KMT2A-MAML2 rearrangement emerged and regressed during neuroblastoma therapy without leukemia after 12.8-year follow-up.

Twelvepatients without therapy-related leukemia were studied after completing TOP2 poison chemotherapy in a high-risk neuroblastoma regimen. One patient harbored an inv(11) that was a KMT2A rearrangement. The KMT2A-MAML2 transcript was expressed at low level. The patient was prospectively followed. The inv(11) was undetectable in ensuing samples. Leukemia never developed after a 12.8-year follow-up period. Enriched etoposide-induced TOP2A cleavage in the relevant MAML2 genomic region supports a TOP2A DNA damage mechanism. After completing TOP2 poison chemotherapies, covert KMT2A-R clones may occur in a small minority of patients; however, not all KMT2A rearrangements herald a therapy-related leukemia diagnosis.

Identification and characterization of a novel heparan sulfate-binding domain in Activin A longest variants and implications for function.

Activins regulate numerous processes including inflammation and are synthesized as precursors consisting of a long N-terminal pro-region and a mature protein. Genomic human databases currently list three activin A (Act A) variants termed X1, X2 and X3. The X3 variant is the shortest, lacks N-terminal segments present in X1 and X2, and has been the focus of most past literature. Here, we asked whether these variants are expressed by human cells and tissues and what structural features are contained within their pro-regions. Human monocytic-like cells THP1 and U937 expressed X1 and X2 variants after exposure to phorbol ester or granulocyte-macrophage colony-stimulating factor, while X2 transcripts were present in placenta. Expression vectors encoding full length X2 or X3 variants resulted in production and secretion of biologically active Act A from cultured cells. Previous studies reported a putative HS-binding domain (HBD) in the X3 pro-region. Here, we identified a novel HBD with consensus HS-binding motifs near the N-terminal end of X1 and X2 pro-regions. Peptides encompassing this new domain interacted with substrate-bound HS with nanomolar affinity, while peptides from putative X3 HBD did not. In good agreement, full length X2 pro-region interacted with heparin-agarose, while the X3 pro-region did not. In sum, our study reveals that Act A variants are expressed by inflammatory cells and placenta and yield biological activity. The high affinity HBD in X1 and X2 pro-region and its absence in X3 could greatly influence overall Act A distribution, availability and activity in physiological and pathological circumstances.

Correction to: HnRNPA2 is a novel histone acetyltransferase that mediates mitochondrial stress-induced nuclear gene expression.

[This corrects the article DOI: 10.1038/celldisc.2016.45.].

Genome-wide TOP2A DNA cleavage is biased toward translocated and highly transcribed loci.

Type II topoisomerases orchestrate proper DNA topology, and they are the targets of anti-cancer drugs that cause treatment-related leukemias with balanced translocations. Here, we develop a high-throughput sequencing technology to define TOP2 cleavage sites at single-base precision, and use the technology to characterize TOP2A cleavage genome-wide in the human K562 leukemia cell line. We find that TOP2A cleavage has functionally conserved local sequence preferences, occurs in cleavage cluster regions (CCRs), and is enriched in introns and lincRNA loci. TOP2A CCRs are biased toward the distal regions of gene bodies, and TOP2 poisons cause a proximal shift in their distribution. We find high TOP2A cleavage levels in genes involved in translocations in TOP2 poison-related leukemia. In addition, we find that a large proportion of genes involved in oncogenic translocations overall contain TOP2A CCRs. The TOP2A cleavage of coding and lincRNA genes is independently associated with both length and transcript abundance. Comparisons to ENCODE data reveal distinct TOP2A CCR clusters that overlap with marks of transcription, open chromatin, and enhancers. Our findings implicate TOP2A cleavage as a broad DNA damage mechanism in oncogenic translocations as well as a functional role of TOP2A cleavage in regulating transcription elongation and gene activation.

HnRNPA2 is a novel histone acetyltransferase that mediates mitochondrial stress-induced nuclear gene expression.

