scholarly journals DIPG-39. NOVEL PROTEOMIC ANALYSIS REVEALS EPIGENETIC THERAPEUTIC TARGETS IN PEDIATRIC GLIOMA

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii294-iii294
Author(s):  
Daphne Li ◽  
Tina Huang ◽  
Jeannie Camarillo ◽  
Andrea Piunti ◽  
Jin Qi ◽  
...  
Keyword(s):  
Treatment Response ◽  
Cell Lines ◽  
Histone H3 ◽  
Therapy Response ◽  
Post Translational Modifications ◽  
Pediatric Glioma ◽  
Epigenetic Signatures ◽  
Monitoring Treatment ◽  
The Impact ◽  
H3k27m Mutation

Abstract INTRODUCTION Diffuse midline glioma is a highly morbid pediatric cancer. Up to 80% harbor Histone H3K27M mutation, which alters Histone H3 post-translational modifications (PTMs) and genomic enrichment patterns, affecting chromatin structure and transcription. We previously identified tumorigenic patterns of H3K27Ac/bromodomain co-enrichment and pre-clinical efficacy of bromodomain inhibition (JQ1) in DMG. Here, we employ a novel proteomics approach developed at our institution to further elucidate the impact of H3K27M mutation on glioma epigenetic signatures and treatment response. METHODS Epiproteomic analysis was performed on pediatric glioma cells (H3K27 WT n=2, H3K27M n=2) to characterize 95 distinct Histone H3 N-terminal tail modification states. Cells were treated with JQ1 or DMSO, and collected at 0h, 24h, 48h, Histones extracted from isolated nuclei and immunopurified, then analyzed by LC-MS/MS. Results were integrated with RNA-Seq and ChIP Seq (H3.3K27M, H3.3, H3K27Ac, H3K27me3, H3K4me1, H3K4me3) from the same cell lines. Pediatric glioma tissues (H3K27M WT n=3, H3K27M n= 9) were similarly analyzed to validate cell line results. RESULTS Cell PTM profiles cluster by H3 mutation status on unsupervised analysis. Significant differential PTM abundance and genomic enrichment H3K27M, H3.3 WT, H3K27Me3 and H3K27Ac was observed between mutant and wild type cell lines with epigenetic-targeted therapy, correlating with cell transcriptomes. CONCLUSIONS Histone H3 tail analysis reveals the effects of H3K27M mutation and bromodomain inhibition on the tumor epigenetic landscape, providing insight into mechanisms of tumorigenesis and therapy response. Further investigation of the utility of these signatures as biomarkers for diagnosis and monitoring treatment response are therefore underway.

scholarly journals PDTM-39. CRACKING THE HISTONE CODE: REVEALING THERAPEUTIC EPIGENETIC TARGETS THROUGH HISTONE H3 TAIL ANALYSIS OF DIFFUSE INTRINSIC PONTINE GLIOMA

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi196-vi196
Author(s):  
Daphne Li ◽  
Tina Huang ◽  
Jeannie Camarillo ◽  
Jin Qi ◽  
Hannah Weiss ◽  
...  
Keyword(s):  
Cell Lines ◽  
Histone H3 ◽  
Diffuse Intrinsic Pontine Glioma ◽  
Tumor Formation ◽  
Wild Type ◽  
Pontine Glioma ◽  
Pediatric Glioma ◽  
Mutation Status ◽  
Histone Codes ◽  
The Impact

