TPL2 enforces RAS-induced inflammatory signaling and is activated by point mutations

Abstract

NF-κB transcription factors, driven by the IRAK/IKK cascade, confer treatment resistance in pancreatic ductal adenocarcinoma (PDAC), a cancer characterized by near-universal KRAS mutation. Through reverse-phase protein array and RNA sequencing we discovered that IRAK4 also contributes substantially to MAPK activation in KRAS-mutant PDAC. IRAK4 ablation completely blocked RAS-induced transformation of human and murine cells. Mechanistically, expression of mutant KRAS stimulated an inflammatory, autocrine IL-1β signaling loop that activated IRAK4 and the MAPK pathway. Downstream of IRAK4, we uncovered TPL2 (also known as MAP3K8 or COT) as the essential kinase that propels both MAPK and NF-κB cascades. Inhibition of TPL2 blocked both MAPK and NF-κB signaling, and suppressed KRAS-mutant cell growth. To counter chemotherapy-induced genotoxic stress, PDAC cells upregulated TLR9, which activated prosurvival IRAK4/TPL2 signaling. Accordingly, a TPL2 inhibitor synergized with chemotherapy to curb PDAC growth in vivo. Finally, from TCGA we characterized 2 MAP3K8 point mutations that hyperactivate MAPK and NF-κB cascades by impeding TPL2 protein degradation. Cancer cell lines naturally harboring these MAP3K8 mutations are strikingly sensitive to TPL2 inhibition, underscoring the need to identify these potentially targetable mutations in patients. Overall, our study establishes TPL2 as a promising therapeutic target in RAS- and MAP3K8-mutant cancers and strongly prompts development of TPL2 inhibitors for preclinical and clinical studies.

Keywords: Inflammation; NF-kappaB; Oncogenes; Oncology; Protein kinases.

Figure 1. IRAK signaling dictates NF-κB activity in PDAC and is essential for RAS oncogenesis.

(A) Classification of patients with low, medium, and high RELA expression based on mRNA Z score from TCGA. (B) Progression-free survival (PFS) and overall survival (OS) of PDAC patients with high versus low RELA expression. (C) Heatmap comparing mRNA expression of NF-κB signature genes from the Broad Institute Molecular Signatures Database (gene sets are listed in Supplemental Table 3) in RELA^High vs. RELA^Low patients. (D and E) PFS and disease-free status, respectively, of PDAC patients with high MYD88 and/or high IRAK1 expression (“MYD88^High, IRAK1^High”) versus low MYD88 and/or low IRAK1 expression (“MYD88^Low, IRAK1^Low”). Eight patients overlapping between 2 groups were excluded. (F and G) OS and vital status of patients as in D and E. (H) Graph of IRAK4 expression in normal human pancreas versus PDAC. Data for normal tissue is from the Genotype-Tissue Expression (GTEx) project and PDAC expression from TCGA PanCancer Atlas. P values are from unpaired 2-sided t test. (I) Soft-agar colonies formed by Irak4-KO and rescue (KO + Irak4^WT) MEF cells transformed with 3 pairs of oncogenes. Data show 9 replicates from 3 independent experiments. (J) Number of tumors formed in nude mice from subcutaneous implantation of WT and IRAK4-KO human and murine RAS-mutant and/or PDAC cell lines. n = 8 tumors per condition. (K) Quantification of soft-agar colonies formed by WT versus Irak4-KO KP2 cells. Data show 6 replicates from 2 independent experiments. (L) Soft-agar colonies formed by KP2 cells treated with IRAK4i or vehicle (V). (M) Soft-agar colonies formed by WT and KRAS^G12D HPNE cells ectopically expressing WT IRAK4 and treated with IRAK4i. For L and M, 3 independent experiments were performed, each in technical triplicate, and one set of data is shown. All error bars indicate mean ± SEM. ****P < 0.0001; ***P < 0.0002; **P < 0.0021; *P < 0.0332.

Figure 2. IRAK4 is crucial for oncogenic RAS-driven MAPK signaling.

