image J software. Sample means (horizontal bars) were compared by students t-test (two-tailed). *** represents p-value

Supplementary Table S2. Multivariable Cox regression analysis of DOT1L expression in tumor tissues as a factor prognostic for outcome in 476 neuroblastoma patients

Supplementary Table S1 (Excel file). List of transcription factor target motifs around transcription start sites of genes down-regulated by DOT1L shRNA, as identified by gene set enrichment analysis.

Supplementary Figure S6 G-K G, Overall patient survival using Kaplan-Meier survival probability plots from the Cologne data set (http://r2.amc.nl) for PA2G4 mRNA expression subdivided around the median PA2G4 expression level among 477 neuroblastoma patients. H, Kaplan-Meier plot for event-free survival of 649 neuroblastoma patients from the kocak dataset (R2 microarray analysis and visualization platform, http://r2.amc.nl) I, Real-time PCR mRNA expression of PA2G4 among 40 neuroblastoma patient tumors treated at Sydney Children's Hospital, subdivided by MYCN amplification status. J, Multivariate event-free survival analysis using cox regression modelling. The p-values were obtained from the cox-regression analysis. K, Incidence of PA2G4 amplification among a range of different human cancer types within The Cancer Genome Atlas (TCGA). Results were generated using cBioportal for Cancer Genomics (https://cancergenome.nih.gov/). NEPC, neuroendocrine prostate cancer; CS, carcinosarcoma.

Supplementary Materials and Methods, Figures, Figure Legends. Fig. S1, N-Myc up-regulates ODC1 and E2F2. Fig. S2, DOT1L and N-Myc do not regulate HIF1α and VEGF. Fig. S3, knocking down DOT1L gene expression does not induce neuroblastoma cell death. Fig. S4, knocking down DOT1L reduces ODC1 and E2F2 expression in neuroblastoma tissues in mice. Fig. S5, DOT1L expression correlates with ODC1 and E2F2 expression in human neuroblastoma tissues.

Supplementary Figure S5. PA2G4 expression enhances the malignant neuroblastoma phenotype in vitro. A, Neurite formation in SH-SY5Y and BE(2)-C cells transfected with PA2G4 siRNA and then treated with 2 µM 13-cis-retinoic acid. B, Immunoblots assessing PA2G4-p48 and PA2G4-p42 protein expression levels using an anti-PA2G4 antibody against whole cell protein lysates from a panel of neuroblastoma and non-malignant myofibroblast cell lines. C, Immunoblots assessing the effect of PA2G4-p48 knockdown on PA2G4 and MYCN protein levels in BE(2)-C and CHP-134 cells. D, Immunoblots confirming transfection of PA2G4-p48 siRNA and MYCN expression plasmid DNA for 72 hours. E and F Cell proliferation measured by BrdU incorporation (E), and, cell viability measured by the Alamar Blue assay.

Supplementary Figure S3. Competitive chemical inhibition of MYCN-PA2G4 binding. A, Chemical structure of WS6. B, Cycloheximide chase assay measuring the half-life of MYCN protein in BE(2)-C and Kelly cells following 0.8 µM WS6 treatment for 24 hours, then treatment with 100 µg/µl CHX for up to 60 minutes. C, BE(2)-C and Kelly cells treated with 0.2 µM or 0.4 µM WS6, followed by colony formation assessment, compared to untreated cells. D, Immunoblotting with an anti-MYC-Tag antibody to quantify PA2G4 protein expression from SH-SY5Y and Kelly cells transfected with the MYC-tagged PA2G4 vector. E, Densitometric quantification of MYCN and PA2G4 protein levels of immunoblots using tumor tissues of TH-MYCN mice treated with either DMSO (n=5) or WS6 (n=5), and quantified by Image J software. Sample means were compared by students t-test. * Represents p-value

Supplementary Table 1: siRNA sequences Supplementary Table 2: qRT-PCR primers Supplementary Table 3: Primers for chromatin immunoprecipitation Supplementary Table 4: Multivariate analysis of event free survival and overall survival of neuroblastoma patients

List of N-Myc-binding gene promoters at which H3K4me3 was reduced by WDR5 siRNA.

List of N-Myc non-binding gene promoters at which H3K4me3 was reduced by WDR5 siRNA.