Reduced mitochondrial DNA copy number, mitochondrial DNA mutations or disruption of electron transfer chain complexes induce mitochondria-to-nucleus retrograde signaling, which induces global change in nuclear gene expression ultimately contributing to various human pathologies including cancer. Recent studies suggest that these mitochondrial changes cause transcriptional reprogramming of nuclear genes although the mechanism of this cross talk remains unclear. Here, we provide evidence that mitochondria-to-nucleus retrograde signaling regulates chromatin acetylation and alters nuclear gene expression through the heterogeneous ribonucleoprotein A2 (hnRNAP2). These processes are reversed when mitochondrial DNA content is restored to near normal cell levels. We show that the mitochondrial stress-induced transcription coactivator hnRNAP2 acetylates Lys 8 of H4 through an intrinsic histone lysine acetyltransferase (KAT) activity with Arg 48 and Arg 50 of hnRNAP2 being essential for acetyl-CoA binding and acetyltransferase activity. H4K8 acetylation at the mitochondrial stress-responsive promoters by hnRNAP2 is essential for transcriptional activation. We found that the previously described mitochondria-to-nucleus retrograde signaling-mediated transformation of C2C12 cells caused an increased expression of genes involved in various oncogenic processes, which is retarded in hnRNAP2 silenced or hnRNAP2 KAT mutant cells. Taken together, these data show that altered gene expression by mitochondria-to-nucleus retrograde signaling involves a novel hnRNAP2-dependent epigenetic mechanism that may have a role in cancer and other pathologies.

The type 2 diabetes presumed causal variant within TCF7L2 resides in an element that controls the expression of ACSL5.

AIMS/HYPOTHESIS: One of the most strongly associated type 2 diabetes loci reported to date resides within the TCF7L2 gene. Previous studies point to the T allele of rs7903146 in intron 3 as the causal variant at this locus. We aimed to identify the actual gene(s) under the influence of this variant. METHODS: Using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease, we generated a 1.4 kb deletion of the genomic region harbouring rs7903146 in the HCT116 cell line, followed by global gene expression analysis. We then carried out a combination of circularised chromosome conformation capture (4C) and Capture C in cell lines, HCT116 and NCM460 in order to ascertain which promoters of these perturbed genes made consistent physical contact with this genomic region. RESULTS: We observed 99 genes with significant differential expression (false discovery rate [FDR] cut-off:10%) and an effect size of at least twofold. The subsequent promoter contact analyses revealed just one gene, ACSL5, which resides in the same topologically associating domain as TCF7L2. The generation of additional, smaller deletions (66 bp and 104 bp) comprising rs7903146 showed consistently reduced ACSL5 mRNA levels across all three deletions of up to 30-fold, with commensurate loss of acyl-CoA synthetase long-chain family member 5 (ACSL5) protein. Notably, the deletion of this single-nucleotide polymorphism region abolished significantly detectable chromatin contacts with the ACSL5 promoter. We went on to confirm that contacts between rs7903146 and the ACSL5 promoter regions were conserved in human colon tissue. ACSL5 encodes ACSL5, an enzyme with known roles in fatty acid metabolism. CONCLUSIONS/INTERPRETATION: This 'variant to gene mapping' effort implicates the genomic location harbouring rs7903146 as a regulatory region for ACSL5.

The next generation of target capture technologies - large DNA fragment enrichment and sequencing determines regional genomic variation of high complexity.

BACKGROUND: The ability to capture and sequence large contiguous DNA fragments represents a significant advancement towards the comprehensive characterization of complex genomic regions. While emerging sequencing platforms are capable of producing several kilobases-long reads, the fragment sizes generated by current DNA target enrichment technologies remain a limiting factor, producing DNA fragments generally shorter than 1 kbp. The DNA enrichment methodology described herein, Region-Specific Extraction (RSE), produces DNA segments in excess of 20 kbp in length. Coupling this enrichment method to appropriate sequencing platforms will significantly enhance the ability to generate complete and accurate sequence characterization of any genomic region without the need for reference-based assembly. RESULTS: RSE is a long-range DNA target capture methodology that relies on the specific hybridization of short (20-25 base) oligonucleotide primers to selected sequence motifs within the DNA target region. These capture primers are then enzymatically extended on the 3'-end, incorporating biotinylated nucleotides into the DNA. Streptavidin-coated beads are subsequently used to pull-down the original, long DNA template molecules via the newly synthesized, biotinylated DNA that is bound to them. We demonstrate the accuracy, simplicity and utility of the RSE method by capturing and sequencing a 4 Mbp stretch of the major histocompatibility complex (MHC). Our results show an average depth of coverage of 164X for the entire MHC. This depth of coverage contributes significantly to a 99.94 % total coverage of the targeted region and to an accuracy that is over 99.99 %. CONCLUSIONS: RSE represents a cost-effective target enrichment method capable of producing sequencing templates in excess of 20 kbp in length. The utility of our method has been proven to generate superior coverage across the MHC as compared to other commercially available methodologies, with the added advantage of producing longer sequencing templates amenable to DNA sequencing on recently developed platforms. Although our demonstration of the method does not utilize these DNA sequencing platforms directly, our results indicate that the capture of long DNA fragments produce superior coverage of the targeted region.