(A) Linear fold-change for all targets evaluated by reverse-phase protein array (RPPA) performed on HPNE-KRAS^G12D overexpressing (OE) IRAK4 and treated with IRAK4i. Targets with fold-change >2 upon IRAK4 overexpression are identified. (B) Heatmap showing relative expression of ERK-regulated targets in RPPA shown in A. (C) Immunoblots of 293T cells transfected with AU1 epitope–tagged WT or kinase-dead IRAK4. (D) Heatmap depicting fold-change for MAPK-, RAS-, and cell growth–related Gene Ontology (GO) signatures upon Irak4 knockout (KO) and rescue (KO + Irak4^WT) in murine KP2 cells. Comparisons are KO vs. WT and rescue vs. KO. Signatures significantly (P < 0.05) depleted (blue) or enriched (red) are marked with an asterisk (*). (E) Immunoblots of WT and Irak4-KO KP2 cells. (F and G) Gene set enrichment plot and normalized enrichment scores (NES), respectively, for signatures associated with oncogenic KRAS in WT, Irak4-KO, and rescue KP2 cells, compared as in D. “Hallmark” and “C6: Oncogenic Signatures” databases were used. Negative NES indicates downregulation and signatures significantly (P < 0.05) depleted (blue) or enriched (red) are marked with an asterisk (*). (H) Gene set enrichment plot for PDAC signature in Irak4-KO and -rescue KP2 cells. PDAC signature gene list is provided in Supplemental Table 3. Barcode plots under curves in F and H depict the enrichment clustering for individual genes in the respective gene signatures interrogated for KO vs. WT (top barcode) and rescue vs. KO (bottom barcode) cells. (I) Immunoblots of KP2 and KI cells treated with IRAK4i (PF06650833) or vehicle (V) for 24 hours in serum-free condition. (J) Immunoblots of PDAC cells treated with IRAK4i for 16 hours. (K) Immunoblots of HPNE-KRAS^G12D and 293T-KRAS^G12V cells treated with IRAK4i for 24 hours in serum-free media. For D and F–H, RNA sequencing was performed on n = 2 independent samples for each condition.

Figure 3. TPL2 mediates signaling between IRAK4 and the MAPK pathway.

(A) Immunoblot of Pa01C cells overexpressing HA epitope–tagged TPL2 WT that were treated with IRAK4i or vehicle (V) for 6 hours in serum-free condition. (B) Immunoblots of 293T cells transfected with WT IRAK4 for 48 hours. (C) Leading-edge analysis performed using data generated by gene set enrichment analysis in order to identify alterations in individual genes within each gene set tested. Significantly downregulated (P < 0.05) gene signatures were analyzed and a clustered heatmap was generated. Section of heatmap depicting change in MAP3K8 (TPL2) and MAP2K1 (MEK1) expression is shown with original clustering preserved. TPL2-associated gene set list is provided in Supplemental Table 3. (D) Immunoblot of various commercially available and patient-derived (Pa01C–Pa16C) human PDAC cell lines and 1 normal human pancreatic cell line (HPNE). (E) Correlation plot of p-ERK and TPL2 intensities for PDAC cell lines in D. Two-tailed Pearson correlation (r) analysis was performed. (F) Representative H&E and IHC images of human and murine normal pancreas and PDAC tissue for p-ERK, TPL2, and p-IRAK4. n = 6 sections per stain. Scale bars: 50 μm (for full image [×400 magnification]) and 10 μm (insets).

Figure 4. High TPL2 expression is poorly prognostic in PDAC.

