Therapeutic Targeting of RNA Splicing Catalysis through Inhibition of Protein Arginine Methylation

Abstract

Cancer-associated mutations in genes encoding RNA splicing factors (SFs) commonly occur in leukemias, as well as in a variety of solid tumors, and confer dependence on wild-type splicing. These observations have led to clinical efforts to directly inhibit the spliceosome in patients with refractory leukemias. Here, we identify that inhibiting symmetric or asymmetric dimethylation of arginine, mediated by PRMT5 and type I protein arginine methyltransferases (PRMTs), respectively, reduces splicing fidelity and results in preferential killing of SF-mutant leukemias over wild-type counterparts. These data identify genetic subsets of cancer most likely to respond to PRMT inhibition, synergistic effects of combined PRMT5 and type I PRMT inhibition, and a mechanistic basis for the therapeutic efficacy of PRMT inhibition in cancer.

Keywords: AML; Arginine methylation; MDS; MS023; PRMT1; PRMT5; SF3B1; SRSF2; Splicing factor mutations; U2AF1.

Figure 1.. Spliceosomal Interacting Proteins Are Targetable Vulnerabilities in Spliceosomal Mutant Cells

(A) Molecular interaction network generated by Cytoscape 3.4.0 (Shannon et al., 2003) displaying proteins involved in RNA splicing, snRNP assembly, and/or mutated in acute myeloid leukemia (AML) and their nearest neighbors of a given physical entity (e.g., genes or proteins). Genes and proteins are illustrated by nodes, which are connected by lines to nodes based on physical or functional interaction. Nodes that are RNA SFs mutated in cancer are displayed in yellow while those that are druggable targets are indicated in red. (B) Heatmap of the relative viability of (MLL-AF9/Vav-cre Srsf2^WT and MLL-AF9/Vav-cre Srsf2^P95H to the indicated compounds following 7 days of growth scored by MTS assay and reported as a ratio to control DMSO-treated cells. Blue indicates a reduction, while red indicates an increase in cell viability, relative to DMSO-treated cells. The experiment was conducted in biological triplicate, and each individual run was repeated in technical triplicate. See also Table S1.

Figure 2.. Preferential Effects of PRMT5 or Type I PRMT Inhibition on SF-Mutant AML over WT Counterparts In Vitro

(A–C) Relative cell viability of (MLL-AF9/Vav-cre Srsf2^WT and MLL-AF9/Vav-cre Srsf2^P95H treated with GSK591 (A), MS023 (B), or E7107 (C), and normalized to control (SGC2096a for PRMT5, MS094 for PRMT1, and DMSO for E7107). Samples were prepared in four to six replicates and averages were calculated, error bars represent SD. Student’s t test was used for statistical analysis. (D) Relative viable cell counts of AML patient samples to GSK591 based on spliceosomal gene mutation status. Primary AML cells with SF-mutations (n = 16) or WT for SRSF2, U2AF1, and SF3B1 (n = 16) were incubated with DMSO or GSK591 (0.5 mM) for 6 days. Cells were subjected to flow cytometry to detect 7-AAD-negative, YO-PRO1-negative, viable cells. Relative viable cell numbers were compared with Welch’s t test. Boxplot top line of whisker denotes the highest value in dataset and bottom line of whisker denotes the lowest value in dataset, box spans interquartile range and line in box indicates median. *p = 0.01–0.05, **p = 0.001–0.01, ***P = 0.0001–0.001, ***p < 0.0001. See also Figure S1 and Table S2.

Figure 3.. Preferential Effects of PRMT5 or Type I PRMT Inhibition on SF-Mutant AML over WT Counterparts In Vivo

(A and B) Kaplan-Meier survival curve of mice treated with vehicle or EPZ015666. Survival comparison by Mantel-Cox log-ranked test (WT vehicle n = 12; WT drug n = 13, Srsf2^P95H vehicle n = 14, Srsf2^P95H drug n = 8) (A). Western blot of PRMT5, symmetric dimethyl arginine (SDMA; both a short exposure and a long exposure are shown), and actin in spleens of mice from (A) at time of death. Organs collected were 24 h after the last dose. Each column represents tissue from a distinct individual representative animal (B). (C and D) Kaplan-Meier survival curve of mice treated with vehicle or MS023. Survival comparison by Mantel-Cox log-ranked test (WT vehicle n = 9, WT drug n = 9, Srsf2^P95H vehicle n = 9, Srsf2^P95H drug n = 11). (C) Western blot of PRMT1, asymmetric dimethylarginine (ADMA) (both a low exposure and a high exposure are shown), SDMA, and actin in spleens of mice from (C) at time of death. Organs collected were 24 h after the last dose. Each column represents tissue from a distinct individual representative animal (D). *p = 0.01–0.05, **p = 0.001–0.01, ***p = 0.0001–0.001, ****p < 0.0001. See also Figure S2.

Figure 4.. Synergistic Effects of Combined PRMT5, Type I PRMT, and/or SF3B Inhibition on Primary Mouse AMLs

(A–C) Heatmap of half maximal inhibitory concentration values of WT or Srsf2^P95H MLL-AF9 cells grown in increasing doses of MS023 plus GSK591 (A), MS023 plus E7107 (B), or GSK591 plus E7107 (C) for 7 days. The combination index (Cl) in each cell type for each drug pair is shown. (D) Kaplan-Meier curve of mice transplanted with MLL-AF9 cells followed by combined treatment with MS023 plus EPZ015666 in vivo. Survival comparison by Mantel-Cox log-ranked test (WT vehicle n = 11, WT drug n = 12, Srsf2^P95H vehicle n = 15, Srsf2^P95H drug n = 16). *p = 0.01–0.05, **p = 0.001–0.01, ***p = 0.0001–0.001, ****p < 0.0001. See also Figure S2.

