Targeting PRMT9-mediated arginine methylation suppresses cancer stem cell maintenance and elicits cGAS-mediated anticancer immunity

Abstract

Current anticancer therapies cannot eliminate all cancer cells, which hijack normal arginine methylation as a means to promote their maintenance via unknown mechanisms. Here we show that targeting protein arginine N-methyltransferase 9 (PRMT9), whose activities are elevated in blasts and leukemia stem cells (LSCs) from patients with acute myeloid leukemia (AML), eliminates disease via cancer-intrinsic mechanisms and cancer-extrinsic type I interferon (IFN)-associated immunity. PRMT9 ablation in AML cells decreased the arginine methylation of regulators of RNA translation and the DNA damage response, suppressing cell survival. Notably, PRMT9 inhibition promoted DNA damage and activated cyclic GMP-AMP synthase, which underlies the type I IFN response. Genetically activating cyclic GMP-AMP synthase in AML cells blocked leukemogenesis. We also report synergy of a PRMT9 inhibitor with anti-programmed cell death protein 1 in eradicating AML. Overall, we conclude that PRMT9 functions in survival and immune evasion of both LSCs and non-LSCs; targeting PRMT9 may represent a potential anticancer strategy.

Fig. 1. PRMT9 levels are elevated in AML.

a, PRMTs mRNA levels in the most deadly cancer types from the TCGA PanCancer Atlas. AML (n = 173), lung adenocarcinoma (LUAD) (n = 510), lung squamous cell carcinoma (LUSC) (n = 484), colon adenocarcinoma (COAD) (n = 438), pancreatic adenocarcinoma (PAAD) (n = 177), breast cancer (BRCA) (n = 1,082), prostate adenocarcinoma (PRAD) (n = 493), liver hepatocellular carcinoma (LIHC) (n = 366), glioblastoma (GBM) (n = 160) and bladder carcinoma (BLCA) (n = 407). Z-scores were determined based on the average expression of each PRMT. PRMT8 was undetectable (n represents the number of tissue samples; https://www.cbioportal.org/). b, PRMT9 protein levels in AML relative to other cancer lines. Data were from the DepMap portal (https://DepMap.org/portal/). AML (n = 10), lung cancer (n = 75), COAD (n = 29), PAAD (n = 17), BRCA (n = 29), PRAD (n = 5), LIHC (n = 12), GBM (n = 11), BLCA (n = 9) and acute lymphoblastic leukemia (ALL) (n = 8). The P value was determined using an unpaired two-sided t-test (n represents the number of different cancer cell lines. c, Top: PRMTs mRNA levels in normal hematopoietic subsets from healthy donors (n = 7) or leukemia subsets from patients with AML (n = 21) in GSE63270. Z-scores were determined based on the average expression of each PRMT. Bottom: the violin plots show PRMT9 expression in LSCs versus normal HSCs and in LSCs versus leukemia blasts. The LSC versus HSC P value was determined using an unpaired two-sided t-test. The LSC versus blast P value was determined using a paired two-sided t-test (n represents the number of patients). CMP, common myeloid progenitor; GMP, granulocyte-monocyte progenitor; LMPP, lympho-myeloid primed progenitor; MEP, megakaryocytic-erythroid progenitor; MPP, multipotent progenitor. d, Fifty representative genes from the MA9 mouse LSC signature. scRNA-seq of MA9 mouse bone marrow (same dataset in Fig. 5h). e, LSC and blast clusters from d. Shown are representative LSC gene (Cbx5) and Prmt9 levels. f, Average fold change in all Prmt levels in an LSC versus a blast cluster based on e. g, Intracellular staining of PRMT9 in CD34⁺CD38⁻ or CD34⁺CD38⁺ populations in PBSCs from individuals with AML (n = 3 individuals) or normal PBSCs. h, PRMT9 protein levels in AML CD34⁺ (n = 30) versus normal PBSC (n = 10) counterparts. i, Quantitative summary of h. The P value was determined using an unpaired two-sided t-test (n represents the number of patients or healthy donors). j, Kaplan–Meier survival analysis of the in-house AML cohort (Supplementary Table 2; n = 94) after dichotomization for median PRMT9 mRNA levels. k, Kaplan–Meier survival analysis of another cohort (GSE12417) after dichotomization for PRMT9 levels below (black, n = 74) or above (red, n = 89) 9.62 log₂-transformed intensity. The threshold was discovered by classifying patients into two clusters using the partitioning around medoids algorithm. P values were determined using a log-rank (Mantel–Cox) test (n represents the number of patients). Source data

Fig. 2. PRMT9 ablation impairs cancer cell survival.

a, Prmt9 levels in cKit⁺ bone marrow cells from MA9 and CMM mice relative to normal counterparts (n = 2 independent experiments). b,c, CFC of MA9 and MA9-ITD cells after Prmt9 KO (n = 5 independent cultures). b, Colony number after Prmt9 deletion induction, as described previously. Data represent the mean ± s.d. The P value was determined using an unpaired two-sided t-test. c, KO efficiency. d,e, MA9-ITD-luciferase cells (0.5 × 10⁶ cells per transplant) were injected into irradiated recipients (n = 5 mice per group). After engraftment, mice were treated with pIpC and assessed for engraftment using imaging (e). d, Quantitative results. Data represent the mean ± s.e.m. P values were established using a two-way analysis of variance (ANOVA). Color bar, luminescence radiance (photons s⁻¹ cm⁻² sr⁻¹). f,g, Another cohort established as d, at the endpoint, spleen (f) of Prmt9 KO and control mice was checked, and engraftment was evaluated based on the percentage of CD45.2⁺ cells (g) (n = 5 mice per group). Data represent the mean ± s.e.m. g, The P value was determined using an unpaired two-sided t-test. h, Survival analysis of MA9-ITD transplants (n = 5 mice per group) on Prmt9 KD. The P value was determined using a log-rank (Mantel–Cox) test. i–k, Molm13 were transduced with mock or PRMT9 WT or PRMT9 mutant vectors resistant to shPRMT9; PRMT9 expression was detected after endogenous PRMT9 was knocked down (i) (n = 2 independent experiments). Cell viability (j) using an MTS assay, and apoptosis (k), based on annexin V staining (j,k, n = 6 independent experiments). j,k, Data represent the mean ± s.d. The P value was determined using a one-way ANOVA. l–o, AML CD34⁺ (n = 11 patients) or PBSC CD34⁺ (n = 3 healthy donors) cells were transduced with shPRMT9. Cell viability (l) and apoptosis (m,n) are shown. PRMT9 levels were evaluated using quantitative PCR (qPCR) analysis (o). l,n,o, Data represent the mean ± s.d. l,n, P values were determined using an unpaired, two-sided t-test. p,q, Molm13 cells transduced with a DOX-inducible shPRMT9 were transplanted into NSG mice (control, n = 8 mice; PRMT9 KD, n = 7 mice). KD efficiency was evaluated (p). After engraftment, mice were treated with DOX to induce PRMT9 KD and engraftment was evaluated based on the percentage of human CD45 cells (q). Data represent the mean ± s.e.m. q, The P value was determined using an unpaired two-sided t-test. r, In parallel, survival was analyzed. r, P was determined using a log-rank (Mantel–Cox) test. Source data

