Pancreatic Ductal Adenocarcinoma Model Research Articles

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 B006: The role of tumor specific IGFBP-3 in the onset and progression of skeletal muscle wasting in a murine model of pancreatic cancer

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer- related death and is highly associated with skeletal muscle wasting (SMW) that leads to treatment intolerance and reduced survival. Research substantiates the role of increased levels of Insulin-like Growth Factor Binding Protein-3 (IGFBP-3) in PDAC-related SMW. Canonically, supraphysiological levels of IGFBP-3 block insulin-like growth factor-1 (IGF-1) binding, thus downregulating the activation of its downstream targets Akt and mTOR. Non-canonical signaling via TGF-βRI results in aberrant FoxO1 signaling, which upregulates the ubiquitin proteasome pathway (UPP) and induces autophagy. Dysregulated UPP and autophagy activation have been shown to promote muscle degradation. Recent findings from our lab implicate canonical and non-canonical signaling of tumor associated IGFBP-3 in increased protein catabolism and decreased anabolism, resulting in SMW in a syngeneic murine model of PDAC. We tested the hypothesis that genetic ablation of IGFBP-3 in PDAC tumor cells attenuates SMW via regulation of Akt and FoxO1, leading to decreases in UPP and autophagy in skeletal muscle.Methods: C57BL/6J female mice (6-8 weeks) were randomized to one of three groups: 1) no tumor control (NTC) (n=10), 2) KCKO-Luc parental cells (PDAC) (n=14), or 3) IGFBP-3-/- KCKO-Luc (n=14). Tumor cells were injected orthotopically. Tumor and lean mass were assessed longitudinally via dual energy x-ray absorptiometry (DEXA). At sacrifice, quadriceps muscles were collected for protein and transcriptional analysis, Tibialis Anterior (TA) muscles for histology and serum for ELISA. TA frozen sections were stained with Oil Red O (ORO) to highlight intramuscular adipogenesis.Results: PDAC mice experience significantly reduced survival (T1/2=56 days) compared to IGFBP-3-/- mice that maintained 100% survival at day 100 (p<0.0001). IGFBP-3-/- mice lost significantly less lean mass from baseline to day 56 compared to PDAC mice (p<0.0001). PDAC mice exhibited 3-fold elevated serum levels of IGFBP-3 compared to NTC and IGFBP-3-/- mice (p<0.01). Transcriptionally, PDAC mice displayed increased expression of igfbp3, tgfbr1, and UPP associated genes fbxo32, and trim63 compared to NTC and IGFBP-3-/- mice. Further, the ratio of pAkt (Ser473), pS6 (Ser235/236), and pFoxO1 (Ser253) to total protein levels were significantly reduced in PDAC mice (p<0.05 vs NTC and IGFBP-3-/-). Additionally, protein levels of ULK1 and the ratio of LC3bII over LC3bI, markers of autophagy, are elevated in PDAC mice compared to NTC and IGFBP-3-/- mice (p<0.05). Lastly, there is a significant increase in ORO positive staining in PDAC mice compared to NTC and IGFBP-3-/- mice (p<0.0001).Conclusion: These findings suggest that PDAC-related SMW is driven by tumor derived IGFBP-3, which acts as an endocrine factor on skeletal muscle cells to inhibit protein synthesis and activate FoxO1 signaling to upregulate the UPP and autophagy. Lastly, we identify skeletal muscle adipogenesis as a biomarker for autophagy upregulation and SMW. Citation Format: Zachary R Sechrist, Calvin L Cole. The role of tumor specific IGFBP-3 in the onset and progression of skeletal muscle wasting in a murine model of 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 B006.