(A) IHC images representing high and low staining H-scores for TPL2 and p-IRAK4 with and without HALO analysis markup. H-score = 3 × (% of strongly stained area) + 2 × (% of moderately stained area) + 1 × (% of weakly stained area). (B) Spearman (r) correlation plot of TPL2 and p-IRAK4 H-scores from tissue microarray (TMA) analysis of 313 tissue specimens from 157 PDAC patients, represented in A. (C) Kaplan-Meier plot comparing survival of patients with high vs. low TPL2 protein expression based on analysis of TMA above. Of the patients with survival data, those who died within 1 month of surgery were excluded from the analysis. (D) Graph depicting MAP3K8 (TPL2) expression in normal human pancreas versus PDAC tissue. Data for normal pancreas tissue is from the Genotype-Tissue Expression (GTEx) project and PDAC expression was from the pancreatic adenocarcinoma TCGA PanCancer Atlas study. P values are from unpaired, 2-sided t test. Outliers (5 in normal, 3 in PDAC) were removed by ROUT, Q = 0.1%. (E) Graph of overall survival (OS) of TCGA PDAC patients separated into short- and longer-surviving cohorts using median OS of approximately 15.5 months. (F) Graph comparing MAP3K8 mRNA expression in longer- vs. short-surviving patients defined in E. P values are from unpaired, 2-sided t test. All 178 (out of 185) TCGA PDAC samples with mRNA expression data were analyzed. (G) Graph comparing months of OS of patients with high (Z score > 1, n = 22) versus low (Z score < 1, n = 28) MAP3K8 expression based on analysis of TCGA Firehose Legacy data set. P values from Kruskal-Wallis test. ****P < 0.0001; ***P < 0.0002.

Figure 5. TPL2 drives both MAPK and NF-κB signaling in PDAC.

(A) Gene set enrichment plots for patients with high (Z score > 1, n = 22) and low (Z score < 1, n = 28) MAP3K8 (TPL2) expression from TCGA Firehose Legacy study. (B) Immunoblots of KRAS-mutant human PDAC cell lines treated with TPL2 inhibitor (TPL2i) or vehicle (V) for 36 hours in serum-free media. (C) Immunoblots of 293T and 293T-KRAS^G12V cells treated with TPL2i for 24 hours in serum-free condition. (D) Immunoblots of Pa01C cells treated with incremental doses of TPL2i. (E) Immunoblots of PDAC cell lines treated with TPL2i. (F) Serum-response element (SRE) reporter assay of HPAC cells treated with TPL2i, BRAFi, MEKi, or ERKi. Data show 3 independent experiments, each done with triplicate samples. (G) Quantification of soft-agar colonies formed by PDAC cell lines treated with TPL2i. Data represent n = 3 (n = 2 for CFPAC-1) for each cell line. One data point is shown per biological replicate, each consisting of 3 technical replicates. P values from 2-way ANOVA with Dunnett’s multiple-comparisons test. (H) Immunoblots of WT or MAP3K8-knockdown HPAC cells. (I) Quantification of HPAC and Pa01C cell proliferation after TPL2 knockdown with shRNA (shMAP3K8). Each data point is the average of 6 replicates. P values by 2-way ANOVA with Dunnett’s multiple-comparisons test. (J) Representative crystal violet–stained images of 2D colony formation of TPL2-knockdown HPAC and Pa01C cells. (K) Light microscopic images of organoids formed by HPAC and Pa01C cells with TPL2 knockdown. Scale bars: 100 μm (full images) and 25 μm (insets). Graph on right is quantification of number of organoids formed in 3 independent wells. P values by 2-way ANOVA with Dunnett’s multiple-comparisons test. All data presented as mean ± SEM. ****P < 0.0001; ***P < 0.0002; *P < 0.0332.

Figure 6. KRAS induces autocrine IL-1β signaling, which activates IRAK4 and TPL2.

