U2AF1 mutations induce oncogenic IRAK4 isoforms and activate innate immune pathways in myeloid malignancies

Abstract

Spliceosome mutations are common in myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML), but the oncogenic changes due to these mutations have not been identified. Here a global analysis of exon usage in AML samples revealed distinct molecular subsets containing alternative spliced isoforms of inflammatory and immune genes. Interleukin-1 receptor-associated kinase 4 (IRAK4) was the dominant alternatively spliced isoform in MDS and AML and is characterized by a longer isoform that retains exon 4, which encodes IRAK4-long (IRAK4-L), a protein that assembles with the myddosome, results in maximal activation of nuclear factor kappa-light-chain-enhancer of B cells (NF-κB) and is essential for leukaemic cell function. Expression of IRAK4-L is mediated by mutant U2 small nuclear RNA auxiliary factor 1 (U2AF1) and is associated with oncogenic signalling in MDS and AML. Inhibition of IRAK4-L abrogates leukaemic growth, particularly in AML cells with higher expression of the IRAK4-L isoform. Collectively, mutations in U2AF1 induce expression of therapeutically targetable 'active' IRAK4 isoforms and provide a genetic link to activation of chronic innate immune signalling in MDS and AML.

Figure 1.. Differential RNA isoform usage correlates with AML prognosis and oncogenic IRAK4 isoforms.

(A) Scatterplot of cumulative and isoform variances of genes in AML samples from TCGA (n = 160). Every point represents a gene, colored by the expression correlation of the most negatively correlated pair of isoforms for that gene. Blue-colored genes have at least one pair of isoforms with mutually exclusive expression pattern. (B) Hierarchical clustering analysis and relative expression of mRNA isoforms in AML samples. Relative expression is such that a value of 1 indicates that the isoform is the only isoform expressed for the given gene, and 0 indicates that the isoform is not expressed. (C) Pathway analysis of genes in Group 2 (from Figure 1B and Supplemental Figure 1C) associated with worse clinical outcome (n = 347 genes) determined by hypergeometric distribution test. (D) Kaplan-meier analysis of AML patients stratified on IRAK4-L (exon 4 included) or IRAK4-S (exon 4 excluded) expression. (E) Exon architecture of IRAK4 and protein domains (below). Sashimi plots represent junction reads in representative AML samples. Exon 4 reads were normalized to the total number of reads in each sample and plotted as a ratio (inclusion/total and exclusion/total). (F) Cumulative expression of IRAK4-L and IRAK4-S in individual AML patients (TCGA). Patients are ordered according the relative expression of IRAK4-L versus IRAK4-S. (G) Relative expression of IRAK4-L to IRAK4-S in normal BM (n = 4) and AML samples (TCGA; n = 160) is shown as box plots including the center (mean) and top and bottom quartiles. Wilcoxon Test, P = 0.07. (H) RT-PCR of IRAK4-L/S using primers flanking exon 4. Densitometric quantification of IRAK4 exon 4 inclusion calculated as the ratio of IRAK4-L versus both isoforms is shown below (three independent experiments). (I) Immunoblot of IRAK4 using an N-terminal antibody that recognizes IRAK4-L and a C-terminal antibody that recognizes IRAK4-L and IRAK4-S (three independent experiments). (J) Immunoblot of IRAK4 in AML patient samples (AML1 and AML2) and healthy samples (cord blood (CB), bone marrow CD34+ (BM-CD34+), BM mononuclear cells (BM-MNC)). The healthy samples are collected from independent donors. The percent of IRAK4-L of total IRAK4 isoforms is shown below.

Figure 2.. IRAK4-L results in Myddosome assembly and maximal activation of innate immune signaling.

