Targeting an RNA-Binding Protein Network in Acute Myeloid Leukemia

Abstract

RNA-binding proteins (RBPs) are essential modulators of transcription and translation frequently dysregulated in cancer. We systematically interrogated RBP dependencies in human cancers using a comprehensive CRISPR/Cas9 domain-focused screen targeting RNA-binding domains of 490 classical RBPs. This uncovered a network of physically interacting RBPs upregulated in acute myeloid leukemia (AML) and crucial for maintaining RNA splicing and AML survival. Genetic or pharmacologic targeting of one key member of this network, RBM39, repressed cassette exon inclusion and promoted intron retention within mRNAs encoding HOXA9 targets as well as in other RBPs preferentially required in AML. The effects of RBM39 loss on splicing further resulted in preferential lethality of spliceosomal mutant AML, providing a strategy for treatment of AML bearing RBP splicing mutations.

Keywords: AML; CRISPR; DCAF15; RBM39; RNA-binding proteins; alternative splicing; leukemia; spliceosome; sulfonamides.

Figure. 1:. A CRISPR Domain-Targeted Screen Identifies RBP Dependencies in AML.

(A) Volcano plot of differentially expressed classical RBPs (483 genes) in AML patient samples (n=195) compared to normal human CD34⁺ hematopoietic stem and progenitor cells (n=28). Blue vertical lines indicate log₂ fold-change (FC)= 0.5/−0.5 cutoff (p value > 0.05). (B) Schematic depicting pooled RBP-focused CRISPR screen. RBD, RNA-binding domains. RRM, RNA- recognition motifs; zf, zinc finger; DEAD, DEAD-box ATPase; KH, KH-homology; dsRBD, double-stranded RBD; PWI, PWI-motif; CSD, Cold-shock; La, La-motif; PUA, PseudoUridine synthase and Archaeosine transglycosylase; R3H, R-x3-H domain. (C) Scatter plot comparison of CRISPR RBP-domain screen in AML and T-ALL, T-cell acute lymphoblastic leukemia. Plotted is the log₂ fold change of sgRNA abundance (day 4/ day 20) for each cell line. Each dot represents the average of all sgRNAs targeting a RBP. Red dots indicate RBPs that are significantly overexpressed in AML and preferentially required in AML. (D) Scatter plot representation of CRISPR RBP-domain screen in AML and LUAD, lung adenocarcinoma. Red dots indicate RBPs that are significantly overexpressed in AML and preferentially required in AML. (E) Fold-change (day 4/day 20) in sgRNA abundance in pooled RBP-focused negative selection screen in MOLM-13 AML cells. Red dots indicate RBPs that are overexpressed in AML patients and exhibit greater than 3-fold depletion in CRISPR negative selection screen. Each dot represents the average of all sgRNAs targeting a RBP. (F and G) Screen validation of RBP candidates using a competition-based proliferation assay in MOLM-13 (F) and THP-1 (G) AML cell lines. Plotted are GFP percentages measured during 20 days in culture and normalized to Day 4. Negative control, sgRosa and two independent sgRNAs targeting each RBP are shown in the graphs (mean ± SD, n=3). See also Figure S1 and Table S1.

Figure 2:. RBM39 is Required to Sustain AML Growth In Vitro and In Vivo.

(A) Violin plot of RBM39 normalized expression in AML patients (red) and normal human CD34⁺ hematopoietic stem and progenitor cells (blue). Horizontal line inside the box represent the Mean, 25^th-75^th percentiles, showing all data points. Statistical analysis was performed using Wilcoxon Rank Sum test. (B) CRISPR mutagenesis of RBM39 exons in MOLM-13 AML cells using a competition-based assay. Green boxes represent annotated RNA-binding domains of RBM39. (C) Bioluminescent images of mice transplanted with MLL-AF9 Nras^G12D cells transduced with sgRosa (n=4) or two independent sgRbm39 (n=7/group). Representative images of 3 mice per sgRNA construct is shown. The same mice are depicted at each time-point. (D) Quantification of bioluminescent imaging in sgRosa negative control and two independent sgRbm39 at the indicated time points. Box-and-whisker plot, Min. to Max. show all points, 25^th-75^th percentiles, Median (horizontal line). Statistical analysis was performed using unpaired Student’s t test by Prism Graphpad (**p< 0.01, ***p< 0.001). (E) Flow cytometry analysis of GFP positive sgRNA- expressing leukemia cells in peripheral blood of MLL-AF9 Nras^G12D leukemia recipient mice at indicated time points. Box-and-whisker plot, Min. to Max. show all points, 25^th-75^th percentiles, Median (horizontal line). Statistical analysis was performed using unpaired Student’s t test by Prism Graphpad (*p< 0.05, **p< 0.01). (F) Kaplan-Meier survival curves of recipient mice transduced with sgRosa negative control and two independent Rbm39 sgRNAs are plotted. The p values were determined using Log rank Mantel-Cox test. Data with statistical significance are as indicated, *p< 0.05, **p< 0.01, ***p< 0.001. See also Figure S2.

