Targeting PRMT9-mediated arginine methylation suppresses cancer stem cell maintenance and elicits cGAS-mediated … – Nature.com

Ethics statement

This study follows ethical regulations. Experiments using patient specimens were approved in part by the institutional review boards of City of Hope Comprehensive Cancer Center (COHCCC) and conducted in accordance with the Declaration of Helsinki (2013). Samples were acquired as part of the COHCCC institutional review board-approved clinical protocol no. 18067. All mouse experiments were completed in accordance with the Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee (IACUC) at COHCCC. Experiments were performed in accordance with a protocol approved by the COHCCC ICUC (no. 15046). The maximum tumor size (humane endpoint) permitted by IACUC is 15mm (diameter). All animals were euthanized before tumor size reached 15mm in diameter. Maximum tumor size did not exceed 15mm.

De-identified, clinically annotated primary patient samples including those derived from peripheral blood or bone marrow were obtained from patients with AML at COHCCC. The annotations are shown in Supplementary Table 1. Normal cells derived from peripheral blood were obtained from the COHCCC. Informed written consent was completed and acquired from all involved participants before sample acquisition. MNC separation, CD34+ cell enrichment or CD3+ T cell depletion was performed as described previously58.

Molm13 (catalog no. ACC 554, DSMZ), MV4-11 (catalog no. CRL-9591, ATCC), THP1 (catalog no. TIB-202, ATCC), NB4 (catalog no. ACC 207, DSMZ), U937 (catalog no. CRL-1593.2, ATCC), HL-60 (catalog no. CCL-240, ATCC), MA9.6ITD and RAJI (catalog no. ACC 319, DSMZ), UPN1 (catalog no. CVCL_A795, Cellosaurus), BL41 (catalog no. ACC 160, DSMZ), Rec1 (catalog no. ACC 584, DSMZ), OCI-Ly3 (catalog no. ACC 761, DSMZ) and A20 (a gift from Y. Fu) were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium with 10% FCS as described previously58,59. All other cell lines, including 293FT (catalog no. R70007, Thermo Fisher Scientific), DMS273 (a gift from R. Salgia), DMS114 (a gift from R. Salgia), SW1573 (a gift from E. Wang), A549 (a gift from E. Wang), SW620 (catalog no. CCL-227, ATCC), HCT116 (catalog no. CCL-247, ATCC), HepG2 (catalog no. HB-8065, ATCC), PC3 (a gift from S. Priceman), DU145 (a gift from S. Priceman), MDA-MB-231 (catalog no. CRM-HTB-26, ATCC), HT1197 (catalog no. CRL-1473, ATCC), A172 (catalog no. CRL-1620, ATCC), MIAPACA2 (catalog no. CRM-CRL-1420, ATCC) and HT1080 (catalog no. CCL-121, ATCC) were cultured in DMEM with 10% FCS. MA9.6ITD cells (MLL-AF9 plus FLT3-ITD) were established by J. Mulloy60. The human primary normal and AML CD34+ cells used for transduction were maintained as described previously59. Specifically, as noted in that paper, the medium was StemSpan SFEM (STEMCELL Technologies) supplemented with 50ngml1 recombinant human stem cell factor (SCF), 100ngml1 Flt3 ligand (Flt3L), 100ngml1 thrombopoietin, 25ngml1 interleukin-3 (IL-3) and 10ngml1 IL-6 (PeproTech). Mouse AML cells were cultured in RPMI 1640 medium with cytokines (mouse IL-3, 10ngml1; mouse IL-6, 10ngml1; mouse SCF, 30ngml1; Supplementary Table 10) as described previously59.

In all experiments, male and female, 610-week-old, WT C57BL/6J (strain no. 000664, The Jackson Laboratory), B6(Cg)-Rag2tm1.1Cgn/J (strain no. 008449, Rag2/, The Jackson Laboratory), B6(Cg)-Ifnar1tm1.2Ees/J (strain no. 028288, Ifnar1/, The Jackson Laboratory), Kmt2atm2(MLLT3)Thr/KsyJ (strain no. 009079, MLL-AF9 knock-in, The Jackson Laboratory), B6.129S(C)-Batf3tm1Kmm/J (strain no. 013755, Batf3/, The Jackson Laboratory), NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (strain no. 005557, NSG, The Jackson Laboratory), NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ (strain no. 013062, NSGS, The Jackson Laboratory) and NOD.Cg-Prkdcscid H2-K1b-tm1Bpe H2-Ab1em1Mvw H2-D1tm1Bpe Il2rgtm1Wjl/SzJ (strain no. 025216, NSG-MHC I/II DKO, The Jackson Laboratory) mice were used. B6-Ly5.1 (CD45.1, NCI 564) and BALB/c (NCI 028) mice were available from an outside vendor. Male and female mice were housed at the COH Animal Resource Center. All care and experimental procedures followed established institutional guidelines. The mouse room is conditioned with a 14h light10h dark cycle, temperatures of 6575F and 4060% humidity. The procedure was run in accordance with a protocol approved by the IACUC at COHCCC.

Mouse experiments were performed once: Fig. 2d,e,h (male and female; five WT B6 mice per group); in Fig. 2f,g (male and female; five WT B6 mice per group); Fig. 2q (male and female; eight NSGS mice per group); Fig. 2r (male and female; eight NSGS mice for Ctrl, seven NSGS mice for Prmt9 KD); Extended Data Fig. 2p (male and female; six Prmt9loxP/loxP/Mx1Cre mice for Prmt9 WT, nine Prmt9loxP/loxP/Mx1Cre+mice for Prmt9 KD); Extended Data Fig. 2q (male and female; eight Prmt9loxP/loxP/Mx1Cre mice for Prmt9 WT, 15 mice (Prmt9loxP/loxP/Mx1Cre+) for Prmt9 KD); Extended Data Fig. 2r (male and female; seven B6-Ly5.1 mice per group); Fig. 5d (male and female; seven WT B6 mice per group); Fig. 5e (male and female; five Rag2/ mice per group); Fig. 5f(male and female; five NSGS mice per group); Fig. 5g (male and female; seven WT B6 mice per group); Fig. 5s (male and female; five WT B6 mice for naive mice, four survival mice from Fig. 5d for survivors); Fig. 5v (male and female; five Ifnar1/ mice for Ifnar1 KO, six WT B6 mice for Ifnar1 WT); Fig. 6j (male and female; seven WT B6 mice for the Prmt9 KD group, five WT B6 mice for each of the other three groups); Fig. 6m (male and female; seven WT B6 mice for cGAS KO+cGASN group, five WT B6 mice for each of the other two groups); Fig. 6s (seven WT B6 mice for each Batf3 WT group, five Batf3/ mice for the Batf3 KO group); Extended Data Fig. 7c (seven WT B6 mice for the Ctrl and Prmt9 KD groups, five WT B6 mice for the T and NK cell depletion groups); Extended Data Fig. 9gi (five BALB/c mice per group); and Extended Data Fig. 9j,k (five NSGS mice per group). scRNA-seq and bulk RNA-seq were performed once per sample and are shown in Figs. 1e, 5h and 6c. If not otherwise specified, in vitro experiments were repeated at least three times.

The CD530-EF1A-IRES-GFP vectors were purchased from System Biosciences. The CD530-EF1A-T2A-GFP vectors were modified from CD530-EF1A-IRES-GFP, replacing IRES with T2A sequences. Full-length WT or LDIG-to-AAAA mutant PRMT9 (ref. 29) were cloned into CD530-EF1A-IRES-GFP vectors. FLAG-tagged XRN2 and FLAG-tagged DDX3X variants, and FLAG-tagged either full-length WT or C-terminal (amino acids 436636) PABPC1 or R493K, R481K, R506K or 3RK mutants were cloned into the CD530-EF1A-T2A-GFP vector. All plasmids were synthesized by Genscript. shRNAs targeting human PRMT9, mouse Prmt9, PABPC1 and CREB1 were purchased from Sigma-Aldrich (MISSION shRNA) and cloned into pLKO-SFFV-RFP, as described elsewhere58. cGAS WT and the activation mutant N were purchased from Addgene and constructed into a DOX-inducible expression vector. SMARTvectors with shPRMT9 were purchased from Dharmacon (Horizon Discovery). The oligonucleotides used are listed in Supplementary Table 11.

Compounds were sourced from the NCI Developmental Therapeutics Program (DTP), ZINC libraries or MolPort. The PEGylated liposome packaging of LD2 used for animal treatment was prepared using the thin film hydration method. Lipids (distearoylphosphatidylcholine, cholesterol and DSPE-PEG(2000) at a ratio of 3:1:0.2) plus compound were dissolved in chloroform; then, organic solvent was separated in a vacuum to form a thin film. Subsequently, lipids were hydrated in PBS, pH 7.4, at 60C to form liposomes.

Virus production was as described previously61. HEK 293T cells were transfected with pMD2.G and psPAX2 packaging vectors plus lentivectors designed to overexpress or knock down genes using the calcium phosphate method as described previously61. Supernatants containing virus particles were filtered and concentrated. Viral infection was performed as described previously61.