Unique Familial MLL(KMT2A)-Rearranged Precursor B-Cell Infant Acute Lymphoblastic Leukemia in Non-twin Siblings.

BACKGROUND: Infant acute lymphoblastic leukemia (ALL) has never occurred in families except for the ∼100% concordant cases in monozygous twins attributed to twin-to-twin metastases. We report the first kindred with infant ALL in non-twin siblings. The siblings were diagnosed with MLL-rearranged (MLL-R) ALL 26 months apart. The second affected sibling had an unaffected dichorionic monozygous co-twin. Both had fatal outcomes. PROCEDURES: Translocations were characterized by karyotype, FISH, multiplex FISH, and MLL breakpoint cluster region (bcr) Southern blot analysis. Breakpoint junctions and fusion transcripts were cloned by PCR. TP53 mutation and NADPH quinone oxidorecuctase 1 (NQO1) C609T analyses were performed, and pedigree history and parental occupations were ascertained. The likelihood of chance occurrence of infant ALL in non-twin siblings was computed based on a binomial distribution. Zygosity was determined by single nucleotide polymorphism (SNP) array. RESULTS: The translocations were not related or vertically transmitted. The complex karyotype of the proband's ALL had chromosome 2, 3, 4, and 11 abnormalities causing a 5'-MLL-AFF1-3' fusion and a non-productive rearrangement of 3'MLL with a chromosome 3q intergenic region. The affected twin's ALL exhibited a simple t(4;11). The complex karyotype of the proband's ALL suggested a genotoxic insult, but no exposure was identified. There was no germline TP53 mutation. The NQO1 C609T risk allele was absent. The likelihood of infant ALL occurring in non-twin siblings by chance alone is one in 1.198 × 10(9) families. CONCLUSIONS: Whether because of a deleterious transplacental exposure, novel predisposition syndrome, or exceedingly rare chance occurrence, MLL-R infant ALL can occur in non-twin siblings. The discordant occurrence of infant ALL in the monozygous twins was likely because they were dichorionic.

A de novo whole gene deletion of XIAP detected by exome sequencing analysis in very early onset inflammatory bowel disease: a case report.

BACKGROUND: Children with very early-onset inflammatory bowel disease (VEO-IBD), those diagnosed at less than 5 years of age, are a unique population. A subset of these patients present with a distinct phenotype and more severe disease than older children and adults. Host genetics is thought to play a more prominent role in this young population, and monogenic defects in genes related to primary immunodeficiencies are responsible for the disease in a small subset of patients with VEO-IBD. CASE PRESENTATION: We report a child who presented at 3 weeks of life with very early-onset inflammatory bowel disease (VEO-IBD). He had a complicated disease course and remained unresponsive to medical and surgical therapy. The refractory nature of his disease, together with his young age of presentation, prompted utilization of whole exome sequencing (WES) to detect an underlying monogenic primary immunodeficiency and potentially target therapy to the identified defect. Copy number variation analysis (CNV) was performed using the eXome-Hidden Markov Model. Whole exome sequencing revealed 1,380 nonsense and missense variants in the patient. Plausible candidate variants were not detected following analysis of filtered variants, therefore, we performed CNV analysis of the WES data, which led us to identify a de novo whole gene deletion in XIAP. CONCLUSION: This is the first reported whole gene deletion in XIAP, the causal gene responsible for XLP2 (X-linked lymphoproliferative Disease 2). XLP2 is a syndrome resulting in VEO-IBD and can increase susceptibility to hemophagocytic lymphohistocytosis (HLH). This identification allowed the patient to be referred for bone marrow transplantation, potentially curative for his disease and critical to prevent the catastrophic sequela of HLH. This illustrates the unique etiology of VEO-IBD, and the subsequent effects on therapeutic options. This cohort requires careful and thorough evaluation for monogenic defects and primary immunodeficiencies.

