Posttranslational modifications (PTMs) of tubulin specify microtubules for specialized cellular f... more Posttranslational modifications (PTMs) of tubulin specify microtubules for specialized cellular functions and comprise what is termed a "tubulin code." PTMs of histones comprise an analogous "histone code," although the "readers, writers, and erasers" of the cytoskeleton and epigenome have heretofore been distinct. We show that methylation is a PTM of dynamic microtubules and that the histone methyltransferase SET-domain-containing 2 (SETD2), which is responsible for H3 lysine 36 trimethylation (H3K36me3) of histones, also methylates α-tubulin at lysine 40, the same lysine that is marked by acetylation on microtubules. Methylation of microtubules occurs during mitosis and cytokinesis and can be ablated by SETD2 deletion, which causes mitotic spindle and cytokinesis defects, micronuclei, and polyploidy. These data now identify SETD2 as a dual-function methyltransferase for both chromatin and the cytoskeleton and show a requirement for methylation in maintenance of genomic stability and the integrity of both the tubulin and histone codes.
Clinical cancer research : an official journal of the American Association for Cancer Research, Jan 2, 2016
DNA methylation is a heritable covalent modification that is developmentally regulated and is cri... more DNA methylation is a heritable covalent modification that is developmentally regulated and is critical in tissue-type definition. Although genotype-phenotype correlations have been described for different subtypes of renal cell carcinoma (RCC), it is unknown if DNA methylation profiles correlate with morphological or ontology based phenotypes. Here we test the hypothesis that DNA methylation signatures can discriminate between putative precursor cells in the nephron. We performed deep profiling of DNA methylation and transcriptome in diverse histopathological RCC subtypes and validated DNA methylation in an independent dataset as well as in The Cancer Genome Atlas Clear Cell and Chromophobe Renal Cell Carcinoma Datasets. Our data provide the first mapping of methylome epi-signature and indicates that RCC subtypes can be grouped into two major epi-clusters: C1 which encompasses clear-cell RCC, papillary RCC, mucinous and spindle cell carcinomas and translocation RCC; C2 which compris...
Over 90% of chondroblastomas contain a heterozygous mutation replacing lysine 36 with methionine ... more Over 90% of chondroblastomas contain a heterozygous mutation replacing lysine 36 with methionine (K36M) in the histone H3 variant H3.3. Here, we show that H3K36 methylation is reduced globally in chondroblastomas and in chondrocytes harboring the same genetic mutation due to inhibition of at least two H3K36 methyltransferases, MMSET and SETD2, by the H3.3K36M mutant proteins. Genes with altered expression as well as H3K36 di- and tri-methylation in H3.3K36M cells are enriched in cancer pathways. In addition, H3.3K36M chondrocytes exhibit several hallmarks of cancer cells including increased ability to form colonies, resistance to apoptosis and defects in differentiation. Thus, H3.3K36M proteins reprogram H3K36 methylation landscape and contribute to tumorigenesis in part through altering the expression of cancer-associated genes.
Clear cell renal cell carcinomas (ccRCCs) harbor frequent mutations in epigenetic modifiers inclu... more Clear cell renal cell carcinomas (ccRCCs) harbor frequent mutations in epigenetic modifiers including SETD2, the H3K36me3 writer. We profiled DNA methylation (5mC) across the genome in cell line-based models of SETD2 inactivation and SETD2 mutant primary tumors because 5mC has been linked to H3K36me3 and is therapeutically targetable. SETD2 depleted cell line models (long-term and acute) exhibited a DNA hypermethylation phenotype coinciding with ectopic gains in H3K36me3 centered across intergenic regions adjacent to low expressing genes, which became upregulated upon dysregulation of the epigenome. Poised enhancers of developmental genes were prominent hypermethylation targets. SETD2 mutant primary ccRCCs, papillary renal cell carcinomas, and lung adenocarcinomas all demonstrated a DNA hypermethylation phenotype that segregated tumors by SETD2 genotype and advanced grade. These findings collectively demonstrate that SETD2 mutations drive tumorigenesis by coordinated disruption of t...
