Niraj S. Trivedi

Vector design influences hepatic genotoxicity after adeno-associated virus gene therapy

The use of adeno-associated virus (AAV) as a gene therapy vector has been approved recently for clinical use and has demonstrated efficacy in a growing number of clinical trials. However, the safety of AAV as a vector has been challenged by a single study that documented hepatocellular carcinoma (HCC) after AAV gene delivery in mice. Most studies have not noted genotoxicity following AAV-mediated gene delivery; therefore, the possibility that there is an association between AAV and HCC is controversial. Here, we performed a comprehensive study of HCC in a large number of mice following therapeutic AAV gene delivery. Using a sensitive high-throughput integration site-capture technique and global expressional analysis, we found that AAV integration into the RNA imprinted and accumulated in nucleus (Rian) locus, and the resulting overexpression of proximal microRNAs and retrotransposon-like 1 (Rtl1) were associated with HCC. In addition, we demonstrated that the AAV vector dose, enhancer/promoter selection, and the timing of gene delivery are all critical factors for determining HCC incidence after AAV gene delivery. Together, our results define aspects of AAV-mediated gene therapy that influence genotoxicity and suggest that these features should be considered for design of both safer AAV vectors and gene therapy studies.

Targeting proximal tubule mitochondrial dysfunction attenuates the renal disease of methylmalonic acidemia

Isolated methylmalonic acidemia (MMA), caused by deficiency of the mitochondrial enzyme methylmalonyl-CoA mutase (MUT), is often complicated by end stage renal disease that is resistant to conventional therapies, including liver transplantation. To establish a viable model of MMA renal disease, Mut was expressed in the liver of Mut−/− mice as a stable transgene under the control of an albumin (INS-Alb-Mut) promoter. Mut−/−;TgINS-Alb-Mut mice, although completely rescued from neonatal lethality that was displayed by Mut−/− mice, manifested a decreased glomerular filtration rate (GFR), chronic tubulointerstitial nephritis and ultrastructural changes in the proximal tubule mitochondria associated with aberrant tubular function, as demonstrated by single-nephron GFR studies. Microarray analysis of Mut−/−;TgINS-Alb-Mut kidneys identified numerous biomarkers, including lipocalin-2, which was then used to monitor the response of the GFR to antioxidant therapy in the mouse model. Renal biopsies and biomarker analysis from a large and diverse patient cohort (ClinicalTrials.gov identifier: NCT00078078) precisely replicated the findings in the animals, establishing Mut−/−;TgINS-Alb-Mut mice as a unique model of MMA renal disease. Our studies suggest proximal tubular mitochondrial dysfunction is a key pathogenic mechanism of MMA-associated kidney disease, identify lipocalin-2 as a biomarker of increased oxidative stress in the renal tubule, and demonstrate that antioxidants can attenuate the renal disease of MMA.

Global genome splicing analysis reveals an increased number of alternatively spliced genes with aging

Alternative splicing (AS) is a key regulatory mechanism for the development of different tissues; however, not much is known about changes to alternative splicing during aging. Splicing events may become more frequent and widespread genome‐wide as tissues age and the splicing machinery stringency decreases. Using skin, skeletal muscle, bone, thymus, and white adipose tissue from wild‐type C57BL6/J male mice (4 and 18 months old), we examined the effect of age on splicing by AS analysis of the differential exon usage of the genome. The results identified a considerable number of AS genes in skeletal muscle, thymus, bone, and white adipose tissue between the different age groups (ranging from 27 to 246 AS genes corresponding to 0.3–3.2% of the total number of genes analyzed). For skin, skeletal muscle, and bone, we included a later age group (28 months old) that showed that the number of alternatively spliced genes increased with age in all three tissues (P < 0.01). Analysis of alternatively spliced genes across all tissues by gene ontology and pathway analysis identified 158 genes involved in RNA processing. Additional analysis of AS in a mouse model for the premature aging disease Hutchinson–Gilford progeria syndrome was performed. The results show that expression of the mutant protein, progerin, is associated with an impaired developmental splicing. As progerin accumulates, the number of genes with AS increases compared to in wild‐type skin. Our results indicate the existence of a mechanism for increased AS during aging in several tissues, emphasizing that AS has a more important role in the aging process than previously known.

