Pancreatic Ductal Adenocarcinoma Cancer Research Articles

Abstract 4271: The ITGA2 (CD49b) collagen receptor excludes the CD8+ T cell infiltration in pancreatic ductal adenocarcinoma

Abstract BACKGROUND: Pancreatic ductal adenocarcinoma cancer (PDAC) is characterized by an enriched extracellular matrix and limited T cell infiltration, which poses challenges for immunotherapy. The significant accumulation of collagen type I (COL1) in PDAC tumor microenvironment raises questions about its functional role in PDAC progression and its interactions with the cellular compartments. OBJECTIVE: The objective of this study was to investigate whether the collagen receptors expressed by cancer cells modulate the anti-tumor activity of CD8+ tumor-infiltrated lymphocytes (CD8+ TILs).METHODS: Collagen receptor expression patterns in PDAC were examined using flow cytometry, western blot, multiplex immunofluorescence, RNA seq, and scRNA seq databases in KPC and treatment-naïve PDAC that were resected from patients. The KPC cell line, known as Hy15549, was established from KPC mouse model (Ptf1-Cre; LSL-KRAS-G12D; Trp53 Lox/+). Using CRISPR-Cas9, we conducted targeted deletions for either ITGA2(CD49b) or ITGB1(CD29), as well as generating double knockouts for both CD49b and CD29. Growth of these cell lines implanted into the flanks of immunocompetent, immunodeficient mice was measured, as well as after CD8+ T cell depletion in immunocompetent mice. We analyzed TILs using FACs. We used multiplex immunofluorescence to analyze tumors microenvironment including collagen fiber alignment. Downstream signaling pathways were assessed using RNA seq and western blot. RESULTS: CD49b is highly expressed specifically in PDAC cancer cells, as assessed in both KPC cell flank tumors and resected human PDAC. Using adhesion assay, we found that CD49b specifically binds to Col1 rather than ColIV. KPC CD29b KO exhibited a complete loss of adhesion to both Col1 and ColIV. Tumor growth of the KPC CD49b KO, KPC CD29 KO, KPC CD49b CD29 KO was observed to be significantly slower than KPC WT despite identical in vitro growth kinetics. Knockout of CD49b in tumors promoted intratumoral infiltration of CD8+ T cells and significantly impeded tumor growth. Tumor growth by KPCCD49bKO was rescued by depletion of CD8+ T cells. In contrast to observations in immune-competent mice, KPCCD49bKO tumor growth was similar to KPCwt in immunodeficient nude mice. Analysis of downstream signaling in KPCCD49bKO tumors revealed a decrease of FAK as well as pFAk expression pathway. The proteomic profiling of KPCCD49bKO revealed an increase of CXCL10 chemokine. Moreover, In the absence of CD49b, the orientation of collagen fiber alignment appeared more scattered, with thinner fibers. This altered alignment could potentially contribute to immune cell infiltration.Conclusion:These findings reveal a previously unreported function of CD49b in PDAC cancer progression and the immune response within the TME. Together, our study reveals a mechanism underlying immune exclusion and suggests a novel immunotherapeutic target in PDAC. Citation Format: Ibrahim Ragab Eissa, Eric D. Abston, Darshini Kuruppu, Yongtao Wang, Guoliang Qiao, Motaz Qadan, Michael Lanuti, Kenneth K. Tanabe. The ITGA2 (CD49b) collagen receptor excludes the CD8+ T cell infiltration in pancreatic ductal adenocarcinoma [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 4271.

Abstract 7291: Detection of a truncated form of multidrug resistance protein 1 (MRP1) in plasma sEVs derived from cancer patients

Abstract Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP dependent transmembrane efflux pump that confers to drug resistance. Overexpression of MRP1 has been observed in both pancreatic ductal adenocarcinoma (PDAC) and breast cancer tissues. Numerous proteomics studies have revealed the presence of MRP1 in small extracellular vesicles (sEVs) released from cancer cells. Cancer-derived sEVs are known to be released into the circulation, thus becoming attractive candidates for non-invasive biomarker development. However, whether the MRP1 level in plasma sEVs is indicative of human cancer has not been evaluated. This study examined the MRP1 expression in plasma sEVs derived from patients with PDAC, breast and colon cancer, and age- and gender-matched healthy subjects. The patient plasma samples were obtained primarily from the NCI-sponsored Cooperative Human Tissue Network and Stephenson Cancer Center. Healthy plasma samples were provided by the Oklahoma Blood institute. Human pancreatic cancer cell lines PANC-1, MIA PaCa-2 and BxPC-3, and the pancreatic ductal cell line hTERT-HPNE were tested for MRP1 expression prior to examination of plasma samples. Cellular and plasma sEVs were isolated using the double filtration followed by polymer precipitation method. Western blot was performed to assess the expression level of MRP1 and proteomics was applied to confirm the detection of a truncated form of MRP1 in plasma sEVs. MRP1 expression was detected in the sEVs derived from all three pancreatic cancer cell lines but absent in the sEVs from the HPNE line. Overexpression of a truncated MRP1 protein was detected in plasma sEVs derived from patients with early stage (stage I-IIA) and late stage (III-IV) PDAC, compared with that from matched healthy subjects (n=17 for each group). Similar findings were evident in plasma sEVs from patients with breast cancer (n=7), while the results remained inconclusive in plasma sEVs from patients with colon cancer (n=7). Protein structure analysis indicated that the truncated form of MRP1 likely consists of the regions between the QCRL-1 motif and the C-terminus of the protein. Proteomic analysis detected the peptides derived from the C-terminus region, consistent with the structural analysis. Furthermore, the truncated form of MRP1 can only be detected using antibodies against the epitopes in the C-terminus and QCRL-1 region of MRP1 protein. In summary, we have detected a truncated form of MRP1 protein in plasma sEVs derived from cancer patients, and the level of its expression is significantly elevated in patients with PDAC and breast cancer. Citation Format: Kritisha Bhandari, Jeng Shi Kong, Chao Xu, Ajay Jain, Wei-Qun Ding. Detection of a truncated form of multidrug resistance protein 1 (MRP1) in plasma sEVs derived from cancer patients [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 7291.