Generation of a mouse model of atypical teratoid/rhabdoid tumor of the central nervous system through combined deletion of Snf5 and p53.

Malignant rhabdoid tumors arise in several anatomic locations and are associated with poor outcomes. In the brain, these tumors are known as atypical teratoid/rhabdoid tumors (AT/RT). While genetically engineered models for malignant rhabdoid tumors exist, none of them recapitulate AT/RT, for which preclinical models remain lacking. In the majority of AT/RT, LOH occurs at the genetic locus SNF5 (Ini1/BAF47/Smarcb1), which functions as a subunit of the SWI/SNF chromatin-remodeling complex and a tumor suppressor in familial and sporadic malignant rhabdoid tumors. Therefore, we generated mice in which Snf5 was ablated specifically in nestin-positive and/or glial fibrillary acid protein (GFAP)-positive progenitor cells of the developing central nervous system (CNS). Snf5 ablation in nestin-positive cells resulted in early lethality that could not be rescued by loss of p53. However, Snf5 ablation in GFAP-positive cells caused a neurodegenerative phenotype exacerbated by p53 loss. Notably, these double mutants exhibited AT/RT development, associated with an earlier failure in granule neuron migration in the cerebellum, reduced neuronal projections in the hippocampus, degeneration of the corpus callosum, and ataxia and seizures. Gene expression analysis confirmed that the tumors that arose in Snf5/p53 mutant mice were distinct from other neural tumors and most closely resembled human AT/RT. Our findings uncover a novel role for Snf5 in oligodendrocyte generation and survival, and they offer evidence of the first genetically engineered mouse model for AT/RT in the CNS.

RNA Biosignatures in Adolescent Patients in a Pediatric Emergency Department With Pelvic Inflammatory Disease.

BACKGROUND: Adolescents are at high risk for pelvic inflammatory disease (PID). Because accurate diagnosis of PID is difficult, and complications of untreated PID are significant, novel methods to improve diagnosis are essential. OBJECTIVES: To determine if patients with PID have unique RNA expression patterns compared to controls. METHODS: Peripheral blood was collected from adolescent females with PID in the emergency department, and from control patients in the operating room. RNA was isolated, and microarray analysis was performed. Initial analysis involved a training set of 18 patients (9 PID patients with either Neisseria gonorrhoeae or Chlamydia trachomatis infection and 9 control patients). Supervised and unsupervised cluster analyses were performed, followed by network analysis. The training set was used to classify a set of 15 additional PID patients and 2 controls. RESULTS: Supervised cluster analysis of the training set revealed 170 genes which were differentially expressed in PID patients versus controls. Network analysis indicated that several differentially expressed genes are involved in immune activation. Analysis of additional PID patients based on the training set findings revealed that patients with positive testing for Trichomonas vaginalis partitioned with the PID group, whereas patients with no organism identified partitioned with both groups. CONCLUSIONS: RNA sample collection from adolescents in the emergency department is feasible. Genes were identified which were differentially expressed in PID patients versus controls, many of which are involved in inflammation. Future studies should confirm the training set findings on a larger sample and may lead to improved accuracy of PID diagnosis.

Exome sequencing analysis reveals variants in primary immunodeficiency genes in patients with very early onset inflammatory bowel disease.

