Reciprocity between p63 and p21 via TNFα/NF-κB signaling in p63-positive salivary duct adenocarcinoma cell.

Introduction:Recently, more and more total pancreatectomy (TP) has been performed for central-located pancreatic ductal cell adenocarcinoma (PDCA), which abuts or involves both gastroduodenal and splenic arteries and demands transaction of both of them for complete resection. Spiked by Warshaw’s procedure (spleen-preserving distal pancreatectomy with excision of splenic vessels), the authors developed a new procedure “Whipple over the splenic artery (WOTSA)” to replace TP by leftward extension of pancreatic parenchyma transaction line and preservation of pancreatic tail and spleen after excision of the splenic artery. This uncontrolled before and after study assesses the safety and efficacy of a new technique “Whipple over the splenic artery (WOTSA)” as a treatment for pancreatectomy for ductal adenocarcinoma (PDAC), which traditionally requires TP for a complete excision.Methods:The study group comprised 40 consecutive patients who underwent WOTSA for PDAC between August 2019 and September 2022. Their clinicopathological characteristics and survival were compared with those of a historical control group comprising 30 consecutive patients who underwent TP between January 2016 and July 2019.Results:None of the 40 patients in the WOTSA group required reoperation due to infarction of the pancreas and/or spleen remnant. Diabetes mellitus (DM) medication after WOTSA were none in 19, oral hypoglycemic agents in 19, and insulin preparations in 2 patients. Compared with TP, patients who underwent WOTSA exhibited similar rates of major operative complications, clear pancreatic parenchyma transaction margin, and a number of harvested positive lymph nodes, but a higher rate of adjuvant chemotherapy completion and a trend toward better median disease-free survival (14 vs. 7.5 months, P=0.023).Conclusions:Compared to TP, WOTSA can be safely performed and have much better postoperative glycemic status without cost of higher operative risk or impaired surgical radicality. These findings indicate that most TPs for PDAC potentially can be replaced by WOTSAs.

MP74-19 HIGH GLUT1 MEMBRANE EXPRESSION AND LOW PSMA MEMBRANE EXPRESSION IN DUCTAL ADENOCARCINOMA AND INTRADUCTAL CARCINOMA OF THE PROSTATE

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with poor prognosis due to its highly metastatic profile. Intercellular communication between cancer and stromal cells via extracellular vesicles (EVs) is crucial for the premetastatic microenvironment preparation leading to tumour metastasis. This study shows that under the influence of bioactive peptides derived from the extracellular matrix microenvironment, illustrated here by the AG‐9 elastin‐derived peptide (EDP), PDAC cells secrete more tumour‐derived EVs. Compared to PDAC‐derived EVs, tumour‐derived EVs resulting from AG‐9 treatment (PDAC AG‐9‐derived EVs) significantly stimulated cell proliferation. At constant amount, tumour‐derived EVs were similarly taken up by PDAC and HMEC‐1 cells. Tumour‐derived EVs stimulated cell proliferation, migration, proteinase secretion, and angiogenesis. Bioluminescence imaging allowed tumour‐derived EV/FLuc+ tracking in vivo in a PDAC mouse model. The biodistribution of PDAC AG‐9‐derived EVs was different to PDAC‐derived EVs. Our results demonstrate that the microenvironment, through EDP release, may not only influence the genesis of EVs but may also affect tumour progression (tumour growth and angiogenesis), and metastatic homing by modifying the in vivo biodistribution of tumour‐derived EVs. They are potential candidates for targeted drug delivery and modulation of tumour progression, and they constitute a new generation of therapeutic tools, merging oncology and genic therapy.

