Preclinical Models Of Pancreatic Ductal Adenocarcinoma Research Articles

KRAS-Driven Tumorigenesis and KRAS-Driven Therapy in Pancreatic Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is associated with significant morbidity and mortality and is projected to be the second leading cause of cancer-related deaths by 2030. Mutations in KRAS are found in the vast majority of PDAC cases and plays an important role in the development of the disease. KRAS drives tumor cell proliferation and survival through activating the MAPK pathway to drive cell cycle progression and to lead to MYC-driven cellular programs. Moreover, activated KRAS promotes a protumorigenic microenvironment through forming a desmoplastic stroma and by impairing antitumor immunity. Secretion of granulocyte-macrophage colony-stimulating factor and recruitment of myeloid-derived suppressor cells and protumorigenic macrophages results in an immunosuppressive environment while secretion of secrete sonic hedgehog and TGFβ drive fibroblastic features characteristic of PDAC. Recent development of several small molecules to directly target KRAS marks an important milestone in precision medicine. Many molecules show promise in preclinical models of PDAC and in early phase clinical trials. In this review, we discuss the underlying cell intrinsic and extrinsic roles of KRAS in PDAC tumorigenesis, the pharmacologic development of KRAS inhibition, and therapeutic strategies to target KRAS in PDAC.

Abstract A001: An open label, phase II trial of ERK inhibition alone and in combination with autophagy inhibition in patients with metastatic cancreatic cancer

Abstract Background: Approximately 92% of pancreatic ductal adenocarcinoma (PDAC) harbor activating mutations in the KRAS oncoprotein. Therapeutic targeting of key elements of the MAPK pathway, including RAF, MEK and ERK, have failed to yield clinical benefit in patients with metastatic PDAC. Autophagy is a putative mechanism of resistance to ERK MAPK inhibition. Hydroxychloroquine (HCQ) is an inhibitor of autophagy and functions by inhibiting acidification of lysosomes. LY3214996 is an inhibitor of ERK 1/2 and has shown anti-tumor activity in pre-clinical models of PDAC. In this study, we evaluate the clinical efficacy of LY3214996 alone and in combination with HCQ. Methods: Eligible patients had metastatic PDAC or poorly differentiated carcinoma of the pancreas. Patients had at least one, but no more than two prior lines of systemic therapy. Twelve patients were included in the safety lead-in. One patient experienced grade 3 acute kidney injury and another experienced grade 3 nausea, thus dose level -1 was chosen as the tolerable dose for combination therapy. Patients were randomized in a 1:1 fashion to receive either LY3214996 200mg PO daily + HCQ 600mg PO BID (Arm 1) or LY3214996 400mg PO daily (Arm 2). The primary endpoint was disease control rate (DCR), defined as the proportion of patients with complete responses (CR), partial responses (PR) or stable disease (SD) that persisted for at least 4 months. Secondary endpoints included overall survival (OS) and progression-free survival (PFS). Point estimates and exact binomial 95% confidence interval (CI) were used for binary endpoints and the Kaplan Meier method was used for survival analysis. With 20 subjects in each treatment arm, there is 79% power using a one-sided alpha of 0.1 to differentiate between an unacceptable DCR of 5% and a desirable DCR of 20%. Results: A total of 39 patients met criteria for analysis (20 in Arm 1, 19 in Arm 2). The DCR rate in Arm 1 was 5% and 5.3% in Arm 2. One patient in each arm had SD. No patients achieved a CR or PR. The median PFS was 1.31 months in Arm 1 (95% CI 0.79-1.77) and 1.87 months in Arm 2 (95% CI 1.64-2.36). The median OS was 2.43 months in Arm 1 (95% CI 1.34-5.77) and 4.56 months in Arm 2 (95% CI 3.08-5.67). The most frequently observed toxicities in both arms included nausea, diarrhea, elevated creatine phosphokinase (CPK), anorexia and cytopenias. Exploratory analysis using patient-derived organoids did not show evidence of synergistic anti-tumor activity of LY3214996 in combination with chloroquine. Conclusion: LY3214996 alone or in combination with HCQ did not result in clinically meaningful activity in patients with metastatic pancreatic cancer. Further studies are needed to evaluate optimal strategies for autophagy inhibition, as well as combination strategies with other ERK MAPK targets (i.e., KRAS inhibitors) to fully elucidate the role of autophagy as a driver of resistance to ERK MAPK inhibition. Citation Format: Rishi Surana, Hui Zheng, Junning Wang, Micaela Morgado, Lauren K Brais, Kirsten L Bryant, Ashwin Somasundaram, Colin D Weekes, Bruno Bockorny, Mary Linton B Peters, Andrea J Bullock, Jessica A Zerillo, Ahmed A Rattani, Hanna K Sanoff, Jeffrey W Clark, Aparna R Parikh, Matthew B Yurgelun, Anuj Patel, Robert J Mayer, James M Cleary, Peter C Enzinger, Douglas A Rubinson, Nadine J McCleary, Andrea C Enzinger, Sarah E Slater, Kimmie Ng, Thomas A Abrams, Leah Biller, Srivatsan Raghavan, Joseph D Mancias, Jonathan O Nowak, Andrew J Aguirre, Channing J Der, Brian M Wolpin, Kimberley Perez. An open label, phase II trial of ERK inhibition alone and in combination with autophagy inhibition in patients with metastatic cancreatic 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 A001.

