CREB activation drives acinar to ductal reprogramming and promote pancreatic cancer progression in animal models of alcoholic chronic pancreatitis.

BACKGROUND AND AIMS: In vivo induction of alcoholic chronic pancreatitis (ACP) causes significant acinar damage, increased fibroinflammatory response, and heightened activation of cyclic response element binding protein 1 (CREB) when compared with alcohol (A) or chronic pancreatitis (CP) mediated pancreatic damage. However, the study elucidating the cooperative interaction between CREB and the oncogenic Kras G12D/+ (Kras*) in promoting pancreatic cancer progression with ACP remains unexplored. METHODS: Experimental ACP induction was established in multiple mouse models, followed by euthanization of the animals at various time intervals during the recovery periods. Tumor latency was determined in these mice cohorts. Here, we established CREB deletion (Creb fl/fl ) in Ptf1a CreERTM/+ ;LSL-Kras G12D+/-(KC) genetic mouse models (KCC-/-). Western blot, phosphokinase array, and qPCR were used to analyze the pancreata of Ptf1a CreERTM+/-, KC and KCC -/- mice. The pancreata of ACP-induced KC mice were subjected to single-cell RNA sequencing (scRNAseq). Further studies involved conducting lineage tracing and acinar cell explant cultures. RESULTS: ACP induction in KC mice had detrimental effects on the pancreatic damage repair mechanism. The persistent existence of acinar cell-derived ductal lesions demonstrated a prolonged state of hyperactivated CREB. Persistent CREB activation leads to acinar cell reprogramming and increased pro-fibrotic inflammation in KC mice. Acinar-specific Creb ablation reduced advanced PanINs lesions, hindered tumor progression, and restored acinar cell function in ACP-induced mouse models. CONCLUSIONS: Our findings demonstrate that CREB cooperates with Kras* to perpetuate an irreversible ADM and PanIN formation. Moreover, CREB sustains oncogenic activity to promote the progression of premalignant lesions toward cancer in the presence of ACP.

Cell-specific nanoengineering strategy disrupts tolerogenic signaling from myeloid-derived suppressor cells to invigorate antitumor immunity in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by intratumoral abundance of neutrophilic/polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) which inhibit T-cell function through JAK2/STAT3-regulated arginase activity. To overcome limitations of systemic inhibition of PMN-MDSCs in cancer-bearing patients-i.e., neutropenia and compensatory myelopoietic adaptations-we develop a nanoengineering strategy to target cell-specific signaling exclusively in PMN-MDSCs without provoking neutropenia. We conjugate a chemically modified small-molecule inhibitor of MDSC-surface receptor CXCR2 (AZD5069) with polyethylene glycol (PEG) and chemically graft AZD5069-PEG constructs onto amphiphilic polysaccharide derivatives to engineer CXCR2-homing nanoparticles (CXCR2-NP). Cy5.5 dye-loaded CXCR2-NP showed near-exclusive uptake in PMN-MDSCs compared with PDAC tumor-cells, cancer-associated fibroblasts, and macrophages. Encapsulation of JAK2/STAT3i Ruxolitinib (CXCR2-NP Ruxo ) resulted in more durable attenuation in STAT3-regulated arginase activity from PMN-MDSCs and induction of cytolytic T-cell activity vs. free Ruxolitinib in-vitro and in-vivo . Cell-specific delivery of payloads via CXCR2-homing immunonanoparticles represents a novel strategy to disrupt MDSC-mediated immunosuppression and invigorate antitumor immunity in PDAC.

