A NR2E1‐interacting peptide of LSD1 inhibits the proliferation of brain tumour initiating cells

Abstract Objectives Elimination of brain tumour initiating cells (BTICs) is important for the good prognosis of malignant brain tumour treatment. To develop a novel strategy targeting BTICs, we studied NR2E1(TLX) involved self‐renewal mechanism of BTICs and explored the intervention means. Materials and Methods NR2E1 and its interacting protein‐LSD1 in BTICs were studied by gene interference combined with cell growth, tumour sphere formation, co‐immunoprecipitation and chromatin immunoprecipitation assays. NR2E1 interacting peptide of LSD1 was identified by Amide Hydrogen/Deuterium Exchange and Mass Spectrometry (HDX‐MS) and analysed by in vitro functional assays. The in vivo function of the peptide was examined with intracranial mouse model by transplanting patient‐derived BTICs. Results We found NR2E1 recruits LSD1, a lysine demethylase, to demethylate mono‐ and di‐methylated histone 3 Lys4 (H3K4me/me2) at the Pten promoter and repress its expression, thereby promoting BTIC proliferation. Using Amide Hydrogen/Deuterium Exchange and Mass Spectrometry (HDX‐MS) method, we identified four LSD1 peptides that may interact with NR2E1. One of the peptides, LSD1‐197‐211 that locates at the LSD1 SWIRM domain, strongly inhibited BTIC proliferation by promoting Pten expression through interfering NR2E1 and LSD1 function. Furthermore, overexpression of this peptide in human BTICs can inhibit intracranial tumour formation. Conclusion Peptide LSD1‐197‐211 can repress BTICs by interfering the synergistic function of NR2E1 and LSD1 and may be a promising lead peptide for brain tumour therapy in future.

Deuterium Exchange and Mass Spectrometry (HDX-MS) method, we identified four LSD1 peptides that may interact with NR2E1. One of the peptides,  that locates at the LSD1 SWIRM domain, strongly inhibited BTIC proliferation by promoting Pten expression through interfering NR2E1 and LSD1 function. Furthermore, overexpression of this peptide in human BTICs can inhibit intracranial tumour formation.
Conclusion: Peptide LSD1-197-211 can repress BTICs by interfering the synergistic function of NR2E1 and LSD1 and may be a promising lead peptide for brain tumour therapy in future.

