Mouse Model Of Pancreatic Ductal Adenocarcinoma Research Articles

Combined Autophagy Inhibition and Dendritic Cell Recruitment Induces Antitumor Immunity and Enhances Immune Checkpoint Blockade Sensitivity in Pancreatic Cancer.

The effect of immune checkpoint inhibitors is extremely limited in patients with pancreatic ductal adenocarcinoma (PDAC) due to the suppressive tumor immune microenvironment (TIME). Autophagy, which has been shown to play a role in anti-tumor immunity, has been proposed as a therapeutic target for PDAC. Here, single-cell RNA-sequencing of autophagy-deficient murine PDAC tumors revealed that autophagy inhibition in cancer cells induced dendritic cell (DC) activation. Analysis of human PDAC tumors substantiated a negative correlation between autophagy and DC activation signatures. Mechanistically, autophagy inhibition increased intracellular accumulation of tumor antigens, which could activate DCs. Administration of chloroquine (CQ), an autophagy inhibitor, in combination with Flt3 ligand (Flt3L)-induced DC infiltration inhibited tumor growth and increased tumor-infiltrating T lymphocytes. However, autophagy inhibition in cancer cells also induced CD8+ T cell exhaustion with high expression of immune checkpoint LAG3. A triple therapy comprising CQ, Flt3L, and an anti-LAG3 antibody markedly reduced tumor growth in orthotopic syngeneic PDAC mouse models. Thus, targeting autophagy in cancer cells and activating DCs sensitizes PDAC tumors to immune checkpoint inhibitor therapy, warranting further development of this treatment approach to overcome immunosuppression in pancreatic cancer.

Abstract A060: Dysregulation of PTEN expression in a subset of Cancer Associated Fibroblasts by Tumor Secreted Factors in PDAC

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) is a deadly malignancy. Cancer associated fibroblasts (CAFs) play a complex role in the PDAC tumor microenvironment (TME), with a subset contributing significantly to tumor progression. Phosphatase and tensin homolog (PTEN) is a tumor suppressor and its loss is associated with increased tumor aggressiveness. Our group previously demonstrated PTEN loss in SMA+ positive CAFS correlated with worsened outcomes in patients and in mouse PDAC models. Our group previously demonstrated the loss of PTEN in CAFS led to activation of Stat3, resulting in an immunosuppressive microenvironment (Lefler et al, 2022 PMID: 35803738). However, little is known of the signals produced by tumor cells or other cell types in the TME that trigger loss of PTEN in CAFs. This study aims to to elucidate the molecular mechanisms underlying PTEN loss in CAFs in PDAC. To address this gap, we employed single cell RNA sequencing of tumors from a mouse PDAC model and identified a (PDRGFRa+Lys6+Stat3+Pten-low) CAF subpopulation. Comparing normal pancreas epithelial cells to tumor cells in this data set, we identified the IL6 family of cytokines, (IL6; Oncostatin-M, OSM; and Leukemia Inhibitory Factor, LIF) and Transforming Growth Factor beta 1 (TGFb1) as potential ligands that regulated PTEN, while myeloid cells were a source of the IL6 family member Oncostatin M (OSM). Treatment of isolated CAFs with these factors revealed no significant difference in PTEN mRNA expression. Thus, we developed an in vitro reporter system in which a PTEN-GFP fusion protein is expressed in CAFs. Treating these CAFs with IL6, OSM and TGFb1 (but not LIF) led to a significant decrease in GFP fluorescence. Analysis of endogenous PTEN by western blot confirmed these results. We posited that expression of PTEN E3 ligases might be increased by treatment with these factors, leading to destruction of PTEN. In support of this hypothesis, the expression of E3 ligases WWP1 and WWP2 was increased by treatment with IL6 and OSM. Additionally, preliminary data has revealed that PTEN is oxidized in isolated CAFs, which causes loss of PTEN activity and potentially increased protein turnover. Current experiments are aimed at testing these potential mechanisms. To conclude, PTEN is dysregulated post-transcriptionally in CAFs. The mechanism of PTEN dysregulation could be via increased expression of E3 ligases that target PTEN and/or PTEN oxidation. Future efforts will be aimed at testing whether inhibition of these pathways can restore PTEN expression in mouse PDAC models. These pathways and interactions could be further exploited for therapeutic benefit in curbing PDAC progression. Citation Format: Ivo N. Woogeng, Lu Han, Samaneh Saberi, Cameron Bumbleburg, Joseph Beaudet, Sudarshana Sharma, Michael Ostrowski. Dysregulation of PTEN expression in a subset of Cancer Associated Fibroblasts by Tumor Secreted Factors in PDAC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A060.

Abstract A059: Understanding the events that promote pancreatic cancer metastasis