Tumor Cell-Intrinsic p38 MAPK Signaling Promotes IL1α-Mediated Stromal Inflammation and Therapeutic Resistance in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a KRAS-driven inflammatory program and a desmoplastic stroma, which contribute to the profoundly chemoresistant phenotype. The tumor stroma contains an abundance of cancer-associated fibroblasts (CAF), which engage in extensive paracrine cross-talk with tumor cells to perpetuate protumorigenic inflammation. IL1α, a pleiotropic, tumor cell-derived cytokine, plays a critical role in shaping the stromal landscape. To provide insights into the molecular mechanisms regulating IL1A expression in PDAC, we performed transcriptional profiling of The Cancer Genome Atlas datasets and pharmacologic screening in PDAC cells and identified p38α MAPK as a key positive regulator of IL1A expression. Both genetic and pharmacologic inhibition of p38 MAPK significantly diminished IL1α production in vitro. Chromatin- and coimmunoprecipitation analyses revealed that p38 MAPK coordinates the transcription factors Sp1 and the p65 subunit of NFκB to drive IL1A overexpression. Single-cell RNA sequencing of a highly desmoplastic murine PDAC model, Ptf1aCre/+; LSL-KrasG12D/+; Tgfbr2flox/flox (PKT), confirmed that p38 MAPK inhibition significantly decreases tumor cell-derived Il1a and attenuates the inflammatory CAF phenotype in a paracrine IL1α-dependent manner. Furthermore, p38 MAPK inhibition favorably modulated intratumoral immunosuppressive myeloid populations and augmented chemotherapeutic efficacy to substantially reduce tumor burden and improve overall survival in PKT mice. These findings illustrate a cellular mechanism of tumor cell-intrinsic p38-p65/Sp1-IL1α signaling that is responsible for sustaining stromal inflammation and CAF activation, offering an attractive therapeutic approach to enhance chemosensitivity in PDAC. SIGNIFICANCE: Inhibition of p38 MAPK suppresses tumor cell-derived IL1α and attenuates the inflammatory stroma and immunosuppressive tumor microenvironment to overcome chemotherapeutic resistance in pancreatic cancer.

Remodeling of Stromal Immune Microenvironment by Urolithin A Improves Survival with Immune Checkpoint Blockade in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a significant contributor to cancer-related morbidity and mortality, and it is known for its resistance to conventional treatment regimens, including chemotherapy and immune checkpoint blockade (ICB)-based therapies. We have previously shown that Urolithin A (Uro A), a gut microbial metabolite derived from pomegranates, can target and inhibit KRAS-dependent PI3K/AKT/mTOR signaling pathways to overcome therapeutic resistance and improve survival in PDAC. However, the effect of Uro A on the tumor immune microenvironment and its ability to enhance ICB efficacy has not been explored. This study demonstrates that Uro A treatment reduces stromal fibrosis and reinvigorates the adaptive T-cell immune response to overcome resistance to PD-1 blockade in a genetically engineered mouse model (GEMM) of PDAC. Flow cytometric-based analysis of Uro A-treated mouse tumors revealed a significant attenuation of immunosuppressive tumor-associated M2-like macrophages with a concurrent increase in the infiltration of CD4+ and CD8+ T cells with memory-like phenotype along with reduced expression of the exhaustion-associated protein, PD-1. Importantly, the combination of Uro A treatment with anti-PD-1 immunotherapy promoted enhancement of the antitumor response with increased infiltration of CD4+ Th1 cells, ultimately resulting in a remarkable improvement in overall survival in GEMM of PDAC. Overall, our findings provide preclinical evidence for the potential of Uro A as a novel therapeutic agent to increase sensitivity to immunotherapy in PDAC and warrant further mechanistic exploration in preclinical and clinical studies. Significance: Immunotherapeutic agents are ineffective against pancreatic cancer, mainly due to the immunosuppressive tumor microenvironment and stromal desmoplasia. Our current study demonstrates the therapeutic utility of a novel gut microbial metabolite, Uro A, to remodel the stromal-immune microenvironment and improve overall survival with anti-PD-1 therapy in pancreatic cancer.

Multi-cancer analysis reveals universal association of oncogenic LBH expression with DNA hypomethylation and WNT-Integrin signaling pathways.

