Edited by: Christian Ostheimer, Martin Luther University of Halle-Wittenberg, Germany
Reviewed by: Limin Zheng, Sun Yat-sen University, China; Avery August, Cornell University, United States
This article was submitted to T Cell Biology, a section of the journal Frontiers in Immunology
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Regulatory T cells (Tregs) are a subpopulation of T cells that are specialized in suppressing immune responses. Here we show that the arginine methyl transferase protein PRMT5 can complex with FOXP3 transcription factors in Tregs. Mice with conditional knock out (cKO) of PRMT5 expression in Tregs develop severe scurfy-like autoimmunity. In these PRMT5 cKO mice, the spleen has reduced numbers of Tregs, but normal numbers of Tregs are found in the peripheral lymph nodes. These peripheral Tregs that lack PRMT5, however, display a limited suppressive function. Mass spectrometric analysis showed that FOXP3 can be di-methylated at positions R27, R51, and R146. A point mutation of Arginine (R) 51 to Lysine (K) led to defective suppressive functions in human CD4 T cells. Pharmacological inhibition of PRMT5 by DS-437 also reduced human Treg functions and inhibited the methylation of FOXP3. In addition, DS-437 significantly enhanced the anti-tumor effects of anti-erbB2/neu monoclonal antibody targeted therapy in Balb/c mice bearing CT26Her2 tumors by inhibiting Treg function and induction of tumor immunity. Controlling PRMT5 activity is a promising strategy for cancer therapy in situations where host immunity against tumors is attenuated in a FOXP3 dependent manner.
Regulatory T cells (Tregs) limit autoimmune processes directed at self-antigens. While the Treg's immunosuppressive functions are beneficial to limit autoimmune processes, studies examining certain syngeneic tumors in animals have confirmed a detrimental role for Tregs in cancer (
FOXP3 is a dimeric and/or tetrameric transcription factor in Tregs and fulfills an important role in both Treg development and function (
All human PRMT cDNAs were purchased from OpenBiosystem and GE Dharmacon, and subcloned into the mammalian expression vectors
To generate the PRMT5fl/fl mouse, PRMT5 conditionally targeted ES cells were obtained from the International Mouse Phenotyping Consortium (Prmt5tm2a(EUCOMM)Wtsi). In the targeted cells, Exon 6, which encodes the catalytic domain, is sandwiched by two loxp sites, and lacZ reporter and Neomycin genes are inserted upstream together with two FRT sequences. We injected the ES cells into C57BL/6 blastocysts and obtained chimeric animals. The founder animals were mated with flippase transgenic mice (B6.Cg-Tg (ACTFLPe)9250Dym/J, 005703, Jackson Lab) to delete lacZ and Neomycin genes. Foxp3Creyfp (B6.129(Cg)-Foxp3tm4(YFP/Cre)Ayr/J, 016959) and CD4cre (Tg(Cd4-cre)1Cwi/BfluJ, 017336) mice were obtained from Jackson Laboratory. All animals were housed and bred in a specific pathogen-free animal facility of the University of Pennsylvania. All the experiments were performed following national, state, and institutional guidelines. Animal protocols were approved by the University of Pennsylvania Institutional Animal Care and Use Committee.
293T cells were grown in DMEM supplemented with 10% heat inactivated fetal bovine serum and antibiotics (1% penicillin/streptomycin; Invitrogen) at 37°C in a humidified incubator with 5% CO2 (v/v). Cells were grown to 80% confluency in 6-well plates, and transient transfection was carried out using a mixture of 6 μg DNA and 18 μl FuGENE 6 (Roche) according to manufacturer's instructions. Twenty-four hours after transfection, the cells were lysed with high salt lysis buffer [20 mM Tris-Cl pH 7.5, 420 mM NaCl, 1% TritonX-100, and complete mini protease inhibitor cocktail (Roche)], then prepared for western blot analysis. For the PRMT5 inhibitor treatments cells were transfected with HA-Foxp3 vector and cultured for 24 h. Then inhibitors were added to the cells with indicated concentrations of CMP5 (IC50: unavailable, Millipore), DS-437 (IC50: 5.9 μM, Sigma), HLCL-61 (IC50: 7.21-21.46 μM for acute myeloid leukemia cell line), EPZ004777 [IC50: 50 μM for PRMT5 (
293T cells were transfected with FLAG-Foxp3 or empty vectors, lysed with high salt lysis buffer, and then immunoprecipitated with anti-FLAG agarose beads (Sigma) overnight at 4°C. The precipitates were then washed three times with lysis buffer and boiled for 5 min in SDS loading buffer. Samples were analyzed by SDS-PAGE and specific bands were cut and subjected to mass spectrometry by the University of Pennsylvania Proteomics and System Biology Core. For the methylation analysis, 293T cells were transfected with HA-Foxp3 vector and immune precipitated with anti-HA magnetic beads (Thermo FIsher). Proteins were eluted with elution buffer (Thermo Fisher) and concentrated by vivaspin 500 (GE Healthcare). Samples were analyzed by SDS-PAGE and subjected for mass spectrumtry by the CHOP Proteome Core at the University of Pennsylvania.