Fig. 3. PRMT9-mediated methylation promotes cancer cell growth.

a, SILAC workflow. b, Alteration of sites carrying DMA (red) or MMA (green) on PRMT9 KD. LC–MS/MS, liquid chromatography–tandem mass spectrometry. c, Cytoscape visualization of proteins carrying PRMT9-regulated R-methyl peptides. d, Percentage of hits among all PRMT9-methylated proteins according to Gene Ontology categories. e, Polysome profiling of RNAs from control and PRMT9 KD Molm13 cells. Shown is the representative trace of one of three biological replicates. f, AML (n = 3 patients) or normal PBSC CD34⁺ (n = 3 healthy donors) cells with PRMT9 KD, analyzed for protein synthesis using an OPP assay. Left: representative. Right: summarized results. Data are the mean ± s.d. g, Validation of representative proteins from the SILAC analysis of Molm13 cells (n = 2 independent experiments). h, c-Myc mRNA levels in RNAs extracted from the indicated fractions in ribosome profiling. i, Downregulated translation factors with a methylated R site after PRMT9 KD. j, Schematic model of methylated arginine at the PABPC1 C terminus. k, In vitro methylation assay of GST-tagged PABPC1-CT mixed with PRMT9. Methylation was analyzed using immunoblotting as indicated (n = 2 independent experiments). l, Methylation assay of PABPC1 peptides mixed with PRMT9. Methylation was analyzed as indicated (n = 3 independent experiments). m, Molm13 cells were transduced with mock or PABPC1 (WT, R493K or 3RK) vectors resistant to PABPC1 shRNA; PABPC1 expression was assessed after endogenous PABPC1 KD (n = 2 independent experiments). n, Protein synthesis. o, Cell viability (n = 5 independent experiments). Data are the mean ± s.d. The P value was determined using a one-way ANOVA. NS, not significant. p, Schematic model of the translation function of PABPC1. q, 293T cells were cotransfected with HA-tagged PABPC1 plus FLAG-tagged PABPC1 (WT or R493K). Cell lysates were then subjected to FLAG pull-down and detected using immunoblotting (n = 1). r, 293T cells were transfected with FLAG-tagged PABPC1 and subjected to poly(A) pulldown, then detected using immunoblotting (n = 2 independent experiments). s, 293T cells were cotransfected with Myc-tagged eRF3 and FLAG-tagged PABPC1 and subjected to FLAG pulldown, then detected as indicated (n = 1). t, Indicated amounts of unmodified, SDMA-R493 or ADMA-R493 PABPC1 peptides (amino acids G491–T507) were spotted for a dot blot assay. PABPC1 peptides were detected using anti-R493me-specific or control antibodies. u, R493 methylation. PRMT9 levels after PRMT9 KD in Molm13 (n = 2 independent experiments) cells. v, PRMT9 and R493 methylation levels in CD34⁺ subsets versus the blast (CD34⁻CD33⁺) subset from the cases with AML (n = 7 patients). P values were determined using a paired two-sided t-test. w, Pearson correlation of R493 methylation with PRMT9 levels in AML CD34⁺. The P value was determined using simple linear regression analysis. The immunoblot analysis is shown in Extended Data Fig. 5c. Source data

Fig. 4. Identification of a PRMT9 inhibitor.

a, Screening pipeline. HT, high throughput. b, Docking pose of the top 30 hits. c, Effects of the top 20 compounds on Molm13 viability. d, Screening of nine compounds using the R493 methylation assay. Catalytic activity was assessed using a dot blot assay with an anti-R493-specific antibody. No. 1: LD2. e, Three-dimensional docking model. Left: LD2 in the pocket. Right: LD2 binding sites. f, CPMG NMR for 40 μM LD2 (blue); LD2 in the presence of PRMT9. g, STD NMR. (i) Reference (blue) and saturated (red) spectra. (ii) STD spectrum showing the difference between reference and saturated spectra. Asterisk denotes impurity. h,i, Thermal shift assay (h) and relative PRMT9 protein (i) of WT mutant PRMT9 from Molm13 cells treated with 2.5 μM LD2. The catalysis inhibition curves are based on the gray intensity of blots normalized to intensity at 37 °C (n = 3 independent experiments). A comparison was made between LD2-treated PRMT9 WT versus LD2-treated PRMT9 mutant. i, Data (n = 3 independent replicates) are represented as the mean ± s.d. P values were determined using a two-way ANOVA. j, Half-maximal inhibitory concentration (IC₅₀) of LD2 in the indicated cells. Cells were treated for 4 days with LD2. MV4-11 (n = 6), NB4 (n = 3), U937 (n = 3), PBSC CD34⁺ (n = 6), MA9.6ITD (n = 3), Molm13 (n = 3) and THP1 (n = 4); data are the mean ± s.d. n indicates independent experiments and represents the number of independent experiments. k, Protein synthesis in the indicated cells after treatment with 2.5 μM LD2, based on an OPP assay. Right: results in vehicle versus LD2 (n = 3 independent experiments). Data are presented as the mean ± s.d. l, R493 methylation of Molm13 cells treated as indicated (n = 3 independent experiments). m, CyTOF of AML MNCs after 4 days of treatment with LD2 (2.5 μM). The frequency of T cells and CD34⁺CD45^dim AML blast cells was noted. Color bar: CD34 intensity. n, Flow plots showing T cell and AML populations in the AML01, before and after T cell depletion. o, T cell depleted or bulk MNCs (n = 3 patients) were treated with LD2 (2.5 μM). AML blasts were determined using flow cytometry. Data are the mean ± s.d. from three independent experiments. p, Frequency of PRMT9^hi (n = 43) versus low (n = 67) AML samples displaying the CTL score high versus low signatures in GSE12417. The P value was determined using a two-sided Fisher exact test. n represents the number of patients. Source data

