Pancreatic Ductal Adenocarcinoma Cells Research Articles

Abstract PR-19: Cell polarity proteins as novel regulators of macropinocytosis

Abstract Macropinocytosis has emerged as a nutrient-scavenging pathway that cancer cells exploit to survive the nutrient-deprived conditions of the tumor microenvironment. Cancer cells are especially reliant on glutamine for their survival, and in pancreatic ductal adenocarcinoma (PDAC) cells, glutamine deficiency can enhance the stimulation of macropinocytosis, allowing the cells to escape metabolic stress through the production of extracellular-protein-derived amino acids. Here, we identify the atypical protein kinase C (aPKC) enzymes, PKCz and PKCi, as novel regulators of macropinocytosis. In normal epithelial cells, aPKCs are known to regulate cell polarity in association with the scaffold proteins Par3 and Par6, controlling the function of several targets, including the microtubule-associated Par1 kinases. In PDAC cells, we identify that each of these cell polarity proteins are required for glutamine stress-induced macropinocytosis. Mechanistically, we find that the aPKCs are regulated by EGFR signaling or by the transcription factor CREM to promote the relocation of Par3 to microtubules, facilitating macropinocytosis in a dynein-dependent manner. Importantly, we determine that cell fitness impairment caused by aPKC depletion in glutamine stress is rescued by the restoration of macropinocytosis and that aPKCs support PDAC growth in vivo. These results identify a previously unappreciated role for the cell polarity protein network in the regulation of macropinocytosis and provide a better understanding of the mechanistic underpinnings that control macropinocytic uptake in the context of metabolic stress. Citation Format: Guillem Lambies Barjau, Szu-Wei Lee, Karen Duong-Polk Duong-Polk, David Dawson, Cheska Marie Galapate, Swetha Maganti, Cosimo Commisso. Cell polarity proteins as novel regulators of macropinocytosis [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 PR-19.

Abstract C033: Targeting PIKfyve-driven lipid metabolism in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) subsists in a nutrient-deregulated microenvironment, making it particularly susceptible to treatments that interfere with cancer metabolism. For example, PDAC utilizes and is dependent on high levels of autophagy and other lysosomal processes including macropinocytosis. Although targeting these pathways has shown potential in preclinical studies, progress has been hampered by the challenge of identifying and characterizing favorable targets for drug development. In our study, we aimed to characterize PIKfyve, a lipid kinase integral to lysosomal functioning, as a novel and targetable vulnerability in PDAC. We first confirmed that PIKfyve inhibition disrupted autophagic flux and disrupted lysosomal function. Next, to understand the metabolic effects of PIKfyve inhibition in PDAC cells, we employed a metabolism-focused CRISPR screen. Through this, we identified that PIKfyve inhibition increases PDAC cells’ dependency on de novo fatty acid synthesis through genes such as FASN and ACACA. Accordingly, PIKfyve inhibition triggered an increase in SREBP1-driven transcriptional and metabolic signatures favoring de novo fatty acid synthesis through increasing expression of genes such as FASN and ACACA. Further, employing targeted lipidomics, we determined that PIKfyve inhibition triggered an SREBP1-dependent accumulation of long-chain sphingolipids and that knockout of serine palmitoyl transferase subunits SPTLC1 and SPTLC2 sensitized PDAC cells to PIKfyve inhibition. Taken together, PIKfyve inhibition disrupts PDAC lipid homeostasis such that PDAC cells upregulate de novo synthesis of sphingolipids as a compensatory mechanism, suggesting that co-targeting de novo fatty acid synthesis and PIKfyve would result in synergistic effects. Given the importance of KRAS-MAPK signaling in controlling PDAC metabolic homeostasis, we hypothesized that the KRAS-MAPK signaling pathway is a primary driver of de novo lipid synthesis, specifically enhancing FASN and ACACA levels. Utilizing chromatin immunoprecipitation sequencing, we determined that KRAS or MEK inhibition reduces MYC binding to FASN and ACACA promoters. In line with this observation, KRAS or MEK perturbation decreased FASN and ACACA transcript and protein levels, suggesting that KRAS or MEK inhibition may sensitize PDAC cells to PIKfyve inhibition. Indeed, the simultaneous targeting of PIKfyve and KRAS-MAPK resulted in the elimination of tumor burden in a syngeneic orthotopic model and tumor regression in a xenograft model of PDAC. Taken together, these studies suggest that disrupting lipid metabolism through PIKfyve inhibition induces synthetic lethality in conjunction with KRAS-MAPK-directed therapies for PDAC. Citation Format: Caleb Cheng, Jasmine P Wisniewski, Sydney Peters, Jing Hu, Rahul Mannan, Bailey Jackson, Rüya Pakkan, Pietro Morlacchi, Brian Magnuson, Tongchen He, Rupam Bhattacharyya, Yuanyuan Qiao, Costas A Lyssiotis, Arul M Chinnaiyan. Targeting PIKfyve-driven lipid metabolism 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 C033.