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 C026: Concurrent inhibition of the RAS ERK-MAPK pathway and PIKfyve as a therapeutic strategy for pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is characterized clinically by poor survival and mechanistically by KRAS- and autophagy-dependent growth. We and others previously demonstrated that inhibition of KRAS signaling via downstream inhibition of the RAF-MEK-ERK pathway enhanced autophagic flux and dependency of PDAC on autophagy. Furthermore, we demonstrated that concurrent treatment with the nonspecific autophagy inhibitor chloroquine (CQ) and ERK MAPK inhibitors synergistically blocked PDAC growth. These findings provided rationale for our initiation of Phase I/II clinical trials evaluating the combination of MEKi (binimetinib; NCT4132505) or ERKi (LY3214996; NCT04386057) with HCQ in PDAC. However, a limitation of this approach is that CQ/HCQ is limited in terms of specificity and potency. Thus, we aimed to identify alternative anti-autophagy strategies. We performed a CRISPR-Cas9 loss- of-function screen in PDAC cell lines that identified the lipid kinase PIKfyve as a growth- promoting gene. PIKfyve is a lipid kinase that is essential for the generation of PI(3,5)P2 and critical for the dynamic regulation of lysosomal recycling. Because PIKfyve has been implicated in regulation of autophagy in other cellular contexts, we evaluated the effects of PIKfyve inhibition on growth and autophagic flux in PDAC cell lines and tumors. We demonstrated that PIKfyve inhibition by the small molecule apilimod resulted in durable growth suppression, with much greater potency than CQ treatment. PIKfyve inhibition potently reduced autophagy as indicated by decreased autophagic flux and the robust accumulation of autophagy-related proteins. Additionally, PIKfyve inhibition resulted in the accumulation of LAMP1 positive vacuoles that exhibited impaired cargo degradation, likely due to a lack of required acidity. Next, we hypothesized that PIKfye inhibition would sensitize PDAC cells to RAS pathway inhibition. We demonstrated that PIKfyve inhibition blocked the compensatory increases in autophagic flux associated both with MEK inhibition and with direct RAS inhibition. Accordingly, combined inhibition of PIKfyve and either the RAS ERK-MAPK pathway or RAS itself showed robust growth suppression across a panel of KRAS-mutant PDAC models. Growth suppression was due, in part, to potentiated cell cycle arrest and induction of apoptosis following loss of IAP proteins. These findings implicate PIKfyve as an effective anti-autophagy target when paired with RAS or ERK-MAPK pathway inhibition in pre-clinical models of PDAC. Citation Format: Jonathan M DeLiberty, Mallory K Roach, Clint A Stalnecker, Ryan Robb, Elyse G Schechter, Noah L Pieper, Runying Yang, Scott Bang, Khalilah E Taylor, Kristina Drizyte-Miller, John P Morris IV, Channing J Der, Adrienne D Cox, Kirsten L Bryant. Concurrent inhibition of the RAS ERK-MAPK pathway and PIKfyve as a therapeutic strategy for 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 C026.

Abstract A031: Identification of therapeutic vulnerabilities in the subset of pancreatic ductal adenocarcinoma (PDAC) with homologous recombination deficiency