Abstract 2118: SGN-CEACAM5C/SAR445953, a novel topoisomerase I inhibitor antibody-drug conjugate targeting CEACAM5, has potent anti-tumor activity in CRC, PDAC, GC and lung cancer tumor models

Abstract Carcinoembryonic antigen cell adhesion molecule 5, CEACAM5, is a glycosylphosphatidylinositol-anchored glycoprotein expressed on the cell surface of most of the colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC) and of more than 2/3 of gastric cancer (GC) and non-small cell lung cancer (NSCLC) while normal tissue expression is limited. The high prevalence of CEACAM5 expression prompted us to develop a novel CEACAM5 topoisomerase I inhibitor (Topo1i) antibody drug conjugate (ADC) with a DAR of 8, SGN-CEACAM5C/SAR445953. The anti-CEACAM5 antibody was chosen based on its high selectivity for CEACAM5 and its potential to direct cytotoxic payloads to tumor. The Topo1i payload was optimized for potency and enhanced bystander activity. SGN-CEACAM5C/SAR445953 is rapidly internalized and demonstrates in vitro cytotoxicity with EC50 values in the sub-nM range while it induces no toxicity on CEACAM5-negative cells. The potent anti-tumor activity is mediated by direct cytotoxicity on CEACAM5-expressing tumor cells and by a strong bystander effect due to the diffusion of the payload to the neighboring CEACAM5-negative tumor cells. Accordingly, the ADC incubated in a co-culture of CEACAM5-positive and -negative cells at a ratio of 1/1 induces a ~50% growth inhibition of CEACAM5-negative cells. Interestingly, with incubated ADC, only 1% of CEACAM5-positive cells in co-culture are sufficient to provide 11% growth inhibition of CEACAM5-negative cells. In vivo, SGN-CEACAM5C/SAR445953 is stable in circulation in SCID mice with a t1/2 close to 15 days. In vivo efficacy at 1, 3 and 10 mg/kg (single administration) was evaluated in panels of 4 CRC, 3 PDAC, 4 NSCLC and 3 GC patient-derived xenograft (PDX) models. At 10 mg/kg, the ADC elicits antitumor regression in 14/14 models. At 3 mg/kg, tumor regression occurs in 12/14 models. This potent, specific and dose dependent anti-tumor activity was further confirmed in Single Mouse Trials (SMT) of 20 CRC PDX models, 31 lung cancer PDX models and 19 gastric cancer PDX models. SMT consists in use of one animal per PDX model per treatment arm and for which the evaluation of efficacy is based on RECIST (Response Evaluation Criteria In Solid Tumors) criteria used in clinic. In CRC, gastric and lung cancer SMT, disease control rates are 95%, 84% and 87%, respectively with overall response rates of 55%, 68% and 71% including 15%, 10% and 26% of complete responses, respectively. The high anti-tumor activity across panels of PDX models of several CEACAM5 positive indications supports further evaluation of SGN-CEACAM5C/SAR445953 in patients with CRC, PDAC, GC and lung cancers (NCT06131840). Citation Format: Yves Baudat, Celine Nicolazzi, Johann Petur Sigurjonsson, Celine Amara, Astrid Clarke, Ryan lyski, Dave Meyer, Valeria Fantin, Marielle Chiron, Stephanie Decary. SGN-CEACAM5C/SAR445953, a novel topoisomerase I inhibitor antibody-drug conjugate targeting CEACAM5, has potent anti-tumor activity in CRC, PDAC, GC and lung cancer tumor models [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 2118.

Nanodrug-bacteria conjugates-mediated oncogenic collagen depletion enhances immune checkpoint blockade therapy against pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) cancer cells specifically produce abnormal oncogenic collagen to bind with integrin α3β1 receptor and activate the downstream focal adhesion kinase (FAK), protein kinase B (AKT), and mitogen-activated protein kinase (MAPK) signaling pathway. Collectively, this promotes immunosuppression and tumor proliferation and restricts the response rate of clinical cancer immunotherapies. Here, by leveraging the hypoxia tropism and excellent motility of the probiotic Escherichia coli strain Nissle 1917 (ECN), we developed nanodrug-bacteria conjugates to penetrate the extracellular matrix (ECM) and shuttle the surface-conjugated protein cages composed of collagenases and anti-programmed death-ligand 1 (PD-L1) antibodies to PDAC tumor parenchyma. We found the oncogenic collagen expression in human pancreatic cancer patients and demonstrated its interaction with integrin α3β1. We proved that reactive oxygen species (ROS) in the microenvironment of PDAC triggered collagenase release to degrade oncogenic collagen and block integrin α3β1-FAK signaling pathway, thus overcoming the immunosuppression and synergizing with anti-PD-L1 immunotherapy. Collectively, our study highlights the significance of oncogenic collagen in PDAC immunotherapy, and consequently, we developed a therapeutic strategy that can deplete oncogenic collagen to synergize with immune checkpoint blockade for enhanced PDAC treatment efficacy. This work was supported by the University of Wisconsin Carbone Cancer Center Research Collaborative and Pancreas Cancer Research Task Force, UWCCC Transdisciplinary Cancer Immunology-Immunotherapy Pilot Project, and the start-up package from the University of Wisconsin-Madison (to Q.H.).