Abstract C027: Efficacy advantages of a novel pan-RAS inhibitor, ADT-1004, over mutant- specific KRAS inhibitors for suppressing pancreatic tumor growth in murine models

Abstract Background/Objective: Prognosis remains grim for localized or metastasized pancreatic ductal adenocarcinoma (PDAC) with 5-year survival rates of 3-16%. More than 90% of PDAC cases harbor KRAS mutations. KRASG12C inhibitors recently developed have limited use for PDAC because only 3% of cases express this mutation. We developed a novel pan-RAS inhibitor, ADT-1004 and evaluated antitumor activity in tumor models established from murine or human PDAC cell lines or patient-derived xenografts (PDX). ADT-1004 is an orally bioavailable prodrug of ADT-007, a highly potent and selective pan-RAS inhibitor (doi.org/10.1101/2023.05.17.541233). Methods: Murine PDAC cells harboring the KRASG12D mutation (KPC-Luc or 2838C3-Luc) were orthotopically implanted into the pancreas of C57BL/6J mice. Four PDX PDAC tumors harboring KRAS mutations were implanted orthotopically in NSG mice. To assess potential to overcome RAS inhibitor resistance, parental KRASG12C MIA PaCa-2 PDAC cells and a resistant derivative of MIA PaCa-2 cells were implanted subcutaneously. To assess selectivity, mice were implanted with RASWT BxPC-3 cells SC. Mice were treated orally with ADT-1004 (40mg/kg) 5x/week for 4 weeks. Sotorasib and adagrasib KRASG12C inhibitors were administered at the same dose, route, and schedule as ADT-1004. Results: ADT-007 potently and selectively inhibited the growth of human and mouse PDAC cell lines in vitro. For example, the IC50 value of ADT-007 for KRASG12C MIA PaCa-2 PDAC cells was 2 nM compared to 2500 nM for RASWT BxPC-3 PDAC cells. Growth inhibition by ADT-007 was dependent on activated RAS and associated with reduced GTP-RAS levels and MAPK/AKT signaling. ADT-1004 was well tolerated in mice at doses up to 175 mg/kg bid orally with sustained plasma levels of ADT-007 exceeding growth IC50 values. ADT-1004 displayed robust antitumor activity in mouse tumor models using human- or mouse-derived PDAC cell lines with G12D, G12V, G12C, or G13Q KRAS mutations, causing selective inhibitory effects on ERK phosphorylation in tumors but not in normal tissues. ADT-1004 also inhibited growth of KRASG12C mutant MIA PaCa-2 tumors to a comparable extent as sotorasib and adagrasib. Sotorasib and adagrasib were ineffective against xenografted tumors of resistant cells derived from MIA PaCa-2 cells, while ADT-1004 strongly inhibited tumor growth. Target selectivity of ADT-1004 was confirmed in vivo, as ADT-1004 did not affect the growth of RASWT BxPC-3 tumors. Conclusions: ADT-1004 inhibited tumor growth of KRAS mutant PDAC tumors by blocking activated RAS to disrupt downstream MAPK/AKT signaling. Side-by-side comparisons reveal antitumor efficacy advantages of ADT-1004 over mutant-specific KRAS inhibitors. These results highlight the promise of ADT-1004 for treatment of PDAC and other RAS-driven cancers by addressing the complex genetic landscape of many cancers and limitations from resistance to mutant specific KRAS inhibitions. Citation Format: Dhana Sekhar Reddy Bandi, Ganji P Nagaraju, Jeremy B Foote, Adam B Keeton, Xi Chen, Kristy L Berry, Yulia Y Maxuitenko, Upender Manne, Donald J Buchsbaum, Gary A Piazza, Bassel F El-Rayes. Efficacy advantages of a novel pan-RAS inhibitor, ADT-1004, over mutant- specific KRAS inhibitors for suppressing pancreatic tumor growth in murine models [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C027.

Abstract C039: Investigating ER stress responses in pancreatic ductal adenocarcinoma under lipid imbalance