(A) qRT-PCR of HEK cells expressing empty vector or KRAS^G12V. Data show 6 replicates from 2 independent experiments. ****P < 0.0001 by 2-way ANOVA with Dunnett’s multiple-comparisons test. (B) Heatmap of Hallmark “KRAS signaling up” signature in MAP3K8-high vs. -low patients. IL1B is significantly enriched in MAP3K8^High patients, shown also in box-and-whisker plot on right. Significance tested with unpaired, 2-sided t test. (C) Immunoblots of HEK-KRAS^G12V cells treated with anti–hIL-1β neutralizing antibody for 24 hours. (D) Immunoblots of HEK cells incubated with conditioned media (CM) from HEK-KRAS^G12V cells (called “KRAS^G12V CM”) and anti–hIL-1β neutralizing antibody. (E) Immunoblots of HEK cells incubated with KRAS^G12V CM and TPL2i or vehicle (V) for 16 hours. (F) Immunoblots of HEK and HPAC cells overexpressing HA epitope–tagged WT TPL2 stimulated with 100 ng/mL recombinant hIL-1β for the indicated duration. (G) Immunoblots of HEK cells expressing KRAS^G12V, HA epitope–tagged WT TPL2, and shIL1R1. (H) Kaplan-Meier curve of PDAC TCGA patients with high vs. low IL1B expression. Follow-up censored at 60 months. All data presented as mean ± SEM.

Figure 7. TPL2 inhibition potentiates chemotherapy by curbing MAPK and NF-κB activation.

(A) Immunoblots of 2 PDAC cell lines overexpressing HA epitope–tagged TPL2 WT treated for 16 hours with different chemotherapy agents (10 μM each). (B and C) Quantification of mRNA transcript levels for multiple genes in 3 PDAC cell lines treated with vehicle, gemcitabine-paclitaxel (Gem-PTX), or FIRINOX (10 μM each of 5-FU, SN-38, and oxaliplatin). (D) Duolink proximity ligation assay (PLA) identifying interaction between p-IRAK4 and TLR9 in 3 PDAC cell lines treated with 10 μM SN-38 for 16 hours. Six ×400 magnification fields per condition were analyzed. Scale bars: 10 μm. (E) Immunoblots of 3 PDAC cell lines treated with TPL2i, SN-38, or their combination for 16 hours. FL, full length; V, vehicle. (F) 2D crystal violet clonogenic colony-forming assays of 3 PDAC cell lines treated with TPL2i, SN-38 (2.5 nM, 5 nM, and 10 nM), or their combination in a dose matrix for 3 to 4 weeks. Data show 1 independent experiment out of ≥3 per cell line. (G) Tumor volume curves for patient-derived Pa01C PDAC cells implanted subcutaneously into nude mice followed by treatment with TPL2i, FIRINOX, or combination therapy. Data represent 10 independent tumors (n = 10) for each drug treatment group and 8 independent tumors (n = 8) for vehicle-treated group. P value from 2-way ANOVA followed by Tukey’s multiple-comparisons test. One outlier was removed by Grubb’s test, α = 0.01. (H) Graph depicting final tumor weight (final pancreas weight – 0.1 g [i.e., normal pancreas weight]) after orthoptic injection of murine KI cells and treatment as indicated for 14 days. Images of ex vivo and in vivo tumors detected by ultrasound along with tumor volume are shown on right. P values from 1-way ANOVA with Tukey’s multiple-comparisons test. All data presented as mean ± SEM. ****P < 0.0001; ***P < 0.0002; **P < 0.0021; *P < 0.0332.

Figure 8. E188K mutation in MAP3K8/TPL2 is a gain-of-function mutation.

(A) Lollipop plot adapted from CBioPortal.org (as of August 2019) identifying mutations in MAP3K8/TPL2. (B) Immunoblots of 293T cells transfected with empty expression vector (EV, control) or vector encoding HA epitope–tagged WT TPL2 or TPL2 E188K for 48 hours. (C) Relative serum-response element (SRE) activity and NF-κB reporter activity in 293T cells transfected as in B. Data are from 2 independent experiments. ****P < 0.0001 by 2-way ANOVA followed by Tukey’s multiple-comparisons test. (D) Heatmap representing relative expression in log₂ units of proteins evaluated by reverse-phase protein array (RPPA) of 293T cells transfected with empty vector (EV, control) or vector encoding HA-tagged WT TPL2 or TPL2 E188K in duplicate (n = 2) for 48 hours. (E) Relative abundance (in log₂ units) of the top 15 upregulated targets in RPPA shown in D. p-ERK (in bold text) is the top hit and significantly upregulated targets are indicated with P values from 2-way ANOVA with Tukey’s multiple-comparisons test. All data presented as mean ± SEM.

