Pancreatic Cells Research Articles

Rakicidin J and K, two cytotoxic and antibacterial cyclic depsipeptides from the marine bacterium Micromonospora chalcea

Rakicidin J (1) and rakicidin K (2), two new cyclic depsipeptides, were isolated from culture broth of Micromonospora chalcea FIM-R150103. Their structures were elucidated by extensive analysis of NMR, HR-ESI-MS, and electronic circular dichroism (ECD) data. The two compounds showed strong cytotoxic activity against human colon carcinoma HCT-8 and human pancreatic cancer PANC-1 cells under normoxic and hypoxic conditions in the range of IC50 values from 0.024 to 0.79 μg/mL. Moreover, compounds 1 and 2 also showed moderate antibacterial activity against ten Gram-positive bacterial strains with MIC values ranging from 4 to more than 32 μg/mL. Structure–activity relationship of these two compounds with a close analogue, rakicidin B1, is also discussed.

Transcription factor KLF9 inhibits the proliferation, invasion, and migration of pancreatic cancer cells by repressing KIAA1522.

Pancreatic cancer (PC) has a poor prognosis and high mortality. Kruppel-like factor 9 (KLF9), a transcription factor, is aberrantly expressed in various neoplasms. The current study sought to analyze the functional role of KLF9 in the proliferation, invasion, and migration of PC cells. The expression patterns of KLF9 and KIAA1522 in normal pancreatic cells (HPDE-C7) and PC cells (Panc 03.27, BxPc3, SW1990) were determined by real-time quantitative polymerase chain reaction and Western blot assay. After treatment of KLF9 overexpression, proliferation, invasion, and migration were evaluated by cell counting kit-8, 5-ethynyl-2'-deoxyuridine staining, and Transwell assays. The binding of KLF9 to the KIAA1522 promoter was analyzed by dual-luciferase assay and chromatin immunoprecipitation. The rescue experiment was conducted to analyze the role of KIAA1522. KLF9 was downregulated, while KIAA1522 was upregulated in PC cells. KLF9 overexpression mitigated the proliferation, invasion, and migration of PC cells. Enrichment of KLF9 led to inhibition of the KIAA1522 promoter and repressed KIAA1522 expression. KIAA1522 overexpression neutralized the inhibitory role of KLF9 in PC cell functions. KLF9 is enriched in the KIAA1522 promoter and negatively regulates KIAA1522 expression, thereby mitigating the proliferation, invasion, and migration of PC cells.

Abstract 7134: Treatment of pancreatic cancer cells with tumor treating fields (TTFields) and PARP inhibitors

Abstract Background: Therapeutic options for advanced stage pancreatic cancer are limited. Patients harboring germline BRCA mutations benefit from PARP inhibitors (PARPi); unfortunately, such mutations are rare in pancreatic cancer. Tumor Treating Fields (TTFields), electric fields that disrupt cellular processes crucial for cancer cell viability, have shown effectiveness against pancreatic cancer cells, and are currently under clinical investigation for the treatment of patients with pancreatic cancer. The mechanism of action of TTFields in various cancer types has suggested involvement of BRCA downregulation. The current study examined the potential sensitization of BRCA wild-type pancreatic cancer cell lines to PARPi with TTFields application. Methods: BxPC3 and AsPC1 human pancreatic BRCA wild-type cancer cells were treated for 72 h with TTFields (frequency of 150 kHz; intensity of 1 and 0.5 V/cm RMS, respectively), using the inovitro device. The PARPi niraparib and olaparib were each administered to the cells at various concentrations, with or without co-application of TTFields. At treatment cessation, cell count, colony formation, and apoptosis were measured. Results: Applying PARPi to pancreatic cell lines yielded a dose-dependent cytotoxic and anti-clonogenic effects, and induced apoptosis. TTFields application also induced such effects when applied alone, while delivery of TTFields together with PARPi amplified the effects demonstrated by PARPi alone. Conclusions: Concomitant application of TTFields and PARPi in BRCA wild type pancreatic cancer cells exhibits a potential improvement relative to the mono-therapies. This could be rationalized based on the previously demonstrated ability of TTFields to induce a state of BRCAness in cancer cells, and suggests benefit for TTFields and PARPi co-treatment in the absence of background BRCA mutations. Citation Format: Hila Ene, Kerem Ben Meir, Antonia Martinez-Conde, Roni Frechtel-Gerzi, Hila Gabay, Daria Gerasimova, Rotem Engelman, Eyal Dor-On, Adi Haber, Moshe Giladi, Yoram Palti. Treatment of pancreatic cancer cells with tumor treating fields (TTFields) and PARP inhibitors [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 7134.