Limb-Bud and Heart (LBH) is a developmental transcription co-factor deregulated in cancer, with reported oncogenic and tumor suppressive effects. However, LBH expression in most cancer types remains unknown, impeding understanding of its mechanistic function Here, we performed systematic bioinformatic and TMA analysis for LBH in >20 different cancer types. LBH was overexpressed in most cancers compared to normal tissues (>1.5-fold; p < 0.05), including colon-rectal, pancreatic, esophageal, liver, stomach, bladder, kidney, prostate, testicular, brain, head & neck cancers, and sarcoma, correlating with poor prognosis. The cancer types showing LBH downregulation were lung, melanoma, ovarian, cervical, and uterine cancer, while both LBH over- and under-expression were observed in hematopoietic malignancies. In cancers with LBH overexpression, the LBH locus was frequently hypomethylated, identifying DNA hypomethylation as a potential mechanism for LBH dysregulation. Pathway analysis identified a universal, prognostically significant correlation between LBH overexpression and the WNT-Integrin signaling pathways. Validation of the clinical association of LBH with WNT activation in gastrointestinal cancer cell lines, and in colorectal patient samples by IHC uncovered that LBH is specifically expressed in tumor cells with nuclear beta-catenin at the invasive front. Collectively, these data reveal a high degree of LBH dysregulation in cancer and establish LBH as pan-cancer biomarker for detecting WNT hyperactivation in clinical specimens.

Cell-Autonomous Cxcl1 Sustains Tolerogenic Circuitries and Stromal Inflammation via Neutrophil-Derived TNF in Pancreatic Cancer.

We have shown that KRAS-TP53 genomic coalteration is associated with immune-excluded microenvironments, chemoresistance, and poor survival in pancreatic ductal adenocarcinoma (PDAC) patients. By treating KRAS-TP53 cooperativity as a model for high-risk biology, we now identify cell-autonomous Cxcl1 as a key mediator of spatial T-cell restriction via interactions with CXCR2+ neutrophilic myeloid-derived suppressor cells in human PDAC using imaging mass cytometry. Silencing of cell-intrinsic Cxcl1 in LSL-KrasG12D/+;Trp53R172H/+;Pdx-1Cre/+(KPC) cells reprograms the trafficking and functional dynamics of neutrophils to overcome T-cell exclusion and controls tumor growth in a T cell-dependent manner. Mechanistically, neutrophil-derived TNF is a central regulator of this immunologic rewiring, instigating feed-forward Cxcl1 overproduction from tumor cells and cancer-associated fibroblasts (CAF), T-cell dysfunction, and inflammatory CAF polarization via transmembrane TNF-TNFR2 interactions. TNFR2 inhibition disrupts this circuitry and improves sensitivity to chemotherapy in vivo. Our results uncover cancer cell-neutrophil cross-talk in which context-dependent TNF signaling amplifies stromal inflammation and immune tolerance to promote therapeutic resistance in PDAC. SIGNIFICANCE: By decoding connections between high-risk tumor genotypes, cell-autonomous inflammatory programs, and myeloid-enriched/T cell-excluded contexts, we identify a novel role for neutrophil-derived TNF in sustaining immunosuppression and stromal inflammation in pancreatic tumor microenvironments. This work offers a conceptual framework by which targeting context-dependent TNF signaling may overcome hallmarks of chemoresistance in pancreatic cancer. This article is highlighted in the In This Issue feature, p. 1275.

Urolithin A attenuates severity of chronic pancreatitis associated with continued alcohol intake by inhibiting PI3K/AKT/mTOR signaling.

Heavy alcohol consumption is the dominant risk factor for chronic pancreatitis (CP); however, treatment and prevention strategies for alcoholic chronic pancreatitis (ACP) remains limited. The present study demonstrates that ACP induction in C57BL/6 mice causes significant acinar cell injury, pancreatic stellate cell (PSC) activation, exocrine function insufficiency, and an increased fibroinflammatory response when compared with alcohol or CP alone. Although the withdrawal of alcohol during ACP recovery led to reversion of pancreatic damage, continued alcohol consumption with established ACP perpetuated pancreatic injury. In addition, phosphokinase array and Western blot analysis of ACP-induced mice pancreata revealed activation of the phosphatidylinositol 3 kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) and cyclic AMP response element binding protein (CREB) signaling pathways possibly orchestrating the fibroinflammatory program of ACP pathogenesis. Mice treated with urolithin A (Uro A, a gut-derived microbial metabolite) in the setting of ACP with continued alcohol intake (during the recovery period) showed suppression of AKT and P70S6K activation, and acinar damage was significantly reduced with a parallel reduction in pancreas-infiltrating macrophages and proinflammatory cytokine accumulation. These results collectively provide mechanistic insight into the impact of Uro A on attenuation of ACP severity through suppression of PI3K/AKT/mTOR signaling pathways and can be a useful therapeutic approach in patients with ACP with continuous alcohol intake.NEW & NOTEWORTHY Our novel findings presented here demonstrate the utility of Uro A as an effective therapeutic agent in attenuating alcoholic chronic pancreatitis (ACP) severity with alcohol continuation after established disease, through suppression of the PI3K/AKT/mTOR signaling pathway.