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.

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.

Enhancing Neoadjuvant Virotherapy's Effectiveness by Targeting Stroma to Improve Resectability in Pancreatic Cancer.

About one-fourth of patients with pancreatic ductal adenocarcinoma (PDAC) are categorized as borderline resectable (BR) or locally advanced (LA). Chemotherapy and radiation therapy have not yielded the anticipated outcomes in curing patients with BR/LA PDAC. The surgical resection of these tumors presents challenges owing to the unpredictability of the resection margin, involvement of vasculature with the tumor, the likelihood of occult metastasis, a higher ratio of positive lymph nodes, and the relatively larger size of tumor nodules. Oncolytic virotherapy has shown promising activity in preclinical PDAC models. Unfortunately, the desmoplastic stroma within the PDAC tumor microenvironment establishes a barrier, hindering the infiltration of oncolytic viruses and various therapeutic drugs-such as antibodies, adoptive cell therapy agents, and chemotherapeutic agents-in reaching the tumor site. Recently, a growing emphasis has been placed on targeting major acellular components of tumor stroma, such as hyaluronic acid and collagen, to enhance drug penetration. Oncolytic viruses can be engineered to express proteolytic enzymes that cleave hyaluronic acid and collagen into smaller polypeptides, thereby softening the desmoplastic stroma, ultimately leading to increased viral distribution along with increased oncolysis and subsequent tumor size regression. This approach may offer new possibilities to improve the resectability of patients diagnosed with BR and LA PDAC.

A Novel AMPK Inhibitor Sensitizes Pancreatic Cancer Cells to Ferroptosis Induction.

Cancer cells must develop strategies to adapt to the dynamically changing stresses caused by intrinsic or extrinsic processes, or therapeutic agents. Metabolic adaptability is crucial to mitigate such challenges. Considering metabolism as a central node of adaptability, it is focused on an energy sensor, the AMP-activated protein kinase (AMPK). In a subtype of pancreatic ductal adenocarcinoma (PDAC)elevated AMPK expression and phosphorylation is identified. Using drug repurposing that combined screening experiments and chemoproteomic affinity profiling, it is identified and characterized PF-3758309, initially developed as an inhibitor of PAK4, as an AMPK inhibitor. PF-3758309 shows activity in pre-clinical PDAC models, including primary patient-derived organoids. Genetic loss-of-function experiments showed that AMPK limits the induction of ferroptosis, and consequently, PF-3758309 treatment restores the sensitivity toward ferroptosis inducers. The work established a chemical scaffold for the development of specific AMPK-targeting compounds and deciphered the framework for the development of AMPK inhibitor-based combination therapies tailored for PDAC.

Abstract CT016: Immunomodulation of the tumor microenvironment of pancreatic ductal adenocarcinoma with histone deacetylase inhibition: Results of a phase 2 clinical trial of entinostat in combination with nivolumab

Abstract Background The tumor microenvironment (TME) of pancreatic ductal adenocarcinoma (PDA) is characterized by a paucity of cytotoxic tumor-infiltrating lymphocytes, an abundance of immune-suppressive cell populations, and a general resistance to immune checkpoint inhibitor (ICI) therapy. In preclinical models of PDA, the HDAC inhibitor entinostat modulated the transcriptional programming of myeloid cells, reducing their capacity for immunosuppression and sensitizing tumors to ICI therapy. Methods We conducted a single-center, open-label, Simon two-stage, phase II study of entinostat in combination with the PD1 inhibitor nivolumab in patients with advanced PDA. Patients received oral entinostat 5 mg once a week. After a 14-day lead-in with entinostat monotherapy, patients concurrently receive entinostat 5 mg orally once a week plus nivolumab 240 mg intravenously every 2 weeks. After 4 months, therapy was continued with entinostat 5 mg weekly plus nivolumab at a dose of 480 mg fixed dose every 4 weeks until the time of progression or unacceptable toxicities. The primary endpoint was the objective response rate by Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. Peripheral immune profiles at baseline and on treatment were assessed by high-dimensional mass cytometry by time of flight (CyTOF) and by Luminex of chemokines in the plasma, and changes in the TME were performed on paired baseline and on treatment biopsies by multiplexed immunohistochemistry (mIHC), image cytometry-based quantification, and bulk RNA-seq analysis. Results From November 2017 to November 2020, 27 evaluable patients were enrolled. Three patients had a partial response (PR) by RECIST v1.1 criteria (objective response rate (ORR)) of 11%, 95% CI, 0.024-0.292), with a median duration of response of 10.2 months. Grade ≥3 treatment-related adverse events (TRAEs) were observed in 19 (63%) patients. The most common grade ≥3 TRAEs were decreased lymphocyte count, anemia hypoalbuminemia, and hyponatremia. Dendritic cell (DC) activation, maturation, migration, antigen processing, and presentation were increased in the periphery after entinostat treatment, consistent with HDAC inhibitor mediated myeloid reprogramming. Gene expression analysis of paired baseline and on treatment tumors demonstrated an enrichment in inflammatory response signaling pathways with combination treatment. Conclusions Entinostat and nivolumab demonstrated durable radiological responses in a subset of patients with PDA. Paired tissue and peripheral analysis showed immunomodulation of myeloid cell populations, consistent with the preclinical hypothesis supporting the trial. This study creates a roadmap for this strategy with future combinatorial therapeutic approaches to enhance the clinical benefit in PDA patients further. Citation Format: Marina Baretti, Ludmila Danilova, Jennifer N. Durham, Courtney B. Betts, Leslie Cope, Dimitrios N. Sidiropoulos, Joseph A. Tandurella, Soren Charmsaz, Nicole Gross, Alexei Hernandez, Won J. HO, Chris Thoburn, Rosalind Walker, James Leatherman Leatherman, Sarah Mitchell, Brian Christmas, Daria A. Gaykalova, Srinivasan Yegnasubramanian, Elana J. Fertig, Lisa M. Coussens, Mark Yarchoan, Elizabeth Jaffee, Nilofer S. Azad. Immunomodulation of the tumor microenvironment of pancreatic ductal adenocarcinoma with histone deacetylase inhibition: Results of a phase 2 clinical trial of entinostat in combination with nivolumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT016.