Distinct microRNA expression signatures are associated with melanoma subtypes and are regulated by HIF1A

The complex genetic changes underlying metastatic melanoma need to be deciphered to develop new and effective therapeutics. Previously, genome‐wide microarray analyses of human melanoma identified two reciprocal gene expression programs, including transcripts regulated by either transforming growth factor, beta 1 (TGFβ1) pathways, or microphthalmia‐associated transcription factor (MITF)/SRY‐box containing gene 10 (SOX10) pathways. We extended this knowledge by discovering that melanoma cell lines with these two expression programs exhibit distinctive microRNA (miRNA) expression patterns. We also demonstrated that hypoxia‐inducible factor 1 alpha (HIF1A) is increased in TGFβ1 pathway‐expressing melanoma cells and that HIF1A upregulates miR‐210, miR‐218, miR‐224, and miR‐452. Reduced expression of these four miRNAs in TGFβ1 pathway‐expressing melanoma cells arrests the cell cycle, while their overexpression in mouse melanoma cells increases the expression of the hypoxic response gene Bnip3. Taken together, these data suggest that HIF1A may regulate some of the gene expression and biological behavior of TGFβ1 pathway‐expressing melanoma cells, in part via alterations in these four miRNAs.

Somatic mutational landscape of AML with inv(16) or t(8;21) identifies patterns of clonal evolution in relapse leukemia

Somatic mutational landscape of AML with inv(16) or t(8;21) identifies patterns of clonal evolution in relapse leukemia

Comparative exome sequencing of metastatic lesions provides insights into the mutational progression of melanoma

BackgroundMetastasis is characterized by spreading of neoplastic cells to an organ other than where they originated and is the predominant cause of death among cancer patients. This holds true for melanoma, whose incidence is increasing more rapidly than any other cancer and once disseminated has few therapeutic options. Here we performed whole exome sequencing of two sets of matched normal and metastatic tumor DNAs.ResultsUsing stringent criteria, we evaluated the similarities and differences between the lesions. We find that in both cases, 96% of the single nucleotide variants are shared between the two metastases indicating that clonal populations gave rise to the distant metastases. Analysis of copy number variation patterns of both metastatic sets revealed a trend similar to that seen with our single nucleotide variants. Analysis of pathway enrichment on tumor sets shows commonly mutated pathways enriched between individual sets of metastases and all metastases combined.ConclusionsThese data provide a proof-of-concept suggesting that individual metastases may have sufficient similarity for successful targeting of driver mutations.

Modeling Smith-Lemli-Opitz syndrome with induced pluripotent stem cells reveals a causal role for Wnt/β-catenin defects in neuronal cholesterol synthesis phenotypes

Smith-Lemli-Opitz syndrome (SLOS) is a malformation disorder caused by mutations in DHCR7, which impair the reduction of 7-dehydrocholesterol (7DHC) to cholesterol. SLOS results in cognitive impairment, behavioral abnormalities and nervous system defects, though neither affected cell types nor impaired signaling pathways are fully understood. Whether 7DHC accumulation or cholesterol loss is primarily responsible for disease pathogenesis is also unclear. Using induced pluripotent stem cells (iPSCs) from subjects with SLOS, we identified cellular defects that lead to precocious neuronal specification within SLOS derived neural progenitors. We also demonstrated that 7DHC accumulation, not cholesterol deficiency, is critical for SLOS-associated defects. We further identified downregulation of Wnt/β-catenin signaling as a key initiator of aberrant SLOS iPSC differentiation through the direct inhibitory effects of 7DHC on the formation of an active Wnt receptor complex. Activation of canonical Wnt signaling prevented the neural phenotypes observed in SLOS iPSCs, suggesting that Wnt signaling may be a promising therapeutic target for SLOS.