Abstract 4375: ALPPL2 is involved in MAPK signal pathway of pancreatic cancer

Abstract Background: Alkaline phosphatase placental-like 2 (ALPPL2) is a member of the alkaline phosphatase family. ALPPL2 is currently being investigated as a novel therapeutic target due to its cancer-specific expression. However, our knowledge of ALPPL2 biological function in cancer is limited. Here we describe an interaction between ALPPL2 and MAPK signaling pathway in pancreatic cancer. Materials and Methods: ALPPL2 mRNA expression in a panel of 27 pancreatic cell lines was determined by RNAseq. Protein expression and phosphorylation were measured by Western blot. ALPPL2 or ALPP were overexpressed in pancreas cell lines through a Lentiviral packaging system. MEK inhibitor (trametinib) activity was assessed in the same panel of cell lines using a 6-day cell proliferation assay on a Synentec Cellavista imaging system. MEK inhibitor resistant cell lines were developed by culturing cells in increasing concentrations of MEK162. Results: We hypothesized that pancreatic cancer cells with high ALPPL2 expression may be less sensitive to MEK inhibition. Our study from a panel of 27 pancreatic cancer cell lines showed that there was no correlation between ALPPL2 expression and response to trametinib. Western blot showed that the level of phospho-ERK was reduced in PANC1 and HPAC cells at 6 hours after trametinib treatment. Signaling rebounded at 24 and 48 hours, though it remained below baseline. Surprisingly, ALPPL2 expression increased at 24 and 48 hours after trametinib treatment, however, the mRNA of ALPPL2 and ALPP was down-regulated in three pancreatic cancer cell lines conditioned to be MEK inhibitor resistant. To understand the role of ALPPL2 in the MAPK signaling pathway in response to MEK inhibition, ALPPL2 and ALPP were overexpressed in 2 pancreatic cancer cell lines: YAPC and PSN-1 whose ALPPL2 and ALPP were undetected by Western blot prior to transfection. Overexpression of ALPPL2 or ALPP in YAPC cells resulted in more ERK phosphorylation with no change in total ERK, it also resulted in increased total and phospho-S6, and decreased phospho-STAT3 with no change in total STAT3. The amount of total and phospho-ERK were not changed in PSN-1 with overexpression of ALPPL2 or ALPP, however, it did result in increased total and phospho-S6, decreased phospho-STAT3. Interestingly, the expression of ALPPL2 and ALPP in YAPC cells with ALPPL2 or ALPP overexpression was increased at 48 hours after trametinib treatment. Discussion: To our knowledge, this is the first study to report that ALPPL2 interacts with the MAPK signaling pathway in pancreatic cancer cells. The activated MAPK signaling pathway appears to control ALPPL2 expression. The MAPK signaling pathway may regulate ALPPL2 expression through transcription and protein metabolism. We have also shown that ALPPL2 expression can activate MAPK signaling pathway. Our findings that ALPPL2 expression can be increased after trametinib treatment provide further rationale for targeting ALPPL2 in cancer. Citation Format: Ke Wei Gong, Bilal Hamid, Kenny W. Castro, Martina S. McDermott, Forrest Epstein, Kevin Chau, Chuhong Hu, Jun Zhang, Ming Lu, Benjamin G. Hoffstrom, Neil A. O'Brien, Dennis J. Slamon. ALPPL2 is involved in MAPK signal pathway of pancreatic cancer [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 4375.

Abstract 991: Unraveling the role of nicotinamide adenine dinucleotide precursors in pancreatic cancer biology

Abstract Introduction: Pancreatic cancer has the lowest 5-year survival rates among all cancers because of the challenges with early detection, its aggressive nature, and resistance to chemotherapy; this makes prevention a promising approach for reducing related mortalities. The disease is predominantly diagnosed in individuals over 60, suggesting that age-related physiological decline impacts its onset; therefore, delaying aging hallmarks may delay its onset. Nicotinamide dinucleotide (NAD+) precursors may hold a unique advantage over other geroprotectors because they target multiple aging hallmarks. Heron, we investigated how NAD+ supplementation impacts normal human pancreatic ductal epithelial cells (HPDE) under stress as a surrogate for cancer prevention in vitro. We also examined the paradoxical impact of NAD+ precursor supplementation on pancreatic cancer growth during chemotherapy. Methods: In vitro, we mimicked age-related NAD+ decline in the media. We stressed HPDE cells under these conditions and evaluated the effect of supplementation with the NAD+ precursor, Nicotinamide mononucleotide (NMN). We assessed cell viability using the PicoGreen assay, mitochondrial function, and reactive oxygen species (ROS) using the Seahorse XF Analyzer and the mitoSox kit, respectively, DNA repair and DNA damage using a western blot for Poly ADP ribose (PAR) and gamma H2AX, respectively, and SIRT3 expression was determined using a western blot. To evaluate the impact of NAD+ supplementation on pancreatic cancer cells, we treated pancreatic cancer cells with NMN and oxaliplatin. We then assessed the mitochondrial function, ROS levels, DNA damage, and repair. We implanted MiaPaCa-2 xenografts into athymic nude mice and treated them daily with 300mg/Kg of NMN and biweekly 5mg/kg of Oxaliplatin. Results: Experiments revealed that NMN is not toxic to HPDE cells, even at high concentrations. There was increased cell survival of HPDE cells under stress (nutrient deprivation and alkylating agents) following NMN supplementation. We also noted reduced ROS levels, increased SIRT3 expression, and PARylation. Pancreatic cancer cells were less sensitive to Oxaliplatin following NMN supplementation and showed an increased oxygen consumption rate, increased PARylation, and reduced ROS levels. There was reduced sensitivity to oxaliplatin treatment in the mice with MiaPaCa-2 xenografts treated with NMN. Conclusion: These findings demonstrate a cytoprotective role of NAD+ precursor supplementation in normal pancreatic ductal epithelial cells, with implications for possible cancer prevention. NAD+ supplementation also rescued cancer cells under cytotoxic stress, meaning that geroprotectors like NMN, in conjunction with chemotherapy, may unintentionally promote cancer progression. Future studies will directly test NAD+ precursor supplementation in genetically engineered pancreatic cancer models. Citation Format: Faith Nakazzi, Mehrad Zarei, Hallie J. Graor, Alexander W. Loftus, Omid Hajihassanni, Jordan M. Winter. Unraveling the role of nicotinamide adenine dinucleotide precursors in pancreatic cancer biology [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 991.

Abstract 2979: Pancreatic cancer cells upregulate LPAR4 in response to isolation stress to create a fibronectin-rich extracellular matrix niche and promote tumor initiation

Abstract Tumor initiation is an inefficient process in which tumor cells must develop cell-autonomous mechanisms to survive harsh microenvironments, establish colonies, and thereby overcome “isolation stress.” We report that pancreatic cancer cells transiently upregulate lysophosphatidic acid receptor 4 (LPAR4), a G-protein coupled receptor, in response to microenvironmental stress or chemotherapy, which promotes stress tolerance, drug resistance, and tumor initiation. Even without exogenous LPA, LPAR4 drives the expression of a subset of extracellular matrix (ECM) genes via an AKT/CREB axis, including fibronectin and versican, which are established drivers of cancer stemness. The upregulation of fibronectin is indispensable for LPAR4-induced tumor initiation in vivo and anchorage-independent growth in vitro. Furthermore, the ligation of this fibronectin-containing ECM created by LPAR4-positive cells via integrins α5β1 or αvβ3 can transfer cancer stemness and stress tolerance to LPAR4-negative cells by activating Yes-associated protein 1 (YAP). Our findings reveal that in response to isolation stress, pancreatic tumor cells that upregulate LPAR4 can autonomously generate a fibronectin-enriched ECM niche and thereby initiate tumor formation. Citation Format: Chengsheng Wu, Taha Rakhshandehroo, Hiromi I. Wettersten, Alejandro Campos, Tami Von Schalscha, Shashi Jain, Ziqi Yu, Jiali Tan, Evangeline Mose, Betzaira G. Childers, Andrew M. Lowy, Sara M. Weis, David A. Cheresh. Pancreatic cancer cells upregulate LPAR4 in response to isolation stress to create a fibronectin-rich extracellular matrix niche and promote tumor initiation [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 2979.

