Faculty Bibliography

Atlantic tomcod in the Hudson River Estuary bioaccumulate high hepatic burdens of environmental toxicants. Previously, we demonstrated that Hudson River tomcod developed resistance to TCDD and PCB toxicity probably through strong natural selection during their early life-stages for a variant of the Aryl Hydrocarbon Receptor2 (AHR2). Here, we evaluated the genomic consequences of the resistant genotype by comparing global gene expression in larval tomcod from the Hudson River with expression in larvae from a nearby sensitive population (Shinnecock Bay). We developed an annotated draft tomcod genome to explore the effects of multigenerational exposure to toxicants and a functionally impaired AHR2 on the transcriptome. We used the tomcod genome as a reference in RNA-Seq to compare global gene expression in tomcod larvae from the Hudson River and Shinnecock Bay after experimental exposure of larvae to graded doses of TCDD. We found dramatic differences between offspring from the two populations in the number of genes that were differentially expressed at all doses (0.01, 0.1, and 1 ppb) and even in the vehicle controls. At the two lowest TCDD doses, 250 and 1,141 genes were differentially expressed in Shinnecock Bay larvae compared with 14 and 12, respectively, in Hudson River larvae. At the highest dose (1.0 ppb), 934 genes were differentially expressed in Shinnecock Bay larvae and 173 in Hudson River larvae, but only 28 (16%) of affected genes were shared among both populations. Given the large difference between the two populations in the number and identity of differentially expressed genes, it is likely that the AHR2 pathway interacts directly or indirectly with many genes beyond those known in the AHR2 battery and that other regulatory systems may also respond to TCDD exposure. The effects of chronic multi-generational exposure to environmental toxicants on the genome of Hudson River tomcod are much greater than previously expected.

Aflatoxin B1 (AFB1) is a mutagen and IARC (International Agency for Research on Cancer) Group 1 carcinogen that causes hepatocellular carcinoma (HCC). Here, we present the first whole-genome data on the mutational signatures of AFB1 exposure from a total of >40,000 mutations in four experimental systems: two different human cell lines, in liver tumors in wild-type mice, and in mice that carried a hepatitis B surface antigen transgene-this to model the multiplicative effects of aflatoxin exposure and hepatitis B in causing HCC. AFB1 mutational signatures from all four experimental systems were remarkably similar. We integrated the experimental mutational signatures with data from newly sequenced HCCs from Qidong County, China, a region of well-studied aflatoxin exposure. This indicated that COSMIC mutational signature 24, previously hypothesized to stem from aflatoxin exposure, indeed likely represents AFB1 exposure, possibly combined with other exposures. Among published somatic mutation data, we found evidence of AFB1 exposure in 0.7% of HCCs treated in North America, 1% of HCCs from Japan, but 16% of HCCs from Hong Kong. Thus, aflatoxin exposure apparently remains a substantial public health issue in some areas. This aspect of our study exemplifies the promise of future widespread resequencing of tumor genomes in providing new insights into the contribution of mutagenic exposures to cancer incidence.

Increased intestinal permeability and translocation of gut bacteria trigger various polyaetiological diseases associated with chronic inflammation and underlie a variety of poorly treatable pathologies. Previous studies have established a primary role of the microbiota composition and intestinal permeability in such pathologies. Using a rat model, we examined the effects of exposure to a bacteriophage cocktail on intestinal permeability and relative abundance of taxonomic units in the gut bacterial community. There was an increase in markers of impaired gut permeability, such as the lactulose/mannitol ratio, plasma endotoxin concentrations, and serum levels of inflammation-related cytokines, following the bacteriophage challenge. We observed significant differences in the alpha diversity of faecal bacterial species and found that richness and diversity index values increased following the bacteriophage challenge. There was a reduction in the abundance of Blautia, Catenibacterium, Lactobacillus, and Faecalibacterium species and an increase in Butyrivibrio, Oscillospira and Ruminococcus after bacteriophage administration. These findings provide novel insights into the role of bacteriophages as potentially pathogenic for mammals and their possible implication in the development of diseases associated with increased intestinal permeability.