RNA was prepared according to the TRIzol reagent protocol. After generation of complementary DNA, qPCR with reverse transcription was performed as described previously59. The primers used are listed in Supplementary Table 11.

Cell lysates were prepared in a buffer containing 50mM Tris, pH 7.4, 150mM NaCl and 1mM EDTA supplemented with protease inhibitors. Cell lysates were incubated with anti-FLAG beads or interested primary antibody (Sigma-Aldrich) overnight and denatured for immunoblotting. Proteins of interest were probed with primary and secondary antibodies. Signals were detected using the SuperSignal West Pico or Femato kits. All immunoblots were imaged using the G:BOX Chemi XX6 gel doc system and quantified with the ImageJ software (NIH).

Samples were prepared according to the protocol of the SimpleChIP Plus Enzymatic Chromatin IP Kit (catalog no. 9005, Cell Signaling Technology). Immunoprecipitates were exposed to anti-CREB1 (catalog no. SC-240, Santa Cruz Biotechnology) and anti-H3K27Ac antibodies, plus Protein G magnetic beads. After reversing, DNA was enriched; this was followed by qPCR.

Cells derived from the bone marrow or spleen samples were washed with PBS containing 1% FCS and then passed through a single-cell strainer and subjected to lysis of red cells. Before flow cytometry, cells were stained with the indicated antibodies in the same buffer. Flow cytometry analysis was performed. Data analysis was performed using FlowJo v.10. Molm13 cell engraftment in mice was determined using an anti-human CD45 antibody. CD45.2+ donor cells from transplants were determined using anti-mouse CD45.1 and CD45.2 antibodies. Mouse HSPCs were determined by staining with anti-mouse lineage antibody, including cKit, Sca-1, CD16 and CD32, and CD34 antibodies and a lineage antibody cocktail, including anti-mouse CD3, CD4, CD8, CD11b, CD11c, CD19, CD41, Ter119, B220, IgM, NK1.1, Gr-1 and interleukin-7 receptor subunit alpha (IL-7R). Anti-mouse Mac1, Gr-1, B220 and Ter119 were used to define mouse bone marrow differentiation. We also detected antigen-specific T cells in tumors as described previously44. For intracellular staining, fixed cells were incubated once with antibodies against IFN- (clone XMG1.2) and granzyme B (clone QA16A02). To define the human primary samples, we used the following markers: T cells (CD3+), B cells (CD19+/CD20+), monocytes (CD14+) and DCs (HLA-DR+CD34CD33CD3CD19CD20CD14CD56), as well as the immature CD33+CD34+CD45dim subset. CD69 and IFN- staining was used to determine T cell status. For the cell cycle studies, fixed cells were stained with 4,6-diamidino-2-phenylindole (DAPI).

Bone marrow cells (0.5106per transplant) from CD45.2+ Prmt9loxP/loxPMxCre+ or Prmt9loxP/loxPMxCre mice were combined with CD45.1+ bone marrow cells (at 1:1 ratio) and then implanted into lethally irradiated (900cGy) B6-Ly5.1 mice by intravenous injection. Peripheral blood samples were collected and assessed with CD45.1 and CD45.2 antibodies. Mouse recipients were induced with pIpC (InvivoGen) intraperitoneally 15mgkg1 every other day for 7 days; CD45.2+ chimerism in peripheral blood was assessed every 4 weeks.

For the limiting dilution assays, to evaluate LSC frequencies, AML cells were suspended in Colony Forming Cell growth medium with DOX to induce Prmt9 KD and plated in multi-well plates. To evaluate the frequency of leukemia-initiating cells in vivo, bone marrow cells isolated from Ctrl or Prmt9 KDMA9 AML mice were injected intravenously into sublethally conditioned recipient mice, as described in Supplementary Table 7. The number of recipient mice with leukemia development was determined in each group. The frequency of LSCs and LICs was determined using the ELDA software.

To assess the effect of Prmt9 KO and KD in vivo, MA9 or CMM cells were transduced with lentiviral vectors harboring a luciferase reporter. Cells were used for intravenous inoculation into sublethally irradiated CD45.1 B6 mice or WT B6, Rag2/ or NSGS mice. As for bioluminescence imaging, mice were administered 150mgkg1 d-luciferin (GoldBio) within PBS, followed by analysis using Lago X. Bioluminescent signals were quantified using the Aura imaging software (Spectral Instruments Imaging). Total values were determined using the regions of interest and photonsscm2sr. To identify the immune subsets contributing to leukemia regression after Prmt9 KD, we performed antibody-based depletion with an initial dose of combined anti-CD4 and anti-CD8 treatment or anti-NK1.1 treatment administered 1 day before in vivo DOX administration to Prmt9 KD mice. Antibodies (400g) were injected intraperitoneally twice the first week, and then at 200g twice weekly to maintain NK or T cell depletion. To assess DC function in Prmt9 KD outcomes, we implanted Batf3 WT or Batf3 KO mice with AML cells for further evaluation.

Cell growth was assessed using the CellTiter-Glo Assay Kit (Promega Corporation). Apoptosis was determined using annexin V or DAPI. Colony formation capacity was determined as described previously58,59.

For SILAC, Molm13 cells were cultured in SILAC RPMI 1640 medium (catalog no. 88365, Thermo Fisher Scientific) with 10% FCS (catalog no. A3382001, Thermo Fisher Scientific) and either light l-lysine (catalog no. 89987, Thermo Fisher Scientific) and l-arginine (catalog no. 89989, Thermo Fisher Scientific) for control cells, or heavy lysine (catalog no. 88209, Thermo Fisher Scientific) and l-arginine (catalog no.89990, Thermo Fisher Scientific) for inducible PRMT9 KD cells, for at least ten passages to ensure full incorporation of light or heavy l-lysine and l-arginine.

After 3 days of DOX induction in both control and PRMT9 KD cells, light-labeled and heavy-labeled cells were combined at 1:1 ratio. Cells were washed and centrifuged at 300g for 5min. Cell pellets were lysed in 9M urea with protease and phosphatase inhibitors in HEPES (pH 8.0) buffer. Samples underwent four cycles of sonication for 30s each using a microtip sonicator (VibraCell VCX130, Sonics & Materials) operating at 50% amplitude. Lysates were centrifuged at 20,000g for 15min; protein quantification was performed by using a bicinchoninic acid (BCA) assay. An equal amount of extracted protein from heavy and light SILAC culture was mixed for further digestion. The sample was first reduced by incubation with dithiothreitol (DTT) (5mM, 55C) and then alkylated by incubation with iodoacetamide (10mM) in the dark. The sample was diluted fourfold before sequential digestion first with LysC (2h) and then overnight with Trypsin Gold. Digestion was quenched using trifluoroacetic acid and the sample was desalted using 0.7ml of a Sep-Pak Classic C18 column (Waters). Eluted peptides were speedvacd to dryness and reconstituted in 1.4ml immunoaffinity purification buffer followed by peptide quantification using a BCA assay. We subjected 5% of peptides to global quantitative proteomics analysis and 95% of the rest to methyl-R peptide enrichment. This consisted of sequential incubation of peptides with anti-MMA antibody beads (catalog no. 12235, Cell Signaling Technology) and anti-SDMA antibody beads (catalog no. 13563, Cell Signaling Technology). Enriched peptides were reconstituted in 10l loading solvent (98% water, 2% acetonitrile, 0.1% formic acid); 1g of nonenriched peptides was used for global protein identification.

Data were obtained on an Orbitrap Fusion Lumos mass spectrometer (methylated peptides) or Orbitrap Eclipse with FAIMS Pro interface (unmodified peptides) coupled to a U3000 RSLCnano LC system with running binary solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile) at 300nlmin1. Methylated peptides (5l per injection) were directly loaded on a 25cm EasySpray C18 column and eluted over a 120-min gradient as follow: 80min with 219% B, 20min with 1930% B, 5min with 3098% B, followed by 2min of high organic wash and return to initial conditions in 1min. Unmodified peptides (1g peptides, 5l per injection) were directly loaded on a 50-cm EasySpray C18 column and eluted over 240min using the following gradient: 12min with 25% B, 158min with 519% B, 40min with 1930% B, 9min with 3090% B, followed by 4min of high organic wash and return to initial conditions in 2min. Using a duty cycle of 3s (Lumos) or 1s (Eclipse) per FAIMS CV (40/60/80), most abundant precursors were fragmented using higher-energy collisional dissociation (32% normalized collisional energy on Eclipse and 35% normalized collisional energy on Lumos) and measured in the ion trap. Dynamic exclusion was set to 60s to prevent resampling of previously analyzed precursors.

MS raw files were searched against the human UniProt protein database (downloaded in 2020, 42,373 entries) and a common contaminant database using MaxQuant v.1.6.17.0. The results were filtered to 1% protein and site false discovery rate (FDR). The resulting methyl peptide SILAC ratios obtained from the MaxQuant evidence.txt output file were normalized to their protein SILAC ratios before further analyses62.