Abstract 7308: Induction of apoptosis via the regulation of pro-apoptotic and anti-apoptotic proteins in pancreatic adenocarcinoma BxPC-3 cells by Chinese medicinal herbs Scutellaria barbata and Oldenlandia diffusa

Abstract Apoptosis is a controlled program cell death process that the body uses to combat cancer development. Pancreatic cancer is a devastating disease with a poor prognosis. It is usually symptom-free during development and is advanced at diagnosis. BxPC-3 is a pancreatic adenocarcinoma cell derived from a 61-year-old female with adenocarcinoma. Scutellaria barbata (SB) and Oldenlanda diffusa (OD) have been used in traditional Chinese medicine for treating liver, lung, and rectal cancers. They are included in most herbal cancer treatment formulas in Taiwan hospitals. We previously demonstrated that aqueous extracts of SB and OD selectively induced apoptosis in colon HCT 116 cancer cells but not in colon CCD 841 CoN normal epithelial cells by modulating the pro-apoptotic and anti-apoptotic proteins. They also induced and regulated apoptosis in pancreatic ductal carcinoma PANC-1 Cells. This study used the green/red/blue fluorescent Apoptosis/Necrosis Detection Kit and the Human Apoptosis Antibody Array - Membrane [43 Targets] Test (Abcam). Our fluorescent apoptosis data showed that a 2-hour treatment with 1.5 mg/mL and 3.0 mg/mL aqueous extract of SB and OD induced a statistically significant percentage of apoptosis in BxPC-3 cells (SB: 42.5 ± 3.5%, 48.5 ± 0.5% > 14.0% ± 4.0%, p < 0.05; OD: 72.0 ± 2.0%, 37.5 ± 1.5%> 14.0% ± 4.0%, p < 0.05) as compared to the negative control respectively. Similar results were obtained with the 4-hour incubation period (SB: 46.0 ± 8.0%, 54.5 ± 2.5% > 9.5% ± 2.5%, p < 0.05; OD: 49.0 ± 7.0%, 37.0 ± 0.0%> 9.5% ± 2.5%, p < 0.05). From the antibody array-membrane experiments, significant up-regulation of various pro-apoptotic proteins such as Bid, Bim, Bad, Bax, C3, C8, CD40, HTRA, IGFBP-4, IGFBP-4, p53, TNF-α, and TRAIL-2 as compared to the negative controls were observed; and regulation of anti-apoptotic proteins HSP27, IGF-II, and Survivin were also revealed. These results suggest that SB and OD contain phytochemicals that induce apoptosis in pancreatic adenocarcinoma BxPC-3 cells by regulating various apoptotic protein productions. Further study of their specific modulation effects of apoptosis is warranted to reveal their potential chemopreventive and therapeutic properties against pancreatic and other cancers. Citation Format: Christine Choi, Sarah Wolf, Rekha Isaac, Elim Choi, Abigail Shim, Angel Campana, Padma T. Uppala, Ryan Hayes, Brian Yuen Yau Wong. Induction of apoptosis via the regulation of pro-apoptotic and anti-apoptotic proteins in pancreatic adenocarcinoma BxPC-3 cells by Chinese medicinal herbs Scutellaria barbata and Oldenlandia diffusa [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 7308.

Abstract 1584: The interplay between natural killer cells and pancreatic stellate cells in pancreatic ductal adenocarcinoma

Abstract Natural Killer (NK) cells have been shown to possess potent anti-tumor activity against multiple cancer types and consequently have been harnessed as a cellular cancer immunotherapy. Furthermore, targeting of pancreatic stellate cells has received increasing interest in the treatment of pancreatic ductal adenocarcinoma (PDAC), with several clinical trials testing the hypothesis that rendering activated pancreatic stellate cells/cancer associated fibroblasts (aPSC/CAF) into a quiescent state (qPSC) can enhance anticancer treatment. Despite this interest, our understanding of the interplay between these two cell types remains limited. We hypothesized that PSC/CAF may exhibit a dynamic interaction with NK cells that may be harnessed for therapeutic strategies and patient stratification. Employing a series of in vitro assays, we demonstrate a significant bi-directional interaction between natural killer cells and pancreatic stellate cells in the context of PDAC. NK cells were found to effectively target and eliminate both aPSC and qPSC, with PSC activation conferring some protection from NK-lysis. Furthermore, we highlight a significant myofibroblastic shift in PSCs regardless of previous activation status when cultured with NK cells; an interaction that was direct contact dependent. NK cells were also found to demonstrate cellular modulation of NKG2A and TIM3 in response to PSC co-culture. Drawing on Luminex ELISA analysis, we suggest that engagement of the IFN-γ signaling pathway may be driving phenotypic modulation. Global proteomic analyses demonstrated differential protein changes induced by NK cells in aPSC compared to qPSC, as well as alterations in the NK cell proteome upon such co-culture. Utilizing multiplex immunohistochemical analysis of PDAC patient samples, we highlight differential spatial distribution of phenotypically distinct NK cells and PSC/CAF subtypes in patient samples between long- (n=16) and short- (n=48) survivors. Moreover, NK-PSC proximity, and not total immune infiltrate, was found to play a prognostic role in PDAC. Our work provides a global overview of the cross-talk between NK and PSC in PDAC, demonstrating a significant bi-directional relationship which offers novel insights into potential therapeutic avenues. Furthermore, we demonstrate, for the first time to our knowledge, the prognostic role of NK-PSC proximity in patient survival and suggest its potential for patient stratification. Citation Format: Rachel Elizabeth Fincham, Parthiban Periasamy, Craig Ryan Joseph, Jia Meng, Jeffrey Chun Tatt Lim, Felicia Wee, Konstantinos Stasinos, Michelle R. Goulart, Jiangfeng Ye, Li Yen Chong, Denise Goh, Joe P. Yeong, Hemant M. Kocher. The interplay between natural killer cells and pancreatic stellate cells 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 1584.