Supporting data; Supplementary Figure 1:Correlation analysis of DKC1 mRNA and dyskerin protein quantity in neuroblastoma cell lines Supplementary Figure 2:Correlation analysis of telomerase activity gene expression in neuroblastoma cells Supplementary Figure 3:Correlation analysis of TERT and MYC gene expression in neuroblastoma tumor samples Supplementary Figure 4:High DKC1 expression is a marker of poor prognosis in neuroblastoma Supplementary Figure 5:Analysis of TERT expression in neuroblastoma tumor samples Supplementary Figure 7: Upregulation of p21cip1 in NB69, but not SK-N-BE(2) cells following down regulation of dyskerin Supplementary Figure 8: Cell cycle and viability following downregulation of dyskerin

Supplementary Figures 1-5 from p53 Is a Direct Transcriptional Target of MYCN in Neuroblastoma

List of N-Myc-binding gene promoters at which H3K4me3 was not reduced by WDR5 siRNA.

Supplementary Methods-Figures-Tables

Supplementary Tables 1-4 from p53 Is a Direct Transcriptional Target of MYCN in Neuroblastoma

Genes differentially down- or up-regulated by WDR5 siRNAs by more than 1.5 fold, as shown by Affymetrix microarray, in BE(2)-C neuroblastoma cells 40 hours after siRNA transfection.

Text file containing detailed methods.

List of N-Myc non-binding gene promoters at which H3K4me3 was not reduced by WDR5 siRNA.

The RNA-binding protein dyskerin, encoded by the DKC1 gene, functions as a core component of the telomerase holoenzyme as well as ribonuclear protein complexes involved in RNA processing and ribosome biogenesis. The diverse roles of dyskerin across many facets of RNA biology implicate its potential contribution to malignancy. In this study, we examined the expression and function of dyskerin in neuroblastoma. We show that DKC1 mRNA levels were elevated relative to normal cells across a panel of 15 neuroblastoma cell lines, where both N-Myc and c-Myc directly targeted the DKC1 promoter. Upregulation of MYCN was shown to dramatically increase DKC1 expression. In two independent neuroblastoma patient cohorts, high DKC1 expression correlated strongly with poor event-free and overall survival (P < 0.0001), independently of established prognostic factors. RNAi-mediated depletion of dyskerin inhibited neuroblastoma cell proliferation, including cells immortalized via the telomerase-independent ALT mechanism. Furthermore, dyskerin attenuation impaired anchorage-independent proliferation and tumor growth. Overexpression of the telomerase RNA component, hTR, demonstrated that this proliferative impairment was not a consequence of telomerase suppression. Instead, ribosomal stress, evidenced by depletion of small nucleolar RNAs and nuclear dispersal of ribosomal proteins, was the likely cause of the proliferative impairment in dyskerin-depleted cells. Accordingly, dyskerin suppression caused p53-dependent G1 cell-cycle arrest in p53 wild-type cells, and a p53-independent pathway impaired proliferation in cells with p53 dysfunction. Together, our findings highlight dyskerin as a new therapeutic target in neuroblastoma with crucial telomerase-independent functions and broader implications for the spectrum of malignancies driven by MYC family oncogenes. Cancer Res; 76(12); 3604–17. ©2016 AACR.

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Text file of Figure Legends to Supplementary data

Supporting data; Supplementary Figure 6:Nucleolar dispersal of rpl5

The molecular mechanisms and gene regulatory networks sustaining cell proliferation in neuroblastoma (NBL) cells are still not fully understood. In this tumor context, it has been proposed that anti-proliferative drugs, such as the pan-HDAC inhibitor panobinostat, could be tested to mitigate tumor progression. Here, we set out to investigate the effects of panobinostat treatment at the unprecedented resolution offered by single-cell sequencing. We identified a global senescence signature paired with reduction in proliferation in treated Kelly cells and more isolated transcriptional responses compatible with early neuronal differentiation. Using master regulator analysis, we identified BAZ1A, HCFC1, MAZ, and ZNF146 as the transcriptional regulators most significantly repressed by panobinostat. Experimental silencing of these transcription factors (TFs) confirmed their role in sustaining NBL cell proliferation in vitro.