Figure 3:. Mass Spectrometry of RBM39 Proteome Identifies an RBP Network Required in AML.

(A) Silver staining of endogenous RBM39 immunoprecipitation (IP) in MOLM-13 cells. Arrowhead represents predicted size of RBM39, asterisks denotes non-specific IgG heavy/light chain signals, and M= protein marker. (B) Overlay of peptide-spectrum match (PSM) counts between IgG control and RBM39 IP (left panel). Top RBM39-interacting partners enriched over IgG (right panel). (C) STRING network analysis of RBM39 protein interactome in AML. Black circle, RBM39; red circles, spliceosome complex; blue circles, ribosome, and green circles, ribosome biogenesis. (D) Plot represents data from MOLM-13 AML screen as shown in Figure 1E. Green denotes RBP genes identified in RBM39 IP-MS and overexpressed in TCGA AML patients. Dotted line represents 2-fold depletion. See also Figure S3 and Table S2.

Figure 4:. RBM39 Loss Alters Splicing of mRNAs Essential for AML Cell Growth.

(A) Number of differentially spliced events in MOLM-13 and THP-1 cells treated with RBM39 small- guide RNA (sgRNA) versus control sgRNA. (B) Scatter plot of cassette exons promoted (red circles) and repressed (blue circles) in MOLM13 treated with RBM39 sgRNA versus control sgRNA. Splicing is quantified using a ‘percent spliced in’ value (PSI, or ψ value). Promoted and repressed cassette exons are defined as those whose inclusion levels are increased or decreased by Ψ ≥10%, respectively. Grey circles represent exons for where ΔΨ is <10%. (C) eCLIP analysis of RBM39 binding sites in MOLM-13 cells. Input-normalized peak signals are shown as log₂ fold change. Purple points indicate eCLIP enriched RBM39 peaks (FDR >5%, logFC>1) in biological replicates. (D) Genomic distribution of RBM39 eCLIP-seq peaks. (E) Metagene profile of RBM39-binding sites on exons throughout the transcriptome in MOLM-13 cells relative to 5’ and 3’ splice sites. Grey box represents exonic region. (F) Enriched motifs for RBM39 binding. The top ten motifs are shown. Inset, consensus sequence, deduced from the top 10 motifs. (G) Gene Ontology (GO) enrichment analysis of terms enriched in differentially spliced mRNAs in MOLM13 cells with RBM39 sgRNA versus control. Terms in red font are related to RNA processing and/or splicing. (H) Enrichment plots from Gene Set Enrichment Analysis (GSEA) of HOXA9 target genes that are differentially spliced upon RBM39 knockout versus control sgRNA (normalized enrichment score (NES) is inferred from permutations of the gene set and the false discovery rate (FDR)). (I) RNA-seq coverage plot of BMI1 and Sashimi plots of GATA2 in MOLM-13 cells treated with RBM39 sgRNA or control overlaid with anti-RBM39 eCLIP-seq tracks. Yellow highlighted reads in GATA2 Sashimi plots highlight exon skipping events with RBM39 sgRNA. See also Figure S4 and Tables S3–S5.

Figure 5:. Pharmacological Inhibition of RBM39 Displays Broad Sensitivity Across Diverse AMLs.

