WDR5 is a conserved regulator of protein synthesis gene expression.

WDR5 is a highly-conserved nuclear protein that performs multiple scaffolding functions in the context of chromatin. WDR5 is also a promising target for pharmacological inhibition in cancer, with small molecule inhibitors of an arginine-binding pocket of WDR5 (the 'WIN' site) showing efficacy against a range of cancer cell lines in vitro. Efforts to understand WDR5, or establish the mechanism of action of WIN site inhibitors, however, are stymied by its many functions in the nucleus, and a lack of knowledge of the conserved gene networks-if any-that are under its control. Here, we have performed comparative genomic analyses to identify the conserved sites of WDR5 binding to chromatin, and the conserved genes regulated by WDR5, across a diverse panel of cancer cell lines. We show that a specific cohort of protein synthesis genes (PSGs) are invariantly bound by WDR5, demonstrate that the WIN site anchors WDR5 to chromatin at these sites, and establish that PSGs are bona fide, acute, and persistent targets of WIN site blockade. Together, these data reveal that WDR5 plays a predominant transcriptional role in biomass accumulation and provide further evidence that WIN site inhibitors act to repress gene networks linked to protein synthesis homeostasis.

Characterization of pediatric golf cart injuries to guide injury prevention efforts.

BACKGROUND: Golf cart injuries represent an increasing source of morbidity and mortality in the United States. Characterization of the circumstances of these injuries can inform injury prevention efforts. METHODS: This study retrospectively reviews a prospective trauma registry at a level-one pediatric trauma center for golf cart-related injuries in patients under 18years of age admitted to the hospital between 2008 and 2016. RESULTS: The 40 identified crashes were associated with 82 hospital days, 17 ICU days, and more than $1 million in hospital charges over the study period. The median hospital stay was 1.5days, and the median hospital charge was $20,489. Severe injuries with an Injury Severity Score of >15 were identified in 25% of patients, and moderate injuries with scores between nine and 15 were identified in an additional 30%. The most common injures were head and neck (60%) and external injuries to the body surface (52.5%). Only a single child was wearing a seatbelt, and the vast majority was not using any safety equipment. Children as young as nine years old were driving golf carts, and child drivers were associated with the cart overturning (p=0.007). CONCLUSIONS: Golf cart crashes were a source of substantial morbidity at a level-one trauma center. Increased safety measures, such as higher hip restraints, seatbelts, and front-wheel breaks could substantially increase the safety of golf carts. Increased regulation of driving age as well as driver education may also reduce these injuries.

ML327 induces apoptosis and sensitizes Ewing sarcoma cells to TNF-related apoptosis-inducing ligand.

Ewing sarcomas are rare mesenchymal-derived bone and soft tissue tumors in children. Afflicted children with distant metastases have poor survival despite aggressive therapeutics. Epithelial-to-mesenchymal transition in epithelial carcinomas is associated with loss of E-cadherin and resistance to apoptosis. ML327 is a novel small molecule that we have previously shown to reverse epithelial-to-mesenchymal transition features in both epithelial and neural crest-derived cancers. Herein, we sought to evaluate the effects of ML327 on mesenchymal-derived Ewing sarcoma cells, hypothesizing that ML327 initiates growth arrest and sensitizes to TNF-related apoptosis-inducing ligand. ML327 induced protein expression changes, increased E-cadherin and decreased vimentin, consistent with partial induction of mesenchymal-to-epithelial transition in multiple Ewing Sarcoma cell lines (SK-N-MC, TC71, and ES-5838). Induction of epithelial features was associated with apoptosis, as demonstrated by PARP and Caspase 3 cleavage by immunoblotting. Cell cycle analysis validated these findings by marked induction of the subG0 cell population. In vitro combination treatment with TRAIL demonstrated additive induction of apoptotic markers. Taken together, these findings establish a rationale for further in vivo trials of ML327 in cells of mesenchymal origin both alone and in combination with TRAIL.