| INTRODUCTION
Malignant brain cancers, like glioblastoma (GBM), are highly heterogeneous and aggressive. They are resistant to chemotherapy and radiation therapy and show a high chance of relapse. The patient survival time is only about 15 months after diagnosis. This situation has lasted over the past decades although multiple novel therapeutic means have been employed. 1,2 It has been suggested that cancer initiating cells (CICs) with stem cell properties underlie the heterogeneity of malignant tumours. 3 They are less differentiated and resistant to chemotherapy and radiation treatment. They are thought to be the "root" of tumour occurrence and are responsible for the growth and relapse of tumours. Targeting CICs to treat cancer may help to improve the outcome of clinical therapies. 4 Researches have revealed that the recurrence of high-grade gliomas is due to the existence of brain tumour initiating cells (BTICs).
BTICs were among the first CICs derived from a solid tumour. 5,6 BTICs express the neural stem cell surface marker CD133. And as little as 100 BTICs could initiate phenocopies of the original tumours in a NOD.CB17-Prkdcscid/J (NOD SCID) mouse brain. 6 BTICs and neural stem cells (NSCs) share several similarities, and it has been suggested that BTICs hijack the self-renewal mechanisms of NSCs to support their proliferation. 7 Many studies have shown that the factors important for NSC maintenance also play important roles in brain tumorigenesis. For example, Nestin, which labels NSCs in adult mouse brain, also marks BTICs in glioblastoma and is required for the longterm sustenance of tumour growth. 8 NR2E1(TLX), an orphan nuclear receptor, is essential for the selfrenewal of BTICs. NR2E1-positive glioma cells can initiate brain tumours and form spheres in suspension culture. 3 Depletion of Nr2e1 in mouse primary tumours significantly extended animal survival time. Interestingly, GBM patients express a high level of NR2E1 which is correlated with poor survival time. 3 NR2E1 may therefore be a valuable target for brain tumour therapy. Just like Nestin, NR2E1 is highly expressed at the hippocampal dentate gyrus and the subventricular zone. It is required for the maintenance and self-renewal of neural stem cells. 9 In NSCs, NR2E1 interacts with LSD1, a histone demethylase, and recruits it to the promoter of Pten. LSD1 then demethylates mono-and dimethylated histone 3 Lys 4 (H3K4) and removes these active epigenetic markers from the regions to silence the expression of Pten, a gene that induces apoptosis, regulates the cell cycle and functions as a tumour repressor. Through coordinated repression of Pten, NR2E1 and LSD1 contribute to the proliferation of NSCs and retinoblastoma cells. 9,10 Pten is an important mis-regulated tumour suppressor gene in almost all types of cancers. It is now an open and interesting question whether a similar mechanism is also employed in BTICs.
Lysine-specific histone demethylase LSD1 (also named AOF2 or KDM1A or BHC110) is a FAD dependent lysine demethylase. LSD1 can demethylate mono-and di-methylated H3K4 in a complex with CoREST, but shifts its targets to mono-and di-methylated H3K9 when it partners with the androgen receptor (AR). [11][12][13][14] Thus, by changing partners, LSD1 is involved in both gene activation and gene repression. The N-terminus of LSD1 is a non-structural element and contains a putative nuclear localization signal. Following this region is the Swip3p/Rsc8p/Moira (SWIRM) domain. After the SWIRM domain is an oxidase domain which is involved in demethylating proteins.
LSD1 is also linked to the growth of glioblastoma and its inhibition increases the sensitivity of glioblastoma cells to histone deacetylase (HDAC) inhibitor treatment. 15 Since NR2E1 and LSD1 both play important roles in glioblastoma, we set out to investigate whether BTICs employ the same NR2E1-LSD1 mechanism, as in NSCs, to regulate BTIC proliferation.

| Cell culture
Brain tumour initiating cells (BTICs) derived from Nestin-TV-a mice were received as a gift from Dr. Haikun Liu's lab. 3 To culture BTICs in monolayer, the cell culture plate was coated with laminin and poly-Llysine. Cells were grown in DMEM/F12 medium plus 20 ng/ml epidermal growth factor (EGF), 10 ng/ml fibroblast growth factor (FGF-2), B27 and insulin-transferrin-selenium supplements (ITSS). Cells were digested with accutase for passage. To culture brain tumour initiating cells in sphere, cells were seeded in a low attached cell culture plate (Corning) in the above medium without any coating. Human BTICs were derived from surgical samples of patients who were diagnosed with IV glioblastoma with the approval of the Sun Yat-sen University Cancer Center Ethics Committee in accordance with ICH-GCP principle and related Chinese regulation. The tumour tissue was minced and digested by 0.25% Trypsin-EDTA (GIBCO). The digested sample was passed through 40 μm strainer to collect single cells. The cells were washed by PBS and then grew in mouse BTIC culture medium in suspension and tumour spheres formed 1 day later. The tumour spheres were collected and digested by StemPro Accutase (GIBCO) and then passaged at 1:3 ratio with fresh medium. To induce BTIC differentiation, the culture medium was changed to DMEM/F12 medium plus 0.5% FBS and 1 μM all-trans retinoic acid (Sigma, R2625). Three days later, the cells were harvested for immunostaining of differentiated cell marks.