(A) Violin plot of DCAF15 normalized counts from RNA-seq in AML patients (red) and normal human CD34⁺ hematopoietic stem and progenitor cells (blue). Horizontal line inside the box represent the Mean, 25^th-75^th percentiles, showing all data points. Statistical analysis was performed using Wilcoxon Rank Sum test. (B) Western blot of K562 whole cell lysates treated with escalated doses of indisulam after 48 hr. (C) Representative flow cytometry plots of Annexin V staining performed on MOLM-13 AML cells treated with 500 nM of indisulam or DMSO for 48 hr. (D) Representative flow cytometry plots showing EdU cell cycle analysis of MOLM-13 AML cells exposed to 500 nM indisulam or DMSO for 48 hr. (E) Bioluminescent imaging of mice transplanted with MOLM-13 luciferase cells and treated with vehicle or 25 mg/kg indisulam. Representative images of 5 mice/group are shown. The same mice are depicted at each time-point. (F) Quantification of bioluminescent signals in vehicle versus indisulam-treated leukemic mice at indicated time-points (n=6/group). Unpaired Student’s t test using Prism 7 (Graphpad). Box-and-whisker plot, Min. to Max. show all points, 25^th-75^th percentiles, Median (horizontal line). Statistical analysis was performed using unpaired Student’s t test by Prism Graphpad (**p< 0.01). (G) Flow cytometry analysis of human MOLM-13 cells in peripheral blood in DMSO versus indisulam groups. Gating was performed on human CD45⁺ in DMSO (n=4–5) and Indisulam (n=5) treated mice. Box-and-whisker plot, 25^th-75^th percentiles, Min. to Max. show all points, Median (horizontal line). Statistical analysis was performed using unpaired Student’s t test by Prism Graphpad (*p< 0.05, **p< 0.01). (H) Kaplan-Meier overall survival of indisulam-treated AML mice. Grey represents the exposure time (days) to indisulam. The p values were calculated using Log rank Mantel-Cox test. (I) Schematic of patient-derived xenograft (PDX) generation and treatment with indisulam (25 mg/kg/day). Patient 1 PDX 1 received 3 days of drug treatment. (J) Percentage of human CD45⁺ (hCD45) cells amongst total live bone marrow mononuclear cells by flow cytometry analysis of bone marrow aspirate pre- and post-treatment with indisulam in five primary AML PDX models from 3 patients. Red indicates SF3B1 mutant patients. (K) Representative flow cytometry plots of CD45 in Patient 2 PDX and Patient 3 PDX 2 bone marrow aspirates pre- and post-indisulam treatment. Percentage of hCD45⁺ cells is indicated. Data with statistical significance are as indicated, *p< 0.05, **p< 0.01, ***p< 0.001. See also Figures S5 and S6.

Figure 6:. Preferential Effects of Sulfonamides on Spliceosomal Mutant AML.

(A) Log₁₀ of IC₅₀ values of a panel of human AML cell lines to E7820 annotated for spliceosomal gene mutation status (red versus black bars indicate spliceosomal mutant or wild-type cells respectively) as determined by the CellTiterGlo assay. IC₅₀ values are calculated from at least 4 technical replicates per experiment, each experiment was performed at least twice, and error bars represent standard deviation. Also shown are log₁₀ relative DCAF15 mRNA expression levels for each cell line. (B) Bar plots of IC₅₀ values of isogenic K562 cells with or without knockin of spliceosomal gene mutations to E7820 (left) or indisulam (right). IC₅₀ values calculated from four technical replicates per experiment; one representative experiment of three biological replicates is shown; error bars represent standard deviation. Statistical analysis was performed using unpaired Student’s t test by Prism Graphpad (*p< 0.05). (C) Bar plots of IC₅₀ values of isogenic NALM-6 cells with or without knockin of spliceosomal gene mutations to E7820 (left) or indisulam (right). IC₅₀ values calculated from one representative experiment of three biological replicates is shown; error bars represent standard deviation). Statistical analysis was performed using unpaired Student’s t test by Prism Graphpad (*p< 0.05). (D) Anti-RBM39 Western blot of isogenic K562 cells treated with DMSO or increasing doses of E7820 for 24 hr. Densitometry quantification of Western blot is shown on the right. (E) Competition assay of RBM39 shRNA knockdown in K562 parental vs. K562 SF3B1^K700E. Analysis was determined by unpaired Student’s t test. Data with statistical significance of mutant cells relative to control are as indicated, (**p< 0.01, ***p< 0.001).

Figure 7:. RBM39 Degradation Targets an RBP Network Required for AML Survival.

(A) Schema of drug testing (6 hours) across isogenic K562 cells with or without heterozygous knockin of the SF3B1^K700E or SRSF2^P95H mutation at the endogenous locus for RNA-seq. Each experiment was perform in duplicates. (B) Violin plot of inclusion level differences of introns or cassette exons in SF3B1^K700E/WT cells treated with DMSO versus SF3B1^WT cells treated with DMSO (“DMSO” column) or SF3B1^K700E/WT cells treated with E7820 or E7107 versus the same cells treated with DMSO. Horizontal line inside the box represent the Mean, 25^th–75^th percentiles, points are “outliers” (out of the 99^th percentile line). Statistical analysis was performed using Wilcoxon Rank Sum test. (C) Bar plot of number of differential splicing events across parental, SF3B1^K700E/WT, and SRSF2^P95H/WT K562 cells. The numbers above each bar indicate number of differentially spliced events. (D) RNA-seq coverage plots of SUPT6H, HNRNPH1, SRSF10, and U2AF2 in the isogenic K562 cells from (A). See also Figures S7.