Combined MEK and STAT3 Inhibition Uncovers Stromal Plasticity by Enriching for Cancer-Associated Fibroblasts With Mesenchymal Stem Cell-Like Features to Overcome Immunotherapy Resistance in Pancreatic Cancer.

BACKGROUND & AIMS: We have shown that reciprocally activated rat sarcoma (RAS)/mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) and Janus kinase/signal transducer and activator of transcription 3 (STAT3) pathways mediate therapeutic resistance in pancreatic ductal adenocarcinoma (PDAC), while combined MEK and STAT3 inhibition (MEKi+STAT3i) overcomes such resistance and alters stromal architecture. We now determine whether MEKi+STAT3i reprograms the cancer-associated fibroblast (CAF) and immune microenvironment to overcome resistance to immune checkpoint inhibition in PDAC. METHODS: CAF and immune cell transcriptomes in MEKi (trametinib)+STAT3i (ruxolitinib)-treated vs vehicle-treated Ptf1aCre/+;LSL-KrasG12D/+;Tgfbr2flox/flox (PKT) tumors were examined via single-cell RNA sequencing (scRNAseq). Clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats associated protein 9 silencing of CAF-restricted Map2k1/Mek1 or Stat3, or both, enabled interrogation of CAF-dependent effects on immunologic remodeling in orthotopic models. Tumor growth, survival, and immune profiling via mass cytometry by time-of-flight were examined in PKT mice treated with vehicle, anti-programmed cell death protein 1 (PD-1) monotherapy, and MEKi+STAT3i combined with anti-PD1. RESULTS: MEKi+STAT3i attenuates Il6/Cxcl1-expressing proinflammatory and Lrrc15-expressing myofibroblastic CAF phenotypes while enriching for Ly6a/Cd34-expressing CAFs exhibiting mesenchymal stem cell-like features via scRNAseq in PKT mice. This CAF plasticity is associated with M2-to-M1 reprogramming of tumor-associated macrophages, and enhanced trafficking of cluster of differentiation 8+ T cells, which exhibit distinct effector transcriptional programs. These MEKi+STAT3i-induced effects appear CAF-dependent, because CAF-restricted Mek1/Stat3 silencing mitigates inflammatory-CAF polarization and myeloid infiltration in vivo. Addition of MEKi+STAT3i to PD-1 blockade not only dramatically improves antitumor responses and survival in PKT mice but also augments recruitment of activated/memory T cells while improving their degranulating and cytotoxic capacity compared with anti-PD-1 monotherapy. Importantly, treatment of a patient who has chemotherapy-refractory metastatic PDAC with MEKi (trametinib), STAT3i (ruxolitinib), and PD-1 inhibitor (nivolumab) yielded clinical benefit. CONCLUSIONS: Combined MEKi+STAT3i mitigates stromal inflammation and enriches for CAF phenotypes with mesenchymal stem cell-like properties to overcome immunotherapy resistance in PDAC.

Emerging Role of CREB in Epithelial to Mesenchymal Plasticity of Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive solid malignancy with a high rate of metastasis and therapeutic resistance as its major hallmarks. Although a defining mutational event in pancreatic cancer initiation is the presence of oncogenic KRAS, more advanced PDAC lesions accumulate additional genomic alterations, including loss of tumor suppressor gene TP53. Co-occurrence of mutant KRAS and TP53 in PDAC promotes hyperactivation of cancer cell signaling pathways driving epithelial to mesenchymal plasticity (EMP). The cellular process of EMP influences the biological behavior of cancer cells by increasing their migratory and invasive properties, thus promoting metastasis. Our previous work has demonstrated that oncogenic KRAS-mediated activation of cyclic AMP response element-binding protein 1 (CREB) is one of the critical drivers of PDAC aggressiveness. The therapeutic approach of targeting this key transcription factor attenuates tumor burden in genetically engineered mouse models (GEMMs) of this disease. Herein, we discuss the significant role of CREB in perpetuating disease aggressiveness and therapeutic resistance through the EMP process. Furthermore, this review updates the therapeutic implications of targeting CREB, highlighting the challenges and emerging approaches in PDAC.