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related death, with a five-year survival rate of 12%. Dense desmoplasia results in poor intratumoral perfusion and elevated interstitial fluid pressure, creating regions with severe oxygen and nutrient deprivation. One consequence is a deficiency in unsaturated fatty acids (uFAs), as the de novo synthesis of uFAs requires oxygen. Our results indicate that PDAC cells experience endoplasmic reticulum (ER) stress and activate subsequent ER stress responses under uFA- limited conditions in vitro. ER stress responses are governed by three major stress sensors: inositol-requiring enzyme 1α (IRE1α), PKR-like ER kinase (PERK), and activating transcription factor 6 (ATF6). When IRE1α is activated, XBP1 mRNA is spliced, resulting in an activated transcription factor, XBP1s, which modulates lipid metabolism. XBP1s is upregulated in PDAC compared to PanIN and normal tissue in murine and human samples. PDAC cells have reduced viability under uFA-limited conditions. Moreover, ER stress is activated under uFA deficiency, and the stress response is diminished upon rescue by monounsaturated oleic acids. To study the role of ER stress responses, we genetically depleted PERK, ATF6, or XBP1 in PDAC cells. The depletion of these pathways rescued PDAC cell viability. Interestingly, B-I09, an inhibitor of XBP1 splicing, led to a mild tumor reduction in vivo. We have found that "basal-like" patient tumors are significantly enriched in the ER stress response pathways based on bulk patient RNA-seq datasets. Therefore, we hypothesize that the two PDAC subtypes “basal” and “classical” might have different responses or reliance on the ER stress pathways. We have created isogenic subtype models to represent basal and classical subtypes by modulating epithelial to mesenchymal (EMT) factors and deltaNP63. Signaling studies suggest that there are no differences between the subtypes’ response to uFA-limited conditions. We are currently assessing the role of XBP1s and IRE1α in vivo by implanting the gene-edited cells into subcutaneous and orthotopic tumor models. Furthermore, we are pursuing spatial mass spectrometry to study the lipid environment in PDAC tumors and investigate whether lipid imbalance is the main cause of ER stress activation in the tumor microenvironment (TME). Overall, our studies show that ER stress response is upregulated in PDAC tumors, and stress response promotes cell death under in vitro TME-like conditions. Subtype features such as EMT and deltaNP63 do not alter PDAC cells’ ER stress responses, and had minimal impact on their stress response reliance. Citation Format: Yanqing (Christine) Jiang, Xu Han, John Tobias, Carson Poltorack, Mai Wang, Pamela Burgess-Jones, Nathan Coffey, Brian Keith, Celeste Simon. Investigating ER stress responses in pancreatic ductal adenocarcinoma under lipid imbalance [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 C039.

Abstract B083: The splicing factor SMNDC1 facilitates alternative RNA splicing, contributing to therapy resistance in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is among the most aggressive and lethal cancers, mainly due to its high resistance to existing treatments. The standard treatment for PDAC involves chemotherapy, either with Gemcitabine (GEM) combined with paclitaxel or the FOLFIRINOX regimen (5-fluorouracil (5FU), leucovorin, irinotecan, oxaliplatin). However, PDACs rapidly develop resistance to these therapies. Similar resistance is observed with new treatments targeting the KRAS-MAPK pathway, which is crucial in driving PDAC. Our preliminary studies have shown that Survival Motor Neuron Domain Containing 1 (SMNDC1) promotes PDAC tumor growth by facilitating exon retention, specifically cassette exon 4 (E4) of MAPK3, linking RNA splicing to KRAS-MAPK signaling. To uncover the mechanisms underlying PDAC therapeutic resistance, we have developed novel in vitro models of resistance to chemotherapy and MAPK inhibitors (MAPKi). Using these models, we found that SMNDC1 and MAPK3 E4 retention is significantly increased in GEM, 5FU (chemotherapy agents), Adagrasib, Sotorasib (KRAS inhibitors; KRASi G12C), and Selumetinib (MEK inhibitor; MEKi) resistant PDAC cells. Additionally, PDAC lines acutely treated (24h) with trametinib or selumetinib (MEKi) showed a doubling in the expression of SMNDC1 and MAPK3 E4 retention. Protein expression analysis via western blotting revealed that GEM, 5FU, Adagrasib, MRTX1133 (KRASi G12D), and Selumetinib resistant PDAC cells upregulated SMNDC1 expression compared to their sensitive counterparts. To determine the role of SMNDC1 in MAPK resistance, we tested SMNDC1 proficient and deficient PDAC cells with KRASi or MEKi. SMNDC1-deficient PDAC cells were more sensitive to these inhibitors than isogenic cells expressing SMNDC1. Furthermore, to show that increased E4 retention of MAPK3 drives resistance to KRASi or MEKi, we treated SMNDC1 proficient PDAC cells with these inhibitors and MAPK3 E4 specific antisense morpholino oligonucleotide (ASO), which increased sensitivity compared to cells treated with the inhibitor and control ASO. To determine if increased E4 retention is a resistance mechanism specific to therapies targeting upstream of ERK1, we treated PDAC cells, proficient and deficient in SMNDC1, with ERK inhibitors (MK8353, Temuterkib). No changes in sensitivity were observed, as these inhibitors target the ERK1 protein downstream of MAPK3 splicing. Additionally, in an orthogonal model, we found that mutant EGFR lung adenocarcinoma cells resistant to the third-generation tyrosine kinase EGFR inhibitor (Osimertinib) also had higher incorporation of E4 of MAPK3. This suggests that upstream inhibition of ERK in the MAPK pathway leads to increased MAPK3 E4 retention. These findings indicate that PDACs use a "splicing switch" to enhance ERK kinase activity, thereby resisting targeted therapies and chemotherapy in cancers driven by mutant KRAS-MAPK signaling pathways. Citation Format: Md Afjalus Siraj, Yushan Zhang, Deanne Yugawa, Gilbert Giri, Prabir Chakraborty, Grant Goda, Geeta Rao, Daniel Dominguez, Stefan Kubicek, Resham Bhattacharya, Luisa Escobar-Hoyos, Priyabrata Mukherjee. The splicing factor SMNDC1 facilitates alternative RNA splicing, contributing to therapy resistance 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 B083.