Induced differentiation inhibits sphere formation in neuroblastoma.

Neuroblastoma arises from the neural crest, the precursor cells of the sympathoadrenal axis, and differentiation status is a key prognostic factor used for clinical risk group stratification and treatment strategies. Neuroblastoma tumor-initiating cells have been successfully isolated from patient tumor samples and bone marrow using sphere culture, which is well established to promote growth of neural crest stem cells. However, accurate quantification of sphere-forming frequency of commonly used neuroblastoma cell lines has not been reported. Here, we show that MYCN-amplified neuroblastoma cell lines form spheres more frequently than non-MYCN-amplified cell lines. We also show that sphere formation is directly sensitive to cellular differentiation status. 13-cis-retinoic acid is a clinically used differentiating agent that induces a neuronal phenotype in neuroblastoma cells. Induced differentiation nearly completely blocked sphere formation. Furthermore, sphere formation was specifically FGF-responsive and did not respond to increasing doses of EGF. Taken together, these data suggest that sphere formation is an accurate method of quantifying the stemness phenotype in neuroblastoma.

Silencing gastrin-releasing peptide receptor suppresses key regulators of aerobic glycolysis in neuroblastoma cells.

BACKGROUND: Under normoxic conditions, cancer cells use aerobic glycolysis as opposed to glucose oxidation for energy production; this altered metabolism correlates with poor outcomes in neuroblastoma. Hypoxia-inducible factor-1 alpha (HIF-1α) and pyruvate dehydrogenase kinase 4 (PDK4) regulate aerobic glycolysis, while pyruvate dehydrogenase phosphatase 2 (PDP2) promotes glucose oxidation. Here, we sought to determine whether gastrin-releasing peptide receptor (GRP-R) signaling regulates glucose metabolism. PROCEDURE: Neuroblastoma cell lines, BE(2)-C and SK-N-AS, were used. PCR microararay for glucose metabolism was performed on GRP-R silenced cells. Target protein expression was validated using Western blotting and VEGF ELISA. Cobalt chloride (CoCl2 ) was used to induce chemical hypoxia. Efficacy of targeting PDK regulation in neuroblastoma was assessed using dichloroacetate (DCA) by conducting cell viability assays and Western blotting for apoptotic markers. RESULTS: Silencing GRP-R decreased HIF-1α expression and blocked VEGF expression and secretion in both normoxic and CoCl2 induced hypoxia. PCR array analysis identified that GRP-R silencing reduced PDK4 and increased PDP2 mRNA expression. These findings were validated by Western blotting. CoCl2 induced hypoxia increased VEGF secretion, HIF-1α, and PDK4 expression. PDK4 silencing decreased HIF-1α expression and VEGF expression and secretion. DCA treatment decreased BE(2)-C and SK-N-AS proliferation while promoting cell death. GRP-R silencing and DCA treatment synergistically halted BE(2)-C proliferation. CONCLUSIONS: We report that GRP-R regulates glucose metabolism in neuroblastoma by modulating HIF-1α, PDK4 and PDP2. PDK4 regulates glucose metabolism, in part, via regulation of HIF-1α. Synergistic consequences of GRP-R inhibition and DCA treatment may suggest a novel therapeutic strategy for the treatment of aggressive neuroblastoma.

Whole exome sequencing identifies de novo mutations in GATA6 associated with congenital diaphragmatic hernia.

