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.
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