BACKGROUND & AIMS: Very early onset inflammatory bowel disease (VEO-IBD), IBD diagnosed at 5 years of age or younger, frequently presents with a different and more severe phenotype than older-onset IBD. We investigated whether patients with VEO-IBD carry rare or novel variants in genes associated with immunodeficiencies that might contribute to disease development. METHODS: Patients with VEO-IBD and parents (when available) were recruited from the Children's Hospital of Philadelphia from March 2013 through July 2014. We analyzed DNA from 125 patients with VEO-IBD (age, 3 wk to 4 y) and 19 parents, 4 of whom also had IBD. Exome capture was performed by Agilent SureSelect V4, and sequencing was performed using the Illumina HiSeq platform. Alignment to human genome GRCh37 was achieved followed by postprocessing and variant calling. After functional annotation, candidate variants were analyzed for change in protein function, minor allele frequency less than 0.1%, and scaled combined annotation-dependent depletion scores of 10 or less. We focused on genes associated with primary immunodeficiencies and related pathways. An additional 210 exome samples from patients with pediatric IBD (n = 45) or adult-onset Crohn's disease (n = 20) and healthy individuals (controls, n = 145) were obtained from the University of Kiel, Germany, and used as control groups. RESULTS: Four hundred genes and regions associated with primary immunodeficiency, covering approximately 6500 coding exons totaling more than 1 Mbp of coding sequence, were selected from the whole-exome data. Our analysis showed novel and rare variants within these genes that could contribute to the development of VEO-IBD, including rare heterozygous missense variants in IL10RA and previously unidentified variants in MSH5 and CD19. CONCLUSIONS: In an exome sequence analysis of patients with VEO-IBD and their parents, we identified variants in genes that regulate B- and T-cell functions and could contribute to pathogenesis. Our analysis could lead to the identification of previously unidentified IBD-associated variants.

ALK mutations confer differential oncogenic activation and sensitivity to ALK inhibition therapy in neuroblastoma.

Genetic studies have established anaplastic lymphoma kinase (ALK), a cell surface receptor tyrosine kinase, as a tractable molecular target in neuroblastoma. We describe comprehensive genomic, biochemical, and computational analyses of ALK mutations across 1,596 diagnostic neuroblastoma samples. ALK tyrosine kinase domain mutations occurred in 8% of samples--at three hot spots and 13 minor sites--and correlated significantly with poorer survival in high- and intermediate-risk neuroblastoma. Biochemical and computational studies distinguished oncogenic (constitutively activating) from nononcogenic mutations and allowed robust computational prediction of their effects. The mutated variants also showed differential in vitro crizotinib sensitivities. Our studies identify ALK genomic status as a clinically important therapeutic stratification tool in neuroblastoma and will allow tailoring of ALK-targeted therapy to specific mutations.

Progressive increase in mtDNA 3243A>G heteroplasmy causes abrupt transcriptional reprogramming.

Variation in the intracellular percentage of normal and mutant mitochondrial DNAs (mtDNA) (heteroplasmy) can be associated with phenotypic heterogeneity in mtDNA diseases. Individuals that inherit the common disease-causing mtDNA tRNA(Leu(UUR)) 3243A>G mutation and harbor ∼10-30% 3243G mutant mtDNAs manifest diabetes and occasionally autism; individuals with ∼50-90% mutant mtDNAs manifest encephalomyopathies; and individuals with ∼90-100% mutant mtDNAs face perinatal lethality. To determine the basis of these abrupt phenotypic changes, we generated somatic cell cybrids harboring increasing levels of the 3243G mutant and analyzed the associated cellular phenotypes and nuclear DNA (nDNA) and mtDNA transcriptional profiles by RNA sequencing. Small increases in mutant mtDNAs caused relatively modest defects in oxidative capacity but resulted in sharp transitions in cellular phenotype and gene expression. Cybrids harboring 20-30% 3243G mtDNAs had reduced mtDNA mRNA levels, rounded mitochondria, and small cell size. Cybrids with 50-90% 3243G mtDNAs manifest induction of glycolytic genes, mitochondrial elongation, increased mtDNA mRNA levels, and alterations in expression of signal transduction, epigenomic regulatory, and neurodegenerative disease-associated genes. Finally, cybrids with 100% 3243G experienced reduced mtDNA transcripts, rounded mitochondria, and concomitant changes in nuclear gene expression. Thus, striking phase changes occurred in nDNA and mtDNA gene expression in response to the modest changes of the mtDNA 3243G mutant levels. Hence, a major factor in the phenotypic variation in heteroplasmic mtDNA mutations is the limited number of states that the nucleus can acquire in response to progressive changes in mitochondrial retrograde signaling.

Dielectrophoretic capture and genetic analysis of single neuroblastoma tumor cells.

