Spontaneous Pancreatic Tumors Research Articles

Loss of p53 and SMAD4 induces adenosquamous subtype pancreatic cancer in the absence of an oncogenic KRAS mutation

Pancreatic cancer is associated with an oncogenic KRAS mutation in approximately 90% of cases. However, a non-negligible proportion of pancreatic cancer cases harbor wild-type KRAS (KRAS-WT). This study establishes genetically engineered mouse models that develop spontaneous pancreatic cancer in the context of KRAS-WT. The Trp53loxP/loxP;Smad4loxP/loxP;Pdx1-Cre (PPSSC) mouse model harbors KRAS-WT and loss of Trp53/Smad4. The Trp53loxP/loxP;Tgfbr2loxP/loxP;Pdx1-Cre (PPTTC) mouse model harbors KRAS-WT and loss of Trp53/Tgfbr2. We identify that either Trp53/Smad4 loss or Trp53/Tgfbr2 loss can induce spontaneous pancreatic tumor formation in the absence of an oncogenic KRAS mutation. The Trp53/Smad4 loss and Trp53/Tgfbr2 loss mouse models exhibit distinct pancreatic tumor histological features, as compared to oncogenic KRAS-driven mouse models. Furthermore, KRAS-WT pancreatic tumors with Trp53/Smad4 loss reveal unique histological features of pancreatic adenosquamous carcinoma (PASC). Single-cell RNA sequencing (scRNA-seq) analysis reveals the distinct tumor immune microenvironment landscape of KRAS-WT (PPSSC) pancreatic tumors as compared with that of oncogenic KRAS-driven pancreatic tumors.

Genetic Deletion of Galectin-3 Inhibits Pancreatic Cancer Progression and Enhances the Efficacy of Immunotherapy

BACKGROUND & AIMSPancreatic ductal adenocarcinoma (PDAC) has a desmoplastic tumor stroma and immunosuppressive microenvironment. Galectin-3 (GAL3) is enriched in PDAC, highly expressed by cancer cells and myeloid cells. However, the functional roles of GAL3 in the PDAC microenvironment remain elusive. METHODSWe generated a novel transgenic mouse model (KPPC;Lgals3-/-) that allows the genetic depletion of GAL3 from both cancer cells and myeloid cells in spontaneous PDAC formation. Single-cell RNA-sequencing analysis was used to identify the alterations in the tumor microenvironment upon GAL3 depletion. We investigated both the cancer cell-intrinsic function and immunosuppressive function of GAL3. We also evaluated the therapeutic efficacy of GAL3 inhibition in combination with immunotherapy. RESULTSGenetic deletion of GAL3 significantly inhibited the spontaneous pancreatic tumor progression and prolonged the survival of KPPC;Lgals3-/- mice. Single-cell analysis revealed that genetic deletion of GAL3 altered the phenotypes of immune cells, cancer cells, and other cell populations. GAL3 deletion significantly enriched the anti-tumor myeloid cell subpopulation with high major histocompatibility complex class II (MHCII) expression. We also identified that GAL3 depletion resulted in CXCL12 upregulation, which could act as a potential compensating mechanism upon GAL3 deficiency. Combined inhibition of CXCL12-CXCR4 axis and GAL3 enhanced the efficacy of anti-PD-1 immunotherapy, leading to significantly inhibited PDAC progression. In addition, deletion of GAL3 also inhibited the basal/mesenchymal-like phenotype of pancreatic cancer cells. CONCLUSIONSGAL3 promotes PDAC progression and immunosuppression via both cancer cell-intrinsic and immune-related mechanisms. Combined treatment targeting GAL3, CXCL12-CXCR4 axis, and PD-1 represents a novel therapeutic strategy for PDAC.

Stromal HIF2 Regulates Immune Suppression in the Pancreatic Cancer Microenvironment

BACKGROUND & AIMS:Pancreatic ductal adenocarcinoma (PDAC) has a hypoxic, immunosuppressive stroma that contributes to its resistance to immune checkpoint blockade therapies. The hypoxia-inducible factors (HIFs) mediate the cellular response to hypoxia, but their role within the PDAC tumor microenvironment remains unknown.METHODS:We used a dual recombinase mouse model to delete Hif1α or Hif2α in α-smooth muscle actin–expressing cancer-associated fibroblasts (CAFs) arising within spontaneous pancreatic tumors. The effects of CAF HIF2α expression on tumor progression and composition of the tumor microenvironment were evaluated by Kaplan-Meier analysis, reverse transcription quantitative real-time polymerase chain reaction, histology, immunostaining, and by both bulk and single-cell RNA sequencing. CAF-macrophage crosstalk was modeled ex vivo using conditioned media from CAFs after treatment with hypoxia and PT2399, an HIF2 inhibitor currently in clinical trials. Syngeneic flank and orthotopic PDAC models were used to assess whether HIF2 inhibition improves response to immune checkpoint blockade.RESULTS:CAF-specific deletion of Hif2α, but not Hif1α, suppressed PDAC tumor progression and growth, and improved survival of mice by 50% (n ¼ 21–23 mice/group, Log-rank P ¼ .0009). Deletion of CAF-HIF2 modestly reduced tumor fibrosis and significantly decreased the intratumoral recruitment of immunosuppressive M2 macrophages and regulatory T cells. Treatment with the clinical HIF2 inhibitor PT2399 significantly reduced in vitro macrophage chemotaxis and M2 polarization, and improved tumor responses to immunotherapy in both syngeneic PDAC mouse models.CONCLUSIONS:Together, these data suggest that stromal HIF2 is an essential component of PDAC pathobiology and is a druggable therapeutic target that could relieve tumor microenvironment immunosuppression and enhance immune responses in this disease.