Abstract Background: Both Ductal Adenocarcinoma (DAC) and Intraductal Carcinoma (IDC) of the prostate are generally associated with aggressive clinical behavior and poor prognosis and can be lethal. Furthermore, previous studies linked aggressive clinical behavior and poor prognosis with discordant FDG positivity and low Prostate-specific membrane antigen (PSMA) expression. A recent study only cited a DAC patient with low 68Ga-PSMA-11 PET/CT uptake but high 18F-FDG PET/CT uptake, however, there is lack of directly compared articles nor large data sets. IDC-P has similar cribriform pattern in the pathological structure as a mean differential diagnosis for DAC. Hence, the objective of this study was to investigate the expression of PSMA and GLUT1 in DAC and IDC-P patients. Methods: The study was conducted on 87 DAC or IDCP patients without any treatment and 97 PAC patients with a Gleason score ≥ 8 of prostate biopsies and prostatectomy samples between August 2017 and August 2022. We performed immunohistochemical staining and scoring of various cancer component samples from the patients to reflect the protein expression levels of PSMA and GLUT1. Results: PSMA expression in PAC tissues was significantly higher than in DAC/IDC-P tissues (141.2 vs 78.6, p < 0.001). There was no significant difference in PSMA membrane expression between DAC/IDC-P and adjacent acinar adenocarcinoma (78.6 vs 93.4, p = 0.166). GLUT1 expression was higher in DAC/IDC-P tissue than in acinar adenocarcinoma tissue adjacent to DAC/IDC-P (68.6 vs 51.3, p = 0.007), but was still lower than that in PAC tissue (68.6 vs 93.1, p = 0.0014). It is worth noting that GLUT1 membrane expression in DAC/IDC-P was significantly increased than in PAC (13.0 vs 6.6, p = 0.025), and in acinar adenocarcinoma adjacent to DAC/IDC-P (13.0 vs 2.0, p < 0.001). Conclusions: In DAC and IDC-P tissues, PSMA membrane expression is low, while GLUT1 expression, especially GLUT1 membrane expression is high. These findings imply that DAC and IDC-P cells may have higher glucose metabolic and raise interest in targeting membrane GLUT1 as a novel anticancer strategy for DAC/IDC-P and other prostate cancer with high glucose metabolism. Citation Format: Xingming Wang, Xiaomei Gao, Lin Qi, Ye Zhang, Hongling Yin, Yu Gan, Yi Cai. High GLUT1 membrane expression and low PSMA membrane expression in ductal adenocarcinoma and intraductal carcinoma of the prostate [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C090.

Endoplasmic reticulum (ER) stress and unfolded protein response are cells' survival strategies to thwart disruption of proteostasis. Tumor cells are continuously being challenged by ER stress. The prion protein, PrP, normally a glycosylphosphatidylinositol (GPI)-anchored protein exists as a pro-PrP retaining its GPI-peptide signal sequence in human pancreatic ductal cell adenocarcinoma (PDAC). Higher abundance of pro-PrP indicates poorer prognosis in PDAC patients. The reason why PDAC cells express pro-PrP is unknown. Here, we report that persistent ER stress causes conversion of GPI-anchored PrP to pro-PrP via a conserved ATF6-miRNA449c-5p-PIGV axis. Mouse neurons and AsPC-1, a PDAC cell line, express GPI-anchored PrP. However, continuous culture of these cells with the ER stress inducers thapsigargin or brefeldin A results in the conversion of a GPI-anchored PrP to pro-PrP. Such a conversion is reversible; removal of the inducers allows the cells to re-express a GPI-anchored PrP. Mechanistically, persistent ER stress increases the abundance of an active ATF6, which increases the level of miRNA449c-5p (miR449c-5p). By binding the mRNA of PIGV at its 3'-UTRs, miR449c-5p suppresses the level of PIGV, a mannosyltransferase pivotal in the synthesis of the GPI anchor. Reduction of PIGV leads to disruption of the GPI anchor assembly, causing pro-PrP accumulation and enhancing cancer cell migration and invasion. The importance of ATF6-miR449c-5p-PIGV axis is recapitulated in PDAC biopsies as the higher levels of ATF6 and miR449c-5p and lower levels of PIGV are markers of poorer outcome for patients with PDAC. Drugs targeting this axis may prevent PDAC progression.