Abstract C038: Cancer-associated fibroblasts maintain critical pancreatic cancer cell lipid homeostasis in the tumor microenvironment

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy whose abundant cancer-associated fibroblasts (CAFs) create a hallmark “desmoplasia” that severely limits oxygen and nutrient delivery. Hypoxia inhibits oxygen-dependent metabolic processes. For example, unsaturated lipid biosynthesis relies on oxygen to maintain desaturase enzymatic activities. Here, we investigated the mechanisms by which PDAC cells maintain lipid homeostasis under these conditions. Exogenous unsaturated lipids, rather than de novo synthesis, sustain PDAC cell viability by relieving endoplasmic reticulum (ER) stress under nutrient scarcity. Furthermore, CAFs are less hypoxic than adjacent malignant cells in vivo due to proximal blood vessel accessibility, nominating them as a potential source of these obligatory species. CAF-conditioned medium promoted PDAC cell survival upon nutrient and oxygen deprivation, an effect reversed by delipidation. We determined that lysophosphatidylcholines (LPCs) are particularly enriched in CAF-conditioned medium and preferentially taken up by PDAC cells, where they are converted to phosphatidylcholines (PCs) to sustain membrane integrity. Inhibiting LPC to PC conversion reversed the ability of exogenous unsaturated LPCs to sustain cancer cell survival and restrain ER stress in vitro and in vivo. Collectively, these results reveal a critical lipid cross-feeding mechanism enabling PDAC cell survival and identify an innovative approach to targeting this metabolic dependency. Efforts to target lipid crosstalk between CAFs and PDAC cells will be discussed. Citation Format: Xu Han, Michelle Burrows, Laura C Kim, Jimmy Xu, Will Vostrejs, Tran Ngoc Van Le, Yanqing Jiang, Edna Cukierman, Reiss Binder Kim, Ben Z Stanger, Clementina Mesaros, Brian Keith, Celeste Simon. Cancer-associated fibroblasts maintain critical pancreatic cancer cell lipid homeostasis in the tumor microenvironment [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 C038.

Abstract C019: A novel SDC1-targeted therapeutic antibody inhibits macropinocytosis and induces anti-tumor immunity in pancreatic cancer

Abstract Given that the mutated KRAS oncogene (KRAS*) dominates the genetic landscape of pancreatic ductal adenocarcinoma (PDAC), the development of KRAS* inhibitors represents a significant breakthrough in PDAC therapy. However, emerging evidence suggests that PDAC may eventually overcome its dependency on KRAS* and develop resistance to KRAS* inhibitor treatment. Our laboratory is dedicated to uncovering novel, accessible therapeutic targets that govern the mechanisms essential for KRAS*- driven PDAC cell survival. Our recent work has identified Syndecan1 (SDC1), a major heparan sulfate proteoglycan, as a critical mediator of KRAS* activity, promoting nutrient salvaging and fueling cancer cell growth, even in cases where PDAC cells have evaded KRAS* targeted therapy. Our finding positions SDC1 as a promising new therapeutic target for PDAC therapy. In our pursuit of new therapeutic approaches that can directly and specifically target SDC1 activity, we have successfully developed a monoclonal antibody against SDC1 (22B mAb), which exhibits remarkable sensitivity and specificity for human SDC1. Our results indicate that 22B mAb exerts substantial inhibition of PDAC cell growth in vitro and desirable tumor inhibitory effects in vivo in murine PDAC tumors and human PDAC cell-derived xenograft tumors. Interrogation of mechanism of actions further demonstrates that 22B elicits both direct effect in inhibiting macropinocytosis to block nutrient salvage of PDAC cells and indirect effect in inducing antibody-dependent cellular cytotoxicity (ADCC) to eliminate tumor cells. Notably, our 22B mAb synergizes with KRAS* inhibitors, chemotherapy, or immunotherapy, resulting in significantly enhanced therapeutic effects. In summary, our 22B mAb represents the first in class of anti-SDC1 antibody that not only directly targets the PDAC cells but also elicits immune cell-mediated killing. Additionally, our antibody holds the potential for developing new anti-PDAC therapeutic strategies, including antibody drug conjugates with cytotoxic or radionuclides payloads, for SDC1 targeted therapy/theranostics in PDAC. Thus, our newly developed 22B therapeutic antibody provides an alternate strategy for PDAC treatment and opens up new research horizons in therapeutic targeting of SDC1 in other malignancies characterized by aberrant SDC1 expression. Citation Format: Zecheng Yang, Madelaine S Theardy, Shuaitong Chen, Yongkun Wei, Mitsunobu Takeda, Xiaofei Wang, Jun Yao, Jennifer Li, Jangho Park, Yangxi Zheng, Long T Vien, Laura Bover, Haoqiang Ying, Wantong Yao. A novel SDC1-targeted therapeutic antibody inhibits macropinocytosis and induces anti-tumor immunity 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 C019.