Fig. 5. PRMT9 inhibition eradicates AML in vivo.

a–c, MA9-luciferase cells were injected into B6 (a, n = 7 mice per group), Rag2^−/− (b, n = 5 mice per group) and NSGS (c, n = 5 mice per group) mice. After engraftment, mice were administered DOX water. Engraftment was tracked using imaging; color bars, luminescence radiance (photons s⁻¹ cm⁻² sr⁻¹). d–f, Kaplan–Meier curves showing the survival of B6 (d), Rag2^−/− (e) and NSGS (f) mice. P values were determined using a log-rank (Mantel–Cox) test. g, CMM cells were injected into B6 mice (n = 7 mice per group). Prmt9 KD was induced as above. The Kaplan–Meier curves show the survival of mice. P values were determined using a log-rank (Mantel–Cox) test. h,i, Different populations (h) or markers (i) identified in bone marrow. j, Prmt9 level in the bone marrow populations. k, Cd69, Ifng and Gzmb levels in T cells of Ctrl (n = 249 cells) and Prmt9 KD (n = 231 cells) bone marrow. Right: Ifng levels. Data are presented as the mean ± s.e.m. The P value was determined using an unpaired two-sided t-test. l, Frequency of AML-specific CD8⁺ T cells in Prmt9 KD mice (n = 5 mice) relative to Prmt9 WT controls (n = 5 mice). Data are the mean ± s.e.m. The P value was determined using an unpaired two-sided t-test. m, Indicated MA9/OVA cells were implanted into B6 mice (n = 5 mice per group). After engraftment, Prmt9 KD was induced. MA9-OVA-specific T cells were assessed. Data are the mean ± s.e.m. The P value was determined using a one-way ANOVA test. n, Subpopulations identified among T cells from the spleen in the merged Ctrl and Prmt9 KD groups. o, Expression levels of the indicated genes in the T cell clusters. p, Distribution of the clusters annotated in n. q,r, Percentage of clusters in CD8⁺ (q) or CD4⁺ (r) T cells annotated in n. s, Survivors of Prmt9 KDMA9 cell-challenged mice (n = 4) were rechallenged with 1 × 10⁶ parental MA9 Prmt9 KD cells (without DOX induction). Control naive C57BL/6 mice (n = 5 mice per group) inoculated with the same number of cells. The Kaplan–Meier curves show the survival of mice. The P value was determined using a log-rank (Mantel–Cox) test. t, Upregulated ISGs in T cells. u, GSEA of DEGs in bone marrow T cells after Prmt9 KD. v, MA9-luciferase cells were injected into WT (n = 7 mice) or Ifnar1 KO mice (n = 5 mice). Prmt9 KD was induced as above. The Kaplan–Meier curves show the survival of mice. The P value was determined using a log-rank (Mantel–Cox) test. Source data

Fig. 6. Immunity after PRMT9 inhibition requires cGAS activity.

a, Upregulated ISGs in MA9 cells. b, GSEA of DEGs in Prmt9 KD MA9 cells. c, Overlapped DEGs in the indicated cells (fold change > 2). NES, normalized enrichment score. d, ISG15 expression in Molm13 cells with endogenous PRMT9 KD and after rescuing with PRMT9 WT or a catalytically dead mutant (n = 5 independent experiments). Data are the mean ± s.d. The P value was determined using a one-way ANOVA. e, ISG levels in AML CD34⁺ cells. Data are the mean ± s.d. from three independent experiments. f, Luciferase activity of THP1-IRF cells engineered as indicated (n = 5 independent experiments). Data are the mean ± s.d. The P value was determined using a one-way ANOVA. g, cGAMP levels in engineered THP1 supernatant (n = 3 independent experiments). Data are the mean ± s.d. h, Left: immunostaining for γH2AX in THP1 cells. Right: γH2AX intensity (n = 100 cells per group). Scale bars, 10 μm. The P value was determined using an unpaired two-sided t-test. i, dsDNA using immunostaining in THP1 cells. Right: dsDNA intensity (n = 50 cells per group). Scale bar, 10 μm. The P value was determined using an unpaired two-sided t-test. j, MA9/OVA cells (Ctrl, n = 5 mice), Prmt9 KD (n = 7 mice), Ctrl + cGAS KO (n = 5 mice) and Prmt9 KD + cGAS WT (n = 5 mice)) were transplanted to establish AML. Prmt9 KD was induced. The Kaplan–Meier curves show the survival. P values were determined using a log-rank (Mantel–Cox) test. k, cGAS KO MA9 cells were transduced with inducible HA-tagged cGAS WT or ΔN. Exogenous cGAS was then assessed (n = 1). l,m, cGAS KO (n = 5 mice), cGAS WT (n = 5 mice) or cGAS-ΔN MA9 (n = 7 mice) cells were transplanted. DOX was given to induce expression of cGAS variants. l, AML engraftment was assessed. Data are the mean ± s.e.m. P values were determined using a one-way ANOVA. m, For another cohort, Kaplan–Meier curves show survival. P values were determined using a log-rank (Mantel–Cox) test. n, cGAS levels in BEAT AML cases (n = 451 patients) and healthy donors (n = 19). The P value was determined using an unpaired two-sided t-test. o, cGAMP levels in the bone marrow of mice (n = 3 mice per group). Data are the mean ± s.d. p, Expression of Cd80, Cd86 and H2-ab1 in the DCs of the scRNA-seq of Ctrl (n = 108 cells) and Prmt9 KD (n = 57 cells) bone marrow. P values were determined using an unpaired two-sided t-test. q,r, LD2-pretreated MA9/OVA cells were cocultured with bone marrow-derived DCs. DCs were then cocultured with OT-I transgenic CD8⁺ T cells. q, IFN-γ production by CD8⁺ T cells. r, IFN-β production by DCs. n = 3 independent experiments. Data are the mean ± s.d. s, MA9 AML cells were implanted into Batf3 WT or KO mice: (1) Prmt9 KD/Batf3 KO (n = 5 mice), (2) Prmt9 KD/Batf3 WT (n = 7 mice) and (3) Prmt9 WT/Batf3 WT (n = 7 mice). Kaplan–Meier curves show survival. P values were determined using a log-rank (Mantel–Cox) test. Source data

Fig. 7. Loss of XRN2 methylation underlies cGAS activation.