Figure 5.. Synergistic Effects of Combined PRMT5, Type I PRMT, and/or SF3B Inhibition on Human AML Lines and Patient Samples

(A) Summary of Cl and fraction affected (Fa) values of human AML cell lines treated with MS023 and GSK591. (B and C) Percentage of viable THP1 (B) and U937 (C) cells after 8 days exposure to MS023, GSK591, or the combination relative to DMSO in vitro. (D) Hematopoietic progenitor cells differentiated from the iPSC lines 5–16 Cre20 (SRSF2 P95L) and N-2.12 (isogenic normal) were treated as indicated. Cell viability represents cell counts relative to DMSO-treated cells. (E) Heatmap of cell viability after 6 days of treatment. Cl at IC₅₀ values of MS023 plus GSK591 is indicated at the bottom. (F and G) Percentage of viable K562 WT or SRSF2^P95H knockin mutant cells (F) and K562 WT or SF3B1^K700E knockin mutant cells (G) after 8 days treatment with MS023, GSK591, or the combination relative to DMSO in vitro. (H) Percentage of viable K562 WT or SRSF2^P95H knockin mutant cells after 8 days treatment with MS023, GSK591, E7107 or in combination relative to DMSO in vitro. All data are representative of three independent experiments, error bars represent SD. *p = 0.01–0.05, **P = 0.001–0.01, ***P = 0.0001–0.001, ****p < 0.0001; one-way ANOVA was used to analyze THP1 and U937, while unpaired t test was used for K562 WT and SRSF2^P95H. See also Figures S3 and S4.

Figure 6.. Global Profiling of PRMT Substrates at Single-Site Resolution by Quantitative Liquid Chromatography-Tandem Mass Spectrometry

(A) Workflow of the SILAC methyl-R-proteomic experiments carried out to identify mono- and dimethylarginine substrates regulated by GSK591 and MS023 in NB4 leukemia cells. (B and C) Sequence motif analysis indicates the consensus sequences significantly enriched in the methyl-peptides regulated by MS023 (B) or GSK591 (C). (D) Heatmap of log₂ SILAC ratios of each methyl-peptide identified and quantified from the SILAC experiment diagrammed in (A). For each pharmacological treatment two biological replicates, in forward and reverse SILAC mode, were carried out and different degrees of methylation were enriched and profiled. Only peptides reproducibly regulated in at least one pair of forward-reverse experiment are visualized in the heatmap. (E and F) Network analysis of the proteins displaying methylation changes upon treatment with MS023 (E) or GSK591 (F). RNA-binding proteins are highlighted in red. (G and H) Functional analysis of changing methylated proteins highlight the biological process terms significantly enriched among proteins displaying methylation changes upon treatment with MS023 (G) and GSK591 (H). See also Figure S5 and Table S3.

Figure 7.. Gene Expression Changes and Cell-Cycle Deregulation with PRMT5 and Type I PRMTs Inhibition

(A and B) GO categories of genes upregulated in K562 WT cells (A) and K562 SRSF2^P95H cells (B) upon MS023 and GSK591 combination treatment. (C) Cell-cycle analysis of K562 WT and SRSF2^P95H cells upon MS023 and GSK591 treatment. (D) Caspase-Glo 3/7 changes following 8 days treatment with MS023 and GSK591 in K562 WT and SRSF2^P95H cells. All data are representative of three independent experiments, error bars represent SD. *p = 0.01–0.05, **p = 0.001–0.01, ***p = 0.0001–0.001, ****p < 0.0001. Student’s t test used for statistical analysis.

Figure 8.. PRMT5 and Type I PRMT Inhibition Leads to Synergistic Changes in Alternative Splicing

(A) Bar graphs enumerating numbers of significantly differentially spliced events in WT or SRSF2^P95H K562 cells treated with each of the compounds listed versus DMSO control. A3SS, alternative 3′ splice site; A5SS, alternative 5′ splice site; MXE, mutually exclusive exon; RI, retained intron; SE, cassette exon. (B) Bar graphs enumerating numbers of significantly differentially spliced exons in WT or Srsf2^P95H K562 cells treated with each of the compounds listed versus DMSO control. (C) Changes in levels of splicing events with CCNG, GCNG, CGNG, or GGNG motifs upon MS023 and GSK591 treatment in K562 cells. (D) Heatmap showing change in PSI of exon splicing events normalized to PSI of WT control within the “Cell Cycle” gene ontology category. (E) RT-PCR data showing changes in PSI levels of EZH2 poison exon inclusion in K562 cells with or without knockin of SRSF2^P95H upon MS023 and GSK591 treatment in vitro. (F) Western blot showing changes in EZH2 protein levels upon MS023 and GSK591 treatment in vitro. All data are representative of three independent experiments, error bars represent SD. *p = 0.01–0.05, **p = 0.001–0.01, ***p = 0.0001–0.001, ****P < 0.0001. Student’s t test used for statistical analysis. See also Figures S6 and S7 and Table S5.