| Cloning
shRNA constructs were generated as previously described. 16

| Reverse transcription and real-time PCR
Reverse transcription was performed with 2 μg of total RNA using the PrimeScript RT reagent kit (Takara). Real-time PCR analysis was performed by using the ABI Prism 7900HT machine (Applied Biosystems) with the SYBR Green mixture (Takara). For each primer, only one correct size band was formed. All experiments were repeated three times independently. The final results were normalized against the expression of β-Actin or Gapdh. Student's t-test was used for the statistical significance appraisal.
2.4 | Cell growth assay 10 6 BTICs were transfected with 2 μg plasmids of interests by electroporation with Amaxa cell line Nucleofector Kit V using nucleofector II. The transfected cells were seeded on a poly-L-lysine and laminin treated 6-well plate. After 24 h, the transfected cells were selected with 1 μg/ml puromycin. After 3 days, the floating dead cells were washed away and the viability of cells was quantitated using MTT assay or CCK-8 assay by following manufacturer's protocol. All experiments were repeated three times independently. Student's t-test was used for the statistical significance appraisal.

| Chromatin immunoprecipitation
ChIP assay was carried out as described previously with slight modification. 17 Briefly, cells were fixed with 1% (w/v) formaldehyde for 10 min at room temperature, and 125 mM glycine was used to inactivate formaldehyde. Chromatins were sonicated to generate average fragment sizes from 200 to 600 bp and immunoprecipitated using the anti-NR2E1 (a gift from Liu's lab), anti-LSD1 (ab17721, Abcam), anti-H3K4me (ab8895, Abcam), anti-H3K4me2 (ab7766, Abcam) antibodies and control IgG or control GFP. The ChIP enriched DNA and input were then decrosslinked and proteins were digested by proteinase K. DNAs were purified by phenol: chloroform extractions and followed by ChIP-qPCR analysis using the ABI PRISM 7900 sequence detection system and Kappa SYBR green master mix (Takara). The values of each real-time PCR assay were normalized with its own input value and then compared with the IgG or GFP value to get the enrichment fold. PCR primers were designed to amplify the promoter regions of mouse Pten and control according to previous research. 18 Each experiment was performed three times independently. Student's t-test was used for the statistical significance appraisal.

| Western blot
Western blot was performed by following standard protocols. 19 Total protein was collected by lysing cells with RIPA buffer containing 0.2 M NaCl, 1% SDS, 1 mM PMSF and 0.1 M DTT. Proteins were then separated by SDS-PAGE and transferred to PVDF membrane (Pall).

| Immunofluorescence staining
The cells were fixed with 4% paraformaldehyde (PFA) for 30 min at 4 C, and then washed in cold PBS for 5 min 3 times. The nuclei were subsequently permeabilized with PBS with 0.5% Triton X-100 for 30 min. Next, the cells were blocked with 1% BSA in PBS for 1 h. The cells were incubated with primary antibody overnight at 4 C. Primary antibodies used were cleaved CASPASE 3 antibody (9664S, CST), Ki67 antibody (ab15580, Abcam), NESTIN antibody (Santa Cruz, sc-58813), NR2E1 antibody (Santa Cruz, sc-377240X), GFAP antibody (Millipore, 5541) and TUJ-1 (abcam, ab1445). After wash, the cells were incubated with anti-rabbit secondary antibody conjugated with proper Alexa Fluor label for 1 h at room temperature in darkness. The nuclei were counterstained with DAPI. The cells were imaged with Olympus IX-73 immunofluorescence microscope.

| In vitro limiting dilution assay
Doxycycline inducible control GFP or LSD1-197-211 lentivirus transduced BTICs were seeded at density of 5, 50, 100, 250 or 500 cells per well into a low attached 24-well plate. 5 μg/ml Doxycycline was added into the plate on every other day. The number of spheres were counted at day 7 after seeding. Stem cell frequency was analysed by using extreme limiting dilution analysis as described 20,21 with software ELDA (http://bioinf.wehi.edu.au/software/elda). Each experiment was performed three times independently. Student's t-test was used for the statistical significance appraisal.