Thesis (Ph.D.)--Baylor College of Medicine. Dept. of Molecular and Cellular Biology, 2005. Includ... more Thesis (Ph.D.)--Baylor College of Medicine. Dept. of Molecular and Cellular Biology, 2005. Includes bibliographical references.
Thesis (Fellows Thesis)--Texas A & M University, 1998. Includes bibliographical references: p. 28... more Thesis (Fellows Thesis)--Texas A & M University, 1998. Includes bibliographical references: p. 28-29. "Major Subject: Biochemistry/Chemistry."
BAP1 (10-15%) and PBRM1 (40-50%) are two of the most commonly mutated genes in clear cell renal c... more BAP1 (10-15%) and PBRM1 (40-50%) are two of the most commonly mutated genes in clear cell renal cell carcinoma (ccRCC). The goal of this study is to determine the prognostic significance of PBRM1 and BAP1 expression in ccRCC. We utilized immunohistochemistry (IHC) to assess PBRM1 protein expression in 1479 primary ccRCC tumors we have previously stained for BAP1. A centralized pathologist reviewed all cases and categorized tumors as positive, or deficient for PBRM1 and BAP1. Kaplan-Meier and Cox regression models were used to evaluate association of PBRM1 and BAP1 expression with risk of death from RCC and risk of metastases after adjustment for age and the Mayo SSIGN score (i.e. stage, size, grade, and necrosis). Tumor expression of PBRM1 and BAP1 was as follows: PBRM1+BAP1+ (40.1%), PBRM1-BAP1+ (48.6%), PBRM1+BAP1- (8.7%), and PBRM1-BAP1- (1.8%). Loss of PBRM1 and BAP1 in the same tumor was significantly lower than expected (1.8% actual versus 5.3% expected, p<0.0001). Compared to patients with PBRM1+BAP1+ tumors, those with PBRM1-BAP1+ were more likely to experience RCC death (HR 1.39, p=0.035), followed by PBRM1+BAP1- (HR 3.25, p<0.001) and PBRM1-BAP1- (HR=5.2, p<0.001). PBRM1 and BAP1 expression did not add independent prognostic information to the Mayo SSIGN score. PBRM1 and BAP1 expression identifies four clinical subgroups of ccRCC patients with divergent clinical outcomes. The clinical value of these biomarkers will be fully realized when therapies targeting pathways downstream of PBRM1 and BAP1 are developed.
Angiogenesis plays a role in tumor growth and is partly mediated by factors in both the fibroblas... more Angiogenesis plays a role in tumor growth and is partly mediated by factors in both the fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) pathways. Durable clinical responses with VEGF tyrosine kinase inhibitors (TKIs) may be limited by intrinsic tumor resistance. We hypothesized that FGF signaling may impact clinical responses to sorafenib. Nephrectomy material was available from 40 patients with metastatic renal cell carcinoma (RCC) enrolled in a phase II clinical trial of sorafenib ± interferon (ClinicalTrials.gov Identifier NCT00126594). Fibroblast growth factor receptor 1 (FGFR1) and fibroblast growth factor receptor substrate 2 alpha (FRS2α) expression was assessed by in situ hybridization and immunofluorescence, respectively. The relationship between fibroblast growth factor pathway marker levels and progression-free survival (PFS) was analyzed using Kaplan-Meier and Cox proportional hazards regression methods. Univariate analysis indicated that mor...
Journal of the National Comprehensive Cancer Network : JNCCN, 2014
Hereditary forms of renal cell carcinoma (RCC) have yielded clues regarding the molecular pathoge... more Hereditary forms of renal cell carcinoma (RCC) have yielded clues regarding the molecular pathogenesis of sporadic RCC. The discovery of germline mutations in chromatin-modulating enzymes also defined a new hereditary RCC syndrome. Although histologically distinct RCC subtypes exist, emerging themes shared between hereditary and sporadic RCC include dysregulation of the von Hippel-Lindau tumor suppressor protein/hypoxia inducible factor axis, defective ciliogenesis, and aberrant tumor metabolism. This article describes the most common hereditary RCC syndromes and associated extrarenal manifestations. Recent evidence supports developing screening guidelines for early-onset RCC to identify persons with germline mutations in the absence of secondary clinical manifestations.