BACKGROUND: Congenital diaphragmatic hernia (CDH) is a common birth defect affecting 1 in 3000 births. It is characterised by herniation of abdominal viscera through an incompletely formed diaphragm. Although chromosomal anomalies and mutations in several genes have been implicated, the cause for most patients is unknown. METHODS: We used whole exome sequencing in two families with CDH and congenital heart disease, and identified mutations in GATA6 in both. RESULTS: In the first family, we identified a de novo missense mutation (c.1366C>T, p.R456C) in a sporadic CDH patient with tetralogy of Fallot. In the second, a nonsense mutation (c.712G>T, p.G238*) was identified in two siblings with CDH and a large ventricular septal defect. The G238* mutation was inherited from their mother, who was clinically affected with congenital absence of the pericardium, patent ductus arteriosus and intestinal malrotation. Deep sequencing of blood and saliva-derived DNA from the mother suggested somatic mosaicism as an explanation for her milder phenotype, with only approximately 15% mutant alleles. To determine the frequency of GATA6 mutations in CDH, we sequenced the gene in 378 patients with CDH. We identified one additional de novo mutation (c.1071delG, p.V358Cfs34*). CONCLUSIONS: Mutations in GATA6 have been previously associated with pancreatic agenesis and congenital heart disease. We conclude that, in addition to the heart and the pancreas, GATA6 is involved in development of two additional organs, the diaphragm and the pericardium. In addition, we have shown that de novo mutations can contribute to the development of CDH, a common birth defect.

Non-fecalith-induced appendicitis: etiology, imaging, and pathology.

This study aims to document the imaging and pathology findings in non-fecalith-induced appendicitis. We reviewed the imaging and pathologic findings in 40 patients with histologically proven purulent appendicitis seen over a 2-year period. Findings documented were (1) total appendiceal involvement, (2) predominant appendiceal tip involvement, (3) presence of a fecalith, and (4) presence of lymphoid hyperplasia. There were a total of 40 patients, 28 males and 12 females. The age range was 2-18 years with a mean of 11.5 years. Twenty-two (55 %) patients demonstrated classic purulent appendicitis of the whole appendix, 20 (91 %) of these appendices had a fecalith. Eighteen (45 %) patients demonstrated purulent appendicitis confined to or predominately involving the tip of the appendix, and all 18 (100 %) patients demonstrated marked lymphoid hyperplasia. Only two (11 %) of these appendices had a fecalith. Overall, a fecalith was found in only 55 % of our cases, while 45 % demonstrated no fecalith, but rather marked lymphoid hyperplasia. Lymphoid hyperplasia appeared to be the underlying predisposing cause of purulent appendicitis in these cases.

Social Determinants of Outcomes Disparity among Pediatric Patients with Solid Tumor.

BACKGROUND: Socioeconomic factors have a significant impact on healthcare outcomes. Metrics such as area deprivation index (ADI) are used to quantify the anticipated influence of these factors. Here, we sought to assess the impact of socioeconomic factors on clinical outcomes among pediatric patients with solid tumor in our region. STUDY DESIGN: We identified 3,863 pediatric patients who were diagnosed with a malignant solid tumor in the Texas Cancer Registry between 1995 and 2019. ADI was used to quantify socioeconomic determinants of health. These outcome variables were determined: stage of disease at diagnosis, time between diagnosis and treatment initiation, and overall mortality. Statistical analysis was performed using logistic regression, linear regression, Cox proportional hazards regression, and Kaplan-Meier survival curves. RESULTS: A total of 53.5% of patients were male and the average age at diagnosis was 4.5 years. Forty-seven percent of patients were White, 13.3% were Black, 36.2% were Hispanic, 1.7% were Asian, and other rare minority groups made up 1.8%. On multivariable analysis, increased risk of death was associated with Black race, rare minority race, residence in a border county, and increasing ADI score, with the risk of death at 5 years rising 4% with each increasing ADI point. CONCLUSIONS: Social determinants of health are associated with disparate outcomes among pediatric patients with solid tumor. Our results suggest that patients who are part of racial minority groups and those who reside in socioeconomically disadvantaged neighborhoods or regions near the Texas-Mexico border are at an increased risk of death. This information may be useful in strategizing outreach and expanding resources to improve outcomes in at-risk communities.

Migration, invasion, and metastasis are mediated by P-Rex1 in neuroblastoma.

