Pancreatic Ductal Adenocarcinoma Cell Growth Research Articles

The molecular mechanism underlying KRAS regulation on STK31 expression in pancreatic ductal adenocarcinoma.

KRAS gene mutations are common in pancreatic ductal adenocarcinoma (PDAC), but targeting mutant KRAS is still challenging. Here, an endoribonuclease-prepared small interfering RNA (esiRNA) library was used to screen new kinases that play critical roles in PDAC driven by KRAS gene mutations, and serine/threonine kinase 31 (STK31) was identified and characterized as a potential therapeutic target for KRAS-mutant PDAC. Our results showed that STK31 was upregulated in KRAS-mutant PDAC patients with poor survival and highly expressed in PDAC cell lines with KRASG12D mutation. Inhibition of STK31 in KRAS-mutant cell lines significantly reduced PDAC cell growth invitro and hindered tumor growth invivo. Gain and loss of function experiments revealed that STK31 is a downstream target of KRAS in PDAC. A pharmacological inhibition assay showed MAPK/ERK signaling involved in STK31 regulation. The further mechanistic study validated that c-Jun, regulated by KRAS/MAPK signaling, directly modulates the transcription level of STK31 by binding to its promoter region. Through RNA sequencing, we found that the cell cycle regulators CCNB1 and CDC25C are downstream targets of STK31. Taken together, our results indicate that STK31, which is the downstream target of the KRAS/MAPK/ERK/c-Jun signaling pathway in KRAS-mutant PDAC, promotes PDAC cell growth by modulating the expression of the cell cycle regulators CCNB1 and CDC25C.

Methylation of FAM110C is a synthetic lethal marker for ATR/CHK1 inhibitors in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest malignancies. An epigenetic-based synthetic lethal strategy provides a novel opportunity for PDAC treatment. Finding more DNA damage repair (DDR)-related or cell fate-related molecules with aberrant epigenetic changes is becoming very important. Family with sequence similarity 110C (FAM110C) is a cell fate-related gene and its function in cancer remains unclear. Seven cell lines, 34 cases of intraductal papillary mucinous neoplasm (IPMN), 15 cases of mucinous cystic neoplasm (MCN) and 284 cases of PDAC samples were employed. Methylation-specific PCR, western blot, CRISPR knockout, immunoprecipitation and a xenograft mouse model were used in this study. FAM110C is methylated in 41.18% (14/34) of IPMN, 46.67% (7/15) of MCN and 72.89% (207/284) of PDAC, with a progression trend from IPMN/MCN to pancreatic cancer (P = 0.0001, P = 0.0389). FAM110C methylation is significantly associated with poor overall survival (OS) (P = 0.0065) and is an independent prognostic marker for poor OS (P = 0.0159). FAM110C inhibits PDAC cells growth both in vitro and in vivo, serving as a novel tumor suppressor. FAM110C activates ATM and NHEJ signaling pathways by interacting with HMGB1. Loss of FAM110C expression sensitizes PDAC cells to VE-822 (an ATR inhibitor) and MK-8776 (a CHK1 inhibitor). FAM110C methylation is a potential diagnostic and prognostic marker in PDAC, and its epigenetic silencing sensitizes PDAC cells to ATR/CHK1 inhibitors.

Abstract 4588: Combination of MTA-cooperative PRMT5 inhibitor and direct mutant-selective KRAS inhibitors as a novel therapeutic approach for MTAP-deficient pancreatic cancer