Few data on the perioperative outcomes of cystectomy after neoadjuvant chemotherapy (NAC) exist. ... more Few data on the perioperative outcomes of cystectomy after neoadjuvant chemotherapy (NAC) exist. In this study, we evaluated whether patients who had previously received NAC were at higher risk of developing perioperative complications. The National Surgical Quality Improvement Program (NSQIP) database was searched to identify cystectomies performed between January 1, 2005 and December 31, 2011. Of 1394 patients identified, about one-tenth (n = 122 [8.8%]) received NAC. A propensity-weighted comparative analysis of perioperative morbidity was conducted. In unadjusted comparisons, patients undergoing cystectomy after NAC were more likely to have peripheral nerve deficits (1.6% [2/122] versus 0.2% [3/1272]; p = .01), blood transfusions (37.7% [46/122] versus 27.5% [350/1272]; p = .02), and unplanned readmissions (11.5% [14/122] versus 6.6% [84/1272]; p = .04), but were less likely to require hospitalization longer than 8 days (45.1% [55/122] versus 58.8% [748/1272]; p = .01). Propensi...
Clear-cell renal cell cancer (CRCC) is initiated typically by loss of the tumor-suppressor VHL, d... more Clear-cell renal cell cancer (CRCC) is initiated typically by loss of the tumor-suppressor VHL, driving constitutive activation of hypoxia-inducible factor-1 (HIF1) and HIF2. However, whereas HIF1 has a tumor-suppressor role, HIF2 plays a distinct role in driving CRCC. In this study, we show that the HIF1α E3 ligase hypoxia-associated factor (HAF) complexes with HIF2α at DNA to promote HIF2-dependent transcription through a mechanism relying upon HAF SUMOylation. HAF SUMOylation was induced by hypoxia, whereas HAF-mediated HIF1α degradation was SUMOylation independent. HAF overexpression in mice increased CRCC growth and metastasis. Clinically, HAF overexpression was associated with poor prognosis. Taken together, our results show that HAF is a specific mediator of HIF2 activation that is critical for CRCC development and morbidity.
Posttranslational modifications (PTMs) of tubulin specify microtubules for specialized cellular f... more Posttranslational modifications (PTMs) of tubulin specify microtubules for specialized cellular functions and comprise what is termed a "tubulin code." PTMs of histones comprise an analogous "histone code," although the "readers, writers, and erasers" of the cytoskeleton and epigenome have heretofore been distinct. We show that methylation is a PTM of dynamic microtubules and that the histone methyltransferase SET-domain-containing 2 (SETD2), which is responsible for H3 lysine 36 trimethylation (H3K36me3) of histones, also methylates α-tubulin at lysine 40, the same lysine that is marked by acetylation on microtubules. Methylation of microtubules occurs during mitosis and cytokinesis and can be ablated by SETD2 deletion, which causes mitotic spindle and cytokinesis defects, micronuclei, and polyploidy. These data now identify SETD2 as a dual-function methyltransferase for both chromatin and the cytoskeleton and show a requirement for methylation in maintenance of genomic stability and the integrity of both the tubulin and histone codes.
Clinical cancer research : an official journal of the American Association for Cancer Research, Jan 2, 2016
DNA methylation is a heritable covalent modification that is developmentally regulated and is cri... more DNA methylation is a heritable covalent modification that is developmentally regulated and is critical in tissue-type definition. Although genotype-phenotype correlations have been described for different subtypes of renal cell carcinoma (RCC), it is unknown if DNA methylation profiles correlate with morphological or ontology based phenotypes. Here we test the hypothesis that DNA methylation signatures can discriminate between putative precursor cells in the nephron. We performed deep profiling of DNA methylation and transcriptome in diverse histopathological RCC subtypes and validated DNA methylation in an independent dataset as well as in The Cancer Genome Atlas Clear Cell and Chromophobe Renal Cell Carcinoma Datasets. Our data provide the first mapping of methylome epi-signature and indicates that RCC subtypes can be grouped into two major epi-clusters: C1 which encompasses clear-cell RCC, papillary RCC, mucinous and spindle cell carcinomas and translocation RCC; C2 which compris...