(A) Pathway analysis of enriched genes in AML patients preferentially expressing IRAK4-L (top) or IRAK4-S (bottom). The pathway scores were calculated by NetWalker based on a random walk method. The scores directly reflect the enrichment of the indicated pathways for high (positive scores) or low (negative scores) expression of the IRAK4 Long isoform. The pathways shown here were the highest-scoring pathways. (B) HEK293 cells transfected with vector (pcDNA3.1), or FLAG-tagged IRAK4-L or IRAK4-S for 24 hours and immunoblotted for the indicated NF-κB and MAPK proteins (three independent experiments). (C) Densiometric analysis of panel (B). One-sided t-tests were used for statistical analyses. (D-E) NF-κB (D) and AP1 (E) activation was measured by κB-site and AP1-site containing reporter assays in HEK293 cells transfected with empty vector, IRAK4-L, or IRAK4-S. Values are normalized to Renilla-luciferase and empty vector (1.0) (3 or 6 independent experiments, respectively). Two- sided t-tests were used for statistical analyses. (F) Representative PCR and immunoblot of THP1 cells following knockdown of IRAK4-L and IRAK4-S (shIRAK4-L/S) or just IRAK4-L (shIRAK4-L) (three independent experiments). (G) NF-κB activation was measured in THP1 cells stably expressing a κB-site containing reporter and shRNAs targeting IRAK4-L (left) or IRAK4-L/S (right) after stimulation with PAM3CSK4 (+, 500 ng/ml or ++, 1000 ng/ml). (3 or 5 independent experiments, respectively). Two-sided t-tests were used for statistical analyses. (H) Overview of IRAK4-L and IRAK4-S downstream signaling based on Figure 2A–E. (I) Sequence-based prediction of protein-protein and domain-domain interactions using all isoforms of IRAK1, IRAK4, and MyD88. Superscripts indicate distinct RNA isoforms encoding the indicated long or short proteins. (J) HEK293 cells transfected with HA-MyD88 and either FLAG-IRAK4-L or FLAG-IRAK4- S were immunoblotted for HA-MyD88 after immunoprecipitation of FLAG. (representative of two independent experiments). (K) TF1 and HL60 cells were immunoblotted for IRAK4 with C-terminal antibody after immunoprecipitation of MyD88 (representative of two independent experiments). *, indicate IgG bands. All data represent the mean ± s.e.m.

Figure 3.. IRAK4-L is required for leukemic cell function.

(A) Overview of experimental design. (B) Colony formation of THP1 cells expressing shIRAK4-L, shIRAK4-L/S, or a control shRNA (shControl) (three independent experiments). Two-sided t-tests were used for statistical analyses. (C-D) THP1 cells expressing shIRAK4-L (n = 9 animals), shIRAK4-L/S (n = 5 animals), or a control shRNA (shControl; n = 6 animals) were engrafted into sublethally-irradiated NSG mice. After 6 weeks, leukemic burden (GFP+ cells) in the BM (C) and PB (D) was determined. Two-sided t-tests were used for statistical analyses. (E) Cord-blood CD34+ cells were transduced with shIRAK4-L, shIRAK4-L/S, or a control shRNA (shControl) and examined for IRAK4 knockdown (left; two independent experiments) and progenitor colony formation in methylcellulose (right; six independent experiments). Two-sided t-tests were used for statistical analyses. (F) THP1 cells transduced with empty retroviral vector (MSCV-IRES-GFP as a control) or THP1 IRAK4-KO cells transduced with retroviral vectors encoding IRAK4-L (left panel) or IRAK4-S (right panel) and then immunoblotted with the N-terminal IRAK4 antibody (left panel) and C-terminal IRAK4 antibody (right panel). (G-H) Representative images of parental and IRAK4-KO THP1 cells examined for leukemic progenitor function in methylcellulose (three biological replicates). Scale bar, 700 microns. Two-sided t-tests were used for statistical analyses. (I) Colony formation of MDS/AML cell lines and control CD34+ cells (2 independent donors) treated with DMSO or CA-4948 for 7–10 days (three independent experiments). Two-sided t-tests were used for statistical analyses. (J) Graph representing the IC50 relative to the ratio of IRAK4-L to IRAK4-S expression in the indicated cell lines and CD34+ cells summarized from Figure 1I and 3I. (K) THP1 cells were xenografted in NSG mice (n = 10) and treated with IRAK4 inhibitor (CA-4948) or vehicle at 12.5 mg/kg/5d/week for 5 weeks. (L) Mice xenografted with THP1 cells were assessed for leukemic engraftment in BM, and spleen and liver weight. Two-sided t-tests were used for statistical analyses. (M) Representative spleens are shown isolated from mice xenografted with THP1 cells at time of death after treatment with vehicle or CA-4948. All data represent the mean ± s.e.m.