Abstract 3141: LCB02A an antibody drug conjugate (ADC) targeting CLDN18.2 expressing solid tumors

Abstract Claudin18.2 (CLDN18.2) is an isoform of CLDN18 with very limited expression in gastric epithelial cells in normal tissues, and aberrant overexpression in gastric, pancreatic, esophageal, ovarian, lung, and other solid tumors. This expression pattern presents a significant hurdle in developing an ADC targeting CLDN18.2 due to potential on-target toxicity to the gastrointestinal (GI) tract. LCB02A is an antibody-drug conjugate (ADC) that targets CLDN18.2, composed of a topoisomerase 1 inhibitor (TOPO1i) as payload (DAR4) linked to the CLDN18.2 specific binding PR301839 hClaudin18.2 (CLDN18.2) is an isoform of CLDN18 with very limited expression in gastric epithelial cells in normal tissues, and aberrant overexpression in gastric, pancreatic, esophageal, ovarian, lung, and other solid tumors. This expression pattern presents a significant hurdle in developing an ADC targeting CLDN18.2 due to potential on-target toxicity to the gastrointestinal (GI) tract. LCB02A is an antibody-drug conjugate (ADC) that targets CLDN18.2, composed of a topoisomerase 1 inhibitor (TOPO1i) as payload (DAR4) linked to the CLDN18.2 specific binding PR301839 humanized IgG1 antibody utilizing LegoChem Biosciences’ ConjuAllTM technology. LCB02A showed potent in vitro cytotoxicity in CLDN18.2 expressing gastric and pancreatic cancer cell lines. LCB02A also showed excellent anti-tumor efficacy in various gastric and pancreatic cell line-derived xenograft (CDX) models and patient-derived xenograft (PDX) models. Preliminary toxicity assessment in cynomolgus monkeys (CLDN18.2 cross-reactive species) demonstrated that LCB02A was well tolerated, with repeat doses of up to 60 mg/kg. In conclusion, LCB02A is a promising ADC for the treatment of CLDN18.2 expressing solid tumors including gastric cancer (GC) and pancreatic ductal adenocarcinoma (PDAC). umanized IgG1 antibody utilizing LegoChem Biosciences’ ConjuAllTM technology. LCB02A showed potent in vitro cytotoxicity in CLDN18.2 expressing gastric and pancreatic cancer cell lines. LCB02A also showed excellent anti-tumor efficacy in various gastric and pancreatic cell line-derived xenograft (CDX) models and patient-derived xenograft (PDX) models. Preliminary toxicity assessment in cynomolgus monkeys (CLDN18.2 cross-reactive species) demonstrated that LCB02A was well tolerated, with repeat doses of up to 60 mg/kg. In conclusion, LCB02A is a promising ADC for the treatment of CLDN18.2 expressing solid tumors including gastric cancer (GC) and pancreatic ductal adenocarcinoma (PDAC). Citation Format: Eun ji Baek, Chul-Woong Chung, Changsik Park, Taeik Jang, Yeojin Jeong, Sang-Ah Lee. LCB02A an antibody drug conjugate (ADC) targeting CLDN18.2 expressing solid tumors [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 3141.

Abstract 5692: Posttranscriptional molecular network facilitates KRAS-linked tumorigenesis via XRN1-miR-21 signaling in pancreatic cancers

Abstract Aberrant expression of mutant KRAS and miRNA-21 (miR-21) are commonly attribute to malignancy and tumorigenic progression of pancreatic cancers. However, few studies have explored the molecular mechanism linking these two prognostic factors. In this study, we explored the underlying molecular bridge between KRAS and miR-21 regulation in pancreatic cancer. Functional integrative analysis of KRAS, an essential oncogenic driver in pancreatic cancer progression, and together with miR-21 revealed XRN1, a 5′-3′ exoribonuclease, as RNA-linked top-scored molecule with a strong survival correlation and malignancy in pancreatic cancer patients. Depletion of XRN1 led to severe reduction in mature miR-21 expression, while unaltered precursor miR-21 levels; indicating that the XRN1 is critical in miR-21 maturation at post-Drosha step in conjunction with Dicer. Moreover, miR-21 sensor-based experiment also confirmed the critical role of XRN1 on miR-21 biogenesis. Depletion experiments showed that XRN1 positively regulates proliferation and self-renewal capacity of pancreatic cancer cells via its 5′-3′ exoribonuclease activity and concomitantly elevated the expression of PTEN and PDCD4 which are the key miR-21 cellular downstream targets. shRNA-mediated XRN1 depletion also yielded smaller tumor volume and longer survival in pancreatic cancer xenografts and syngeneic model demonstrating the therapeutic potential of targeting XRN1. Subsequent silencing of KRAS reduced XRN1 expression in pancreatic cancer cells, suggesting that KRAS plays a key role in this XRN1-miR-21 axis. Furthermore, drug-resistant pancreatic cells showed elevated expression of both KRAS and XRN1-miR-21 indicating that this axis is also associated with drug sensitivity. In sum, these results suggest that a novel KRAS-XRN1 posttranscriptional regulatory axis plays a pivotal role in tumor progression by mediating miR-21-directed signaling, and thus may suggest a novel targeting strategy for pancreatic cancer therapeutics. Significance: This study identifies molecular regulatory interplay between KRAS and XRN1-miR-21 signaling in pancreatic cancers, elucidates how KRAS-XRN1 modulates tumorigenesis via miR-21, and demonstrates that targeting this signaling can contribute to therapeutic strategies for pancreatic cancers. [Y. Seo and S. E. Oh contributed equally to this work.] Citation Format: Yoona Seo, Seung Eon Oh, Jong Heon Kim. Posttranscriptional molecular network facilitates KRAS-linked tumorigenesis via XRN1-miR-21 signaling in pancreatic cancers [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 5692.

