Molecular characterization of SETD2, a histone methyltransferase, in clear cell renal cell carcinoma (ccRCC).

Abstract

4624 Background: Although the von Hippel-Lindau (VHL) tumor suppressor is mutated in 60% of ccRCC, deletion of VHL in mice is insufficient for tumorigenesis. Sequencing of ccRCC tumors identified mutations in SETD2, a histone H3 lysine 36 (H3K36) trimethyltransferase. We hypothesize that loss of SETD2 methyltransferase activity decreases H3K36 trimethylation (H3K36Me3) in ccRCC, and contributes to the cancer phenotype. Methods: H3K36Me3 immunohistochemical (IHC) staining was quantitated in wild-type and mutant SETD2 nephrectomy tissue. To establish frequency of SETD2 loss of heterozygosity (LOH), genomic DNA was isolated from 51 VHL deficient ccRCC specimens and analyzed with Affymetrix single-nucleotide polymorphism (SNP) arrays. To evaluate alterations of histone modifications in advanced ccRCC, H3K36Me3 IHC was quantitated on tissue microarrays (TMAs) representing 28 paired ccRCC specimens with unaffected kidney parenchyma and 40 metastases. To identify kidney-specific H3K36Me3 binding sites, chromatin immunoprecipitation (ChIP) sequencing was performed on nephrectomy specimens. Results: Nephrectomy specimens with SETD2 truncating mutations have decreased H3K36Me3 (>50%) compared to uninvolved kidney tissue. Using SNP arrays, LOH at chromosome 3p (location of VHL and SETD2 genes) was detected in >90% of the tested tumors. IHC reveals a 27.4% and 52.0% decrease in H3K36Me3 positive nuclei in primary ccRCC and metastases, respectively, when compared to matched adjacent unaffected kidney tissue (p <0.0001). Using ChIP sequencing, 421 and 1,718 genomic regions were upregulated in the tumor and unaffected kidney tissue. Conclusions: SETD2 truncating mutations in patient-derived tissue have decreased H3K36Me3 indicating that SETD2 is a non-redundant H3K36 methyltransferase. LOH of VHL and SETD2 occurs in >90% of tested ccRCC tumors. Genomic regions enriched for H3K36Me3 binding are being validated in additional patient-derived tissues. H3K36Me3 is decreased in primary ccRCC and even more in metastases, suggesting that SETD2 methyltransferase activity is progressively misregulated in RCC. Loss of SETD2 activity may cooperate with VHL silencing to promote tumorigenesis.