Abstract The genetic landscape of pancreatic ductal adenocarcinoma (PDAC) is characterized by mutations in the KRAS, TP53, CDKN2A and SMAD4 genes alongside multiple low-frequency mutations, which may offer opportunities for precision medicine. The success of poly-ADP-ribose polymerase (PARP) inhibitors in metastatic PDAC characterized by germline mutations in the Breast Cancer (BRCA) genes has led to the hypothesis that mutations in other BRCAness genes may sensitize PDAC to PARP inhibitors. To test this hypothesis, we used KPC mice as a backbone (Pdx1-Cre; LSL-KrasG12D/+; LSL-Trp53R172H/+) to generate models of PDAC driven by the loss of Ataxia Telangiectasia mutated (Atm, KPCA) or Brca1 (KPCB). Loss of Atm or Brca1 resulted in homologous recombination deficiency and genomic instability in vivo. In vitro cultures of KPCA and KPCB PDAC cells had higher numbers of micronuclei and were more radiosensitive than KPC cells. Next, we performed bulk and single-cell RNA sequencing to identify therapeutic vulnerabilities associated with Atm or Brca1 loss. We found that loss of Atm upregulated ATR signaling, suggesting ATR as a therapeutic target in KPCA mice. In agreement, ATR inhibition improved the survival of KPCA mice, with evidence of tumor growth delay. However, though PARP inhibition significantly improved the survival of KPCB mice, with delayed tumor growth, it was ineffective in KPCA mice. In addition, transcriptomic analysis showed an enrichment of immune cells in KPCB PDAC compared to KPC and KPCA PDAC. Notably, we found greater levels of STING signaling, a more diverse and abundant cytokine profile and more CD8 T cells in KPCB PDAC, suggesting sensitivity to immune checkpoint blockade (ICB). However, ICB was ineffective in KPCB mice as a single agent or in combination with PARP inhibition. Based on our transcriptomic data, we hypothesized that boosting antigen priming using a CD40 agonist (CD40a) in combination with FLT3-L may improve ICB efficacy in our mouse models of BRCAness. Immunotherapy consisting of combined ICB, CD40a and FLT-3L had little efficacy in either KPCA or KPCB mice. However, ATR inhibition improved the efficacy of combined ICB, CD40a and FLT-3L in KPCA mice, as indicated by increased CD8 T cell infiltration in the tumor, delayed tumor growth and longer survival. In contrast, we found that PARP inhibition combined with ICB, CD40a and FLT-3L was ineffective in KPCB mice. In summary, we present evidence to suggest that PDAC characterized by BRCAness is not a homogeneous group with respect to sensitivity to PARP inhibition, composition of the tumor microenvironment and response to immunotherapy. We highlight ATM loss as a potential predictive biomarker of ATR inhibitor efficacy and immunotherapy in PDAC. Future directions include the evaluation of inflammation as a therapeutic vulnerability in murine PDAC with loss of Brca1. Ultimately, we aim to inform clinical trials to refine precision medicine of PDAC characterized by BRCAness. Citation Format: Mathias Tesson, Kay Kong, Peter Repsicak, Aldo Bader, Ya-Ching Hsieh, Craig Nourse, Kristina Kirschner, Crispin Miller, Ed Roberts, David Chang, Jen Morton. Identification of therapeutic vulnerabilities in the subset of pancreatic ductal adenocarcinoma (PDAC) with homologous recombination deficiency [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 A031.

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.

Abstract A011: Enhanced Radiosensitivity of Pancreatic Cancer Achieved through Inhibition of Cyclin-Dependent Kinase 1

Abstract Introduction: One of the major challenges in treating Pancreatic Ductal Adenocarcinoma (PDAC) is overcoming radioresistance. This study investigates the targeting of Cyclin-dependent kinase-1 (CDK1) through both genetic and pharmaceutical inhibition as a strategy to increase the radiosensitivity of PDAC cells. Methods: We conducted mass spectrometry and phosphoproteomics analyses to compare engineered radiation-resistant PDAC cell lines (MIA PaCa-2 and PANC-1) with their parental controls. Additionally, we examined the TCGA PDAC database to correlate clinical outcomes, such as overall survival (OS), with CDK1 mRNA expression levels in PDAC patients. Furthermore, we developed a microRNA-based TET-on inducible shRNA to specifically inhibit CDK1 expression in PDAC cell lines. In an orthotopic PDAC model, we evaluated the radiation sensitivity of PDAC tumors with or without doxycycline treatment. We also employed a selective CDK1 inhibitor, RO-3306, to target CDK1 activation, followed by in vitro experiments using immunoblotting, immunocytochemistry, and clonogenic assays. Results: Our phosphoproteomics analysis revealed a significant increase in phospho-CDK1 (Tyr15) levels in the radiation-resistant cell lines. High CDK1 expression was associated with poorer OS in PDAC patients. Additionally, we observed heightened CDK1 phosphorylation at the threonine tyrosine 14 (Tyr14) residue following radiation exposure. Through our in vivo and in vitro experiments, we demonstrated that CDK1 inhibition, in conjunction with radiation therapy, delayed tumor growth and enhanced radiosensitivity in PDAC cells. Treatment with RO-3306 caused a substantial shift of cells into the G2/M phase and disrupted DNA repair post-radiation exposure, further enhancing radiosensitivity. Conclusion: Our findings underscore the critical role of CDK1 in PDAC radioresistance. Targeting CDK1 in combination with radiotherapy shows promise for future exploration as a potential treatment strategy in PDAC. Citation Format: Stetson Van Matre, Saaimatul Huq, Lokesh Akana, Oscar Zuniga, Henrique Rodrigues, Adam Wolfe. Enhanced Radiosensitivity of Pancreatic Cancer Achieved through Inhibition of Cyclin-Dependent Kinase 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 A011.