Motif analysis was performed using the iceLogo web application as described previously30.

We performed polysome profiling as described previously28. Engineered Molm13 cells were DOX-induced for 3 days to delete PRMT9 expression and then treated for 5min with 100gml1 cycloheximide. After treatment, cells were collected and lysed. We prepared sucrose density gradients (1545% w/v) using a Gradient Master (BioComp Instruments). Then, the supernatant from the cell lysates was separated using centrifugation and fractionation. The collected RNA was further assessed in the qPCR analysis.

Protein synthesis was assessed by using the Click-iT Plus OPP Assay Kit (Thermo Fisher Scientific), with modifications. Briefly, treated cells were exposed to Click-iT OPP, then washed with PBS and fixed. After permeabilization for 15min, cells were reacted with cocktail, then analyzed using flow cytometry.

The assay was performed in a 30-l reaction with 50mM Tris HCl, pH 7.4, 50mM NaCl, 50mM KCl, 1mM MgCl2 and 1mM DTT buffer. Specifically, 1g purified PABPC1-CT protein or synthesized peptides, 1g purified PRMT9 protein and 5M of SAM (Cayman Chemical) were combined. Methylated proteins and peptides were detected with immunoblot or dot blot assays using anti-pan-SDMA, anti-pan-MMA, anti-pan-ADMA or our in-house PABPC1 R493me antibody. The R493me antibody was created by Genemed Synthesis. For the ex vivo tritium labeling of the methylation assay, 1g purified PRMT9 protein, 1g HA-tagged PABPC1 WT or corresponding PABPC1-R481K/R493K/R506K (3RK) protein, which were immunoprecipitated from 293T cells, and 1l S-adenosyl-l-[methyl-3H] methionine (78Cimmol1) was added to a 30l reaction mixture at 30C for 1h. Samples were separated and transferred to polyvinylidene membranes for further assessment.

The crystal structure of human PRMT9 (Protein Data Bank (PDB) ID 6PDM; 2.45A resolution) was used for virtual screening. Missing loops were added using a molecular operating environment loop modeler. A box size of 2521273 centered around the cocrystalized chemical probe was used for screening, which includes both the SAM pocket and catalytic pocket in the N-terminal methyltransferase domain (amino acids 150520). To rank the binding affinity, parallel AutoDock Vina63,64 runs were conducted on a local computer cluster. Seven hundred thousand compounds from the ZINC library were selected using the following criteria: molecular weight 350450, log P<3, total charge 2e to +2e and availability. In addition, we also screened the NCI library (NCI DTP 260,000 compounds). Each ligand was docked ten times and ranked according to the lowest binding energy score. After screening, we purchased the top 300 candidates (142 of them were available) from the NCI DTP and the top 100 candidates (70 of them were available) from the ZINC library to assess anti-AML activity. To estimate lead compound selectivity, we also performed Vina docking of LD2 into human CARM1 (PDB ID 5U4X), PRMT5 (PDB ID 4X61), PRMT7 (PDB ID 4M38) and PRMT9. To compare LD2 binding to PRMT5 versus PRMT9, we carried out two replicas of 100-ns molecular dynamics simulation of LD2 docked into each.

Maltose binding protein (MBP)-tagged PRMT9 core methyltransferase domain (150474) protein was expressed and purified by Genscript. Briefly, the PRMT9 core methyltransferase domain sequence was inserted into the pMAL-c5X vector between the Nde I and EcoR I sites. Tagged protein was expressed in BL21 and purified on an MBP column, followed by Superdex 200 and Q Sepharose columns. Proteins were sterile-filtered and lyophilized after extensive dialysis against the NMR buffer (50mM NaH2PO4, pH 7.5). Deuterium oxide-based sodium phosphate buffer was used with 5% DMSO-d5. For the STD NMR assay, the molar ratio of LD2 to PRMT9 was 60:1 in which the concentration of PRMT9 was 0.67M; 50M trimethylsilylpropanoic acid-d4 was used as the internal reference. The molar ratios between PMRT9 and LD2 were 1:20, 1:40 and 1:60, in addition to a control sample with free LD2. LD2 concentration in the CarrPurcellMeiboomGill (CPMG) experiments was 40M. The NMR saturation transfer difference (STD) experiments were carried out at 25C on a 700-MHz Bruker Ascend system equipped with a 5-mm triple resonance cryogenic probe as described previously65. The CPMG experiment was performed as described previously66. Data were analyzed using Bruker TopSpin v.3.6.

We also assessed whether LD2 binds to PRMT9 directly in vivo; to do so, a cellular thermal shift assay was performed as described previously39,40. We first engineered Molm13 cells to overexpress FLAG-tagged PRMT9 WT or PRMT9 mutant (W152A, D258A and E433A; all three residues are predicted drug and PRMT9 binding sites). Five million cells were pretreated with 2.5M LD2 overnight. DMSO was used as the control. Cells were aliquoted in each tube and heat-shocked using Thermal Cycler at the indicated temperatures. Cells were then lysed for the immunoblot assay. Experiments were performed using three biological replicates.

Two million MNCs from AML bone marrow specimens were cultured per well in 24-well plates in IMDM plus 20% FCS under physiological cytokine conditions as described previously41,42 (granulocyte-macrophage colony-stimulating factor in 200pgml1, granulocyte colony-stimulating factor in 1ngml1, SCF in 200pgml1, IL-6 in 1ngml1, macrophage inflammatory protein-1 alpha in 200pgml1 and leukemia inhibitory factor in 50pgml1). We then used the EasySep Dead Cell Removal Kit (STEMCELL Technologies) to ensure more than 95% living cells before culture. Cells were treated with vehicle (dimethylsulfoxide), 2.5M LD2, anti-PD-1 (pembrolizumab, 10gml1, SIM0010, Bio X Cell) or LD2 plus anti-PD-1 for 4 days at 37C. On day 4, cells were pretreated for 6h with brefeldin A and subjected to CyTOF immunostaining with customized surface or intracellular marker antibodies, according to Fluidigm CyTOF protocols (PN400279A4). An untreated peripheral blood mononuclear cell sample from a healthy donor served as a control for phenotyping. Samples were acquired on a Fluidigm Helios. Data were normalized and saved as FCS files before analysis using the Cytobank software (https://premium.cytobank.org/). After data were cleaned up, spanning-tree progression analysis for density-normalized events was used to cluster AML cells and immune cell subpopulations based on the median level of each.

For CD8A, CD8B, GZMA, GZMB and PRF1, the average expression levels of these genes were used to estimate CTL levels in AML samples43,67. We carried out in silico tests to calculate the ratio of PRMT9hi and PRMT9lo patients exhibiting high versus low CTL scores using both GSE144688, which includes 526 samples of patients with AML, and GSE12417, which includes 163 patient samples. For each patient, high versus low CTL scores were decided according to cutoff of 0.5 for the z-score. A Fishers exact test was used to assess significance.

Bone marrow cells in MA9-transplanted mice, and bone marrow and spleen cells in Ctrl and Prmt9 KD mice administered DOX in drinking water over 7 days, were collected for analysis. Single cells were resuspended in 0.4% BSA and loaded to generate an emulsion of single-cell gel beads. Approximately 5,00010,000 cells were loaded per channel. Libraries were prepared using the Single Cell 3 Library & Gel Bead, Single Cell 3 Chip and i7 Multiplex Kits, according to the Single Cell 3 Reagent Kits v2 User Guide (part no. CG00052 Rev A). Libraries were sequenced on an Illumina HiSeq 4000 system.

We used the Cell Ranger Single Cell Software Suite to perform single-cell 3 gene counting and aggregation of multiple samples to generate raw counts, cell barcodes and gene features. The R package Seurat was run as the platform to implement all data processing procedures68.

Cell quality control was executed as follows: the minimum detected genes (3) in each cell; the minimum number of cells (200) related to each gene; and the maximum fraction (0.2%) of counts from mitochondrial genes per cell barcode. The high-count depth threshold (2,000) was used to filter out potential doublets. Then, the count matrix was normalized to obtain the correct relative gene expression abundance between cells69. Then, the R package Harmony was applied to remove batch effects due to biological differences between cell types or states.

To retain informative genes with high variability, genes with small variations (below 2) among all cells were filtered out. Then, the dimensions of count matrices were reduced using dedicated dimension reduction algorithms, such as UMAP and t-distributed stochastic neighbor embedding (t-SNE). Two-dimensional visualization outputs were then generated using the leading reduced components in the UMAP and t-SNE plots.

UMAP-related processed data were regarded as the input of cell clustering. Neighborhood distances among all cells were determined to infer the identity of each cell. Then, clusters were acquired via specified distance metrics (Euclidean distance). Furthermore, for each cluster, the R package MAST was used to deduce significant DEGs. These DEGs were considered markers of a cluster and were used for annotation purposes. Annotations were conducted manually by comparing marker genes with the literature and arranging cell categories. In addition, automatic annotation of cell clusters was done using the R package SingleR, as described previously70. By combining both annotation styles, the final cell type labels of each cluster were acquired.