Neuroblastoma accounts for approximately 15% of pediatric cancer-related deaths despite intensive multimodal therapy. This is due, in part, to high rates of metastatic disease at diagnosis and disease relapse. A better understanding of tumor biology of aggressive, pro-metastatic phenotypes is necessary to develop novel, more effective therapeutics against neuroblastoma. Phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1 (P-Rex1) has been found to stimulate migration, invasion, and metastasis in several adult malignancies. However, its role in neuroblastoma is currently unknown. In the present study, we found that P-Rex1 is upregulated in pro-metastatic murine models of neuroblastoma, as well as human neuroblastoma metastases. Correspondingly, silencing of P-Rex1 was associated with decreased migration and invasion in vitro. This was associated with decreased AKT-mTOR and ERK2 activity, dysregulation of Rac, and diminished secretion of matrix metalloproteinases. Furthermore, increased P-Rex1 expression was associated with inferior relapse-free and overall survival via tissue microarray and Kaplan-Meier survival analysis of a publicly available clinical database. Together, these findings suggest that P-Rex1 may be a novel therapeutic target and potential prognostic factor in neuroblastoma.

The impact of margins and re-resection in pediatric synovial sarcoma.

INTRODUCTION: Synovial sarcoma is one of the most common soft tissue sarcomas in children. Guidelines regarding the adequate extent of resection margins and the role of re-resection are lacking. We sought to evaluate the adequate resection margin and the role of re-resection in predicting outcomes in children with synovial sarcomas. METHODS: A cohort of 36 patients less than 18 years of age at diagnosis who were treated for localized synovial sarcoma at three tertiary pediatric hospitals between January 2004 and December 2020 were included in this study. Patient and tumor demographics, treatment information, and margin status after surgical resection were collected from the medical record. Clinical, treatment, and surgical characteristics, as well as outcomes including hazard ratios (HRs), event-free survival (EFS), and overall survival (OS) were compared by resection margins group and re-resection status. RESULTS: Patients in the R1 resection group were significantly more likely to relapse or die compared to patients in the R0 resection group. However, there was no significant difference in EFS (HR 0.52, p = 0.54) or OS (HR 1.56, p = 0.719) in R0 patients with less than 5 mm margins compared to R0 patients with more than 5 mm margins. Patients with R1 on initial or re-resection had significantly worse OS than patients who had R0 resection on initial or re-resection (HR = 10.12, p = 0.005). CONCLUSION: This study re-affirms that R0 resection is an independent prognostic predictor of better OS/EFS in pediatric synovial sarcoma. Second, our study extends this finding to report negative margins on initial resection or re-resection is associated with better OS/EFS than positive margins on initial resection or re-resection. Lastly, we found that there is no difference in outcomes associated with re-resection or <5 mm margins for R0 patients, indicating that re-resection and <5 mm margins are acceptable if microscopic disease is removed.

Autophagy mediates paracrine regulation of vascular endothelial cells.

Gastrin-releasing peptide (GRP) is a proangiogenic ligand secreted by tumors and acts directly upon binding to GRP receptor in endothelial cells. Angiogenesis plays a critical role in the pathology of various diseases, including cancer, as the formation of new blood vessels potentiates the rate of tumor growth and dissemination. GRP increases the migration of endothelial cells, but much is unknown about its role on endothelial cell proliferation and survival, as well as the signaling pathways involved. In the present study, we showed that GRP increases endothelial cell proliferation and tubule formation. There was a time-dependent increase in the levels of phosphorylated AKT, mammalian target of rapamycin (mTOR), and S6R in human umbilical vein endothelial cells treated with GRP. Interestingly, GRP treatment decreased the expression of proautophagic factors, ATG5, BECN1, and LC3 proteins. GRP also attenuated rapamycin-induced formation of autophagosomes. Moreover, overexpression of ATG5 or BECN1 significantly decreased tubule formation induced by exogenous GRP, whereas siRNA against ATG5 or BECN1 resulted in increased tubule formation with GRP treatment. Our results show that GRP inhibits the process of autophagy in vascular endothelial cells, thereby increasing endothelial cell proliferation and tubule formation. Here, we describe a novel role of GRP in the regulation of autophagy of endothelial cells, thereby providing a potential new therapeutic strategy in targeting angiogenesis during cancer progression.