Abstract 6247: Comprehensive characterization of cancers with epithelial mesenchymal transition phenotype through long-read sequencing

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) and linitis-plastica type gastric cancer (STAD) are the most representative refractory cancers even in the era of molecularly targeted therapy and immunotherapy. Extensive characterization of these tumors, especially regarding the genomic and epigenomic characteristics of pure cancer cells, has been hampered by the high stromal content in the primary tumor samples. Here we took advantage of cancer cells purified from malignant ascites and established corresponding cell lines to overcome the limitations associated with the analysis and to discover their vulnerabilities. Methods: We developed cell lines from malignant ascitic fluid samples obtained from 60 patients with PDAC and 59 patients with linitis-plastica type STAD. We performed comprehensive multi-omic analyses of these cell lines using long-read whole-genome sequencing (WGS) for complex somatic genomic alterations and for methylation aberration. We also conducted long-read RNA sequencing (iso-seq) for full-length isoform exploration, and chromatin immunoprecipitation coupled with sequencing (ChIP-seq) for enhancer analysis. Complementary short-read whole-genome sequencing and RNA sequencing were also performed. Results: Comprehensive transcriptome analyses identified a cluster with high epithelial-mesenchymal transition (EMT) characteristics, consisting of the majority of the PDAC cell lines and a half of the STAD cell lines. ChIP-seq analysis against H3K27ac revealed the distinct transcriptional circuit that generated EMT cluster-specific super-enhancers governed by SMAD3, independent of cancer type. Most of the transcriptional activity was controlled by the gene methylation. We also identified the differential expression levels of the splicing factors between the EMT-activated and the non-activated groups. Comprehensive isoform analysis by iso-seq revealed that the isoform usage was completely different between the EMT group and the other, especially in genes of the actomyosin pathway which confer characteristic features of the EMT cluster in the cell morphology and cell motility. Using the CHM13 reference genome, long-read WGS analyses revealed the recurrent structural alterations specific to the EMT group such as those involving CDKN2A. We also identified PDAC-specific structural alterations. Conclusion: We identified the distinct molecular characteristics of the EMT-activated group in PDAC and linitis-plastica type STAD which may confer the therapeutic resistance. Our findings provide an interesting insight for the further innovation of the treatment strategy for these intractable cancers. Citation Format: Yosuke Tanaka, Hiroyuki Mano. Comprehensive characterization of cancers with epithelial mesenchymal transition phenotype through long-read sequencing [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 6247.

Abstract 2093: Co-treatment with KRAS G12D inhibitor MRTX1133 plus TTFields against human pancreatic and Colorectal cancer cell lines results in synergistic up-regulation of cleaved PARP in KRAS G12D & unexpectedly in KRAS G12V as well

Abstract Background: Both colorectal and pancreatic cancers are major global health issues and among deadliest cancers with great disease burden. The most commonly mutated oncogenic alteration in human cancers is KRAS, which is prevalent in pancreatic malignancies. The KRAS G12D mutation subtype is present in more than 40% of pancreatic ductal adenocarcinoma (PDAC). To this date there is no available targeted therapy options for patients with KRAS subtype mutations. MRTX1133 has been identified as a non-covalent, potent, and selective inhibitor of KRASG12D. This small molecule inhibitor has been shown to suppress KRASG12D signaling in cells and in vivo. Tumor Treating Fields (TTFields) therapy is a novel approach to treating cancer, the electric fields alter the behavior of cancer cells and prevent them from growing and dividing. We hypothesize that the co-application of MRTX1133 with TTFields will enhance the effects of MRTX1133 in PDAC and colorectal cancer (CRC). Martials and Methods: PDAC cell line CFPAC1 with KRAS G12 V mutation and LS513 CRC cell line with KRAS G12D Mutation were treated with same dose of MRTX1133 (500 nM) and were co-treated with 150kHz TTFields. After 48 hours western blot was used to probe for cleaved PARP, cleaved C3, phosphor-ERK and DUSP6. Results: In both KRAS G12D & KRAS G12V cell lines, synergistic upregulation of cPARP was observed following 48-hour co-treatment. Synergistic inhibition of pERK happened in KRAS G12D cell line but no change in pERK level following TTFields or MRTX1133 treatment was seen. Upregulation of cC3 in KRAS G12D happened in only co-treatment conditions. DUSP6 levels increased following MRTX1133 treatment in KRAS G12V cell line. Conclusions: The surprising synergistic upregulation of cPARP in both KRAS G12V and G12D cells following co-treatment of KRAS G12D inhibitor MRTX1133 and TTFields can bring hope for other KRAS mutation subtypes as well as G12D. the increase in cPARP in KRAS G12V with no effect on pERK is particularly interesting and demonstrates the need of more studies to investigate the mechanisms of this synergy. Citation Format: Vida Tajiknia, Praveen Srinivasan, Maximilian PInho-Schwermann, William MacDonald, Connor Purcell, Wafik El-Deiry. Co-treatment with KRAS G12D inhibitor MRTX1133 plus TTFields against human pancreatic and Colorectal cancer cell lines results in synergistic up-regulation of cleaved PARP in KRAS G12D & unexpectedly in KRAS G12V as well [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 2093.

Abstract 1021: Defining total protease activity profiles in pancreatic cancer using a FRET substrate array