Cells were lysed in lysis buffer and the soluble fractions were collected and incubated with anti-HA angarose, anti-FLAG agarose (Sigma-Aldrich), or anti-symmetric dimethyl arginine antibody Sym10 (Upstate) conjugated with Dynabeads protein G magnetic beads (Invitrogen) for 2 h at 4°C. The precipitates were then washed three times with lysis buffer and boiled for 5 min in SDS loading buffer. Samples were analyzed by SDS-PAGE, transferred to Immobilon-P (Millipore) PVDF membrane, and probed with anti-Flag M2-Peroxidase (Sigma), or anti-HA Peroxidase (3F10; Roche). For the detection of tag proteins, immunocomplexes were detected using Immobilon Western Chemiluminescent horseradish peroxidase (HRP) Substrate (Millipore). For human Tregs, expanded cells were harvested and lysed on ice for 1 h with RIPA buffer (50 mM Tris-HCl (pH7.5), 150 mM NaCl, 1% NonidetP-40, 0.25% NaDOC, 1 mM MgCl2, and 10% (vol/vol) glycerol) containing protease inhibitor (1:100; P8340; Sigma-Aldrich), NaF (10 mM), and PMSF (1 mM). Cell lysates were cleared by centrifugation, and the supernatants were incubated with anti-FOXP3 (1 μg, eBio7979), anti-PRMT5 (1 μg, Millipore O14744), or IgG (1 μg, 5415S; Cell Signaling) at 4°C overnight, and then immunoprecipated with Protein G-Sepharose beads (P3296; Sigma) for 1 h at 4°C. The immunocomplexes then were washed with RIPA buffer and examined by Western blotting.
Spleen, axillary and inguinal lymph nodes, and thymus of 18–21 day-old male mice were collected, and single-cell suspensions were made. The cells were stained with L/D aqua (Thermo Fisher Scientific) per manufacturer's instruction for eliminating dead cells. Then their membranes were stained with anti-CD4-percp (100432, Biolegend), CD8-APC/R700 (564983, BD), TCRβ-BV605 (109241, Biolegend), CD25-PE (553866, BD Bioscience), GITR-PE (120208, Biolegend), CD134-PE/Cy7 (119415, Biolegend), CD304-PE-Cy7 (145211, Biolegend), and/or ICOS-PE/Cy7 (313519, Biolegend), CD44-APC-Fire750 (103062, Biolegend), and/or CD62L-PE-Cy7 (104418, Biolegend). After 20 min of incubation on ice, the cells were then fixed with a Foxp3 staining buffer set (eBioscience) and intracellularly stained with CTLA4-BV421 (106312, Biolegend), KI67-BV421 (652411, Biolegend), Foxp3-APC (17-5773-82, eBioscience) and/or Helios-PE (137206, Biolegend), and subjected to flow cytometry using the fluorescence-activated cell sorter (FACS) LSR (BD Biosciences). FACS data were analyzed with FlowJo software (Tree Star). For separating YFP+/− cells, the cells were fixed with 1% paraformaldehyde in PBS for 5 min on ice after membrane staining, then re-fixed using the Foxp3 staining kit, followed by intracellular staining as described above.
For the cytokine expression studies, the spleen cells were harvested and stimulated with PMA (50 ng/ml) and ionomycin (10 μM) and anti-CD3 (0.5 μg/ml) for 6 h with Protein Transport Inhibitor Cocktail (× 500, eBioscience). The cells were then membrane stained with L/D aqua followed by anti-CD4-percpCy5.5 (100434, Biolegend) CD8-AF488 (100723, Biolegend) and fixed with 1% paraformaldehyde in PBS for 10 min on ice and then re-fixed using the Foxp3 staining kit, followed by intracellular staining as described above. After fixation, the cell were stained with anti-IL-4-BV711 (504133, Biolegend), IL-17a-BV421 (506926, Biolegend), IFNγ-PE-cy7 (505826, Biolegend), and Foxp3-APC as described above and analyzed with FACS LSR.
