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SETD2 loss-of-function promotes renal cancer branched evolution through replication stress and impaired DNA repair.

Kanu N, Grönroos E, Martinez P, Burrell RA, Yi Goh X, Bartkova J, Maya-Mendoza A, Mistrík M, Rowan AJ, Patel H, Rabinowitz A, East P, Wilson G, Santos CR, McGranahan N, Gulati S, Gerlinger M, Birkbak NJ, Joshi T, Alexandrov LB, Stratton MR, Powles T, Matthews N, Bates PA, Stewart A, Szallasi Z, Larkin J, Bartek J, Swanton C - Oncogene (2015)

Bottom Line: Distinct SETD2 mutations have been identified in spatially separated regions in ccRCC, indicative of intratumour heterogeneity.In this study, we have addressed the consequences of SETD2 loss-of-function through an integrated bioinformatics and functional genomics approach.Consistent with these data, we observe chromosomal breakpoint locations are biased away from H3K36me3 sites in SETD2 wild-type ccRCCs relative to tumours with bi-allelic SETD2 aberrations and that H3K36me3-negative ccRCCs display elevated DNA damage in vivo.

View Article: PubMed Central - PubMed

Affiliation: UCL Cancer Institute, Paul O'Gorman Building, London, UK.

ABSTRACT
Defining mechanisms that generate intratumour heterogeneity and branched evolution may inspire novel therapeutic approaches to limit tumour diversity and adaptation. SETD2 (Su(var), Enhancer of zeste, Trithorax-domain containing 2) trimethylates histone-3 lysine-36 (H3K36me3) at sites of active transcription and is mutated in diverse tumour types, including clear cell renal carcinomas (ccRCCs). Distinct SETD2 mutations have been identified in spatially separated regions in ccRCC, indicative of intratumour heterogeneity. In this study, we have addressed the consequences of SETD2 loss-of-function through an integrated bioinformatics and functional genomics approach. We find that bi-allelic SETD2 aberrations are not associated with microsatellite instability in ccRCC. SETD2 depletion in ccRCC cells revealed aberrant and reduced nucleosome compaction and chromatin association of the key replication proteins minichromosome maintenance complex component (MCM7) and DNA polymerase δ hindering replication fork progression, and failure to load lens epithelium-derived growth factor and the Rad51 homologous recombination repair factor at DNA breaks. Consistent with these data, we observe chromosomal breakpoint locations are biased away from H3K36me3 sites in SETD2 wild-type ccRCCs relative to tumours with bi-allelic SETD2 aberrations and that H3K36me3-negative ccRCCs display elevated DNA damage in vivo. These data suggest a role for SETD2 in maintaining genome integrity through nucleosome stabilization, suppression of replication stress and the coordination of DNA repair.

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Reduced H3K36me3 is associated with elevated DNA damage in primary ccRCC tumours. (a) Images of tumour cells from a ccRCC case stained negatively for SETD2 and H3K36me3 but strongly positive for γH2AX (scale bar: 50 μm). (b) Images of tumour cells from a ccRCC case stained positive for SETD2 and H3K36me3, but weakly stained for γH2AX (scale bar: 50 μm).
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fig3: Reduced H3K36me3 is associated with elevated DNA damage in primary ccRCC tumours. (a) Images of tumour cells from a ccRCC case stained negatively for SETD2 and H3K36me3 but strongly positive for γH2AX (scale bar: 50 μm). (b) Images of tumour cells from a ccRCC case stained positive for SETD2 and H3K36me3, but weakly stained for γH2AX (scale bar: 50 μm).

Mentions: In light of the apparent protective role for SETD2 and the selection pressure for SETD2 inactivation in ccRCC as an intermediate event that occurs around the time of detectable branched ccRCC evolution, we explored the relationship between SETD2 mutation and genomic instability in vivo. We assessed SETD2 expression, H3K36me3 levels and DNA damage in a cohort of 202 samples from 100 RCC tumours, assayed by immunohistochemistry on tissue microarrays (Figures 3a and b and Supplementary Table S5). SETD2 mutation status was not available for this cohort. However, as SETD2 is the main H3K36 trimethylase8 and the majority of mutations identified to date result in frameshifts or premature stop codons in the N-terminal half of the gene, we used a C-terminal SETD2 antibody that would not detect SETD2 affected by protein truncations distal to the reported mutations, the majority of which occur before the C-terminal SRI domain (Figure 1b). Therefore, SETD2 and H3K36me3 immunohistochemical staining were used as surrogate markers for SETD2 loss-of-function. A highly significant correlation was observed between SETD2 and H3K36me3 immunohistochemical staining (P=3.8e-14, Cochran-Armitage test, Supplementary Figure S3a), suggesting that absence of H3K36me3 staining is a good surrogate for SETD2 loss-of-function.8 DNA damage was assessed by staining for phosphorylated histone 2 A.X (γH2AX), which marks ongoing DNA damage signalling and DNA double-strand breaks.19 Both SETD2 and H3K36me3 levels were significantly anti-correlated with γH2AX staining (P<0.05, Supplementary Figures S3b and c), suggesting that SETD2 loss-of-function may contribute to DNA damage and double-strand breaks. These data further suggest a role for SETD2 and H3K36me3 in the maintenance of genome stability.