Abstract Proteases are an important component of many biological processes in the human body, and as such makes them a well-known subject to explore when investigating a variety of disease states from cancer to cardiovascular disease. In all stages of cancer, including early development, proteases play critical roles in almost every aspect for processes such as invasion, promotion of tumor growth, and metastasis. This makes screening of circulating proteases a potentially powerful tool for early detection and prognosis for a variety of cancers. The human proteome, however, is very complex, with around 600 proteases in total, and at least 234 of them found in circulation. Additionally, proteases often act on more than one substrate, cross reactively cleaving several substrates at different rates, further adding to the complexity of cleavage patterns of the human proteome. Previous liquid biopsies for cancer detection based on protease cleavage patterns focused mainly on detecting the activities of specific proteases, using single substrates to examine the effects of well-studied cancer-related proteases such as MMPs and cathepsins. In this study, we developed a method for screening cleavage patterns of proteases active in plasma samples that deviated from this norm. Due to the complexity of the human proteome, rather than developing a specific substrate for a specific protease, we utilized a large array of substrates to screen for cleavage patterns in plasma samples of cancer patients aimed at not one specific protease, but a wide range that may be present. In this proof of concept study, we used a commercially available internally quenched set of 360 substrates composed of 8 amino acids and a FRET pair. Initially, we optimized this assay to measure protease activity in the complex plasma environment. Next, we screened several plasma samples collected from patients with pancreatic ductal adenocarcinoma (PDAC) or pancreatitis, and screen negative samples using the 360 substrate set assay and identified 5 top substrates that were proficient in identifying PDAC from other conditions. Finally, we performed a validation study with a larger cohort (45 screen negative, 26 PDAC, 19 pancreatitis) using the selected substrates to determine the diagnostic performance of the assay. All five of the selected substrates showed 1.3-1.8-fold increase in fluorescence signal in PDAC compared to screen negative and pancreatitis. Amongst the cancer samples, the substrates showed no significant difference between stage, supporting possible applications for early detection. Additionally, area under the curve analysis of the five substrates gave a score of 0.8. In summary, in this study, we reported a novel approach for the detection of cancer exploiting the protease cleavage patterns in plasma samples. We believe that this approach holds great potential for detecting substrates and proteases for early detection of PDAC and other cancers. Citation Format: Morgan Stewart, Arnaud Quentel, Jose Montoya Mira, Elise Manalo-Hall, Srivathsan Ranganathan, Bruce Branchaud, Fehmi Civitci, Eugene Tu, Jared Fischer, Yu-Jui Chiu, Adem Yildirim. Defining total protease activity profiles in pancreatic cancer using a FRET substrate array [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 1021.

Abstract 1597: CD144+cancer-associated fibroblasts drive the malignancy of pancreatic cancer via stimulating inflammatory paracrine

Abstract Purpose: Cancer-associated fibroblasts (CAFs) are highly heterogeneous in pancreatic ductal adenocarcinoma (PDAC). We previously reported that CAFs can acquire an endothelial phenotype under stress. Thus, we hypothesized that there are CAFs highly expressing endothelial markers and substantiated a novel subtype of CAFs promoting tumor progression. Experimental design: We performed single-cell RNA sequencing to identify the existence of CD144+CAFs. The prospective and retrospective analysis were combined to figure out the correlation between CD144+CAFs proportion and clinical outcome. The proliferation and invasion assay of cell lines, organoids and pancreatic cancer xenograft model were used to detect the effect of CD144+CAFs on tumor progression. Cytokine array assay, RNA-sequencing, IP-mass spectrum, Ch-IP and luciferase analysis were conducted to elucidate the underlying mechanism. siRNA delivery nanosystem was designed and administrated to precisely target CD144+CAFs in vivo. Results: CD144+CAFs were present in tumor microenvironment of PDAC and breast cancer and patients with a higher CD144+CAFs proportion have worse prognosis in PDAC. CD144+CAFs can promote the metastasis of PDAC through enhancing epithelial-mesenchymal transition. CD144-β-catenin-STAT3 signaling axis was activated and downstream inflammatory secreted cytokines were transcriptionally upregulated to maintain the functions and phenotype of CD144+CAFs. A novel CAF-targeting siRNA delivery nanosystem, loading FAPα and siCDH5, was designed and administrated to precisely target CD144+CAFs, which substantially inhibited the protumoral roles in vivo. Conclusion: CD144+CAFs can promote the growth and metastasis of PDAC by activating CD144-β-catenin-STAT3 signaling axis and stimulating inflammatory secretion. CAF-targeting siRNA delivery nanosystem can inhibit tumor progression by precisely targeting CD144+CAFs. Citation Format: Xugang Sun, Jun Yu, Tiansuo Zhao, Jihui Hao. CD144+cancer-associated fibroblasts drive the malignancy of pancreatic cancer via stimulating inflammatory paracrine [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 1597.

Abstract 4574: Next generation nuclear export blocker KPT8602 synergizes with KRASG12D inhibitor MRTX1133 resulting in improved antitumor effects against pancreatic ductal adenocarcinoma

Abstract Introduction: A novel KRAS inhibitor, MRTX1133, specifically targeting the G12D isoform, prevalent in pancreatic ductal adenocarcinoma (PDAC), holds promise for a substantial PDAC patient population. Nevertheless, given the clinical experience with sotorasib (a KRASG12C inhibitor) in lung cancer, it's anticipated that resistance to the KRASG12D inhibitor will eventually emerge in most PDAC patients. This underscores the need to identify strategies that can enhance the efficacy of KRAS inhibitors in PDAC. Emerging evidence suggests that the nuclear export protein exportin 1 (XPO1) is a druggable vulnerability in KRAS mutant cancers. XPO1 plays a pivotal role in shuttling various critical tumor suppressors, genome surveillance proteins, and transcription factors out of the nucleus and is frequently overexpressed in various cancers, including PDAC. In this study, we employed both in vitro and in vivo models to assess the potential of KPT8602, an investigational and next-generation selective XPO1 inhibitor, to synergize with the KRASG12D inhibitor MRTX1133 and sensitize MRTX1133-resistant PDAC cells. Methods: We examined the cytotoxic and molecular effects of KRASG12D inhibitor MRTX1133 in combination with nuclear export blocker KPT8602 in KRASG12D mutant 2D and 3D cellular/spheroid models. The preclinical antitumor efficacy of the combination was evaluated in a KRASG12D mutant PDAC cell derived xenograft (CDX) model. Results: KPT8602 synergized with MRTX1133 in vitro yielding suppressed proliferation of KRASG12D mutant PDAC cells. Similar synergistic effects were also seen in 3D cultures of KRASG12D mutant PDAC cancer cell lines and patient derived primary tumor cells where the combination suppressed spheroid formation and reduced spheroid viability. Also, the combination inhibited the clonogenic potential of KRASG12D mutant PDAC cells. In addition, we developed a PDAC cell line resistant to MRTX1133 and observed that such MRTX1133-resistant cell line was acutely sensitive to treatment with KPT8602. Mechanistically, KPT8602 and MRTX1133 combination led to reduction in protein expression of KRAS downstream effectors and cell cycle markers. Furthermore, a combined administration of KPT8602 and MRTX1133 at sub-optimal doses resulted in remarkable tumor growth inhibition in a KRASG12D mutant HPAC CDX mice model. Moreover, KPT8602 as a maintenance therapy also prevented tumor relapse. Conclusion: This is the first study demonstrating that nuclear export blocker KPT8602 can synergize with KRASG12D inhibitor MRTX1133 and has the potential to target MRTX1133-resistant PDAC cells. This study provides a rationale for combining MRTX1133 with KPT8602 for the treatment of PDAC patients with KRASG12D mutant tumors. Citation Format: Husain Yar Khan, Amro Aboukameel, Md Hafiz Uddin, Sahar F. Bannoura, Khaled Keffri, Medha Jasti, Rachael Virga, Sarah Motorwala, Khalil Choucair, Tarik Hadid, Mohammad Najeeb Al-Hallak, Eliza Beal, Miguel Tobon, Rafic Beydoun, Greg Dyson, Steve Kim, Misako Nagasaka, Philip A. Philip, Basel El-Rayes, Herbert Chen, Anthony Shields, Ramzi Mohammad, Boris Pasche, Asfar Azmi. Next generation nuclear export blocker KPT8602 synergizes with KRASG12D inhibitor MRTX1133 resulting in improved antitumor effects against pancreatic ductal adenocarcinoma [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 4574.

