DIPG-76. HISTONE H3 PHOSPHORYLATION IN H3K27M MIDLINE GLIOMAS

Abstract Diffuse midline gliomas (DMG) patients have a dire prognosis despite radiation therapy and there is an urgent need to develop more effective treatments. DMG are characterized by heterozygous mutations in select H3 genes resulting in the replacement of lysine 27 by methionine (K27M) that leads to global epigenetic reprogramming and drives tumorigenesis. We previously reported that pharmacological inhibition of aurora kinase (AKI) may represent a targeted approach for treating tumors with this mutation. Our analysis with both published dataset and patient samples showed that patients with higher aurora kinase A (AKA) expression were associated with worse survival. AKA phosphorylates H3S10 and H3S28 during mitosis. Intriguingly, phosphorylation of the H3S28 (H3S28ph) by AKA blocks PRC2 methyltransferase activity and decreases global H3K27me3 in certain stem cells. We propose that a similar mechanism occurs in H3K27M DMG tumors, where there is a reciprocal relationship between H3S28ph and H3K27me3. We found that AKI significantly decreases H3S28ph while increasing H3K27me3 specifically in H3K27M tumors. To further evaluate the link between the H3K27M mutation and H3 serine phosphorylation, we used CRISPR/Cas9-directed gene editing to silence H3S28ph by replacing serine with alanine (H3S28A) in DIPG cell lines. Ectopic expression of histone H3S28A leads to a prominent epigenetic changes in H3K27M tumors and is similar to AKA inhibition. Overall, this study highlights H3S28ph, one of the targets of AK, is a key driver of epigenetic changes in H3K27M tumors through both direct and indirect changes to H3K27me3 and H3K27ac across the genome.

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DIPG-78. REVERTANCE OF THE H3K27M MUTATION RESCUES CHROMATIN MARKS NECESSARY FOR ONCOGENESIS IN DIFFUSE MIDLINE GLIOMA

Neuro-Oncology ◽

10.1093/neuonc/noaa222.120 ◽

2020 ◽

Vol 22 (Supplement_3) ◽

pp. iii302-iii302

Author(s):

Cody Nesvick ◽

Charles Day ◽

Liang Zhang ◽

Edward Hinchcliffe ◽

David Daniels

Keyword(s):

Critical Role ◽

Aurora Kinase ◽

Epigenetic Changes ◽

Methionine Residue ◽

H3 Phosphorylation ◽

Diffuse Midline Glioma ◽

H3k27m Mutation

Abstract Diffuse midline glioma (DMG) is a lethal brain tumor that typically occurs in children. Numerous studies have demonstrated the central role of the H3K27M mutation and secondary loss of H3K27 trimethylation (H3K27me3) in DMG tumorigenesis. Understanding how the H3K27M mutation alters the epigenetic landscape of the cell is necessary for revealing molecular targets that are critical to tumorigenesis. To investigate the epigenetic effects of H3K27M mutation in DMG, we developed revertant DMG cell lines with the mutant methionine residue reverted to wildtype (i.e., M27K). Revertant cells were analyzed for epigenetic changes and phenotypic differences in vitro and in vivo. H3M27K DMG cells grew in culture but displayed diminished proliferative capacity. H3M27K cells demonstrated total loss of H3K27M expression and restored trimethylation of H3K27 and H3K4. Furthermore, consistent with the hypothesis that the H3K27M mutation impacts H3 phosphorylation via expression of Aurora Kinase during mitosis, H3M27K cells demonstrated reduced expression of both Aurora Kinase A and phosphorylation of H3 serine residues 10 and 28. In line with the critical role of H3S10 phosphorylation in chromatin segregation, H3M27K cells also demonstrated restored chromosome segregation compared to H3K27M cells. In vivo data will be discussed. Revertance of the H3K27M mutation reduces tumorigenesis in DMG tumors. Isogenic H3M27K cells display reversal of key epigenetic changes associated with oncogenesis in DMG. The revertant H3M27K DMG model is a useful tool to investigate the downstream epigenetic reprogramming specific to H3K27M mutation in these tumors.