For TILs, tumors were dissected and minced in digestion buffer (0.025 mg/ml liberase TM, 0.05 mg/ml DNAse I (Roche) in RPMI) and then incubated at 37°C with rotating for 30 min. Then cells were filtered through a cell strainer (Falcon). Cells were then stained with anti-CD4-BV785 (100453, Biolegend), CD8-APC/R700 (564983, BD), PD-1-BV421 (135221 BIolegend), TCRβ-BV605, PD-L1-PE (124308, Biolegend), Ly6G-Percp (127654, Biolegend), CD206-PE/Cy7 (141720, Biolegend), CD45-AF488 (103122, Biolegend), F4/80-BUV395 (565614, BD), NKp46-BUV737 (565085, BD), and Foxp3-APC as described above and analyzed with FACS LSR.
TCRβ+ CD4+ CD45RBlow CD25high cells from CD4Cre/+ or CD4+/+ PRMT5fl/fl mice were sorted using the FACS Aria II. A total of 100,000 cells per sample were used for mRNA extraction using Trizol (Invitrogen) and RNeasy micro kit (Qiagen) according to manufacturer's instructions. The extracted mRNA was subjected for RNA sequencing and statistics were analyzed by the High Throughput Sequencing Core at CHOP at the University of Pennsylvania. Differentially expressed gene (DEG) was defined as fold change ≥2 and post-probability of equally expressed (PPEE) < 0.05 calculated by EBSeq method.
CD4+ T cells were provided by the Human Immunology Core at the University of Pennsylvania from healthy donors. CD4+ CD25high CD127low Tregs were sorted using the FACS Aria II (BD Bioscience) to a purity >98%. For
Lentivirus was produced using the ViraSafeTM lentivirus packaging system (Cell Biolabs) per manufacturer's instructions. Viral supernatants were concentrated by adding 50% PEG8000 and 1.5 M NaCl (Final concentration: 5% PEG8000 and 0.15 M NaCl), rotated overnight at 4°C, then centrifuged for 30 min at 3,300 g. The pellets were suspended in T cell media and used for lentivirus transfection. For lentivirus transfection to human Tregs, expanded human Tregs (3 × 105 cells) were harvested and suspended in lentivirus-containing medium with IL-2 (200 IU/ml) and anti-CD3/CD28 beads in 5 ml round bottom tubes (Fisher) and then cenreifuged for 2.5 h at 450 g at RT. The cells were then re-suspended in T cell medium supplemented with IL-2 (200 IU/ml) and cultured in a 12 well-plate. After 2 days of culture, medium was changed and puromycin (1 μg/ml) was added for selecting transfected cells. After 6 days, live cells were sorted and used for the
Foxp3-introduced MIGR1 vectors were used for retrovirus production with pMD2. G and pUMVC (Addgene) at 7.5:5:2.5 ratio using 293T cells. Viral supernatants were concentrated as described above. CD4+ T cells were transfected with the same method as lentivirus transfection as described above. Two days after transfection, GFP+ cells were sorted and expanded for 5 days. Then GFP+ cells were re-sorted and subjected to the suppression assay as described below.
For the mouse cells, CD4+ T cells were enriched from splenocytes using a mouse CD4+ T cell isolation kit (Stem Cells). CD4+ CD25− CD45RBhigh Teff cells and CD4+ CD25+ YFP+ CD45RBlow Treg cells were separated from CD4+ cells using the FACSAria II (BD Biosciences). Teff cells were labeled with Cell Trace Violet (CTV, Molecular Probes) and mixed with Tregs and Dynabeads® Mouse T-Activator CD3/CD28 (Life Technologies) in a V-bottom 96-well plate (20,000 cells/well of Teffs, indicated ratio of Tregs, and 0.2 μl/well of CD3/CD28 beads). For the human cells, healthy donor PBMC were provided by the Human Immunology Core of the University of Pennsylvania, and CD4+ T cells were enriched using the MACS CD4+ isolation kit (Miltenyi Biotech). Then CD4+ CD25− CD127high Teff cells and CD4+ CD25+ CD127low Treg cells were separated from CD4+ cells using the FACSAria II (BD Biosciences). Teff cells were labeled with Cell Trace Violet (Molecular Probes), and mixed with Tregs and irradiated (2000 rad) CD3− PBMC, and then stimulated with anti-human CD3 OKT3 (eBioscience) in a V-bottom 96-well plate (20,000 cells/well of Teffs, indicated ratio of Tregs, 50,000/well of irradiated PBMC from which CD3+ cells were eliminated by human CD3+ T cell isolation kit (Stem Cells), and 0.1 μg/ml of anti-CD3 antibody). After 3 days of culture, cell proliferation was analyzed by flow cytometry. For the human transfected cells, anti-CD3/CD28 beads (0.02 μl/well, Thermo Fisher) were used instead of anti-CD3 and irradiated PBMC.
