Abstract-Standard sediments are required for prospective risk assessments so that comparable fate and effects data can be generated within and between laboratories. One approach is to use artificial media that can be easily reproduced and characterized. However, a concern is that these media may not simulate natural sediments in terms of binding and toxicological properties. In this study, the acute and chronic effects of permethrin were compared in two artificial and one natural sediment using larvae of the midge Chironomus riparius. The influence of clay and carbon content and organic matter type on permethrin toxicity was also investigated. The toxic response measured in natural sediment was lower than that in the two artificial sediments, one of which contained peat and the other which contained ␣-cellulose as the organic carbon source. Of the two, the peat-based medium gave a more comparable response to the natural sediment. Acute and chronic permethrin toxicity was influenced by all sediment factors investigated with a consistently lower toxicity measured in the peat-based sediment compared with the ␣-cellulose sediment. A decrease in toxicity, coupled with an increase in bulk sediment pemethrin concentration, was seen as both clay and organic carbon contents increased. Consideration should be given to improving the environmental realism of simple artificial formulations if the intention is to represent effects measured in natural sediments.

No abstract

One of the most frequently genetically altered chromatin modifiers in melanoma is the Enhancer of Zeste Homolog 2 (EZH2), the catalytic component of the Polycomb Repressive Complex 2 (PRC2), which methylates lysine 27 on histone 3 (H3K27me3), a chromatin mark associated with transcriptional repression. Genetic alterations in EZH2 in melanoma include amplifications and activating point mutations at tyrosine 641 (Y641). The oncogenic role of EZH2 in melanoma has previously been determined; however, its downstream oncogenic mechanisms remain underexplored. Here, we found that in genetically engineered mouse models, expression of Ezh2Y641F causes up-regulation of a subset of interferon-regulated genes in melanoma cells, suggesting a potential role of the immune system in the pathogenesis of these mutations. Expression of these interferon genes was not a result of changes in H3K27me3, but through a direct and non-canonical interaction between Ezh2 and Signal Transducer And Activator of Transcription 3 (Stat3). We found that Ezh2 directly binds Stat3, and that in the presence of Ezh2Y641F mutant, Stat3 protein is hypermethylated. Expression of Stat3 was required to maintain an anti-tumor immune response and its depletion resulted in faster melanoma progression and disease recurrence. Molecularly, Stat3 and Ezh2 bind together at many genomic loci, and, in association with the rest of the PRC2 complex, repress gene expression. These results suggest that one of the oncogenic mechanisms of Ezh2-mediated melanomagenesis is through evasion of the anti-tumor immune response, and that the immunomodulatory properties of Stat3 are context dependent.

EZH2 (Enhancer of Zeste Homolog 2) is the catalytic component of the Polycomb Repressive Complex group 2 (PRC2), which establishes the repressive epigenetic mark histone 3 lysine 27 trimethylation (H3K27me3), resulting in silencing of gene expression. EZH2 was originally identified as a tumor suppressor since loss-of-function events were observed in myelodysplastic syndrome, acute myeloid, and T-cell leukemias. However, next-generation sequencing identified recurrent activating point mutations at tyrosine residue 641 (Y641) in 25% of germinal center diffuse large B-cell lymphoma and 15% of follicular lymphomas, suggesting that EZH2 also functions as an oncogene in a cell type-dependent manner. Using a faithful genetically engineered mouse model, we previously demonstrated that the Ezh2Y641F mutation drives formation of B-cell lymphoma as a single event and in cooperation with Bcl2 amplification or p53 loss. Unexpectedly, at the chromatin level, expression of Ezh2Y641F did not monotonically increase abundance of global H3K27me3 but rather resulted in redistribution of this mark across the genome, exhibiting loss of H3K27me3 at many loci, with direct effects on transcription. Others have shown that EZH2 mediates germinal center proliferation via repression of p21 and cooperation with Bcl6; however, given the global effect of Ezh2Y641F on chromatin, the oncogenic mechanisms of these mutations remain underexplored. These include the timing of the mutation during hematopoietic development, its role during differentiation, and the oncogenic activity of downstream targets. In order to investigate the effect of Ezh2Y641 mutations on hematopoietic development and differentiation, we used Cre alleles to drive expression of the mutant protein at different stages of hematopoietic development. Our data demonstrate that expression of Ezh2Y641 in early B cells is sufficient to drive oncogenic transformation. However, expression of Ezh2Y641 in hematopoietic stem cell (HSC) is not sufficient to drive oncogenic transformation despite the presence of mature healthy B cells. Additionally, expression of Ezh2Y641F in HSCs results in loss of self-renewal, differentiation bias towards the lymphoid lineages, and a partial block at the pre-pro B-cell stage. With regards to direct downstream targets, we identified upregulation of several HoxC cluster genes in Ezh2Y641F-mutant B cells prior to transformation. Upregulation of these genes is mediated via loss of H3K27me3 at the HoxC cluster and focal gains of H3K27 acetylation (H3K27ac). Overall, our findings underline the complicated biology and oncogenic activity of EZH2Y641 mutations in lymphoma, shed light on the effect of these mutations during hematopoietic development, and underscore some of the consequences of the paradoxical loss of H3K27me3 at certain loci. Citation Format: Samantha J. Nixon, Sarah Zimmerman, Jeremy M. Simon, George P. Souroullas. Understanding the properties and oncogenic mechanisms of EZH2 Y641 mutations in lymphoma [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-08.