a,b, Phospho-CHK1, CHK2 and γH2AX levels after PRMT9 KD (a) or LD2 (b) in THP1 (n = 2 independent experiments). c, Comet assay of THP1 after PRMT9 KD for 48 and 72 h. Right: summary of each group (n = 50 cells). Scale bar, 50 μm. The P value was determined using an unpaired two-sided t-test. d, Luciferase activity of THP1-IRF cells after KO of the indicated genes. Data are the mean ± s.d. from three independent experiments. e, Methylation assay of KHDRBS1 (amino acids 326–339), XRN2 (amino acids 937–950) or DDX3X (amino acids 80–92) peptides. Methylation was analyzed using an anti-MMA antibody (n = 2 independent experiments). f, XRN2 and DDX3X levels after respective KO (n = 2 independent experiments). g, Luciferase activity of WT and cGAS KO THP1-IRF cells. gRNA-resistant XRN2 WT and R946K constructs were ectopically expressed in THP1-IRF cells (n = 5 independent experiments). A reporter assay was performed using the cells with KO endogenous XRN2. Data are the mean ± s.d. The P value was determined using a one-way ANOVA. h, Luciferase activity of THP1-IRF cells (n = 5 independent experiments). gRNA-resistant DDX3X WT or R88K constructs were ectopically expressed in THP1-IRF cells. A reporter assay was performed using the cells with KO endogenous DDX3X. Data are the mean ± s.d. The P value was determined using an unpaired two-sided t-test. i,j, In vitro methylation of XRN2 peptides with PRMT9 (i) or PRMT5 (j) with increased dose of LD2 (i) or EPZ015666 (j) (n = 1). k, XRN2-engineered THP1 cells were prepared for IP using anti-FLAG beads; interactors were detected as indicated (n = 2 independent experiments). l,m, R-loop signals by dot blots (l, n = 2) or immunostaining (m) in THP1 cells. Scale bar, 10 μm. ssDNA, single-stranded DNA. n,o, R-loop signals in RNASEH1-overexpressed THP1 cells treated with LD2 (2.5 μM) (n) or PRMT9 KD (o) (n = 2 independent experiments). p, Cell cycle of THP1 cells treated for 48 h with LD2 (2.5 μM), n = 5 independent experiments. Right: statistics. Data are the mean ± s.d. Right: P values were determined using a one-way ANOVA. q, Phospho-CHK1 in engineered THP1 cells treated with LD2 (2.5 μM) (n = 2 independent experiments). r, Luciferase activity of THP1-Lucia luciferase cells treated with (2.5 μM) LD2 (n = 5 independent experiments). Data are the mean ± s.d. The P value was determined using a one-way ANOVA. Source data

Fig. 8. Combining LD2 with an ICI ablates AML.

a,b, Uniform manifold approximation and projection (UMAP) (a) and histogram (b) showing Cd274 (PD-L1) and Pdcd1lg2 (PD-L2) expression in MA9 cells from the scRNA-seq analysis of Ctrl (n = 1,827 cells) and Prmt9 KD (n = 1,124 cells) leukemic cells. Data are the mean ± s.e.m. P values were determined using unpaired two-sided t-tests. c, CyTOF of AML MNCs treated with LD2 (2.5 µM for 4 days), colored according to the expression of PD-L1 based on the CD34⁺CD45^dim subsets (n = 3 patients). d, CyTOF of AML MNCs after treatment. The frequency of CD3⁺ T cells and CD34⁺CD45^dim AML blasts were noted. The color bar shows the intensity of CD34 expression. e, Relative leukemia cell (CD34⁺CD45^dim) frequencies of AML01 in d and Fig. 4m. f,g, CD69 (f) and IFN-γ (g) levels in CD8⁺ T cells in AML01. h–j, MA9 cells were transplanted (i, n = 5 mice per group). We treated AML-bearing mice for 3 weeks with vehicle, a PD-1 inhibitor (10 mg kg⁻¹ intraperitoneally every other day), LD2 (10 mg kg⁻¹ intravenously twice a day) or LD2 plus PD-1 inhibitor. After treatment, leukemic progenitor (GFP⁺cKit⁺) engraftment was assessed (h). MA9-specific CD8⁺ T cells were assessed (representative plots are shown in j). Cytomegalovirus (CMV)-specific pentamers were the negative control. i, Histograms. j, Data summary. Data are the mean ± s.e.m. h,i, P values were determined using a one-way ANOVA. k, As in h, Kaplan–Meier curves show the survival of mice (n = 5 mice per group). P values were determined using a log-rank (Mantel–Cox) test. l, Secondary transplantation (n = 5 mice per group) based on bone marrow cells from the first transplants (h); MA9 (GFP⁺) cells in the bone marrow were assessed. Data are the mean ± s.e.m. P values were determined using a one-way ANOVA. m–p, Two million AML MNCs were implanted intrafemorally into an irradiated MHC class I and 2 DKO mouse (n,o,p, n = 6 mice per group). After engraftment, mice were treated with vehicle or LD2 (10 mg kg⁻¹ intravenously twice a day). After 3 weeks of treatment, the number and frequency of leukemic CD34⁺ cells (m,n) and frequencies of CD8⁺ T cells expressing CD69 (o) and IFN-γ (p) were assessed. Data are the mean ± s.e.m. n,p, P values were determined using an unpaired two-sided t-test. q,r, PRMT9 KD gene signature levels in the indicated ICI-treated cohorts of patients enrolled in clinical trials against melanoma (q) and BLCA (r) cancer with CR and PD^,,. Single-sample GSEA was applied. Violin plots were used to compare the distribution of NES between groups. Statistical comparisons were carried out using unpaired two-sided t-tests. n; represents the number of patients. Source data

Extended Data Fig. 1. PRMT9 levels are elevated in AML.