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy due to overwhelming metastatic burden. PDAC cells migrate and invade by two distinct epithelial-to-mesenchymal transition phenotypes: collective (partial-EMT) or single-cell (complete-EMT). The mechanism(s) by which these invasive phenotypes intravasate is unknown. The Tumor Microenvironment of Metastasis (TMEM) doorway, which is formed by a tumor cell, macrophage and an endothelial cell in direct contact, causes opening of the vasculature in which tumor cells intravasate. Intravasation via TMEM doorway is inhibited by Tie2 blockade. Whether this occurs in PDAC and whether the EMT phenotype affects intravasation is unknown. We hypothesize that PDAC cells intravasate through TMEM-dependent mechanisms regardless of EMT phenotype. Tumor samples from murine models were stained for TMEM doorways using a triple immunohistochemistry (IHC) stain (tumor cells expressing the actin regulatory protein Mena, macrophages expressing CD68, and endothelial cells expressing CD31). TMEM doorway opening was assessed in murine models of PDAC treated with or without Tie-2 inhibitor by analyzing TMEM-mediated extravasation of high-molecular weight dextran (155kD-TMR dextran) by Intravital Imaging, IHC and immunofluorescence (IF) of serial slides and circulating tumor cells. An intravasation trans-endothelial migration assay was also performed to assess the intravasation event by TMEM doorway in vitro. A student t-test was used for the analyses. TMEM doorways were observed in all PDAC murine models. For the first time in PDAC, focal extravasation of dextran events followed by a tumor cell intravasation was visualized by intravital imaging. PDAC tumor bearing mice treated with Tie2 inhibitor had a 2-fold decrease in the number of circulating tumor cells compared to control (958 ± 499.8 vs 534 ± 479.9 cells/mL blood, p=0.0257). The intravasation trans-endothelial migration assay showed that C-EMT cells have a higher migration rate than P-EMT cells (17.8 ± 5.53 vs 10.94 ± 3.84 migrating cells p=0.01), and this ratio increases with the presence of macrophages (23.75 ± 5 vs 15.54 ± 3.09 migrating cells p=0.005). The Tie-2 inhibition decreases the number of C-EMT and P-EMT migrating cells (7.69 ± 1.3 vs 3.67 ± 1.2 migrating cells p=0.01). Orthotopic tumors from C-EMT phenotype grow at a faster rate than the P-EMT (0.49mg ± 0.09 vs 0.12mg ± 0.05 p<0.05). Tumors from C-EMT and P-EMT cells will be analyzed for TMEM doorway score, TMEM activity and stemness signatures around TMEM doorway to determine if TMEM doorway is a niche for stemness in PDAC. TMEM doorways are present in PDAC mouse model. Inhibition of Tie2 leads to decreased TMEM doorway function and circulating tumor cells in PDAC. In addition, regarding the EMT phenotype, TMEM doorways are functional mechanisms of intravasation in vitro. This is a novel mechanism in PDAC and suggests a promising therapeutic target for decreasing intravasation and potential metastasis in PDAC. Citation Format: Erika Pereira Zambalde, Lina Ariyan, Francisco Puerta-Martinez, Yuqin Zhu, John Condeelis, Ben Stanger, John McAuliffe. Understanding the events that promote pancreatic cancer metastasis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A059.

Abstract A038: The hypoxic regulation of macrophage function in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is marked by an extensive fibroinflammatory stroma and a low-oxygen (hypoxic) microenvironment, which contribute to disease progression and therapeutic resistance. Within the PDAC stroma, macrophages and cancer-associated fibroblasts (CAFs) are the predominant cell types. Macrophages are a major immunosuppressive population in PDAC. These cells are highly plastic and, as a result, their environment plays an important role in regulating their function. Macrophages adapt to hypoxia primarily by stabilizing hypoxia-inducible factors (HIFs), which are oxygen-labile transcription factors. Although PDAC is profoundly hypoxic and both the tumor cells and stromal cells experience hypoxia, the effects of hypoxia and HIFs on macrophages and their interactions with other stromal cells in PDAC are not yet fully understood. We found that hypoxia promotes the acquisition of inflammatory cancer-associated fibroblasts (inflammatory CAFs), a fibroblast subtype that produces high levels of cytokines and chemokines. Utilizing a hypoxia marker in mouse models of PDAC, we have observed that inflammatory CAFs and macrophages are predominantly situated in hypoxic tumor regions compared with normoxic regions, while T cells are largely absent from these oxygen-poor regions. These preliminary data raise the possibility that inflammatory fibroblasts induced by hypoxia promote macrophage infiltration into hypoxic tumor regions and facilitate an immunosuppressive macrophage phenotype. We have also found that when macrophages are exposed to conditioned media derived from fibroblast cultures under either hypoxic or normoxic conditions, factors secreted from the hypoxia-exposed fibroblasts promote an immunosuppressive macrophage phenotype. We are currently identifying the factors released from hypoxic fibroblasts that drive the immunosuppressive macrophage phenotype. Future work will focus on evaluating the effects of macrophage-intrinsic responses to hypoxia through HIFs on pancreatic tumorigenesis in vivo. Citation Format: Sean Hannifin, Ashley M Mello, Tenzin Ngodup, Katelyn L Donahue, Marina Pasca di Magliano, Kyoung Eun Lee. The hypoxic regulation of macrophage function in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A038.

Abstract B075: Functional characterization of TGF-β-dependent cancer-associated fibroblasts in pancreatic cancer using novel in vitro and in vivo models

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is characterized by abundant cancer- associated fibroblasts (CAFs) which contribute to a tumor-promoting, chemotherapy-resistant and immunosuppressive tumor microenvironment (TME). However, CAF-ablating therapeutic strategies have thus far failed in clinical trials. One potential reason is that PDAC CAFs are heterogeneous and plastic. It was recently discovered that myofibroblastic CAFs (myCAFs) and inflammatory CAFs (iCAFs) can originate from pancreatic stellate cells (PSCs) via TGF-β and IL-1 signaling, respectively. However, their functional heterogeneity remains to be elucidated. We hypothesize that distinct CAF subtypes exhibit different tumor-promoting roles mediated by targetable signaling mechanisms. To this end, we aimed at characterizing the functions of TGF- β-dependent myCAFs in shaping the TME and influencing PDAC progression. Methods: We generated novel myCAF-depleted PDAC mouse models by orthotopically injecting PDAC organoids derived from KPC (Kras LSL-G12D/+ ; Trp53 LSL-R172H/+ ; Pdx1-Cre) mice into Fap-Tgfbr2 knockout (Fap-tTA; Rosa26 LSLtdTom/+ ; tet-O-Cre; Tgfbr2 fl/fl ) mice. The pancreatic tumors were analyzed by flow cytometry, histology and single-cell RNA-sequencing (scRNAseq). In a complementary approach, we generated myCAF-enriched PDAC mouse models by orthotopically co-injecting KPC PDAC organoids and novel TGF-β-driven ‘myCAF-locked’ murine PSCs. In addition, to further dissect myCAF-malignant cell signaling crosstalk, KPC PDAC organoids and myCAF-locked PSCs were co-cultured and then flow-sorted for bulk RNA- sequencing. Results: MyCAF-depleted PDAC tumors from Fap-Tgfbr2 knockout mice had a significant decrease in myCAFs, and a significant expansion of iCAFs, compared to control mice. Additionally, Masson’s trichrome staining indicated significant reduction in collagen content in the TME upon myCAF depletion. Moreover, scRNAseq analysis revealed that myCAF depletion shaped the composition of innate immune cells, including macrophages and neutrophils. Notably, although primary tumor growth was not altered, liver and lung metastases decreased, while diaphragm metastases and ascites increased, in myCAF-depleted PDAC mice. Conversely, liver and lung metastases increased, while diaphragm metastases and ascites decreased in myCAF-enriched PDAC mouse models, suggesting that TGF-β-dependent myCAFs play a role in promoting distant metastases. In line with this, transcriptomic analysis of PDAC organoids co-cultured with myCAF-locked PSCs showed enrichment of pathways in epithelial-mesenchymal transition, hypoxia, VEGF, WNT and cell-matrix interactions. Conclusion: Our study demonstrates that TGF-β-dependent CAFs are not only largely responsible for collagen deposition in PDAC, but also possess immune-modulating properties. Moreover, our study supports a role of TGF-β-dependent CAFs in promoting PDAC metastasis to the liver and the lungs, potentially through upregulating pro-metastatic pathways in the malignant cells. Citation Format: Priscilla S.W. Cheng, Muntadher Jihad, Judhell S. Manansala, Weike Luo, Gianluca Mucciolo, Sara Pinto Teles, Giulia Biffi. Functional characterization of TGF-β-dependent cancer-associated fibroblasts in pancreatic cancer using novel in vitro and in vivo models [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B075.

