Periodic Reporting for period 3 - ONCOFUM (Integrating the tissue-specificity and chronology of hereditary renal cancer predisposition)
Periodo di rendicontazione: 2022-04-01 al 2023-09-30
During the first period of ONCOFUM, we studied the consequences of Fh1 loss in mouse and newly generated human models. We found that the loss of FH in kidney tubules promotes Atf4 translation and activates the integrated stress response in murine models, both in vitro and in vivo. This same signature is also observed in human FH-KO kidney tubules. Since the engagement of this pathway is key for metabolic adaptation to the loss of FH enzymatic activity and subsequent mitochondrial dysfunction, it may play an important role in the tissue-specific tumorigenesis in type II papillary renal cell carcinoma. The molecular mechanisms that lead to increased Atf4 translation upon FH loss are currently under investigation and whether activation of this pathway is also observed in SDHX-deficient renal proximal tubular cells. We also confirmed that upon Fh1 loss cells engage into a profound epigenome rewiring. Importantly, we identified changes in chromatin structure and function that are specifically associated with Fh1 loss. Our results indicate that the epigenetic rewiring triggered by FH loss is mediated by the pioneering factor Foxa2. We are currently performing RIME experiments to identify the partners of Foxa2 and how it is activated by FH loss.
• Capitalizing on cellular models generated in the lab, we took an unbiased multi-omics approach (RNA sequencing, metabolomics and proteomics) to investigate signatures activated upon FH loss. Geneset enrichment analysis (GSEA) performed on RNAseq datasets from Fh1fl/fl, Fh1-/-CL1, Fh1-/-CL19 and Fh1-/-+Fh1 renal proximal tubular cells identified a significant increase in genes linked to mitochondrial integrated stress response (ISR) and stress-induced transcription factors (TFs), including Atf4, Nrf2 and Chop. A subset of ISR genes were chosen and experimentally validated as significantly increasing in both Fh1-KO clones using qPCR. Analysis of tandem mass tagging (TMT) proteomics performed in Fh1fl/fl, Fh1-/-CL1and Fh1-/-CL19 renal proximal tubular cells using Perseus bioinformatics software confirmed that a significant proportion of ISR genes were also significantly increased at protein level, including Atf4. Increased Atf4 and Atf4-target asparagine synthetase (Asns) levels were validated via WB, while increased Atf4 gene expression was also observed in the Fh1-KO clones using qPCR. Many genes of the ISR are involved in one carbon metabolism, amino acid uptake and synthesis, specifically the neutral amino acids, such as serine, glycine, threonine and cysteine, and in the glutathione cycle. Metabolomic analysis of the above indicated cellular models found a significant in increase in the intracellular levels of neutral amino acids and this increase was diminished upon reconstitution of Fh1. Similarly, an increase in GSH and GSSG levels indicate regulation of GSH synthesis and the glutathione cycle. Using ISRIB, which specifically targets the integrated stress response by overcoming phosphorylation of the eukaryotic translation initiation factor, eIF2, significantly decreased Atf4 translation and ISR target gene expression, including the Atf4 transcript itself in the Fh1-KO clones.
• Using publicly available microarray data from Fh1-KO murine kidneys a significant enrichment in the ISR and ISR-associated TFs was also found. GSEA analysis of RNAseq data from human FH-KO renal tubular cells generated by CRISPR-Cas9 gene editing technology also identified a significant increase in the ISR in FH-KO cells. To determine relevance of this pathway to kidney cancer biology, we utilised the cancer genome atlas (TCGA) and gene expression profiling interactive analysis (GEPIA) bioinformatic tool and demonstrated that increased expression of ISR-linked metabolic genes are associated with decreased overall and disease-free survival in papillary renal cell carcinoma (KIRP). This association was specific to type II papillary renal cell carcinoma (KIRP.C2) the molecular subtype associated with both FH loss and NRF2 activation, but not type I papillary renal cell carcinoma (KIRP.C1). This suggests that this pathway may be important for tumorigenesis.
In summary, with this part of the project we discovered that the loss of FH in kidney tubules promotes Atf4 translation and activates the ISR in murine models, both in vitro and in vivo. This same signature is also observed in human FH-KO kidney tubules. Current data suggests engagement of this pathway may be key for metabolic adaptation to the loss of FH enzymatic activity and subsequent mitochondrial dysfunction. This pathway may also play an important role in tissue-specific tumorigenesis in type II papillary renal cell carcinoma.