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 477: Tumor treating fields (TTFields) targeted self assembling nanoparticles for pancreatic cancer treatment: In vitro and in vivo assessment

Abstract Tumor Treating Fields (TTFields) is a novel, non-invasive and safe treatment modality, which is prescribed to be used in conjunction with classical anticancer chemotherapeutic drugs. TTFields when used alone are safe, but their use in combination with classical chemotherapy continues to offer drug related adverse reactions which ultimately impacts the patient compliance. To address this challenge, we earlier reported meticulously optimized self-assembling cationic-anionic polymer nanoparticles (S-CAP NPs) with average particle size of about 200 nM, encapsulation efficiency of 55-65 % and sustained drug release over 60 h. We also reported that, TTFields were capable of destabilizing the S-CAP NPs leading to targeted drug release. The results were also corroborated with in vitro cytotoxicity and colony formation studies in presence of TTFields using multiple pancreatic cancer cell lines. After establishing the proof-of-concept, the objective was to assess the in vitro cellular uptake of S-CAP NPs. For this, fluorescent dye loaded S-CAP NPs were developed and subjected to cellular uptake analysis. The in vitro cellular uptake studies confirmed the enhanced uptake of S-Cap NPs by passive diffusion in the pancreatic cancer cell lines compared to normal pancreatic epithelial cell line. The studies corroborated preferential cellular uptake of S-Cap NPs by the pancreatic tumor. Among all the formulations, the chitosan- bovine serum albumin S-CAP NPs was most optimum and was used for in vivo safety studies. For this, in vivo acute and repeated dose 28-day toxicity of the optimum placebo S-CAP NPs was conducted in Sprague Dawley rats (Western University of Health Sciences Protocol #R20IACUC031) and OECD guidelines. The in vivo studies showed a LD50 value above 2000 mg/kg and NOAEL value of 1000 mg/kg. These high doses safety of S-CAP NPs confirmed the potential of this approach for future preclinical efficacy studies followed by clinical translation. Lastly, the in vitro stability studies were conducted as per the ICH guidelines for a period of 36 months and the formulations were concluded to be stable. The tumor targeting potential, in vivo safety and stability of S-CAP NPs, establish their translational potential for TTFields triggered targeted drug release in cancer treatment. This approach will not only reduce the adverse effect associated with combination of TTFields and chemotherapy but can also be explored as a platform technology for management of other cancers. This study is supported by AACR-Novocure tumor treating fields research fellowship. Citation Format: Preshita Prafulla Desai, Sunil Prabhu. Tumor treating fields (TTFields) targeted self assembling nanoparticles for pancreatic cancer treatment: In vitro and in vivo assessment [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 477.

Abstract 2895: ILRAP blockade mediates anti-fibrotic effects in pancreatic cancer-associated fibroblasts

Abstract Introduction: Pancreatic cancer (PDAC) patients have poor prognosis partly due to excessive activity of cancer-associated fibroblasts (CAFs). CAFs drive the fibrosis that causes excessive type III collagen and extracellular matrix deposition that in turn reduces drug response resulting in poor survival. In support, high levels of the type III collagen serum biomarker PRO-C3 correlates with poor survival in PDAC. TGF-β is thought to be the main driver of PRO-C3 and tumor fibrosis. Cytokines such as Interleukin 1 (IL-1) play a key role in the pancreatic tumor microenvironment and may play a role in tumor fibrosis as well. In this study, we first investigated the potential of IL-1 in activating fibroblasts to drive fibrosis and produce PRO-C3. Subsequently, we established a co-culture of pancreatic cancer cells and pancreatic CAFs to investigate the anti-fibrotic properties of nadunolimab, an antibody that blocks IL-1 signaling by targeting IL-1 Receptor Accessory Protein (IL1RAP). Nadunolimab is currently in phase I/IIa clinical development for treatment of pancreatic cancer (CANFOUR, NCT03267316). Methods: Human primary pancreatic cancer-associated fibroblasts (CAFs) were cultured in Ficoll-media (Scar-in-a-jar, SiaJ) supplemented with TGF-β, IL-1α or IL-1β. The fibrotic activity of the fibroblasts was investigated by measuring the formation of type III collagen (PRO-C3) at days 3, 6, 9 and 12. Then, pancreatic cancer cells (BxPc3) and pancreatic CAFs were cultured either alone or in a co-culture. Nadunolimab or isotype control were added at the start of these cultures and supernatants were collected after three days. The level of PRO-C3 was measured by ELISA. Results: Both IL-1α or IL-1β were equipotent to TGF-β in inducing PRO-C3 in the SiaJ monoculture, indicating that IL-1 is pro-fibrotic. In addition, when cancer cells and CAFs were co-cultured, PRO-C3 levels increased compared to single-cell cultures (1.5-6-fold). When co-cultures were treated with nadunolimab, the induction of PRO-C3 was blocked to levels similar to monocultures, whereas the isotype control had no effect on PRO-C3 levels. Conclusion: IL-1 activated fibroblasts and induced type III collagen formation (PRO-C3), suggesting that IL-1 is a driver of tumor fibrosis. In support, pancreatic tumor cells induced collagen type III formation (PRO-C3) in pancreatic CAFs and blockade of IL1RAP with nadunolimab inhibited this collagen formation. Thus, nadunolimab may have anti-fibrotic properties and PRO-C3 could potentially be used for prognostic/predictive enrichment and as a pharmacodynamic marker in future studies evaluating anti-IL-1 modalities in PDAC. Citation Format: Neel I. Nissen, Nils Hansen, Elin Jaensson Gyllenbäck, Camilla R. Millrud, Marcus Järås, David Liberg, Morten A. Karsdal, Nicholas Willumsen. ILRAP blockade mediates anti-fibrotic effects in pancreatic cancer-associated fibroblasts [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 2895.

Abstract 4441: The epigenetic factor BMI1 regulates metabolism and tumorigenesis in human pancreatic cancer cells