Abstract 6167: Spatial transcriptomic analysis identifies pancreatic cancer cell surface markers for imaging and therapy

Abstract Pancreatic cysts and precancer lesions have been reported in up to 50% of adults; however, few of the precancer lesions develop into aggressive pancreatic ductal adenocarcinoma (PDAC). Strategies for the differentiation of precancers and PDAC are needed. Recently proposed molecular imaging targets to accomplish this goal include integrin αvβ6 and fibroblast activation protein (FAP). To discover and quantitatively evaluate potential imaging and therapeutic targets, we performed Visium spatial transcriptomic (ST) sequencing on human surgical sections of normal tissue adjacent to precancers, intraductal papillary mucinous neoplasms and PDAC. All human tissues were collected in accordance with the Stanford Institutional Review Board. The resulting data were correlated with immunohistochemistry (IHC) and combined with analysis with publicly available spatial and single cell PDAC datasets to create a quantitative PDAC cancer surfaceome score. Pseudotime and receiver operating characteristic (ROC) analyses were also performed. We found that clusters with high cancer surfacesome scores were spatially correlated with a cellular morphology consistent with cancer. In the cancer cluster, the expression of surface markers CLDN4, GPRC5A, TSPAN8, and CEACAM5 was enhanced more than 13-fold compared with the normal pancreas, while FAP and ITGB6 were differentially expressed by less than 4-fold. Within cancer clusters, the fraction of spots overexpressing CLDN4, S100P, ITGB6, MUC5AC, TFF1, CEACAM5, and FAP was 88%, 87%, 78%, 66%, 59%, 47%, and 38%, respectively. The Pearson’s correlation across space and expression intensity between CLDN4, S100P, GPRC5A, TSPAN8, MUC1 and TFF1 was greater than 0.6 for all combinations. The correlation of mRNA encoding ITGB6 and FAP with cancer surfaceome markers was smaller, e.g. 0.37 and 0.01 versus S100P, respectively. The area under the curve (AUC) for the differentiation of PDAC from normal or precancerous tissue for CLDN4, S100P, MUC5AC, TFF1, CEACAM5, and TSPAN8 was 0.9, 0.5, 0.3, 0.5, 0.8, and 0.4, respectively. From pseudotime trajectory analysis, we found that MUC5AC and TFF1 increased at a precancerous pseudotime whereas CLDN4 increased in time and space in spots corresponding to a PDAC-specific surfaceome score. This work demonstrates the feasibility of using spatial transcriptomics and IHC in human pancreatic cancer samples to discover and compare the expression of cell surface markers that can be employed in imaging and therapeutic protocols. Citation Format: James Wang, Aris J. Kare, Martn K. Schneider, Jai Woong Seo, Andrei Iagaru, Gregory W. Charville, Walter G. Park, Katherine W. Ferrara. Spatial transcriptomic analysis identifies pancreatic cancer cell surface markers for imaging and therapy [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 6167.

Abstract 1583: Neutrophil pro-tumorigenic role in progression and therapy resistance in pancreatic ductal adenocarcinoma

Abstract Pancreatic cancer (PC) continues to be a challenge for today’s therapeutics, with a meager 5-year survival rate of 12%. Due to the pancreas’ location, patients often receive diagnosis late, leaving them with limited treatment options. Often, they fail to respond to chemotherapy regimens or develop resistance to those therapies. Tumor-promoting inflammation is a hallmark of cancer, contributing to tumor cells’ survival and proliferation. Infiltrating leukocytes and pro-inflammatory cytokines released into the tumor microenvironment (TME) often cause this inflammation. My lab has previously linked increases in pancreatic ductal adenocarcinoma (PDAC) progression and chemotherapy resistance to inflammation and the CXCR2 pathway. In the present study, we examined the role of neutrophil recruitment in PDAC progression using patient samples and KRAS-dependent KC and KPC murine models. Our data suggest a positive association between tumor-associated neutrophils (TANs) and PDAC progression. In vitro, we observed neutrophil-PDAC interaction enhancing the survival of both neutrophils and PDAC cell lines. Moreover, this survival was mediated by cellular aggressiveness and therapy resistance. Concerning survival, we saw increased expression of anti-apoptotic genes in the neutrophils upon PDAC cancer cell conditioned media treatment. We also observed modulated expression of immunoregulatory molecules. Our data suggest that increased recruitment of TANs supports PDAC progression and survival, and PDAC cell-neutrophil interaction modulates neutrophil survival and immunoregulatory phenotype. Citation Format: Reegan Sturgeon, Esther Johnson, Caitlin Molczyk, Paran Goel, Parker Tinsley, Lauren Abrahams, Michael Maher, Rakesh K. Singh. Neutrophil pro-tumorigenic role in progression and therapy resistance in pancreatic ductal adenocarcinoma [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 1583.