e16311 Background: Patients with mutations in BRCA 1/2 genes are at increased risk for pancreatic ductal cell adenocarcinoma (PDAC) in addition to breast and ovarian cancer. In 2019, the NCCN updated its guidelines recommending gene testing for all patients with PDAC to include associated genes: ATM, BRCA1, BRCA2, CDKN2A, MSH2, MLH1, MSH6, EPCAM, PALB2, STK11, TP53. This retrospective, observational study reports on adherence to genetic screening recommendations among patients with newly diagnosed PDAC who received oncologic care in rural Northeast Georgia (NEGA). Methods: Chart abstraction was performed utilizing the EPIC database from hospital electronic medical records of patients who were diagnosed with PDAC between 1/1/20-12/31/21 and received oncologic care in rural NE GA. This abstraction was completed to include information on gene testing recommendations, testing completion, and time from diagnosis to testing. The deidentified dataset was then analyzed using appropriate descriptive and associative statistical testing. Results: Of the cohort of 109 patients diagnosed with PDAC over these two years, only 32 (29.4%) received recommendations and completed genetic screening. Among this segment of patients, 16 (14.7%) were screened within 10 days of diagnosis, 8 (7.3%) within 11-30 days, and 6 (5.5%) were screened over 30 days after being diagnosed with PDAC. Among the 77 (70.6%) patients who did not receive screening, 45 of them (41.3%) did not receive any discussion regarding screening despite planning for other standard of care treatment. The remaining 32 patients (29.4% of the no screening cohort) did not receive screening due to desire to pursue palliative care/hospice/or due to terminal illness that prevented further workup. Of the 45 patients who did not receive screening despite PDAC treatment intent, majority (43; 95.5%) did not receive recommendations regarding the importance of genetic screening. Gene testing of the 32 patients who had screening completed showed that 4 (12.5%) had a pathogenic mutation (3 with CFTR; 1 ATM) and 16 (50%) had variants of unknown significance (VUS). Conclusions: This study highlights a gap in adherence to guideline-directed genetic testing in PDAC patients. Notable findings include increased likelihood to complete the test when recommendations and discussions of genetic screening occurred early in the course after initial diagnosis of PDAC. Moreover, 12.5% of those screened were found to have pathogenic mutation and half were found to have at least one VUS suggesting that genetic screening may potentially identify causative mutations. The findings also highlight the consideration for gene testing recommendation for the large proportion of patients who are terminally ill at diagnosis, as genetic screening would potentially benefit the family members and inform that they receive genetic screening as well.

Abstract The compound SOP1812, a tetra-substituted naphthalene diimide derivative has been previously disclosed to have single-digit nM anti-proliferative activity in a panel of human pancreatic ductal adenocarcinoma (PDAC) cell lines (Ahmed et al., ACS Med Chem Lett, 2020, 11, 1634-1644). It also has significant anti-tumor activity in the MIA-PACA2 xenograft model for PDAC as well as in the more demanding KPC model. We now report that this compound also shows significant anti-tumor activity in three patient-derived xenograft (PDX) models for PDAC. One is from a primary PDAC tumor at stage I and the two others from patients with stage IV PDAC (PDX tumor studies undertaken under contract by Champions Oncology). Transcriptome studies on cellular responses to SOP1812 have previously demonstrated that expression of a number of oncogenes and related signaling pathways are down-regulated by this agent. The promoter regions of these genes have an over-representation of quadruplex-containing sequences and several have also been shown by us to be also down-regulated in treated tumor xenografts. Our working hypothesis that the mechanism of action of SOP1812 in vivo involves targeting of quadruplex-containing genes, is also supported by the PDX data since these tumors have elevated levels of several quadruplex-containing genes previously identified by us in cells and in vivo. SOP1812 is bio-available at therapeutic doses and is well tolerated at these levels in these animal models. It is currently being evaluated as a candidate for clinical development by Qualigen Therapeutics Inc. Citation Format: Stephen Neidle, Ahmed Ahmed, Richard Angell, Sally Oxenford. The potent quadruplex-binding compound SOP1812 shows anti-tumor activity in patient-derived in vivo models of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4069.

Abstract Biofield therapies have gained popularity and are being explored as possible treatments for cancer. In some cases, devices have been developed that mimic the electromagnetic fields (EMF) that are emitted from people delivering biofield therapies. However, it is not clear if EMFs are the mechanism of action. We previously reported that biofield therapy significantly inhibited the growth of lung cancer cells in vitro and their relevant animal model mediated through inflammatory and immune pathways. We expanded this research to examine the effects of human biofield therapy on the growth of pancreatic ductal adenocarcinoma (PDAC) cells and explored relevant mechanisms. Cell viability of human pancreatic cancer Panc-1 and mouse pancreatic cancer Panc02 cells was measured by PrestoBlue assay. Cell cycle was measured by PI staining and cell voltage potentials were assessed using DiBAC4 staining. It is well-established that cancer cells have distinct bioelectrical properties and most have depolarized cell voltage potentials that support cell proliferation. Expression of cell signaling proteins was determined by Western blot. We found that Panc-1 and Panc02 cells exposed to biofield treatment for 15 and 30 mins, respectively, resulted in significantly lower viability compared to that of sham control. These experiments were replicated 12 times for Panc-1 cells and 6 times for Panc02 cells. We further observed that the experimental exposure significantly increased G1 phase population of Panc-1 cells (sham control=43.7% (0.8) vs treatment=55.0% (0.8%), p<0.001). In contrast, Panc02 cells exposed to biofield therapy had significantly higher population of G2/M cells than that of sham control group (sham control=22.5% (0.5) vs treatment=27.7% (1.1), p<0.001). We found that biofield therapy resulted in a 36.7% reduction in cell voltage potentials as measured by the intensity of DiBAC4 staining in Panc-1 cells compared to that of sham controls (p<0.01). Finally, the treatment down-regulated pAkt expression and the pAkt/Akt ratio by 45.3% and 43.8%, respectively, in Panc-1 cells in comparison with the sham control group. The effect of biofield therapy on cell signaling proteins measured by Reverse Phase Proteomic Array in Panc-1 cells and the growth of human Panc-1 mouse orthotopic model is ongoing and the results will be presented at the meeting. Together, these findings suggest that exposure to biofield therapy results in reduced growth of pancreatic cancer cells which might be in part mediated through modification of the cell cycle - differentially for different cell types, reductions in cell voltage potentials suggesting hyperpolarization of cells, and down regulation of PI3K/Akt pathways. Citation Format: Peiying Yang, Sharmistha Chakraborty, Phuong Nguyen, Meng Cui, Andrew Cusimano, Daoyan Wei, Sarah Prinsloo, Lorenzo Cohen. Biofield therapy suppressed the growth of human pancreatic cancer cells by modulation of cell cycle and cell voltage potentials [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5382.