Abstract Dysregulation of epigenetic factors is a key component of tumorigenesis. BMI1, a member of the polycomb repressor complex 1 (PRC1), is an oncogenic factor studied in many types of cancer, including pancreatic ductal adenocarcinoma (PDAC). PDAC is the third most common cause of cancer-related death in the United States and investigation of the biology involved in transformation is critical for improving outcomes in this devastating disease. In PDAC, BMI1 is required for initiation of pancreatic cancer in mice and enhances in vitro growth of human and murine tumor cell lines. CRISPR-mediated knock out of BMI1 results in reduced subcutaneous and orthotopic tumor growth in mice and decreased expression of genes related to metabolism, specifically glycolysis, and cell proliferation. BMI1 has also been implicated in regulation of metabolism in other epithelial tumor types, including ovarian cancer. Given these preliminary findings, we hypothesized that loss of BMI1 in multiple human PDAC cell lines would result in altered metabolic activity, reducing the growth and tumorigenic properties of these cells. We demonstrated that BMI1 expression is higher in PDAC cell lines compared to normal pancreatic epithelial cells and normal human donor pancreatic tissue. Following CRISPR-mediated BMI1 knock out in human PDAC cell lines, we investigated the alterations to metabolism using metabolic flux analysis. Loss of BMI1 resulted in lower basal and compensatory rates of glycolysis and increased oxygen consumption rates compared to wild-type cells. Metabolomic analysis demonstrated reduction in all the enzymes involved in the glycolysis pathway with loss of BMI1. Cell growth was reduced by loss of BMI1 in vitro, and orthotopic injection into mice of human cells with BMI1 knock out resulted in decreased tumor size compared to wild-type, recapitulating these findings in vivo. Together, these data identify a role of BMI1 in promotion of tumor growth through regulation of metabolism in pancreatic cancer. Experiments are ongoing to determine the underlying mechanism of this regulation and implications on therapeutic targeting with BMI1/PRC1 complex inhibitors in conjunction with traditional chemotherapy. Citation Format: Jamie N. Mills, Dominik Awad, Heather K. Schofield, Joyce K. Thompson, Hannah Watkoske, Damien Sutton, Nicholas Nedzesky, Donovan Drouillard, Zeribe Nwosu, Carlos Espinoza, Yaqing Zhang, Annachiara Del Vecchio, Christopher J. Halbrook, Marina Pasca di Magliano, Costas A. Lyssiotis, Filip Bednar. The epigenetic factor BMI1 regulates metabolism and tumorigenesis in human pancreatic cancer cells [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 4441.

Abstract 7495: Validation of microfluidic isolation of circulating pancreatic tumor cells from blood

Abstract Background. Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis and is the fourth leading cause of cancer-related deaths in the US. PDAC is diagnosed in the late stages, which limits treatment options and yields poor clinical outcomes. Recurrence is a significant issue for patients undergoing surgery for PDAC. Circulating tumor cells (CTCs) are thought to be a potential mediator of recurrence, facilitating the formation of secondary tumor sites after surgery. Thus, there is a need to isolate CTCs to improve understanding of disease recurrence, to ultimately improve outcomes. Herein, we aim to validate our inertial microfluidic (iMF) system for isolating pancreatic CTCs from blood. We also benchmark our iMF system to a commercially available immunomagnetic negative selection system, EasySep™, and quantify target cell recovery and cell enrichment. Methods. Our iMF system employs the differential cell sizes of CTCs from blood cells and inertial migration of such cells in a straight microchannel (150 µm × 50 µm × 24 mm) to enrich CTCs from blood. The iMF system sample flow rate used is 100 µL/min, with a 200 µL/min PBS buffer flow rate. The EasySep™ Direct Human CTC Enrichment kit targets CD2, CD14, CD16, CD19, CD45, CD61, CD66b, and Glycophorin to remove blood cells from the sample using magnetic particles, thereby separating them from CTCs. We spiked low cell concentrations of 50, 100, and 500 Hoechst-stained pancreatic carcinoma cells, PANC1, in 1 mL of healthy blood then captured using either EasySep™ or iMF. Cell enumeration was performed after centrifugation or Cytospin. Results. PANC1 cells and WBCs have average sizes of 18 µm and 8.4 µm, respectively. We characterized our iMF device to capture cells ≥ 12 µm, which yields capture of most PANC1 cells. Indeed, our iMF system achieved 82.8±19.2%, 72.5±16.3%, and 59.3±3.8% recovery for 50, 100, and 500 cell-spikes in lysed blood, respectively. For the same 50, 100, and 500 cell-spikes, the total iMF CTC yields after Cytospin were 46.3±11.8, 44.3±9.1, and 32.7±2.1%, while the total CTC yields from EasySep™ were 4.6±3, 5.3±2.1, 8.3±1%, respectively. When we processed whole blood directly, EasySep™ had lower cell recovery, 3.8±0.2, 4.6±1, and 3.3±0.4%, respectively. Our system also demonstrated 8 × higher target cell enrichment compared to EasySep™. Blood lysis before iMF separation did not drastically affect the recovery of target cells. Conclusions. We determined that our system has improved performance over the commercial isolation system, EasySep™, thereby validating the effectiveness of microfluidic isolation for PDAC. In future studies, we can then use our iMF system in CTC isolation from routine blood draws from PDAC patients undergoing surgical resection. We may then isolate real patient samples and study relevant phenotypes of PDAC CTCs which may therefore be used for identifying certain mechanisms of PDAC progression. Citation Format: Celine Macaraniag, Ifra Khan, Jian Zhou, Pier Cristoforo Giulianotti, Alain Borgeat, Gina Votta-Velis, Ian Papautsky. Validation of microfluidic isolation of circulating pancreatic tumor cells from blood [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 7495.

Abstract 2903: CTHRC1 induces PSCs (pancreatic stellate cells) into myCAFs (myofibroblast-like cancer-associated fibroblasts)

Abstract Collagen triple helix repeat containing-1 (CTHRC1) is a secreted protein that contributes to the progression of various cancers, including pancreatic cancer. The higher expression of CTHRC1 in tumor tissues is associated with poorer survival outcomes. However, its specific roles in tumor extracellular matrix (ECM) remodeling remain unclear. Our study aims to investigate the influences of CTHRC1 on pancreatic stellate cells (PSCs), a main source of ECM production in pancreatic cancer. The analyses of the publicly available pancreatic cancer patient data revealed that CTHRC1 is mainly expressed in cancer stroma and highly correlated with ECM-related genes. An in vitro study showed that more than 40% of these genes can be upregulated by CTHRC1. CTHRC1 specifically activated PSC into myofibroblast-like cancer-associated fibroblasts (myCAFs), which are characterized by a significantly upregulated POSTN gene expression. Periostin (coded by the POSTN gene) has a central role in the CTHRC1-PSCs-cancer metastasis axis. Furthermore, CTHRC1 promoted pancreatic cancer cell proliferation through PSC activation to a greater extent than via direct stimulation. Proof-of-concept experiments showed that the long-term (4-week) inhibition of CTHRC1 led to significant tumor suppression and ECM reduction, and also resulted in an unexpected shift in the CAF subtype from myCAFs to inflammatory CAFs (iCAFs). PSC activation was demonstrated to be the key molecular mechanism responsible for the tumor-promoting effects of CTHRC1, and CTHRC1 has a critical role in CAF subtype differentiation and tumor microenvironment (TME) remodeling. The inhibition of CTHRC1 as a therapeutic strategy for the treatment of pancreatic cancer warrants further investigation. Citation Format: Jin Gyeong Lee, Min Kyung Kang, Fen Jiang, Kyoungjin Ryu, Juwon Kim, Yun-Yong Park, Sang Seok Koh. CTHRC1 induces PSCs (pancreatic stellate cells) into myCAFs (myofibroblast-like cancer-associated fibroblasts) [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 2903.