a–h, PRMTs mRNA levels in cancers from TCGA PanCancer Atlas. AML (n = 173), Lung adenocarcinoma (LUAD, n = 510), Lung squamous cell carcinoma (LUSC, n = 484), Colon Adenocarcinoma (COAD, n = 438), Pancreatic adenocarcinoma (PAAD, n = 177), Breast Cancer (BRCA, n = 1082), Prostate adenocarcinoma (PRAD, n = 493), Liver Hepatocellular Carcinoma (LIHC, n = 366), Glioblastoma (GBM, n = 160), Bladder Carcinoma (BLCA, n = 407). PRMT expression in AML (red) was compared with other cancers (only with significant difference were indicated). The violin plot in gray indicates PRMTs (except PRMT8 which is undetectable) level was significantly higher in AML than indicated cancer type. P values (a-h) were determined by unpaired two-sided t-tests. ‘n’ represents the number of patients. i–n, PRMTs protein levels in AML lines relative to those seen in lines representing the other deadly cancers. AML (n = 14), Lung Cancer (LC, n = 77), COAD (n = 29), PAAD (n = 17), BRCA (n = 29), PRAD (n = 5), LIHC (n = 12), GBM (n = 11), BLCA (n = 9) and Acute Lymphoblastic Leukemia (ALL, n = 8). Data was based on DepMap. P values were determined by unpaired two-sided t test. ‘n’ represents number of cancer cell lines. o, PRMT9 levels in AML (n = 10) and B-NHL (n = 8) lines relative to lines from other deadly cancers (n = 330), based on DepMap. P values were determined by one-way ANOVA. ‘n’ represents number of cell lines. p, PRMT9 protein levels in lines from most-deadly cancers, (n = 1). q, Expression level of LSC-signature-genes in LSC (n = 383 cells) and Blast (n = 1191 cells) based on MA9 scRNAseq analyses. P value was determined by unpaired two-sided t test. r, Q-PCR analysis of PRMT9 levels in AML CD34+ cells from an in-house cohort (n = 94 patients) and from PBSCs from healthy donors (n = 19). PRMT9 levels were normalized to β-actin. P value was determined by unpaired two-sided t test. Source data

Extended Data Fig. 2. PRMT9 function is dispensable for normal hematopoiesis.

a, PRMT9 mRNA levels from BEAT AML dataset of mononuclear cells (MNCs) from AML cases (n = 451 patients) and healthy donors (n = 12). P value was determined by unpaired two-sided t test. b, PRMT9 levels in AML cases with different cytogenetic karyotypes from BEAT AML. NK: n = 107, t (8;21): n = 11, 11q23: n = 15, t (15;17): n = 15, inv (16): n = 25, Complex: n = 32. ‘n’ represents the number of patients. c, d, PRMT9 levels in BEAT AML of FLT3-ITD (c) and NPM1 (d) mutated subsets relative to their WT counterparts. FLT3wt n = 346, FLT3-ITD: n = 105; NPM1wt: n = 340, NPM1mut: n = 108. P value was determined by unpaired two-sided Mann–Whitneyt test. ‘n’ represents the number of patients. e, PRMT9 protein levels in B-NHL lines relative to PBMC control, (n = 1). f, Kaplan-Meier survival analysis of a cohort (TARGET-AML) after dichotomization for PRMT9 levels below (green, n = 99) or above (red, n = 57) 3.55 log2-transformed intensity. The threshold is discovered via Partitioning Around Medoids (PAM). P value was determined by log-rank (Mantel–Cox) test. ‘n’ represents number of patients. g, h, Pearson’s correlation coefficient of CREB1, STAT3, STAT5A, and GATA2 with PRMT9 levels across TCGA AML cohort (g). Correlation of CREB1 with PRMT9 levels across TCGA DLBCL cohort (h). Statistics were determined by pairwise gene correlation analysis as described by GEPIA. Data was sourced from GEPIA. i, Expression level of Creb1 in LSC (n = 383 cells) and Blast (n = 1444 cells) based on MA9 AML scRNAseq analyses. P value was determined by unpaired two-sided t test. j, PRMT9 levels after CREB1 KD. n = 1. k, Anti-CREB1 and anti-H3K27Ac ChIP-seq analysis in Molm13. l, Upper panel, diagram showing predicted CREB1 binding sites on TSS site in the PRMT9 promoter. Red bars indicate regions representing CBS (CREB1 Binding Site) and Ctrl sites after ChIP assay. Lower panel, one representative result of ChIP-qPCR analysis of enrichment of CREB1 and H3K27Ac at the CBS site and at a distal control site in Molm13 and normal PBSC CD34+ cells. Similar results were generated from three independent experiments. m, n, PRMT9 mRNA levels in human (m) or murine (n) hematopoietic subset as indicated. Human HSCs are from GSE17054; GMP and MEP cells are from GSE11864; monocytes are from GSE11864 and E-MEXP-1242. Muirne Prmt9 levels were normalized to Myeloid (Gr1+Mac1+) Prmt9 mRNA levels and log2-transformed. Data was sourced from Bloodspot. o, Schema of the Prmt9 targeting strategy. The lower left panel shows representative genotyping results of Prmt9 wt, floxed and KO alleles. Lower right panel, Prmt9 protein levels in BM cells from Prmt9 WT and KO mice. p–q, Frequency of hematopoietic progenitors (p) and mature lineage cells (q) in mouse BM at 16 weeks after pIpC administration. (p), Prmt9 WT (n = 6), Prmt9 KO (n = 9); (q), Prmt9 WT (n = 8), Prmt9 KO (n = 15). ‘n’ represents mice number in each group. Data were presented as mean ± SEM. P values were determined by unpaired two-sided t test. r, Competitive transplantation of CD45.2 Prmt9 KO BM cells with normal CD45.1 BM cells in recipient mice, n = 7 mice/group. The percentage of CD45.2 in PB was assessed. Results represent the mean ± SEM. P was determined by two-way ANOVA. Source data

Extended Data Fig. 3. PRMT9 ablation impairs cancer cell survival.

a, Representative image of colonies in MA9-ITD cells (n = 5 biological replicates). Scale bar: 1000 µm. b, Representative plots show the gating for MA9 cells transduced with Dox-inducible shCtrl and shPrmt9. c, d, Prmt9 KD efficiency in indicated cells, were shown (c). CFC of indicated cells after Prmt9 KD (d). Results represent the mean ± SD from 3 independent experiments. e–g, in vitro limiting dilution assay (LDA) assay to evaluate the LSC frequency in Prmt9 KD engineered MA9/FLT3-ITD+ (e), MA9 (f) and CMM (g) AML cells. LSC frequency and p value were calculated using Extreme Limiting Dilution Analysis (ELDA). LSC frequencies are presented as mean ± 95% confidence interval are shown; ELDA were used to analyze χ2 test with 1 degree of freedom. h–k, Cancer lines as indicated were transduced with shPRMT9 to KD endogenous PRMT9. Cell viability (j) by an MTS assay. Apoptosis (k) by Annexin V staining. PRMT9 expression levels were detected (h, i). Results (j, k) represent the mean ± SD from at least 3 (J, n = 4; K, n = 3) independent experiments. l, Primary AML CD34+ cells were transduced with shPRMT9-1 and analyzed for apoptosis as above. m, Schema of PRMT9 inducible KD Molm13 xenografted in NSG mice. Briefly, Molm13 cells were transduced with a DOX-inducible shPRMT9 or control vector and transplanted into mice. Once engraftment was confirmed (>1% in PB), mice were treated with DOX to induce PRMT9 KD. At endpoint, engraftment was evaluated based on percentage of hCD45 cells in BM. In parallel analysis, mouse survival was analyzed. Source data