Mouse CD4+ CD25low CD45RBhigh naïve T cells were sorted as described above. The cells were labeled with CTV, then seeded in a V-bottom 96-well plate (20,000 cells/well) and incubated with the indicated amount of TGFβ (Peprotec), 20 IU/ml of IL-2 and a 1:1 ratio of anti-CD3/CD28 magnetic beads (0.5 μl/well, Invitrogen) for 3 days. After the incubation, the cells were fixed and stained with anti-Foxp3-APC as described above, then analyzed using a FACS Canto (BD).
Jurkat cells (1,000,000 cells/ml) were transfected with IL-2 promoter (1.2 μg, Panomics), pRL-TK (0.03 μg, Promega), and indicated vectors (0.8 μg) using Fugene 6 per manufacturer's instructions. Twenty Four hours after transfection, cells were stimulated with PMA (50 ng/ml) and ionomycin (10 μM) for 6 h. A luciferase assay was then performed using Dual luciferase assay kit (Promega). Fold changes were calculated as (P.I. stimulated Firefly Luciferase/Renilla Luciferase)/(Non-stimulated Firefly lusciferase/Renilla Luciferase).
Tissues were fixed with 10% neutral buffered formalin and embedded in paraffin. Sections were de-paraffinized and stained with H & E by the Cell Imaging Core of the Abramson Family Cancer Research Institute.
Six to Ten weeks old female Balb/c mice (
Lymph node Tregs from Foxp3Creyfp and Foxp3Creyfp-PRMT5fl/fl mice (male, 18–21 days old) were collected using a FACSAria II as described above and then subjected to bisulfite sequencing analysis using the EZ-DNA Methylation Kit (Zymo Research) and primers described previously (
Statistical analysis was performed using the Student's
To define the molecular interactions of FOXP3, we transfected and expressed FLAG-FOXP3 in 293T cells and then immunoprecipitated FOXP3 proteins with anti-FLAG agarose beads for identifying FOXP3-interacting proteins. We immunoprecipitated FOXP3 proteins with anti-FLAG agarose beads (
Identification of PRMT5 as a binding partner of FOXP3.
To reveal the role and function of PRMT5 on Tregs, we generated transgenic mice which have conditional PRMT5 deletion in Tregs since PRMT5 constitutive deficiency results in early embryonic lethality (
Foxp3Creyfp-PRMT5fl/fl mice display scurfy-like symptoms.
To study the Treg populations that still exist in the PRMT5fl/fl Foxp3Creyfp mice, we isolated CD4+ Foxp3+ YFP+ cells from spleen, lymph node and thymus (
We next investigated whether T cells in lymph nodes are activated and differentiated into the effector cells as deduced by staining for CD44 and CD62L. In the PRMT5fl/fl Foxp3Creyfp mice, the population of CD8+ and CD4+ Foxp3− cells contained significant amounts of effector cells (the CD44high CD62Llow population in
We further analyzed the suppressive function of Tregs from lymph nodes by suppression assays. As shown in
We further investigated the molecules that are expressed in Tregs and associated with their function and maturation. In Tregs from PRMT5fl/fl Foxp3Creyfp mice, many function related surface markers such as CD25 and GITR were up-regulated (
To exclude the possibility that scurfy-like autoimmune phenotype affected the expression of these molecules, we used Foxp3Creyfp heterozygous mice (Foxp3Creyfphet). In those heterozygous mice there is no scurfy-like phenotype as half of the Tregs are Foxp3Creyfp positive because of random inactivation of the X chromosome (
Populations and phenotypes of Tregs in Foxp3Creyfp+/−-PRMT5fl/+ or -PRMT5fl/fl mice.
