Periodic Reporting for period 3 - ONCOFUM (Integrating the tissue-specificity and chronology of hereditary renal cancer predisposition)
Período documentado: 2022-04-01 hasta 2023-09-30
• 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.