Distinct mechanisms of innate and adaptive immune regulation underlie poor oncologic outcomes associated with KRAS-TP53 co-alteration in pancreatic cancer.

Co-occurrent KRAS and TP53 mutations define a majority of patients with pancreatic ductal adenocarcinoma (PDAC) and define its pro-metastatic proclivity. Here, we demonstrate that KRAS-TP53 co-alteration is associated with worse survival compared with either KRAS-alone or TP53-alone altered PDAC in 245 patients with metastatic disease treated at a tertiary referral cancer center, and validate this observation in two independent molecularly annotated datasets. Compared with non-TP53 mutated KRAS-altered tumors, KRAS-TP53 co-alteration engenders disproportionately innate immune-enriched and CD8+ T-cell-excluded immune signatures. Leveraging in silico, in vitro, and in vivo models of human and murine PDAC, we discover a novel intersection between KRAS-TP53 co-altered transcriptomes, TP63-defined squamous trans-differentiation, and myeloid-cell migration into the tumor microenvironment. Comparison of single-cell transcriptomes between KRAS-TP53 co-altered and KRAS-altered/TP53WT tumors revealed cancer cell-autonomous transcriptional programs that orchestrate innate immune trafficking and function. Moreover, we uncover granulocyte-derived inflammasome activation and TNF signaling as putative paracrine mediators of innate immunoregulatory transcriptional programs in KRAS-TP53 co-altered PDAC. Immune subtyping of KRAS-TP53 co-altered PDAC reveals conflation of intratumor heterogeneity with progenitor-like stemness properties. Coalescing these distinct molecular characteristics into a KRAS-TP53 co-altered "immunoregulatory program" predicts chemoresistance in metastatic PDAC patients enrolled in the COMPASS trial, as well as worse overall survival.

Interleukin-1 signaling in solid organ malignancies.

As inflammation plays a critical role in the development and progression of cancer, therapeutic targeting of cytokine pathways involved in both tumorigenesis and dictating response to clinical treatments are of significant interest. Recent evidence has highlighted the importance of the pro-inflammatory cytokine interleukin-1 (IL-1) as a key mediator of tumor growth, metastatic disease spread, immunosuppression, and drug resistance in cancer. IL-1 promotes tumorigenesis through diverse mechanisms, including the activation of oncogenic signaling pathways directly in tumor cells and via orchestrating crosstalk between the cellular constituents of the tumor microenvironment (TME), thereby driving cancer growth. This review will provide an overview of IL-1 signaling and physiology and summarize the disparate mechanisms involving IL-1 in tumorigenesis and cancer progression. Additionally, clinical studies targeting IL-1 signaling in the management of solid organ tumors will be summarized herein.

Targeting PI3K Pathway in Pancreatic Ductal Adenocarcinoma: Rationale and Progress.

Pancreatic ductal adenocarcinoma (PDAC) remains among the deadliest solid tumors that remain treatment-refractory and show a dismal prognosis. More than 90% of PDAC tumors harbor mutations in the K-Ras that exert a strong pro-tumorigenic effect by activating several downstream effector pathways, including phosphatidylinositol-3-kinase (PI3K)-Akt. The role of frequently activated PI3K/Akt pathway in promoting PDAC aggressiveness is well established. Therapeutic approaches targeting PI3K and downstream signaling components in different cellular compartments, including tumor, stromal and immune cells, have directly impacted the tumor burden in this cancer type. Our previous work has demonstrated that targeting the PI3K/Akt/mTOR pathway reduced tumor growth and improved survival in the genetic mouse model of PDAC. Here, we discuss the significance of targeting PI3K signaling and the biological impact of PI3K inhibition in modulating the tumor-stromal immune crosstalk within the microenvironment of pancreatic cancer. Furthermore, this review updates on the current challenges involving the therapeutic implications of targeting this pathway in PDAC.