Abstract B014: NF-kB Regulates Innate Immunity in the Muscle Microenvironment to Control Distinct States of Wasting in Pancreatic Cancer-Induced Cachexia

Abstract Cancer cachexia is a debilitating syndrome characterized by unintentional weight loss largely due to the depletion of adipose and skeletal muscle mass. Cachexia occurs in at least half of all patients with cancer, with increasing incidences in more advanced cases, and is estimated to be responsible for greater than 20% of all cancer related deaths. In pancreatic ductal adenocarcinoma (PDAC), which has a 5-year survival rate of 13%, the incidence of cachexia can be as high as 80% and is associated with poor disease outcomes. Mitigating the effects of cachexia has the potential to increase both quality of life and survival for patients with PDAC. However, there are currently no effective means of treating cachexia, thereby underscoring the need to further understand the pathology of this disease. Although cancer cachexia is classically characterized as a systemic inflammatory disorder, emerging evidence indicates that weight loss also associates with local tissue inflammation. We queried the regulation of this inflammation and its causality to cachexia by exploring skeletal muscle, whose atrophy strongly associates with poor outcomes. Using both a genetically engineered mouse model of PDAC- induced cachexia named KPP and patient samples, we show that cachectic muscle is marked by enhanced innate immunity. Accumulation of macrophages is regulated by an NF-kB activity that localizes to multiple cells within the muscle microenvironment. In addition, this accumulation of macrophages derives from an equal contribution of infiltrating monocytes and resident cells. Moreover, NF-kB activated cells and macrophages undergo a crosstalk; whereas NF-kB+ cells signal and recruit macrophages to inhibit muscle regeneration and promote atrophy, but interestingly at the same time can also protect myofibers, macrophages signal back to NF-kB+ cells to sustain an innate immune response in a feed-forward loop. Together, we propose that NF-kB functions in multiple cells in the muscle microenvironment to regulate innate immunity that both promotes and protects against muscle wasting in cancer. Citation Format: Denis C Guttridge, Benjamin R Pryce, Alexander Oles, Erin E Talbert, Katherine A Morgan, David A Mahvi, Michael C Ostrowski, Teresa A Zimmers, James G Tidball, David J Wang. NF-kB Regulates Innate Immunity in the Muscle Microenvironment to Control Distinct States of Wasting in Pancreatic Cancer-Induced Cachexia [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B014.

Abstract B007: Optimization of an Orthotopic Mouse Model of Pancreatic Cancer to Simulate Liver Metastasis and Cancer Cachexia

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with a poor prognosis due to two key features. The first is metastasis at presentation, occurring in approximately 80% of PDAC patients, with 90% of these metastases occurring in the liver. The second is cachexia, which compromises treatment tolerance and reduces the quality of life for patients. Although various mouse models of PDAC exist, recapitulating both metastatic and cachectic features have been challenging. In this study, we optimized an orthotopic mouse model of PDAC by altering implantation sites, subcloning parental murine PDAC cells, adjusting the number of transplanted cells, and varying the age of recipient mice to study liver metastasis and cancer cachexia. These modifications significantly increased the latency of tumor development and the time course of the disease, concurrently increasing the rate of liver metastasis to approximately 70%. Furthermore, reliable cachexia endpoints were also achieved in both PDAC mice with and without metastases, mirroring the condition in patients. We also found that cachectic muscles from PDAC mice with metastasis exhibit a similar transcriptional profile to muscles derived from mice and patients without metastasis. Together, this model is likely to be advantageous in both advancing our understanding of the mechanisms of PDAC cachexia and in evaluating novel therapeutics. Citation Format: David J Wang, Victoria Spadafora, Benjamin R Pryce, Alexander Oles, Erin E Talbert, Micheal C Ostrowski, Denis C Guttridge. Optimization of an Orthotopic Mouse Model of Pancreatic Cancer to Simulate Liver Metastasis and Cancer Cachexia [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B007.

Abstract B080: A porcine model of pancreatic ductal adenocarcinoma for human scale translational cancer research

Abstract Pancreatic ductal adenocarcinoma (PDAC) remains a highly fatal form of cancer, requiring urgent advancements in diagnostic and therapeutic strategies. Although PDAC mouse models have been crucial in advancing our comprehension of the disease, they present some limitations in replicating key human PDAC aspects. In this study, we present the development of the first large animal model of pancreatic cancer via Cre activatable mutations in the endogenous KRAS and TP53 genes 1,2. In order to activate these mutations in the pancreas, two Cre driver pig lines expressing Cre recombinase from PTF1A 3 and PDX1 were created. Crossing both Cre lines with pigs carrying latent KRAS G12D and TP53 R167H alleles produced double (KC) and triple (KPC) genetically modified animals. Pathological analyses revealed the presence of inter- and intralobular fibrosis with multifocal acinar-to-ductal metaplasia (ADM), some low-grade PanINs, dysplasia, inflammation and duct obstruction in most of the KPC pigs between 3 to 6 months of age. Interestingly, the majority of these pigs already exhibited local metastasis in the lymph nodes, suggesting that the metastatic potential is established early in the tumorigenic process. Age-matched KC pigs exhibited mainly ADM with dysplasia. Flow cytometry and RNA expression analyses of the established pancreatic tumour primary cells revealed a pronounced epithelial-to-mesenchymal transition (EMT). Moreover, both bulk tumour samples and primary tumour cells showed high expression of the pancreatic cancer stem cell marker CD44, with differential expression of its isoforms, including the major isoform and variants 6 and 9. In addition, histological and molecular analyses revealed notable similarities between pig and human PDAC, including molecular subtypes, neuronal invasion and loss of acinar identity. These findings highlight the unique advantage of the pig PDAC model, that allows longitudinal and multi-regional tumour sampling in the absence of any therapeutic interventions. This makes it a valuable tool for developing new diagnostic and therapeutic approaches that can greatly improve patient outcomes in the long run.