SETD2 loss-of-function promotes renal cancer branched evolution through replication stress and impaired DNA repair.

Kanu N, Grönroos E, Martinez P, Burrell RA, Yi Goh X, Bartkova J, Maya-Mendoza A, Mistrík M, Rowan AJ, Patel H, Rabinowitz A, East P, Wilson G, Santos CR, McGranahan N, Gulati S, Gerlinger M, Birkbak NJ, Joshi T, Alexandrov LB, Stratton MR, Powles T, Matthews N, Bates PA, Stewart A, Szallasi Z, Larkin J, Bartek J, Swanton C - Oncogene (2015)

Reduced H3K36me3 is associated with elevated DNA damage in primary ccRCC tumours. (a) Images of tumour cells from a ccRCC case stained negatively for SETD2 and H3K36me3 but strongly positive for γH2AX (scale bar: 50 μm). (b) Images of tumour cells from a ccRCC case stained positive for SETD2 and H3K36me3, but weakly stained for γH2AX (scale bar: 50 μm).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4660036&req=5

fig3: Reduced H3K36me3 is associated with elevated DNA damage in primary ccRCC tumours. (a) Images of tumour cells from a ccRCC case stained negatively for SETD2 and H3K36me3 but strongly positive for γH2AX (scale bar: 50 μm). (b) Images of tumour cells from a ccRCC case stained positive for SETD2 and H3K36me3, but weakly stained for γH2AX (scale bar: 50 μm).
Mentions: In light of the apparent protective role for SETD2 and the selection pressure for SETD2 inactivation in ccRCC as an intermediate event that occurs around the time of detectable branched ccRCC evolution, we explored the relationship between SETD2 mutation and genomic instability in vivo. We assessed SETD2 expression, H3K36me3 levels and DNA damage in a cohort of 202 samples from 100 RCC tumours, assayed by immunohistochemistry on tissue microarrays (Figures 3a and b and Supplementary Table S5). SETD2 mutation status was not available for this cohort. However, as SETD2 is the main H3K36 trimethylase8 and the majority of mutations identified to date result in frameshifts or premature stop codons in the N-terminal half of the gene, we used a C-terminal SETD2 antibody that would not detect SETD2 affected by protein truncations distal to the reported mutations, the majority of which occur before the C-terminal SRI domain (Figure 1b). Therefore, SETD2 and H3K36me3 immunohistochemical staining were used as surrogate markers for SETD2 loss-of-function. A highly significant correlation was observed between SETD2 and H3K36me3 immunohistochemical staining (P=3.8e-14, Cochran-Armitage test, Supplementary Figure S3a), suggesting that absence of H3K36me3 staining is a good surrogate for SETD2 loss-of-function.8 DNA damage was assessed by staining for phosphorylated histone 2 A.X (γH2AX), which marks ongoing DNA damage signalling and DNA double-strand breaks.19 Both SETD2 and H3K36me3 levels were significantly anti-correlated with γH2AX staining (P<0.05, Supplementary Figures S3b and c), suggesting that SETD2 loss-of-function may contribute to DNA damage and double-strand breaks. These data further suggest a role for SETD2 and H3K36me3 in the maintenance of genome stability.

Bottom Line: Distinct SETD2 mutations have been identified in spatially separated regions in ccRCC, indicative of intratumour heterogeneity.In this study, we have addressed the consequences of SETD2 loss-of-function through an integrated bioinformatics and functional genomics approach.Consistent with these data, we observe chromosomal breakpoint locations are biased away from H3K36me3 sites in SETD2 wild-type ccRCCs relative to tumours with bi-allelic SETD2 aberrations and that H3K36me3-negative ccRCCs display elevated DNA damage in vivo.

View Article: PubMed Central - PubMed

Affiliation: UCL Cancer Institute, Paul O'Gorman Building, London, UK.

ABSTRACT
Defining mechanisms that generate intratumour heterogeneity and branched evolution may inspire novel therapeutic approaches to limit tumour diversity and adaptation. SETD2 (Su(var), Enhancer of zeste, Trithorax-domain containing 2) trimethylates histone-3 lysine-36 (H3K36me3) at sites of active transcription and is mutated in diverse tumour types, including clear cell renal carcinomas (ccRCCs). Distinct SETD2 mutations have been identified in spatially separated regions in ccRCC, indicative of intratumour heterogeneity. In this study, we have addressed the consequences of SETD2 loss-of-function through an integrated bioinformatics and functional genomics approach. We find that bi-allelic SETD2 aberrations are not associated with microsatellite instability in ccRCC. SETD2 depletion in ccRCC cells revealed aberrant and reduced nucleosome compaction and chromatin association of the key replication proteins minichromosome maintenance complex component (MCM7) and DNA polymerase δ hindering replication fork progression, and failure to load lens epithelium-derived growth factor and the Rad51 homologous recombination repair factor at DNA breaks. Consistent with these data, we observe chromosomal breakpoint locations are biased away from H3K36me3 sites in SETD2 wild-type ccRCCs relative to tumours with bi-allelic SETD2 aberrations and that H3K36me3-negative ccRCCs display elevated DNA damage in vivo. These data suggest a role for SETD2 in maintaining genome integrity through nucleosome stabilization, suppression of replication stress and the coordination of DNA repair.

Show MeSH
Related in: MedlinePlus