Targeting Tumor-Stromal IL6/STAT3 Signaling through IL1 Receptor Inhibition in Pancreatic Cancer.

A hallmark of pancreatic ductal adenocarcinoma (PDAC) is the presence of a dense, desmoplastic stroma and the consequent altered interactions between cancer cells and their surrounding tumor microenvironment (TME) that promote disease progression, metastasis, and chemoresistance. We have previously shown that IL6 secreted from pancreatic stellate cells (PSC) stimulates the activation of STAT3 signaling in tumor cells, an established mechanism of therapeutic resistance in PDAC. We have now identified the tumor cell-derived cytokine IL1α as an upstream mediator of IL6 release from PSCs that is involved in STAT3 activation within the TME. Herein, we show that IL1α is overexpressed in both murine and human PDAC tumors and engages with its cognate receptor IL1R1, which is strongly expressed on stromal cells. Further, we show that IL1R1 inhibition using anakinra (recombinant IL1 receptor antagonist) significantly reduces stromal-derived IL6, thereby suppressing IL6-dependent STAT3 activation in human PDAC cell lines. Anakinra treatment results in significant reduction in IL6 and activated STAT3 levels in pancreatic tumors from Ptf1aCre/+;LSL-KrasG12D/+; Tgfbr2flox/flox (PKT) mice. Additionally, the combination of anakinra with cytotoxic chemotherapy significantly extends overall survival compared with vehicle treatment or anakinra monotherapy in this aggressive genetic mouse model of PDAC. These data highlight the importance of IL1 in mediating tumor-stromal IL6/STAT3 cross-talk in the TME and provide a preclinical rationale for targeting IL1 signaling as a therapeutic strategy in PDAC.

Combined Blockade of MEK and CDK4/6 Pathways Induces Senescence to Improve Survival in Pancreatic Ductal Adenocarcinoma.

Activating KRAS mutations, a defining feature of pancreatic ductal adenocarcinoma (PDAC), promote tumor growth in part through the activation of cyclin-dependent kinases (CDK) that induce cell-cycle progression. p16INK4a (p16), encoded by the gene CDKN2A, is a potent inhibitor of CDK4/6 and serves as a critical checkpoint of cell proliferation. Mutations in and subsequent loss of the p16 gene occur in PDAC at a rate higher than that reported in any other tumor type and results in Rb inactivation and unrestricted cellular growth. Therefore, strategies targeting downstream RAS pathway effectors combined with CDK4/6 inhibition (CDK4/6i) may have the potential to improve outcomes in this disease. Herein, we show that expression of p16 is markedly reduced in PDAC tumors compared with normal pancreatic or pre-neoplastic tissues. Combined MEK inhibition (MEKi) and CDK4/6i results in sustained downregulation of both ERK and Rb phosphorylation and a significant reduction in cell proliferation compared with monotherapy in human PDAC cells. MEKi with CDK4/6i reduces tumor cell proliferation by promoting senescence-mediated growth arrest, independent of apoptosis in vitro We show that combined MEKi and CDK4/6i treatment attenuates tumor growth in xenograft models of PDAC and improves overall survival over 200% compared with treatment with vehicle or individual agents alone in Ptf1acre/+ ;LSL-KRASG12D/+ ;Tgfbr2flox/flox (PKT) mice. Histologic analysis of PKT tumor lysates reveal a significant decrease in markers of cell proliferation and an increase in senescence-associated markers without any significant change in apoptosis. These results demonstrate that combined targeting of both MEK and CDK4/6 represents a novel therapeutic strategy to synergistically reduce tumor growth through induction of cellular senescence in PDAC.

Stroma secreted IL6 selects for "stem-like" population and alters pancreatic tumor microenvironment by reprogramming metabolic pathways.