Abstract A051: Innate immune cell dynamics and pancreatic tumor progression in alcoholic pancreatitis

Abstract Introduction: Persistent inflammation due to alcoholic chronic pancreatitis (ACP) is associated with pancreatic ductal adenocarcinoma (PDAC). Myeloid cell infiltration within the pancreas, particularly with oncogenic mutant KrasG12D/+ (*Kras), is crucial for driving PDAC carcinogenesis. Our previous work demonstrated that hyperactivation of cyclic AMP response element binding protein (CREB) accelerates ADM/PanIN progression with heightened fibroinflammation. However, the association between CREB in precursor neoplastic lesions and innate myeloid cells remains unclear. Methods: We established an ACP mouse model using an alcohol diet along with prolonged caerulein administration in Ptf1aCreERTM/+; LSL-KrasG12D/+ (KC), and CREB deleted [Crebfl/fl in KC (KCC-/-)] mice. Single-cell RNA sequencing (scRNA-seq) of the mouse pancreas, multiparametric immunophenotyping of tumor-infiltrating myeloid cells and chromatin immunoprecipitation (ChIP-seq) with CREB pulldown were performed. Cytokine arrays and qPCR studies followed co-culture assays using mouse neutrophils (J774M) and macrophages (RAW 264.7) with epithelial cell lines harboring *Kras with in vitro ACP induction were conducted. Results: ACP induction in KC mice led to increased infiltration of neutrophils and alternatively activated macrophages with the pancreas. Conversely, Creb-deleted mice exhibited significantly reduced myeloid cell infiltration despite ACP induction. ChIP-Seq and qPCR identified CREB-regulated chemokines and immunomodulators as mediators of myeloid cell trafficking. Co-culture of mouse neutrophils and macrophages with KC-ACP conditioned media significantly increased IL-1 receptor antagonist (Il-1ra) expression and secretion. Single-cell transcriptomic analysis revealed heightened Il1rn mRNA levels in neutrophils and macrophages in KC-ACP when compared with KC pancreatic tumors. Creb-deleted PDAC models showed strong downregulation of Il1rn in macrophages, confirming CREB’s role in modulating myeloid cell interactions via epithelial cell signaling. Conclusions: Our findings highlight that CREB activation in epithelial cells under ACP conditions facilitates crosstalk with myeloid cells, promoting IL-1ra upregulation in neutrophils and macrophages. This CREB-driven regulation of IL-1ra in myeloid cells contributes to inflammation-associated tumorigenesis. Targeting this axis may offer a novel therapeutic approach to curb pancreatic cancer progression. Citation Format: Siddharth Mehra, Sudhakar Jinka, Varun Krishnamoorthy, Supriya Srinivasan, Anna Bianchi, Andrew Adams, Haleh Amirian, Manan Patel, Jashodeep Datta, Nipun Merchant, Nagaraj Nagathihalli. Innate immune cell dynamics and pancreatic tumor progression in alcoholic pancreatitis [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 A051.