Our understanding of the diversity of cells that escape the primary tumor and seed micrometastases remains rudimentary, and approaches for studying circulating and disseminated tumor cells have been limited by low throughput and sensitivity, reliance on single parameter sorting, and a focus on enumeration rather than phenotypic and genetic characterization. Here, we utilize a highly sensitive microfluidic and dielectrophoretic approach for the isolation and genetic analysis of individual tumor cells. We employed fluorescence labeling to isolate 208 single cells from spiking experiments conducted with 11 cell lines, including 8 neuroblastoma cell lines, and achieved a capture sensitivity of 1 tumor cell per 10(6) white blood cells (WBCs). Sample fixation or freezing had no detectable effect on cell capture. Point mutations were accurately detected in the whole genome amplification product of captured single tumor cells but not in negative control WBCs. We applied this approach to capture 144 single tumor cells from 10 bone marrow samples of patients suffering from neuroblastoma. In this pediatric malignancy, high-risk patients often exhibit wide-spread hematogenous metastasis, but access to primary tumor can be difficult or impossible. Here, we used flow-based sorting to pre-enrich samples with tumor involvement below 0.02%. For all patients for whom a mutation in the Anaplastic Lymphoma Kinase gene had already been detected in their primary tumor, the same mutation was detected in single cells from their marrow. These findings demonstrate a novel, non-invasive, and adaptable method for the capture and genetic analysis of single tumor cells from cancer patients.

S(+)-ibuprofen destabilizes MYC/MYCN and AKT, increases p53 expression, and induces unfolded protein response and favorable phenotype in neuroblastoma cell lines.

Neuroblastoma is a common pediatric solid tumor that exhibits a striking clinical bipolarity: favorable and unfavorable. The survival rate of children with unfavorable neuroblastoma remains low among all childhood cancers. MYCN and MYC play a crucial role in determining the malignancy of unfavorable neuroblastomas, whereas high-level expression of the favorable neuroblastoma genes is associated with a good disease outcome and confers growth suppression of neuroblastoma cells. A small fraction of neuroblastomas harbors TP53 mutations at diagnosis, but a higher proportion of the relapse cases acquire TP53 mutations. In this study, we investigated the effect of S(+)-ibuprofen on neuroblastoma cell lines, focusing on the expression of the MYCN, MYC, AKT, p53 proteins and the favorable neuroblastoma genes in vitro as biomarkers of malignancy. Treatment of neuroblastoma cell lines with S(+)-ibuprofen resulted in a significant growth suppression. This growth effect was accompanied by a marked decrease in the expression of MYC, MYCN, AKT and an increase in p53 expression in neuroblastoma cell lines without TP53 mutation. In addition, S(+)-ibuprofen enhanced the expression of some favorable neuroblastoma genes (EPHB6, CD44) and genes involved in growth suppression and differentiation (EGR1, EPHA2, NRG1 and SEL1L). Gene expression profile and Ingenuity pathway analyses using TP53-mutated SKNAS cells further revealed that S(+)-ibuprofen suppressed molecular pathways associated with cell growth and conversely enhanced those of cell cycle arrest and the unfolded protein response. Collectively, these results suggest that S(+)-ibuprofen or its related compounds may have the potential for therapeutic and/or palliative use for unfavorable neuroblastoma.

Histone H2AX suppresses translocations in lymphomas of Eµ-c-Myc transgenic mice that contain a germline amplicon of tumor-promoting genes.

The DNA damage response (DDR) can restrain the ability of oncogenes to cause genomic instability and drive malignant transformation. The gene encoding the histone H2AX DDR factor maps to 11q23, a region frequently altered in human cancers. Since H2ax functions as a haploinsufficient suppressor of B lineage lymphomas with c-Myc amplification and/or translocation, we determined the impact of H2ax expression on the ability of deregulated c-Myc expression to cause genomic instability and drive transformation of B cells. Neither H2ax deficiency nor haploinsufficiency affected the rate of mortality of Eµ-c-Myc mice from B lineage lymphomas with genomic deletions and amplifications. Yet H2ax functioned in a dosage-dependent manner to prevent unbalanced translocations in Eµ-c-Myc tumors, demonstrating that H2ax functions in a haploinsufficient manner to suppress allelic imbalances and limit molecular heterogeneity within and among Eµ-c-Myc lymphomas. Regardless of H2ax copy number, all Eµ-c-Myc tumors contained identical amplification of chromosome 19 sequences spanning 20 genes. Many of these genes encode proteins with tumor-promoting activities, including Cd274, which encodes the PD-L1 programmed death ligand that induces T cell apoptosis and enables cancer cells to escape immune surveillance. This amplicon was in non-malignant B and T cells and non-lymphoid cells, linked to the Eµ-c-Myc transgene, and associated with overexpression of PD-L1 on non-malignant B cells. Our data demonstrate that, in addition to deregulated c-Myc expression, non-malignant B lineage lymphocytes of Eµ-c-Myc transgenic mice may have constitutive amplification and increased expression of other tumor-promoting genes.