Pancreatic adenocarcinoma is a devastating disease with an abysmal survival rate of 9%. A robust fibro-inflammatory and desmoplastic stroma, characteristic of pancreatic cancer, contribute to the challenges in developing viable therapeutic strategies in this disease. Apart from constricting blood vessels and preventing efficient drug delivery to the tumor, the stroma also contributes to the aggressive biology of cancer along with its immune-evasive microenvironment. In this study, we show that in pancreatic tumors, the developing stroma increases tumor initiation frequency in pancreatic cancer cells in vivo by enriching for CD133 + aggressive "stem-like" cells. Additionally, the stromal fibroblasts secrete IL6 as the major cytokine, increases glycolytic flux in the pancreatic tumor cells, and increases lactate efflux in the microenvironment via activation of the STAT signaling pathway. We also show that the secreted lactate favors activation of M2 macrophages in the tumor microenvironment, which excludes CD8 + T cells in the tumor. Our data additionally confirms that the treatment of pancreatic tumors with anti-IL6 antibody results in tumor regression as well as decreased CD133 + population within the tumor. Furthermore, inhibiting the lactate efflux in the microenvironment reduces M2 macrophages, and makes pancreatic tumors more responsive to anti-PD1 therapy. This suggests that stromal IL6 driven metabolic reprogramming plays a significant role in the development of an immune-evasive microenvironment. In conclusion, our study shows that targeting the metabolic pathways affected by stromal IL6 can make pancreatic tumors amenable to checkpoint inhibitor therapy.

Combined Src/EGFR Inhibition Targets STAT3 Signaling and Induces Stromal Remodeling to Improve Survival in Pancreatic Cancer.

Lack of durable response to cytotoxic chemotherapy is a major contributor to the dismal outcomes seen in pancreatic ductal adenocarcinoma (PDAC). Extensive tumor desmoplasia and poor vascular supply are two predominant characteristics which hinder the delivery of chemotherapeutic drugs into PDAC tumors and mediate resistance to therapy. Previously, we have shown that STAT3 is a key biomarker of therapeutic resistance to gemcitabine treatment in PDAC, which can be overcome by combined inhibition of the Src and EGFR pathways. Although it is well-established that concurrent EGFR and Src inhibition exert these antineoplastic properties through direct inhibition of mitogenic pathways in tumor cells, the influence of this combined therapy on stromal constituents in PDAC tumors remains unknown. In this study, we demonstrate in both orthotopic tumor xenograft and Ptf1acre/+;LSL-KrasG12D/+;Tgfbr2flox/flox (PKT) mouse models that concurrent EGFR and Src inhibition abrogates STAT3 activation, increases microvessel density, and prevents tissue fibrosis in vivo. Furthermore, the stromal changes induced by parallel EGFR and Src pathway inhibition resulted in improved overall survival in PKT mice when combined with gemcitabine. As a phase I clinical trial utilizing concurrent EGFR and Src inhibition with gemcitabine has recently concluded, these data provide timely translational insight into the novel mechanism of action of this regimen and expand our understanding into the phenomenon of stromal-mediated therapeutic resistance. IMPLICATIONS: These findings demonstrate that Src/EGFR inhibition targets STAT3, remodels the tumor stroma, and results in enhanced delivery of gemcitabine to improve overall survival in a mouse model of PDAC.

Increased MTH1-specific 8-oxodGTPase activity is a hallmark of cancer in colon, lung and pancreatic tissue.