Over 90% of chondroblastomas contain a heterozygous mutation replacing lysine 36 with methionine ... more Over 90% of chondroblastomas contain a heterozygous mutation replacing lysine 36 with methionine (K36M) in the histone H3 variant H3.3. Here, we show that H3K36 methylation is reduced globally in chondroblastomas and in chondrocytes harboring the same genetic mutation due to inhibition of at least two H3K36 methyltransferases, MMSET and SETD2, by the H3.3K36M mutant proteins. Genes with altered expression as well as H3K36 di- and tri-methylation in H3.3K36M cells are enriched in cancer pathways. In addition, H3.3K36M chondrocytes exhibit several hallmarks of cancer cells including increased ability to form colonies, resistance to apoptosis and defects in differentiation. Thus, H3.3K36M proteins reprogram H3K36 methylation landscape and contribute to tumorigenesis in part through altering the expression of cancer-associated genes.
Clear cell renal cell carcinomas (ccRCCs) harbor frequent mutations in epigenetic modifiers inclu... more Clear cell renal cell carcinomas (ccRCCs) harbor frequent mutations in epigenetic modifiers including SETD2, the H3K36me3 writer. We profiled DNA methylation (5mC) across the genome in cell line-based models of SETD2 inactivation and SETD2 mutant primary tumors because 5mC has been linked to H3K36me3 and is therapeutically targetable. SETD2 depleted cell line models (long-term and acute) exhibited a DNA hypermethylation phenotype coinciding with ectopic gains in H3K36me3 centered across intergenic regions adjacent to low expressing genes, which became upregulated upon dysregulation of the epigenome. Poised enhancers of developmental genes were prominent hypermethylation targets. SETD2 mutant primary ccRCCs, papillary renal cell carcinomas, and lung adenocarcinomas all demonstrated a DNA hypermethylation phenotype that segregated tumors by SETD2 genotype and advanced grade. These findings collectively demonstrate that SETD2 mutations drive tumorigenesis by coordinated disruption of t...
Thesis (Ph.D.)--Baylor College of Medicine. Dept. of Molecular and Cellular Biology, 2005. Includ... more Thesis (Ph.D.)--Baylor College of Medicine. Dept. of Molecular and Cellular Biology, 2005. Includes bibliographical references.
Thesis (Fellows Thesis)--Texas A & M University, 1998. Includes bibliographical references: p. 28... more Thesis (Fellows Thesis)--Texas A & M University, 1998. Includes bibliographical references: p. 28-29. "Major Subject: Biochemistry/Chemistry."
BAP1 (10-15%) and PBRM1 (40-50%) are two of the most commonly mutated genes in clear cell renal c... more BAP1 (10-15%) and PBRM1 (40-50%) are two of the most commonly mutated genes in clear cell renal cell carcinoma (ccRCC). The goal of this study is to determine the prognostic significance of PBRM1 and BAP1 expression in ccRCC. We utilized immunohistochemistry (IHC) to assess PBRM1 protein expression in 1479 primary ccRCC tumors we have previously stained for BAP1. A centralized pathologist reviewed all cases and categorized tumors as positive, or deficient for PBRM1 and BAP1. Kaplan-Meier and Cox regression models were used to evaluate association of PBRM1 and BAP1 expression with risk of death from RCC and risk of metastases after adjustment for age and the Mayo SSIGN score (i.e. stage, size, grade, and necrosis). Tumor expression of PBRM1 and BAP1 was as follows: PBRM1+BAP1+ (40.1%), PBRM1-BAP1+ (48.6%), PBRM1+BAP1- (8.7%), and PBRM1-BAP1- (1.8%). Loss of PBRM1 and BAP1 in the same tumor was significantly lower than expected (1.8% actual versus 5.3% expected, p<0.0001). Compared to patients with PBRM1+BAP1+ tumors, those with PBRM1-BAP1+ were more likely to experience RCC death (HR 1.39, p=0.035), followed by PBRM1+BAP1- (HR 3.25, p<0.001) and PBRM1-BAP1- (HR=5.2, p<0.001). PBRM1 and BAP1 expression did not add independent prognostic information to the Mayo SSIGN score. PBRM1 and BAP1 expression identifies four clinical subgroups of ccRCC patients with divergent clinical outcomes. The clinical value of these biomarkers will be fully realized when therapies targeting pathways downstream of PBRM1 and BAP1 are developed.
