Constitutive IRAK4 Activation Underlies Poor Prognosis and Chemoresistance in Pancreatic Ductal Adenocarcinoma

To date, the prognosis of pancreatic ductal adenocarcinoma (PDAC) remains dismal with an overall 5-year survival rate of about 8%, which has not changed in the last 4 decades (1, 2). In addition to the universal presence of strong oncogenic events, such as activating mutation of KRas, the constitutive activation of the NF-κB transcription factors is another major mechanism that drives the aggressive behavior of PDAC (3–6). Understanding how NF-κB is activated in PDAC is clinically important and has been an area of intense interest (5). For instance, expression of oncogenic KRas upregulates IL1α and GSK3α production to activate the NF-κB pathway in a feedforward manner (7, 8). High miR-301a level suppresses translation of NF-κB–repressing factor protein, thereby activating NF-κB, which in turn enhances miR-301a expression to create a positive feedforward loop (9). However, direct inhibitors of NF-κB or miRNAs have yet to be successful in clinic, indicating the need to explore other strategies to curb this pathway.

Another element that contributes to the recalcitrant nature of PDAC, in addition to its strong intrinsic survival signaling, is the uniquely inflamed and immunosuppressive extrinsic microenvironment, which is believed to hamper cytotoxic T-lymphocyte infiltration and drug delivery (10, 11). In normal tissue, inflammation is triggered by the engagement of the Toll-like receptors (TLR), which activate the innate immune response through the NF-κB and p38/MAPK signaling cascades (12). Several lines of evidence indicate that stimulation of the TLRs could drive NF-κB activity in PDAC. For instance, ligation of TLR4 enhances the invasiveness of PDAC cells in an NF-κB–dependent manner (13). In genetically engineered mouse models, ligation of TLR7 or TLR9 accelerates stromal inflammation and progression of PDAC activation of the NF-κB and MAPK pathways (14, 15). However, the intrinsic role of TLR signaling in neoplastic PDAC cells and how it affects the extrinsic tumor microenvironment have not been thoroughly described.

In this study, we demonstrate for the first time that the interleukin-1 receptor-associated kinase 4 (IRAK4), the master kinase that relays signaling downstream of TLRs (16, 17), is constitutively activated in PDAC cell lines, patient-derived cell lines, and tumor samples. Activated IRAK4 staining positively correlates with activated NF-κB in human PDAC samples, and importantly, portends high postoperative relapse and poor patient survival. We showed that pharmacologic blockade or silencing of IRAK4 potently suppressed NF-κB activity, abolished the tumorigenic potential of human and murine PDAC cells, and greatly sensitized PDAC cells to various chemotherapeutics in vivo and in vitro. Our data lay a solid foundation for testing IRAK4 inhibitors as a novel class of therapeutic agents for PDAC treatment.

Human PDAC lines AsPC-1, BxPC-3, Capan-1, Capan-2, CFPAC-1, HPAF-II, HPAC, MIA Paca-2, PANC-1, SW1990, and HPNE cells were purchased from the ATCC, which performs authentication on its own cell lines. All cells were used for fewer than 6 months after receipt or resuscitation from cryopreservation. HPDE, MPanc96, and Hs766T were kind gifts from Dr. David Linehan (University of Rochester, NY). KRas-Ink4a cells were a kind gift from Dr. David DeNardo (Washington University, MO) and were published (18). Patient-derived PDAC cell lines (PDCL) Pa01c, Pa02c, and Pa14c were kind gifts from Dr. Channing Der (University of North Carolina, NC) and published (19, 20). HPNE and HPDE cells are immortalized, normal human pancreatic epithelial cells.

Human cancer-associated fibroblasts (CAFs) were isolated from the PDAC specimen of a patient who underwent the Whipple procedure and expanded using a published method (21). CAFs were confirmed to be α-SMA–positive and pan-cytokeratin–negative by immunofluorescence (IF).

FFPE PDAC and matched adjacent normal pancreatic tissues diagnosed between years 2000 and 2010 were retrieved from the Department of Pathology and Immunology at Washington University, MO, under Institutional Review Board–approved protocol (#201404143). All selected patients had documented follow-up to 6 years after surgery. None received neoadjuvant treatment prior to surgery, but all received adjuvant treatment containing either 5-fluorouracil (5-FU) or gemcitabine. Pancreatic tissue array (catalog # PA961B) was purchased from US Biomax.