Abstract 489: Unlocking immune resistance and metastasis of PDAC via dual acting circularized nanodisc

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and deadliest forms of cancer. While major progress in immunotherapy has been made in recent years, it remains poorly effective in this malignancy. Two key players stand behind immune resistance of PDAC and inadequate efficacy of immunotherapy to suppress its metastasis. First, the immune-resistant pancreatic cancer stem cells (CSCs) evade immune surveillance acting as a source for tumor relapse and metastasis. This immune resistance stems from low immunogenicity of PDAC cells as well as the inaccessible CSCs that reside deep in the tumor core. Second, pancreatic cancer associated fibroblasts (CAFs) fuel metastasis of PDAC cells by suppressing their immune recognition and establishing pre-metastatic niche at distant organs to help their colonization. Our hypothesis is to overcome immune resistance of PDAC and inhibit early steps of its metastasis via simultaneous immunogenic eradication of immune resistant CSCs and switching immunosuppressive and pro-metastatic CAFs into quiescent ones. Therefore, we engineered ultrasmall circularized nanodiscs (cND) that encapsulate oncolytic peptide LTX-315 and fibroblast remodeling Notch signaling inhibitor. The engineered oncolytic LTX-315 loaded cND successfully induced immunogenic cell death (ICD) of both bulk PANC1 and pancreatic CSCs. Compared to non-treated and free LTX-315 treated cells, LTX-315 cND resulted in enhanced release of DAMPs including ATP and HMGB-1 as well as surface translocation of calreticulin. The ultrasmall size (9 nm) of cND enabled its deep tumor penetration to eradicate resistant CSCs, as revealed using 3D PDAC heterospheroids. In parallel, the Notch blocking cND promoted quiescence of the activated pancreatic stellate cells as denoted by downregulation of SMA, FAP and collagen. In KPC syngeneic PDAC mouse model, the dual oncolytic/Notch blocking cND combined with aPD-L1 therapy significantly suppressed tumor growth and prevented its metastasis to liver compared to vehicle-treated mice. Immunophenotyping study showed marked reduction in the abundance of CSCs and CAFs in the tumor treated with the cNDs. Moreover, the tumor infiltration of CD86+ MHCII+ dendritic cells and cytotoxic CD8+ T cells was enhanced after treatment with the cND. Overall, our dual strategy of targeting pancreatic CSCs and CAFs via oncolytic/Notch blocking cND boosted the anti-tumor and anti-metastatic efficacy of aPD-L1 therapy to eliminate primary PDAC tumor and suppress its metastasis. Citation Format: Ahmed Elzoghby, Hagar Emam, Seungbin Yim, Cuiyan Xin, Mahmoud Nasr. Unlocking immune resistance and metastasis of PDAC via dual acting circularized nanodisc [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 489.

Abstract 1403: The role of Dclk1-expression cells in normal pancreas and in pancreatic tumor progression

Abstract Background: Tuft cells, marked by Doublecortin-like kinase-1 (Dclk1), are rare in the healthy pancreas but increase markedly during pancreatic tumorigenesis. While Group 2 innate lymphoid cells (ILC2s) are known to modulate inflammation and immunity, their specific roles in cancer-related immunity and immunotherapy interactions with pancreatic tuft cells remain unknown. Methods: To investigate tuft cells during early pancreatic neoplasia, we used a Dclk1 reporter mouse model and single-cell RNA-seq to study Dclk1-expressing cells in normal pancreas and neoplasia using Mist1/Kras mouse. We treated mice with IL-13 peptides and anti-IL-13 antibodies to explore the IL-13-ILC2 axis in tuft cell regulation. Results: Within the normal pancreas, Dclk1 expression marked several specialized epithelial cell types, including tuft-like cells, neuroendocrine cells, and a small number of acinar progenitor cells. A notable increase in Dclk1-expressing cells was observed at the onset of early PanIN lesions, with a subsequent decline in advanced PanIN stages and PDAC. Using flow cytometric analysis, we observed an increase in ILC2 cells in the Mist1/Kras pancreas in response to IL33 produced by pancreatic cancer-associated fibroblasts (CAFs). Blocking ILC2 activation with anti-IL33 antibodies reduced Dclk1 cell hyperplasia during PanIN progression. Given the known association between tuft cells, ILC2 cells, and the IL13/IL4 signaling pathways in the intestine, we discovered that Dclk1-expressing cells highly expressed the IL4 receptor (IL4ra). IL-13 levels increased during PanIN progression, and we found that ILC2s comprised the primary source of IL-13 within pancreatic tumors. In vitro, studies showed that the presence of IL-13 maintained the expansion of Dclk1-expressing tuft-like cells in pancreatic organoids derived from Dclk1-DTR-ZsGreen mice. Correspondingly, the in vivo administration of anti-IL-13 resulted in reduced Dclk1 expression. Conclusion: Previous studies have shown that pancreatic tuft cells inhibit pancreatic cancer progression. Our study reveals a novel regulatory mechanism wherein IL-13 secreted by ILC2s modulates Dclk1 tuft cell expression during pancreatic tumorigenesis. The IL13-ILC2 axis appears to be a potential therapeutic target to regulate Dclk1 cell activity, which could influence the course of pancreatic tumorigenesis. Future investigations will further explore the interaction between the IL13/ILC2 axis and Dclk1 in pancreatic cancer development. Citation Format: Giovanni Valenti, Pasquale Laise, Feijing Wu, Hiroki Kobayashi, Quin T. Waterbury, Ryota Takahashi, Tuo Ruan, Zhengyu Jiang, Yosuke Ochiai, Leah B. Zamechek, Timothy C. Wang. The role of Dclk1-expression cells in normal pancreas and in pancreatic tumor progression [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 1403.