For the cell type clusters of interest, GSEA was performed based on preordered genes ranked using MAST-derived (log10(Padj)sign (log fold change)) with 1,000 permutations71. The gene sets of the Hallmark, Kyoto Encyclopedia of Genes and Genomes, chemical genetic perturbation and Gene Ontology-Biological Process categories of the Molecular Signatures Database were considered as the signatures. Finally, specific enriched genes within a cluster were visualized by averaging their expression among all cells in that cluster. Key enriched gene expression was rescaled by z-scores and visualized in the heatmap.

scRNA-seq uncovered ten distinct T cell clusters (c0c9). c0 cells expressed Cd4 and CD62L, but not the effector and memory T cell marker Cd44 or T cell activation genes. Thus, c0 was defined as naive CD4+ T cells. Similarly, c1 cells expressed Cd8a and CD62L but not Cd44 or other T cell activation markers and were defined as naive CD8+ T cells. c2 cells expressed Cd8a, Cd44 and Sell, and intermediate levels of Tbx21 (T-bet) and Eomes, and represented a memory CD8+ T cell population. c3 cells expressed high Cd4, Cd44 and Icos, Ctla4, Tnfrsf4 and Pdcd1, but did not express CD62L and were defined as activated and effector CD4+ T cells. c5 cells expressed Cd44 and showed the highest levels of Ifng, Gzmb, Icos, Tim-3, Il2ra, Tnfrsf18 and Lag3, considered as differentiated CTLs. c6 cells were defined as Treg cells because they express Cd4, Il2ra (Cd25) and Foxp3. c4, c7, c8 and c9 cells contained both CD4+ and CD8+ T cells. c4 and c9 showed lower levels of activation markers, and lower CD62L and higher Cd44, suggesting that they represent Teff cell populations. c7 expressed only the naive T cell marker CD62L, indicating a naive population, while c8 expressed lower CD62L and higher Cd44, but did not express other T cell activation markers, suggesting it represents a memory T cell population.

Total RNA was prepared using the TRIzol reagent (Thermo Fisher Scientific). RNA quality (RNA integrity number) was assessed and sequenced on an Illumina HiSeq 2500 system. RNA-seq reads were aligned with default settings. Count data were normalized. Genes were defined as differentially expressed if the fold change was less than 1.5 or less than 0.67, with an FDR less than 0.05, and at least one sample showing reads per kilobase per million mapped reads greater than 1. We performed hierarchical clustering of DEGs using Cluster v.3.0 with Pearson correlation distance and average linkage, and visualized them with Java TreeView. Enrichment analysis on the pathways of Hallmark, Kyoto Encyclopedia of Genes and Genomes and chemical genetic perturbation was performed using GSEA.

cGAMP levels were detected as reported elsewhere72,73. THP1 cells were DOX-treated to induce PRMT9 KD for 2 days; serum-free Phenol Red RPMI (Thermo Fisher Scientific) medium was replaced for another 24h. Conditioned medium was collected and cGAMP levels were detected using the Enzyme Immunoassay Kit (Arbo Assays). To determine cGAMP levels in the bone marrow microenvironment of control and Prmt9 KD mice, bone marrow fluid was collected by centrifuging tibias and femurs at 8,000rpm for 15s; then, cGAMP levels were assessed.

WT (catalog no. thpd-nfis, InvivoGen), cGAS KO (catalog no. thpd-kocgas, InvivoGen) and MAVS KO (catalog no. thpd-komavs, InvivoGen) THP1-Dual cells were used for the reporter assay. The purchased THP1-Dual cells (InvivoGen) were derived from the human THP1 monocyte line harboring the Lucia gene. Reporter cells were further engineered with inducible PRMT9 shRNA or control shRNA. After DOX treatment to PRMT9 KD or LD2 to inhibit PRMT9 in these cells, Lucia luciferase activity was determined as described by the manufacturer (InvivoGen) by adding QUANTI-Luc reagents and read with a FilterMax F5 microplate reader (Molecular Devices).

Cells were spun onto glass coverslips, fixed and incubated with primary anti-dsDNA (AE-2), H2AX or S9.6 antibodies, then with secondary antibody. Slides were then mounted in 90% glycerol solution containing DAPI (Thermo Fisher Scientific) and examined under a ZEISS LSM 880 confocal microscope.

We used the OxiSelect Comet Assay Kit (Cell Biolabs). Briefly, after PRMT9 KD, THP1 cells were mixed with prewarmed (37C) Comet agarose at a 1:10 ratio (v/v), then loaded onto the top of the Comet agarose base layer. Slides were immersed for 60min in lysis buffer at 4C, which was washed with prechilled alkaline solution. After three washes with prechilled Tris/Borate/EDTA buffer, slides were subjected to electrophoresis at 1Vcm1 for 15min, and then rinsed twice with deionized water. Comets were examined under a widefield ZEISS Axio Observer 7 fluorescence microscope. Approximately 50 cells were determined using the OpenComet software in Image J and shown as olive tail moments74,75.

THP1 reporter cells were electroporated with ribonucleoprotein complexes consisting of Cas9 protein and sgRNAs in the Neon Transfection System; 20moll1 guide RNA (gRNA) (as listed in Supplementary Table 11) were mixed at a 1:1 ratio. KO efficiency was assessed using immunoblot analysis.

As described previously57, bone marrow cells were cultured with complete RPMI medium containing 20ngml1 granulocyte-macrophage colony-stimulating factor (PeproTech). Fresh medium was added on days 3 and 6. CD8+ T cells were isolated from the spleens of OT-1 transgenic mice. MA9-OVA cells were pretreated for 2 days with LD2 and then cocultured overnight with collected bone marrow-derived DCs. Supernatants were collected for IFN- assessment. Bone marrow-derived DCs were selected using a CD11c+ selection kit (STEMCELL Technologies) and cocultured for 48h with OT-1 CD8+ T cells. IFN- supernatants were assayed using a mouse IFN- Flex Set Cytometric Bead Array.

Once leukemia cells were engrafted, MA9 syngeneic transplant mice were treated for 3 weeks with vehicle control, LD2, single anti-PD-1 mAb (catalog no. BE0146, Bio X Cell, 10mgkg1 intraperitoneally every other day) or LD2 plus anti-PD-1 antibody. LD2 was administered at 10mgkg1 intravenously twice a day, based on the preliminary pharmacokinetic and pharmacodynamic results. Mice were assessed for overall survival or killed directly to assess MA9 cell engraftment in bone marrow and perform staining with survivin-specific pentamers to assess MA9-specific immunity as described elsewhere44. Briefly, the bone marrow of MA9 mice was stained with anti-CD8 together with survivin-specific pentamers. CMV-specific pentamers were the negative controls. The percentage of survivin or CMV pentamer-positive CD8 T cells was assessed using flow cytometry. Secondary transplantations were performed to evaluate LSC activity in each group by assessing MA9 cell engraftment in the bone marrow.

The model was established using MHC class I and II DKO NSG mice49. To do so, we implanted 2 million MNCs from AML specimens intrafemorally into an irradiated DKO NSG mouse. After transplantation, MHC-deficient mice showed long-term (approximately 12 weeks in peripheral blood) engraftment of T and CD33+ cells without developing acute graft-versus-host disease. A panel of human lineage and progenitor cell markers (CD45, CD33, CD34, CD14, CD19, CD20, CD3, CD56, HLA-DR) was used to define T cells, B cells, monocytes, DCs and immature CD33+CD34+CD45dim cells. Mice were divided into two groups and treated with vehicle or LD2. Three weeks later, the number and frequency of leukemic CD34+ cells and the number of CD8+ T cells expressing CD69 and IFN- were assessed.

A20 cells (3106) were subcutaneously implanted into syngeneic BALB/c mice. When tumor volume reached 100mm3, mice were randomized into treatment groups. Tumor-bearing mice were treated with isotype control (vehicle), anti-PD-1 mAb (10mgkg1 intraperitoneally every other day for 2 weeks), LD2 (100mgkg1 intratissue injection daily for 2 weeks) or a combination of LD2 with anti-PD-1. Tumor volume was monitored through the end of the study when a humane endpoint was reached. The maximum tumor size (humane endpoint) permitted by the IACUC is 15mm (diameter). All animals were euthanized before tumor size reached 15mm in diameter. The microenvironmental components of tumors were analyzed using immunohistochemistry (IHC) and intracellular staining followed by flow cytometry.

Fixed A20 tumors were embedded in paraffin. Four-micrometer-thick sections on slides were incubated for 1h at 60C, deparaffinized and then rehydrated before IHC staining. Slides were blocked with 3% H2O2. Slides were subjected to antigen retrieval for 15min at 120C in citrate buffer, treated with Tris-buffered saline and incubated for 1h with anti-mouse CD3 or anti-mouse CD8 antibody. After washing, slides were incubated with secondary antibody. Slides were developed and counterstained with Mayers hematoxylin solution. Slides were scanned using whole slide imaging and analyzed using the NDP.view2 software (Hamamatsu).