Immunohistochemistry (IHC) was performed using p-IRAK4 (T345S; A8A8; ABNOVA; 1:200), p-NF-κB/p65 (S536; Abcam ab86299; 1:200), and p-TAK1 (S184/187; Bioss bs-3439R; 1:200). IHC scores were determined by the sum of intensity (0: none, 1: faint, 2: weak, 3: moderate, 4: strong) and distribution (0: none, 1: <25%, 2: 25%–50%, 3: 50%–75%, 4: >75%) sub-scores. All samples were scored two times in a blinded manner. Further details were provided in Supplementary Data.

Stable knockdowns were generated using pSuper-Retro-Puro or neo/GFP plasmids encoding scramble sequence, or indicated shRNAs as described (22). Further details included in Supplementary Data.

5-FU, gemcitabine, and paclitaxel were purchased from the Siteman Cancer Center Pharmacy. The IRAK1/4 inhibitor, N-(2-Morpholinylethyl)-2-(3-nitrobenzoylamido)-benzimidazole, was purchased from Sigma (I5409). IMD-0354 was purchased from Selleckchem.

Viability was assayed using Resazurin colorimetric analysis; synergism studies were performed as described (23); soft-agar assay was performed as described (24). Further details were provided in Supplementary Data.

CAFs were added to rehydrated 8-μm pore, 24-well cell culture inserts (with or without collagen; BD Bioscience). Serum-free conditioned media (200 μL) from PANC-1 or Capan-1 cells were added to lower chambers. After 24 hours, uninvaded CAFs were removed from the upper chamber, and invaded cells were fixed with 4% paraformaldehyde, stained with toluidine blue overnight, and counted under microscope.

Details were provided in Supplementary Data.

Human Cytokine Antibody Array C3 (catalog # AAH-CYT-3) was purchased from Raybio. Membranes were incubated with serum-free conditioned media collected from 70% to 80% confluent Capan-1 cells treated with DMSO or 10 μmol/L of IRAK1/4i overnight (about 16 hours) and processed according to the manufacturer's protocol. Identical results were obtained from a repeat experiment using different sets of Capan-1–conditioned media.

NF-κB reporter activity was performed using Dual-Glo Luciferase Assay System and read with Synergy H4 Hybrid Multi-Mode Microplate Reader. All experiments were done three times in triplicate and data represented as mean ± SEM.

All animal experiments were conducted according to Institutional Animal Care and Use Committee protocol (#20130191). Details were provided in Supplementary Data.

Normal distributions were compared by two-tailed Student t test or ANOVA; tumor growth curves were compared using Mann–Whitney U tests; survivals were analyzed using Kaplan–Meier method and Log-rank test. Statistical analyses were performed with Graphpad Prism v6.0. P values <0.05 were considered as statistically significant.

To explore the intrinsic role of TLR signaling in PDAC, we first investigated the phosphorylation/activation status of IRAK (25, 26), particularly IRAK1 and IRAK4 in a panel of well-characterized human PDAC lines. We found IRAK1 to be constitutively phosphorylated in 9, and IRAK4 in 11, of 12 lines in the absence of TLR ligand stimulation. In contrast, IRAK4 phosphorylation was barely detected in both, whereas IRAK1 was weakly phosphorylated in one, of two nontransformed pancreatic ductal cell lines, HPNE and HPDE (Fig. 1A). Consistent with published literature, the majority of PDAC lines also showed constitutive phosphorylation of NF-κB/p65 and iκB (5, 6). Similarly, p-IRAK4 is detectable in four early-passaged patient-derived cell lines (PDCLs), along with increased p-NF-κB/p65 and p-iκBα (Supplementary Fig. S1A).

