News Release
Title
Aberrant active cis-regulatory elements associated with downregulation of RET finger protein
overcome chemoresistance in glioblastoma
Key Points○RET Finger Protein and histone deacetylase1 make a complex which modulates H3K27
acetylation and cis-regulatory elements in glioma cells○RET Finger Protein knockdown disrupts the RFP-HDAC1 complex, and alters thedeterminants of oxidative stress and base excision repair○RET Finger Protein knockdown enhances temozolomide chemosensitivity both in vitro andin vivo in glioblastoma
Summary
Glioblastoma (GBM), the most common primary brain tumor, is the most aggressive humancancers, with a median survival rate of only 14.6 months. Temozolomide (TMZ) is the frontline
chemotherapeutic drug in GBM. Drug resistance is the predominant obstacle in TMZ therapy.
Drug resistance occurs via multiple pathways. Histone3 Lysin27 residue (H3K27)-acetylation
status regulates cis-regulatory elements, which increases the likelihood of gene transcription.
Histone deacetylase complex (HDAC)s deacetylate lysine residues on core histones, leading to adecrease in gene transcription. In cis-regulatory element regions, complexes with HDAC
repress histones by H3K27ac deacetylation. RET Finger Protein (RFP) is a protein that isexpressed in many kinds of cancer. RFP forms a protein complex with HDAC1. The disruptionof the RFP-HDAC1 complex has resulted in increased drug sensitivity in other cancers (Figure1).

(Figure 1)

A research teams led by Dr. Atsushi Natsume, Associate professor in the Department ofNeurosurgery (Dean: Kenji Kadomatsu, M.D., Ph.D.) in collaboration with Dr. Takuya Kato,Assistant Professor in the Department of Pathology in Kitasato University School of Medicine(Dean: Toshiyuki Miyashita, M.D., Ph.D.) and Drs. Atsushi Enomoto, Associate professor, andMasahide Takahashi, Professor in the Department of Pathology in Nagoya University School ofMedicine, Drs. Melissa Ranjit and Masaki Hirano studied the effects of downregulation of RFP
in glioma cells, and its combination with TMZ treatment. RFP depletion with TMZ treatment
showed a considerable decrease in glioma cell growth and increased the survival time ofintracranial tumor bearing mice. RFP depletion abated numerous cis-regulatory elements anddecreased the expression of genes with functions of apoptosis, mitosis, cell cycle and DNA
replication. The RNA for PARPBP, a protein that increases TMZ resistance, was also
diminished, while those of FOXO1 and TBP-2, both of which enhance oxidative stress, wereincreased. RFP plays an augmentative role in TMZ resistance by H3K27 deacetylation anddisorganization of cis-regulatory elements related to RFP.
RFP shows promise as a suitable target for an effective combination therapy with TMZ.

Research Background
New treatment strategies to improve glioblastoma patient survival have been a pressing
need in recent years. MGMT demethylation is a known factor in the resistance ofTemozolomide(TMZ), which is the mainstream drug. PARP1 also contributes to TMZ resistanceby the BER pathway. PARP-binding protein (PARPBP) is increased in various types of cancer.
(Figure 2)(Figure 2)
The RET Finger Protein and its role in various types of cancer has been explored in recentyears. Also known as TRIM27, RFP is a transcription factor that becomes oncogenic upon
fusion with RET tyrosine kinase. RFP is involved in cellular processes like cell growth andapoptosis, and is expressed in many types of cancer. RFP forms a tripartite complex along withHistone deacetylase 1(HDAC1) and NF-Y.

It was previously made apparent that the breakdown of the RFP-HDAC1 complex increases thechemosensitivity of cisplatin in other cancers. The RFP-HDAC1 complex binds to thethioredoxin binding protein-2 promoter, resulting in inhibited TBP-2 expression. Thioredoxin isa protein that lessens oxidative stress by scavenging Reactive oxygen species. Thioredoxin
binding protein-2 (TBP-2) binds to thioredoxin, leading to an increase in reactive oxygen
species and oxidative stress, and resulting in cell death. But the mechanism and effects of thedisruption of the RFP-HDAC1 complex in TMZ sensitivity in glioma remained to be
characterized. In this study, we determined whether RFP-KD causes TMZ sensitivity inTMZ-resistant glioma and whether the RFP-HDAC1 complex breakdown causes aberration of
H3K27ac- controlled cis- regulatory elements.
Research Results

RFP-KD increases the efficacy of TMZ in RFP-expressing glioma cells.
We performed cell viability assessments of high RFP-expressing TMZ-resistant glioma celllines T98, U87-MGMT and TGS-01 transfected with siRFP treated with TMZ. We found thatthe combination of siRFP and TMZ treatment yielded the lowest cell viability. The combined
treatment with siRFP and TMZ also had a consistent effect on the survival of mice with theU87-MGMT orthotropic xenograft (Figure 3).