Feasibility of administering human pancreatic cancer chemotherapy in a spontaneous pancreatic cancer mouse model

BackgroundBoth modified FOLFIRINOX (mFFX) and gemcitabine/nab-paclitaxel chemotherapy regimens have been shown to improve clinical outcomes in patients with pancreatic cancer, and are often used interchangeably as the standard of care. Preclinical studies often do not use these regimens, since administering these multiagent approaches can be difficult. In this study, we assessed the feasibility of administering these two chemotherapy regimens in spontaneous pancreatic tumors using KPC mice with the ultimate goal of advancing preclinical studies.MethodsKPC mice were created by breeding KrasLSL−G12D/+ to Trp53fl/fl;Ptf1αCre/+, resulting in KrasLSL−G12D/+;p53fl/+;Ptf1αCre/+ mice. At 14 weeks of age, mice were palpated for spontaneous tumor growth that was verified using ultrasounds. Mice with tumors under 15 mm in diameter were used. The mice were assigned to one of seven treatment regimens: 1 cycle of mFFX (FFX X1), 2 cycles of mFFX (FFX X2), 1 cycle of mFFXwith 40 Gy SBRT (FFX SBRT), 1 cycle of gemcitabine/nab-paclitaxel (GEM/AB X1), 2 cycles of gemcitabine/nab-paclitaxel (GEM/AB X2), 2 cycles of gemcitabine/nab-paclitaxel with 40 Gy SBRT (GEM/AB SBRT), or saline only (control).ResultsIn total, 92 mice were included. The median OS in the FFX X2 group was slightly longer that the median OS in the FFX X1 group (15 days vs 11 days, P = 0.003). Mice in the GEM/AB X2 group had longer OS when compared to mice in the GEM/AB X1 group (33.5 vs 13 days, P = 0.001). Mice treated with chemotherapy survived longer than untreated control animals (median OS: 6.5 days, P < 0.001). Moreover, in mice treated with chemotherapy, mice that received 2 cycles of GEM/AB X2 had the longest survival, while the FFX X1 group had the poorest OS (P < 0.001). The addition of chemotherapy was associated with reduced number of myeloid and lymphoid cell types, except for CD4 + cells whose levels were largely unaltered only in tumors treated with gemcitabine/nab-paclitaxel. Lastly, chemotherapy followed by consolidative SBRT trended towards increased local control and survival.ConclusionsWe demonstrate the utility and feasibility of clinically relevant mFOLFIRINOX and gemcitabine/nab-paclitaxel in preclinical models of pancreatic cancer.

Oncolytic Vaccinia Virus Gene Modification and Cytokine Expression Effects on Tumor Infection, Immune Response, and Killing.

Oncolytic vaccinia viruses have promising efficacy and safety profiles in cancer therapy. Although antitumor activity can be increased by manipulating viral genes, the relative efficacy of individual modifications has been difficult to assess without side-by-side comparisons. This study sought to compare the initial antitumor activity after intravenous administration of five vaccinia virus variants of the same Western Reserve backbone and thymidine kinase gene deletion in RIP-Tag2 transgenic mice with spontaneous pancreatic neuroendocrine tumors. Tumors had focal regions of infection at 5 days after all viruses. Natural killer (NK) cells were restricted to these sites of infection, but CD8+ T cells and tumor cell apoptosis were widespread and varied among the viruses. Antitumor activity of virus VV-A34, bearing amino acid substitution A34K151E to increase viral spreading, and virus VV-IL2v, expressing a mouse IL2 variant (mIL2v) with attenuated IL2 receptor alpha subunit binding, was similar to control virus VV-GFP. However, antitumor activity was significantly greater after virus VV-A34/IL2v, which expressed mIL2v together with A34K151E mutation and viral B18R gene deletion, and virus VV-GMCSF that expressed mouse GM-CSF. Both viruses greatly increased expression of CD8 antigens Cd8a/Cd8b1 and cytotoxicity genes granzyme A, granzyme B, Fas ligand, and perforin-1 in tumors. VV-A34/IL2v led to higher serum IL2 and greater tumor expression of death receptor ligand TRAIL, but VV-GMCSF led to higher serum GM-CSF, greater expression of leukocyte chemokines and adhesion molecules, and more neutrophil recruitment. Together, the results show that antitumor activity is similarly increased by viral expression of GM-CSF or IL2v combined with additional genetic modifications.

Single cell dissociation from mouse tumor samples and analysis on BD FACSLyric Flow Cytometer.

e15245 Background: Single cell multiomics analysis of tumor cells and tumor infiltrating immune cells using high parameter flow cytometry and single cell genomics technologies is a powerful approach in the investigation of tumor biology and in the discovery of new drug targets and biomarkers. The preparation of single cells from solid tumor samples often involves a physical dissociation step in combination with an enzymatic digestion step. Methods: The effect of the tumor cell dissociation process on the phenotypic properties of single cells isolated from solid tumor samples were analyzed using a 12-color reagent panel on the BD FACSLyric flow cytometer. Specifically, spontaneous pancreatic tumor samples from genetically modified mice were processed using a manual dissociation process with a scalpel blade as well as an integrated tumor sample dissociation process with the prototype Singulator instrument with various protocol settings. The BD Horizon Dri Tumor &amp; Tissue Dissociation Reagent (TTDR) reagent was used for the enzymatic step in all sample preparations. The yield, viability, and frequency of tumor cells and different subsets of leukocytes were measured for each tumor-derived single cell sample preparation. On the Singulator system, cell yield reached a plateau with highest viability at the 30-min process time and a higher percentage of tumor cells was obtained compared to the manual sample processing method. Results: The relative frequencies of T cells, B cells, myeloid cells and macrophages also changed in different sample preparation methods. However, the spatial heterogeneity in the tumor section and cell composition may contribute to these differences and would require more in-depth analysis. In summary, these results suggest that there are many variables in the tumor sample single cell dissociation process that could impact the downstream study results, particularly for multiomics studies. Conclusions: This study highlights the need for optimization and standardization of the sample processing methodologies for downstream analysis. Disclaimers: For Research Use Only. Not for use in diagnostic or therapeutic procedures. Class 1 Laser Product. BD, the BD Logo, FACSLyric, and Horizon are trademarks of Becton, Dickinson and Company. © 2020 BD and its subsidiaries. All rights reserved.