We next investigated the phosphorylation status of IRAK4 (p-IRAK4) in 103 resected human PDAC samples by IHC using a phospho-antibody that was previously reported in melanoma (27). We found p-IRAK4 to be present and significantly stronger in the ductal epithelia of 20 out of 30 (or 66.7%) PDAC samples (Fig. 1B and C), compared with their matched normal pancreatic tissues. In all 103 PDAC samples analyzed, p-IRAK4 expression is present in 58.8% of cases at various intensities and extent in the neoplastic ductal cells (Fig. 1C), which is significantly higher than normal pancreatic tissue (P = 0.0002). As a control, total IRAK4 protein is detected in both normal pancreatic and PDAC tissues (Fig. 1B). We next validated this finding using a commercially available pancreatic tissue microarray and found p-IRAK4 IHC staining to be present at various intensities in 63.5% of PDAC samples, which is comparable to our findings in surgical samples (Supplementary Fig. S1B and S1C).

To gain an initial insight of whether p-IRAK4 contributes to NF-κB activity in PDAC, we next determined the correlation between the staining intensities of p-IRAK4 and p-NF-κB/p65. We found complete overlap of p-IRAK4 and p-NF-κB/p65 IHC staining in the neoplastic epithelia of surgical PDAC samples (Fig. 1D). IHC analysis of 85 PDAC samples from tissue microarray showed various degrees of p-NF-κB/p65 staining intensities in 87% of samples (Supplementary Fig. S1B and S1D), and we noted strong correlation between the staining intensities of p-IRAK4 and p-NF-κB/p65 (Spearman R 0.87, P < 0.0001; Fig. 1E). In a genetically engineered p48-Cre;p53^Flox/WT;LSL-KRas^G12D (or KPC) mouse model of PDAC, both p-IRAK4 and p-NF-κB/p65 stainings are present at a very low level starting from early pancreatic intraepithelial neoplasia (PanIN-1) and become increasingly stronger during progression to higher grade PanIN-2, PanIN-3, and PDAC (Fig. 1F), indicating a potential role of IRAK4 and NF-κB activity during PDAC development.

Because p-IRAK4 staining is detectable in the majority but not all PDAC samples, we determined whether p-IRAK4 staining is associated with prognosis. Remarkably, we found that patients with positive p-IRAK4 PDAC had a significantly higher chance of postsurgical relapse and worse overall survival despite adjuvant treatment, compared with patients with negative p-IRAK4 tumors (6-year median relapse-free survival: 12.56 vs. 32.95 months, log-rank P = 0.0122; 6-year median overall survival: not reached vs. 19.05 months, log-rank P = 0.0065). We observed no difference in tumor size, nodal status, tumor grade, age, and gender distribution between p-IRAK4–positive versus negative tumor (not shown), establishing IRAK4 activation as an independent prognostic factor in PDAC. Together, our findings conclude that IRAK4 is frequently activated and associated with aggressive behavior of PDAC, and provide a strong rationale to investigate the pathogenic role of IRAK4 in PDAC.

To determine whether IRAK4 contributes to NF-κB activity in PDAC cells, we treated PDAC lines with a widely described dual IRAK1/4 inhibitor (28). We found dose-dependent suppression of phosphorylated IRAK1, IRAK4, iκBα, and NF-κB/p65 in all four PDAC lines tested (Fig. 2A). Consistently, IRAK1/4i significantly suppressed NF-κB–driven luciferase activity in five of six PDAC lines, but not in BxPC-3 which has barely detectable p-IRAK1 and p-IRAK4 (Fig. 2B). Furthermore, IRAK1/4i dose-dependently suppressed the anchorage-independent (AI) growth of eight of 10 established PDAC lines in soft agar, and all three PDCLs tested, but had no significant suppression on the viability of HPNE, HPDE, and most PDAC cells in monolayer culture except for mild effect in PANC-1 (Fig. 2C; Supplementary Fig. S2A–S2C). To exclude the nonspecific effect of IRAK1/4i, ectopic expression of a constitutively active IKKβ mutant (S177E/S181E) rendered IRAK1/4i ineffective in suppressing the NF-κB–driven reporter activity and AI growth of PANC-1 cells (Fig. 2D and E). As a complementary experiment, although the NF-κB activity of BxPc-3 line is independent of IRAK1/4 inhibition and can be suppressed by the IKKβ inhibitor IMD-0354, overexpression of wild-type, but not kinase-dead, IRAK4 significantly increased p-NF-κB, AI growth, and rendered BxPC-3 cells sensitive to IRAK1/4i (Fig. 2F and G). These results suggest that the NF-κB activity of PDAC is dependent on IRAK1/4.