Abstract B058: Rewiring CD8+ T cell responses to PD-1 immune checkpoint blockade in PDAC via the inhibitory receptor PSGL-1

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an aggressive, poorly immunogenic cancer and non-responsive to all currently available treatments, including immune checkpoint blockade (ICB) therapies. As survival rates remain low (13%), there is an urgent need for new therapeutic options. A hallmark of PDAC is the limited infiltration of anti-tumor cytotoxic CD8+ T cells. Analysis of single-cell RNA-sequencing data of PDAC tumors from treatment naïve patients reveals high expression of P-selectin glycoprotein ligand 1 (PSGL-1) on tumor-infiltrating CD8+ T cells. PSGL-1 is an intrinsic negative regulator of CD8+ T cells that promotes the development of functional exhaustion. We therefore hypothesized that PSGL-1-mediated immune suppression is a critical barrier to effective anti-tumor immunity in PDAC. To test this, we used preclinical models of orthotopic injection PDAC tumor cells into pancreata, recapitulating patient responses to PDAC with limited T cell infiltrates and uncontrolled tumor growth in C57BL/6 (wildtype) control mice and compared anti-tumor immune responses in PSGL-1KO (C57BL/6 background) mice. At 28 days post-injection of KPC.4662 PDAC tumor cells, tumor burden in PSGL-1KO mice was reduced by >50% compared to controls, with 45% of PSGL-1KO animals exhibiting >70% reduction. Total immune infiltration (CD45+) was 2-fold greater in tumors from PSGL-1KO mice, with significant increases in infiltrating CD4+ and CD8+ T cells. Detailed analyses revealed that infiltrating CD8+ T cells from PSGL-1KO mice were less terminally differentiated towards exhaustion. PSGL-1KO PD-1+ CD8+ T cells expressed less TOX expression and fewer cells co-expressed CD38 and CD101 while more expressed CX3CR1 compared to controls. Intratumoral PSGL-1KO CD8+ T cells also retained greater functionality as well as indicated by inflammatory cytokine production (IFNg, TNFa) and Granzyme B expression. Moreover, PSGL-1KO mice showed protection against metastatic disease to the lungs. We find that protection by PSGL-1KO mice is T cell dependent as transient depletion of CD4+ and CD8+ T cells prior to tumor cell implantation results in the loss of PDAC tumor growth inhibition by PSGL-1KO mice. Less-differentiated, proliferation-competent precursors of exhausted CD8+ T cells (TPEX) are essential to PD-1 ICB responsiveness in patients and preclinical models. Given that PSGL-1-deficiency limits terminal differentiation of CD8+ T cells in PDAC, we hypothesized that PSGL-1-deficiency would promote responsiveness to PD-1 ICB, which is ineffective as a monotherapy in PDAC patients and mouse models. While therapeutic treatment with anti-PD-1 had no effect on the tumor growth in the control animals, PD-1 ICB resulted in tumor ablation in 85% of PSGL-1KO animals, demonstrating a profound synergistic effect of PD-1 inhibition in the absence of PSGL-1. From these studies, we conclude that PSGL- 1 is a critical inhibitor anti-tumor T cell immunity in PDAC and represents a novel ICB target with high translational potential for promoting immune responses to PDAC tumors. Citation Format: Jennifer L Hope, Yijuan Zhang, Hannah A. F. Hetrick, Evelyn S. Sanchez Hernandez, Gabriele Romano, Sreeja Roy, Michelle Lin, Ashley B Palete, Swetha Maganti, Dennis C Otero, Katelyn T Byrne, Cosimo Commisso, Linda M Bradley. Rewiring CD8T cell responses to PD-1 immune checkpoint blockade in PDAC via the inhibitory receptor PSGL-1 [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B058.

Abstract IA-08: Syndecan 1 is a therapeutic target for KRAS-driven pancreatic cancer

Abstract KRAS inhibitors targeting G12C mutation have been approved by FDA for the treatment of non-small cell lung cancers and have shown promising clinical responses in pancreatic ductal adenocarcinoma (PDAC). RAS inhibitors targeting additional KRAS mutations or multiple RAS isoforms are under clinical development. Although targeting KRAS exhibits anti-tumor effect in PDAC, majority of tumors inevitably develop resistant disease, as is the case for most targeted therapies. In this new era of targeting KRAS driven cancer, understanding mechanisms driving this resistance to RAS targeted therapies and further devising combination strategies to overcome it have become a necessary, though challenging, task to achieve long term disease control. To tackle this problem, we employed various in vitro and in vivo preclinical model systems to explore the development of resistance to both genetic and pharmacological KRAS blockade. Our previous work identified the membrane proteoglycan Syndecan 1 (SDC1) as a KRAS downstream surrogate whose cell surface localization is tightly controlled by oncogenic KRAS signaling and is critical for KRAS-driven tumor development. Here, our analysis of KRAS driven genetically engineered PDAC mouse model and human pancreatic or colorectal cancer (CRC) cells with KRASG12C mutation discovered that, while cell surface SDC1 is reduced upon acute genetic or pharmacological inhibition of oncogenic KRAS, plasma membrane SDC1 is recovered in tumor cells that become resistant to KRAS inhibition (KRAS-bypass cells). Surface localization of SDC1 leads to the activation of multiple receptor tyrosine kinases (RTKs) and macropinocytosis, resulting in the resistance to KRAS blockade. We further identified that the recovery of plasma membrane SDC1 in KRAS-bypass cells is driven by YAP1 oncogene, which induces the downregulation of ARF6 GAPs, leading to the activation of ARF6, a small GTPase required for SDC1 membrane recycling. To therapeutically targeting SDC1, we have successfully developed a monoclonal antibody (22B mAb), which exhibits remarkable sensitivity and specificity for human SDC1. Our results indicate that 22B mAb exerts substantial inhibition of PDAC cell growth in vitro and desirable tumor inhibitory effects in vivo in murine PDAC tumors and human PDAC cell-derived xenograft tumors. Importantly, our 22B mAb synergizes with KRAS inhibitors, chemotherapy, or immunotherapy, resulting in significantly enhanced therapeutic effects. Taken together, our study identified SDC1 as a functional driver for the bypass of KRAS-dependence and therapeutically targeting SDC1 with a novel mAb will overcome the acquired resistance to KRAS targeted therapy. As such, our research yields important new insights in the fields of cancer biology regarding mechanisms of resistance employed by human cancers, and identifies new actionable therapeutic targets and approaches to overcome resistance to KRAS inhibition. Citation Format: Mitsunobu Takeda, Zecheng Yang, Madeline S Theardy, Alexey Sorokin, Oluwadara Coker, Shuaitong Chen, Long T Vien, Laura Bover, Haoqiang Ying, Scott Kopetz, Wantong Yao. Syndecan 1 is a therapeutic target for KRAS-driven pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr IA-08.