| Animals and intracranial transplantation
The animal experiments were performed with the approval of Animal Experimentation Ethics Committee (AEEC) in the Sixth Affiliated Hospital of Sun Yat-sen University. 10 5 doxycycline inducible GFP or LSD1-197-211-GFP transduced human BTICs were intracranially transplanted into the frontal lobe of 6 to 8-week-old female nude mice (Beijing HFK Bioscience Co. Ltd, China). The cells were suspended in 5 μl PBS and injected into the right frontal lobe at 2 mm lateral and 1 mm anterior to bregma with 2.5 mm depth from the skull base. The mice were fed with water with or without fresh 2 mg/ml Doxycycline (DOX) daily from the second day after transplantation. The brain tumour growth was monitored by IVIS Spectrum imaging (PerkinElmer) after transplantation. The mouse brain was harvested after sacrificed by CO 2 suffocation for hematoxylin-eosin staining.
Mouse tumour volume was calculated as previously described by the formula V = ab 2 /2, where a and b are the length and width of the tumour. 22 The experiments have been performed twice independently with each group contained no less than five mice. Mouse survival curve was plotted by Graphpad prism 7.0 software.

| Amide Hydrogen/Deuterium Exchange and Mass Spectrometry (HDX-MS)
Human NR2E1 (GenScript) was cloned into pET22b to express protein with C-terminal His tag. The protein was purified with Ni-NTA beads and followed by gel filtration. Human LSD1 (ATCC) was cloned into pGEX6 to express protein with N-terminal GST tag. The protein was purified with glutathione agarose and the GST tag was removed by precision protease digestion at 4 C overnight. The eluted protein was further purified by gel filtration. To study the peptide of LSD1 Native SWIRM protein crystallizes in the space groups P2 1 2 1 2 1 and P2 1 2 1 2 whereas SeMet SWIRM protein crystallized in space group I222. Diffraction data of native crystals and SeMet crystals were collected at beamline A1 and beamline F2 respectively in Cornell High Energy Synchrotron Source. All data were processed with DENZO/SCALEPACK. The structure was determined by multiwavelength anomalous dispersion (MAD) methods using SHELX and SHARP and refined using CNS. 25

| Luciferase Assay
Pten promoter containing LSD1 and NR2E1 binding site was cloned into pGL3-basic plasmid to drive the expression of a luciferase gene. Renilla

| NR2E1 and LSD1 synergistically repress PTEN to promote BTIC proliferation
It is reported that NR2E1 interacts with LSD1 and promotes neural stem cell proliferation via repressing Pten expression by demethylating H3K4me and H3K4me2 at Pten promoter. 18 To examine whether the same mechanism was adopted in BTICs, we first performed coimmunoprecipitation assay using the whole cell lysate. An anti-NR2E1 antibody could pull down endogenous LSD1, but control IgG could not, suggesting that NR2E1 and LSD1 form a complex in BTICs ( Figure 2A). Next, we examined the expression of PTEN in Nr2e1 and Lsd1 knockdown BTICs. The downregulation of NR2E1 and LSD1 led to the upregulation of PTEN at both mRNA and protein levels ( Figure 2B,C). This is in line with the luciferase activity of Pten promoter being upregulated in Nr2e1 knockdown, Lsd1 knockdown as well as Nr2e1 and Lsd1 double knockdown BTICs ( Figure S2A).
To examine whether PTEN is one of the major downstream effectors of NR2E1 and LSD1, we adopted Sigma approved shRNA of Pten for the rescue assay. This shRNA alone could efficiently downregulate Pten expression ( Figure S2B,C). Downregulation of Pten by shRNA led to increased cell viability ( Figure S2D). We then co-transfected Pten shRNA with Nr2e1 shRNA or Lsd1 shRNA respectively into BTICs.
We found that the reduced cell viability caused by Nr2e1 or Lsd1 H3K4me and H3K4me2 at its promoter ( Figure 2G).
Therefore, our data proved that BTICs adopted the same NR2E1/ LSD1-Pten regulatory axis as neural stem cells.