RFP-KD impairs cis-regulatory-elements-mediated regulation of genes related to cell division,
the cell cycle, DNA replication and apoptosis
We located regions of enrichment (RoEs) with the marker H3K27ac of active cis-regulatory
elements via ChIP in U87-MGMT and T98 cells that had undergone RFP-KD. We determinedthe corresponding gene expression levels by RNA-seq. Upon GO analysis, we found thatRFP-KD upregulates apoptosis and downregulates the pathways for cell division, the cell cycle
and DNA replication in glioma cells. We also found that the gene expression levels andH3K27ac peaks of thioredoxin binding protein-2 (TBP-2, TXNIP) and FOXO1 were bothactivated upon RFP-KD, while that of PARPBP was significantly decreased (Figure 4).

RFP and PARPBP expression correlates with poor prognosis in patients with glioma
We analyzed the overall survival of 633 patients with glioma which was stratified byexpression of RFP [log2(RPKM + 0.001), cutoff = 10.39 (median)) by means of a Kaplan-Meierplot. Also, we found that PARPBP expression correlates with poor prognosis in patients withglioma log2(RPKM + 0.001), cutoff = 5.115 (median) through analysis via a Kaplan-Meier plot.
This shows that RFP and PARPBP expression correlates with poor prognosis in patients withglioma (Figure 5).

RFP-KD induces oxidative stress leading to apoptosis and inhibition of cell division.

U87-MGMT cells treated with siRFP and/or TMZ showed fluorescence in a CellROX Orange
assay that indicated an increase in oxidative stress. Cell cycle analysis suggested that siRFP
alone decreased the percentage of cells in the S and G2/M phase, and increased the percentageof cells in the G0/G1 phase in U87-MGMT cells. TMZ alone increased the percentage of cells inthe G2/M phase. TUNEL assay showed that the combination of siRFP and TMZ inducedapoptosis in U87-MGMT cells (Figure 6).

Figure 3. RFP-KD increases the efficacy of TMZ in RFP-expressing glioma cells.

Figure 4. RFP-KD impairs cis-regulatory-elements-mediated regulation of genes related to cell
division, the cell cycle, DNA replication and apoptosis

Figure 5. RFP and PARPBP expression correlates with poor prognosis in patients with gliomaApoptotic cells

Figure 6. RFP-KD induces oxidative stress leading to apoptosis and inhibition of cell division.

Research Summary and Future Perspective
Downregulation of RFP in glioma cells causes changes in histone H3K27 modification
patterns, which further leads to increase in oxidative stress levels, inhibition in cell divisionand cell cycle, apoptosis, and depletion of genes that abet TMZ resistance. The precise
mechanism by which the RFP-HDAC1 complex directly controls the “H3K27ac gain” andupregulated genes, but controls the “H3K27ac loss” and downregulated genes indirectly
remains to be explained. Although further studies on some of these changes are necessary, RFP
has proven to be a pertinent target in combined therapy with temozolomide in glioblastoma.
Publication
Melissa Ranjit, Masaki Hirano, Kosuke Aoki, Yusuke Okuno, Fumiharu Ohka, Akane
Yamamichi, Sachi Maeda, Kazuya Motomura, Keitaro Matsuo, Atsushi Enomoto, Yasushi Ino,
Tomoki Todo, Masahide Takahashi, Toshihiko Wakabayashi, Takuya Kato and Atsushi
Natsume. Aberrant active cis-regulatory elements associated with downregulation of RET
finger protein complex overcome chemoresistance in glioblastoma. Cell Reports, published
online on February 26, 2019.
DOI：10.1016/j.celrep.2019.01.109