Extended Data Fig. 4. PRMT9-mediated methylation promotes cancer cell growth.

a, iceLogo motif analysis of R-methyl peptides regulated by PRMT9. b–d, Protein synthesis in normal BM cKit + , MA9-ITD cells (b), B-NHL (Rec1 and OCI-Ly3) (c) and indicated AML lines (d) with shCtrl or shPRMT9. The right panel of (b-d) summarizes the results (n = 3 independent experiments). Data were mean ± SD. e, Indicated gene levels in Molm13 (n = 3 independent experiments). Data were mean ± SD. f, SAMHD1 levels in RNAs extracted from indicated fractions in a ribosome profiling assay. g, h, Spectra of R493, R506 and R481 methylation in LC-MS/MS. i, 293 T were transfected with Flag-tagged PABPC1-CT, then subjected to immunoprecipitation and immunoblot (n = 2 independent experiments). j, Purification of Myc-tagged PRMT9 by the Myc-Trap. Purity-check with indicated PRMTs antibodies, (n = 1). k, In vitro methylation assay of GST-tagged PABPC1-CT mixed with PRMT1 protein, SAM, then analyzed by immunoblot (n = 2 independent experiments). l, In vitro methylation assay of GST-tagged PABPC1-CT mixed with PRMT5/MEP50, SAM, then analyzed by immunoblot (n = 2 independent experiments). m, Ex-vivo tritium methylation assay using PABPC1-WT and -3RK (R481K/R493K/R506K) immunoprecipitated from 293 T (n = 2 independent experiments). n, 293 T were co-transfected with HA-tagged eIF4G plus Flag-tagged PABPC1, then subjected to pull-down and immunoblot (n = 1). o, 293 T were transfected with Flag-tagged PABPC1-CT, then subjected to pull-down and immunblot (n = 2 independent experiments). p, PABPC1 R455/R460 methylation, after PRMT9 KD in Molm13 (n = 2 independent experiments). q, PABPC1 R493 methylation, after PRMT9 overexpression in Molm13 (n = 2 independent experiments). r, PABPC1 methylation, in Molm13 treated with EPZ056544 at 5 µM for 48 hr (n = 2 independent experiments). s, PABPC1 R493 methylation, in Molm13 cells treated with PRMT1i (MS023, 5 µM, 48 hr), PRMT5i (EPZ015666, 5 µM, 96 hr) or PRMT7i (SGC3027, 5 µM, 48 hr). H4R3me2A, HSP70me (IP HSP70 and detected with anti-MMA antibody), SDMA (SYM10 antibody) were positive controls for MS023, SGC3027, and EPZ015666 respectively (n = 2 independent experiments). Source data

Extended Data Fig. 5. PRMT9 expression is correlated with R493 methylation.

a, representative gating strategy of human AML sample excluding T cells and B cells, and sorting CD34+ subset and CD34-CD33+ leukemia blasts (Blasts). After sorting, the purity of LSC and Blast cells were checked again. b, Sorting strategy of six primary AML samples for the LSC and Blasts (n = 6 samples). c, PABPC1 R493me, PRMT9 levels in CD34+ subset and Blasts (n = 7 patient samples). d, Sorting strategy of cKit+ and cKit- indicated leukemia cells from BM of AML developed mice. e, Levels of PABPC1 R493me, Prmt9 in cKit+ and cKit- leukemia cells as indicated (n = 3 independent experiments). Source data

Extended Data Fig. 6. Identification of a PRMT9 inhibitor.

a, Effects of hits (142 from NCI-DTP and 70 from ZINC library) on Molm13 viability. Cells were treated for 4 days with 1 or 5 μM compounds. For each compound, the number represents its library ID. Experiments were triplicated; P values were derived from t tests. b, R493 methylation assay. PABPC1 peptides (G491-T507) were incubated with PRMT9, and SAM. Catalytic activity was assessed by anti-R493 antibody. Synthesized R493me peptide as positive control. c, Catalysis screen of top 20 compounds. PRMT9 protein was pretreated with indicated compound (10 μM), then incubated with PABPC1 peptides and SAM. d, e, Catalytic activity of indicated compounds based on R493 methylation assay (d). Inhibition curves were calculated based on intensity of dots and normalized to DMSO (e). Data is mean ± SD (n = 3 independent experiments). f, g, NSC641396, NSC661221 and NSC645330 share a carbazole ring scaffold. LD2 is designed based on NSC641396 (g). Docking model of NSC641396 in the catalytic pocket (f). h, IC50 analysis of LD2 in B-NHL lines. Cells were treated for 4 days with LD2. Data were mean ± SD (n = 6 independent experiments). i, Substrates of PRMT1 (H4R3me2A, FLT3 R972/R973me), CARM1 (PABPC1 R455/R460me), PRMT5 (H3R8me2S), PRMT7 (HSP70me) were assessed in Molm13 treated 2 days with 2.5 μM LD2 (n = 2 independent experiments). j, Dose-dependent inhibition of PABPC1 R493 methylation level after 2 days of treatment with LD2 in Molm13 cells (n = 1). Cells were treated with a dose titration of 0.5–40 μM LD2 for 48 hr. The inhibition activity of LD2 to PRMT9 (right red curve) was evaluated by calculating the normalized PABPC1 R493 methylation signal. The inhibition activity of LD2 to PRMT5 (right blue curve) was evaluated by calculating the normalized SmB’B’ methylation signal (supplementary Fig. 2). k, Vina docking of LD2 with PRMT9, PRMT5, CARM1 or PRMT7 was performed, and the average docking scores (kcal/mol, with standard deviation) were shown. l, Molecular dynamics simulation analyses of LD2 with PRMT9 or PRMT5. Smaller root-mean-square-fluctuation (RMSF) of ligand (LD2) in the binding pocket of PRMT9 compared with PRMT5 was shown. m, Ctrl and PRMT9 KD Molm13 were treated for 4 days with LD2 (2.5 uM), and viability was assessed. Data were mean ± SD from 4 independent experiments. n, CyTOF of AML MNCs after treatment for 4 days with vehicle or LD2 (2.5 μM). The frequency of CD3 + T cells and CD34+CD45dim AML blasts was noted. o, Relative leukemia (CD34+CD45dim) and T cell frequencies before and after LD2 treatment based on CyTOF from 3 samples. p, Enriched T cells were treated for 4 days with LD2 (2.5 μM) for 4 days, and cell viability was assessed (n = 3 independent experiments). Data were mean ± SD. q, Frequency of PRMT9 high versus low AML samples displaying the Cytotoxic T Lymphocyte (CTL) score high versus low signatures in cohort GSE14468. P value was calculated using two-sided Fisher exact chi-squared test. ‘n’ represents number of patients. Source data