Abstract Pancreatic ductal adenocarcinoma (PDAC) cells derive their resistance to therapy and aggressive clinical course from the symbiotic signaling and metabolic interactions with cancer-associated fibroblasts (CAFs). CAFs have been shown to provide nutrients to "parasitic" PDAC cells including water soluble glucose and amino acids. In hypoxic tumor microenvironments, aggressive PDAC are functional auxotroph for lipids, and scavenge exogenous lipids to build cellular membranes. However, the mechanism by which PDAC cells obtain these water-insoluble essential membrane building materials remains poorly understood. To gain insights into the mechanism of lipid scavenging, we generated cholesterol-auxotrophic human and mouse PDAC PDAC cells since cholesterol is the major constituent of lipid cellular membranes. Here, we discovered that PDAC cells utilize CAFs as a main source of lipids in vivo through direct heterotypic cellular contacts. In this process, PDAC cells directly acquire the CAF plasma membrane (PM) via trogocytosis. In response to yet unidentified paracrine “feed me” signals activating CAF-dependent trogocytosis, CAFs upregulate phosphatidylserine (PtdSer) on the outer leaflet of the PM. Consequently, blockade of PtdSer on CAFs partially deterred trogocytic transfer of CAF membranes to PDAC cells in vitro. Furthermore, Ca2+-dependent phospholipid scramblase TMEM16F is a critical regulator of PtdSer externalization. TMEM16F is highly expressed in human PDAC CAFs compared to fibroblasts isolated from the matching adjacent non-malignant pancreatic tissues CAFs deficient in TMEM16F scramblase exhibited markedly reduced ability to support the growth of cholesterol-auxotrophic PDAC cells cultured in lipid depleted media. We propose trogocytosis as a new mode of lipid scavenging by PDAC cells from CAFs involving activation of Ca2+-dependent phospholipid scramblase TMEM16F in CAFs and increased expression of PtdSer as “eat me” signals on CAF PM. To inactivate trogocytosis, we nominate TMEM16F as a plausible PDAC therapy target using clinically available TMEM16 inhibitors with a potential for impact on treatment of PDAC patients in the near term. Citation Format: Charline Ogier, Alena Klochkova, Battuya Bayarmagnai, Linara Gabitova, Diana Restifo, Aizhan Surumbayeva, Janusz Franco-Barraza, Debora Vendramini Costa, Ralph Francescone, Jaye Gardiner, Emmanuelle Nicolas, Elizabeth A. Handorf, Kathy Q. Cai, Edna Cukierman, Igor Astsaturov. Cancer associated fibroblasts sustain critical dependency of pancreatic cancer cells on exogenous lipids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1571.