Abstract 4604: Imipridones ONC201 and ONC212 exhibit anti-tumor activity in biliary tract cancers and synergy when combined with trametinib and olaparib in vitro

Abstract Background: Biliary tract cancers (BTCs) are an aggressive group of malignancies affecting 220,000 new individuals globally each year. Despite advances in immunotherapy and targeted therapies, 5-year survival remains at 2% for those with metastatic disease. Thus, new therapies are desperately needed. The novel imipridone class of antineoplastic agents show promising activity in preclinical pancreatic ductal adenocarcinoma models; however, have not been investigated in BTC. Dordaviprone (ONC201) is a dopamine receptor D2 and mitochondrial protease ClpP modulator that induces apoptosis in cancer cells through both upregulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and the integrated stress response. ONC212 is a second-generation imipridone that targets orphan GPCR 132 and ClpP, leading to reduced oxidative phosphorylation from ClpX suppression. We hypothesized that the ONC201 and ONC212 would demonstrate antitumor effects in BTCs, both alone, and in combination with MEK and PARP inhibition. Methods: Two established BTC cell lines, RBE and HuCCT1, were used for these experiments. Sensitivity of RBE and HuCCT1 cells to ONC201, ONC212, MEK inhibition (trametinib), and PARP inhibition (olaparib) was assessed using CellTiter-Glo® luminescent cell viability assay. Each cell line was plated on 96-well plates and treated with ONC201 concentrations ranging from 0uM to 40uM for RBE and 0uM to 32uM for HuCCT1; ONC212 ranging from 0nM to 752nM for RBE and 0nM to 640nM for HuCCT1; trametinib ranging from 0nM to 24nM for RBE and 0nM to 16nM for HuCCT1; and olaparib ranging from 0uM to 256uM for RBE and 0uM to 124uM for HuCCT1. Results were analyzed after 72 hours of incubation. Synergy studies were conducted in both cell lines, combining each imipridone with either trametinib or olaparib, and SynergyFinder® was used to evaluate synergy. Western blotting was performed on lysates generated from treated cell lines. Results: Both ONC201 and ONC212 demonstrated antineoplastic effects in two BTC cell lines. Half maximal inhibitory concentrations (IC50) of ONC201 in RBE and HuCCT1 cell lines were 2.5uM and 2.0uM, respectively. The IC50 of ONC212 in RBE and HuCCT1 was 47nM and 39nM, respectively, which was similar to IC50s demonstrated with cytotoxic chemotherapy (gemcitabine IC50 = 7.57nM (RBE) and 9.84nM (HuCCT1)). Both lines also demonstrated sensitivity to trametinib (IC50 = 5.9nM (RBE) and 3.7nM (HCCT1)), and to a lesser extent olaparib (IC50 = 64uM (RBE) and 31uM (HuCCT1)). Although synergy was noted with several combinations, this was most striking with the combination of ONC212 and either trametinib or olaparib. Further analysis of key signal transduction pathways, including MAPK and PI3K, as well as analysis of mechanisms of cell-death through apoptosis, autophagy, ferroptosis, and necroptosis is underway Citation Format: Grace Sun, Alexis J. Lannigan, Jasper Chan, Maximilian Schwermann, Varun V. Prabhu, Lanlan Zhou, Wafik S. El-Deiry, Alexander Grenander Raufi. Imipridones ONC201 and ONC212 exhibit anti-tumor activity in biliary tract cancers and synergy when combined with trametinib and olaparib in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4604.

A phase 1 trial of combined MEK, STAT3 and PD-1 inhibition in metastatic pancreatic ductal adenocarcinoma (PDAC).