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 B034: Myeloperoxidase limits tumor-associated neutrophils mediated immunosuppression in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease with a survival rate under 12%. Treatments like immunotherapy and chemotherapy are ineffective in PDAC due to the immunosuppressive tumor microenvironment, in which tumor-associated neutrophils (TANs) are a major component. TAN immunosuppression has been shown to depend upon increased production of reactive oxygen species, with myeloperoxidase (MPO) being a major source of it. We investigated the contribution of MPO in mediating TAN immunosuppression using orthotopic PDAC models in wild-type and syngeneic MPO-deficient mice. We observed a significant delay in tumor growth and improved clinical scores when MPO was deficient. Additionally, treatment with the MPO-specific inhibitor Verdiperstat also delayed tumor growth. Flow cytometry analysis of the tumor microenvironment further confirmed a significant increase in CD8 T and NK cells, along with an increase in granzyme B level in both subsets, when MPO was deficient. Additionally, there was an increase in activation marker (CD25) and memory subset among both CD8 and CD4 T cells in MPO-deficient mice. Ex vivo studies with isolated TANs from tumor-bearing mice showed reduced CD8 T and NK cell suppression, and reactive oxygen species level in absence of MPO, supporting the contribution of MPO in TAN immunosuppression. These data suggest that loss of MPO in TANs has decreased immunosuppressive functions resulting in increased cytotoxic and activated T cells and NK cells with increased memory response within the tumor microenvironment. Our results demonstrate the contribution of MPO in regulating tumor progression, TAN immunosuppression, and immune cell infiltration using an orthotopic PDAC model. Citation Format: Angisha Basnet, Brain Boone, Tracy Liu. Myeloperoxidase limits tumor-associated neutrophils mediated immunosuppression 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 B034.

Abstract B071: Antigen-presenting cancer-associated fibroblasts are required for ICI-sensitivity in pancreatic ductal adenocarcinoma

Abstract Cancer-associated fibroblasts (CAFs) are a hallmark of many cancers and are particularly prevalent in pancreatic ductal adenocarcinoma (PDAC). This highly fibrotic environment is thought to play a role in resistance to therapy; however, attempts to target CAFs have been unsuccessful in clinical trials. Deeper insights into CAF biology have revealed that these cells are heterogeneous in origin, gene expression, and function. RNA sequencing studies have identified a subset of CAFs, antigen-presenting CAFs (apCAFs), which are defined by MHC-II and interact with CD4 T cells. Given the importance of CD4 T cells in the antitumor response in PDAC, understanding apCAF biology could demonstrate ways to promote their differentiation and enhance T cell activity.Methods: Two clones differing in sensitivity to ICIs, sKPC (sensitive) and rKPC (resistant), derived from the autochthonous KPC murine PDAC model, were implanted orthotopically into the pancreas of syngeneic C57BL/6 mice. Mice were untreated, treated with dual checkpoint inhibition, or immunized with a KRAS peptide vaccine. Tumors were harvested for TME characterization, functional assays, single-cell RNA sequencing (scRNAseq), and imaging mass cytometry (IMC). apCAFs, CD11c+, and CD4+ T cells were isolated for functional assays. Peptide-pulsed apCAFs and CD11c+ cells were cultured with naïve OT-II CD4+ T cells or sKPC tumor infiltrating CD4+ T cells. apCAF KO mice were generated by crossing PDGFRαCre and MHC-IIflx mice.Results: Immunosensitive sKPC tumors are characterized by higher immune cell and apCAF infiltration than rKPC tumors. These apCAFs activate tumor-infiltrating CD4 T cells and induce regulatory T cell (Treg) differentiation, but not production of immunosuppressive cytokines. IMC analysis shows greater proximity of apCAFs to CD4 T cells in sKPC tumors compared with rKPC tumors. Remarkably, apCAF KO mice implanted with sKPC tumors lose sensitivity to ICI. scRNAseq has uncovered a unique transcriptome of apCAFs depending on tumor of origin and time following tumor implantation, revealing potential targets by which to quantitatively and qualitatively modulate apCAFs and enhance the antitumor response. Furthermore, scRNAseq of CD4 T cells cocultured with apCAFs has shown a unique pattern of CD4 T cell gene upregulation depending on the TME from which apCAFs were isolated, which could yield strategies to modulate not only apCAFs but also CD4 T cell activity.Conclusions: apCAFs are more abundant and more proximal to CD4 T cells in immunosensitive tumors, and apCAF KO sKPC tumor-bearing mice lose sensitivity to ICI. Together these data implicate apCAFs as essential mediators of the antitumor immune response. The unique transcriptome of apCAFs and cocultured CD4 T cells depending on tumor of origin suggests multiple strategies to modulate their interactions. Although a small proportion of the TME, modulating apCAF number or function could provide a solution to the currently unmet need of new and more effective treatments for PDAC patients. Citation Format: Saumya Maru, Meredith Wetzel, Jacob T Mitchell, Nicole Gross, Lalitya Andaloori, Kathryn Howe, Emma Kartalia, Guanglan Mo, James Leatherman, Won Jin Ho, Luciane Kagohara, Elana Fertig, Edward J Pearce, Elizabeth M Jaffee. Antigen-presenting cancer-associated fibroblasts are required for ICI-sensitivity in pancreatic ductal adenocarcinoma [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 B071.