| Prediction of LSD1 peptides involved in the NR2E1-LSD1 interaction
To understand how NR2E1 and LSD1 synergistically function, we has been solved. 29   AO domains of LSD1. 10 We have predicted the interaction between NR2E1 LBD (PDB code: 4XAI) and LSD1 (PDB code: 3ZMU) using the ZDOCK program. 26  were set as block residues. As a result, 3600 docking complexes were generated and clustered into 650 groups using the MMSTB clustering method 27 with a root-mean-square deviation (RMSD) cutoff at 8 Å.

| Specificity of peptide LSD1-197-211
Although LSD1-197-211 could interfere with the synergistic function of NR2E1 and LSD1 and inhibit the proliferation of BTICs, the specificity of this peptide is unclear. Both NR2E1 and LSD1 are highly expressed in 293T cells ( Figure 5A). Knockdown of Nr2e1 by shRNA did not, however, lead to the upregulation of Pten at the mRNA level, suggesting that the NR2E1-LSD1 mechanism is not involved in the proliferation of 293T cells ( Figure 5B). To test whether LSD1-197-211 had any effect on the cells that do not rely on the NR2E1-LSD1 based cell proliferation, we overexpressed GFP and LSD1 peptides in 293T cells separately with puromycin selection for 3 days. Western blot showed that the PTEN protein level was similar in the GFP and LSD1 peptide overexpressed 293T cells ( Figure 5C).  Figure 5D). Besides 293T cells, we also examined whether LSD1-197-211 peptide had any effect in glioma cell lines LN299, T98G and U251. CCK8 assays revealed that LSD1-197-211 peptide could not inhibit these cells ( Figure S5A). These results suggest that the LSD1-197-211 peptide selectively inhibits BTICs.
To further characterize the specificity of LSD1-197-211, we determined the crystal structure of the human LSD1 SWIRM domain, residues 183-267 (Table S1). The SWIRM structure mainly contains a long central helix separating two smaller helix-loop-helix motifs at both sides ( Figure 5E 3.6 | LSD1 197-211 inhibits the brain tumour formation of human BTIC Human and mouse NR2E1 protein sequences share more than 97% similarity, so does LSD1. In addition, human and mouse LSD1-197-211 peptide sequences are exactly the same. As compared to other histone H3K4 demethylase, LSD1 is highly expressed in BTICs ( Figure S6A), manifesting its importance in BTICs. Hence, we deduced that LSD1-197-211 peptide should be able to repress human BTICs (hBTICs) as well. To test this hypothesis, we derived two hBTICs from surgical tissue of glioblastoma patients who were diagnosed of grade IV glioblastoma in Sun Yat-sen University Cancer Center ( Figure S6B). The cells expressed NESTIN and NR2E1, the neural stem cell markers ( Figure S6C). They differentiated into GFAP positive astrocytes and TUJ1 positive neuronal cells by retinoid acid treatment ( Figure S6D,E). Western blot further confirms that neural stem cell marker NESTIN and NR2E1 were highly enriched in mouse and human BTICs as compared to the glioma cell lines U251, T98G and LN229 ( Figure S6F). Cancer stem cell marker CD133 was also expressed at a significantly higher level in BTICs than glioma cell lines ( Figure S6G).
We then knocked down Nr2e1 and Lsd1 by shRNA in hBTICs.
Western blot revealed that either NR2E1 or LSD1 downregulation led to the increment of PTEN protein ( Figure S6H,I), indicating that hBTICs adopt the same regulatory mechanism as mouse BTICs.  Figure S5A). This might be due to that these glioma cells do not rely on NR2E1 for cell growth ( Figure S6F).
Hence, LSD1-197-211 peptide should be used together with chemotherapy reagent such as TMZ in brain tumour treatment for sound prognosis.
LSD1 is broadly expressed in most organ tissue with relatively high level in the brain and liver. While NR2E1 is mainly expressed in the brain ( Figure S8A)