TPS713 Background: PDAC is characterized by its innate and acquired resistance to both MAPK pathway inhibition and immune checkpoint (e.g. PD-1/PD-L1) inhibition (ICI) via multiple mechanisms. In preclinical models of PDAC, combined MEK and STAT3 inhibition (MEKi+STAT3i) uncovers stromal plasticity by attenuating cancer-associated fibroblasts (CAF) with IL-6/CXCL1-secretory phenotypes while enriching for Ly6a/CD34-expressing CAF phenotypes with mesenchymal stem cell-like features. This remodeling of CAF heterogeneity is associated with a striking attenuation in and reprogramming of tumor-associated macrophages (TAMs) as well as enhanced trafficking of CD8 T cells, which exhibit a distinct effector and anti-apoptotic transcriptional program. The addition of MEKi+STAT3i to PD-1 blockade overcomes ICI resistance by significantly enhancing the recruitment, degranulating capacity, and functional cytotoxicity of CD8+ T-cells, thereby augmenting antitumor responses and dramatically improving survival in these models. Furthermore, a patient with refractory PDAC treated off-label with this combination achieved a meaningful response. Based on this strong rationale, a phase 1 trial was initiated to test the combination of MEKi+STAT3 and PD1 inhibition in patients with metastatic PDAC. Methods: NCT05440942 is an open-label, prospective, single-institution phase 1 trial testing the safety, preliminary efficacy, and biomarkers of response to the combination of trametinib (MEKi), ruxolitinib (JAK2/STAT3 inhibitor) and retifanlimab (PD-1 inhibitor) in patients with metastatic PDAC. Patients with metastatic PDAC who have had disease progression on at least one line of prior therapy, with good organ function, preserved performance status, and without major intercurrent illness are eligible. Patients must have an accessible lesion for biopsy and must be willing to undergo this research biopsy at baseline and on treatment. Part 1 of the study is a dose-escalation phase with 3 dose levels and a target dose of trametinib 2mg orally daily, ruxolitinib 15mg orally twice daily, and retifanlimab 500mg intravenously every 28 days. The dose escalation is being done using the novel Bayesian keyboard design and 9-15 patients will be treated to get to the potential maximum tolerated dose (MTD). Dose level 1 has been completed without any dose-limiting toxicities seen. Part 2 is an expansion phase which will accrue an additional 20 patients. All patients in part 1 and 2 will have core-needle biopsies pre-treatment and after 4 weeks. Serial blood samples will be collected at baseline and on treatment. Biopsies are being analyzed by multiparameter immune profiling using mass cytometry and bulk RNA sequencing; blood is being analyzed for circulating tumor DNA and immune profiling. These results will be correlated with clinical response to therapy to determine biomarkers of response and resistance. Clinical trial information: NCT05440942 .

Abstract A022: Novel nanoparticle delivery of toll like receptor agonists alters the tumor immune landscape and improves response to standard of care therapy in preclinical PDAC models

Abstract The tumor microenvironment (TME) of pancreatic ductal adenocarcinoma (PDAC) is becoming an increasingly complex system, driven by the interactions between multiple different cell types. These drivers of cancer progression can suppress local immune responses, promote resistance to therapies, and reinforce a pro-tumorigenic TME. Despite advances in other tumor types with immune checkpoint blockade, minimal progress has been made in the treatment of PDAC with immunotherapies targeted at adaptive responses. As such, with a majority of the TME being made up of innate immune cells being skewed towards immunosuppressive phenotypes, there remains an underutilized immunologic target. Of the potential subset of targets, toll like receptors (TLR) have garnered significant interest given the key role they have in controlling the innate immune response in a number of different immune cells. On their own, agonists of these family of receptors lack efficacy, either through poor tumor penetration, lack of target specificity or toxicity. To address this unmet need, we have developed a nano-delivery approach for TLR agonists for target immune cell uptake. Thus far, we have established two conjugates, a protein-based nanoparticle termed FN-TLR7 for the delivery of a CL307-based conjugate and a DNA nanoparticle termed CAGE-TLR9 for the delivery of a CpG-based agonist. We have found that these conjugates can activate target immune cells within the TME via an advantageous pharmacokinetic profile which significantly reduces nonspecific toxicities compared to free agonists. When paired with the current standard of care options in PDAC, we see significant improvements in tumor control. The implications of nano-delivery of TLR agonists for treating PDAC tumors will be discussed. Citation Format: Nathan Beals, Anthony Sorrentino, Dafna Bar-Sagi. Novel nanoparticle delivery of toll like receptor agonists alters the tumor immune landscape and improves response to standard of care therapy in preclinical PDAC models [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A022.

Abstract C025: Fibroblast-specific IL1R1-p38 MAPK signaling sustains stromal inflammation and contributes to therapeutic resistance in pancreatic cancer