Auranofin repurposing for lung and pancreatic cancer: low CA12 expression as a marker of sensitivity in patient-derived organoids, with potentiated efficacy by AKT inhibition

BackgroundThis study explores the repurposing of Auranofin (AF), an anti-rheumatic drug, for treating non-small cell lung cancer (NSCLC) adenocarcinoma and pancreatic ductal adenocarcinoma (PDAC). Drug repurposing in oncology offers a cost-effective and time-efficient approach to developing new cancer therapies. Our research focuses on evaluating AF's selective cytotoxicity against cancer cells, identifying RNAseq-based biomarkers to predict AF response, and finding the most effective co-therapeutic agents for combination with AF.MethodsOur investigation employed a comprehensive drug screening of AF in combination with eleven anticancer agents in cancerous PDAC and NSCLC patient-derived organoids (n = 7), and non-cancerous pulmonary organoids (n = 2). Additionally, we conducted RNA sequencing to identify potential biomarkers for AF sensitivity and experimented with various drug combinations to optimize AF's therapeutic efficacy.ResultsThe results revealed that AF demonstrates a preferential cytotoxic effect on NSCLC and PDAC cancer cells at clinically relevant concentrations below 1 µM, sparing normal epithelial cells. We identified Carbonic Anhydrase 12 (CA12) as a significant RNAseq-based biomarker, closely associated with the NF-κB survival signaling pathway, which is crucial in cancer cell response to oxidative stress. Our findings suggest that cancer cells with low CA12 expression are more susceptible to AF treatment. Furthermore, the combination of AF with the AKT inhibitor MK2206 was found to be particularly effective, exhibiting potent and selective cytotoxic synergy, especially in tumor organoid models classified as intermediate responders to AF, without adverse effects on healthy organoids.ConclusionOur research offers valuable insights into the use of AF for treating NSCLC and PDAC. It highlights AF's cancer cell selectivity, establishes CA12 as a predictive biomarker for AF sensitivity, and underscores the enhanced efficacy of AF when combined with MK2206 and other therapeutics. These findings pave the way for further exploration of AF in cancer treatment, particularly in identifying patient populations most likely to benefit from its use and in optimizing combination therapies for improved patient outcomes.

Xevinapant Combined with Pembrolizumab in Patients with Advanced, Pretreated, Colorectal and Pancreatic Cancer: Results of the Phase Ib/II CATRIPCA Trial.

Xevinapant is an orally available inhibitor of apoptosis proteins (IAP) inhibitor. Preclinical data suggest that IAP antagonism may synergize with immune checkpoint blockers by modulating the NFκB pathway in immune cells. Adult patients with non-high microsatellite instability advanced/metastatic pancreatic ductal adenocarcinoma (PDAC) or colorectal cancer were enrolled in this phase Ib/II study and received pembrolizumab 200 mg every 3 weeks intravenously, and ascending doses of oral xevinapant (100, 150, and 200 mg daily for 14 days on/7 days off). Dose escalation followed a 3+3 design with a 21-day dose-limiting toxicity (DLT) evaluation period. Following the determination of the recommended phase II dose (RP2D), 14 patients with PDAC and 14 patients with colorectal cancer were enrolled in expansion cohorts to assess preliminary efficacy. Forty-one patients (26 males) with a median age of 64 years were enrolled: 13 in the dose escalation and 28 in the two expansion cohorts. No DLT was observed during dose escalation. The RP2D was identified as xevinapant 200 mg/day + pembrolizumab 200 mg every 3 weeks. The most common adverse events (AE) were fatigue (37%), gastrointestinal AE (decreased appetite in 37%, nausea in 24%, stomatitis in 12%, and diarrhea and vomiting in 10% each), and cutaneous AE (pruritus, dry skin, and rash seen in 20%, 15%, and 15% of patients, respectively). The best overall response according to RECIST1.1 was partial response (confirmed) in 1 (3%), stable disease in 4 (10%), and progressive disease in 35 (88%). Xevinapant combined with pembrolizumab was well tolerated with no unexpected AEs. However, antitumor activity was low.

Risk perception and surveillance uptake in individuals at increased risk for pancreatic ductal adenocarcinoma

BackgroundSurveillance for pancreatic ductal adenocarcinoma (PDAC) is recommended for high-risk individuals with genetic variants in PDAC-associated genes and/or family history. Surveillance uptake and adherence may depend on the perception of...

Exploring the therapeutic potential of focal adhesion kinase inhibition in overcoming chemoresistance in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is among the leading causes of cancer-related deaths worldwide. Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase often overexpressed in PDAC. FAK has been linked to cell migration, survival, proliferation, angiogenesis and adhesion. This review first highlights the chemoresistant nature of PDAC. Second, the role of FAK in PDAC cancer progression and resistance is carefully described. Additionally, it discusses recent developments of FAK inhibitors as valuable drugs in the treatment of PDAC, with a focus on diamine-substituted-2,4-pyrimidine-based compounds, which represent the most potent class of FAK inhibitors in clinical trials for the treatment of PDAC disease. To conclude, relevant computational studies performed on FAK inhibitors are reported to highlight the key structural features required for interaction with the protein, with the aim of optimizing this novel targeted therapy.