Variants in GATA4 are a rare cause of familial and sporadic congenital diaphragmatic hernia.

Congenital diaphragmatic hernia (CDH) is characterized by incomplete formation of the diaphragm occurring as either an isolated defect or in association with other anomalies. Genetic factors including aneuploidies and copy number variants are important in the pathogenesis of many cases of CDH, but few single genes have been definitively implicated in human CDH. In this study, we used whole exome sequencing (WES) to identify a paternally inherited novel missense GATA4 variant (c.754C>T; p.R252W) in a familial case of CDH with incomplete penetrance. Phenotypic characterization of the family included magnetic resonance imaging of the chest and abdomen demonstrating asymptomatic defects in the diaphragm in the two "unaffected" missense variant carriers. Screening 96 additional CDH patients identified a de novo heterozygous GATA4 variant (c.848G>A; p.R283H) in a non-isolated CDH patient. In summary, GATA4 is implicated in both familial and sporadic CDH, and our data suggests that WES may be a powerful tool to discover rare variants for CDH.

Differential regulation of cyclin-dependent kinase inhibitors in neuroblastoma cells.

Gastrin-releasing peptide (GRP) and its receptor (GRP-R) are highly expressed in undifferentiated neuroblastoma, and they play critical roles in oncogenesis. We previously reported that GRP activates the PI3K/AKT signaling pathway to promote DNA synthesis and cell cycle progression in neuroblastoma cells. Conversely, GRP-R silencing induces cell cycle arrest. Here, we speculated that GRP/GRP-R signaling induces neuroblastoma cell proliferation via regulation of cyclin-dependent kinase (CDK) inhibitors. Surprisingly, we found that GRP/GRP-R differentially induced expressions of p21 and p27. Silencing GRP/GRP-R decreased p21, but it increased p27 expressions in neuroblastoma cells. Furthermore, we found that the intracellular localization of p21 and p27 in the nuclear and cytoplasmic compartments, respectively. In addition, we found that GRP/GRP-R silencing increased the expression and accumulation of PTEN in the cytoplasm of neuroblastoma cells where it co-localized with p27, thus suggesting that p27 promotes the function of PTEN as a tumor suppressor by stabilizing PTEN in the cytoplasm. GRP/GRP-R regulation of CDK inhibitors and tumor suppressor PTEN may be critical for tumoriogenesis of neuroblastoma.

De novo copy number variants are associated with congenital diaphragmatic hernia.

BACKGROUND: Congenital diaphragmatic hernia (CDH) is a common birth defect with significant morbidity and mortality. Although the aetiology of CDH remains poorly understood, studies from animal models and patients with CDH suggest that genetic factors play an important role in the development of CDH. Chromosomal anomalies have been reported in CDH. METHODS: In this study, the authors investigated the frequency of chromosomal anomalies and copy number variants (CNVs) in 256 parent-child trios of CDH using clinical conventional cytogenetic and microarray analysis. The authors also selected a set of CDH related training genes to prioritise the genes in those segmental aneuploidies and identified the genes and gene sets that may contribute to the aetiology of CDH. RESULTS: The authors identified chromosomal anomalies in 16 patients (6.3%) of the series including three aneuploidies, two unbalanced translocation, and 11 patients with de novo CNVs ranging in size from 95 kb to 104.6 Mb. The authors prioritised the genes in the CNV segments and identified KCNA2, LMNA, CACNA1S, MYOG, HLX, LBR, AGT, GATA4, SOX7, HYLS1, FOXC1, FOXF2, PDGFA, FGF6, COL4A1, COL4A2, HOMER2, BNC1, BID, and TBX1 as genes that may be involved in diaphragm development. Gene enrichment analysis identified the most relevant gene ontology categories as those involved in tissue development (p=4.4×10(-11)) or regulation of multicellular organismal processes (p=2.8×10(-10)) and 'receptor binding' (p=8.7×10(-14)) and 'DNA binding transcription factor activity' (p=4.4×10(-10)). CONCLUSIONS: The present findings support the role of chromosomal anomalies in CDH and provide a set of candidate genes including FOXC1, FOXF2, PDGFA, FGF6, COL4A1, COL4A2, SOX7, BNC1, BID, and TBX1 for further analysis in CDH.