PET/MRI enables simultaneous in vivo quantification of β-cell mass and function.

Non-invasive imaging of β-cells represents a desirable preclinical and clinical tool to monitor the change of β-cell mass and the loss of function during pre-diabetic stages. Although it is widely accepted that manganese (Mn) ions are actively gated by voltage-dependent calcium channels (VDCC) in response to glucose metabolism, little is known on its specificity in vivo for quantification of islet β-cell function using Mn and magnetic resonance imaging (MRI). On the other hand, glucagon-like-peptide-1 receptor (GLP-1R) represents a validated target for the estimation of β-cell mass using radiolabeled exendin-4 (Ex4) and positron emission tomography (PET). However, a multiparametric imaging workflow revealing β-cell mass and function quantitatively is still missing.Methods: We developed a simultaneous PET/MRI protocol to comprehensively quantify in vivo changes in β-cell mass and function by targeting, respectively, GLP-1R and VDCC coupled with insulin secretion. Differences in the spatial distribution of Mn and radiolabeled Ex4 were monitored overtime in native and transgenic pancreata, characterized by spontaneous pancreatic neuroendocrine tumor development. Follow-up with mass spectrometry imaging (MSI) and autoradiography allowed the ex vivo validation of the specificity of Mn and PET tracer uptake and the detection of endogenous biometals, such as calcium and zinc, throughout the endocrine and exocrine pancreas.Results: Our in vivo data based on a volumetric PET/MRI readout for native pancreata and insulinomas connects uptake of Mn measured at early imaging time points to high non-specific binding by the exocrine tissue, while specific retention was only found 24 h post injection. These results are supported by cross-validation of the spatial distribution of exogenous 55Mn and endogenous 44Ca and 64Zn as well with the specific internalization of the radiolabeled peptide targeting GLP-1R.Conclusion: Simultaneous PET/MR imaging of the pancreas enabled the comprehensive in vivo quantification of β-cell function and mass using Mn and radiolabeled Ex4. Most important, our data revealed that only late time-point measurements reflect the Mn uptake in the islet β-cells, while early time points detect non-specific accumulation of Mn in the exocrine pancreas.

Abstract 4012: SAG/RBX2/ROC2 regulates pancreatic cancer initiation, cystic formation in mice driven by KrasG12D

Abstract BACKGROUND &amp; AIMS: SAG, a small ring finger protein, a component of CRLs, might be involved in pancreatic tumorigenesis. SAG overexpression in human pancreatic ductal adenocarcinomas indicating that SAG could be promotes pancreatic tumorigenesis. We investigated how manipulated SAG (overexpression or disruption of SAG) cooperate KRAS to regulate pancreatic cancer progression in mice. METHODS: We disrupted or overexpressed SAG specifically in pancreata of mice (Sagfl/fl;P48-Cre or SagTG;P48-Cre mice) and crossed them with LSL-KRASG12D mice, which express an activated form of KRAS and develop spontaneous pancreatic tumors. The resulting Sagfl/fl;LSL-KRASG12D;P48-Cre or SagTG;LSL-KRASG12D;P48-Cre mice were monitored; Pancreatic tissues were collected and analyzed by histology and immunohistochemical analyses. Protein was isolated from mouse tissues and primary acinar cells were prepared and cultured in 3D condition. Human PDA TMA was analyzed by immunohistochemistry. RESULTS: Overexpression of Sag with KRASG12D in pancreata of mice increased the proliferation of pancreatic epithelial cells, lead to formation of acinar to ductal metaplasia and pancreatic intraepithelial neoplasias (mPanINs) and shorten mice survival. Disruption of Sag in LSL-KRASG12D;P48-Cre mice accelerated the growth of pancreatic cystic lesions compared to LSL-KRASG12D;P48-Cre mice, but did not change mouse survival. In mechanism, Sag promotes pancreatic tumorigenesis by regulating Deptor/mTOR/S6K axis and EGFR signaling pathway. CONCLUSION: Sag is an oncogenic cooperator of activated KRAS for pancreatic tumorigenesis. Targeting SAG E3 ligase may, therefore, have therapeutic value for the treatment of prostate cancer associated with mutant KRAS. Citation Format: Mingjia Tan, Qiang Zhang, Yi Sun. SAG/RBX2/ROC2 regulates pancreatic cancer initiation, cystic formation in mice driven by KrasG12D [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4012.