Next we compared the expression level of other known Treg-associated molecules. YFP+ cells from PRMT5fl/fl Foxp3Creyfphet mice showed decreased expression of Nrp-1, CTLA4, ICOS, and Ki67 (
We also investigated whether PRMT5 deletion affects iTreg generation from naïve T cells by using CD4cre-PRMT5fl/fl mice. PRMT5 deletion reduced the iTreg development compared with WT littermates (
To reveal how PRMT5 deletion affects overall mRNA profiles expressed in Tregs, we collected PRMT5 deleted Tregs and subjected them to RNA sequencing analysis. For this experiment, we used the Tregs from CD4+/+ and CD4cre/+ -PRMT5fl/fl mice because CD4cre/+ PRMT5fl/fl mice do not display scurfy-like symptoms, principally because of reduced function of CD8+T and CD4+T cells (Tanaka et al. in preparation), to avoid the effect of endogenous inflammation in their bodies on their Tregs. In addition, CD4cre enables an earlier deletion of PRMT5 expression in the T cells' developmental stage in the thymus that differs from Foxp3cre. This event possibly defines the complete phenotypic changes in generated Tregs. As shown in
Functional and RNA sequencing analysis of PRMT5-deleted Tregs.
Since we found a dramatic reduction of Tregs in Foxp3creyfp PRMT5fl/fl mouse spleen, we next studied the stability of PRMT5 deleted Tregs by analyzing epigenetic modifications of the CNS2 region, which mediates an important role in sustained expression of Foxp3 during Treg activation (
To test whether FOXP3 can be methylated by PRMT5, we first analyzed if PRMT5 knockdown decreases symmetric arginine di-methylation signals by immunoprecipitation with anti-symmetric arginine di-methylation antibody sym10. PRMT5 deletion by shRNA (vector 86) could reduce PRMT5 expression, and symmetric arginine di-methylation signals (
Analysis of methylation sites in human FOXP3.
Next, we sought to identify the symmetric methylation sites. First we computationally analyzed the methylation sites on FOXP3 using the on- line web tool first reported by Kumar et al. (
We also confirmed by mass spectrometry that FOXP3 is di-methylated. Since 293T expresses high levels of PRMT5 proteins and is efficient for gene transfection, we used 293T transfected with HA-FOXP3 and purified the HA-FOXP3 using anti-HA magnetic beads. We found that there are several distinct dimethylation sites on FOXP3: R27 and R146, in addition to R51 (
We next analyzed the effect of the R51 point mutation on Foxp3 functions. Human CD4+ T cells were retrovirally transfected with the empty vector, FOXP3 WT or a mutated FOXP3 R51K gene and subjected to suppression assays. We found that the R51K mutation dramatically decrease its suppressive functions on T effector cells compare with FOXP3 WT (
The R51 mutation by itself did not alter FOXP3 suppressive activity on IL-2 promoter activity. On the other hand, the FOXP3 R397 mutation to W, which is known as a natural IPEX mutation and cannot bind to the promoter DNA sequences (
Pharmacologic PRMT5 inhibition should also impair Treg functions. There are several reported small molecule PRMT5 inhibitors including EPZ015666, CMP5, HLCL61, EPZ004777, and DS-437. DS-437 and EPZ004777 that are S-adenosylmethionine (SAM) competitive inhibitors (
Effect of PRMT5 inhibitors on FOXP3 methylation and Treg function.
The effect of those inhibitors was examined in assays of Treg suppressive function to Teff cells. Treg suppression assays with mouse T cells revealed inhibition of mouse Treg functions with DS-437 (
Our laboratory previously established many of the features of p185erbB2/neu ectodomain targeted monoclonal antibody therapy. Our laboratory had also identified a contribution of Treg activity in dampening tumor elimination (
We next sought to determine whether inhibition of PRMT5 could improve anti-p185erbB2/neu targeted therapy in the resistant tumor model. CT26-Her2 cells were used. CT26 cells were established by engineering the Balb/c syngeneic tumor line CT26 to express human Her2 (
Effect of PRMT5 inhibitor DS-437 on targeted therapy.