Angiogenesis plays a role in tumor growth and is partly mediated by factors in both the fibroblas... more Angiogenesis plays a role in tumor growth and is partly mediated by factors in both the fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) pathways. Durable clinical responses with VEGF tyrosine kinase inhibitors (TKIs) may be limited by intrinsic tumor resistance. We hypothesized that FGF signaling may impact clinical responses to sorafenib. Nephrectomy material was available from 40 patients with metastatic renal cell carcinoma (RCC) enrolled in a phase II clinical trial of sorafenib ± interferon (ClinicalTrials.gov Identifier NCT00126594). Fibroblast growth factor receptor 1 (FGFR1) and fibroblast growth factor receptor substrate 2 alpha (FRS2α) expression was assessed by in situ hybridization and immunofluorescence, respectively. The relationship between fibroblast growth factor pathway marker levels and progression-free survival (PFS) was analyzed using Kaplan-Meier and Cox proportional hazards regression methods. Univariate analysis indicated that mor...
Journal of the National Comprehensive Cancer Network : JNCCN, 2014
Hereditary forms of renal cell carcinoma (RCC) have yielded clues regarding the molecular pathoge... more Hereditary forms of renal cell carcinoma (RCC) have yielded clues regarding the molecular pathogenesis of sporadic RCC. The discovery of germline mutations in chromatin-modulating enzymes also defined a new hereditary RCC syndrome. Although histologically distinct RCC subtypes exist, emerging themes shared between hereditary and sporadic RCC include dysregulation of the von Hippel-Lindau tumor suppressor protein/hypoxia inducible factor axis, defective ciliogenesis, and aberrant tumor metabolism. This article describes the most common hereditary RCC syndromes and associated extrarenal manifestations. Recent evidence supports developing screening guidelines for early-onset RCC to identify persons with germline mutations in the absence of secondary clinical manifestations.
Few data on the perioperative outcomes of cystectomy after neoadjuvant chemotherapy (NAC) exist. ... more Few data on the perioperative outcomes of cystectomy after neoadjuvant chemotherapy (NAC) exist. In this study, we evaluated whether patients who had previously received NAC were at higher risk of developing perioperative complications. The National Surgical Quality Improvement Program (NSQIP) database was searched to identify cystectomies performed between January 1, 2005 and December 31, 2011. Of 1394 patients identified, about one-tenth (n = 122 [8.8%]) received NAC. A propensity-weighted comparative analysis of perioperative morbidity was conducted. In unadjusted comparisons, patients undergoing cystectomy after NAC were more likely to have peripheral nerve deficits (1.6% [2/122] versus 0.2% [3/1272]; p = .01), blood transfusions (37.7% [46/122] versus 27.5% [350/1272]; p = .02), and unplanned readmissions (11.5% [14/122] versus 6.6% [84/1272]; p = .04), but were less likely to require hospitalization longer than 8 days (45.1% [55/122] versus 58.8% [748/1272]; p = .01). Propensi...
Clear-cell renal cell cancer (CRCC) is initiated typically by loss of the tumor-suppressor VHL, d... more Clear-cell renal cell cancer (CRCC) is initiated typically by loss of the tumor-suppressor VHL, driving constitutive activation of hypoxia-inducible factor-1 (HIF1) and HIF2. However, whereas HIF1 has a tumor-suppressor role, HIF2 plays a distinct role in driving CRCC. In this study, we show that the HIF1α E3 ligase hypoxia-associated factor (HAF) complexes with HIF2α at DNA to promote HIF2-dependent transcription through a mechanism relying upon HAF SUMOylation. HAF SUMOylation was induced by hypoxia, whereas HAF-mediated HIF1α degradation was SUMOylation independent. HAF overexpression in mice increased CRCC growth and metastasis. Clinically, HAF overexpression was associated with poor prognosis. Taken together, our results show that HAF is a specific mediator of HIF2 activation that is critical for CRCC development and morbidity.
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