Abstract 3026: Role of AnnexinA2, Sema3D and PlexinD1 in mediating perineural invasion as a mechanism of metastasis in pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDA) is a devastating malignant disease. Upon diagnosis, eighty percent of patients present with metastatic disease. Perineural invasion, the spreading of cancer to the space surrounding or invading the nerve, is often found in surgically removed pancreatic tumors and is associated with a worse overall prognosis including high risk of recurrence and metastasis. Perineural invasion is thought to represent the initial steps of metastasis; however, the mechanism that mediates the role of neurons in precancerous inflammation and pancreatic cancer initiation is not known. We have previously found that Annexin A2 (AnxA2), is a metastasis-associated protein in PDA and is not required for primary PDA growth but is essential for the metastasis in genetically engineered spontaneous pancreatic tumor producing KPC mice. Knock-down of AnxA2 results in diminished metastasis and prolonged survival in mouse liver metastasis models of PDA. Moreover, the presence of perineural invasion is AnxA2 dependent in this model. By comparing tumor cells of AnxA2 wild type vs. knock-out KPC mice, we identified Semaphorin 3D (Sema3D) and PlexinD1 (PlxnD1) were among the most differentially expressed genes. We found that AnxA2 regulates the secretion of Sema3D. Originally characterized as axon guidance genes, ligand Sema3D along with its receptor PlxnD1 and co-receptor, Neuropilin 1 (NP1), are among the cellular pathways most frequently altered at the genetic level in PDA. Moreover, we found that knock down of Sema3D or PlxnD1 suppresses metastasis formation in mouse models of PDA (Foley et al. Science Signaling 2015). In the present study, our lab has found that PNI was present in 9/10 human PDA samples with abundant Sema3D expression compared to only 3/10 human PDA samples with low Sema3D expression. Using a Dorsal Root Ganglion (DRG) invasion assay, we found paracrine signaling between DRG and pancreatic cancer cells is important for increasing tumor cell invasion toward DRGs. Using small interfering RNA for AnxA2 in tumor cells, we find that decrease in AnxA2 significantly decreases tumor invasion. Furthermore, knock down of Sema3D from tumor cells, using shRNA, shows significant decrease in tumor cell invasion toward DRGs. We have also tested the role of the Sema3D receptor, PlexinD1, on DRGs in regulating the invasion of tumor cells towards nerves. A neutralizing antibody for PlexinD1 treated on DRGs caused significant decrease in tumor cell invasion in comparison to DRGs treated with IgG isotype control. These results demonstrate that AnxA2, Sema3D and PlexinD1 are involved in paracrine signaling between the tumor cells and nerves. Taken together, we conclude AnxA2/Sema3D/PlexinD1 signaling may aid in mediating perineural invasion of tumor cells towards nerves as a mechanism of metastasis in pancreatic ductal adenocarcinoma. Citation Format: Noelle R. Jurcak, Kenji Fujiwara, Agnieszka A. Rucki, Kelly Foley, Adrian Murphy, Stephen Muth, Abigail Brittingham, Elizabeth M. Jaffee, Lei Zheng. Role of AnnexinA2, Sema3D and PlexinD1 in mediating perineural invasion as a mechanism of metastasis in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3026.

Effect of stereotactic radiation (SBRT) on survival in a preclinical model of spontaneous pancreatic cancer with induction gemcitabine/nab-paclitaxel.

273 Background: There are no useful preclinical models that resemble relevant fractionated radiation, such as SBRT, with relevant chemotherapy. Most preclinical studies use a single fraction of radiation to large fields, causing significant toxicity and/or insufficient dose which leads to accelerated repopulation and worsened outcomes. Here, we employ mice with spontaneous pancreatic tumors, and treat them in a preclinical trial utilizing gemcitabine/nab-paclitaxel +/-SBRT. Methods: Mice with an activated KRas (G12D) allele and heterozygous for p53 expression only in the pancreas (KPC) were created and spontaneous tumors were diagnosed via weekly palpation and ultrasound. Mice with single tumors that were between 3-7mm at diagnosis were sequentially randomized to received either no treatment, gemcitabine/nab-paclitaxel x 2 weeks, or gemcitabine/nab-paclitaxel x 2 weeks followed by 8Gy x 5 daily fractions of SBRT to the pancreatic tumor. SBRT was delivered using X-RAD 225Cx (Precision X-Ray, Inc) small animal irradiator under image guidance by cone beam micro CT using AP/PA technique with a 10mm collimator. Every mouse was subjected to necropsy and a cause of death assigned. Tumor growth was monitored by small animal ultrasound. Kaplan-Meier survival analysis was performed to assess efficacy of treatments. Results: A total of 40 mice were enrolled with 12 receiving chemotherapy alone, 13 receiving chemotherapy +SBRT and 15 mice had no further treatment. The addition of induction chemotherapy improves lifespan compared to untreated mice. The addition of SBRT further improved survival. Causes of death in untreated animals of the gemcitabine/nab-paclitaxel group were largely from local progression, whereas the addition of SBRT provided local control causing most of the mice in this cohort to die from metastatic progression. Conclusions: SBRT improves survival when added to gem/nab-paclitaxel in a preclinical model, which may reflect favorable biology for this approach. Our novel system employs similar SBRT fields and doses, and may thus be useful for the evaluation of other chemotherapeutic regimens with SBRT.

Ultrasensitive MRI detection of spontaneous pancreatic tumors with nanocage-based targeted contrast agent

Contrast agents with greater specificity and sensitivity are required for the diagnosis of pancreatic cancers by magnetic resonance imaging (MRI). In this study, small heat shock protein 16.5 (Hsp16.5)-based nanocages conjugated to gadolinium(III)-chelated contrast agents and iRGD peptides (which target neuropilin-1 expressed on pancreatic cancer cells) were developed. To investigate whether template size influences relaxivity, nanocages with one to four hydrophobic domains were designed. MRI data showed that larger nanocages had higher T1 relaxivity than smaller nanocages, which resulted from a reduction in molecular tumbling rates caused by an increase in nanocage size, and a robust cage structure resulting from the introduction of hydrophobic domains. For in vivo MRI studies, the engineered nanocages were evaluated using the KrasG12D; Trp53R172H; Pdx-1Cre (KPC) transgenic mouse models, which develop clinically relevant pancreatic tumor under normal processes of angiogenesis, immune function and inflammation. Molecular MRI with protein nanocages was enabled to detect neuropilin-1-positive cells and to produce strong signal enhancement of spontaneous pancreatic tumors in KPC genetically engineered mouse models. Novel iRGD-modified nanocages displayed potential as a specific and sensitive MRI contrast agent for the diagnosis of pancreatic tumors for clinical translation.