To further dissect the individual role of IRAK1 and IRAK4 in NF-κb activation, we performed shRNA knockdown of IRAK1, IRAK4, or both in PDAC lines. Stable knockdown of IRAK1 or IRAK4 by shRNAs, and more so with both, potently inhibits p-IKKα/β and p-NF-κB in human PDAC lines and a murine PDAC cell line (KRas-Ink4a) derived from transgenic KRas^G12D; Ink4a/Arf^−/− mice (Fig. 2H; Supplementary Fig. 2D). Interestingly, knockdown of IRAK1 had little effect on p-IRAK4 in two PDAC lines tested (Fig. 2H), suggesting either insufficient IRAK1 knockdown or that IRAK4 is activated independent of IRAK1 in PDAC. Consistent with our findings with IRAK1/4i, knockdown of IRAK4 or both IRAK1 and IRAK4, and less so of IRAK1, reduce AI growth of human and murine PDAC lines (Fig. 2I and J; Supplementary Fig. S2E). In PANC-1 cells, the suppressive effect of shIRAK1+4 on AI growth could be rescued with re-introduction of constitutively-active IKKβ mutant (Supplementary Fig. S2F), again supporting the notion that the IRAKs function predominantly through the IKK-NF-κB cascade in PDAC. Furthermore, genetic ablation of IRAK4 with CRISPR/Cas9n technique significantly reduced AI growth of PANC-1 and a PDCL line, Pa01c (Fig. 2K).

We next performed knockdown-rescue experiments to delineate the requirement of kinase function of IRAK1 and IRAK4 in PDAC cells growth. We found that the decreased soft-agar growth in shIRAK4-transduced Capan-1 and PANC-1 cells could be rescued by re-introduction of wild-type, but not kinase-dead IRAK4 (KK213AA), whereas the suppressive effect of shIRAK1 could be rescued with both wild-type and kinase-dead IRAK1 (K239A). Supporting this finding and consistent with published reports (29–31), the kinase activity of IRAK4, but not IRAK1, is essential in activating IKKα/β and NF-κb in PANC-1 cells (Fig. 2M). Resonating this observation, we also found that TLR7-induced NF-κB activation in HEK293T cells requires the kinase activity of IRAK4, but not IRAK1 (Supplementary Fig. S2G). Together, our data concluded that IRAK4 kinase activity is essential in driving NF-κb activity in PDAC and should be explored as a therapeutic target.

Activated NF-κB is a major mechanism that underlies chemoresistance in PDAC (5, 6, 32). Because the IRAK kinases drive NF-κB activity in PDAC cells, we next determined whether suppression of IRAK1/4 augments the cytotoxic effect of chemotherapeutics. We found that exposure of Capan-1 and PANC-1 cells to gemcitabine significantly upregulates NF-κB activity, as reported (32). However, such induction could be stifled by IRAK1/4i (Fig. 3A). Mechanistically, knockdown of IRAK1 and/or IRAK4 was sufficient to induce PARP cleavage, an indicator of apoptosis, which was further enhanced by exposure to gemcitabine (Fig. 3B). The chemosensitization effect of shIRAK1+4 could be partially rescued by reintroduction of constitutively active IKKβ mutant (Fig. 3C), again supporting IRAK1/4 as therapeutic targets upstream of NF-κB. Consistently, PDAC cells stably expressing shIRAK1 or shIRAK4, and to a much greater extent, both, were significantly more sensitive to the killing effect of gemcitabine and 5-FU, two commonly used chemotherapeutics in PDAC treatment (Fig. 3D), as shown by significant reductions in IC₅₀ of both chemotherapeutic agents compared with scramble cells (Supplementary Table S1). To translate these findings, we perform synergism analysis by treating Capan-1, MIA Paca-2 and PANC-1 with combination mixtures of IRAK1/4i plus each chemotherapeutic agent at constant ratios, as described (23). In all three PDAC lines, we observed various degrees of synergism between IRAK1/4i and each chemotherapeutic agent (Fig. 3E). Overall, our findings provide a strong rationale for testing IRAK1/4i plus chemotherapy in vivo.