Abstract Periodic fasting enhances the activity of several chemotherapeutics, molecularly-targeted drugs, endocrine agents and immune checkpoint inhibitors. Identifying drugs that are approved for non-oncological conditions, but acquire antitumor properties through fasting could pinpoint new cancer cell liabilities and define new treatment options. By screening over 800 approved drugs in PK9 pancreatic ductal adenocarcinoma (PDAC) cells, we identified several azoles (antifungal agents), including clotrimazole (CTZ), as agents whose cytotoxic activity against cancer cells is synergistically enhanced by starvation conditions. Since azoles inhibit 14alpha-demethylase, which is a key enzyme for cholesterol (CE) biosynthesis, we hypothesized that starvation and azoles would cooperate by blunting CE production in PDAC cells. Consistent with this notion, we found that simvastatin (an HMG-CoA reductase inhibitor) and terbinafine (TRB), which obstruct squalene epoxidase (SQLE), another key enzyme from the CE biosynthetic pathway, also had their antitumor effects strongly enhanced by starvation. Combined starvation and CTZ or starvation and TRB reduced intracellular CE in Capan-1 cells both in vitro and in vivo. Methyl-beta-cyclodextrin, which depletes intracellular CE, and starvation also showed a synergistic interaction in Capan-1 and in MiaPaCa2 cells (another PDAC cell line). Finally, culture media supplementation with a water-soluble CE formulation prevented the synergistic interaction between starvation and CE biosynthesis inhibitors. TRB potentiation through starvation was countered through supplementation with insulin, IGF1 and leptin, which are downregulated during fasting. Consistent with CE being an essential constituent of membrane lipid rafts, that harbor the growth- and survival-promoting PI3K/AKT signaling cascade, we found combined CTZ or TRB and starvation to markedly downregulate phosphorylated AKT in Capan-1 xenografts. Similar results were obtained in MiaPaCa2 cells. Capan-1 cell transduction with constitutively active, myristoylated AKT protected them from combined CTZ or TRB and starvation, indicating that AKT inhibition mediates the synergistic interaction between CE production inhibitors and fasting. Weekly 48h fasting enhanced CTZ and TRB antitumor effects in vivo in Capan-1 xenograft-bearing mice and, when combined with TRB, lowered circulating LDL (while increasing HDL) cholesterol as compared to TRB alone. Overall, these findings support the potential of fasting-based diets in combination with CE biosynthesis inhibitors against PDAC. Citation Format: Amr Khalifa, Ana Guijarro, Asmaa Namatalla, Moustafa Ghanem, Matteo Lambertini, Alessio Nencioni, Irene Caffa. Periodic fasting and cholesterol biosynthesis inhibitors achieve a synergistic antitumor activity in gastrointestinal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3020.

Abstract LCB84 is a human Trop-2-targeting antibody drug conjugate (ADC) composed of monomethyl auristatin E (MMAE) as payload and the Hu2G10 (by Mediterranea Theranostic) humanized IgG1 antibody that selectively targets the ADAM10-activated Trop-2 protein selectively expressed in transformed cancer cells (1). LCB84 was prepared using ConjuAll࣪, a proprietary site-directed conjugation technology of LegoChem Biosciences, which incorporates a conjugation ‘handle’ joined by enzymatic prenylation to a specifically engineered recognition sequence (CaaX) on antibody light chains. This conjugation handle facilitates simple versatile chemical conjugation to the linker-payload. A proprietary plasma-stable cleavable linker that is recognized and cleaved by a cancer-associated lysosomal enzyme, β-glucuronidase, was used to enable efficient and traceless payload release in a cancer-specific manner. LCB84 has been evaluated for anti-tumor activity and showed superior anticancer efficacy in triple-negative breast cancer (TNBC), pancreatic ductal adenocarcinoma (PDAC), gastric cancer and non-small cell lung cancer (NSCLC) cell line-derived xenograft (CDX) models compared to the ADC competitors Trodelvy and DS-1062. The LCB84 treatments were well tolerated, with no changes in body weight compared to control animals, for all dosing groups. LCB84 has robust cross-reactivity against primate Trop-2, which allows rigorous toxicity studies in monkeys. Remarkably, preliminary toxicity studies using cynomolgus monkeys showed that LCB84 is well tolerated, with calculated therapeutic index (TI, MTD/MED) of ~30 for single dosing and ~40 for repeat dosing. In conclusion, LCB84 is highly effective against Trop-2-positive CDX models in mice at doses that are well tolerated in mice and in primate models. Use of this proprietary plasma-stable cancer-selective linker technology and the Hu2G10 anti-Trop-2 monoclonal antibody that targets cancer-activated Trop-2 has led to a greatly improved next generation ADC for the treatment of various Trop-2-positive solid cancers including TNBC, PDAC, NSCLC and gastric cancer. Citation Format: Hyejung Kim, Emanuela Guerra, Eunji Baek, Yeojin Jeong, Hyogeun You, Byeongjun Yu, Taeik Jang, Alberti Saverio, Chul-Woong Chung, Changsik Park. LCB84, a TROP2-targeted ADC, for treatment of solid tumors that express TROP-2 using the hu2G10 tumor-selective anti-TROP2 monoclonal antibody, a proprietary site-directed conjugation technology and plasma-stable tumor-selective linker chemistry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 328.