Abstract 22: Investigating the functional contribution of TANK binding kinase 1 to inflammation induced disease progression

Abstract Initial stages of human pancreatic ductal adenocarcinoma (PDA) are commonly characterized by an activating mutation in K-RAS along with extensive immune cell infiltration. Direct inhibition of K-RAS through pharmacological means remains a challenge; however targeted inhibition of TANK Binding Kinase 1 (TBK1), a critical downstream effector of mutant active K-RAS is an attractive alternative. High levels of active TBK1 are associated with inflammatory disease and cancer progression. TBK1 and homolog, IKKe activate the immune response transcription factor NF-κB. In metabolically challenged mice, IKKe regulates energy balance by sustaining chronic, low-grade inflammation. We hypothesize that TBK1 signaling is also critical in metabolic regulation and is required for progression of inflammation-induced diseases such as PDA. A kinase dead, Tbk1Δ/Δ mouse was used to determine the contribution of TBK1 to metabolic disease and PDA progression. Metabolic phenotyping experiments were performed with Tbk1Δ/Δ and Tbk1+/+ mice including a high fat diet weight study, body composition and metabolic chamber analyses. Tbk1+/+ and Tbk1Δ/Δ mice were crossed into a genetically engineered mouse model of PDA to determine the consequences of genetically removing Tbk1 on tumor development and overall survival. Here we report that Tbk1Δ/Δ mice are significantly smaller, leaner and have less fat than Tbk1+/+ mice on high fat chow diets (HFD) for 14 weeks. Tbk1Δ/Δ mice are more active and have smaller and more abundant adipocytes relative to Tbk1+/+ mice. Additionally, Tbk1Δ/Δ mice are protected from HFD induced hypercholesterolemia and liver steatosis. White adipose tissue (WAT) from HFD fed Tbk1Δ/Δ mice exhibit an induction of brown fat gene expression suggesting that thermogenesis is a contributor to their healthier and more active phenotype. Macrophage infiltration is fairly low in WAT from Tbk1+/+ and Tbk1Δ/Δ mice on normal chow. However on HFD, WAT from Tbk1+/+ mice display a significant increase in macrophage marker expression compared to Tbk1Δ/Δ mice. These findings indicate that HFD fed Tbk1Δ/Δ mice are protected from classic phenotypes of metabolic syndrome. In PDA, TBK1 is expressed and more active in human PDA cell lines relative to immortalized pancreatic epithelial lines and fibroblasts. Human PDA cell lines are sensitive to a small molecule inhibitor of TBK1 (compound II) in the low micromolar range. In a K-Ras driven genetic mouse model of PDA, TBK1 supports spontaneous pancreatic tumor growth as evidenced by smaller tumors in Tbk1Δ/Δ: PDA mice relative to Tbk1+/+: PDA mice. Our results suggest that TBK1 contributes to metabolic regulation and demonstrate the therapeutic potential of targeting TBK1 in pancreatic malignancies. Current work is focused on delineating the inflammatory and metabolic dysregulation in these animals and determining the precise mechanism by which TBK1 supports the progression of PDA. Citation Format: Victoria H. Burton, Rolf A. Brekken. Investigating the functional contribution of TANK binding kinase 1 to inflammation induced disease progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 22.

Evaluation of triptolide pro-drug (Minnelide) as an anti-stromal and anti-tumoral therapeutic option for pancreatic cancer.

262 Background: In pancreatic cancer, the stromal microenvironment is considered to be the major reason behind the failure of conventional and targeted therapy. The desmoplastic stroma is responsible for compression of vasculature in the tumor resulting in impaired drug delivery. Pancreatic stellate cells contribute extensively to this desmoplastic reaction. Our group has recently evaluated a water-soluble pro-drug of triptolide (Minnelide) in pre-clinical studies for pancreatic cancer with very promising results. The current study evaluates the ability of Minnelide to deplete the stromal architecture and the stellate cell population in pancreatic cancer resulting in increased drug delivery. Methods: Patient tumor xenografts in SCID mice (PDX) and KRASG12DTP53-PDX-Cre spontaneous pancreatic tumor mice (KPC) were treated with 0.42mg/kg Minnelide. Vascular patency was studied by terminal perfusion of mice with 4% PFA following injection with tomato lectin. Drug delivery was determined by quantitating accumulated doxorubicin in the tumor tissue. Cell viability was studied by MTT assay and the invasion of cells in vitro was studied using Boyden Chamber assay. Results: Minnelide treatment decreased Hyaluronic acid (HA) in PDX (48% of control) as well as in KPC model (52% of control). Expression of HA synthase (HAS) genes were decreased in both tumor models in response to Minnelide (22% of control in PDX and 35% of control in KPC tumors). HAS Enzyme activity also consistently decreased in both the models after treatment with Minnelide (28% of control in PDX and 31% of control in KPC). In vitro, triptolide decreased the invasion of the tumor cells towards the stellate cells indicating an inhibition of crosstalk. Treatment with Minnelide resulted in “open” and functional vessels in the treated animals resulting in increased drug delivery. Median survival of animals increased by 38 days after treatment with Minnelide. Conclusions: Our study showed that Minnelide depleted the stromal architecture leading to increased functional vasculature and enhanced drug delivery in the tumor. This is an extremely promising observation for Minnelide that is currently under clinical trial.