| DISCUSSION
High-grade glioma, including glioblastoma, is the most common primary malignant brain tumour. The general treatment for high-grade glioma includes surgery, radiotherapy and chemotherapy. However, it is virtually impossible to completely resect these infiltrative tumours and concurrent radiotherapy and chemotherapy do not provide any significant survival benefit for patients. Five-year survival ratio of patient is still less than 5%. Therefore, novel treatment strategies are desperately needed for this grim disease. BTICs are responsible for the growth and relapse of brain tumour. Therefore, Elimination of them may improve the clinic outcome. Our study revealed that BTICs not only express cancer stem cell markers Nestin and CD133, [32][33][34][35] they can also differentiate into multiple neural lineages. Importantly, they rely on NR2E1 and LSD1, a transcriptional and epigenetic regulatory mechanism for the cell growth, which provides an interesting target to explore means to inhibit BITCs.
Past clinical research shows that drugs that target epigenetic modifiers yield promising survival benefits in multiple diseases. For example, the use of valproic acid, an HDAC inhibitor, together with radiotherapy, has shown a greater efficacy in GBM patients. 36 cells. In addition, prediction of the interaction between LSD1 and NR2E1 LBD using ZDOCK program also shows that the solvent accessible area of LSD1-354-377 is likely to be smaller than that of LSD1-197-211 ( Figure 3D), indicating that LSD1-354-377 may contribute to a weaker interaction between LSD1 and NR2E1 than LSD1-197-211 (Table S2). This is consistent with the deletion mutant study result. In summary, overexpression of LSD1-197-211 efficiently blocks the function of NR2E1 and LSD1 complex and disrupts the demethylation activity of LSD1 at the Pten promoter and leads to its upregulation, and therefore, inhibits the proliferation of BTICs ( Figure 6G).
However, we should point out that a number of glioblastomas harbour Pten mutation. In this case, LSD1-197-211 peptide is likely ineffective in these Pten mutated glioblastomas. Besides, since the NR2E1 and LSD1 mechanism is also employed by NSCs for selfrenewal, we anticipate that LSD1-197-211 may also inhibit NSC proliferation. If this is the case, neurogenesis disturbance that might result from the use of this peptide for brain glioma treatment would be a concern. It is known that neurogenesis is most active in the foetus and reduces with aging. Elderly people, in whom neurogenesis is very low, have the highest probability of developing high-grade brain glioma. LSD1-197-211 may therefore hold great therapeutic potential Overall, our study revealed that LSD1-197-211 may serve as a leading peptide for peptide drug development for glioblastoma. Of course, numerous difficulties need to be conquered to bring the discovery to application. Among them, how to deliver the peptide through blood-brain barrier (BBB) to impede into glioblastoma cells is a huge impediment. Due to this common difficulty in the field, we are unable to directly apply the peptide to treat brain tumour at the moment. Even we proved in the study that this peptide can inhibit hBTICs in vivo, it is still a long way to go to apply this peptide for medical application. To date, some preclinical and clinical studies regarding peptide application in brain tumour treatment have been carried out. 46 Multiple cell-penetrating peptides have been studied for delivering peptide, nucleic acids or chemicals into brain tumours. 46 For example, Ueda et al. made a D-isomer peptide dPasFHV-p53C 0 peptide, which contains cell penetrating peptide CPP, penetration accelerating sequence (PAS) and apoptosis inducer C-terminus of p53 (p53C 0 ). This peptide could increase the survival rate when administrated to the intracranial GBM mouse model. 47 The kind of studies inspires us to explore the means of peptide delivery for GBM treatment in future. Besides, we will also try different peptide wrapping materials, such as nanoparticles conjugated with cell penetrating peptides to see whether the peptide delivery efficiency could be increased. To facilitate peptide delivery, we will also explore whether a shorter LSD1-197-211 based peptide could be still effective to inhibit BTICs. In summary, further investigation on the peptide delivery, safety and stability optimization will be needed to bring the discovery closer to application.