MYCN activation is a hallmark of advanced neuroblastoma (NB) and a known master regulator of metabolic reprogramming, favoring NB adaptation to its microenvironment. We found that the expression of the main regulators of the molecular clock loops is profoundly disrupted in MYCN-amplified NB patients, and this disruption independently predicts poor clinical outcome. MYCN induces the expression of clock repressors and downregulates the one of clock activators by directly binding to their promoters. Ultimately, MYCN attenuates the molecular clock by suppressing BMAL1 expression and oscillation, thereby promoting cell survival. Reestablishment of the activity of the clock activator RORα via its genetic overexpression and its stimulation through the agonist SR1078, restores BMAL1 expression and oscillation, effectively blocks MYCN-mediated tumor growth and de novo lipogenesis, and sensitizes NB tumors to conventional chemotherapy. In conclusion, reactivation of RORα could serve as a therapeutic strategy for MYCN-amplified NBs by blocking the dysregulation of molecular clock and cell metabolism mediated by MYCN., MYCN is frequently amplified in neuroblastomas. Here, the authors show that MYCN disrupts the molecular clock by downregulating clock activator RORα and that the reactivation of RORα restores BMAL1 activity, and inhibits lipid metabolism and neuroblastoma growth

PRDM (PRDI-BF1 and RIZ homology domain containing) family members are sequence-specific transcriptional regulators involved in cell identity and fate determination, often dysregulated in cancer. The PRDM15 gene is of particular interest, given its low expression in adult tissues and its overexpression in B-cell lymphomas. Despite its well characterized role in stem cell biology and during early development, the role of PRDM15 in cancer remains obscure. Herein, we demonstrate that while PRDM15 is largely dispensable for mouse adult somatic cell homeostasis in vivo, it plays a critical role in B-cell lymphomagenesis. Mechanistically, PRDM15 regulates a transcriptional program that sustains the activity of the PI3K/AKT/mTOR pathway and glycolysis in B-cell lymphomas. Abrogation of PRDM15 induces a metabolic crisis and selective death of lymphoma cells. Collectively, our data demonstrate that PRDM15 fuels the metabolic requirement of B-cell lymphomas and validate it as an attractive and previously unrecognized target in oncology., The transcriptional regulator PRDM15 is expressed at low levels in normal tissues but overexpressed in B-cell lymphomas. Here, the authors show that PRDM15 depletion does not affect adult somatic cell homeostasis but leads to a metabolic crisis which impairs B-cell lymphomagenesis.

In this prospective study, we assessed the effectiveness and acceptability of paliperidone palmitate 1-month (PP1M) and aripiprazole monohydrate (AM) over 1-year follow-up. We included 195 subjects (117 treated with PP1M and 78 with AM) with schizophrenia spectrum disorders from real-world settings. We estimated no differences in hospitalization (Odds Ratio=1.59; p = 0.12), symptoms improvement (p = 0.90 adjusted for baseline severity), and discontinuation (Hazard Ratio=0.72; p = 0.20) at study endpoint. Although current evidence suggests the possible superiority of AM over PP1M, our findings showed comparable effectiveness between these drugs. Additional studies in real-world settings with direct comparisons between these two LAIs are needed.

Background: Fibromyalgia Syndrome (FMS) is characterized by chronic widespread pain, fatigue, unrefreshing sleep and cognitive dysfunction. Depressive and manic symptoms are often reported in FMS patients’ history. The aim of this study was to evaluate the prevalence of bipolar spectrum symptoms (BSS) and to correlate these with quality of life (QoL) scores and antidepressant treatment. Methods: From October 2017 to July 2018, a battery of QoL questionnaires (FIQ, PSQI and SF-12) was administered to 120 FMS patients after a clinical examination. The MOODS-SR lifetime questionnaire was then remotely administered to the patients included in the study. Results: The presence of depressive and manic lifetime symptoms was found, in line with the results of the available literature. A correlation was found between the history of depressive symptoms and the severity of FIQ and SF-12 scores. Despite a low statistical strength, a trend toward a correlation between a history of manic symptoms and SNRI treatment was detected. Conclusions: The correlation between the MOOD—depressive domains and poor QoL is in line with the available literature. Further studies are needed to corroborate these findings and to elucidate the relationship between manic symptoms and SNRI treatment.