Abstract A71: Investigating the functional contribution of TANK Binding Kinase 1 to metabolic regulation in pancreas cancer

Abstract Effective therapies are needed to enhance the long-term survival of patients with pancreatic ductal adenocarcinoma (PDA), the fourth leading cause of cancer-related deaths in the United States. Initial stages of human PDA are commonly characterized by an activating mutation in K-RAS along with extensive immune cell infiltration. Yet, effectively inhibiting K-RAS through pharmacological means has proven difficult. However, high levels of active TANK Binding Kinase 1 (TBK1), a critical downstream effector of mutant active K-RAS in lung cancer, are associated with inflammatory disease. TBK1 and homolog, IKKe can both activate the immune response transcription factor NF-κB. Interestingly, IKKe has been shown to regulate lipid and glucose metabolism by sustaining a state of chronic, low-grade inflammation in obese mice. Interestingly, IKKe has been shown to regulate energy balance by sustaining a state of chronic, low-grade inflammation in obese mice. Given this recent finding, we suspect that TBK1 can also promote glucose and lipid metabolism concerted with inflammation to promote pancreatic tumorigenensis. Therefore, we set out to distinguish TBK1 metabolic function and then determine if and how these TBK1 functions contribute to PDA. Homozygous Tbk1 deletion in a C57BL/6 genetic background is embryonic lethal (E14.5) due to massive liver degeneration and apoptosis. However, conditional deletion of the Tbk1 catalytic domain in the 129S5 mouse strain resulted in viable animals that express low levels of a truncated, enzymatically inactive form of TBK1. Various metabolic phenotyping experiments were performed with these Tbk1Δ/Δ mice to gain insight into the non-redundant metabolic roles of TBK1. In general we've observed that Tbk1Δ/Δ mice are significantly smaller, leaner and have less fat than Tbk1 +/+ mice on both normal chow and high fat chow. Accordingly, Tbk1Δ/Δ mice are more active and have increased thermogenesis relative to Tbk1 +/+ mice. In the PDA setting, TBK1 is expressed and more active in human PDA cell lines relative to immortalized pancreatic epithelial lines (HPNE) and fibroblasts. Human PDA cell lines are sensitive to a small molecule inhibition of TBK1 in the low micromolar range. TBK1 supports the growth of spontaneous pancreatic tumors in a K-Ras driven genetic mouse model of PDA, as evidenced by smaller tumors from Tbk1Δ/Δ: PDA mice relative to Tbk1 +/+: PDA mice at multiple timepoints. So far, our results imply that TBK1 contributes to metabolic regulation and demonstrate the therapeutic potential of targeting TBK1 in pancreatic malignancies. Current work is focused on delineating the inflammatory and metabolic dysregulation in these animals and determining the precise mechanism by which TBK1 supports pancreatic cancer. Citation Format: Victoria H. Burton, Rolf A. Brekken. Investigating the functional contribution of TANK Binding Kinase 1 to metabolic regulation in pancreas cancer. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A71.

Loss of Activin Receptor Type 1B Accelerates Development of Intraductal Papillary Mucinous Neoplasms in Mice With Activated KRAS

Activin, a member of the transforming growth factor-β (TGFB) family, might be involved in pancreatictumorigenesis, similar to other members of the TGFB family.Human pancreatic ductal adenocarcinomas contain somatic mutations in the activin A receptor type IB (ACVR1B) gene, indicating that ACVR1B could be a suppressor of pancreatic tumorigenesis. We disrupted Acvr1b specifically in pancreata of mice (Acvr1b(flox/flox);Pdx1-Cre mice) and crossed them with LSL-KRAS(G12D) mice, which express an activated form of KRAS and develop spontaneous pancreatic tumors. The resulting Acvr1b(flox/flox);LSL-KRAS(G12D);Pdx1-Cre mice were monitored; pancreatic tissues were collected and analyzed by histology and immunohistochemical analyses. We also analyzed p16(flox/flox);LSL-Kras(G12D);Pdx1-Cre mice and Cre-negative littermates (controls). Genomic DNA, total RNA, andprotein were isolated from mouse tissues and primary pancreatic tumor cell lines and analyzed by reverse-transcription polymerase chain reaction, sequencing, and immunoblot analyses. Human intraductal papillary mucinous neoplasm (IPMN) specimens were analyzed by immunohistochemistry. Loss of ACVR1B from pancreata of mice increased the proliferation of pancreatic epithelial cells, led toformation of acinar to ductal metaplasia, and induced focalinflammatory changes compared with control mice. Disruptionof Acvr1b in LSL-KRAS(G12D);Pdx1-Cre mice accelerated thegrowth of pancreatic IPMNs compared with LSL-KRAS(G12D);Pdx1-Cre mice, but did not alter growth of pancreatic intraepithelial neoplasias. We associated perinuclear localization of the activated NOTCH4 intracellular domain to the apical cytoplasm of neoplastic cells with the expansion of IPMN lesions in Acvr1b(flox/flox);LSL-KRAS(G12D);Pdx1-Cre mice. Loss of the gene that encodes p16 (Cdkn2a) was required for progression of IPMNs to pancreatic ductal adenocarcinomas in Acvr1b(flox/flox);LSL-Kras(G12D);Pdx1-Cre mice. We also observed progressive loss of p16 in human IPMNs of increasing grades. Loss of ACVR1B accelerates growth of mutant KRAS-induced pancreatic IPMNs in mice; this process appears to involve NOTCH4 and loss of p16. ACVR1B suppresses early stages of pancreatic tumorigenesis; the activin signaling pathway therefore might be a therapeutic target for pancreatic cancer.