Abstract C050: Vitamin B6 restriction impairs pancreatic cancer progression

Abstract Introduction: Cancer cells utilize altered metabolism to sustain their rapid growth, and understanding how these metabolic changes aid in tumor development could enhance current treatment approaches. Vitamins are essential micronutrients necessary in small quantities to support the proper functioning and metabolism of our organs and systems. Despite the recognized importance of vitamins in maintaining overall health, there is no direct evidence linking vitamin metabolism to pancreatic ductal adenocarcinoma (PDAC). Methods: PDAC animal models, organoid models, 3D-culture models, and LC-MS/MS-based metabolomic analysis were used to explore the role of vitamin B6 (VB6) in pancreatic cancer. Results: Our data showed that 1) Patients with PDAC exhibit lower levels of circulating vitamin B6 compared to healthy individuals of the same age. Additionally, vitamin B6 levels are reduced in the interstitial fluid of KPC (KrasG12D/+; p53R172H/+; Pdx1-Cretg/+) tumors and in the blood of mice with KPC tumors compared to healthy mice of the same age; 2) PDAC cells metabolize VB6 more rapidly than non-cancerous pancreatic epithelial cells; 3) VB6 restriction impairs PDAC cell proliferation in vitro and PDAC tumor growth in vivo; 4) VB6 depletion decreases key metabolites of the one-carbon pathway necessary for PDAC cell growth; 5) Inhibiting the one- carbon pathway mimics the effects of a vitamin B6 deficiency on PDAC cell proliferation. And knocking down VB6-dependent enzymes SHMT1/2 negates the pro-growth impact of VB6. Conclusion: Our research suggests that VB6 plays a key role in regulating various metabolic processes essential for the growth of PDAC cells. By focusing on inhibiting VB6-dependent enzymatic reactions, we may uncover new therapeutic strategies for treating PDAC. Citation Format: Chunbo He, Kamiya Mehla. Vitamin B6 restriction impairs pancreatic cancer progression [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 C050.

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 A076: LINE-1 ORF1p mimics viral innate immune evasion mechanisms in pancreatic cancer

Abstract Aberrant expression of repeat elements displaying viral mimicry is a common feature found in both mouse models and human pancreatic ductal adenocarcinoma (PDAC). However, cancer cells must employ mechanisms to manage this "viral" repeat element load to avoid triggering innate immune responses. Here, we demonstrate that the long interspersed nuclear element 1 (LINE-1) ORF1 protein (ORF1p) in PDAC cells plays a role in shielding repeat RNAs from activating pathogen recognition receptor (PRR)-mediated antiviral responses, independent of retrotransposition. Suppression of ORF1p using lentiviral shRNA induces innate immune responses through the PRRs, RIG-I and MAVS (double-stranded RNA sensors). Interestingly, PDAC cell lines with low ORF1p lack these two receptors, suggesting convergent mechanisms to suppress PRR signaling. Furthermore, RNA immunoprecipitation sequencing (RIP-seq) demonstrates ORF1p association with repeat RNA, including LINE and SINE elements that typically trigger antiviral responses. Subcellular localization of ORF1p in processing bodies (p-bodies) reveals specific interaction with MOV10, a dsRNA helicase crucial for antiviral responses. Loss of ORF1p results in significant growth reduction in vitro and in vivo, which supports ORF1p as a potential immune oncology therapeutic target. Altogether, our results demonstrate a novel function of ORF1p in shielding cytoplasmic repeat RNA from PRR sensing, shedding light on how PDAC cells evade host immune responses triggered by viral-like repeat RNA. Citation Format: Eunae You, Bidish Patel, Alexandra Rojas, Siyu Sun, Natalie Ho, Ildiko Phillips, Michael Raabe, Yuhui Song, Katherine Xu, Joshua Kocher, Peter Richieri, Martin Taylor, Linda Nieman, Benjamin Greenbaum, David Ting. LINE-1 ORF1p mimics viral innate immune evasion mechanisms 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 A076.