Recurrence and Survival Prediction Models for Receiving Preoperative Chemotherapy Pancreatic Ductal Adenocarcinoma Cancer: A Single-center Retrospective Research

Purpose: Regard preoperative chemotherapy as a holistic sequential treatment modality to identify predictive factors associated with recurrence and survival in pancreatic ductal adenocarcinoma (PDAC) and construct models to provide decision support and treatment selection for the candidate patients. Methods: Retrospect PDAC patients who received preoperative chemotherapy from January 2016 to June 2023 at the Second Affiliated Hospital of Zhejiang University School of Medicine. Run a univariate and multivariate analysis of factors associated with recurrence-free survival (RFS) and overall survival (OS). Scoring systems were developed based on the hazard ratios (HR) of all significant predictors. Kaplan–Meier analyses and log-rank tests were used to calculate RFS and OS. Results: A total of 72 PDAC patients with preoperative chemotherapy were analyzed. After preoperative chemotherapy, 21(29.17%) patients achieved any radiologic downstaging with 68(94.44%) patients achieving negative resection margin status (R0 margins). For carbohydrate antigen 19-9(CA19-9) non-secreting patients, shrinkage rate of length diameter of tumor≥20%(P=0.027), resection margin status(P=0.011) were independent predictors of OS. And for CA19-9 secreting patients, CA19-9 serum level at the end of chemotherapy≥1000(P=0.027) and resection margin status (P=0.012) were independent predictors of OS. Points according HR were allocated to these factors in the proposed scoring system, respectively. Recurrence curves and survival curves with statistically significant differences were also presented for patients grouped according to the scoring systems. For CA19-9 non-secreting patients, the OS of patients with a score from 2 to 2.97 was significantly shorter than that of patients with a score from 4.94 to 5.91 (P=0.009). And for CA19-9 secreting patients, the OS of patients with a score of 2 was significantly shorter than that of patients with a score from 3.75 to 4.99 (P<0.001). Conclusion: Simple scoring models for RFS and OS incorporating CA19-9 serum levels or the tumor shrinkage rate and postoperative pathology may assist clinicians in evaluating prognosis of PDAC patients with preoperative chemotherapy.

The prognostic value of mutational signatures in pancreatic cancer.

689 Background: Pancreatic ductal adenocarcinoma (PDAC) is associated with poor overall survival (OS), however there are significant outliers. Understanding the biological factors driving PDAC cancer may reveal new biomarkers and treatment strategies. Mutational signatures, imprinted in the cancer genome, encode the life history of the cancer and may be used to stratify tumors into clinically-relevant subgroups. Methods: In this study we performed mutational signature analysis (MutationalPatterns, COSMIC v3.3) on whole genomes from 434 PDAC patients, comprising 167 patients with resected primary tumors, and 267 advanced PDACs from patients enrolled in the COMPASS trial. We evaluated whether the mutational signatures detected associate with OS using the log-rank test in the overall cohort and stratified by disease stage, adjusting for multiple hypotheses with 5% false discovery rate (FDR). We further characterized these associations in exploratory analyses. Results: Among the 55 COSMIC signatures detected in the overall cohort, the presence of three signatures significantly associated with OS after FDR correction. SBS85, a signature associated with indirect effects of activation-induced cytidine deaminase, was present in 18/434 (4.1%) samples, and was significantly associated with improved OS in the overall study population (log-rank p=6.5e-4, FDR-adjusted-p=0.026). SBS93, a signature of unknown etiology previously seen in other gastrointestinal cancers, was present in 4/434 (0.9%) samples, and was significantly associated with worse OS in the overall study population (log-rank, p=7e-6; FDR-adjusted-p=1.1e-3). SBS33, also of unknown etiology and previously seen in other gastrointestinal cancers, was present in 73/434 (16.8%) samples. It was seen more frequently in advanced disease (Fisher’s-exact p=1.9e-4), but was significantly associated with worse OS only in the resected cohort (log-rank, p=2.3e-4, FDR-adjusted-p=0.012). Conclusions: We identified three mutational signatures—SBS85, SBS93, and SBS33—that have not previously been described in PDAC and associate with OS. Further characterization of these novel signatures in PDAC and their underlying biology may uncover new treatment avenues.

Abstract C057: Delineating tissue-specific oncogene selection with autochthonous CRISPR activation screening

Abstract Background: While the genetic landscapes of pancreatic ductal adenocarcinoma (PDAC) and non-small cell lung cancer (NSCLC) are well described, the contribution of individual genes and transcriptomic programs to tumor initiation, progression and metastasis remain incompletely understood. To quantify the oncogenic potential of individual genes and identify transcriptomic programs that drive progression and metastasis, we have developed a platform for in vivo CRISPR activation (CRISPRa) screening that allows us to delineate tissue-specific oncogene selection. Material and Methods: Our platform consists of a conditional, CRISPRa-competent, murine tumor model (PPKS; P53(F/F), Kras(LSL-G12D/+), R26(LSL-SAM), coupled with targeted delivery of Cre-recombinase/sgRNA library encoding lentivirus [1]. We designed a CRISPRa screening library targeting 452 of the most commonly amplified and mutated genes across human adenocarcinoma. Using this library, we generated pancreatic and lung tumors, and harvested tissues and metastatic lesions at multiple time points (6 days, 4 weeks, 8 weeks, and endpoint). The change in guide distribution over time was then determined using NGS and bioinformatic analysis. To confirm the oncogenic potential of the most highly enriched genes, single gene-activated tumors were compared to non-targeted controls. Results: We found that essentially all guides (99.9%) were represented in vivo at the 6-day time point. The degree of guide selection increased over time and was significantly higher in the pancreas relative to the lung, indicating a higher degree of parallel tumor evolution in the lung. Lung samples were characterized by Egfr, Sox2 and Myc displaying gradually increasing enrichment over time and Egfr displayed the highest level of enrichment in endpoint lung tumors. In stark contrast, pancreas samples were almost completely dominated by Myc activation at the 4-week timepoint, with increased enrichment in the later timepoints, indicating a strong competitive advantage of Myc-activation, specifically in the pancreas. To confirm the pro-tumorigenic role of Myc in the pancreas, we analyzed the survival and phenotype of Myc-activated PPKS tumors, which revealed accelerated tumor progression relative to controls. Conclusions: We present one of the first implementations of autochthonous in vivo CRISPR activation screening, allowing for direct observation and quantification of oncogene selection and competition in vivo. Our results replicate tissue-specific differences in oncogene selection and indicate a dominant role of Myc-activation in pancreatic oncogenesis. Our platform has the potential to be used to address a wide range of inquiries into tumor biology, including drivers of progression, metastasis and resistance to targeted therapies.