Abstract Introduction: The desmoplastic stroma of pancreatic ductal adenocarcinoma (PDAC) perpetuates therapeutic resistance by impairing drug delivery and effector immune cell infiltration and activation. We have identified tumor cell-derived interleukin-1α (IL1α) as a critical activator of the stroma, comprised of cancer-associated fibroblasts (CAF), towards a pro-inflammatory phenotype via the IL1 receptor, IL1R1, and subsequent activation of p38 MAPK. Our hypothesis is that disruption of the CAF-specific IL1α/p38 MAPK signaling axis can improve chemotherapeutic resistance by remodeling the fibrotic stromal landscape and the overall immune microenvironment in PDAC. Methods: p38 MAPK was pharmacologically inhibited with pexmetinib and genetically with an shRNA lentiviral system. Bulk RNA and ATAC sequencing (RNAseq and ATACseq) were performed on human pancreatic stellate cells (hPSC). Gene set enrichment analyses and motif enrichment analyses were performed on the RNAseq and ATACseq datasets, respectively. Tumor cells (KPC-6694), derived from LSL-KrasG12D/+; LSL-Tp53R172H/+; Pdx1Cre/+ (KPC) murine tumors, were injected orthotopically into Col1a2Cre/+; Mapk14f/f and Col1a2Cre/+; Il1r1f/f mice, and tumors were harvested for downstream analysis. Ptf1aCre/+; LSL-KrasG12D/+; Tgfbr2flox/flox (PKT) mice were treated with pexmetinib (PEX, 30mg/kg, daily PO), anakinra (AKA, 50mg/kg IP, BID) or vehicle control for 2.5 weeks prior to sacrifice for downstream analyses and single-cell RNA sequencing (scRNA) on tumors. Results: In vitro inhibition of p38 MAPK prevented CAF activation into an inflammatory fibroblast, when stimulated with IL1α, demonstrated through bulk RNAseq and ATACseq. Bulk ATACseq revealed epigenetic regulation of the inflammatory CAF phenotype by IL1-mediated activation of p38 MAPK. Stromal-specific deletion of Il1r1 or Mapk14 resulted in signification reduction of tumor weight and intratumoral myeloid-derived suppressor cells. Additionally, pharmacologic blockade of both IL1R1 (AKA) and p38 MAPK inhibition (PEX) altered intratumoral immune cell and CAF populations, by flow cytometry and scRNA. Lastly, p38 inhibition or IL1R1 blockade combined with chemotherapy significantly reduced tumor burden and favorably altered the immune landscape in a PDAC genetically engineered mouse model. Conclusions: These findings provide important mechanistic data in preclinical PDAC models to explore p38 MAPK inhibition in combination therapy to target the fibrotic stroma and immunosuppressive tumor microenvironment. Citation Format: Samara P. Singh, Austin R. Dosch, Siddharth Mehra, Iago de Castro Silva, Vanessa Tonin Garrido, Anna Bianchi, Haleh Amirian, Edmond W. Box, Jashodeep Datta, Nagaraj S. Nagathihalli, Nipun B. Merchant. Fibroblast-specific IL1R1-p38 MAPK signaling sustains stromal inflammation and contributes to therapeutic resistance in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C025.

Development and evaluation of 5FUCore, OxaliCore, and IrinoCore transcriptomic signatures for predicting response to individual drugs in the FOLFIRINOX regimen: Towards toxicity reduction and personalized chemotherapy in PDAC.

4141 Background: Recently, we have successfully developed a transcriptomic signature that predicts the response to gemcitabine treatment in patients with localized pancreatic ductal adenocarcinoma (PDAC) (PMID: 36178036) and also in those with metastatic PDAC (PMID: 36496056). This was achieved by using an approach that combined several preclinical PDAC models. The FOLFIRINOX regimen (FFX) consisting of 5-Fluorouracil (5FU), Oxaliplatin, and Irinotecan is the more effective treatment option, but the high toxicity limits its use. In this study, we developed and analyzed three different transcriptomic signatures for the drugs composing FFX separately and evaluated their clinical utility. Methods: We combined the transcriptomic profile of 14 patients derived xenograft (PDX) and 31 patients derived cell cultures (PDC) with their corresponding chemotherapy response profiles. We developed predictive signatures for 5FU ( 5FUCore), Oxaliplatin ( OxaliCore), and Irinotecan ( IrinoCore), as previously reported for the GemCore signature (PMID: 36178036). They were then validated in three independent cohorts of patients: COMPASS (n = 94), Linehan (n = 28), and a new cohort of 87 advanced PDAC patients. Results: Three ICA components that reflect the chemotherapy response profiles for 5FU, oxaliplatin, and irinotecan were evaluated in the COMPASS cohort to determine their ability to identify sensitive patients in the FFX arm. Patients predicted as sensitive to each drug separately had a median overall survival (OS) of 77.6 (95% CI, 67.9-NR, P =0.02), 78.7 (95% CI, 57.6-NR, P= 0.002), and 70.4 (95% CI, 57.6-NR, P= 0.02) months for 5FU, oxaliplatin, and irinotecan, respectively. Notably, the model that integrated the group of sensitive patients to two or three drugs had the highest median OS in the COMPASS and Linehan cohorts with a HR of 0.25 (95% CI, 0.11-0.57; P < 0.001) and 0.11 (95% CI, 0.02-0.70; P= 0.019), respectively. Importantly, they also show a better objective response than the other groups ( P= 0.009). In the advanced PDAC group, the signatures showed a strong ability to discriminate sensitive patients in the FFX arm with a median OS of 20.1 (95% CI, 2.3-NR, P= 0.006), 13.64 (95% CI, 9.57-NR, P= 0.003), and 15.87 (95% CI, 2.9-NR, P= 0.002) months for 5FU, oxaliplatin, and irinotecan, respectively. Finally, we evaluated the best combination of two drugs in the pooled group. Patients sensitive to both 5FU and oxaliplatin showed the best response with 60% of patients achieving partial response. Conclusions: The signatures developed provide a tool for predicting responsiveness to the individual therapeutic drugs of the FFX regimen. They may ultimately lead to the development of a personalized strategy for combined chemotherapy regimens, increasing efficacy and reducing toxicity in PDAC.