Metastasis-Associated Protein Ribosomal RNA Processing 1 Homolog B (RRP1B) Modulates Metastasis through Regulation of Histone Methylation

Overexpression of ribosomal RNA processing 1 homolog B (RRP1B) induces a transcriptional profile that accurately predicts patient outcome in breast cancer. However, the mechanism by which RRP1B modulates transcription is unclear. Here, the chromatin-binding properties of RRP1B were examined to define how it regulates metastasis-associated transcription. To identify genome-wide RRP1B-binding sites, high-throughput ChIP-seq was performed in the human breast cancer cell line MDA-MB-231 and HeLa cells using antibodies against endogenous RRP1B. Global changes in repressive marks such as histone H3 lysine 9 trimethylation (H3K9me3) were also examined by ChIP-seq. Analysis of these samples identified 339 binding regions in MDA-MB-231 cells and 689 RRP1B-binding regions in HeLa cells. Among these, 136 regions were common to both cell lines. Gene expression analyses of these RRP1B-binding regions revealed that transcriptional repression is the primary result of RRP1B binding to chromatin. ChIP-reChIP assays demonstrated that RRP1B co-occupies loci with decreased gene expression with the heterochromatin-associated proteins, tripartite motif-containing protein 28 (TRIM28/KAP1), and heterochromatin protein 1-α (CBX5/HP1α). RRP1B occupancy at these loci was also associated with higher H3K9me3 levels, indicative of heterochromatinization mediated by the TRIM28/HP1α complex. In addition, RRP1B upregulation, which is associated with metastasis suppression, induced global changes in histone methylation. Implications: RRP1B, a breast cancer metastasis suppressor, regulates gene expression through heterochromatinization and transcriptional repression, which helps our understanding of mechanisms that drive prognostic gene expression in human breast cancer. Mol Cancer Res; 12(12); 1818–28. ©2014 AACR.

Necdin is a breast cancer metastasis suppressor that regulates the transcription of c-Myc

Metastasis is the primary cause of death in breast cancer. Earlier studies using a mammary tumorigenesis mouse model identified Necdin (Ndn) as a germline modifier of metastasis. Differential expression of Ndn induces a gene-expression signature that predicts prognosis in human breast cancer. Additionally, a non-synonymous germline single nucleotide polymorphism (T50C; V17A) in Ndn distinguishes mouse strains with differing metastatic capacities. To better understand how hereditary factors influence metastasis in breast cancer, we characterized NDN-mediated transcription. Haplotype analysis in a well-characterized breast cancer cohort revealed that NDN germline variation is associated with both NDN expression levels and patient outcome. To examine the role of NDN in mammary tumor metastasis and transcriptional regulation, mouse mammary tumor cell lines stably over-expressing either the wildtype 50T or variant 50C Ndn allele were generated. Cells over-expressing Ndn 50T, but not Ndn 50C, exhibited significant decrease in cell invasiveness and pulmonary metastases compared to control cells. Transcriptome analyses identified a 71-gene expression signature that distinguishes cells over-expressing the two Ndn allelic variants. Furthermore, ChIP assays revealed c-Myc, a target gene of NDN, to be differentially regulated by the allelic variants. These data demonstrate that NDN and the T50C allele regulate gene expression and metastasis efficiency.

A somatic cell defect is associated with the onset of neurological symptoms in a lysosomal storage disease

Mutations in individuals with the lysosomal storage disorder Niemann-Pick disease, type C1 (NPC1) are heterogeneous, not localized to specific protein domains, and not correlated to time of onset or disease severity. We demonstrate direct correlation of the time of neurological symptom onset with the severity of lysosomal defects in NPC1 patient-derived fibroblasts. This is a novel assay for NPC1 individuals that may be predictive of NPC1 disease progression and broadly applicable to other lysosomal disorders.