Figure 9. E188K mutation stabilizes TPL2 protein.

(A) Immunoblots of HEK T/tH cells stably expressing HA epitope–tagged WT TPL2 or TPL2 E188K treated with 10 μg/mL cycloheximide (CHX). Table below shows half-life (t_1/2) of WT TPL2 and TPL2 E188K protein calculated by measuring HA-TPL2 band intensities, normalizing to t₀ and performing 1-phase exponential decay. Data represent 1 of 3 independent experiments showing similar results. (B) Immunoprecipitation of HA epitope–tagged WT TPL2 or TPL2 E188K treated with 10 μM MG132 (proteasome inhibitor) and immunoblotted as indicated. (C) Sanger sequencing peaks of MAP3K8 (TPL2) locus in Hs695T. Arrow indicates the naturally occurring missense point mutation responsible for the Glu→Lys substitution at codon 188 (E188K) in TPL2 in the Hs695T cell line. (D) Immunoblot of Hs695T cells treated with TPL2i or vehicle (V) for 24 hours in 10% serum–containing media. (E) Proliferation of Hs695T cells treated with TPL2i. P values from 2-way ANOVA with Dunnett’s multiple-comparisons test. (F) Viability of Hs695T cells treated with PLX4032 (BRAFi) alone or in combination with 2 different concentrations of TPL2i. Graph on right depicts GI₅₀ of PLX4032 in log₁₀ units. Data represent 10 replicates from 4 independent experiments. P values are from 1-way ANOVA with Dunnett’s multiple-comparisons test. All data presented as mean ± SEM. ****P < 0.0001; ***P < 0.0002; **P < 0.0021.

Figure 10. R442H is a gain-of-function mutation that curtails proteasomal degradation of TPL2.

(A) Immunoblots of 293T cells transiently transfected with empty vector (EV), or vector encoding HA epitope–tagged WT TPL2 or TPL2 R442H for 48 hours. On right is quantification of p-ERK band intensities for WT TPL2 and TPL2 R442H samples from immunoblots on left. Data represent 2 independent experiments. P values from 2-way ANOVA with Holm-Šidák multiple-comparisons test. (B) Immunoblots of HEK T/tH cells stably expressing vector encoding WT TPL2 or TPL2 R442H and treated with 10 μg/mL cycloheximide (CHX) for indicated durations. Table below states half-life (t_1/2) of TPL2 WT and mutant proteins calculated by measuring HA-TPL2 band intensities, normalizing to t₀ and performing 1-phase exponential decay analysis as shown in graph at the bottom. Experiment was performed 3 times and 1 set of data is shown. (C) Immunoblots of HEK T/tH cells stably expressing empty vector or HA epitope–tagged TPL2 mutants treated with 10 μM MG132 (proteasome inhibitor) for 4 hours. One of 2 or more independent experiments is shown. C-terminally truncated TPL2 (ΔC-term) is used as positive control. Poly-Ub, polyubiquitinated. (D) Immunoblots of IGROV1 cells serum starved for 24 hours and treated with TPL2i for 2 hours. (E) Immunoblots of IGROV1 cells after shRNA-mediated TPL2 depletion. (F and G) Proliferation of IGROV1 cells treated with TPL2i and after shRNA-mediated TPL2 depletion, respectively. P values from 2-way ANOVA with Dunnett’s multiple-comparisons test. (H) 3D organoid growth of IGROV1 cells after shRNA-mediated TPL2 depletion and rescue with either WT TPL2 or R442H mutant. Organoids were counted from 4 independent transfected wells per condition. Scale bars: 100 μm. P values from 1-way ANOVA with Dunnett’s multiple-comparisons test. All data presented as mean ± SEM. ****P < 0.0001; ***P < 0.0002; **P < 0.0021; *P < 0.0332.