Abstract C061: Loss of CaMK2B accelerates tumor formation and enhances metastatic competency in genetically engineered mouse models of PDAC

Abstract Background Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer- related deaths in the United States and is often not diagnosed until it has already metastasized. Our prior work established that calcium signaling pathways facilitate a pro-metastatic phenotype through the induction of an epithelial-to-mesenchymal transition (EMT) program in PDAC tumor cells, but the downstream signaling molecules mediating this phenotype have remained elusive. MethodsUsing a novel genetically engineered model of pancreatic cancer, we demonstrate that loss of Calcium/calmodulin-dependent kinase-II B (CaMK2B), a downstream effector kinase activated by calcium, enhances cellular plasticity, and increases metastasis. To this end, we generated a genetically engineered mouse model where Camk2b was knocked out in tumor cells in a lineage-labeled Kras- and p53-driven PDAC mouse model (herein KPCY-Camk2bcKO). Results Genetic deletion of Camk2b in the KPCY genetically engineered model accelerated tumor formation and dramatically reduced the overall median survival of KPCY- Camk2b cKO mice (78 days) relative to KPCY controls (136 days). Pathological and immunofluorescence analysis of KPCY-Camk2b cKO animals showed an increased number of poorly differentiated tumor regions and a higher metastatic burden, with >85% of KPCY- Camk2b cKO animals presenting with multi-focal metastases. Further analysis of tumor cells in KPCY-Camk2b cKO mice showed enhanced expression of markers of the highly aggressive squamous PDAC subtypes. Strikingly, Camk2b deletion combined with oncogenic Kras G12D (in the absence of p53 alterations), was sufficient to form lethal metastatic PDAC tumors, while age-matched control Kras G12D tumors with intact Camk2b only progressed to the acinar-to-ductal metaplasia (ADM) or Pancreatic Intraepithelial Neoplasia (PanIN) stage. Collectively, these results establish Camk2b loss as a new oncogenic and pro-metastatic mechanism in PDAC Conclusion This work demonstrates that Camk2b loss enhances tumor cell evolution and plasticity within PDAC to promote metastasis. In this context, we speculate that loss of Camk2b rewires calcium signaling to drive an aggressive subtype of PDAC with increased metastasis. Citation Format: Jessica Peura, Nikita Bhalerao, Calvin Johnson, Yamini Ogoti, Faith Keller, Emma Watson, Zhen Zhao, Jason Pitarresi. Loss of CaMK2B accelerates tumor formation and enhances metastatic competency in genetically engineered mouse models of PDAC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C061.

Abstract B059: Modulating the immunosuppressive pancreas tumor microenvironment through intratumoral delivery of cytokine-encoding mRNAs

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive cancer, with a mere 13% average 5-year survival rate. The hallmark desmoplastic stromal response in PDAC leads to poor vascularization, drug delivery challenges, and impaired cytotoxic immune cell infiltration, contributing to de novo therapy resistance. Our previous research has shown therapeutic induction of cellular senescence with RAS pathway targeting therapies can induce cytokine and chemokine production through the senescence-associated secretory phenotype (SASP) that can effectively activate anti-tumor T cell immunity and responses to anti-PD-1 immune checkpoint blockade. To build on these findings as well as overcome limitations associated with senescence-inducing therapy toxicity and induction of pro-tumorigenic SASP cytokines, here we leveraged mRNA technology to deliver specific SASP cytokines and chemokines we have found to stimulate immune responses directly into the suppressive PDAC TME as an immune oncology platform. We created an in vitro transcription pipeline for mRNA production and multiplexing, and demonstrate we can intratumorally deliver multiple mRNAs simultaneously into the TME of transplanted and autochthonous PDAC mouse models, stimulating the local production of cytokines normally absent in PDAC. We further characterized a novel combination of five cytokines and chemokines that can effectively recruit and activate both innate and adaptive immune responses against PDAC. Through repeated bi-weekly dosing, this mRNA combination also promotes tumor destruction and improves survival outcomes. Considering the recent success of off-the-shelf neoantigen mRNA vaccines in early human PDAC patient trials, we have now developed mRNAs that encode PDAC-specific antigens. By integrating our cytokine mRNA platform with antigen mRNAs, we observe a significant increase in intratumoral antigen-presenting dendritic cell populations and a substantially enhanced T-cell mediated anti- tumor immune response in orthotopic transplant models of PDAC. Overall, this study not only unveils pivotal insights into the cytokines absent in PDAC that are crucial for promoting anti- tumor immunity, but also pioneers an innovative immunotherapy approach. This platform has the potential to be integrated with existing immunotherapy modalities, addressing the longstanding challenge of therapy resistance in PDAC. Citation Format: Chaitanya Naimesh Parikh, Kelly De Marco, Katherine Murphy, Loretah Chibaya, Lin Zhou, Youwei Qiao, Griffin Kane, Li Li, Wen Xue, Marcus Ruscetti. Modulating the immunosuppressive pancreas tumor microenvironment through intratumoral delivery of cytokine-encoding mRNAs [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B059.