Extended Data Fig. 7. PRMT9 inhibition eradicates AML in vivo.

a, Control or inducible Prmt9 KD MA9-lucifase cells were injected into wild-type B6 (1×10⁶ cells per mouse, n = 5/group). After 30 days when leukemia robustly developed, mice were continuously administered with Dox. Kaplan-Meier curves show survival. P value was determined by log-rank (Mantel–Cox) test. b, MA9 AML burden was assessed by bioluminescence imaging over indicated days and the statistics for the quantitative results on day 30 and day 50 from bioluminescence imaging were shown, n = 5 mice/group. Data are presented as mean ± SEM. P values were determined by unpaired two-sided t test. c, MA9 AML cells we implanted into WT mice and evaluated progression: 1) Ctrl (Prmt9 WT), n = 7 mice; 2) Prmt9 KD, n = 7 mice; 3) Prmt9 KD with T cell depletion, n = 5 mice; 4) Prmt9 KD with NK cell depletion, n = 5 mice. Kaplan-Meier curves show survival. P value was determined by log-rank (Mantel–Cox) test. d, following engraftment, 1 day prior to in-vivo DOX administration to KD Prmt9, mice are administered with anti-CD4/CD8 treatment or anti-NK1.1 to deplete T or NK cells. Plot of the depletion in PB was shown. e, Control or Prmt9 KD MA9-lucifase cells were injected into wild-type B6 mice (1×10⁶ cells per mouse, n = 5). Following engraftment, mice were treated with Dox in drinking water for 7 days. The plot shows the frequency of MA9, CD3 + , B220+ and Gr1 + /Mac1+ cells in BM of each mouse at the time of collection analyzed by flow-cytometry. BM and spleen cells from one representative mouse (with the frequency of each subset highlighted in red) in each group were selected and subjected to scRNA-seq analysis. Data are presented as mean ± SEM. P value of each comparison was determined by unpaired two-sided t test. f, identified populations in MA9 BM. g, Frequency of AML cells in BM. h–j, Different populations (h) and markers (i) identified within Ctrl and Prmt9 KD groups merged in spleen cells. identified spleen populations are shown (j). k, T cell frequency in spleen cells. l, Expression of T cell marker genes in spleen. m, Frequency of Cd44+ cells in BM T cells. n, Frequency of Tregs (Foxp3+) in BM CD4 + T cells. o, Plot of leukemic BM in MA9 rechallenge. p, GSEA analysis of BM T cells. Normalized enrichment scores and family-wise error rate P-value was determined by the GSEA permutation method. The Normalized Enrichment Score is calculated by dividing the Enrichment Score from the actual ranking by the means of the random permutations. An enrichment P-value is calculated by comparing the observed frequency of an annotation term with the frequency expected by chance; individual terms beyond cut-off (p-value ≤ 0.05) are deemed enriched. q–u, (q-r) UMAP (q) and histogram (r) showing Isg15 expression in indicated BM subpopulation; (s-t) UMAP and histogram showing Ifit1 expression in indicated BM population; (u) Cxcl10 expression in indicated BM populations. Ctrl: T cells, n = 249 cells; Monocytes/Macrophages, n = 631 cells; DCs, n = 108 cells; Granulocytes, n = 3378 cells; B cells, n = 413 cells. Prmt KD: T cells, n = 231 cells; Monocytes/Macrophages, n = 469 cells; DCs, n = 57 cells; Granulocytes, n = 3906 cells; B cells, n = 134 cells. For (o, q, r), Data were mean ± SEM. P values were determined by unpaired two-sided t-tests. Source data

Extended Data Fig. 8. Immunity following PRMT9 inhibition requires cGAS activity.

a, Ifit1 expression in AML cells from BM scRNA-seq. b, GSEA analysis of BM T cells. Normalized enrichment scores and family-wise error rate P-value was determined by the GSEA permutation method. c, d, RNA-seq of common DEG in PRMT9 KD versus Ctrl cell lines. Heatmap (c) and GSEA (d) show upregulation of IFNα response genes upon PRMT9 KD. The color code represents z scores for differential gene expression. For (b) and (d), the normalized enrichment scores and family-wise error rate P-value was determined by the GSEA permutation method. The Normalized Enrichment Score is calculated by dividing the Enrichment Score from the actual ranking by the means of the random permutations. An enrichment P-value is calculated by comparing the observed frequency of an annotation term with the frequency expected by chance; individual terms beyond cut-off (p-value ≤ 0.05) are deemed enriched. e, IFI44 expression in Molm13 engineered as indicated (n = 5 independent experiments). Data were mean ± SD. P value was determined by one-way ANOVA. f, g, Levels of selected ISG genes (f) in AML and B-NHL lines after 2 days LD2 treatment (n = 3 independent experiments). Data were presented as mean ± SD. h, Lucia activity of engineered THP1 after LD2 treatment (n = 5 independent experiments). Data were mean ± SD. P value was determined by one-way ANOVA. i, Levels of PRMT9 and Flag-tagged ENPP1 in engineered THP1 cells, (n = 1). j, Levels of γH2AX in THP1 cells, (n = 1). k, dsDNA by immunostaining in THP1 cells after LD2 treatment for 72 hr. Violin plots (right) summarized dsDNA intensity (n = 50 cells/group). Scale bar, 10 μm. l, Immunostaining for γH2AX in Ctrl and PRMT9 KD THP1 cells. Violin plots (right) summarized γH2AX intensity (n = 100 cells/group). Scale bar, 10 μm. m, dsDNA by immunostaining in Ctrl and PRMT9 KD THP1 cells. Violin plots (right) summarized dsDNA intensity (n = 50 cells/group). Scale bar, 10 μm. P values (k,l,m) were calculated by unpaired two-sided t test. n, Levels of cGAS in Ctrl and cGAS-KO MA9 cells (n = 2 independent experiments). o, ENPP1 mRNA levels in the BEAT AML (n = 451 AML cases; n = 19 healthy donors). The P value was by unpaired two-sided t test. p-q, cGAS and ENPP1 expression in AML and B-NHL lines compared with other cancer cell lines. AML (n = 14), B-NHL (n = 9), LC (n = 77), COAD (n = 30), PAAD (n = 20), BRCA (n = 30), PRAD (n = 5), LIHC (n = 14), GBM (n = 12), BLCA, (n = 11) and ALL (n = 8). P values were by unpaired two-sided t test. ‘n’ represents number of cell lines. r, Cd80, Cd86, H2-ab1 expression in monocytes/macrophages from BM, from scRNA-seq. Ctrl, n = 631 cells; Prmt9 KD, n = 469 cells. Data were mean ± SEM. P values were determined by unpaired two-sided t-test. s, Representative spectrum of XRN2 R946 methylation in SILAC-based methyl-peptide quantitative LC-MS/MS. t, 293 T were transfected with Flag-tagged XRN2, then for immunoprecipitation and the interactor detected by immunoblot, (n = 1). γH2AX was detected in the input lysate. Source data