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

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

Abstract C018: Targeting KRAS inhibitor resistance by ferroptosis induction in PDAC

Abstract KRAS inhibitors (KRASi) have shown promising antitumor activity in PDAC patients; however, intrinsic and acquired resistance will likely preclude durable monotherapy responses in the clinic. Genetic and pharmacologic targeting of the KRAS pathway is associated with transcriptional and metabolic adaptations, including epithelial-to-mesenchymal transition (EMT) and dysregulated iron homeostasis that contribute to resistance. Therapy resistant mesenchymal cancer cells have a high reliance on iron that increases their susceptibility to ferroptosis, an iron-dependent form of cell death characterized by generation of lipid reactive oxygen species (ROS). We hypothesize that KRASi resistance characterized by EMT can be overcome by ferroptosis induction in PDAC. Here, we use in vitro and ex vivo PDAC models and transcriptomic and proteomic profiling to define critical ferroptotic liabilities in KRASi-treated PDAC. First, we evaluated the effect of inhibiting different ferroptosis defense mechanisms (e.g. SLC7A11, GPX4, FSP1) using newly developed ferroptosis inducers (FINs) in a panel of epithelial and mesenchymal PDAC cells. Mesenchymal cells were more sensitive to ferroptosis induction compared to epithelial cells. Since ferroptotic death is regulated by multiple ferroptosis defense pathways, and in general PDAC are relatively resistant to ferroptosis induction, it is unlikely that a single ferroptosis inducer will be successful. Therefore, targeting multiple nodes with FINs in a combinatorial approach may lead to a more pronounced effect. Combination GPX4 and SLC7A11 inhibition and GPX4 and FSP1 inhibition were the most effective combinations compared to respective monotherapies. Next, we evaluated if combination of KRASi and ferroptosis induction is synergistic. Transcriptomic analysis of KRAS-mutant PDAC cell lines sensitive or with a priori resistance to KRASi revealed that EMT-related gene sets were upregulated in KRASi resistant cell lines. Quantitative proteomics revealed that PDAC cells treated long-term with KRASi undergo EMT and likewise long-term KRASi in in vivo PDAC models induces EMT. A KRASi-anchored CRISPR/Cas9 screen showed that sgRNAs targeting GPX4 dropped out in multiple cell lines indicating that ferroptosis induction may be synergistic upon KRAS inhibition. Remarkably, KRASi induced an increase of lipid peroxidation levels in PDAC cells which was rescued with antioxidant ferrostatin-1. We also observed that previously generated KRASi resistant cells were more sensitive to GPX4 inhibition. Combination of GPX4 inhibitor with KRASi led to a synergistic effect on cell viability and a significantly higher level of lipid peroxidation compared to monotherapy. These findings highlight the importance of investigating ferroptosis induction as a novel strategy to overcome therapeutic resistance in KRAS-mutant PDAC. We aim to further evaluate these combinations in vivo to determine the best combinatorial strategies in PDAC to prevent or circumvent KRASi resistance. Citation Format: Aparna Padhye, Qijia Yu, Nicole Sindoni, Huan Zhang, Julien Dilly, Hanrong Feng, Andrew Aguirre, Joseph Mancias. Targeting KRAS inhibitor resistance by ferroptosis induction 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 C018.

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 A059: Understanding the events that promote pancreatic cancer metastasis

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

Abstract A032: Cancer associated fibroblasts drive classical to basal change associated with increased T cell presence in pancreatic ductal adenocarcinoma