Figure 4.. IRAK4-L expression is associated with U2AF1 mutations in MDS and AML.

(A) Heatmap was generated based on the Z-score correlation of individual exon’s expression to all somatic mutations. Each indicated mutation was regressed against the relative expression (after adjusting for total IRAK4 expression) of each IRAK4 exon (RPKM) in a multiple linear regression model. Shown are z-scores of partial correlations. A z-score of greater than |1.96| corresponds to P < 0.05. Six out of 160 AML patients exhibit U2AF1 mutations (Z = 2.43; *. P = 0.01). (B) Sashimi plots representing IRAK4 exon 4 inclusion or exclusion in healthy CD34+ cells (n = 7 samples), CD34+ cells isolated from MDS patients with no splicing factor (SF^WT) mutations (MDS-SF^WT; n = 6 samples), and from patients with mutation (mut) in U2AF1 (MDS-U2AF1^mut; n = 6 samples) based on RNA-sequencing junction reads. Quantification of the junction reads for exon 4 are shown for healthy controls (n = 7), MDS-SF^WT (n = 6), and MDS-U2AF1^mut (n = 6). Exon 4 inclusion and exclusion reads were normalized to the total number of reads in each sample and plotted as a ratio (inclusion/total and exclusion/total). Two-sided t-tests were used for statistical analyses. (C) RT- PCR analysis of healthy CD34+ cells (n = 7 samples), CD34+ cells isolated from MDS patients with no splicing factor mutations (MDS-SF^WT; n = 17 samples), and from patients with mutation in U2AF1 (MDS-U2AF1^mut; n = 5 samples) using primers flanking IRAK4 exon 4. Densitometric quantification of IRAK4 exon 4 inclusion calculated as the ratio of the long isoform versus both isoforms is shown below each sample. (D) Densitometric quantification of IRAK4 exon 4 inclusion is summarized from panel (C). The number in parentheses indicates the number of samples exhibiting >10% expression of IRAK4-L relative to all IRAK4 isoforms. Two-sided t-tests were used for statistical analyses. (E) Consensus sequence motifs identified at the distal 3’ splice sites of exon exclusion events in U2AF1-S34F cells as compared to U2AF1 using publicly available data and visualized with weblogo software. Above the U2AF1-S34F consensus motifs is the sequence of the distal 3’ splice site of IRAK4 exon 4. All data represent the mean ± s.e.m.

Figure 5.. U2AF1-S34F induces expression of IRAK4-L and increased innate immune pathway activation.