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Basal aurora kinase B activity is sufficient for histone H3 phosphorylation in prophase

Biology Open ◽

10.1242/bio.20133079 ◽

2013 ◽

Vol 2 (4) ◽

pp. 379-386 ◽

Cited By ~ 15

Author(s):

L.-T.-T. Le ◽

H.-L. Vu ◽

C.-H. Nguyen ◽

A. Molla

Keyword(s):

Histone H3 ◽

Aurora Kinase ◽

Aurora Kinase B ◽

H3 Phosphorylation ◽

Histone H3 Phosphorylation

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Aurora kinase-A regulates microtubule organizing center (MTOC) localization, chromosome dynamics, and histone-H3 phosphorylation in mouse oocytes

Molecular Reproduction and Development ◽

10.1002/mrd.21272 ◽

2011 ◽

Vol 78 (2) ◽

pp. 80-90 ◽

Cited By ~ 29

Author(s):

Jun Ding ◽

Jason E. Swain ◽

Gary D. Smith

Keyword(s):

Histone H3 ◽

Aurora Kinase ◽

Microtubule Organizing Center ◽

Aurora Kinase A ◽

Chromosome Dynamics ◽

Mouse Oocytes ◽

H3 Phosphorylation ◽

Organizing Center ◽

Histone H3 Phosphorylation ◽

Kinase A

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O-Linked N-Acetylglucosamine Cycling Regulates Mitotic Spindle Organization

Journal of Biological Chemistry ◽

10.1074/jbc.m113.470187 ◽

2013 ◽

Vol 288 (38) ◽

pp. 27085-27099 ◽

Cited By ~ 24

Author(s):

Ee Phie Tan ◽

Sarah Caro ◽

Anish Potnis ◽

Christopher Lanza ◽

Chad Slawson

Keyword(s):

Mitotic Spindle ◽

Aurora Kinase ◽

Post Translational Modification ◽

Gain Of Function ◽

Aurora Kinase B ◽

H3 Phosphorylation ◽

Cytoplasmic Proteins ◽

New Perspective ◽

Glcnac Transferase ◽

Histone H3 Phosphorylation

Any defects in the correct formation of the mitotic spindle will lead to chromosomal segregation errors, mitotic arrest, or aneuploidy. We demonstrate that O-linked N-acetylglucosamine (O-GlcNAc), a post-translational modification of serine and threonine residues in nuclear and cytoplasmic proteins, regulates spindle function. In O-GlcNAc transferase or O-GlcNAcase gain of function cells, the mitotic spindle is incorrectly assembled. Chromosome condensation and centrosome assembly is impaired in these cells. The disruption in spindle architecture is due to a reduction in histone H3 phosphorylation by Aurora kinase B. However, gain of function cells treated with the O-GlcNAcase inhibitor Thiamet-G restored the assembly of the spindle and partially rescued histone phosphorylation. Together, these data suggest that the coordinated addition and removal of O-GlcNAc, termed O-GlcNAc cycling, regulates mitotic spindle organization and provides a potential new perspective on how O-GlcNAc regulates cellular events.

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Co-relation with novel phosphorylation sites of IκBα and necroptosis in breast cancer cells

BMC Cancer ◽

10.1186/s12885-021-08304-7 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Sung Hoon Choi ◽

Hee-Sub Yoon ◽

Shin-Ae Yoo ◽

Sung Ho Yun ◽

Joo-Hee Park ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Breast Cancer Cells ◽

Phosphorylation Site ◽

Aurora Kinase ◽

Serine Phosphorylation ◽

Flight Mass Spectrometry ◽

Iκb Kinase ◽

Orbitrap Mass Spectrometer ◽

Nf Kappab

Abstract Background Phosphorylation of NF-kappaB inhibitor alpha (IκBα) is key to regulation of NF-κB transcription factor activity in the cell. Several sites of IκBα phosphorylation by members of the IκB kinase family have been identified, but phosphorylation of the protein by other kinases remains poorly understood. We investigated a new phosphorylation site on IκBα and identified its biological function in breast cancer cells. Methods Previously, we observed that aurora kinase (AURK) binds IκBα in the cell. To identify the domains of IκBα essential for phosphorylation by AURK, we performed kinase assays with a series of IκBα truncation mutants. AURK significantly promoted activation of IκBα at serine 32 but not serine 36; by contrast, IκB kinase (IKK) family proteins activated both of these residues. We also confirmed phosphorylation of IκBα by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) and nano-liquid chromatography hybrid quadrupole orbitrap mass spectrometer (nanoLC-MS/MS; Q-Exactive). Results We identified two novel sites of serine phosphorylation, S63 and S262. Alanine substitution of S63 and S262 (S63A and S262A) of IκBα inhibited proliferation and suppressed p65 transcription activity. In addition, S63A and/or S262A of IκBα regulated apoptotic and necroptotic effects in breast cancer cells. Conclusions Phosphorylation of IκBα by AURK at novel sites is related to the apoptosis and necroptosis pathways in breast cancer cells.

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