Abstract Introduction: A complex immunosuppressive tumor microenvironment (TME) is a distinctive feature of pancreatic ductal adenocarcinoma (PDAC). The TME consists of an admixture of cell types including epithelial, endothelial, immune cells of both myeloid and lymphoid origin, and cancer-associated fibroblasts (CAFs). The traditionally defined subtypes of PDAC tumor cells include classical, associated with the retention of epithelial markers and a better prognosis, and basal, which is characterized by a quasi-mesenchymal phenotype with poorer responses to chemotherapy and worse overall survival. Additionally, CAF subtypes are associated with specific roles within the TME. These include inflammatory (iCAF), with the capacity to secrete IL-6, myofibroblastic (myCAF), defined by high levels of alpha smooth muscle actin (αSMA) expression, and antigen presenting (apCAF), defined by expression of HLA-DR and antigen processing and presentation capacity. We hypothesized that interactions between CAFs and tumor cells altered tumor cell phenotype and could be delineated. Methods: Using multi-omics platforms, we evaluated the impact of CAF populations on tumor cell phenotype. Our approach integrated multiplex immunohistochemistry, imaging mass cytometry (IMC) and RNA-scope of patient tumor tissues with functional in vitro studies from matched patient-derived organoid (PDO) and CAF cocultures. The dynamic impact of CAFs on PDOs was examined using a combination of bulk RNA sequencing, multiparametric flow cytometry and secretome analysis, in addition to RT-qPCR and western blot comparing cocultures to matched PDO monocultures. Results: IMC of 15 patient tumors revealed distinct neighborhoods of CAFs and tumor cells. iCAFs were in close proximity to classical tumor cells, while myCAFs were in close proximity to basal tumor cells. Further, in our PDO-CAF co-cultures the PDOs underwent a shift from classical to basal subtype in 42% of co-cultures, a phenomenon also inferred in patient tissues by associating collagen content with classical or basal designation. We also captured a tumor cell population expressing both basal and classical markers that may represent a transition state. The shift from classical to basal tumor cell subtype was accompanied by enhanced epithelial-mesenchymal transition and a loss of IL-8 secretion. Interestingly, when we examined immune cell distribution within tumor tissues, T cells were both the most proximal and the most abundant immune cell type in relation to tumor cells. Moreover, the highest frequency of total and activated T cells was seen in close association with basal tumor cells. Conclusions: Our study underscores the importance of understanding the complex interplay between CAFs and tumor cells in PDAC. Further, we identify a relationship between T cells and tumor cell subtypes. By leveraging high-dimensional data and patient-derived models, we provide new insights into the spatial and functional dynamics of the PDAC TME, paving the way for novel therapeutic strategies to reprogram the TME and enhance patient survival. Citation Format: Jacquelyn W Zimmerman, Samantha Guinn, Mili Ramani, Brayan Perez, Joseph A Tandurella, Daniel J Zabransky, Jae W Lee, Emma Kartalia, Jignasha Patel, Sarah Shin, Alexei Hernandez, Courtney Cannon, Nicole Gross, Soren Charmsaz, Elana J Fertig, Richard A Burkhart, Won J Ho, Elizabeth M Jaffee. Cancer associated fibroblasts drive classical to basal change associated with increased T cell presence 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 A032.

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

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

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

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

Abstract A078: Functional screen for mediators of onco-mRNA translation specificity in pancreatic cancer

Abstract Oncogenic protein dosage is tightly regulated to enable cancer cells to adapt and survive. In healthy cells, translation initiation of onco-mRNA transcripts encoding oncogenes and growth factors is tightly regulated to maintain accurate protein expression levels. However, this process is broken in cancer cells, which rely on the maintenance of high dosage of oncogenic proteins for their survival and to drive cancer development. Whether this is regulated at the level of translational control and the key factors in cis and trans remain unknown. The Myc oncogene is a central paradigm of an exquisitely regulated oncogene and a major driver of pancreatic ductal adenocarcinoma (PDAC). While there has been a tremendous effort to target Myc in cancer, clinically relevant Myc inhibition remains a challenge. Therefore, identifying selective promoters of MYC translation may present a novel therapeutic opportunity. Employing a genome-wide CRISPRi screen for modulators of MYC mRNA translation initiation in PDAC cells, we identified regulators of selective MYC translation and validated four RNA binding proteins (RBPs), including epitranscriptome modifiers. We focused on a top hit, a little studied RBP, RBM42, which is highly expressed in PDAC and predicts poor patient survival. We found that RBM42 binds and selectively regulates the translation of MYC and a precise, yet vital suite of pro-oncogenic transcripts, including JUN and EGFR. Mechanistically, employing immunoprecipitation-mass spectrometry analysis, we found that RMB42 is a novel ribosome-associated protein (RAP). Through RNA structure determination and mutagenesis analysis, we showed that RBM42 directly binds and remodels the MYC 5’UTR RNA structure, facilitating the formation of the translation pre-initiation complex and increasing MYC translation. Importantly, we demonstrated that RBM42 is necessary for human PDAC cell growth and fitness and PDAC tumorigenesis in xenograft mouse models in a Myc-dependent manner in vivo. In PDAC patient samples, RBM42 expression is correlated with Myc protein levels and transcriptional activity. This work transforms our understanding of the translational code in cancer and offers a new therapeutic opening to target the expression of oncogenes. Citation Format: Joanna R Kovalski, Goksu Sarioglu, Vishvak Subramanyam, Grace Hernandez, Gilles Rademaker, Juan A Oses-Prieto, Macey Slota, Kwun Wah Wen, Grace E Kim, Alma L Burlingame, Hani Goodarzi, Rushika M Perera, Davide Ruggero. Functional screen for mediators of onco-mRNA translation specificity 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 A078.