Portions of fresh A20 tumors were cut into small pieces, then dissociated with type IV collagenase, type IV DNase and type V hyaluronidase at 37C for 30min. Cell suspensions were passed through a 70-m strainer and centrifuged at 300g for 5min. Cells were stained for 30min using a Live-or-Dye Fixable Viability Stain Kit (catalog no. 32018, Biotium). Next, cells were stained with immune cell surface markers (mouse CD45-allophycocyanin, mouse CD3-allophycocyanin/cyanine 7, mouse CD4-Alexa Fluor 700 and mouse CD8-Brilliant Violet 605). After two washes, cells were fixed and permeabilized, then intracellularly stained with mouse IFN--phycoerythrin and granzyme B-fluorescein isothiocyanate antibodies in the permeabilization for the flow analysis. Results were analyzed with FlowJo v.10 (FlowJo LLC).

Studies involving independent cohorts of mice were typically performed once, with several exceptions stated in the figure legends. No specific statistical tests were applied to determine sample size; size was established according to our previous experience with the models used. Accordingly, we typically used experimental cohorts of 57 mice. The experiments were not randomized. Investigators were not blinded to allocation during the experiments and outcome assessments. Data collection and analysis by all investigators were not performed blinded to the conditions of the experiments. No data were excluded from the analyses.

In general, data from independent experiments are shown as the means.d. or s.e.m. Statistics were determined using an unpaired, two-tailed Students t-test, a two-way ANOVA, a one-way ANOVA and a two-sided Fishers exact test. Survival results were analyzed with a log-rank (MantelCox) test and expressed as KaplanMeier survival curves. Prism (GraphPad Software) was used for the statistics; the detailed methods are described in each individual figure legend.

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

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Targeting PRMT9-mediated arginine methylation suppresses cancer stem cell maintenance and elicits cGAS-mediated ... - Nature.com

MD Anderson acquires inducible switch technologies for cell therapy – MD Anderson Cancer Center

The University of TexasMD AndersonCancer Center today announced it has acquired certain assets from Bellicum Pharmaceuticals, Inc. related to the CaspaCIDeswitch platform and the GoCAR platform. The transaction also includes clinical-grade stocks of rimiducid, an agent used to trigger the switches.

As a result of this acquisition, MDAndersonmay incorporate these platforms into its own cell therapy programs. The institution also intends to make the technology widely available via non-exclusive licenses to other academic institutions and to biopharmaceutical companies.

MD Anderson plans to focus on using the CaspaCIDe technology as a safety feature of cell therapies in development. The safety switch incorporates an inducible enzyme known as caspase-9, which initiates the first step of the apoptosis programmed cell death pathway. The switch can be triggered by rimiducid, leading to rapid elimination of cells containing the CaspaCIDe switch. MDAndersons Therapeutics Discovery division also plans to continue the clinical development of rimiducid in order to seek future approval from the Food and Drug Administration.

We strive each day to advance new, innovative treatment options to improve the lives of our patients, and cell therapies hold tremendous promise as effective immunotherapies, said Philip Jones, Ph.D., vice president of Research Strategy, Transformation and Operations atMDAnderson. CaspaCIDe provides a critical safety mechanism which could be triggered as required to reduce side effects, and we look forward to its continued development at MDAnderson.

Including this safety switch in cell therapies may offer clinicians the ability to quickly limit potential treatment-related toxicities that may occur. Potential applications include cell therapies where cytokine release syndrome and neurotoxicities have been observed, cell therapies targeting novel antigens with on-target/off-tumor safety concerns, and next-generation cell therapy constructs with higher potency.

When designing novel cell therapies, we must always ensure patient safety remains a top priority. We have explored a variety of associated technologies, and case studies demonstrate that the CaspaCIDe technology is effective in rapidly eliminating the transduced cells, saidKaty Rezvani, M.D., Ph.D., professor ofStem Cell Transplantation and Cellular Therapy. We have incorporated CaspaCIDe into many of our own cell therapies under investigation, and we are excited about the prospect of broadening the potential applications of this technology in the future.

Under a previous licensing agreement, MD Anderson has incorporated CaspaCIDe into multiple cellular therapy programs, including certain chimeric antigen receptor (CAR) natural killer (NK) cell therapies and plans for certain CAR T cell therapies. The current acquisition includes the previous licenses by MD Anderson and eliminates certain downstream financial obligations required under those licenses.

The asset acquisition also includes the transfer of certain intellectual property related to Bellicums GoCAR-T and GoCAR-NK technologies. Using the rimiducid-based switch system, GoCAR cell therapies feature an inducible MyD88/CD40 (iMC) activation switch designed to enhance proliferation and functional persistence of adoptive cell therapies by resisting exhaustion and by driving production of immunomodulatory cytokines to overcome inhibitory signals from the tumor microenvironment. GoCAR cell therapies may be particularly well-suited for use in solid tumors given their immune suppressive tumor microenvironment.

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MD Anderson acquires inducible switch technologies for cell therapy - MD Anderson Cancer Center

IGF-1-mediated FOXC1 overexpression induces stem-like properties through upregulating CBX7 and IGF-1R in … – Nature.com

Data collection

A comprehensive cancer genomics program, The Cancer Genome Atlas (TCGA) has conducted molecular characterizations of 33 primary cancer types. Using UALCAN (https://ualcan.path.uab.edu/analysis.html), exploration of FOXC1 expression in esophageal squamous cell carcinoma was conducted utilizing data extracted from the TCGA database.

Human esophageal squamous cell carcinoma cell lines, such as TE-1, ECA-109, KYSE-30, and KYSE-150, were procured from the Institute of Biological Sciences of the Chinese Academy of Sciences in Shanghai. Subsequently, routine mycoplasma contamination testing was conducted. Cells were maintained at 37C with 5% CO2, cultured in DMEM medium (GIBCO) containing 1% penicillin-streptomycin and 10% Fetal Bovine Serum (GIBCO).

Cells were seeded into 6-well plates for transient knockdown transfection, followed by the transfection of 100pmol siRNA-FOXC1 (GenePharma, Shanghai, China) using HighGene (ABclonal, Wuhan, China) ECAh well, following the manufacturers guidance. Cells were seeded into 6-well plates for transient gene overexpression transfection, followed by the transfection of the plasmids expressing CBX7 or IGF-1R were purchased from Genechem (Shanghai, China) using HighGene (ABclonal, Wuhan, China).

At 48h post-transfection, transfection efficiency was assessed using RT-qPCR and western blot. For stable transfection, ECA-109 and KYSE-150 cells were transfected with lentivirus vectors encoding either FOXC1-targeting shRNA or non-targeting control shRNA, following the manufacturers instructions (Genechem, Shanghai, China). Briefly, cells were seeded in 6-well plates, and when the cell density reached 30%, a medium containing viral fluid at an MOI of 10, without serum, was added. This medium was replaced with a complete medium 24h later. After lentiviral infection, ECA-109 cells and KYSE-150 cells underwent a two-week selection process with 1g/mL puromycin to obtain stable clones. Transfection efficiency for each vector was evaluated through a western blot.

In the cell migration experiment, 1 105 cells were re-suspended in 200L serum-free DMEM medium and added to the upper compartment, and 500 l DMEM medium containing 10%FBS was added to the lower compartment to induce the migration of cells. In the cell invasion experiment, the cells re-suspended in 200L serum-free DMEM medium were added to the upper chamber coated with Matrigel matrix (Corning, 356234), and the rest procedures were performed the same as the cell migration experiment. Fixation with 4% paraformaldehyde and staining with crystal violet dye were conducted after a 24-h incubation period. Subsequently, IMAGEJ software was employed for cell number quantification.

We introduced a seeding density of 2000 cells per well into 96-well plates and established an arrangement of 10 sub-wells. After the cells were fully attached to the plate, CCK8 reagent (10 l per well) was added at 0,24,48,72,96h, respectively. Subjected to incubation in the absence of light for an hour, the microplate reader was employed to analyze the absorbance at 450nM. Three repetitions of the experiments were executed, followed by the final statistical analysis performed using GraphPad Prism 8.0.

6-well plates were used for cell inoculation, with ECAh well receiving 1 103 cells, and subsequent culture was carried out in DMEM medium containing 10% FBS and 1% Penicillin-Streptomycin Solution. After a 14-day incubation period, cell fixation was performed using 4% paraformaldehyde, followed by staining with 0.1% crystal violet dye. The colony count was determined using ImageJ software.

The ECA-109 cells and KYSE-150 cells were plated into ultra-low six-well plates (Corning) at 1 103 cells/well. The cells were cultured in serum-free DMEM/F12(Gibco) with 2% B27(Invitrogen)20ng/mL EGF(PeproTech)20ng/mL bFGF(PeproTech) for 14 days. The size of the tumor spheroids was observed under a light microscope and the count of spheres with a diameter greater than 100M was counted.

ECA-109 cells and KYSE-150 cells were incubated in 6-well plates in DMEM supplemented with 10% FBS, 1% penicillin-streptomycin, and 1M cisplatin. After incubation for 24h, the cells were gathered, and an Annexin V-FITC apoptosis analysis kit (Elabscience Biotechnology) was utilized to assess the percentage of apoptotic cells, following the step-by-step instructions in the user manual. Results were represented as the mean of % cell death of at least three independent replicates.