Abstract B092: PP2A activation alters macropinosome processing in pancreatic cancer cells leading to metabolic stress and cancer cell death

Abstract The majority of pancreatic ductal adenocarcinoma (PDAC) patients present with late-stage, metastatic disease that is often resistant to therapeutics, resulting in the lowest survival rate of all major cancers. Metabolic rewiring in response to oncogenic signals plays a critical role in PDAC cell survival, tumor growth, and metastasis. In contrast to normal epithelial cells, these metabolic alterations make PDAC tumors dependent on glutamine and glucose for survival, highlighting a unique metabolic vulnerability that can be therapeutically exploited. However, during times of nutrient stress, PDAC cells can circumvent this vulnerability by engulfing extracellular fluids to replenish amino acids, including glutamine, in a process called, macropinocytosis. Macropinocytosis occurs downstream of oncogenic KRAS, a small GTPase that is almost universally mutated in PDAC patients. The inhibition of macropinocytosis in vivo reduces PDAC tumor growth, underscoring the importance of this pathway to cancer cell survival. However, the signaling mechanisms that regulate this process and the contribution of macropinocytic signaling to aggressive cancer phenotypes remains poorly understood. Protein phosphatase 2A (PP2A) is a heterotrimeric complex that reguates a wide variety of cell signaling pathways, inlcuding KRAS, and is commonly deregulated in human PDAC tumors. Recently, PP2A has been implicated as an important regulator of macropinocytosis, but the mechanism by which this occurs is unknown. We demosntrate that suppression of PP2A accelerates the formation of KRAS-driven precursor lesions in an in vivo mouse model of PDAC, implicating PP2A as a critical regulator of mutant KRAS phenotypes. Here, we show that acute PP2A activation prevents the fusion of macropinosomes with the lysosome, robbing PDAC cells of critical metabolic nutrients and driving cell death. Furthermore, we find PP2A associates with the lipid kinase, PIKfyve, a key regulator of macropinosome-lysosome fusion. Finally, we determined that PP2A activating compounds can function synergistically with metabolic inhibitors, support a new therapeutic strategy in this aggressive and deadly cancer. Together, our results implicate PP2A as a critical suppressor of PDAC metabolic plasticity and highlight the use of PP2A activating compounds to prevent PDAC nutrient scavenging. Citation Format: Garima Baral, Claire M Pfeffer, Indiraa Doraivel, Sara N Filippelli, Brittany N Heil, Brittany L Allen-Petersen. PP2A activation alters macropinosome processing in pancreatic cancer cells leading to metabolic stress and cancer cell death [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B092.

Abstract A047: Fibroblast-specific IL1 receptor inhibition reprograms the inflammatory stroma to overcome the immunosuppressive tumor microenvironment in pancreatic cancer

Abstract OBJECTIVE: KRAS-driven inflammatory signaling in pancreatic ductal adenocarcinoma (PDAC) promotes cancer-associated fibroblast (CAF) polarization and an immunosuppressive tumor microenvironment (iTME), driving therapeutic resistance. IL1α, a downstream product of KRAS signaling, activates the pro-inflammatory CAF (iCAF) phenotype and correlates with worse survival. Using patient and PDAC mouse model single-cell RNA sequencing (scRNAseq) data, we have shown that the IL1 Receptor (IL1R1) is highly enriched in the fibroblast compartment. However, the role of IL1R1 in modulating iCAFs and the iTME remains poorly understood. We hypothesize that CAF-specific disruption of IL1R1 signaling will reprogram cellular components of the PDAC TME to overcome the immunosuppressive milieu and therapeutic resistance. METHODS: The effects of IL1R1 inhibition on iCAF modulation in vivo were determined in Ptf1aCre/+, LSL-KrasG12D/+, Tgfbr2flox/flox (PKT) mice treated with IL1R1 antagonist. To determine if CAF-specific silencing of IL1R1 recapitulates the TME remodeling after IL1R1 inhibition, we generated a fibroblast-specific IL1R1KO mouse model Col1a2Cre/+;Il1r1flox/flox (CAF-Il1r1KO) and orthotopically injected KPC tumor cells for endpoint analysis. Flow cytometry was performed in primary tumors of CAF-Il1r1KO and littermate controls. Functional experiments evaluating myeloid-derived suppressor cell (MDSC) trafficking were performed using ex vivo bone marrow-derived cells (BMDCs) co-cultured with CAF/tumor-conditioned media (CM) with/without IL1R1 inhibition in a transwell system. MDSC contribution to iTME was assessed by co-culturing MDSCs from CAF-Il1r1KO tumors and splenic T cells from naïve C57BL/6 mice. To investigate the effects of IL1R1 deletion in overcoming therapeutic resistance, survival studies in CAF-Il1r1KO mice with chemotherapy were conducted. RESULTS: Treatment of PKT mice with IL1R1 antagonist diminished transcriptional levels of Il6 and Cxcl1, iCAF-associated cytokines, in the CAF compartment. Compared to littermate controls, orthotopic tumors in CAF-Il1r1KO showed significant reduction in tumor weights, increased CD8+ T cell infiltration, and overall reduction in polymorphonuclear (PMN) MDSC trafficking by flow cytometry. Moreover, migration of BMDCs was reduced when co-cultured with CAF/tumor CM and IL1R1 antagonist. In addition, co-culture of MDSCs from CAF-Il1r1KO mice with naïve T cells showed diminished inhibition of T cell functional activity. Chemotherapy treatment of orthotopic tumors in CAF-Il1r1KO mice showed significantly decreased tumor size and improved survival compared to tumors in littermate controls. CONCLUSION: Our findings illustrate a novel mechanism of CAF-specific disruption of IL1R1 signaling inhibiting trafficking of immunosuppressive MDSCs and increased infiltration of cytotoxic CD8+ T cells in the PDAC TME. These data pave the way for novel combinatorial treatment strategies targeting IL1R1 to overcome chemotherapeutic resistance through reprogramming of the stromal compartment in PDAC. Citation Format: Camille Acevedo, Samara Singh, Austin Dosch, Edmond W Box, Iago C Silva, Anna Bianchi, Siddharth Mehra, Haleh Amirian, Andrew Adams, Nagaraj Nagathihalli, Jashodeep Datta, Nipun B Merchant. Fibroblast-specific IL1 receptor inhibition reprograms the inflammatory stroma to overcome the immunosuppressive tumor microenvironment in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A047.