Abstract A061: A novel genetically engineered mouse model of Myc-driven pancreatic cancer recapitulates phenotypic heterogeneity, metastasis, and therapy resistance seen in clinical populations

Abstract One of the most aggressive cancers in the US is pancreatic ductal adenocarcinoma (PDAc), whose incidence is expected to rise in the coming years. The standard-of-care treatments have improved over the past few decades, but they still primarily involve harsh chemotherapy cocktails and have only marginally increased overall survival, to a 5-year survival rate of 12 percent. c-MYC (MYC), a potent transcription factor, is frequently dysregulated in PDAc. In PDAc and other cancers, MYC plays a significant role in tumor progression, and its dysregulation has been linked to tumor aggressiveness and therapeutic resistance. In various mouse models of cancer, it has been demonstrated that oncogenic MYC expression controls components of the tumor microenvironment (TME). MYC expression in PDAc has been associated with a desmoplastic immune suppressive TME, though the precise mechanism is still unknown. In this study, we introduce a novel genetically modified mouse model (GEMM) of PDAc that can be used to simulate the disease's heterogeneity and investigate how MYC controls the tumor’s immune and stromal microenvironments. In the pancreas of our KMCERT2 model, both mutant Kras and lowly deregulated Myc are expressed through inducible Cre-driven mechanisms. Our research demonstrates how deregulated MYC cooperates with oncogenic KRASG12D in the adult pancreas to promote PDAc, faithfully reproducing the inter- and intra-tumoral heterogeneity observed in clinical PDAc populations. Currently, an embryonic KrasG12D- and p53 loss/mutant-driven PDAc model (KPC) is used for the majority of murine studies of PDAc. To learn more about the mechanisms underlying observed phenotypes in our KMCERT2 model, RNA- and DNA sequencing were performed on microdissected autochthonous tumor specimens as well as KMCERT2 tumor-derived cell lines. In contrast to the KPC model, our inducible KMCERT2 model of PDAc exhibits genetic alterations, such as CDKN2A loss, comparable to human disease. A distinct loss of nuclear p53 expression is also observed. Intriguingly, multiplexed immunohistochemistry analysis of immune cell composition showed that MYC-driven tumors have a higher density of antigen-presenting cells (APCs) when compared to spontaneous KPC tumors. Standard-of-care medications like gemcitabine and FOLFIRINOX are frequently ineffective against human PDAc. Orthotopic therapeutic studies using our KMCERT2 tumor-derived cell lines show a variety of responses to these therapies, with both resistant and sensitive tumor models, allowing us to use this model to better understand the mechanisms causing therapeutic resistance and to test new therapies. Additionally, we observe consistent liver metastasis in both spontaneous and orthotopic transplant settings. Together, these studies investigate the function of dysregulated MYC expression in murine PDAc metastatic behavior, immune phenotypes, and therapeutic response. It also offers mouse models of PDAc that are both spontaneous and orthotopic, recapitulating the complex and highly metastatic nature of the human disease. Citation Format: Isabel A. English, Patrick J. Worth, Kevin A. MacPherson, Michael B. Heskett, Vidhi Shah, Jackie L. Phipps, Courtney Betts, Shamilene Sivagnanam, Carl Pelz, Amy S. Farrell, Brittany L. Allen-Petersen, Meghan M. Joly, Motoyuki Tsuda, Colin J. Daniel, Jenny Eng, Xiaoyan Wang, Ashley L. Kiemen, Laura D. Wood, Terry K. Morgan, Mary C. Thoma, Jennifer M. Chu, Paul T. Spellman, Lisa M. Coussens, Ellen M. Langer, Rosalie C. Sears. A novel genetically engineered mouse model of Myc-driven pancreatic cancer recapitulates phenotypic heterogeneity, metastasis, and therapy resistance seen in clinical populations [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 A061.

NDRG1 overcomes resistance to immunotherapy of pancreatic ductal adenocarcinoma through inhibiting ATG9A-dependent degradation of MHC-1

AimsPancreatic ductal adenocarcinoma (PDAC) is a deadly disease that is resistant to immune checkpoint blockade (ICB) therapies. Emerging evidence suggests that NDRG1 may be an important target for the development of new therapies for PDAC. Herein, we investigated the novel roles of NDRG1 and Combretastatin A-4 (CA-4) in the treatment of PDAC ICB resistance. MethodsEnrichment of MHC class I was detected by RNA sequence and verified by RT-qPCR and immunoblotting in NDRG1-knockdown human pancreatic cancer cell lines. The protein degradation mode was found by stimulation with various inhibitors, and the autophagy degradation pathway was found by immunoprecipitation and immunolocalization. The roles of NDRG1 and MHC-I in immunotherapy were investigated by orthotopic solid tumors, histology, immunohistochemistry, multiplex immunofluorescence staining and flow cytometry. ResultsHere, we identified a previously undescribed role of NDRG1 in activating major histocompatibility complex class 1 (MHC-1) expression in pancreatic ductal adenocarcinoma (PDAC) cells through lysosomal-autophagy-dependent degradation. In mouse models of PDAC, either tumor cell overexpression or pharmacologic activation of NDRG1 leads to MHC-1 upregulation in tumor cells, which in turn promotes the infiltration and activity of CD8 + T cells, enhances anti-tumor immunity, and overcomes resistance to ICB therapy. Moreover, combination therapy of CA-4 and ICB overcomes the drug resistance of pancreatic cancer to ICB therapy. In PDAC patients, NDRG1 expression correlates with high MHC-1 expression and better survival. ConclusionOur results reveal NDRG1 in PDAC cancer cells as a tumor suppressor and suggest that pharmaceutically targeting NDRG1 is a promising way to overcome pancreatic cancer resistance to immunotherapy and provides a potential therapeutic strategy for the treatment of pancreatic cancer patients.