Abstract INTRODUCTION Diffuse intrinsic pontine glioma (DIPG) is a highly morbid pediatric cancer. Up to 80% harbor a Histone H3K27M mutation, which alters wild type Histone H3 protein post-translational modifications (PTMs) and genomic enrichment patterns to impact chromatin structure and transcription regulation. We previously identified tumorigenic patterns of H3K27Ac/bromodomain co-enrichment in DIPG, and demonstrated pre-clinical efficacy of bromodomain inhibition (JQ1). Here, we employ a novel proteomics platform, developed at our institution, to further elucidate the impact of H3K7M mutation on glioma histone codes and response to bromodomain inhibition. METHODS Epiproteomic analysis was performed on pediatric glioma cell lines (H3K27 WT n=2, H3K27M n=2) to characterize 95 distinct Histone H3.3 and H3.1 N-terminal tail modification states. Cells were treated with JQ1 or DMSO, and collected at 0h, 24h, 48h. Histones were extracted from isolated nuclei, immunopurified, and analyzed by LC-MS/MS. Results were integrated with RNA-Seq and ChIP Seq results (H3K27M, H3K27Ac, H3K27me3, H3K4me1, H3K4me3) from the same DIPG cell lines. Pediatric glioma tissues (H3K27M WT n=3, H3K27M n= 9) were similarly analyzed to validate cell line results. RESULTS Cell PTM profiles cluster by H3 mutation status on unsupervised analysis. Relative H3 PTM abundance were compared across cell lines by tumor location, H3 mutation status, and in response to treatment. Significant differential genomic enrichment H3K27M and H3.3 WT proteins, H3K27Me3 and H3K27Ac were observed between mutant and wild type cell lines with epigenetic-targeted therapy, correlating with cell transcriptomes. CONCLUSIONS Histone H3 tail epiproteomic analysis reveals DIPG histone codes in situ, revealing the effects of bromodomain inhibition on the tumor epigenetic landscape and providing new insight to the mechanism of tumor formation and therapy response. Further investigation of the utility of these signatures as biomarkers for diagnosis and longitudinal monitoring of treatment response are therefore currently underway.

scholarly journals Histone tail analysis reveals H3K36me2 and H4K16ac as epigenetic signatures of diffuse intrinsic pontine glioma

2020 ◽  
Author(s):  
Shejuan An ◽  
Jeannie Camarillo ◽  
Tina Huang ◽  
Daphne Li ◽  
Juliette Morris ◽  
...  
Keyword(s):  
Cell Lines ◽  
Radiation Treatment ◽  
Diffuse Intrinsic Pontine Glioma ◽  
Response To Treatment ◽  
Wild Type ◽  
Post Translational Modification ◽  
Pontine Glioma ◽  
Epigenetic Signatures ◽  
The Impact

Abstract Background: Diffuse intrinsic pontine glioma (DIPG) is an aggressive pediatric brainstem tumor. Most DIPGs harbor a histone H3 mutation, which alters histone post-translational modification (PTM) states and transcription. Here, we employed quantitative proteomic analysis to elucidate the impact of the H3.3K27M mutation, as well as radiation and bromodomain inhibition (BRDi) with JQ1, on DIPG PTM profiles.Methods: We performed targeted mass spectrometry on H3.3K27M mutant and wild-type tissues (n=12) and cell lines (n=7).Results: We found 29.2% and 26.4% of total H3.3K27 peptides were H3.3K27M in mutant DIPG tumor cell lines and tissue specimens, respectively. Significant differences in modification states were observed in H3.3K27M specimens, including at H3K27, H3K36, and H4K16. In addition, H3.3K27me1 and H4K16ac were the most significantly distinct modifications in H3.3K27M mutant tumors, relative to wild-type. Further, H3.3K36me2 was the most abundant co-occurring modification on the H3.3K27M mutant peptide in DIPG tissue, while H4K16ac was the most acetylated residue. Radiation treatment caused changes in PTM abundance in vitro, including increased H3K9me3. JQ1 treatment resulted in increased mono- and di-methylation of H3.1K27, H3.3K27, H3.3K36 and H4K20 in vitro. Conclusion: Taken together, our findings provide insight into the effects of the H3K27M mutation on histone modification states and response to treatment, and suggest that H3K36me2 and H4K16ac may represent unique tumor epigenetic signatures for targeted DIPG therapy.

scholarly journals Histone tail analysis reveals H3K36me2 and H4K16ac as epigenetic signatures of diffuse intrinsic pontine glioma

2020 ◽  
Vol 39 (1) ◽  
Author(s):  
Shejuan An ◽  
Jeannie M. Camarillo ◽  
Tina Yi-Ting Huang ◽  
Daphne Li ◽  
Juliette A. Morris ◽  
...  
Keyword(s):  
Cell Lines ◽  
Radiation Treatment ◽  
Diffuse Intrinsic Pontine Glioma ◽  
Response To Treatment ◽  
Wild Type ◽  
Post Translational Modification ◽  
Pontine Glioma ◽  
Epigenetic Signatures ◽  
The Impact