Abstract IA-06: Elucidation of metabolic resistance mechanisms to RAS inhibition

Abstract Pancreatic ductal adenocarcinoma (PDAC) is characterized by KRAS- and autophagy-dependent growth. We previously determined that concurrent inhibition of autophagy, using the lysosomal inhibitor chloroquine (CQ), and of ERK, using a small molecule ERK inhibitor (ERKi), synergistically suppressed the growth of pancreatic ductal adenocarcinoma (PDAC) cell lines and patient xenograft-derived (PDX) organoids in vitro and PDX tumors in vivo. Our findings provided the rationale for our initiation of Phase I and Phase I/II clinical trials evaluating the combination of MEKi (binimetinib; NCT04132505) or ERKi (LY3214996; NCT04386057) with hydroxychloroquine (HCQ) in PDAC. We have extended these findings and determined that the combined inhibition of KRASG12C or KRASG12D and autophagy was effective in KRAS-mutant cancers. Our ongoing studies are centered on developing additional combinations for targeting metabolic resistance mechanisms to KRAS or ERK MAPK targeted therapies. First, we performed a CRISPR/Cas-9 mediated genetic loss-of-function screen in the presence of CQ to determine additional sensitizers to autophagy inhibition. Top sensitizers included multiple facilitators of the DNA damage response, mTOR pathway components, and genes involved in the upstream regulation of the autophagy pathway. Additionally, we identified PIKfyve, a lipid kinase critical for the recycling dynamics of lysosomes, as an essential autophagy-related gene in PDAC cells. PIKfyve inhibition with apilimod resulted in a potent reduction of autophagic flux and growth. Importantly, PIKfyve inhibition blocked the compensatory increases in autophagic flux associated both with MEK inhibition and with direct RAS inhibition. Accordingly, combined inhibition of PIKfyve and either the RAS ERK-MAPK pathway or RAS itself showed robust growth suppression across a panel of KRAS-mutant PDAC models. Growth suppression was due, in part, to potentiated cell cycle arrest and induction of apoptosis following loss of IAP proteins. We conclude that concurrent inhibition of RAS and PIKfyve is a synergistic, cytotoxic combination that may represent a novel therapeutic strategy for PDAC. In a parallel line of investigation, we determined that autophagic signaling is temporally regulated following RAS pathway inhibition, peaking at 48-72 hours, and returning to basal levels by 7 days. Interestingly, we found that as autophagic upregulation subsides, another nutrient scavenging pathway, macropinocytosis, increases. We hypothesize that further dissection of the dynamic regulation of autophagy and macropinocytosis will improve current anti-nutrient scavenging treatment strategies. We conclude that concurrent suppression of multiple metabolic processes, to block compensatory rebound activities, will be needed for effective PDAC treatment. Citation Format: Jonathan M. DeLiberty, Ryan Robb, Mallory K. Roach, Sarah E. Ackermann, Runying Yang, Noah L. Pieper, Khalilah E. Taylor, Scott Bang, Elisa Baldelli, Emanuel F. Petricoin III, John P. Morris IV, Clint A. Stalnecker, Kirsten L. Bryant. Elucidation of metabolic resistance mechanisms to RAS inhibition [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-06.