• In the second part of the programme, we started elucidating the changes in the epigenetic landscape upon fumarate hydratase loss. To this aim, we investigated the changes in chromatin accessibility and subsequent enhancer/promoter activation. We have used Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) to profile the open chromatin of the above-described cellular models. Importantly, this approach enabled us to identify accessible promoters as well as enhancers. We have also used H3K27ac chromatin immunoprecipitation with sequencing (ChIP-seq) to assess how active these promoters and enhancers are. We observed a correlation plot of ATAC-seq signal at all accessible regions. Biological replicates cluster with each other. Importantly Fh1fl/fl and Fh1-/- +pFh cluster with each other, and the two KO clones cluster with each other. MA plot showing differentially accessible regions in Fh1-/- CL1 and Fh1-/- CL19 compared to WT. Similar findings were made using H3K27ac ChIP-seq. Normalised footprinting scores across the various classes of regions identified Nrf2, Atf4, Ddit3 in a specific epigenetic cluster that is reversible upon Fh1 reconstitution. These results are consistent with the preliminary results described in the first part of the programme, where we identified the activation of a reversible ATF4-dependent pathway in Fh1-deficient cells. Overall, these results indicate that our approaches are suitable to identify changes in chromatin accessibility and function upon Fh1 loss.
• Using publicly available microarray data from Fh1-KO murine kidneys a significant enrichment in the ISR and ISR-associated TFs was also found. GSEA analysis of RNAseq data from human FH-KO renal tubular cells generated by CRISPR-Cas9 gene editing technology also identified a significant increase in the ISR in FH-KO cells. To determine relevance of this pathway to kidney cancer biology, we utilised the cancer genome atlas (TCGA) and gene expression profiling interactive analysis (GEPIA) bioinformatic tool and demonstrated that increased expression of ISR-linked metabolic genes are associated with decreased overall and disease-free survival in papillary renal cell carcinoma (KIRP). This association was specific to type II papillary renal cell carcinoma (KIRP.C2) the molecular subtype associated with both FH loss and NRF2 activation, but not type I papillary renal cell carcinoma (KIRP.C1). This suggests that this pathway may be important for tumorigenesis.
In summary, with this part of the project we discovered that the loss of FH in kidney tubules promotes Atf4 translation and activates the ISR in murine models, both in vitro and in vivo. This same signature is also observed in human FH-KO kidney tubules. Current data suggests engagement of this pathway may be key for metabolic adaptation to the loss of FH enzymatic activity and subsequent mitochondrial dysfunction. This pathway may also play an important role in tissue-specific tumorigenesis in type II papillary renal cell carcinoma.
• In the second part of the programme, we started elucidating the changes in the epigenetic landscape upon fumarate hydratase loss. To this aim, we investigated the changes in chromatin accessibility and subsequent enhancer/promoter activation. We have used Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) to profile the open chromatin of the above-described cellular models. Importantly, this approach enabled us to identify accessible promoters as well as enhancers. We have also used H3K27ac chromatin immunoprecipitation with sequencing (ChIP-seq) to assess how active these promoters and enhancers are. We observed a correlation plot of ATAC-seq signal at all accessible regions. Biological replicates cluster with each other. Importantly Fh1fl/fl and Fh1-/- +pFh cluster with each other, and the two KO clones cluster with each other. MA plot showing differentially accessible regions in Fh1-/- CL1 and Fh1-/- CL19 compared to WT. Similar findings were made using H3K27ac ChIP-seq. Normalised footprinting scores across the various classes of regions identified Nrf2, Atf4, Ddit3 in a specific epigenetic cluster that is reversible upon Fh1 reconstitution. These results are consistent with the preliminary results described in the first part of the programme, where we identified the activation of a reversible ATF4-dependent pathway in Fh1-deficient cells. Overall, these results indicate that our approaches are suitable to identify changes in chromatin accessibility and function upon Fh1 loss.
To investigate the response to FH loss we used an unbiased multi-omics approach (RNA sequencing, metabolomics and proteomics). We have also validated these results using a variety of molecular biology tools, namely Western blotting (WB) and quantitative PCR (qPCR), and a highly specific pharmacological inhibitor, ISRIB, to look at ISR pathway activation and target gene expression. For the epigenome analyses, we have used Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq). We have also used H3K27ac chromatin immunoprecipitation with sequencing (ChIP-seq) to assess how active these promoters and enhancers are. We have then integrated these regions into a range of classes, depending on whether these regions changes in accessibility, and whether this change occurs with increased H3K27ac, and vice versa. Finally, motif enrichment analysis and DNA footprinting analysis has been performed to explore what transcription factors may be responsible for these changes.