Bioprinting of hydrogel beads to engineer pancreatic tumor-stroma microtissues for drug screening

Pancreatic ductal adenocarcinoma (PDAC) having features of dense fibrotic stromal and extracellular matrix (ECM) components has poor clinical outcome. In vitro construction of relevant preclinical PDAC models recapitulating the tumor-stroma characteristics is therefore in great need for the development of pancreatic cancer therapy. In this work, a three-dimensional (3D) heterogeneous PDAC microtissue based on a dot extrusion printing (DEP) system is reported. Gelatin methacryloyl (GelMA) hydrogel beads encapsulating human pancreatic cancer cells and stromal fibroblasts were printed, which demonstrated the capacity of providing ECM-mimetic microenvironments and thus mimicked the native cell-cell junctions and cell-ECM interactions. Besides, the spherical structure of the generated hydrogel beads, which took the advantage of encapsulating cells in a reduced volume, enabled efficient diffusion of oxygen, nutrients and cell waste, thus allowing the embedded cells to proliferate and eventually form a dense pancreatic tumor-stroma microtissue around hundred microns. Furthermore, a tunable stromal microenvironment was easily achieved by adjusting the density of stromal cells in the hydrogel beads. Based on our results, the produced heterogeneous pancreatic microtissue recapitulated the features of cellular interactions and stromal-like microenvironments, and displayed better anti-cancer drug resistance than mono-cultured pancreatic cancer spheroids. Together, the DEP system possesses the ability to simply and flexibly produce GelMA hydrogel beads, providing a robust manufacturing tool for the pancreatic cancer drug screening platform fabrication. In addition, the engineered pancreatic tumor-stroma microtissue based on bioprinted GelMA hydrogel beads, other than being ECM-biomimetic and stroma-tunable, can be used for observation in situ and may serve as a new drug screening platform.

Bioprinting of hydrogel beads to engineer pancreatic tumor-stroma microtissues for drug screening.

Pancreatic ductal adenocarcinoma (PDAC) having features of dense fibrotic stromal and extracellular matrix (ECM) components has poor clinical outcome. In vitro construction of relevant preclinical PDAC models recapitulating the tumor-stroma characteristics is therefore in great need for the development of pancreatic cancer therapy. In this work, a three-dimensional (3D) heterogeneous PDAC microtissue based on a dot extrusion printing (DEP) system is reported. Gelatin methacryloyl (GelMA) hydrogel beads encapsulating human pancreatic cancer cells and stromal fibroblasts were printed, which demonstrated the capacity of providing ECM-mimetic microenvironments and thus mimicked the native cell-cell junctions and cell-ECM interactions. Besides, the spherical structure of the generated hydrogel beads, which took the advantage of encapsulating cells in a reduced volume, enabled efficient diffusion of oxygen, nutrients and cell waste, thus allowing the embedded cells to proliferate and eventually form a dense pancreatic tumor-stroma microtissue around hundred microns. Furthermore, a tunable stromal microenvironment was easily achieved by adjusting the density of stromal cells in the hydrogel beads. Based on our results, the produced heterogeneous pancreatic microtissue recapitulated the features of cellular interactions and stromal-like microenvironments, and displayed better anti-cancer drug resistance than mono-cultured pancreatic cancer spheroids. Together, the DEP system possesses the ability to simply and flexibly produce GelMA hydrogel beads, providing a robust manufacturing tool for the pancreatic cancer drug screening platform fabrication. In addition, the engineered pancreatic tumor-stroma microtissue based on bioprinted GelMA hydrogel beads, other than being ECM-biomimetic and stroma-tunable, can be used for observation in situ and may serve as a new drug screening platform.

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Abstract 4187: Combined MEK and STAT3 inhibition reprograms the tumor microenvironment to overcome immunotherapy resistance in pancreatic cancer

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is characterized by immune exclusion, stromal desmoplasia, and resistance to immune checkpoint inhibition (ICI). We have previously demonstrated that reciprocally activated RAS/RAF/MEK/ERK and JAK/STAT3 pathways mediate therapeutic resistance, while combined MEK and STAT3 inhibition (MEKi+STAT3i) overcomes this resistance in preclinical PDAC models. Given the dramatic decrease in tumor burden observed following MEKi+STAT3i, we investigated its effect on the stromal and immune microenvironment. Methods: Ptf1aCre/+KrasG12D/+Tgfbr2flox/flox (PKT) mice were treated with vehicle, trametinib (MEKi, 3.3 mg/Kg, oral gavage three times weekly), ruxolitinib (STAT3i, 20 mg/Kg, oral gavage three times weekly), αPD-1 antibody (200 µg/mouse, i.p. injection twice weekly), combined MEKi+STAT3i or MEKi+STAT3i with αPD-1 beginning at 4-4.5 weeks of age. Mice were sacrificed after four weeks of treatment and tumors were harvested for single cell RNA sequencing, and high-dimensional immune-profiling by mass cytometry and flow cytometry. Results: Single-cell transcriptomic analysis revealed that combined MEKi+STAT3i not only altered stromal architecture but also reprogramed tumor-resident CAFs from Il6/Cxcl1-expressing inflammatory phenotype towards Ly6a/Cd34-expressing fibroblast phenotype with both mesenchymal and hematopoietic progenitor-like properties. This stromal plasticity was associated with a striking attenuation and reprogramming of F4/80+ macrophages, M2-like macrophages (F4/80+CD206+), and MDSCs (CD11b+F4/80-Ly6G+/Ly6C+), as well as enhanced trafficking of CD4+ and CD8+ T-cells which exhibited a distinct effector and anti-apoptotic transcriptional program. The addition of MEKi+STAT3i to PD-1 blockade overcomes immune checkpoint resistance by significantly augmenting anti-tumor responses and dramatically improving survival in PKT mice, compared with vehicle treatment (median 181 vs. 44 days, p<0.0001) or anti-PD-1 monotherapy (median 181 vs. 57 days, p=0.0005). The addition of MEKi+STAT3i to PD-1 blockade not only augmented the recruitment of activated and memory T-cell populations, but also improved their degranulating capacity and functional cytotoxicity compared to PD-1 blockade alone. Importantly, treatment of a patient with chemotherapy-refractory metastatic PDAC with MEKi (Trametinib), STAT3i (Ruxolitinib), and PD-1 inhibitor (Nivolumab) was well-tolerated and yielded significant clinical benefit. Conclusion: These data uncover a novel paradigm in which combined MEKi+STAT3i reprograms stromal inflammation and immune tolerance to overcome immunotherapy resistance in PDAC. The clinical efficacy of combined MEKi+STAT3i and anti-PD1 treatment provides encouraging signals for its translatability and is currently being pursued in a clinical trial. Citation Format: Vanessa Tonin Garrido, Jashodeep Datta, Xizi Dai, Anna Bianchi, Iago De Castro Silva, Purushottam Lamichhane, Siddharth Mehra, Samara P. Singh, Austin R. Dosch, Oliver Umland, Michael n N. VanSaun, Peter J. Hosein, Nagaraj Nagathihalli, Nipun Merchant. Combined MEK and STAT3 inhibition reprograms the tumor microenvironment to overcome immunotherapy resistance in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4187.