Abstract A061: DKK3 in pancreatic cancer – Elucidating the roles of a double-edged sword

Abstract Pancreatic ductal adenocarcinoma (PDAC) remains a highly challenging disease with a poor prognosis due to lack of diagnostic biomarkers and high tumor heterogeneity. Therefore, it is crucial to meticulously examine the intricate molecular mechanisms in PDAC. We previously identified DKK3 (Dickkopf-3) as a novel factor promoting organ regeneration during pancreatitis and established a previously unknown link between DKK3 and the Wnt and Hedgehog signaling. Given that pancreatitis is a significant risk factor for pancreatic cancer, we were motivated to investigate the potential role of DKK3 in PDAC development. We utilized PDAC mouse models driven by LSL-KrasG12D/+; Ptf1aCre/+ with wildtype, homozygous/heterozygous DKK3 deletion in both epithelial and stromal compartment while employing in vitro and ex vivo systems to examine the stage and compartment-specific roles of the same. Through a comprehensive and time-resolved analysis of tumor characteristics, we discovered that the loss of DKK3 led to rapid oncogenic transformation, marked by a significant increase in metaplasia and desmoplasia starting at an early stage (10 weeks). Ex vivo acinar-to-ductal metaplasia assay and rescue experiments with wildtype and DKK3-null acinar cells revealed that secreted DKK3 operates as a tumor suppressive roadblock in PDAC-initiating steps. This advanced preneoplastic stage translated into accelerated progression toward invasive tumors at 36 weeks, a shortened overall survival, and a higher liver metastasis incidence in the DKK3-null animals at the endpoint. Interestingly, transcriptomic analyses further validated our observation and revealed the involvement PI3-AKT signaling pathway as an underlying mechanism. Further characterization of isolated tumor cells confirmed our observations, revealing prominent mesenchymal features and enhanced migratory capacities upon loss of DKK3. Interestingly, DKK3-null PDACs exhibited a pronounced stroma remodeling with a myofibroblastic cancer-associated fibroblast-enriched environment, as well as an immunosuppressive stroma with abundant regulatory T cells and cytotoxic T cell depletion, as substantiated by coculture experiments. Intriguingly, while displaying similar phenotypic features to DKK3-null tumors, Dkk3+/- heterozygotes exhibited the most aggressive outcome. To further dissect this finding, we conducted a set of in silico approaches and immunostainings revealing persistent stromal DKK3 expression in DKK3- proficient PDACs. This suggested a possible dual role of DKK3 that could operate either as a friend or a foe depending on its spatiotemporal expression in PDAC. In line, in vitro systems demonstrated a distinct oncogenic effect of DKK3 on endpoint tumor cells, particularly promoting malignant cell migration. In summary, we propose that epithelial DKK3 exerts cell-autonomous tumor suppressive functions during the onset of PDAC, while stromal DKK3 acts as an oncogenic cue driving cancer progression. These findings highlight DKK3 as a promising biomarker and a novel therapeutic target for PDAC tumors. Citation Format: Dharini Srinivasan, Frank Arnold, Elodie Roger, Anna Härle, Michael K Melzer, Chantal Allgöwer, Patrick C Hermann, Lukas Perkhofer, Johann Gout, Alexander Kleger. DKK3 in pancreatic cancer – Elucidating the roles of a double-edged sword [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A061.

Abstract A049: Collagen-driven kinome reprogramming reveals unique, actionable DDR1-signaling dependencies in pancreatic cancer cells

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDA) is the 3rd most common cause of cancer death in the United States, with a 5-year survival rate of only 13%, in part due to late detection and metastatic dissemination at early stages of progression. Most PDA tumors exhibit a dense fibro-inflammatory stroma consisting of fibroblasts, immune cells, and a dense collagen-rich extracellular matrix (ECM) that regulate disease progression. How distinct collagens (COLs) influence PDA progression remains unclear. COLs signal via specific cell surface receptors, of which the Discoidin Domain Receptors (DDRs) constitute a unique family of collagen-binding receptor tyrosine kinases (RTKs). DDR1 is expressed by PDA cancer cells, while DDR2 is expressed by mesenchymal cells. DDR1 is activated by both basement membrane COL (e.g. COL4) and fibrillar COLs (e.g. COLs 1, 2, 11). Compared to most RTKs, relatively little is understood about differential ligand activation and signaling downstream of DDRs. Our working hypothesis is that switches in ECM collagen composition over the course of PDA progression induce changes in DDR1-intracellular signaling cascades, which may reveal unexplored therapeutic susceptibilities to target and eliminate PDA cancer cells. Methods and Recent Advances: Using genetically engineered mouse models of PDA, we previously found that genetic ablation of Ddr1 impedes tumor progression, blocking the transition from well-differentiated to poorly-differentiated adenocarcinoma and, as a result, metastasis. Moreover, we showed that xenografts of DDR1-expressing human PDA cell lines display enhanced tumor growth exclusively when implanted within a COL1 scaffold. Leveraging an innovative high-throughput kinase-activity mapping (HT-KAM) platform and kinase network resource database (PhosphoAtlas), we started investigating mechanisms of differential activation of DDR1 by distinct COLs, including COL1 and COL11, which is an understudied fibrillar collagen that is prominent in the stroma of late-stage, metastatic PDA. New, Unpublished Findings: Using several PDA cell line models with or without DDR1 expression, our results indicate that DDR1 activates two distinct signaling networks: (1) a conserved, coordinated response that is modestly activated by COL1 but is hyperactivated by COL11 (e.g., ERBB, AKT, MEK/ERK, SRC), and (2) a unique set of phospho-signaling nodes that are only induced by COL11 –and not COL1 (e.g., specific RTKs and PKCs). Several of these kinases are associated with cancer cell proliferation and survival, as well as EMT induction, in part illuminating the malignancy-promoting effects of DDR1 in late-stage PDA in response to changes in COL composition. Conclusion: Differential activation of DDR1 represents a new paradigm on how distinct fibrillar COLs within the TME may drive PDA cell signaling. We are actively exploring how distinct COLs differentially regulate PDA tumor growth and metastatic behavior via DDR1, and how specific kinase-targeting interventions may prevent malignant phenotypes or induce tumor cell death. Citation Format: Anjum Sohail, Yeonjoo Hwang, Denise P Munoz, Yoshihiro Ishikawa, Rafael Fridman, Howard Crawford, Jean-Philippe Coppe. Collagen-driven kinome reprogramming reveals unique, actionable DDR1-signaling dependencies in pancreatic cancer cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A049.