Abstract Spatial transcriptomics (ST) and CO-Detection by indEXing (CODEX) are becoming the leading transformative approaches for studying spatially resolved tumor heterogeneity and immune complexity. To explore the application of these two cutting-edge technologies and to further investigate the complexities of tumor microenvironment, we applied 10x Visium Gene Expression platform and Akoya CODEX platform on surgical resections from pancreatic ductal adenocarcinoma (PDAC), breast cancer (BRCA), and clear cell renal carcinoma (ccRCC) patients. A total of 30+ protein markers were screened, verified, and conjugated as CODEX antibodies for the 3 types of cancers. Tumor microenvironment was vividly revealed by a set of cancer markers, immune markers, and stroma cells markers on a single ST slide and a single CODEX image. Notably, the distinct immune microenvironment was observed between tumor nests in the neighborhood in terms of cell types, number of cells, and cell distribution patterns. This work highlights the importance of direct visualization for tumor heterogeneity. It also provides a significant insight for understanding tumor complexity and a promising opportunity to benefit personalized medicine. Citation Format: Siqi Chen, Andrew Shinkle, Yanyan Zhao, Chia-Kuei Mo, Andrew Houston, Preet Lal, John M. Herndon, Ryan C. Fields, William Gillanders, Feng Chen, Li Ding. Spatial transcriptomics and multiplexed imaging to explore tumor heterogeneity and immune complexity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1710.

Abstract As a fatal disease with short overall survival, pancreatic cancer is among the top five leading cause of cancer-related deaths worldwide, current therapies against pancreatic cancer lack tumor specificity and generally cause toxic side effects on normal and healthy tissues. Thus, exploring and developing novel tumor-specific agents for treating pancreatic cancer is a critical need. NAD(P)H:quinone oxidoreductase 1 (NQO1) is elevated in the pancreatic cancers whereas it lacks in associated normal tissues. Therefore, NQO1 bioactivatable drugs are promising due to tumor-selective killing NQO1-positive cancer cells. Our previous studies have revealed that the novel NQO1 bioactivatable drug - deoxynyboquinone (DNQ) is ten-fold more potent than the prototypic NQO1 bioactivatable drug β-lapachone to efficiently kill NQO1-positive cancer cells. However, DNQ causes a severe side effect action - high-grade methemoglobinemia that limits its clinical usefulness. Here, we developed a novel DNQ derivative, isopentyl-deoxynboquinone (IP-DNQ), which selectively killed pancreatic ductal adenocarcinoma (PDA) cells in an NQO1-dependent way with an extremely low IC50 (0.1 µM). IP-DNQ evoked massive ROS production and oxidative DNA lesions, resulting in PARP1 hyperactivation and mitochondrial catastrophe. Furthermore, we also observed that IP-DNQ induced G2/M phase cell cycle arrest and promoted programmed necrosis and caspase-dependent apoptosis co-existing in IP-DNQ-treated NQO1-positive pancreatic cancer cells. In addition, IP-DNQ treatment caused extremely low side effect of methemoglobinemia, significantly suppressed tumor growth and extended mice lifespan in orthotopic pancreatic cancer xenograft model. In summary, our findings offer a novel potential therapy against NQO1-positive pancreatic cancers and enable mechanism-based synergy with other anticancer drugs. Citation Format: Xiumei Huang, Lingxiang Jiang, Matthew W. Boudreau, Paul J. Hergenrother. A novel NQO1 bioactivatable drug induces mitochondrial dysfunction and G2/M phase cell cycle arrest to selectively kill pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6183.