Targeting the desmoplastic tumor microenvironment (TME) is emerging as another attractive therapeutic strategy in PDAC. The highly inflamed and immunosuppressive TME is widely believed to be driven by cytokines and chemokines secreted by PDAC cells (7, 11, 33–35). Because IRAK activation induces cytokine secretion in normal innate immune response, we speculate that production of inflammatory cytokines/chemokines in PDAC is also regulated by IRAK. Indeed, the abundance of IL1α, IL1β, IL8, PDGF, GM-CSF, CCL2, CXCL1, and CXCL2 in the conditioned medium of Capan-1 cells was reduced following IRAK1/4i treatment (Fig. 4A). As controls, secretion of TNFα, TNFβ, and EGF was slightly increased, whereas other factors including TGFβ and VEGF were unchanged following IRAK1/4i treatment. These changes were confirmed by qRT-PCR using Capan-1 cells treated with IRAK1/4i or transduced with IRAK1 or/and IRAK4 shRNAs (Fig. 4B and C). Interestingly, knockdown of IRAK1 had less pronounced effect on the transcription of these factors compared with IRAK4- or double-knockdown cells, suggesting IRAK4 as the predominant driver in cytokine/chemokine production in PDAC (Fig. 4C). As confirmation, we observed very similar, but not identical, changes in production of these secreted factors in another PDAC line, PANC-1 (Fig. 4B). Notably, the suppressive of shIRAK4 on cytokine/chemokine production in PANC-1 could be partially reversed by re-introduction of activated IKKβ mutant (Fig. 3D).

Because many of these affected chemokines/cytokines are known to induce desmoplasia, we next asked whether IRAK1/4 inhibition would impair the ability of PDAC cells to stimulate the growth and behavior of CAFs (Fig. 4E). Indeed, condition media collected from IRAK1, IRAK4, or IRAK1/4 knocked-down Capan-1 and PANC-1 cells were greatly defective in inducing invasion, migration, and proliferation of CAFs (Fig. 4F–H). Based on these results, we speculate that, in addition to curbing the survival of neoplastic PDAC cells, IRAK1/4 inhibition can attenuate the fibrotic TME of PDAC and is an attractive therapeutic strategy.

We next tested whether inhibition of IRAK1 and/or IRAK4 can impede PDAC growth in vivo. Consistent with our findings in soft agar, knockdown of IRAK1 or IRAK4 significantly thwarted the tumorigenic potential of PANC-1 cells and extended the survival of tumor-bearing mice (Fig. 5A and B). Strikingly, shIRAK1+shIRAK4 or IRAK4-ablated PANC-1 cells completely lost tumorigenic potential in vivo, although one mouse from each group died unexpectedly from unknown causes (Fig. 5A and B). Consistent with our previous findings (Fig. 2L), the suppressive effect of shIRAK4 in vivo could be rescued by re-expression of wild-type, but not kinase-dead, IRAK4 (Fig. 5C). We next confirmed these findings using another PDAC line, Capan-1, which stably expresses firefly luciferase and either scramble shRNA (group 1) or shIRAK4 (group 2). We chose to focus on IRAK4 because it is now the target of interest. Treatment was started once tumor establishment was confirmed by palpation and positive bioluminescence signal. Group 1 mice were randomized to receive vehicle, gemcitabine, IRAK1/4 inhibitor, or both; whereas group 2 mice were treated with vehicle or gemcitabine. In group 1 mice, combined gemcitabine and IRAK1/4i significantly impeded tumor growth, although IRAK1/4 inhibitor alone showed modest yet statistically significant reduction in tumor growth compared with vehicle (Fig. 5E). Consistently, Capan-1 tumors treated with both gemcitabine and IRAK1/4i, and less so with IRAK1/4i alone, had significantly lower bioluminescence signals compared with vehicle- or gemcitabine-treated tumors (Fig. 5E). Notably, mice in all four cohorts showed no sign of distress or body weight loss during treatment (Supplementary Fig. S3A). Contrary to scramble tumors, shIRAK4 (group 2) tumors could be suppressed by gemcitabine alone (Fig. 5D), resonating previous findings that shIRAK4 sensitizes PDAC cells to gemcitabine in vitro (Fig. 3D).