DYRK1B blockade promotes tumoricidal macrophage activity in pancreatic cancer

ObjectiveHighly malignant pancreatic ductal adenocarcinoma (PDAC) is characterised by an abundant immunosuppressive and fibrotic tumour microenvironment (TME). Future therapeutic attempts will therefore demand the targeting of tumours and stromal compartments...

20(S)-Ginsenoside Rh2 overcomes gemcitabine resistance in pancreatic cancer by inhibiting LAMC2-Modulated ABC transporters

IntroductionGemcitabine (GEM) is the first-line drug for pancreatic ductal adenocarcinoma (PDAC), but drug resistance severely restricts its chemotherapeutic efficacy. Laminin subunit γ2 (LAMC2) plays a crucial role in extracellular matrix formation in the development of GEM-resistance. However, the biological function of LAMC2 in GEM resistance and its molecular mechanisms are still unclear. 20(S)-Ginsenoside Rh2 (Rh2), one of the principal active components isolated from Ginseng Radix et Rhizoma, possesses strong anti-tumor effects. However, the effects of Rh2 on overcoming GEM resistance and its action mechanisms remain to be elucidated. ObjectivesThis study aimed to determine the efficacy of Rh2 on overcoming GEM resistance and to explore its underlying molecular mechanisms. MethodsClinical study, Western blotting, publicly available databasesand bioinformatic analyses were performed to investigate the protein expression of LAMC2 in the GEM-resistant PDAC patients and the acquired GEM-resistant PDAC cells. Then, the effects of Rh2 on overcoming the GEM resistance in PDAC were evaluated both in vitro and in vivo. Stable silencing or overexpression of LAMC2 in the GEM-resistant PDAC cells were established for validating the role of LAMC2 on Rh2 overcoming the GEM resistance in PDAC. ResultsThe protein expression of LAMC2 was markedly increased in the GEM-resistant PDAC patient biopsies compared to the sensitive cases. The protein expression of LAMC2 was significantly higher in the acquired GEM-resistant PDAC cells than that in their parental cells. Rh2 enhanced the chemosensitivity of GEM in the GEM-resistant PDAC cells, and inhibited the tumor growth of Miapaca-2-GR cell-bearing mice and Krastm4TyjTrp53tm1BrnTg (Pdx1-cre/Esr1*) #Dam/J (KPC) mice. Rh2 effectively reversed the GEM resistance in Miapaca-2-GR and Capan-2-GR cells by inhibiting LAMC2 expression through regulating the ubiquitin–proteasome pathway. Knockdown of LAMC2 enhanced the chemosensitivity of GEM and the effects of Rh2 on overcoming the GEM resistance in PDAC cells and the orthotopic PDAC mouse model. Conversely, LAMC2 overexpression aggravated the chemoresistance of GEM and abolished the effects of Rh2 on overcoming GEM resistance via modulating ATP-binding cassette (ABC) transporters leading to the active GEM efflux. ConclusionsLAMC2 plays an important role in the GEM resistance in PDAC, and Rh2 is a potential adjuvant for overcoming the chemoresistance of GEM in PDAC.

Nanoparticle delivery of innate immune agonists combined with senescence-inducing agents promotes T cell control of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) has quickly risen to become the third leading cause of cancer-related death in the United States. This is in part because of its fibrotic tumor microenvironment (TME) that contributes to poor vascularization and immune infiltration and subsequent chemo- and immunotherapy failure. Here, we investigated an immunotherapy approach combining delivery of stimulator of interferon genes (STING) and Toll-like receptor 4 (TLR4) innate immune agonists by lipid-based nanoparticle (NP) coencapsulation with senescence-inducing RAS-targeted therapies, which can remodel the immune suppressive PDAC TME through the senescence-associated secretory phenotype. Treatment of transplanted and autochthonous PDAC mouse models with these regimens led to enhanced uptake of NPs by multiple cell types in the PDAC TME, induction of type I interferon and other proinflammatory signaling pathways, increased antigen presentation by tumor cells and antigen-presenting cells, and subsequent activation of both innate and adaptive immune responses. This two-pronged approach produced potent T cell-driven and type I interferon-mediated tumor regression and long-term survival in preclinical PDAC models dependent on both tumor and host STING activation. STING and TLR4-mediated type I interferon signaling was also associated with enhanced natural killer and CD8+ T cell immunity in human PDAC samples. Thus, combining localized immune agonist delivery with systemic tumor-targeted therapy can orchestrate a coordinated type I interferon-driven innate and adaptive immune response with durable antitumor efficacy against PDAC.

Inhibition of SUMOylation Induces Adaptive Antitumor Immunity against Pancreatic Cancer through Multiple Effects on the Tumor Microenvironment.

Improvement of outcome in patients with pancreatic ductal adenocarcinoma (PDAC) requires exploration of novel therapeutic targets. Thus far, most studies of PDAC therapies, including those inhibiting small ubiquitin-like modifications (SUMOylation), have focused on PDAC epithelial cell biology, yet SUMOylation occurs in a variety of cell types. The mechanisms by which SUMOylation impacts PDAC in the context of its tumor microenvironment are poorly understood. We used clinically relevant orthotopic PDAC mouse models to investigate the effect of SUMOylation inhibition using a specific, clinical-stage compound, TAK-981. In contrast to its inhibition of PDAC cell proliferation in vitro, the survival benefit conferred by TAK-981 in vivo is dependent on the presence of T cells, suggesting that induction of adaptive antitumor immunity is an important antitumor effect of SUMOylation inhibition in vivo. To understand how this adaptive antitumor immunity is promoted, we investigated how SUMOylation inhibition in vivo alters major cell types/subtypes and their communications in the PDAC tumor microenvironment by performing transcriptomic analyses at single-cell resolution, which allowed mapping of cells in our orthotopic mouse model to cells in human PDAC tumors based on gene expression profiles. Findings are further validated by flow cytometry, immunofluorescence, IHC, western blots, and qPCR. The single-cell transcriptome dataset provided here suggests several combination strategies to augment adaptive immune responses that are necessary for durable disease control in patients with PDAC.