High-Risk Neuroblastoma: A Surgical Perspective.

High-risk neuroblastoma requires multimodal treatment including systemic chemotherapy, surgical resection, radiation therapy, stem cell transplant, and immunotherapy. Surgeons play a vital role in obtaining local control of neuroblastoma and must therefore be knowledgeable about this complex pathology. This article provides a review of the optimal timing and extent of resection, the impact of various image-defined risk factors on surgical planning, and surgical approaches and techniques to enhance the resection of tumors in different anatomic locations.

Improving Billing and Collections in a High-Volume Pediatric Surgery Practice: Denials-Based Approach.

BACKGROUND: Despite minimal coding and billing training, surgeons are frequently tasked with both in clinical practice. This often results in denials for reimbursement based on incorrect or insufficient documentation, and reduced collections for work performed. We sought to evaluate how to correct these deficits while improving reimbursement for the most frequently rejected procedures at a high-volume academic center. STUDY DESIGN: Hospital billing data were analyzed for a 4-year period (2018 to 2021) to determine the CPT code denials with the largest overall cost. The denials were then stratified according to payor, reason for denial, and preventability. Assigned ICD-10 codes were categorized based on specificity as related to the procedure. The distribution of denials according to ICD-10 specificity was evaluated using the chi-square test. RESULTS: A total of 8,833 denials representing $11,009,108 in billing were noted during the study period. The CPT code 44970 (laparoscopic appendectomy) was the code associated with the largest financial impact, representing 12.8% of the total denied amount ($1.41M). Of the 823 denials for CPT 44970, 93.3% were associated with nonspecific ICD-10 codes, whereas only 42.0% had been associated with procedure-specific ICD-10 codes. Of the patients with nonspecific codes, 80.7% of denials were due to criteria that could be remedied with supplemental information or timely filing, representing $1,059,968 in collections. CONCLUSIONS: This is the first study to systematically evaluate a pathway for using denial data to improve collections for work performed at a high-volume academic pediatric surgery practice. Using this methodology, targets for improvement in coding and/or documentation can be identified to improve the financial performance of a surgical department. This study also provides evidence that association with nonspecific diagnostic codes is correlated with initial denial of payment by insurance companies.

WDR5 facilitates recruitment of N-MYC to conserved WDR5 gene targets in neuroblastoma cell lines.

Collectively, the MYC family of oncoprotein transcription factors is overexpressed in more than half of all malignancies. The ability of MYC proteins to access chromatin is fundamental to their role in promoting oncogenic gene expression programs in cancer and this function depends on MYC-cofactor interactions. One such cofactor is the chromatin regulator WDR5, which in models of Burkitt lymphoma facilitates recruitment of the c-MYC protein to chromatin at genes associated with protein synthesis, allowing for tumor progression and maintenance. However, beyond Burkitt lymphoma, it is unknown whether these observations extend to other cancers or MYC family members, and whether WDR5 can be deemed as a "universal" MYC recruiter. Here, we focus on N-MYC amplified neuroblastoma to determine the extent of colocalization between N-MYC and WDR5 on chromatin while also demonstrating that like c-MYC, WDR5 can facilitate the recruitment of N-MYC to conserved WDR5-bound genes. We conclude based on this analysis that N-MYC and WDR5 colocalize invariantly across cell lines at predicted sites of facilitated recruitment associated with protein synthesis genes. Surprisingly, we also identify N-MYC-WDR5 cobound genes that are associated with DNA repair and cell cycle processes. Dissection of chromatin binding characteristics for N-MYC and WDR5 at all cobound genes reveals that sites of facilitated recruitment are inherently different than most N-MYC-WDR5 cobound sites. Our data reveals that WDR5 acts as a universal MYC recruiter at a small cohort of previously identified genes and highlights novel biological functions that may be coregulated by N-MYC and WDR5 to sustain the neuroblastoma state.