In this study, we defined a FOXP3 post-translational modifier, PRMT5. As far as we know, this is the first report that Foxp3 interacts with and can be methylated by PRMT5. Acuto et al. have shown that FOXP3 is methylated at R51, however they did not specify which member of the PRMT family methylates FOXP3 (
Post-translational arginine modification of proteins is known to regulate many biological processes. PRMT proteins transfer methyl groups from SAM to arginine residues. There are 3 types of PRMTs; and PRMT5 belongs to the Type 2 PRMT set, which can symmetrically di-methylate arginine residues of target proteins (
PRMT5 proteins were identified as histone methylating enzymes that led to suppression of gene expression. However, more recently, PRMT5 has been found to methylate and alter the activity of transcription factors. Di-methylation of the NF-kB p65 subunit enhances its function (
RNA sequencing results and Ki-67 staining clearly indicate that PRMT5 deletion affects Treg proliferation. In addition, those Tregs show increased inflammatory cytokine and cytokine receptor expression. These results also suggest that PRMT5 deletion weakens the Foxp3 suppressive function of expression of those inflammatory cytokines. Indeed, several Foxp3 regulating genes reported by Kwon et al. (
Zhao et al. showed that the symmetrical methylation of histone H4 at R3 position specifically recruits the DNA methyltransferase DNMT3a to suppress the targeted genes (
In our experimental conditions, we have not noted increased expression of Foxp3 by inhibiting PRMT5. Those studies include genetic knock outs, shRNA, and inhibitor experiments in both mice and humans. On the other hand, a recent publication by Zheng et al. found that PRMT5 negatively regulates FOXP3 expression in human Tregs, especially in ulcerative colitis patients (
PRMT5 is known to be expressed universally but is overexpressed in a large number of cancers, including breast cancer (
Our results suggest that treatment with a PRMT5 inhibitor in a syngeneic mouse erbB2/neu breast tumor model shows only a slight effect on tumor growth by itself, but significantly enhanced anti-erbB2 targeted antibody therapy. This model only demonstrated a small number of tumor-infiltrating Tregs; but, importantly and unexpectedly, the level of Tregs actually increases with targeted antibody treatment. This inhibitor limits antibody-dependent Treg infiltration.
In human patients, increased frequency of Tregs during trastuzumab therapy coincided with disease progression (
We did not see increased Tregs in humanized 4D5-treated tumors in our CT26Her2 tumor model (data not shown). This difference probably arises because CT26Her2 malignant growth is driven by the RAS oncogene and resistant to erbB2/neu targeted therapy. Rather, we observed inhibited Treg functions in DS-437 treated mouse lymph nodes and increased total CD8+ T cells, CD8+ PD-1+ T cells, and NKp46+ cells in their TIL. CD8+ PD-1+ T cells are known to be tumor-reactive T cells (
We found that the combination of 4D5 and DS-437 also increased the NKp46+ cell population. NKp46 is a marker of NK cells, suggesting that the combination therapy also induced NK cell activity. We noted DS-437 does not affect the tumor associated macrophage's phenotype, suggesting that PRMT5 inhibition during anti-erbB2 mAb therapy would not be dependent on the macrophage activity.
Our studies identify PRMT5 as a FOXP3 binding partner and epigenetic modulator, and potentially important for Treg-targeting small molecules. PRMT5 deletion in Tregs caused scurfy-like symptoms and severe autoimmunity in the mice. PRMT5 deleted Tregs clearly showed proliferation abnormality and decreased suppressive functions. In this study we used many different inhibitors of PRMT5. Most of the inhibitors appeared to inhibit FOXP3 methylation. However, DS-437 and EPZ004777, which are SAM competitive inhibitors, displayed the most efficient suppression of FOXP3 methylation and greater activity in limiting Treg function.
Our experiments indicate that limiting PRMT5 function may promote tumor immunity by inhibiting Treg function and limiting Treg migration into tumors. Interestingly, PRMT5 levels and activity is reported to be elevated in several tumor cells and abnormal PRMT5 functions may contribute to some aspects of the malignant phenotype (
YN performed
MMG, is an employee and shareholder in Macrophage Therapeutics, MIG, is an advisor to Macrophage Therapeutics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
This work was supported by grants from the Breast Cancer Research Foundation and the National Institutes of Health to MIG (BCRF-17-061, R01CA219034), TK (R01HL111501, R01AI121250) and YN and TK (R21A135359). BL is a recipient of He Yu Scholar, Shanghai leading talent program and the National Science Foundation of China Distinguished Young Scholars 31525008. Flow cytometry was performed at the Abramson Cancer Center Flow Cytometry and Cell Sorting Shared Resource, a member of Path BioResource, in the Perelman School of Medicine of the University of Pennsylvania, which was established in part by equipment grants from the NIH Shared Instrument Program, and receives support from NIH P30 CA016520 from the National Cancer Institute.
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