Animal models of spontaneous pancreatic neuroendocrine tumors

Pancreatic neuroendocrine tumors (PNETs) are usually low-grade neoplasms derived from the endocrine pancreas. PNETs can be functioning and cause well-described hormonal hypersecretion syndromes or non-functioning and cause only tumor mass effect. PNETs appear to be more common recently likely due to incidental detection by imaging. Although the diagnosis and management of PNETs have been evolving rapidly, much remains to be studied in the areas of molecular pathogenesis, molecular markers of tumor behavior, early detection, and targeted drug therapy. Unique challenges facing PNETs studies are long disease course, the deep location of pancreas and difficult access to pancreatic tissue, and the variety of tumors, which make animal models valuable tools for PNETs studies. Existing animal models of PNETs have provided insights into the pathogenesis and natural history of human PNETs. Future studies on animal models of PNETs should address early tumor detection, molecular markers of tumor behavior, and novel targeted therapies.

Abstract A17: The role of post-translational activation of MYC in pancreatic cancer development and progression

Abstract Pancreatic cancer is the 4th leading cause of cancer-related deaths in the United States, with 46,420 new cases and 39,590 deaths estimated in 2014. Pancreatic cancer is a disease with a high rate of mortality, as it is generally diagnosed in the advanced stages when no curative treatments are available. Despite increased understanding of the morphology, biology, and molecular aberrations in pancreatic cancer, the 5-year survival rate has not improved significantly in the past 30 years. The c-MYC (MYC) oncoprotein is a potent regulator of cell fate decisions that drives nearly all aspects of tumorigenesis, including tumor cell proliferation and tumor microenvironment remodeling. High levels of MYC expression occur in a wide variety of human tumors, and animal models demonstrate that MYC overexpression can drive tumorigenesis in many tissues. The MYC gene is frequently amplified in human pancreatic cancer and MYC mRNA and/or protein are often overexpressed. However, the role of MYC in pancreatic cancer development and progression is not well understood. Our laboratory has demonstrated that MYC is post-translationally activated by KRAS signaling. Specifically, the RAS-RAF-MEK-ERK signaling pathway leads to phosphorylation of MYC at Serine 62 (pS62-MYC), which increases MYC’s protein stability as well as its activation of oncogenic target gene expression. Furthermore, we have observed high expression of pS62-MYC in primary pancreatic tumors, starting as early as PanIN 1, compared to normal adjacent tissue. Since activating mutations in KRAS are an early and nearly universal event in the development of pancreatic ductal adenocarcinoma (PDAC), these data suggest that MYC may be a critical downstream effector of KRAS-driven pancreatic cancer. We have generated unique c-MYC knock-in mice in which MYC is expressed from the ROSA26 locus following tissue-specific expression of Cre recombinase (ROSA-LSL-MYC). These mice express transcriptionally deregulated, but physiological levels of MYC. At these levels, however, MYCWT knock-in is not sufficient to drive tumors in any tested tissue. Since KRAS can post-translationally activate MYC, we hypothesized that low-level expression of deregulated MYC would increase KRAS driven pancreatic tumors in mice that co-express KRASG12D and MYCWT . We crossed ROSA-LSL-MYC mice with LSL-KRASG12D;Pdx1-Cre (KC) mice to generate the LSL-KRASG12D;ROSA-LSL-MYC;Pdx1-Cre (KMC) model. We found that co-expression of MYC and activated KRAS induces acinar to ductal metaplasia (ADM) and accelerates both PanIN precursor lesion formation and its conversion to PDAC relative to KRAS only. Tumors from KMC mice histologically resemble human PDAC, with a 20% metastatic rate to the liver. Together, these results suggest that MYC is important for both the initiation and progression of pancreatic tumors. In addition to ductal adenocarcinoma, the MYC/KRAS expressing mice also developed metastatic, poorly differentiated carcinomas with neuroendocrine features. While activation of KRAS is thought to be one of the earliest oncogenic changes in PDAC-precursor cells, studies suggest that several other genetic events occur during the evolution of PanIN lesions to PDAC, including loss of p16, mutation/loss of p53, and loss of SMAD4. Importantly, tumors arising in our KMC model spontaneously exhibit these stage-appropriate molecular aberrations, suggesting that this model accurately recapitulates the genetic evolution of human pancreatic cancers. We also observe a strong desmoplastic reaction with prominent stroma and immune cell infiltration in these spontaneous pancreatic tumors. Thus, our novel KMC mice may provide a physiologically relevant model of human pancreatic cancer for the testing of new therapeutic agents. Citation Format: Amy S. Farrell, Colin J. Daniel, Zina Jenny, Brett Sheppard, Jody Hooper, Rosalie C. Sears. The role of post-translational activation of MYC in pancreatic cancer development and progression. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A17.