Repurposing HDAC and mTOR inhibitors for pancreatic cancer.

e16234 Background: Pancreatic ductal adenocarcinoma (PDAC) will be the second leading cause of cancer death by 2030 through early onset dedifferentiation and metastasis that results in limited treatments. FDA-approved targeted therapies–including proteasome, mitogen-activated protein kinase (MEK), histone deacetylase (HDAC), and mammalian target of rapamycin (mTOR) inhibitors–have shown preclinical benefit but failed in clinic. However, combination targeted therapy has been largely unexplored in clinical trials. We hypothesized based on prior drug screening efforts in the lab on aggressive subtypes of pancreatic cancer that combinations of these targeted therapies could be synergistically active in preclinical PDAC models. Methods: We optimized combinations of proteasome, MEK, mTOR, and HDAC inhibitors by performing cell viability experiments in human PDAC lines in 2D and 3D cell culture conditions in vitro. Optimized drug combinations from these experiments were then trialed in two different in vivo animal models–human FG xenografts in immunocompromised mice (NSG) and spontaneous primary PDAC tumors from transgenic pancreatic-specific Kras-overexpressing, P53-deficient mice (KPC). RNA-sequencing and Seahorse metabolic assays were utilized to analyze molecular mechanisms of drug synergy. Dedifferentiation was assessed by histology of KPC tumors. Results: The HDAC inhibitor Panobinostat and mTOR inhibitor Everolimus synergistically killed human pancreatic cancer cell lines grown in 2D and 3D cell culture in vitro. Synergy was highest at low concentrations of Panobinostat. In vivo, low-dose Panobinostat and Everolimus synergistically blocked growth of human FG xenografts in NSG mice. In vivo synergy of low-dose Panobinostat and Everolimus was highest in the KPC mouse model, where only combination therapy could significantly reduce tumor growth, stall dedifferentiation, and improve survival. Mechanistically, RNA-sequencing of Panobinostat/Everolimus-treated FG cells revealed that Panobinostat increased tumor suppressor genes opposing dedifferentiation such as p21, EGR1, and CCN2. Everolimus complemented Panobinostat by decreasing expression of enzymes metabolizing acetyl groups such as fatty acid synthase, stearoyl-CoA desaturase, and sterol response element binding factors. Functionally, Everolimus’ activity to reduce oxidative consumption increased histone acetylation specifically in the presence of Panobinostat. Conclusions: A combination of FDA-approved, targeted oral therapies (low-dose Panobinostat/Everolimus) is synergistically active in preclinical models of pancreatic cancer in vitro and in vivo. mTOR inhibitors create drug synergy by specifically increasing histone acetylation in HDAC inhibitor-treated cells by reversing HDAC inhibitor de-repression of acetyl/oxidative metabolism, illustrating a novel connection between cancer epigenetics and metabolism.

Molecular regulation of CD8 T cell activity and heterogeneity in pancreatic cancer

Abstract T cell dysfunction or ‘exhaustion’ is a hallmark of solid malignancies. Despite the critical importance of CD8 T lymphocytes in controlling tumor growth and immunotherapy efficacy, the mechanisms governing T cell exhaustion remain unclear. Here, we focused on resolving the heterogeneity and molecular programming of exhausted CD8 T cell states in the context of pancreatic cancer. Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with a discouraging 5-year survival rate of approximately 8%. PDAC tumors commonly present as immunologically ‘cold’, devoid of protective T cell populations, and are typically resistant to promising immunotherapies such as immune checkpoint blockade or adoptive cell therapy. Therefore, understanding the unique microenvironmental and CD8 T cell-intrinsic signals governing T cell exhaustion and accumulation in PDAC tumors may reveal crucial insight for engineering CD8 T cells for optimal immunotherapy efficacy. Here, we developed preclinical PDAC models for profiling tumor-specific murine CD8 T cell responses. Through application of spectral flow cytometry and multimodal single-cell sequencing approaches, we have uncovered impressively diverse antigen-specific CD8 T cell states in PDAC tumors. Select CD8 T cell populations identified within the PDAC tumor microenvironment included canonical populations of terminally-exhausted and progenitor-exhausted CD8 T cells as well as unexpected and uncharacterized CD8 T cell populations. Accordingly, we also have identified critical transcription factors controlling key features of CD8 T cell heterogeneity and persistence in PDAC tumors. These findings may ultimately provide insight for tailoring CD8 T cell fate and function in PDAC. Supported by 2022 AAI Intersect Fellowship for Computational Scientists and Immunologists

Orthotopic Models of Pancreatic Cancer to Study PDT.