Cellular homeostasis is dependent on a balance between DNA damage and DNA repair mechanisms. Cells are constantly assaulted by both exogenous and endogenous stimuli leading to high levels of reactive oxygen species (ROS) that cause oxidation of the nucleotide dGTP to 8-oxodGTP. If this base is incorporated into DNA and goes unrepaired, it can result in G > T transversions, leading to genomic DNA damage. MutT Homolog 1 (MTH1) is a nucleoside diphosphate X (Nudix) pyrophosphatase that can remove 8-oxodGTP from the nucleotide pool before it is incorporated into DNA by hydrolyzing it into 8-oxodGMP. MTH1 expression has been shown to be elevated in many cancer cells and is thought to be a survival mechanism by which a cancer cell can stave off the effects of high ROS that can result in cell senescence or death. It has recently become a target of interest in cancer because it is thought that inhibiting MTH1 can increase genotoxic damage and cytotoxicity. Determining the role of MTH1 in normal and cancer cells is confounded by an inability to reliably and directly measure its native enzymatic activity. We have used the chimeric ATP-releasing guanine-oxidized (ARGO) probe that combines 8-oxodGTP and ATP to measure MTH1 enzymatic activity in colorectal cancer (CRC), non-small cell lung cancer (NSCLC) and pancreatic ductal adenocarcinoma (PDAC) along with patient-matched normal tissue. MTH1 8-oxodGTPase activity is significantly increased in tumors across all three tissue types, indicating that MTH1 is a marker of cancer. MTH1 activity measured by ARGO assay was compared to mRNA and protein expression measured by RT-qPCR and Western blot in the CRC tissue pairs, revealing a positive correlation between ARGO assay and Western blot, but little correlation with RT-qPCR in these samples. The adoption of the ARGO assay will help in establishing the level of MTH1 activity in model systems and in assessing the effects of MTH1 modulation in the treatment of cancer.

Src kinase inhibition restores E-cadherin expression in dasatinib-sensitive pancreatic cancer cells.

The Src family of non-receptor tyrosine kinases are frequently activated in pancreatic ductal adenocarcinoma (PDAC), contributing to disease progression through downregulation of E-cadherin and induction of epithelial-to-mesenchymal transition (EMT). The purpose of this study was to examine the efficacy of Src kinase inhibition in restoring E-cadherin levels in PDAC. Immunohistochemical analysis of human PDAC samples showed Src activation is inversely correlated with E-cadherin levels. Protein and mRNA levels of E-cadherin, the gene expression of its various transcriptional repressors (Zeb1, Snail, Slug, LEF-1, TWIST), and changes in sub-cellular localization of E-cadherin/ß-catenin in PDAC cells were characterized in response to treatment with the Src inhibitor, dasatinib (DST). DST repressed Slug mRNA expression, promoted E-cadherin transcription, and increased total and membranous E-cadherin/ß-catenin levels in drug-sensitive PDAC cells (BxPC3 and SW1990), however no change was observed in drug-resistant PANC1 cells. BxPC3, PANC1, and MiaPaCa-2 flank tumor xenografts were treated with DST to examine changes in E-cadherin levels in vivo. Although DST inhibited Src phosphorylation in all xenograft models, E-cadherin levels were only restored in BxPC3 xenograft tumors. These results suggest that Src kinase inhibition reverses EMT in drug-sensitive PDAC cells through Slug-mediated repression of E-cadherin and identifies E-cadherin as potential biomarker for determining response to DST treatment.

Urolithin A, a Novel Natural Compound to Target PI3K/AKT/mTOR Pathway in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy and is highly resistant to standard treatment regimens. Targeted therapies against KRAS, a mutation present in an overwhelming majority of PDAC cases, have been largely ineffective. However, inhibition of downstream components in the KRAS signaling cascade provides promising therapeutic targets in the management of PDAC and warrants further exploration. Here, we investigated Urolithin A (Uro A), a novel natural compound derived from pomegranates, which targets numerous kinases downstream of KRAS, in particular the PI3K/AKT/mTOR signaling pathways. We showed that treatment of PDAC cells with Uro A blocked the phosphorylation of AKT and p70S6K in vitro, successfully inhibited the growth of tumor xenografts, and increased overall survival of Ptf1aCre/+;LSL-KrasG12D/+;Tgfbr2flox/flox (PKT) mice compared with vehicle or gemcitabine therapy alone. Histologic evaluation of these Uro A-treated tumor samples confirmed mechanistic actions of Uro A via decreased phosphorylation of AKT and p70S6K, reduced proliferation, and increased cellular apoptosis in both xenograft and PKT mouse models. In addition, Uro A treatment reprogrammed the tumor microenvironment, as evidenced by reduced levels of infiltrating immunosuppressive cell populations such as myeloid-derived suppressor cells, tumor-associated macrophages, and regulatory T cells. Overall, this work provides convincing preclinical evidence for the utility of Uro A as a therapeutic agent in PDAC through suppression of the PI3K/AKT/mTOR pathway.