Abstract B090: Evaluating the molecular determinants of PDAC racial health disparities

Abstract Pancreatic cancer is the 3rd leading cause of cancer-related deaths, with a 5-year survival rate of just 13%. Pancreatic ductal adenocarcinoma (PDAC), accounting for over 90% of pancreatic malignancies, has the highest incidence rate, mortality rate, and shortest survival times in non- Hispanic Black (defined here as African and/or African American ancestry) patients compared to other races. To identify molecular features that may contribute to racial health disparities in PDAC, we used quantitative mass spectrometry to determine the proteomic signatures unique to tumor racial origin in a cohort of 30 Caucasian and 12 African American patients. This analysis of the bulk tumor proteome revealed 183 proteins were differentially expressed between these groups. The protein with the highest expression in tumors from Black patients relative to Whites was Ring Finger Protein 2 (RNF2) that acts as an epigenetic transcriptional repressor in developmental processes pertaining to cellular differentiation. To determine the molecular pathways in PDAC regulated by RNF2, chromatin immunoprecipitation and sequencing (ChIP-seq) was performed on MIA-PaCa2 and a primary PDAC cell line derived from a Black patient to identify binding sites for RNF2 in PDAC cells. From the 1,142 replicated peaks identified, RNF2 peaks were localized over the transcription start site of 644 unique transcripts, including GATA6, which is an important determinant in PDAC subtype delineation with higher GATA6 expression associated with the less aggressive and more chemosensitive Classical subtype. Modulating RNF2 levels/activity by exogenous expression, short hairpin RNA (shRNA) knockdown, and small molecule inhibition confirms that GATA6 is a target of RNF2 repression in PDAC. Multi-omic (transcriptomic, proteomic, and phosphoproteomic) characterization was performed on MIA-PaCa2 cells with CRISPR-Cas9 knockout (KO) of RNF2. Differential gene expression (DE) analysis revealed changes in platinum drug resistance, EMT, and innate immune response, which are characteristic of the aggressive Inflammatory PDAC subtype we previously reported using proteomics. Total and phospho-proteomics corroborated these results identifying changes in DNA repair, inflammation response, TNFα mediated signaling, and apoptotic signaling. These results reflect the enrichment of Inflammatory subtype markers in tumors originating in Black patients. In agreement with these results, RNF2 KO shows a significant reduction in migration, as well as an increase in sensitivity to oxaliplatin. Our work identifies differences in the underlying proteome of PDAC tumors associated with tumor racial origin and describes how elevated expression of RNF2 in PDACs from Black patients contribute to inflammation and subtype delineation. This study delineates the associations between tumor racial origin, subtype specification, and response to therapy that could mitigate PDAC health disparities and be broadly applied across all PDAC patients. Citation Format: Anthony E Johansen-Sallee, Alexa M Barber, Henry C.-H. Law, Vignesh Vudatha, Jose Trevino, Nicholas T Woods. Evaluating the molecular determinants of PDAC racial health disparities [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 B090.

Abstract C012: Overcoming Cell Subtype-Specific Adaptive Resistance to KRAS G12D Inhibition in KRAS G12D-Mutant Pancreatic Cancer

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) has a 5-year survival rate of only 12%, with limited treatment options. Oncogenic KRAS mutations drive PDAC tumor progression, which occur in the majority of PDAC tumors. KRAS G12D is the most common KRAS mutation, found in 36% of PDAC cases, which is associated with the worst clinical outcomes. Mutant-selective KRAS G12D inhibitors (KRASi G12D), such as MRTX1133, have recently demonstrated initial responses in KRAS G12D-mutant preclinical models. However, mechanisms of adaptive resistance to KRAS G12D inhibition are largely under-investigated and novel therapeutic strategies to overcome drug resistance are urgently needed. Results: Adaptive RAS signaling reactivation was found to dampen KRAS G12D inhibition in a library of KRAS G12D PDAC cell lines, thereby minimizing drug efficacy. Quantitative downstream analyses of drug combination screening using a KRAS G12D cell line library further identified this adaption to KRAS inhibition was cell subtype specific. Fibroblast growth factor receptor (FGFRs) drove adaptive RAS reactivation specifically in mesenchymal PDAC cells, which are aggressive, and intrinsically resistant to KRASi. In contrast, epithelial growth factor receptor (EGFR) reactivated RAS G12D signaling in epithelial cells, which are sensitive to KRASi G12D. Genetic and pharmacological inhibition of FGFR1 or FGFR2 improved drug efficacy by decreasing FGFR-mediated RAS reactivation specifically in mesenchymal PDAC lines. Pharmacological blockade of EGFR enhanced drug efficacy by inhibiting EGFR-mediated RAS reactivation specifically in epithelial PDAC lines. Furthermore, low-concentration of a novel pan- RAS inhibitor, which inhibits both wild-type and mutant RAS, completely abrogated MAPK signaling and significantly improved efficacy, when combined with KRASi G12D, suggesting a pro-survival dependency on wild-type RAS as a compensatory mechanism in response to KRAS G12D inhibition. Conclusion: Upstream RTK-mediated adaptive RAS signaling re-activation in response to KRAS inhibition in PDAC is cell subtype specific. Complete inhibition of RAS signaling by overcoming adaptive RAS rebound improves efficacy of KRASi G12D. Citation Format: Qingxiang (Nick) Lin, Alvin Morales, Haley Barnes, Ryan Bruce Corcoran. Overcoming Cell Subtype-Specific Adaptive Resistance to KRAS G12D Inhibition in KRAS G12D-Mutant 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 C012.