A hallmark of pancreatic ductal adenocarcinoma (PDAC) is its poor prognosis that stems from a marked resistance to therapy, an invasive nature, and a high metastatic potential. Photodynamic therapy (PDT) is a promising modality for effectively managing PDAC both preclinically and clinically. While clinical trials of PDT for PDAC are still in their early stages, a plethora of elegant preclinical studies are supporting the translation and clinical adoption of PDT-based treatment regimens, many of which leverage orthotopic preclinical models of PDAC. Given the aggressiveness of the disease that is largely dependent on the localization of PDAC tumors, it is imperative that preclinical models used to evaluate PDT-based treatment regimens recapitulate elements of the natural pathogenesis in order to design treatment regimens tailored to PDAC with the highest potential for clinical success. In light of the importance of clinically relevant models of PDAC, this chapter details and discusses the methodologies developed over the last three decades to leverage orthotopic PDAC models in order to evaluate PDT-based treatment regimens. The shortcomings of these are also discussed, in addition to the future directions that the field is headed to establish the most relevant orthotopic models of PDAC.

Abstract PO-066: High uptake, retention, and in vivo activity of L-Annamycin in pancreatic cancer models

Abstract Introduction: Annamycin (ANN) is a non-cardiotoxic potent topoisomerase II poison that is structurally related to doxorubicin (Dox) but it displays significantly different biological properties. ANN is formulated in multilamellar liposomes to enable effective in vivo administration as L-ANN. Pancreatic ductal adenocarcinoma (PDAC) represents a major challenge and is the deadliest cancer in decades. Pancreas drug uptake and retention remains a significant factor contributing to the chemoresistance of PDAC. Additionally, drug resistance on a cellular level further limits the therapeutic options for pancreatic cancer patients. Based on unique biological properties, we select L-ANN for detailed characterization of anti-tumor potency in preclinical PDAC models. Objective: The objective of this study was to validate the L-ANN exceptional uptake in pancreatic cancer cells and tumors and explore its high pancreas uptake to target PDACs by assessing the efficacy of L-ANN in the preclinical mouse models. Methods: The uptake, cytotoxicity, and L-ANN-induced apoptosis were studied in human MDA-PATC50 and MDA-PATC53 cell lines. L-ANN pharmacokinetics and tissue-organ distribution were analyzed in CD-1 mice after bolus administration of L-ANN, ANN or DOX. Subcutaneous and orthotopic MDA-PATC50 and MDA-PATC53 mouse models were used to assess activity of L-ANN in vivo. Results: Fluorescent microscopy and FACS analysis showed significantly higher uptake of ANN in MDA-PATC50 and MDA-PATC53 when compared to DOX-treated cells. Interestingly, ANN displayed distinct subcellular distribution with predominantly cytosolic localization. Average IC50 of ANN or DOX treated MDA-PATC50 and MDA-PATC53 cells were 25.1 nM and 41.9 nM for ANN and 32.8 nM and 130.4 nM for DOX respectively (72h exposure). Biodistribution study showed rapid L-Ann absorption in the pancreas. The Cmax of L-Ann was 15-fold higher than Dox after administration of L-ANN or Dox (both at 4 mg/kg) in pancreatic tissue. The subcutaneous models demonstrated dose-dependent reduction in tumor volumes after 3 doses of L-Ann in both PDAC models. Additionally, efficacy of L-ANN was assessed in two orthotopic PDAC models. MRI confirmed tumor volume reduction in L-Ann 3 mg/kg and 4 mg/kg doses in MDA-PATC50 model (p<0.05 for both 3 and 4 mg), and in MDA-PATC53 model (p<0.001 and p<0.0001, respectively). The median survivals (MS) were 135 and 103 days for L-Ann at 3 mg/kg and 4 mg/kg respectively (MS for vehicle was 49 days, p<0.0001). MDA-PATC50 orthotopic model demonstrated the similar results (ongoing study). Conclusion: Annamycin exhibits high and rapid uptake in the pancreatic cancer cells and in pancreas itself when compared to Dox. Subsequently, ANN appeared to be highly cytotoxic against tested pancreatic cancer cell lines and as L-ANN potently inhibits tumor growth and increase survival in experimental models of pancreatic cancer. These experiments demonstrate remarkable activity of L-ANN in PDAC models and support future clinical studies. Citation Format: Ya'an Kang, Rafal Zielinski, Roberto Cardenas Zuniga, Radjendirane Venugopal, Maria Poimenidou, Magdalena Remiszewski, Shaohua Peng, Edd Felix, Krzysztof Grela, Stanislaw Skora, Van N. Nguyen, Izabela Fokt, Waldemar Priebe. High uptake, retention, and in vivo activity of L-Annamycin in pancreatic cancer models [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-066.