Exclusion of T Cells From Pancreatic Carcinomas in Mice Is Regulated by Ly6Clow F4/80+ Extratumoral Macrophages

Immunotherapies that induce T-cell responses have shown efficacy against some solid malignancies in patients and mice, but these have little effect on pancreatic ductal adenocarcinoma (PDAC). We investigated whether the ability of PDAC to evade T-cell responses induced by immunotherapies results from the low level of immunogenicity of tumor cells, the tumor's immunosuppressive mechanisms, or both. Kras(G12D/+);Trp53(R172H/+);Pdx-1-Cre (KPC) mice, which develop spontaneous PDAC, or their littermates (controls) were given subcutaneous injections of a syngeneic KPC-derived PDAC cell line. Mice were then given gemcitabine and an agonist of CD40 to induce tumor-specific immunity mediated by T cells. Some mice were also given clodronate-encapsulated liposomes to deplete macrophages. Tumor growth was monitored. Tumor and spleen tissues were collected and analyzed by histology, flow cytometry, and immunohistochemistry. Gemcitabine in combination with a CD40 agonist induced T-cell-dependent regression of subcutaneous PDAC in KPC and control mice. In KPC mice given gemcitabine and a CD40 agonist, CD4(+) and CD8(+) T cells infiltrated subcutaneous tumors, but only CD4(+) T cells infiltrated spontaneous pancreatic tumors (not CD8(+) T cells). In mice depleted of Ly6C(low) F4/80(+) extratumoral macrophages, the combination of gemcitabine and a CD40 agonist stimulated infiltration of spontaneous tumors by CD8(+) T cells and induced tumor regression, mediated by CD8(+) T cells. Ly6C(low) F4/80(+) macrophages that reside outside of the tumor microenvironment regulate infiltration of T cells into PDAC and establish a site of immune privilege. Strategies to reverse the immune privilege of PDAC, which is regulated by extratumoral macrophages, might increase the efficacy of T-cell immunotherapy for patients with PDAC.

Abstract B23: Preclinical evaluation of a PARP inhibitor in mice representing genetically different subtypes of pancreatic cancers

Abstract The incredibly short survival of patients diagnosed with pancreatic cancer underscores one of the greatest failures of the medical community: our inability to identify effective treatments for pancreatic ductal adenocarcinoma. Most new compounds in clinical trials fail because of highly toxic side effects in normal tissues and limited efficacy in tumor response. An ideal drug would kill only tumor cells and spare the normal cells. In this regard, poly(ADP-ribose) polymerase (PARP) inhibitors have demonstrated a promising therapeutic potential, particularly in patients with BRCA-deficient tumors. BRCA-deficient cells have an impaired homology-directed repair (HDR) pathway, a mechanism critical for resolving DNA double strand breaks (DSBs). When PARP1 is inhibited, DNA single strand breaks are not repaired and they mature to double strand breaks, which are very toxic for the cell. Hence, inhibition of PARP1 is selectively toxic to cells with an impaired HDR pathway, such as BRCA-deficient tumor cells, but not to normal cells. Because up to 10% of pancreatic cancer patients carry mutations in either BRCA2 or other proteins involved in the HDR pathway, PARP inhibitors may also prove effective in this setting. However, a number of subtleties remain that call this conclusion into question, including poor drug delivery, loss of heterozygosity, and BRCA-independent alteration of the HDR pathway. We designed a preclinical study to evaluate the efficacy of niraparib, a potent PARP inhibitor currently in clinical trials, in three different genetically engineered mouse models representing different pancreatic cancer patient populations. The KPC mice carry mutations in genes that are frequently mutated in pancreatic cancer patients and develop tumors that fully recapitulate the biology of human pancreatic ductal adenocarcinoma. These mice will represent the broad population of patients with spontaneous pancreatic tumors. A second model with heterozygous mutations in BRCA2 (KPCB2+/-) will represent patients with hereditary BRCA2 mutations. A third model with homozygous conditional deletion of BRCA2 (KPCB2f/f) will represent spontaneous tumors that have completely lost HDR functions. We show that BRCA2-deficient KPC tumor cells strongly respond to treatment with niraparib in vitro and in vivo. Niraparib efficiently inhibits PARP activity, increases double strand break formation and induces M-phase cell cycle arrest in tumors of KPCB2f/f mice. In the future, we will use pre- and post-treatment tumor biopsies to determine the molecular mechanisms of response to PARP inhibition. The ultimate goal of this study is to identify genetic determinants of PARP sensitivity and validate our results in a clinical setting. Identification of a biomarker would be an extremely valuable clinical tool to classify pancreatic cancer patients that could respond to the treatment with PARP inhibitors, a tool that is currently not available. Citation Format: Barbara Orelli, Stephen A. Sastra, Carmine F. Palermo, Thomas Ludwig, Kenneth P. Olive. Preclinical evaluation of a PARP inhibitor in mice representing genetically different subtypes of pancreatic cancers. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr B23.

Systemic siRNA delivery to a spontaneous pancreatic tumor model in transgenic mice by PEGylated calcium phosphate hybrid micelles

Efficient systems for delivery of small interfering RNA (siRNA) are required for clinical application of RNA interference (RNAi) in cancer therapy. Herein, we developed a safe and efficient nanocarrier comprising poly(ethylene glycol)-block-charge-conversional polymer (PEG-CCP)/calcium phosphate (CaP) hybrid micelles for systemic delivery of siRNA and studied their efficacy in spontaneous bioluminescent pancreatic tumors from transgenic mice. PEG-CCP was engineered to provide the siRNA-loaded hybrid micelles with enhanced colloidal stability and biocompatibility due to the PEG capsule and with endosome-disrupting functionality due to the acidic pH-responsive CCP segment where the polyanionic structure could be converted to polycationic structure at acidic pH through cis-aconitic amide cleavage. The resulting hybrid micelles were confirmed to have a diameter of <50nm, with a narrow size distribution. Intravenously injected hybrid micelles significantly reduced the luciferase-based luminescent signal from the spontaneous pancreatic tumors in an siRNA sequence-specific manner. The gene silencing activity of the hybrid micelles correlated with their preferential tumor accumulation, as indicated by fluorescence imaging and histological analysis. Moreover, there were no significant changes in hematological parameters in mice treated with the hybrid micelles. These results demonstrate the great potential of the hybrid micelles as siRNA carriers for RNAi-based cancer therapy.