Abstract 620: Screening of small-molecule MAPK pathway inhibitors on a panel of 130 human cancer cell lines identifies determinants of response in RAS-mutant subgroups

Abstract Oncogenic mutations in members of the RAS family, HRAS, NRAS, and KRAS, are frequently identified in various cancers and often lead to increased signaling of the mitogen-activated protein kinase (MAPK) pathway. KRAS is the most frequently mutated RAS isoform, with a mutation incidence of >10% across all cancers, and up to 70% and 20% in pancreatic and non-small cell lung cancer (NSCLC), respectively. RAS has long been considered to be undruggable, and direct inhibition of RAS with small molecule inhibitors has only recently been achieved, with the approval of the KRAS G12C-mutant selective inhibitors sotorasib and adagrasib for treatment of NSCLC. Indirect targeting of RAS-mutated cancers by inhibitors of downstream components such as MEK and ERK has shown variable responses. Novel insights into MAPK signaling have renewed interest into targeting the MAPK pathway in RAS-mutant cancer, with pan-RAF dimer inhibitors and dual RAF/MEK inhibitors reaching clinical trials. To improve the chances of success, it is important to determine which patient populations are most likely to respond to these inhibitors. To gain more insight into the cellular targeting of MAPK pathway inhibitors, eighteen inhibitors of the MAPK components KRAS, RAF, MEK, and ERK were profiled on a panel of 130 cell lines in cell viability assays (the Oncolines® panel). The cell lines represented a diverse range of tumor tissues and had different genomic backgrounds. Many of the cell lines had mutations in KRAS, NRAS, or BRAF. Drug responses were determined by exposing the cell lines to nine-point duplicate dose ranges of the inhibitors. Using bioinformatics, cell line responses to the inhibitors were related to genomic and transcriptomic features, to identify determinants of drug response in both tissue-agnostic and tissue-specific contexts. The results of our analyses revealed variable responses of KRAS-mutant cell lines to the inhibitors, reflecting clinical responses. Tissue-focused analyses showed that the different responses could be traced back to the different KRAS amino acid substitutions. For instance, in pancreatic cancer cell lines, the KRAS Q61H mutation predicted increased sensitivity to MAPK pathway inhibitors compared to other KRAS amino acid substitutions. Additionally, in tissue groups with a more heterogenous mutation profile, co-mutations explained the decreased effectivity of MAPK pathway inhibitors in KRAS-mutant cell lines, such as PIK3CA mutations in colorectal cancer cell lines. Lastly, a gene expression-based signature of MAPK activity predicted response to MAPK pathway inhibitors, independent of mutation status. Our findings have uncovered determinants of response to MAPK pathway inhibitors in RAS-mutant cell lines. These determinants can serve as markers to select patients who are most likely to benefit from MAPK inhibitors. Citation Format: Jeffrey J. Kooijman, Awan Al Koerdi, Daphne J. Kluitmans, Esmee van den Bossche, Jelle Dylus, Jeroen A. de Roos, Janneke J. Melis, Guido J. Zaman. Screening of small-molecule MAPK pathway inhibitors on a panel of 130 human cancer cell lines identifies determinants of response in RAS-mutant subgroups [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 620.

Abstract 4463: Metabolic reprogramming through Notch1 signaling promotes a survival advantage in pancreatic neuroendocrine tumor cells

Abstract Background: Cancer cells utilize both oxidative phosphorylation (OXPHOS) and glycolysis to generate energy. Switching between OXPHOS and glycolysis can promote tumor progression. The mechanisms governing oncogenic metabolic reprogramming are largely unknown, but recent data has suggested that Notch1 dysregulation in cancer cells can contribute to altered metabolic phenotypes. We hypothesized that Notch1 signaling supports metabolic reprogramming in pancreatic neuroendocrine tumor (pNET) cells. Methods: We established a Notch1-knockout pNET cell line (BON-N1-KO) by deleting Notch1 at exon 3 in a pNET cell line (BON) using CRISPR/Cas9. Proliferation over time was measured using a trypan blue exclusion test. Seahorse Glycolytic Rate Assay and Mitochondria Stress Test were used to measure the glycolytic and mitochondrial activities of cells. A glycolysis deprivation assay was employed to determine cell viability at varying glucose concentrations. Single end RNAseq was performed using the Illumina NGS platform, to a read depth of 50M. Results: Over time, pNET cells lacking Notch1 (BON-N1-KO) proliferated slower than wildtype (WT) cells (p=0.034). Compared to WT BON, the BON-N1-KO cells had reduced basal oxygen consumption rate (28.2 ± 1.3 vs. 40.7 ± 1.4; p=0.02, ATP production (21.9 ± 1.0 vs. 29.6 ± 0.98; p=0.04), and maximal respiration (39.4 ± 1.7 vs. 63.7 ± 2.1; p=0.004). BON-N1-KO cells also had a reduction in basal glycolysis, as measured by proton efflux rate, compared to WT (48.7 ± 2.6 vs. 57.7 ± 6.2; p=0.2). To further test metabolic reprogramming, WT and BON-N1-KO cells were starved of glucose (0mM), compared to normal glucose (17.5mM) and viability was measured over time. By day 5, the BON-N1-KO group had a viability of 0%, whereas 12.8% of WT cells were alive (p=0.038). To determine if Notch1 loss was associated with altered expression of established metabolic genes, we performed RNAseq. Differential gene expression analysis found that OXPHOS-related genes (UQCC2, COX15, COX20) and glycolysis-related genes (Slc1a1, Slc2a4, Hk1, Hk2) were significantly down-regulated in BON-N1-KO cells. Conclusions: Our study shows that Notch1 signaling facilitates metabolic reprogramming as a survival advantage in pNET cells. Targeting Notch1 signaling to mediate cellular metabolism may be a novel therapeutic strategy in pNETs. Citation Format: Rachael Guenter, Weisheng Chen, Brendon Herring, Yuvasri Golivi, Melissa Sammy, Jason Whitt, Renata Jaskula-Sztul, Herbert Chen, J. Bart Rose. Metabolic reprogramming through Notch1 signaling promotes a survival advantage in pancreatic neuroendocrine tumor cells [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 4463.