Abstract Background Diffuse intrinsic pontine glioma (DIPG) is an aggressive pediatric brainstem tumor. Most DIPGs harbor a histone H3 mutation, which alters histone post-translational modification (PTM) states and transcription. Here, we employed quantitative proteomic analysis to elucidate the impact of the H3.3K27M mutation, as well as radiation and bromodomain inhibition (BRDi) with JQ1, on DIPG PTM profiles. Methods We performed targeted mass spectrometry on H3.3K27M mutant and wild-type tissues (n = 12) and cell lines (n = 7). Results We found 29.2 and 26.4% of total H3.3K27 peptides were H3.3K27M in mutant DIPG tumor cell lines and tissue specimens, respectively. Significant differences in modification states were observed in H3.3K27M specimens, including at H3K27, H3K36, and H4K16. In addition, H3.3K27me1 and H4K16ac were the most significantly distinct modifications in H3.3K27M mutant tumors, relative to wild-type. Further, H3.3K36me2 was the most abundant co-occurring modification on the H3.3K27M mutant peptide in DIPG tissue, while H4K16ac was the most acetylated residue. Radiation treatment caused changes in PTM abundance in vitro, including increased H3K9me3. JQ1 treatment resulted in increased mono- and di-methylation of H3.1K27, H3.3K27, H3.3K36 and H4K20 in vitro. Conclusion Taken together, our findings provide insight into the effects of the H3K27M mutation on histone modification states and response to treatment, and suggest that H3K36me2 and H4K16ac may represent unique tumor epigenetic signatures for targeted DIPG therapy.

scholarly journals PDTB-14. INVESTIGATING HISTONE H3 POST-TRANSLATIONAL MODIFICATIONS USING PARAFFIN-EMBEDDED PEDIATRIC GLIOMA TISSUE SAMPLES

2016 ◽  
Vol 18 (suppl_6) ◽  
pp. vi152-vi153
Author(s):  
Amanda Saratsis ◽  
Tina Huang ◽  
Andrea Piunti ◽  
Rintaro Hashizume ◽  
Elizabeth Bartom ◽  
...  
Keyword(s):  
Histone H3 ◽  
Glioma Tissue ◽  
Tissue Samples ◽  
Post Translational Modifications ◽  
Pediatric Glioma

scholarly journals Histone tail analysis reveals H3K36me2 and H4K16ac as epigenetic signatures of diffuse intrinsic pontine glioma

2020 ◽  
Author(s):  
Shejuan An ◽  
Jeannie Camarillo ◽  
Tina Huang ◽  
Daphne Li ◽  
Juliette Morris ◽  
...  
Keyword(s):  
Cell Lines ◽  
Radiation Treatment ◽  
Diffuse Intrinsic Pontine Glioma ◽  
Response To Treatment ◽  
Wild Type ◽  
Post Translational Modification ◽  
Pontine Glioma ◽  
Epigenetic Signatures ◽  
The Impact

Abstract Background: Diffuse intrinsic pontine glioma (DIPG) is an aggressive pediatric brainstem tumor. Most DIPGs harbor a histone H3 mutation, which alters histone post-translational modification (PTM) states and transcription. Here, we employed quantitative proteomic analysis to elucidate the impact of H3.3K27M mutation, as well as radiation and bromodomain inhibition (BRDi) with JQ1, on DIPG PTM profiles. Methods: We performed targeted mass spectroscopy on H3.3K27M mutant and wild-type tissues (n=12) and cell lines (n=7). Results: We found 29.2% and 26.4% of total H3.3K27 peptides were H3.3K27M in mutant DIPG tumor cell lines and tissue specimens, respectively. Significant differences in distinct PTMs were observed in H3.3K27M specimens, including at H3K27, H3K36, and H4K16 amino acid residues. In addition, H3.3K27me1 and H4K16ac were the most significantly distinct modifications in H3.3K27M mutant tumors relative to wild-type. Further, H3.3K36me2 was the most abundant modification co-occurring on the H3.3K27M mutant peptide in DIPG tissue, while H4K16ac was the most acetylated residue. Radiation treatment caused changes in PTM abundance in vitro , including increased H3K9me3. BRDi with JQ1 resulted in increased mono- and di-methylation of H3.1K27, H3.3K27, H3.3K36 and H4K20 in vitro . Conclusion: Taken together, our findings provide insight into the effects of the H3K27M mutation on Histone modification states and response to treatment, and suggest H3K36me2 and H4K16ac in DIPG may represent unique tumor epigenetic signatures for targeted therapy.

scholarly journals Histone Tail Analysis Reveals H3K36me2 And H4K16ac As Epigenetic Signatures of Diffuse Intrinsic Pontine Glioma