Simple SummaryHow stromal cells control tumor progression is an active area of investigation. Cancer associated fibroblasts (CAFs) have been suggested to both limit, or promote cancer dissemination in various contexts, but a systemic understanding of stromal influence in determining cancer metastasis is lacking. This study takes a closer look at the diversity of fibroblasts that emerge in different metastatic stages of PDAC (Pancreatic Ductal Cell Adenocarcinoma), a cancer type with high stromal infiltration. Specifically, we found that the CAFs respond to the growing (but yet not metastasized) tumor by transition to a myofibroblast like stage, potentially resisting cancer invasion. As the cancer disseminates, these fibroblasts are nearly entirely replaced by highly diverse set of subpopulations with distinct functions. We have previously advanced a framework, termed Evolved Levels of Invasability (ELI), explaining the vast differences in cancer metastasis and placental invasion among mammals. We found that PDAC’s metastatic transition and stromal trespass is accompanied by the downregulation of pro-resistive and the upregulation of pro-invasability ELI signatures in CAF subpopulations. Cancer-associated fibroblasts (CAFs) are now appreciated as key regulators of cancer metastasis, particularly in cancers with high stromal content, e.g., pancreatic ductal cell carcinoma (PDAC). However, it is not yet well understood if fibroblasts are always primed to be cooperative in PDAC transition to metastasis, if they undergo transformation which ensures their cooperativity, and if such transformations are cancer-driven or intrinsic to fibroblasts. We performed a fibroblast-centric analysis of PDAC cancer, as it transitioned from the primary site to trespass stromal compartment reaching the lymph node using published single-cell RNA sequencing data by Peng et al. We have characterized the change in fibroblast response to cancer from a normal wound healing response in the initial stages to the emergence of subclasses with myofibroblast and inflammatory fibroblasts such as signatures. We have previously posited “Evolved Levels of Invasibility (ELI)”, a framework describing the evolution of stromal invasability as a selected phenotype, which explains the large and correlated reduction in stromal invasion by placental trophoblasts and cancer cells in certain mammals. Within PDAC samples, we found large changes in fibroblast subclasses at succeeding stages of PDAC progression, with the emergence of specific subclasses when cancer trespasses stroma to metastasize to proximal lymph nodes (stage IIA to IIB). Surprisingly, we found that the initial metastatic transition is accompanied by downregulation of ELI-predicted pro-resistive genes, and the emergence of a subclass of fibroblasts with ELI-predicted increased invasibility. Interestingly, this trend was also observed in stellate cells. Using a larger cohort of bulk RNAseq data from The Cancer Genome Atlas for PDAC cancers, we confirmed that genes describing this emergent fibroblast subclass are also correlated with lymph node metastasis of cancer cells. Experimental testing of selected genes characterizing pro-resistive and pro-invasive fibroblast clusters confirmed their contribution in regulating stromal invasability as a phenotype. Our data confirm that the complexity of stromal response to cancer is really a function of stage-wise emergence of distinct fibroblast clusters, characterized by distinct gene sets which confer initially a predominantly pro-resistive and then a pro-invasive property to the stroma. Stromal response therefore transitions from being tumor-limiting to a pro-metastatic state, facilitating stromal trespass and the onset of metastasis.

The transcriptional regulator nuclear factor of activated T-cells, cytoplasmic 3 (NFATc3) is constitutively activated in several cancer types and plays important roles in cancer development and progression. Heavily phosphorylated NFATc3 resides in the cytoplasm of resting cells, and dephosphorylated NFATc3 translocates to the nucleus to activate expression of target genes in cells exposed to stimuli, for instance, hypoxia. Apart from phosphorylation, various post-translational modifications have been reported to regulate NFAT transcriptional activity. However, the mechanisms remain elusive. Here, we have demonstrated that NFATc3 is activated in human pancreatic ductal adenocarcinoma (PDAC) cells and that excessive activation of NFATc3 is correlated to advanced stages of PDAC and short survival time of PDAC patients. NFATc3 is deSUMOylated at K384 by SENP3 under hypoxia, which impairs the interaction between NFATc3 and phosphokinase GSK-3β, subsequently decreases NFATc3 phosphorylation and increases its nuclear occupancy. Knockdown of SENP3 greatly decreased hypoxia-induced NFATc3 nuclear occupancy. Our results highlight that SENP3-mediated deSUMOylation acts as an essential modulator of NFATc3, which is instrumental in PDAC tumor progression under hypoxia.

The volume regulated anion channel (VRAC) is known to be involved in different aspects of cancer cell behaviour and response to therapies. For this reason, we investigated the effect of DCPIB, a presumably specific blocker of VRAC, in two types of cancer: pancreatic duct adenocarcinoma (PDAC) and melanoma. We used patch-clamp electrophysiology, supported by Ca2+ imaging, gene expression analysis, docking simulation and mutagenesis. We employed two PDAC lines (Panc-1 and MiaPaCa-2), as well as a primary (IGR39) and a metastatic (IGR37) melanoma line. DCPIB markedly increased whole-cell currents in Panc-1, MiaPaca2 and IGR39, but not in IGR37 cells. The currents were mostly mediated by KCa 1.1 channels, commonly known as BK channels. We confirmed DCPIB activation of BK channels also in HEK293 cells transfected with α subunits of this channel. Further experiments showed that in IGR39, and to a smaller degree also in Panc-1 cells, DCPIB induced a rapid Ca2+ influx. This, in turn, indirectly potentiated BK channels and, in IGR39 cells, additionally activated other Ca2+ -dependent channels. However, Ca2+ influx was not required for activation of BK channels by DCPIB, as such activation involved the extracellular part of the protein and we have identified a residue crucial for binding. DCPIB directly targeted BK channels and, also, acutely increased intracellular Ca2+ . Our findings extend the list of DCPIB effects that should be taken into consideration for future development of DCPIB-based modulators of ion channels and other membrane proteins.