Next, to better mimic the native TME and to understand the consequence of systemic IRAK inhibition in an immunocompetent setting, we resorted to the murine KRas-Ink4a line, which can be grown in syngeneic immunocompetent FVB/NJ mice (18). Strikingly, mice orthotopically inoculated with shIRAK1+shIRAK4 KRas-Ink4a cells are completely healthy and showed no signs of distress for up to 6 months, whereas mice inoculated with shLacZ KRas-Ink4a cells all died by 40 days from large palpable tumors (Fig. 5F). On necropsy, none of the 10 pancreases inoculated with shIRAK1+shIRAK4 KRas-Ink4a cells showed any macro- or microscopic trace of tumor growth (not shown). This striking result again supports a critical role of IRAK in PDAC tumorigenesis. Next, we treated FVB/NJ mice with either vehicle, gemcitabine, IRAK1/4i, or both, 10 days after orthotopic inoculation with KRas-Ink4a cells. All mice were sacrificed when the vehicle-treated mice reached moribund status due to large palpable tumor or ascites. We found that mice treated with both gemcitabine and IRAK1/4i showed significantly reduced pancreatic tumor weight, compared with vehicle or gemcitabine-treated cohorts (Fig. 5G). Again, no mice showed any sign of toxicity or body weight loss during treatment (Supplementary Fig. S3A). Together, our data support testing IRAK4 inhibitor in combination with chemotherapy in PDAC treatment in clinical trials.

To study the in vivo effect of IRAK1/4i on PDAC, we analyzed tumors harvested from both the Capan-1 (Fig. 5D) and KRas-Ink4a (Fig. 5G) experiments. At lower magnifications, combo-treated tumors showed larger areas of necrosis compared with the other cohorts (Fig. 6A and Supplementary Fig. S3B). By multicolor IF, combo-treated tumors showed significantly less proliferative cells (double Ki-67⁺, cytokeratin⁺), higher apoptotic cells (cleaved-caspase-3⁺; cytokeratin⁺; Fig. 6A and Supplementary Fig. S3B), and less stromal fibroblasts (by α-SMA area) and fibrosis by trichrome staining (Fig. 6B and Supplementary Fig. S3B). Importantly, tumors treated with IRAK1/4i showed weaker, but not loss, of p-IRAK4 and p-TAK1 (a substrate of IRAK4) staining, compared with vehicle- or gemcitabine-treated tumors (Fig. 6C), indicating on-target effect. However, this observation also explains why knockdown of IRAK1 and IRAK4, or knockout of IRAK4, was much more effective in blocking tumorigenesis, underscoring the need to develop more potent IRAK4 inhibitor for clinical testing in the future.

Overcoming the pathogenic NF-κB signaling is an actively pursued therapeutic strategy in PDAC (5). In this study, we demonstrate that PDAC commonly activates NF-κB through the innate immune IRAK kinases. Notably, the presence of p-IRAK4 is strongly associated with a higher chance of relapse and poor overall prognosis. However, it should be pointed out that our analysis using surgical PDAC tumors is confined to patients with resectable disease, which represents only 10% to 15% of all PDAC cases (2). Patients with advanced or metastatic disease are routinely diagnosed with fine-needle aspiration, which does not allow for this kind of histologic study. Nonetheless, 80% of patients who underwent surgical resection eventually succumb to disease relapse despite adjuvant therapy (2), so identifying the mechanisms that affect patient survival is crucial for the development of effective adjuvant regimen to improve patient outcome. Herein, we provide proof of principle that suppression of IRAK4 can greatly cripple the growth of PDAC and augment the effect of chemotherapy in vitro and in vivo. However, in contrast to the dramatic effect of genetic ablation of IRAK4, the relatively modest antitumor effect of IRAK1/4i used in this study underscores the need to develop more potent IRAK4 inhibitors for clinical testing.