Introduction: Chronic pain patients who undergo to stressful events may experience worsening in pain, sleep, and quality of life (QoL). The primary objective of this observational study was to compare QoL and sleep parameters before and after the COVID-19 lockdown in patients with Fibromyalgia Syndrome (FMS). The psychological impact of lockdown was also assessed, as well as the emotional impact of the pandemic and its correlations with patient socio-demographics. Methods: Patients aged ≥18 years with FMS diagnosed according to ACR (American College of Rheumatology) 2016 Criteria with at least one pre-pandemic QoL and sleep evaluation were included. QoL and sleep disturbances were analyzed by comparing scores on the Fibromyalgia Impact Questionnaire-Revised (FIQ-R), the 12-item Short Form Survey (SF-12), and the Pittsburgh Sleep Quality Index (PSQI) before and after the first lockdown in Italy (March to May 2020). Psychological impact was investigated via a 52-item survey of daily life changes in FMS management during the lockdown and emotional impact with the Impact Event Scale-Revised (IES-R) tool during the onset period after the lockdown. Questionnaire responses were correlated with patients’ socio-demographics. Results: Questionnaires were submitted via email to 54 patients; 37/54 patients (63.7%) returned them. QoL and sleep disturbances showed no statistically significant worsening. However, the psychological impact survey revealed that 3 out of 5 patients feared very much for their family members’ lives during lockdown. The emotional impact survey disclosed 72.7% of patients with psychological distress. Conclusions: While questionnaire responses showed no significant changes in QoL and sleep after the COVID-19 lockdown in this sample of FMS patients, the emotional investigation revealed moderate/severe psychological distress not detected by commonly used QoL tests in FMS.

Neuroblastoma (NB) is one of the primary causes of death for pediatric malignancies. Given the high heterogeneity in NB’s mutation landscape, optimizing individualized therapies is still challenging. In the context of genomic alterations, MYCN amplification is the most correlated event with poor outcomes. MYCN is involved in the regulation of several cellular mechanisms, including cell cycle. Thus, studying the influence of MYCN overexpression in the G1/S transition checkpoint of the cell cycle may unveil novel druggable targets for the development of personalized therapeutical approaches. Here, we show that high expression of E2F3 and MYCN correlate with poor prognosis in NB despite the RB1 mRNA levels. Moreover, we demonstrate through luciferase reporter assays that MYCN bypasses RB function by incrementing E2F3-responsive promoter activity. We showed that MYCN overexpression leads to RB inactivation by inducing RB hyperphosphorylation during the G1 phase through cell cycle synchronization experiments. Moreover, we generated two MYCN-amplified NB cell lines conditionally knockdown (cKD) for the RB1 gene through a CRISPRi approach. Indeed, RB KD did not affect cell proliferation, whereas cell proliferation was strongly influenced when a non-phosphorylatable RB mutant was expressed. This finding revealed the dispensable role of RB in regulating MYCN-amplified NB’s cell cycle. The described genetic interaction between MYCN and RB1 provides the rationale for using cyclin/CDK complexes inhibitors in NBs carrying MYCN amplification and relatively high levels of RB1 expression.

Background: Long-acting injectable (LAI) antipsychotics are efficacious in managing psychotic symptoms in people affected by severe mental disorders, such as schizophrenia and bipolar disorder. The present study aimed to investigate whether attitude toward treatment and treatment adherence represent predictors of symptoms changes over time.Methods: The STAR Network “Depot Study” was a naturalistic, multicenter, observational, prospective study that enrolled people initiating a LAI without restrictions on diagnosis, clinical severity or setting. Participants from 32 Italian centers were assessed at three time points: baseline, 6-month, and 12-month follow-up. Psychopathological symptoms, attitude toward medication and treatment adherence were measured using the Brief Psychiatric Rating Scale (BPRS), the Drug Attitude Inventory (DAI-10) and the Kemp's 7-point scale, respectively. Linear mixed-effects models were used to evaluate whether attitude toward medication and treatment adherence independently predicted symptoms changes over time. Analyses were conducted on the overall sample and then stratified according to the baseline severity (BPRS < 41 or BPRS ≥ 41).Results: We included 461 participants of which 276 were males. The majority of participants had received a primary diagnosis of a schizophrenia spectrum disorder (71.80%) and initiated a treatment with a second-generation LAI (69.63%). BPRS, DAI-10, and Kemp's scale scores improved over time. Six linear regressions—conducted considering the outcome and predictors at baseline, 6-month, and 12-month follow-up independently—showed that both DAI-10 and Kemp's scale negatively associated with BPRS scores at the three considered time points. Linear mixed-effects models conducted on the overall sample did not show any significant association between attitude toward medication or treatment adherence and changes in psychiatric symptoms over time. However, after stratification according to baseline severity, we found that both DAI-10 and Kemp's scale negatively predicted changes in BPRS scores at 12-month follow-up regardless of baseline severity. The association at 6-month follow-up was confirmed only in the group with moderate or severe symptoms at baseline.Conclusion: Our findings corroborate the importance of improving the quality of relationship between clinicians and patients. Shared decision making and thorough discussions about benefits and side effects may improve the outcome in patients with severe mental disorders.