2020 ◽  
Author(s):  
Shejuan An ◽  
Jeannie Camarillo ◽  
Tina Huang ◽  
Daphne Li ◽  
Juliette Morris ◽  
...  
Keyword(s):  
Cell Lines ◽  
Radiation Treatment ◽  
Diffuse Intrinsic Pontine Glioma ◽  
Response To Treatment ◽  
Wild Type ◽  
Post Translational Modification ◽  
Pontine Glioma ◽  
Epigenetic Signatures ◽  
The Impact

Abstract Background: Diffuse intrinsic pontine glioma (DIPG) is an aggressive pediatric brainstem tumor. Most DIPGs harbor a histone H3 mutation, which alters histone post-translational modification (PTM) states and transcription. Here, we employed quantitative proteomic analysis to elucidate the impact of H3.3K27M mutation, as well as radiation and bromodomain inhibition (BRDi) with JQ1, on DIPG PTM profiles.Methods: We performed targeted mass spectroscopy on H3.3K27M mutant and wild-type tissues (n=12) and cell lines (n=7).Results: We found 29.2% and 26.4% of total H3.3K27 peptides were H3.3K27M in mutant DIPG tumor cell lines and tissue specimens, respectively. Significant differences in distinct PTMs were observed in H3.3K27M specimens, including at H3K27, H3K36, and H4K16 amino acid residues. In addition, H3.3K27me1 and H4K16ac were the most significantly distinct modifications in H3.3K27M mutant tumors relative to wild-type. Further, H3.3K36me2 was the most abundant modification co-occurring on the H3.3K27M mutant peptide in DIPG tissue, while H4K16ac was the most acetylated residue. Radiation treatment caused changes in PTM abundance in vitro, including increased H3K9me3. BRDi with JQ1 resulted in increased mono- and di-methylation of H3.1K27, H3.3K27, H3.3K36 and H4K20 in vitro. Conclusion: Taken together, our findings provide insight into the effects of the H3K27M mutation on Histone modification states and response to treatment, and suggest H3K36me2 and H4K16ac in DIPG may represent unique tumor epigenetic signatures for targeted therapy.

scholarly journals CBMT-16. EGFRvIII EXPRESSION CONFERS CHEMOSENSITIVITY BY INCREASING DNA MISMATCH REPAIR PROTEIN EXPRESSION AND REPLICATION STRESS

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi36-vi36
Author(s):  
Nina Struve ◽  
Zev A Binder ◽  
Lucy F Stead ◽  
Tim Brend ◽  
Stephen J Bagley ◽  
...  
Keyword(s):  
Dna Damage ◽  
Protein Expression ◽  
Treatment Response ◽  
Mismatch Repair ◽  
Cell Lines ◽  
Dna Mismatch Repair ◽  
Replication Stress ◽  
Dna Mismatch ◽  
Mgmt Promoter ◽  
The Impact

Abstract MGMT promoter methylation is the only accepted biomarker with prognostic role in GBM but its routine implementation is limited partly response to TMZ is heterogeneous, but also due to lack of effective alternative treatment options. Therefore, additional biomarkers are needed to enable better prediction of survival and to improve individualized treatment of GBM patients. A potential new biomarker is the epidermal growth factor receptor variant III (EGFRvIII). This constitutively activated deletion variant is present in approximately one third of all IDH wildtype GBM, but its relevance to treatment response is poorly understood. The aim of the present study was to analyze the impact of endogenous EGFRvIII expression on chemosensitivity and the mechanisms underlying any differential treatment response. EGFRvIII expression was associated with prolonged median overall survival but only for GBM patients with MGMT promoter methylated tumors. In line with this, we observed increased TMZ sensitivity of EGFRvIII+ and MGMT promoter methylated cells, which translated into improved survival in xenograft experiments. The increased TMZ sensitivity was associated with an elevated DNA damage induction accompanied by an increased expression of DNA mismatch repair (MMR) proteins in EGFRvIII+ cell lines and EGFRvIII+ GBM patient samples. Subsequently, only a moderate reduction in MMR protein expression resulted in a dramatic TMZ resistance, suggesting that EGFRvIII expression specifically sensitized MGMT deficient cells to TMZ treatment by upregulating MMR. Furthermore, EGFRvIII expression in GBM cell lines was accompanied by increased DNA damage, replication fork slowing, stalling and enhanced origin firing, implying replication stress. Targeting of EGFRvIII-dependent replication stress by irinotecan led to hypersensitivity of EGFRvIII+ cells. Taken together this study illustrates that EGFRvIII-induced upregulation of MMR and replication stress increases chemosensitivity thereby highlighting the vulnerability of EGFRvIII+ GBM to available treatments. These important data may also guide the development of new and more effective personalized strategies.