Aside from enhancing the survival of PDAC cells, IRAK4 appears to be a master regulator of a multitude of cytokines and chemokines including GM-CSF, CCL2, CXCL1, CXCL2, IL8, and IL-β, which are all known to modulate the intensely fibrotic and immune-suppressive TME of PDAC. For instance, PDAC-derived GM-CSF promotes recruitment of Gr1⁺CD11b⁺ myeloid cells, which inhibits CD8⁺ T cells (36). The abundance of CCL2 in the PDAC microenvironment engages CCR2 receptor on tumor-associated macrophages to suppress CD8⁺ cells and enhances tumor-initiating stem cells (18, 37). As such, addition of CCR2 inhibitor to chemotherapy FOLFIRINOX achieved an objective response of 49% in patients with metastatic PDAC (38). Necroptosis-induced CXCL1 secretion fuels PDAC progression by promoting macrophage-induced immune suppression (39). In an elegant study by Ling and colleagues (7), expression of oncogenic KRas activates NF-κB through enhanced secretion of IL1α. Although we did observe increased expression of IL1α following expression of KRas^G12D in HPNE and HPDE cells, as published (7), we failed to detect upregulation of p-IRAK4 compared with vector control cells (not shown), indicating that the expression of oncogenic KRas, at least in cell culture model, is insufficient to induce IRAK4 phosphorylation. Therefore, further work using genetically engineered mouse models is warranted to clearly establish the signaling interplay between oncogenic KRas and the IRAK-IKK-NF-κB cascades in PDAC development.

Aside from PDAC, targeting the IRAK1/4 cascade has been shown to be a promising strategy in other malignancies including DLBCL (29, 40), myelodysplastic syndrome (41), T-cell ALL (42), melanoma (27), breast cancer (43), and head and neck cancer (44), further underscoring the urgent need to develop potent IRAK inhibitors for clinical testing. Another study showed that TAK1 inhibition could augment the effect of chemotherapy in PDAC (45). Supporting this study, we found that treatment of PDAC lines with TAK1 inhibitor 5Z-7-oxozeaenol significantly reduced AI growth and p-NF-κB/p65 (not shown), and that IRAK1/4i significantly reduced p-TAK1 (Fig. 6Cand not shown).

How IRAK4 is activated in PDAC is currently unknown. Because activating mutations of IRAK4 or MyD88, the adaptor protein upstream of IRAK4, are not reported in PDAC from analysis of TCGA, COSMIC database and from our own Sanger sequencing of PDAC lines (not shown), we hypothesize that IRAK4 is activated by engagement of the interleukin receptors or the TLRs by pathogen- or damage-associated molecular patterns in the TME. To test this, we are currently conducting loss-of-function analysis of individual TLRs, as well as IL1R and IL18R, in multiple PDAC cells.

In summary, our study provides a strong rationale for targeting IRAK4 as a novel therapeutic approach in PDAC. We showed that p-IRAK4 is not only a strong prognostic marker for poor outcomes in patients with PDAC, but also a major driver of NF-κB activity and could be therapeutically exploited to improve chemotherapy response. Our work also suggests the need to develop and test more potent IRAK4 inhibitors in combination with chemotherapy in preclinical models. Furthermore, understanding how the IRAKs are activated in PDAC, using cell line and genetically engineered mouse models, will add invaluable knowledge to the biology of PDAC.

No potential conflicts of interest were disclosed.

Conception and design: D. Zhang, D.G. DeNardo, K.-H. Lim

Development of methodology: D. Zhang, L. Li, M.B. Ruzinova, K.-H. Lim

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): L. Li, H. Jiang, B. Knolhoff, M.B. Ruzinova, K.-H. Lim

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): D. Zhang, L. Li, A. Wang-Gillam, M.B. Ruzinova, K.-H. Lim

Writing, review, and/or revision of the manuscript: A.C. Lockhart, A. Wang-Gillam, D.G. DeNardo, M.B. Ruzinova, K.-H. Lim

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): D. Zhang, K.-H. Lim

Study supervision: K.-H. Lim

The content is solely the responsibility of the authors and does not necessarily represent the official view of the NIH.

We wish to thank Dr. Ron Bose and Jeff Guo for critical reading of the article, Maureen K. Highkin for technical advice, Julie Prior at the Molecular Imaging Center (funded under P50 CA094056 from the NCI) for assistance with animal imaging and analysis, and the Digestive Disease Research Core Center (funded by grant P30DK052574 from the NIDDK) for providing technical support.

This study was supported by the NCI Cancer Center Support Grant # P30 CA091842 (K.-H. Lim), Washington University Institute of Clinical and Translational Sciences grant UL1 TR000448 from the NCATS of the NIH (K.-H. Lim), and Elsa Pardee Foundation Award (K.-H. Lim and M.B. Ruzinova).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.