PI3K/AKT and ERK regulate retinoic acid-induced neuroblastoma cellular differentiation.

Neuroblastoma, the most common extra-cranial solid tumor in infants and children, is characterized by a high rate of spontaneous remissions in infancy. Retinoic acid (RA) has been known to induce neuroblastoma differentiation; however, the molecular mechanisms and signaling pathways that are responsible for RA-mediated neuroblastoma cell differentiation remain unclear. Here, we sought to determine the cell signaling processes involved in RA-induced cellular differentiation. Upon RA administration, human neuroblastoma cell lines, SK-N-SH and BE(2)-C, demonstrated neurite extensions, which is an indicator of neuronal cell differentiation. Moreover, cell cycle arrest occurred in G1/G0 phase. The protein levels of cyclin-dependent kinase inhibitors, p21 and p27(Kip), which inhibit cell proliferation by blocking cell cycle progression at G1/S phase, increased after RA treatment. Interestingly, RA promoted cell survival during the differentiation process, hence suggesting a potential mechanism for neuroblastoma resistance to RA therapy. Importantly, we found that the PI3K/AKT pathway is required for RA-induced neuroblastoma cell differentiation. Our results elucidated the molecular mechanism of RA-induced neuroblastoma cellular differentiation, which may be important for developing novel therapeutic strategy against poorly differentiated neuroblastoma.

Combination bromo- and extraterminal domain and poly (ADP-ribose) polymerase inhibition synergistically enhances DNA damage and inhibits neuroblastoma tumorigenesis.

PURPOSE: JQ1 is a bromo- and extraterminal (BET) domain inhibitor that downregulates MYC expression and impairs the DNA damage response. Poly (ADP-ribose) polymerase (PARP) inhibitors prevent DNA damage sensing and repair. We hypothesized that JQ1 would promote a DNA repair-deficient phenotype that sensitizes neuroblastoma cells to PARP inhibition. METHODS: Four human neuroblastoma cell lines were examined: two MYCN-amplified (BE(2)-C and IMR-32), and two non-MYCN-amplified (SK-N-SH and SH-SY5Y). Cells were treated with JQ1 (BET inhibitor), Olaparib (PARP inhibitor), or in combination to assess for therapeutic synergy of JQ1 and Olaparib. Treated cells were harvested and analyzed. Quantitative assessment of combination treatment synergy was performed using the median effect principle of Chou and Talalay. RESULTS: Combination treatment with Olaparib decreased the IC50 of JQ1 by 19.9-fold, 2.0-fold, 12.1-fold, and 2.0-fold in the BE(2)-C, IMR-32, SK-N-SH, and SH-SY5Y cell lines, respectively. In the MYCN-amplified cell lines, BE(2)-C and IMR-32, combination treatment decreased gene expression of MYCN relative to single-drug treatment alone or control. Combination treatment decreased protein expression of DNA repair proteins Ku80 and RAD51, led to accumulation of DNA damage marker phospho-histone H2A.X, and increased caspase activity. In the non-MYCN-amplified cell lines, SK-N-SH and SH-SY5Y, combination treatment induced G0/G1 cell cycle arrest. CONCLUSIONS: Combination BET and PARP inhibition synergistically inhibited neuroblastoma tumorigenesis in vitro. In MYCN-amplified neuroblastoma cells, this effect may be induced by downregulation of MYCN transcription, defects in DNA repair, accumulation of DNA damage, and apoptosis. In non-MYCN-amplified cell lines, combination treatment induced cell cycle arrest.