Extended Data Fig. 9. Combining LD2 with an ICI ablates cancers.

a, b, UMAP (a) and histogram (b) showing indicated gene expression in T cells from scRNA-seq of Ctrl (n = 249 cells) and Prmt9 KD (n = 231 cells) BM T cells. Data were mean ± SEM. P values were unpaired two-sided t-test. c, Frequency of PD-L1+ cells among leukemic subset (CD34+CD45dim) following treatment LD2 (2.5 µM, 4 days), n = 3 samples. d, e, CyTOF of AML MNCs after indicated treatment. The frequency of CD3 + T and CD34+CD45dim AML blasts were noted (d). Relative leukemia (CD34+CD45dim) cell frequencies of (d and right panel of Fig. 4m) were shown in (e). f, Prmt9 levels in normal mouse PBMCs or A20 (n = 2 independent experiments). g, h, Balb/C mice were inoculated with A20 cells (n = 5 mice/group). Tumor bearing mice were treated with isotype control (VEH), anti-PD1 mAb (10 mg/kg/i.p./q.o.d./2 wks), LD2 (100 mg/kg/ i.t./q.d./2 wks) or combination (n = 5 mice/group). Tumor volume was monitored (g) and pictures were acquired (h). i, Weight of A20 tumors in each group (n = 5 mice/group); j, k, NSGS mice (n = 10 mice) were inoculated with A20 cells. Tumor bearing mice were treated with vehicle or LD2 (100 mg/kg/i.t./ q.d./2 wks, n = 5 mice/group). Final tumor weights are shown (k). Data (g, i, j,k) were mean ± SEM. P values were determined by two-way ANOVA (g, j), one-way ANOVA (i) or unpaired two-sided t test (k). l, Levels of R493 methylation in A20 cells treated with LD2 (n = 2 independent experiments). m, n, Levels of Cd274 (m) and mIfit1 (n) cells in A20 treated with LD2 (n = 3 independent experiments). Data were mean ± SD. o, Representative image of anti-mCD3 IHC staining in indicated groups. Scale bar, 100 μm. p, Quantification of CD3 T cells, based on the number of cells per gram of tumor in indicated treatment groups (n = 5 mice/group). Data are presented as mean ± SD. P values were determined by one-way ANOVA. q, r, Representative image of anti-mCD8. Scale bar, 100 μm. (r) Quantification of CD8 T cells in indicated groups (n = 5 mice/group). s–v, Representative plots showing IFNγ (s) and GZMB (u) expression in CD8 + T cells in indicated treatment groups. Quantification of IFNγ + (t) and GZMB + (v) cells among CD8 + T cells in indicated groups (t, v- n = 5 mice/group). Data (r, t, v) are mean ± SEM. P values (r, t, v) were determined by one-way ANOVA. Source data

Extended Data Fig. 10. Humanized AML mouse model establishment.

a, b, Two million AML MNCs were implanted intra-femorally into an irradiated MHCI/II1/2 double-KO (DKO) NSG or regular NSG mice (n = 3 mice/group). After transplant, engraftment of CD3 + T cells, CD33+ cells in PB were monitored for 12 weeks. Then, BM engraftment of human hematopoietic subsets including T cells, monocytes and DCs, as well as the immature CD33 + CD34+CD45dim subset were assessed. Data were mean ± SEM. c, Representative gating of hematopoietic subsets from DKO mouse BM. d, Single sample gene set enrichment analysis (ssGSEA) of AML trial dataset GSE183415 which contains samples with clinical responses to PD-1 inhibitors (Complete Response [CR, n = 10] vs. No Response [NR, n = 12]) using the following gene signature: PRMT9 KD signature, 390 ISG signature, Reactome DNA repair signature, Reactome Double strand break repair signature, and Reactome G2M DNA damage checkpoint signature. ‘n’ represents patient numbers in indicated group. Data are presented as mean ± SD, statistical comparisons were performed using unpaired two-sided t-test, ‘ns’ indicates no significance. e, Proposed model. f, ‘REACTOME’ signature ‘DNA Repair’, ‘DNA Double Strand Break Repair’, ‘G2M DNA Damage Checkpoint’ plots of GSEA of MA9 AML cells (Prmt9 KD vs. Ctrl). g, 390 ISGs genes signature plot of GSEA of BM DCs (Prmt9 KD vs. Ctrl). h, ‘Hallmarks’ signature Interferon Gamma Response plot of GSEA of BM T cells (Prmt9 KD vs. Ctrl). i, ‘Hallmarks’ signature Interferon Gamma Response plot of GSEA of BM MA9 AML cells (Prmt9 KD vs. Ctrl). For GSEA analysis (g-i), Normalized enrichment scores and family-wise error rate P-value was determined by the GSEA permutation method. The Normalized Enrichment Score is calculated by dividing the Enrichment Score from the actual ranking by the means of the random permutations. An enrichment P-value is calculated by comparing the observed frequency of an annotation term with the frequency expected by chance; individual terms beyond cut-off (p-value ≤ 0.05) are deemed enriched. Source data