Enhanced radiosensitivity of pancreatic cancer achieved through inhibition of Cyclin-dependent kinase 1

Background and purposeOvercoming radioresistance is a critical challenge in pancreatic ductal adenocarcinoma (PDAC). Our study investigates the targeting of Cyclin-dependent kinase-1 (CDK1) through genetic and pharmaceutical inhibition to radiosensitize PDAC cells. Materials and MethodsMass spectrometry and phosphoproteomics were used to analyze engineered radiation-resistant PDAC cell lines (MIA PaCa-2 and PANC-1) compared to parental controls. The TCGA PDAC database was queried for clinical outcomes and patients were dichotomized based on the median CDK1 mRNA expression. We generated a microRNA-based TET-on inducible shRNA to inhibit CDK1 expression in two PDAC cell lines. We used an orthotopic model of PDAC to test the radiation sensitivity of PDAC tumors with or without doxycycline treatment. We targeted CDK1 activation with a selective CDK1 inhibitor, RO-3306, followed by in vitro experiments employing immunoblotting, immunocytochemistry, and clonogenic assays. ResultsPhosphoproteomics analysis revealed that phospho-CDK1 (Tyr15) was significantly elevated in the resistant clones. We found that high CDK1 expression was associated with worse OS in PDAC patients. Radiation exposure increased CDK1 phosphorylation. In MIA PaCa-2 and PANC-1 cells, CDK1 inhibition synergized with radiation therapy to delay tumor growth in vivo. CDK1 inhibition via. RO-3306 resulted in a significant shift of cells into the G2/M phase and disrupted DNA repair after radiation exposure. In vitro, pre-treatment with RO-3306 led to enhanced radiosensitivity of PDAC cells. ConclusionCDK1 plays a crucial role in PDAC radioresistance. Targeting CDK1 with radiotherapy holds promise for further investigation in PDAC treatment.

A Photoactivatable Self-Assembled Nanoagonist for Synergistic Therapy against Pancreatic Ductal Adenocarcinoma.

Immunotherapy has revolutionized the cancer treatment paradigm, yet efficient immunotherapeutic responses against immune-cold/desert cancers remain challenging. Herein, we report that photoactivatable nanoagonists yield a potent antitumor synergy of photoimmunotherapy against pancreatic ductal adenocarcinoma (PDAC). The nanoagonist was fabricated by assembling an amphiphilic boron dipyrromethene-derived polymer conjugated with a Toll-like receptor agonist via a photocleavable linker and stimulator of interferon genes agonist. The nanoagonist enables light-induced generation of reactive oxygen species and on-demand release of the agonists to yield synergistic photoimmunotherapy. The produced tumor antigens promote dendritic cell maturation, which is further amplified by these agonists for eliciting adaptive immunity, accompanied by apparently abscopal and long-term memory effects. The nanoagonist further alleviates the fibrosis of tumor stroma and the immunosuppressive microenvironment, leading to the deep infiltrations of clinically used therapeutics and immune cells to yield preferable combinational treatments against PDAC models. These results provide valuable insights into activatable nanoparticles for cancer therapy against immune-desert cancers.

Hypoxia Abrogates Tumor-Suppressive Activities of C/EBPδ in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a dismal disease with a low 5-year survival rate of only 13%. Despite intense research efforts, PDAC remains insufficiently understood. In part, this is attributed to opposing effects of key players being unraveled, including the stroma but also molecules that act in a context-dependent manner. One such molecule is the transcription factor C/EBPδ, where we recently showed that C/EBPδ exerts tumor-suppressive effects in PDAC cells in vitro. To better understand the role of C/EBPδ in different contexts and the development of PDAC, we here build on these findings and assess the effect of C/EBPδ in a PDAC model in mice. We establish that the lack of oxygen in vivo-hypoxia-counteracts the tumor-suppressive effects of C/EBPδ, and identify a reciprocal feedback loop between C/EBPδ and HIF-1α. RNA sequencing of C/EBPδ-induced cells under hypoxia also suggests that the growth-limiting effects of C/EBPδ decrease with oxygen tension. Consequently, in vitro proliferation assays reveal that the tumor-suppressive activities of C/EBPδ are abrogated due to hypoxia. This study demonstrates the importance of considering major physiological parameters in preclinical approaches.

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.

BP1003 Decreases STAT3 Expression and Its Pro-Tumorigenic Functions in Solid Tumors and the Tumor Microenvironment.