(A) Experimental design to analyze RNA splicing changes in normal CD34+ cells expressing U2AF1 or U2AF1-S34F (left). Exon 3–4 junction reads of IRAK4 in U2AF1 and U2AF1-S34F CD34+ cells (three independent samples) (right). Two-sided t-tests were used for statistical analyses. (B) Schematic of IRAK4 exon 4 splicing reporter. Wild-type IRAK4 exon 4 (AAG at −3 position) or mutant IRAK4 exon 4 (TAG at −3 position) and 100 bp of flanking introns were cloned into a splicing reporter (pFlare5A) and expressed into HEK293 cells (HEK293-IRAK4exon4). (C) Representative PCR of IRAK4 exon 4 splicing in HEK293-IRAK4exon4 expressing U2AF1 and U2AF1-S34F (five independent experiments for AAG; two independent experiments for TAG). (D) (Top) Experimental design to measure IRAK4 RNA and protein isoform expression in K562 cell lines that have stably integrated doxycycline-inducible FLAG-U2AF1 or FLAG-U2AF1-S34F. (Bottom) IRAK4 RNA isoform expression was measured using primers flanking exon 4 in doxycycline (DOX)-induced K562 cells expressing FLAG-U2AF1 or FLAG- U2AF1-S34F. Top band indicates IRAK4-L and bottom band indicates IRAK4-S (five independent experiments). (E) IRAK4 protein isoform expression analyzed with a C-terminal IRAK4 antibody in DOX-induced K562 cell expressing FLAG-U2AF1 or FLAG-U2AF1-S34F (three independent experiments). (F) The indicated NF-κB and MAPK protein expression was measured in DOX- induced K562 cells expressing FLAG-U2AF1 or FLAG-U2AF1-S34F by immunoblotting (two independent experiments). (G) Expression of the indicated NF-κB (TNFAIP3, IL6, CXCL2, and CXCL8) and MAPK (Jun and Egr1) target genes was measured in DOX-induced K562 cells expressing FLAG-U2AF1 or FLAG-U2AF1-S34F after IL-1β stimulation (10 ng/ml) for 30 or 60 min (three independent experiments). Two-sided t-tests were used for statistical analyses. All data represent the mean ± s.e.m.

Figure 6.. U2AF1-S34F AML cells are sensitive to IRAK4 inhibitors.

(A) K562-U2AF1-S34F cells were treated with 10 mM IRAK1/4-inhibitor for 1 or 2 hours and immunoblotted for NF-κB and MAPK activation. (B) Densitometric analysis of panel (A) summarized from three independent biological replicates. Two-sided t-tests were used for statistical analyses. (C) K562-U2AF1 cells were treated with DMSO, 10 μM IRAK1/4-Inh, or 10 μM CA-4948 for 7 days and assessed for viability by Trypan Blue exclusivity (three independent experiments). One-sided t-tests were used for statistical analyses. (D) Representative images of K562 cells expressing wild-type U2AF1 or U2AF1-S34F were treated with DMSO or 10 μM CA-4948 for 48 hours days and then analyzed by flow cytometry for AnnexinV and Propidium Iodide (PI) staining (three independent experiments). (E) Summary of (D). Two-sided t-tests were used for statistical analyses.(F) K562 cells treated with 10 mM IRAK1/4-Inh (five independent experiments) or CA-4948 (three independent experiments) were evaluated after 7 days for colony formation in methylcellulose. Two-sided t-tests were used for statistical analyses. (G) Schematic of experimental design. (H) MDS patient-derived BM cells were evaluated after 7 days for colony formation in methylcellulose treated with 0.5 μM CA-4948 or vehicle (two independent experiments). (I) MDS patient-derived BM cells were transfected with siIRAK4 or control siRNA and then evaluated after 7 days for colony formation in methylcellulose (two independent experiments). (J) NSG mice were xenografted with U2AF1-mutant MDS BM cells. After engraftment, mice were treated with CA-4948 or vehicle and human cell engraftment was assessed by flow cytometry on BM aspirates. (K) Summary of BM cell engraftment (hCD45) for independent mice and patient-derived samples. Two-sided t-tests were used for statistical analyses. (L) MDS BM cells were isolated from mice treated with vehicle (PBS) or CA-4948 treatment after 3 weeks (panel J,K) and then xenografted into secondary NSG mice. Human cell engraftment was assessed by flow cytometry on BM aspirates. (M) Summary of BM cell engraftment (hCD45) for mice and 2 independent patient-derived samples. Data represent the mean ± s.e.m. for all panels except H and I where data represent the mean ± s.d.