scholarly journals Pharmacological Inhibition of Histone Demethylase JMJD3 Reduces Leukemia Cell Survival and Represses Production of the Cytokine CCL2 in MDS/AML

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5219-5219
Author(s):  
Yue Wei ◽  
Yu Jia ◽  
Hong Zheng ◽  
Hui Yang ◽  
Zachary S. Bohannan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Inflammatory Cytokines ◽  
Conflicts Of Interest ◽  
Histone H3 ◽  
Leukemia Cell ◽  
Normal Peripheral Blood ◽  
Inflammatory Genes ◽  
Pharmacological Inhibition ◽  
The Impact

Abstract Deregulation of pro-inflammation signals plays an important role in MDS and AML. We have previously demonstrated that the histone H3 demethylase JMJD3 (KDM6B), a key regulator of inflammatory genes, is significantly overexpressed in the bone marrow progenitor cell population of patients with MDS (Wei et al, 2013). GSK-J4, a novel selective inhibitor against JMJD3, has been shown to modulate pro-inflammatory signals and affect cell growth in T-ALL and glioma. We therefore sought to evaluate the effect of GSK-J4 in MDS and AML. First, we observed that GSK-J4 significantly reduces the survival of MDS and AML cell lines (MDS-L and TF1) by 70% at concentrations 1-3 uM. These doses have no obvious cytotoxic effect on primary normal peripheral blood cells. The repression of survival was associated with induction of apoptosis, cell cycle blockade, and inhibition of p38MAPK activation. Second, we evaluated the impact of GSK-J4 on histone H3 methylation and found that the compound did not affect overall histone H3K4 and K27 methylation, which is consistent with previous findings (Ntziachristos et al, 2014). To pinpoint functionally relevant targets of JMJD3 inhibition in AML/MDS, we sought to identify disease-relevant inflammatory cytokines because JMJD3 binds and modulates promoters of many pro-inflammatory genes, particularly cytokines. We analyzed 38 inflammatory cytokines in bone marrow plasma specimens of patients with MDS (N=37) using a Luminex-based cytokine array, which revealed that levels of the cytokines CCL2 (also called MCP-1) and CCL11 (also called eotaxin-1) are significantly elevated in patients compared to healthy controls by 700 and 330 fold respectively (p=0.002 and 0.04, respectively). Both CCL2 and 11 are c-c motif chemokines whose encoding genes are located in the human chromosome 17q cytokine gene cluster. Furthermore, compared to pre-treatment samples, CCL2 was significantly elevated in paired plasma samples that were collected after hypomethylating agent (HMA) treatment (2.5 fold increase, N=11 pairs, p=0.04). Luminex results were confirmed by cytokine-specific ELISA. Previous studies have shown that JMJD3 binds to CCL2 gene promoter in mouse macrophage cells (De Santa et al, 2009). Together these results implicate that CCL2 may be a relevant pro-inflammatory signal modulated by JMJD3 in MDS/ AML. We therefore sought to evaluate impact of JMJD3 inhibition on CCL2 production by MDS/AML cells. In MDS-L and TF1 cells, GSK-J4 significantly repressed RNA expression and cytokine release of CCL2. Furthermore, in HMA-resistant MDS-L and TF1 derivative cell lines, productions of CCL2 are elevated by 1.8 and 2.5 fold respectively compared to their parental lines. Treatment of HMA-resistant lines with GSK-J4 improved the anti-leukemia effect of decitabine and had a synergistic effect with HMA. In summary, we have demonstrated that pharmacological inhibition of JMJD3 by GSK-J4 has anti-leukemia effects in MDS/AML cells and has the potential to improve hypomethylation-based therapy in this disease. A possible mechanism for this effect is through the modulation of the inflammatory cytokine CCL2. To further evaluate this hypothesis, we will characterize the effect of JMJD3 and its inhibition on histone methylation of the CCL2 promoter in future work. Disclosures No relevant conflicts of interest to declare.

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