Primary respiratory chain disease causes tissue-specific dysregulation of the global transcriptome and nutrient-sensing signaling network.

Primary mitochondrial respiratory chain (RC) diseases are heterogeneous in etiology and manifestations but collectively impair cellular energy metabolism. Mechanism(s) by which RC dysfunction causes global cellular sequelae are poorly understood. To identify a common cellular response to RC disease, integrated gene, pathway, and systems biology analyses were performed in human primary RC disease skeletal muscle and fibroblast transcriptomes. Significant changes were evident in muscle across diverse RC complex and genetic etiologies that were consistent with prior reports in other primary RC disease models and involved dysregulation of genes involved in RNA processing, protein translation, transport, and degradation, and muscle structure. Global transcriptional and post-transcriptional dysregulation was also found to occur in a highly tissue-specific fashion. In particular, RC disease muscle had decreased transcription of cytosolic ribosomal proteins suggestive of reduced anabolic processes, increased transcription of mitochondrial ribosomal proteins, shorter 5'-UTRs that likely improve translational efficiency, and stabilization of 3'-UTRs containing AU-rich elements. RC disease fibroblasts showed a strikingly similar pattern of global transcriptome dysregulation in a reverse direction. In parallel with these transcriptional effects, RC disease dysregulated the integrated nutrient-sensing signaling network involving FOXO, PPAR, sirtuins, AMPK, and mTORC1, which collectively sense nutrient availability and regulate cellular growth. Altered activities of central nodes in the nutrient-sensing signaling network were validated by phosphokinase immunoblot analysis in RC inhibited cells. Remarkably, treating RC mutant fibroblasts with nicotinic acid to enhance sirtuin and PPAR activity also normalized mTORC1 and AMPK signaling, restored NADH/NAD(+) redox balance, and improved cellular respiratory capacity. These data specifically highlight a common pathogenesis extending across different molecular and biochemical etiologies of individual RC disorders that involves global transcriptome modifications. We further identify the integrated nutrient-sensing signaling network as a common cellular response that mediates, and may be amenable to targeted therapies for, tissue-specific sequelae of primary mitochondrial RC disease.

Transient treatment with epigenetic modifiers yields stable neuroblastoma stem cells resembling aggressive large-cell neuroblastomas.

Cancer stem cells (CSCs) are plastic in nature, a characteristic that hampers cancer therapeutics. Neuroblastoma (NB) is a pediatric tumor of neural crest origin, and half of the cases are highly aggressive. By treating NB cell lines [SKNAS, SKNBE(2)C, CHP134, and SY5Y] with epigenetic modifiers for a short time, followed by sphere-forming culture conditions, we have established stem cell-like NB cells that are phenotypically stable for more than a year. These cells are characterized by their high expression of stemness factors, stem cell markers, and open chromatin structure. We referred to these cells as induced CSCs (iCSCs). SKNAS iCSC and SKNBE(2)C iCSC clones (as few as 100 cells) injected s.c. into SCID/Beige mice formed tumors, and in one case, SKNBE(2)C iCSCs metastasized to the adrenal gland, suggesting their increased metastatic potential. SKNAS iCSC xenografts showed the histologic appearance of totally undifferentiated large-cell NBs (LCNs), the most aggressive and deadly form of NB in humans. Immunohistochemical analyses showed that SKNAS iCSC xenografts expressed high levels of the stem cell marker CXCR4, whereas the SKNAS monolayer cell xenografts did not. The patterns of CXCR4 and MYC expression in SKNAS iCSC xenografts resembled those in the LCNs. The xenografts established from the NB iCSCs shared two common features: the LCN phenotype and high-level MYC/MYCN expression. These observations suggest both that NB cells with large and vesicular nuclei, representing their open chromatin structure, are indicative of stem cell-like tumor cells and that epigenetic changes may have contributed to the development of these most malignant NB cells.