Half of high-risk neuroblastoma patients have MYCN amplification. Here, the authors show that MYCN induces fatty acid uptake and synthesis to support neuroblastoma and inhibition of a fatty acid transporter impairs tumor progression in preclinical models.Neuroblastoma (NB) is a childhood cancer arising from sympatho-adrenal neural crest cells. MYCN amplification is found in half of high-risk NB patients; however, no available therapies directly target MYCN. Using multi-dimensional metabolic profiling in MYCN expression systems and primary patient tumors, we comprehensively characterized the metabolic landscape driven by MYCN in NB. MYCN amplification leads to glycerolipid accumulation by promoting fatty acid (FA) uptake and biosynthesis. We found that cells expressing amplified MYCN depend highly on FA uptake for survival. Mechanistically, MYCN directly upregulates FA transport protein 2 (FATP2), encoded by SLC27A2. Genetic depletion of SLC27A2 impairs NB survival, and pharmacological SLC27A2 inhibition selectively suppresses tumor growth, prolongs animal survival, and exerts synergistic anti-tumor effects when combined with conventional chemotherapies in multiple preclinical NB models. This study identifies FA uptake as a critical metabolic dependency for MYCN-amplified tumors. Inhibiting FA uptake is an effective approach for improving current treatment regimens.

Chromosome 17q21-ter is commonly gained in neuroblastoma, but it is unclear which gene in the region is important for tumorigenesis. The JMJD6 gene at 17q21-ter activates gene transcription. Here we show that JMJD6 forms protein complexes with N-Myc and BRD4, and is important for E2F2, N-Myc and c-Myc transcription. Knocking down JMJD6 reduces neuroblastoma cell proliferation and survival in vitro and tumor progression in mice, and high levels of JMJD6 expression in human neuroblastoma tissues independently predict poor patient prognosis. In addition, JMJD6 gene is associated with transcriptional super-enhancers. Combination therapy with the CDK7/super-enhancer inhibitor THZ1 and the histone deacetylase inhibitor panobinostat synergistically reduces JMJD6, E2F2, N-Myc, c-Myc expression, induces apoptosis in vitro and leads to neuroblastoma tumor regression in mice, which are significantly reversed by forced JMJD6 over-expression. Our findings therefore identify JMJD6 as a neuroblastoma tumorigenesis factor, and the combination therapy as a treatment strategy., Although the gain in chromosome 17q21-ter is commonly associated with neuroblastoma, it is not clear which gene of this region mediates tumorigenesis. Here, the authors are showing that JMJD6, which locates in that region, is a neuroblastoma tumorigenic factor.