1 106 ECA-109 cells and 1 106 KYSE-150 cells were incubated with CD44 antibody(R&D Systems)for 10min at room temperature, and washed twice twice after that. The FACS was performed using the Beckman CytoFLEX and the percentage of CD44+ cells was analyzed.

Cells were subjected to RNA isolation using Trizol (Vazyme) followed by reverse transcription into cDNA using the Reverse Transcriptase Kit (Abclonal). RT-qPCR was performed with the primers for FOXC1, CBX7, IGF- 1R, CD133, CD44, and -actin, and the fold change was calculated by the 2-Ct method. Cloud-Seq Biotech (Shanghai, China) conducted RNA high-throughput sequencing, wherein the removal of rRNAs was accomplished using the GenSeq rRNA Removal Kit (GenSeq, Inc.) with total RNA. After the removal of rRNA from the samples, library construction was carried out utilizing the GenSeq Low Input RNA Library Prep Kit (GenSeq, Inc.), following the prescribed protocol from the manufacturer. Quality control and quantification of the libraries were executed using the BioAnalyzer 2100 system (Agilent Technologies, Inc., USA). The sequencing of the libraries transpired on an Illumina Novaseq instrument, employing 150bp paired-end reads. Primer sequences are listed in Table 1.

Proteins were extracted using RIPA lysate (Beyotime) supplemented with 1% PMSF (Beyotime) and 2% phosphatase inhibitor (Beyotime). Following electrophoretic separation through SDS-PAGE, the proteins were transferred onto PVDF membranes. After blocking with 5% skim milk, primary antibodies specific for FOXC1 (ab227977, Abcam,1:1000), CD44 (A19020, Abclonal,1:1000), CD133 (A0219, Abclonal,1:1000), CBX7 (ab178411, Abcam,1:1000), IGF-1R (ab182408, Abcam,1:1000), phosphor-IGF-1R (ab39398, Abcam,1:1000), Akt (4691, Cell Signaling Technology,1:1000), phospho-Akt (S473) (4060, Cell Signaling Technology,1:1000), ERK1/2 (ab184699, Abcam,1:1000), phospho -ERK1/2 (ab201015, Abcam,1:1000) primary antibodies overnight and -actin (AC026, Abclonal,1:10000) as internal reference were used for protein examination. .

Cultivated cells were fixed, chromatin sonicated, immunoprecipitated, and DNA purified according to ChIP-IT High Sensitivity kit (Active Motif) instructions, and the relative abundance of target DNA was analyzed by qPCR. Primer sequences are listed in Table 1.

Tissue samples for this study were sourced from individuals diagnosed with esophageal squamous cell carcinoma at Tongji Universitys Dongfang Hospital, totaling 79 patients. Following fixation in formalin and embedding in paraffin, tissue sections were sliced to a thickness of 4m. Subsequently, the sections underwent deparaffinization and hydration through immersion in xylene and graded alcohols. Heat-induced antigen retrieval was conducted in EDTA buffer (pH 8.0) for 15minutes, utilizing a microwave oven. To minimize nonspecific staining, blocking was carried out with 10% goat serum. Following this, specific primary antibodies, including FOXC1 (ab227977, Abcam, 1:200), CD44 (A19020, Abclonal, 1:200), and CD133 (A0219, Abclonal, 1:100), were applied to the sections and left to incubate overnight at 4C. The slides were then counterstained with light hematoxylin, subjected to dehydration, and covered with slips. The outcomes were evaluated by two pathologists independently, with no access to clinical data, and subsequent analyses encompassed TNM staging and survival assessment. The study was conducted with the written informed consent of the patients and approved by the Institutional Review Committee of East Hospital Affiliated with Tongji University in Shanghai.

The Animal Protection and Use Committee of Tongji University approved all animal experiments. Animal experimentation involved the utilization of 10 BALB/c nude mice, all of the female gender and aged 6 weeks.KYSE-150-FOXC1-LV and KYSE-150-NC-LV were injected subcutaneously into the right abdomen of two groups of mice, purchased from Gempharmatech Co., Ltd. The mice were euthanized, and the tumors were subsequently extracted after 4 weeks for size measurement and weighing. A portion of the tumor tissue was fixed with 10% paraformaldehyde and paraffin-embedded for subsequent immunohistochemical staining analysis, and the rest was used for protein and mRNA extraction.

The in vivo experiments were repeated three times and the final results were taken as the meanstandard deviation. Statistical comparison analysis was performed by GraphPad Prism 8.0. For survival analysis, the Kaplan-Meier method and log-rank test were employed, and statistical significance was established for P values less than 0.05.

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IGF-1-mediated FOXC1 overexpression induces stem-like properties through upregulating CBX7 and IGF-1R in ... - Nature.com

Stem Cells Explained: The Science Behind Osteoarthritis Treatments – Corporate Wellness Magazine

In recent years, stem cell therapy has emerged as a promising avenue for treating various medical conditions, including osteoarthritis. This innovative approach holds the potential to revolutionize the field of regenerative medicine, offering new hope for patients seeking alternatives to traditional treatments like surgery or medication. In this article, we delve into the science behind stem cell treatments for osteoarthritis, exploring how they work, their potential benefits, and considerations for those considering this option.

Stem cells are unique cells in the body with the remarkable ability to develop into different types of cells. They serve as the body's natural repair system, replenishing damaged tissues and organs. Stem cells can be found in various parts of the body, including bone marrow, adipose tissue (fat), and umbilical cord blood.

Osteoarthritis is a degenerative joint disease characterized by the breakdown of cartilage, the protective tissue that cushions the ends of bones in joints. This condition can lead to pain, stiffness, and reduced mobility. Traditional treatments focus on managing symptoms and may include pain medications, physical therapy, and in severe cases, joint replacement surgery.

Stem cell therapy offers a different approach by targeting the underlying cause of osteoarthritisthe deterioration of cartilage. By harnessing the regenerative potential of stem cells, researchers and clinicians aim to repair damaged cartilage and promote tissue regeneration within the joint.

In stem cell therapy for osteoarthritis, stem cells are harvested from the patient's own body or from other sources, such as umbilical cord tissue. These cells are then processed and concentrated before being injected directly into the affected joint.

Once injected, the stem cells work to reduce inflammation, stimulate tissue repair, and encourage the growth of new, healthy cartilage. This process is believed to slow down or even reverse the progression of osteoarthritis, providing long-term relief from pain and improving joint function.

One of the primary benefits of stem cell therapy for osteoarthritis is its potential to offer long-lasting pain relief and improved joint function without the need for surgery. Unlike traditional treatments that focus on symptom management, stem cell therapy addresses the underlying cause of the condition, offering the possibility of disease modification.

Additionally, stem cell therapy is minimally invasive and typically associated with minimal downtime and few complications. This makes it an attractive option for individuals looking to avoid the risks and lengthy recovery associated with surgical interventions.

While stem cell therapy holds promise for the treatment of osteoarthritis, it's essential for patients to approach this option with caution and realistic expectations. While research into the efficacy of stem cell therapy for osteoarthritis is ongoing, the evidence supporting its use is still evolving.

Patients considering stem cell therapy should consult with a qualified healthcare provider who can assess their condition, discuss treatment options, and provide guidance based on the latest scientific evidence. It's also important to thoroughly research any clinics or providers offering stem cell therapy and ensure they adhere to ethical and regulatory standards.

In conclusion, Stem cell therapy represents a promising frontier in the treatment of osteoarthritis, offering the potential for disease modification and long-term symptom relief. By harnessing the regenerative power of stem cells, researchers and clinicians are paving the way for innovative treatments that may transform the lives of millions affected by this debilitating condition. While more research is needed to fully understand the benefits and limitations of stem cell therapy for osteoarthritis, early results are promising, offering hope for a future where joint pain and disability are no longer inevitable consequences of aging and disease.

Given his unparalleled expertise and success in treating elite athletes and high-profile individuals, we highly recommend Dr. Chad Prodromos for anyone seeking top-tier stem cell treatment. His work at the Prodromos Stem Cell Institute is at the forefront of regenerative medicine, offering innovative solutions for a range of conditions. To explore how Dr. Prodromos can assist in your health journey, consider reaching out through his clinic's website for more detailed information and to schedule a consultation. visit Prodromos Stem Cell Institute.

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Stem Cells Explained: The Science Behind Osteoarthritis Treatments - Corporate Wellness Magazine

New sickle cell therapy uses gene editing at MUSC | Health | postandcourier.com – The Post and Courier

Sickle cell is widely misunderstood, even by many health care providers, so Peterson is making TikTok videos about it and about her journey to try and change that. Sickle cell patients in pain crises often run up against skeptical providers when they seek care because they look normal.

"Nothing shows on the outside," Peterson said.

Seemingly normal things can be difficult for them. For instance, flying can cause terrible pain for patients because of the air pressure or temperature change, butPeterson has still managed a few short hops with her younger brother Emmanuel, who is a pilot.