Plakophilin-2 (PKP2) is a component of the desmosome and known for its role in cell-cell adhesion. Mutations in human PKP2 associate with a life-threatening arrhythmogenic cardiomyopathy, often of right ventricular predominance. Here, we use a range of state-of-the-art methods and a cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout mouse to demonstrate that in addition to its role in cell adhesion, PKP2 is necessary to maintain transcription of genes that control intracellular calcium cycling. Lack of PKP2 reduces expression of Ryr2 (coding for Ryanodine Receptor 2), Ank2 (coding for Ankyrin-B), Cacna1c (coding for CaV1.2) and Trdn (coding for triadin), and protein levels of calsequestrin-2 (Casq2). These factors combined lead to disruption of intracellular calcium homeostasis and isoproterenol-induced arrhythmias that are prevented by flecainide treatment. We propose a previously unrecognized arrhythmogenic mechanism related to PKP2 expression and suggest that mutations in PKP2 in humans may cause life-threatening arrhythmias even in the absence of structural disease.It is believed that mutations in desmosomal adhesion complex protein plakophilin 2 (PKP2) cause arrhythmia due to loss of cell-cell communication. Here the authors show that PKP2 controls the expression of proteins involved in calcium cycling in adult mouse hearts, and that lack of PKP2 can cause arrhythmia in a structurally normal heart.

Exposure to aristolochic acid (AA, IARC Group 1 carcinogen) present in some traditional herbal medicines leads to aristolochic acid nephropathy (AAN), often complicated by development of multiple urothelial carcinomas of sequential onset. We used genome-scale mutational signature analysis of multiple urinary tract tumors of AAN cases from a unique patient group to determine the relationships of the patients' late-onset second cancers to the AA exposure as well as to the first cancers. Aristolactam-DNA adduct-positive AAN patients (n=4) who developed cancer within 8 years following the initial exposure to AA were chosen for analysis of their first cancers (upper tract urothelial carcinomas, UTUC) and second cancers of delayed onset (1-9 years after first-cancer diagnosis, involving the bladder or ureteral meatus). All patients had received a kidney transplant before developing the second cancers and had a functional renal graft prior to prophylactic nephroureterectomy. Genomic DNAs were isolated from FFPE sections of the renal cortex and from the upper and lower tract tumors of each patient using laser capture micro-dissection or macrodissection of the tumor areas. Low-coverage (~15x) exome 100-bp paired-end sequencing was performed using Illumina HiSeq2500. Customized variant calling identified somatic variants absent in non-tumor tissues. Non-negative matrix factorization was applied to extract mutational signatures in the tumor tissues. In all cases, we established the mutational signature of AA (the COSMIC signature 22) in the first UTUC as well as second cancers involving the bladder or lower ureter (meatus). Additionally, the first and second cancers harbored considerable overlaps in exposure-specific (A>T) somatic mutations. This finding provides evidence that the delayed onset of bladder urothelial carcinomas in AAN patients is likely due to distal seeding of cancer cells originating from the primary UTUC tumors. Our first-of-its-kind study addresses the risk as well as mechanistic factors leading to the second, lateonset bladder urothelial carcinomas following kidney transplantation and primary UTUC development. Our results underline the importance of long-term bladder follow-up in high-risk populations with established or suspected AA exposure

Background: Recently, tumor mutation burden (TMB) has been shown to increase the presentation of neoantigens that stimulate immune tumor recognition, resulting in improved immunotherapy (IT) outcomes in melanoma and other cancers. As melanoma is highly immunogenic, here we tested whether TMB associates with immune recognition during tumor progression, hence impacting melanoma overall survival (OS), independently of IT treatment. Methods: We have generated somatic mutation data from 314 IT-naive metastatic melanomas from The Cancer Genome Atlas (TCGA). In the TCGA cohort, TMB has been calculated for 210 genes (200GS) previously established from TMB studies of anti-CTLA4 and anti-PD1/PD-L1 IT. For validation, we have sequenced exonic regions of 20 genes (20GS) with the highest TMB among 200GS in 89 IT-naive metastatic melanomas ascertained at New York University Langone Medical Center. The TMB was defined using total number of somatic, non-synonymous mutations in either 200GS (TCGA discovery) or 20GS (validation), respectively. For discovery and validation cohorts, OS from primary diagnosis of samples with high TMB was compared against low TMB, using thresholds established in previous studies. Results: We found that total TMB predicts better OS (p = 0.03, HR = 2.64) in TCGA melanomas. Restricting the analysis only to the established 200GS, this association became more significant in all patients (p = 0.01, HR = 2.67) as well as in patients without IT (p = 0.01, HR = 2.67). In the validation stage of 89 melanomas without prior IT treatment, a high TMB in a subset of 20GS accurately determined favorable OS (p = 0.02, HR = 2.69) and confirmed TCGA observations from the 200GS. Conclusions: Here we show, for the first time, that in addition to IT, high TMB predicts more favorable OS in patients that never received IT, potentially serving as a novel marker of prognosis of melanoma and likely other immunogenic tumors at early stages. In addition, our study suggests that TMB test can be robust when applied to only a small subset of genes that trigger significantly higher immunogenicity. This may also eventually assist with accurate sub-selection of early stage patients likely to respond to IT regimens