Overexpression and aberrant activation of signal transducer and activator of transcription 3 (STAT3) contribute to tumorigenesis, drug resistance, and tumor-immune evasion, making it a potential cancer therapeutic target. BP1003 is a neutral liposome incorporated with a nuclease-resistant P-ethoxy antisense oligodeoxynucleotide (ASO) targeting the STAT3 mRNA. Its unique design enhances BP1003 stability, cellular uptake, and target affinity. BP1003 efficiently reduces STAT3 expression and enhances the sensitivity of breast cancer cells (HER2+, triple negative) and ovarian cancer cells (late stage, invasive ovarian cancer) to paclitaxel and 5-fluorouracil (5-FU) in both 2D and 3D cell cultures. Similarly, ex vivo and in vivo patient-derived models of pancreatic ductal adenocarcinoma (PDAC) show reduced tissue viability and tumor volume with BP1003 and gemcitabine combination treatments. In addition to directly affecting tumor cells, BP1003 can modulate the tumor microenvironment. Unlike M1 differentiation, monocyte differentiation into anti-inflammatory M2 macrophages is suppressed by BP1003, indicating its potential contribution to immunotherapy. The broad anti-tumor effect of BP1003 in numerous preclinical solid tumor models, such as breast, ovarian, and pancreatic cancer models shown in this work, makes it a promising cancer therapeutic.

Targeting MYC effector functions in pancreatic cancer by inhibiting the ATPase RUVBL1/2

ObjectiveThe hallmark oncogene MYC drives the progression of most tumours, but direct inhibition of MYC by a small-molecule drug has not reached clinical testing. MYC is a transcription factor that...

Insights into gemcitabine resistance in pancreatic cancer: association with metabolic reprogramming and TP53 pathogenicity in patient derived xenografts

BackgroundWith poor prognosis and high mortality, pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies. Standard of care therapies for PDAC have included gemcitabine for the past three decades, although resistance often develops within weeks of chemotherapy initiation through an array of possible mechanisms.MethodsWe reanalyzed publicly available RNA-seq gene expression profiles of 28 PDAC patient-derived xenograft (PDX) models before and after a 21-day gemcitabine treatment using our validated analysis pipeline to identify molecular markers of intrinsic and acquired resistance.ResultsUsing normalized RNA-seq quantification measurements, we first identified oxidative phosphorylation and interferon alpha pathways as the two most enriched cancer hallmark gene sets in the baseline gene expression profile associated with intrinsic gemcitabine resistance and sensitivity, respectively. Furthermore, we discovered strong correlations between drug-induced expression changes in glycolysis and oxidative phosphorylation genes and response to gemcitabine, which suggests that these pathways may be associated with acquired gemcitabine resistance mechanisms. Thus, we developed prediction models using baseline gene expression profiles in those pathways and validated them in another dataset of 12 PDAC models from Novartis. We also developed prediction models based on drug-induced expression changes in genes from the Molecular Signatures Database (MSigDB)’s curated 50 cancer hallmark gene sets. Finally, pathogenic TP53 mutations correlated with treatment resistance.ConclusionOur results demonstrate that concurrent upregulation of both glycolysis and oxidative phosphorylation pathways occurs in vivo in PDAC PDXs following gemcitabine treatment and that pathogenic TP53 status had association with gemcitabine resistance in these models. Our findings may elucidate the molecular basis for gemcitabine resistance and provide insights for effective drug combination in PDAC chemotherapy.

Ex-vivo 3D cellular models of pancreatic ductal adenocarcinoma: from embryonic development to precision oncology.

Pancreas is a vital gland of gastro-intestinal system with exocrine and endocrine secretory functions, interweaved into essential metabolic circuitries of the human body. Pancreatic ductal adenocarcinoma (PDAC) represents one of the most lethal malignancies, with a five-year survival rate of 11%. This poor prognosis is primarily attributed to the absence of early symptoms, rapid metastatic dissemination, and the limited efficacy of current therapeutic interventions. Despite recent advancements in understanding the etiopathogenesis and treatment of PDAC, there remains a pressing need for improved individualized models, the identification of novel molecular targets, and the development of unbiased predictors of disease progression. Here we aim to explore the concept of precision medicine utilizing three-dimensional, patient-specific cellular models of pancreatic tumors and discuss their potential applications in uncovering novel druggable molecular targets and predicting clinical parameters for individual patients.