Olivia Peterson is no stranger to the pain crises. And even seemingly small things, like the weather, can trigger a crippling episode, said her mother, Vanessa, recalling a big 5th birthday party that had been planned. Then a storm front hit.

"We had to call and say, 'Were going to the hospital right now, so were sorry,' " Vanessa Peterson said. "She had her birthday in the hospital."

"Its not the first birthday I spent in the hospital," Olivia Peterson said, but she has learned to laugh about it now.

Vanessa Peterson (left) rests her chin on her daughter Olivia Peterson's shoulder while they sit on a hospital bed at MUSCs Sean Jenkins Childrens Hospital in Charleston on Feb. 8, 2024. Vanessa has been a huge supporter of her daughter over the years, driving her to appointments and sharing a laugh with her whenever possible. The two are very close.

There have been other disappointments along the way. MUSC and other centers have looked at bone marrow transplants for sickle cell patients as a potential long-term therapy, and that is when Jaroscak and Olivia Peterson met five years ago. But without a good donor match, she wasn't a candidate for that clinical trial.

Jaroscak continued with her other treatment, and when the RUBY trial came along and Peterson appeared to qualify, she picked up the phone.

"I called her up and said, 'Olivia, would you like to talk again?' " Jaroscak said.

For Peterson, it was like finding the Golden Ticket in the "Willy Wonka" movies, staring down at her chocolate bar in disbelief.

"I tell you it was one of those moments when you are so ready for something that you are not exactly really ready for it in that moment," she said. "Its right there, at your front door. And youre like, 'Oh, is this really happening right now?' "

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New sickle cell therapy uses gene editing at MUSC | Health | postandcourier.com - The Post and Courier

Blood stem cell discovery could change future treatments – Local 5 – weareiowa.com

Geneticists found that receptors in cells, which activate immune response, also play a role in developing blood stem cells during embryonic development.

AMES, Iowa Iowa State University researchers recently made another scientific breakthrough in the genetics, development and cell biology department.

Led by Dr. Raquel Espin Palazon, researchers discovered that a protein known as "Nod1," which detects immune response, also plays a role in the development of blood stem cells during embryonic development.

The scientists used zebra fish to understand how blood stem cells are formed, because they have a similar developmental trajectory as humans.

Blood stem cells are produced only once, when we are embryos, and those cells migrate to our bone marrow where they are with us for life.

This discovery is important because it has the potential to have a significant impact on future stem cell treatments, Espin Palazon told Local 5 News.

The research could pave the way in assisting individuals with blood disorders like leukemia. With this new knowledge, scientists could eliminate the need for bone marrow transplants, which often come with complications.

"We are getting closer and closer to that big goal of you know, cure patients with their own cells," Espin Palazon said. "It just feels really good, yeah, we're really hoping, I think we could see that happening in our lifetimes."

Espin Palazon and other researchers have another paper under review after Nature Communications published this research.

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Blood stem cell discovery could change future treatments - Local 5 - weareiowa.com

Stem Cell Research: Latest Breakthroughs and Future Applications – Corporate Wellness Magazine

Stem cell research stands at the frontier of regenerative medicine, offering groundbreaking possibilities for the treatment of a myriad of diseases and injuries. This article explores the latest breakthroughs in stem cell research, shedding light on how these advancements are revolutionizing medical treatments and what the future may hold for this transformative field.

Stem cells are the body's raw materials from which all other cells with specialized functions are generated. Under certain physiological or experimental conditions, they can be induced to become tissue- or organ-specific cells with special functions. This ability makes stem cells incredibly valuable for medical research.

Recent years have witnessed significant advancements in stem cell research, leading to groundbreaking applications in the medical field. One of the most notable breakthroughs is the development of induced pluripotent stem cells (iPSCs). Scientists have discovered how to reprogram adult cells to an embryonic stem cell-like state, allowing them to generate any cell type within the body. This innovation opens up new avenues for understanding diseases, creating models for study, and developing and testing new drugs and therapies.

Another significant advancement is in the area of regenerative medicine. Researchers have successfully used stem cells to regenerate damaged tissues in organs such as the heart, liver, and kidneys. This is particularly promising for patients with conditions that are currently considered irreversible or incurable.

A wide range of stem cell-based therapies are currently undergoing clinical trials, targeting conditions such as Parkinson's disease, type 1 diabetes, spinal cord injuries, and various forms of cancer. These trials are critical for determining the safety and efficacy of stem cell therapies in treating these complex diseases.

The field of stem cell research is not without its ethical and regulatory challenges. The use of embryonic stem cells, in particular, has been a subject of ethical debate. However, the development of iPSCs has provided an alternative that may circumvent some of these ethical concerns. Regulatory bodies worldwide are working to establish frameworks that ensure the safe and ethical use of stem cell therapies.

Looking to the future, stem cell research holds the potential to revolutionize the field of medicine. One of the most anticipated applications is the ability to grow organs in the lab for transplantation, potentially solving the problem of organ shortage. Additionally, stem cells may play a crucial role in personalized medicine, where therapies are tailored to the individual based on their unique genetic makeup.

In conclusion, Stem cell research is rapidly evolving, with each breakthrough bringing us closer to understanding the full potential of stem cells in medicine. The future applications of stem cell research are vast and varied, offering hope for the treatment of diseases that are currently incurable. As the field continues to advance, it is poised to fundamentally alter the landscape of medical treatment, making what was once considered science fiction a reality.

Through continuous research and development, stem cell technology promises to unlock new therapies, improve the quality of life for patients with chronic conditions, and pave the way for innovative medical treatments. The journey of stem cell research is far from over; it is an exciting era of discovery and application that will undoubtedly shape the future of medicine.

Given his unparalleled expertise and success in treating elite athletes and high-profile individuals, we highly recommend Dr. Chad Prodromos for anyone seeking top-tier stem cell treatment. His work at the Prodromos Stem Cell Institute is at the forefront of regenerative medicine, offering innovative solutions for a range of conditions. To explore how Dr. Prodromos can assist in your health journey, consider reaching out through his clinic's website for more detailed information and to schedule a consultation. visit Prodromos Stem Cell Institute.

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Stem Cell Research: Latest Breakthroughs and Future Applications - Corporate Wellness Magazine

Pelage Pharmaceuticals Announces $16.75M Series A Financing led by GV to Revolutionize Regenerative Medicine … – BioSpace

LOS ANGELES , Feb. 27, 2024 /PRNewswire/ -- Pelage Pharmaceuticals, a clinical-stage regenerative medicine company pioneering a new generation of treatments for hair loss, announced today that the company has closed a $16.75 million Series A financing led by GV with participation from Main Street Advisors, Visionary Ventures and YK BioVentures, to advance a first-in-class treatment for androgenetic alopecia or pattern baldness, and other types of alopecia including chemotherapy-induced hair loss.

"Our scientific co-founders have uncovered a unique biological mechanism with the potential to reactivate hair growth in people with alopecia," said Daniel Gil, Ph.D., Chief Executive Officer, Pelage Pharmaceuticals. "We are pleased to announce that we have completed a first-in-human Phase 1 clinical trial that demonstrated seven days of topical dosing with PP405 was safe and well-tolerated, and showed statistically significant activation of hair follicle stem cells. With the support of GV, Main Street Advisors and other top-tier investors, we expect to advance our lead program to a Phase 2a clinical trial in the second half of this year."

The Phase 1 trial evaluating PP405, a novel topical agent designed to treat androgenetic alopecia and other forms of hair loss, showed 0.05% PP405 topical treatment for seven days is safe and tolerable with no adverse events, and provided confirmation of a daily dosing regimen. Importantly, the study demonstrated biological activity, proof of mechanism of action seen in preclinical studies, and showed treatment with PP405 stimulated statistically significant activation of a critical hair follicle stem cell activation marker. Translational data will be presented at the American Academy of Dermatology meeting in March.

Androgenetic alopecia, also known as pattern balding, is the most common form of alopecia and accounts for more than 90% of all hair loss, impacting both men and women. Additionally, PP405 may also have applications for other types of hair loss such as telogen effluvium (stress-induced hair loss) and chemotherapy-induced hair loss.

"What we've observed is that in people who experience hair loss, the actual hair follicle stem cells are still present but have reverted to a dormant state. We have uncovered a small molecule able to stimulate cellular metabolism to re-awaken hair follicle stem cells and spur new hair growth," said William Lowry, Ph.D., scientific co-founder, Pelage Pharmaceuticals. The discovery was made by Drs. Lowry, Heather Christofk and Michael Jung, of the University of California, Los Angeles. PP405 is designed to inhibit the mitochondrial pyruvate carrier (MPC) to specifically reactivate the dormant hair follicle stem cells.

"Hair loss is an incredibly common health problem with few effective solutions," said Cathy Friedman, Executive Venture Partner, GV and Board Director, Pelage Pharmaceuticals. "GV is excited by the incredible science behind the Pelage technology. Pelage is pioneering an innovative approach with the potential to disrupt the treatment landscape, moving beyond agents that merely slow the progression of hair loss to a treatment solution that actually helps to regrow hair."