Simple Summary Neuroblastoma is a devasting childhood cancer in which multiple copies (amplification) of the cancer-causing gene MYCN strongly predict poor outcome. Neuroblastomas are reliant on high levels of cellular components called polyamines for their growth and malignant behavior, and the gene regulating polyamine synthesis is called ODC1. ODC1 is often coamplified with MYCN, and in fact is regulated by MYCN, and like MYCN is prognostic of poor outcome. Here we studied a naturally occurring genetic variant or polymorphism that occurs in the ODC1 gene, and used gene editing to demonstrate the functional importance of this variant in terms of ODC1 levels and growth of neuroblastoma cells. We showed that this variant impacts the ability of MYCN to regulate ODC1, and that it also influences outcome in neuroblastoma, with the rarer variant associated with a better survival. This study addresses the important topic of genetic polymorphisms in cancer. Ornithine decarboxylase (ODC1), a critical regulatory enzyme in polyamine biosynthesis, is a direct transcriptional target of MYCN, amplification of which is a powerful marker of aggressive neuroblastoma. A single nucleotide polymorphism (SNP), G316A, within the first intron of ODC1, results in genotypes wildtype GG, and variants AG/AA. CRISPR-cas9 technology was used to investigate the effects of AG clones from wildtype MYCN-amplified SK-N-BE(2)-C cells and the effect of the SNP on MYCN binding, and promoter activity was investigated using EMSA and luciferase assays. AG clones exhibited decreased ODC1 expression, growth rates, and histone acetylation and increased sensitivity to ODC1 inhibition. MYCN was a stronger transcriptional regulator of the ODC1 promoter containing the G allele, and preferentially bound the G allele over the A. Two neuroblastoma cohorts were used to investigate the clinical impact of the SNP. In the study cohort, the minor AA genotype was associated with improved survival, while poor prognosis was associated with the GG genotype and AG/GG genotypes in MYCN-amplified and non-amplified patients, respectively. These effects were lost in the GWAS cohort. We have demonstrated that the ODC1 G316A polymorphism has functional significance in neuroblastoma and is subject to allele-specific regulation by the MYCN oncoprotein.

PRDM12 is a member of the PRDI-BF1 (positive regulatory domain I-binding factor 1) homologous domain (PRDM)-containing protein family, a subfamily of Kruppel-like zinc finger proteins, controlling key processes in the development of cancer. PRDM12 is expressed in a spatio-temporal manner in neuronal systems where it exerts multiple functions. PRDM12 is essential for the neurogenesis initiation and activation of a cascade of downstream pro-neuronal transcription factors in the nociceptive lineage. PRDM12 inactivation, indeed, results in a complete absence of the nociceptive lineage, which is essential for pain perception. Additionally, PRDM12 contributes to the early establishment of anorexigenic neuron identity and the maintenance of high expression levels of pro-opiomelanocortin, which impacts on the program bodyweight homeostasis. PRDMs are commonly involved in cancer, where they act as oncogenes/tumor suppressors in a “Yin and Yang” manner. PRDM12 is not usually expressed in adult normal tissues but its expression is re-activated in several cancer types. However, little information is currently available on PRDM12 expression in cancers and its mechanism of action has not been thoroughly described. In this review, we summarize the recent findings regarding PRDM12 by focusing on four main biological processes: neurogenesis, pain perception, oncogenesis and cell metabolism. Moreover, we wish to highlight the importance of future studies focusing on the PRDM12 signaling pathway(s) and its role in cancer onset and progression.

The oncoprotein BCR-ABL1 triggers chronic myeloid leukemia. It is clear that the disease relies on constitutive BCR-ABL1 kinase activity, but not all the interactors and regulators of the oncoprotein are known. We describe and validate a Drosophila leukemia model based on inducible human BCR-ABL1 expression controlled by tissue-specific promoters. The model was conceived to be a versatile tool for performing genetic screens. BCR-ABL1 expression in the developing eye interferes with ommatidia differentiation and expression in the hematopoietic precursors increases the number of circulating blood cells. We show that BCR-ABL1 interferes with the pathway of endogenous dAbl with which it shares the target protein Ena. Loss of function of ena or Dab, an upstream regulator of dAbl, respectively suppresses or enhances both the BCR-ABL1-dependent phenotypes. Importantly, in patients with leukemia decreased human Dab1 and Dab2 expression correlates with more severe disease and Dab1 expression reduces the proliferation of leukemia cells. Globally, these observations validate our Drosophila model, which promises to be an excellent system for performing unbiased genetic screens aimed at identifying new BCR-ABL1 interactors and regulators in order to better elucidate the mechanism of leukemia onset and progression.

Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Amplification of the MYCN oncogene is associated with an aggressive phenotype and poor outcome in childhood neuroblastoma. Polyamines are highly regulated essential cations that are frequently elevated in cancer cells, and the rate-limiting enzyme in polyamine synthesis, ornithine decarboxylase 1 (ODC1), is a direct transcriptional target of MYCN. Treatment of neuroblastoma cells with the ODC1 inhibitor difluoromethylornithine (DFMO), although a promising therapeutic strategy, is only partially effective at impeding neuroblastoma cell growth due to activation of compensatory mechanisms resulting in increased polyamine uptake from the surrounding microenvironment. In this study, we identified solute carrier family 3 member 2 (SLC3A2) as the key transporter involved in polyamine uptake in neuroblastoma. Knockdown of SLC3A2 in neuroblastoma cells reduced the uptake of the radiolabeled polyamine spermidine, and DFMO treatment increased SLC3A2 protein. In addition, MYCN directly increased polyamine synthesis and promoted neuroblastoma cell proliferation by regulating SLC3A2 and other regulatory components of the polyamine pathway. Inhibiting polyamine uptake with the small-molecule drug AMXT 1501, in combination with DFMO, prevented or delayed tumor development in neuroblastoma-prone mice and extended survival in rodent models of established tumors. Our findings suggest that combining AMXT 1501 and DFMO with standard chemotherapy might be an effective strategy for treating neuroblastoma.

The MYC family of transcription factors is a major driver of human cancer and potential therapeutic target. However, no clinically viable drugs have been yet developed that are able to directly tackle MYC oncoproteins. In our laboratory, we are exploring alternative approaches aiming to disturb signalling downstream of MYC. MYCN is frequently activated in neuroblastoma, a paediatric solid malignancy that, in its metastatic form, has a very poor prognosis. An important pathway regulated by MYC is the CKS1/SKP2/p27kip1 axis. In this study, we have repurposed the anti-psychotic drug Prozac to disrupt CKS1/SKP2/p27Kip1 signalling and assess its potential as an anti-neuroblastoma agent in vitro and in vivo. Using DNA editing technology, we show that stabilisation of p27Kip1 operated by Prozac in MYC-activated cells is essential for the anti-neuroblastoma activity of the drug. Furthermore, dosing mice with a concentration of Prozac equivalent to that used in long-term clinical trials in children with psychiatric disorders caused a significant reduction of metastatic disease in two models of high-risk neuroblastoma. The favourable toxicity profile of Prozac suggests that long-term treatments might be implemented in children with MYC/CKS1high neuroblastomas.

Histone deacetylases (HDACs) are evolutionary conserved enzymes which operate by removing acetyl groups from histones and other protein regulatory factors, with functional consequences on chromatin remodeling and gene expression profiles. We provide here a review on the recent knowledge accrued on the zinc-dependent HDAC protein family across different species, tissues, and human pathologies, specifically focusing on the role of HDAC inhibitors as anti-cancer agents. We will investigate the chemical specificity of different HDACs and discuss their role in the human interactome as members of chromatin-binding and regulatory complexes.

This study focuses on a lightweight syntactic foam constituted by an epoxy matrix filled with polydispersed Glass Microballoons (GMs) up to 0.75 volume fraction. We present experimental results on hydrostatic loading which demonstrate the possibility of different failure modes depending on whether the surface of the composite is painted/coated or not. In order to explain this surprising behaviour, we propose a three-dimensional Finite Element (FE) micromechanical model. First, we develop a cubic MultiParticle Unit Cell (MPUC) which includes 100 randomly placed GMs and accounting for their polydispersion, in terms of both size and radius ratio. This model is validated by subjecting it to effective uniaxial stress and comparing its predictions of the elastic moduli with experimental findings and an analytical homogenisation technique. Second, towards modelling failure, we implement a structural criterion proposed by our group, which posits that any GM undergoes brittle failure when its average elastic energy density reaches a critical value. We then utilise our measurements of the effective strength under uniaxial compressive stress to identify different critical values for selected types of GMs. Third, on the basis of the MPUC, we reach our goal by developing a larger FE model, including 300 GMs, which enables the study of the stress diffusion from the external surface through an appropriately thick layer of composite, under macroscopic uniform pressure. This micromechanical model allows us to demonstrate the influence of the paint/coating on the syntactic foam failure mode through a detailed analysis of the collapsed GMs and the matrix stress state.