575 Background: SBP-101, a polyamine metabolic inhibitor, inhibited growth in 6 human pancreatic ductal adenocarcinoma (PDA) cell lines and 3 murine xenograft tumor models of human PDA. SBP-101 monotherapy in heavily pre-treated PDA patients (> 2 prior regimens) showed a median survival of 5.9 months at the optimal dose level. Purpose: To assess the PK, safety and efficacy of SBP-101 in combination with gemcitabine (G) and nab-paclitaxel (A) in patients with previously untreated metastatic PDA. Methods: In a modified 3+3 dose escalation scheme, subcutaneous injections of SBP-101 were dosed at 0.2, 0.4 or 0.6 mg/kg days 1-5 of each 28-day cycle. G (1000 mg/m2) and A (125 mg/m2) were administered intravenously on Days 1, 8, and 15 of each cycle. PK was evaluated on day 1 of cycle 1 in cohorts 1-3. Safety was evaluated by clinical and laboratory assessments. Efficacy was assessed by CA19-9 levels, objective response using RECIST criteria, progression-free survival (PFS) and overall survival (OS). A 4th cohort using a modified dosing schedule of 0.4 mg/kg SBP-101 days 1-5 for cycles 1-2 and days 1, 8, and 15 every cycle thereafter was added to mitigate hepatic toxicity, and that dose and schedule were recommended for Phase 1b expansion. Results: Fifty patients were enrolled (N=25, Phase 1a and N=25, Phase 1b) and received up to 13 treatment cycles. SBP-101 plasma Cmax and AUC0-t increased in a slightly more than dose proportional manner and were unchanged by the addition of G and A. PK parameters of G and A were unaltered by increasing doses of SBP-101. The most common nonserious adverse events related to SBP-101 (>10%) are fatigue (N=14), LFT/transaminase abnormalities (N=15), vision abnormalities (N=10), injection site pain (N=13), dehydration (N=7), nausea (N=7). Serious adverse events related to SBP-101 observed in some subjects include hepatic toxicity (N=6) and retinal toxicity (N=8) both occurring after prolonged treatment and requiring dose reduction or discontinuation. At the recommended dose and schedule (N=30), CA19-9 levels decreased 60-99% in 19 of 29 evaluable patients, with 1/29 (3%) achieving a complete remission 13/29 achieving partial responses (45%) and 10/29 achieving stable disease at 8 weeks (35%). PFS was confounded by SBP-101 dosing holds implemented to investigate potential toxicity. Sixteen subjects are in survival follow-up. Median OS is 10.1 months and is not final. Conclusions: Interim results suggest SBP-101 may enhance first-line treatment with G and A in patients with metastatic PDA. Hepatic toxicity can be mitigated with dose reduction or discontinuation. A vision screening program will be used in future studies to mitigate retinal toxicity. Clinical trial information: NCT03412799.

The VRAC blocker DCPIB directly gates BK channels and increases intracellular Ca2+ in melanoma and pancreatic duct adenocarcinoma cell lines

Due to challenges in early-stage detection, aggressive behavior, and poor response to systemic therapy, pancreatic cancer is one of the most fatal cancer types globally. The role of RNA-binding protein (RBP) transcription and translation of cancer cells has been well demonstrated, although their roles in pancreatic cancer is less well understood. In this study, we found that heterogeneous nuclear ribonucleoprotein C (hnRNPC), a RBP, is highly expressed in pancreatic ductal adenocarcinoma (PDAC) tissues and cells. In addition, we discovered that overexpression of hnRNPC in PDAC cells in vitro increased cell proliferation, migration, invasion, and metastasis. The presence of hnRNPC promoted tumorigenesis of pancreatic cells in metastatic in vivo models, which was also validated. In silico analyses revealed that hnRNPC is a strong positive regulator of IQ Motif Containing GTPase Activating Protein 3 (IQGAP3) activity. The experimental confirmation of this association revealed a direct interaction of IQGAP3 and hnRNPC to induce cell growth and invasion in PDAC cells by activating the epithelial-mesenchymal transition. In light of the findings that hnRNPC accelerates PDAC progression by interfering with IQGAP3, it appears that this technique for diagnosis and treatment of PDAC may have promise.