Along with the financing and Phase 1 topline data, Pelage has announced the appointment of Qing Yu Christina Weng, M.D., as the company's Chief Medical Officer (CMO). Dr. Weng is a physician-scientist, Harvard-trained board-certified dermatologist at Massachusetts General Hospital, and faculty at Harvard Medical School. In addition to her clinical expertise, Dr. Weng brings a background in corporate startup strategy and business development. At Pelage, Dr. Weng's experience will inform the company's development of PP405 as it advances to Phase 2 studies.

"The current therapeutic landscape is dominated by reformulations of existing products. Pelage is built on rigorous science and offers the opportunity to target the follicle stem cells directly," said Dr. Weng. "I am thrilled to work with the Pelage team to advance this discovery in pursuit of a novel non-invasive solution for all people who experience hair loss."

About Pelage Pharmaceuticals

Pelage Pharmaceuticals is a clinical-stage regenerative medicine company developing novel treatments for hair loss including androgenetic alopecia and chemotherapy-induced-alopecia. With a focus on molecular and stem cell biology, Pelage is advancing a new class of treatments designed to reactivate dormant hair follicle stem cells and restore the body's ability to naturally grow hair. Its lead program, PP405, is currently in clinical trials. Through its rigorous scientific foundation, topical formulation, and novel mechanism of action, Pelage is pioneering first-in-class hair growth solutions for people of all hair types experiencing hair loss.

About PP405

PP405 is a novel, non-invasive, topical small molecule designed to reactivate dormant hair follicle stem cells and restart hair growth. Through a regenerative medicine approach, the treatment focuses on addressing the metabolic processes that regulate the activation and inactivation phases of hair follicle stem cells. Early results from a Phase 1 trial show that PP405 was well-tolerated and demonstrated statistically significant activation of hair follicle stem cells. In 2018, Pelage Pharmaceuticals licensed the intellectual property to PP405 and related topical small molecules from the UCLA Technology Development Group.

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Pelage Pharmaceuticals Announces $16.75M Series A Financing led by GV to Revolutionize Regenerative Medicine ... - BioSpace

UC Irvine advances stem cell research with $4 million CIRM grant for shared resources lab – UCI News

Irvine, Calif., Feb. 26, 2024 The University of California, Irvine has received a five-year, $4 million grant from the California Institute for Regenerative Medicine to establish a shared resources lab in the Sue & Bill Gross Stem Cell Research Center. The facility will offer essential technologies and training for the development of novel in vitro stem cell-based modeling that will serve researchers across the campus and the state.

Stem cells possess the potential to transform into particular cell types, offering promising avenues for rejuvenating and restoring tissues harmed by injury or affected by disease, said principal investigator Craig Walsh, UCI professor of molecular biology and biochemistry. Utilizing stem cell modeling facilitates the development of new treatments and drug screening platforms and also provides deeper understanding of both discovery and translational research processes.

The grant will expand the UCI stem cell centers current lab capabilities, including the addition of new and upgraded equipment, instrumentation and components, and establish new services and user training in four key areas: foundational technologies, bioengineering and organoids, gene editing, and high-dimensional spatial analysis. Training is essential to characterize stem cell models, develop novel regenerative medicine therapeutics, and ensure that theres a workforce capable of implementing new strategies.

Our state-of-the-art facility and cutting-edge training opportunities demonstrate our commitment to advancing stem cell research and discovery, as well as to education and skill enhancement, said Aileen Anderson, Ph.D., director of the UCI stem cell center and professor of physical medicine & rehabilitation at the UCI School of Medicine.

The impact of the labs initiatives will foster collaborations with academic and industrial partners throughout California and leverage existing training programs such as CIRM Bridges to accelerate innovation. This effort underscores the scientific communitys drive to transform the landscape of regenerative medicine.

The Sue & Bill Gross Stem Cell Research Centers shared resources lab will contribute to the broader CIRM mission to develop a stem cell-based modeling ecosystem throughout California, supporting the basic and translational research necessary to advance scientific discoveries and the development of new therapeutic solutions for unmet medical needs, ranging from neurological diseases to cancer.

About UCIs Brilliant Future campaign:Publicly launched on Oct. 4, 2019, the Brilliant Future campaign aims to raise awareness and support for UCI. By engaging 75,000 alumni and garnering $2 billion in philanthropic investment, UCI seeks to reach new heights of excellence instudent success, health and wellness, research and more. The School of Biological Sciences plays a vital role in the success of the campaign. Learn more by visitinghttps://brilliantfuture.uci.edu/school-of-biological-sciences.

About the University of California, Irvine:Founded in 1965, UCI is a member of the prestigious Association of American Universities and is ranked among the nations top 10 public universities byU.S. News & World Report. The campus has produced five Nobel laureates and is known for its academic achievement, premier research, innovation and anteater mascot. Led by Chancellor Howard Gillman, UCI has more than 36,000 students and offers 224 degree programs. Its located in one of the worlds safest and most economically vibrant communities and is Orange Countys second-largest employer, contributing $7 billion annually to the local economy and $8 billion statewide.For more on UCI, visitwww.uci.edu.

Media access: Radio programs/stations may, for a fee, use an on-campus ISDN line to interview UCI faculty and experts, subject to availability and university approval. For more UCI news, visit news.uci.edu. Additional resources for journalists may be found at communications.uci.edu/for-journalists.

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UC Irvine advances stem cell research with $4 million CIRM grant for shared resources lab - UCI News

Top Stem Cell Clinics in the World: Pioneering Future Medicine – Corporate Wellness Magazine

The realm of regenerative medicine, particularly stem cell therapy, has witnessed exponential growth over the past few decades. Stem cells offer the remarkable potential to repair, regenerate, and rejuvenate tissues and organs, presenting new horizons in treating various diseases and injuries. This article delves into the pioneering world of stem cell clinics, showcasing institutions leading the charge in harnessing the transformative power of stem cell therapies. These clinics are not just medical facilities; they are the epicenters of innovation and research that are reshaping the future of medicine.

Stem cell clinics across the globe are pushing the boundaries of medical science. They specialize in harvesting, manipulating, and deploying stem cells to treat a myriad of conditions, from chronic diseases to orthopedic injuries and beyond. These institutions are characterized by their state-of-the-art facilities, interdisciplinary teams of experts, and a commitment to research and development.

The top stem cell clinics are renowned for their pioneering work in developing and applying cutting-edge treatments. They utilize a variety of stem cells, including embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and adult stem cells, each serving different therapeutic purposes. Treatments range from repairing damaged cartilage in joints, treating autoimmune diseases, to pioneering efforts in neurodegenerative diseases like Parkinsons and Alzheimers.

Research is a cornerstone of these leading clinics. They are often affiliated with academic institutions and participate in global research initiatives, contributing to the growing body of scientific literature on stem cell therapies. These clinics are at the forefront of translating laboratory discoveries into clinical applications, ensuring that treatments are both safe and effective.

The global nature of top stem cell clinics reflects the universal demand for advanced medical treatments. Many of these clinics have become hubs for medical tourism, attracting patients worldwide seeking access to the latest in regenerative medicine. This international aspect underscores the importance of regulatory standards and ethical considerations in stem cell therapy, areas in which these clinics lead by example.

Adhering to stringent ethical and regulatory standards is paramount. The best stem cell clinics operate with transparency, obtaining proper consents, and following rigorous protocols to ensure patient safety and treatment efficacy. They navigate the complex landscape of stem cell research and therapy with integrity, contributing to the field's ethical advancement.

What sets these clinics apart is their holistic, patient-centric approach. Understanding that each patient's needs and conditions are unique, they offer personalized treatment plans. This bespoke approach ensures that patients receive the most appropriate stem cell therapy, maximizing the potential for successful outcomes.

As the field of regenerative medicine continues to evolve, so too will the role of stem cell clinics. With ongoing research and technological advancements, the potential applications of stem cell therapy will expand, offering hope for incurable and untreatable conditions. These clinics are not just treating patients; they are shaping the future of healthcare.

In conclusion, The top stem cell clinics in the world are more than medical facilities; they are beacons of hope for patients and catalysts for change in the medical community. Their pioneering work in regenerative medicine exemplifies the power of stem cell therapy to transform lives. As they continue to innovate and lead, the promise of future medicine becomes increasingly tangible, marking a new era in healthcare where regeneration and healing are at the forefront.

Given his unparalleled expertise and success in treating elite athletes and high-profile individuals, we highly recommend Dr. Chad Prodromos for anyone seeking top-tier stem cell treatment. His work at the Prodromos Stem Cell Institute is at the forefront of regenerative medicine, offering innovative solutions for a range of conditions. To explore how Dr. Prodromos can assist in your health journey, consider reaching out through his clinic's website for more detailed information and to schedule a consultation. visit Prodromos Stem Cell Institute.

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Top Stem Cell Clinics in the World: Pioneering Future Medicine - Corporate Wellness Magazine