Abstract KRAS is mutationally activated in 95% of pancreatic ductal adenocarcinoma (PDAC) patients. Direct KRAS inhibitors are under intense preclinical and clinical development, with two KRASG12C mutant-selective inhibitors (G12Ci) now approved. However, treatment-associated resistance to KRAS inhibitors has been reported in the clinic highlighting an urgent need to identify both additional therapeutic targets and novel combination treatment strategies with KRAS inhibitors. To that end, we performed a CRISPR/Cas9 loss-of-function screen using a library targeting ~2,500 druggable genes in KRASG12C/D-mutant PDAC cell lines. We identified multiple genes that were essential for PDAC cell growth and selected PRMT5, a protein arginine methyltransferase, to assess as a novel therapeutic target for pancreatic cancer. We found that suppression of PRMT5 activity using two distinct first-generation clinical candidate small molecule inhibitors (JNJ-64619178 and GSK3326595) demonstrated single agent activity and reduced PDAC cell growth. We further assessed a mechanistically distinct MTA-cooperative clinical candidate PRMT5 inhibitor, MRTX1719, that is selective for MTAP-deleted tumors, which occur in ~25% of PDAC patients. We determined that MRTX1719 exhibited low nanomolar GI50 activities in MTAP-deficient but not MTAP wild-type KRAS-mutant cell lines. Unexpectedly, PDAC cells treated with MRTX1719 exhibited a paradoxical activation of ERK, suggesting that combined inhibition of PRMT5 and KRAS might be therapeutically advantageous. In support of our hypothesis, we demonstrated that combination treatment with MRTX1719 and mutant selective KRAS inhibitors (G12Ci/MRTX849 and G12Di/MRTX1133) further sensitized KRASG12C/D-mutant PDAC cells to KRAS inhibition in short- and long-term growth assays as well as in vivo xenograft studies. Our ongoing studies are evaluating the consequences of co-targeting PRMT5 and KRAS on gene expression changes using RNA-Seq and cancer cell signaling pathways using RPPA analyses. In summary, our data support concurrent inhibition of PRMT5 and KRAS as a promising therapeutic strategy for MTAP-deficient KRAS-mutant pancreatic cancer. Citation Format: Kristina Drizyte-Miller, Wen-Hsuan Chang, Andrew M. Waters, Clint A. Stalnecker, Junning Wang, Andrew J. Aguirre, Adrienne D. Cox, Channing J. Der. Combination of MTA-cooperative PRMT5 inhibitor and direct mutant-selective KRAS inhibitors as a novel therapeutic approach for MTAP-deficient 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 4588.

Abstract 445: Determining the molecular and biologic effects of HER3 and IDH1 antagonism on liver endothelium-PDAC crosstalk

Abstract INTRODUCTION: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy due to the lack of early diagnosis and limited response to treatments. Austere and nutrient-deprived conditions typically present in the PDAC tumor microenvironment (TME) induce chemotherapy resistance. Therefore, novel therapeutic strategies are needed. We have previously demonstrated the primary tumor expresses IDH1 which supports the pro-survival oxidative phosphorylation (OXPHOS) metabolism, and has been successfully targeted by ivosidenib, an IDH1 inhibitor, in preclinical studies. Meanwhile, endothelial cells (ECs) from the liver, the most common site of PDAC metastases, secrete soluble factors in the conditioned medium (CM) that activate HER3, a receptor tyrosine kinase, involved in cell proliferation and metabolism. OBJECTIVES: We sought to (1) uncover the mechanisms involved in driving the metabolic differences between primary and metastatic tumors through the utilization of ECs from the liver (2) conduct metabolic analyses to determine metabolic effects of HER3 activation, and (3) examine potential synergism of combination therapy against HER3 and IDH1 in primary and metastatic niches. METHODS: The effect of CM on PDAC cell growth was determined by PICO Green Assay. The effects of ECs HER3/IDH1 inhibitions on cellular metabolism were measured by multiple metabolic assays including Seahorse, CellTiter-Glo, lactate ELISA, and TMRE assays. HER3-specific antibody Seribantumab, HER3 inhibitor Sapitanib, and IDH1 inhibitor ivosidenib were used to block HER3 and/or IDH1. Moreover. A cell viability-based synergy study was performed to assess the effects of HER3-IDH1 combination. Effects of HER3 and IDH1 inhibitions on PDAC tumor growth and mouse survival in vivo were determined in syngeneic, orthotopic models with PDAC primary tumors and liver metastases. RESULTS: CM from liver ECs significantly increased cell growth in PDAC cells in vitro and increased glycolytic tropism in multiple HER3 positive PDAC cell lines measured by metabolic assays mentioned above. HER3 antibody and HER3 inhibitor both increased oxidative consumption rate (OCR), an indirect measure of OXPHOS, and decreased extracellular acidification rate (ECAR), a measure of glycolysis measured by Seahorse. Combination therapy utilizing HER3 and IDH1 inhibitors revealed synergy. In our in vivo tumor model, mice treated with combination therapy had significantly longer overall survival than those given either monotherapy or no treatment. CONCLUSIONS: Our results demonstrated a paracrine role of liver ECs in promoting cell growth via activating HER3 in PDAC cells as well as reprograming cellular metabolism from OXPHOS towards glycolysis. Identification of the metabolic shift that takes place in metastatic PDAC to the liver due to HER3 activation gives way for the development of a potentially lethal drug combination. Citation Format: Christina S. Boutros, Alexander W. Loftus, Moeez Rathore, Mehrdad Zarei, Kimberly Curry, Jordan M. Winter, Rui Wang. Determining the molecular and biologic effects of HER3 and IDH1 antagonism on liver endothelium-PDAC crosstalk [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 445.

Abstract 1667: The role of CCN1 in mutant K-RAS addiction in pancreatic cancer

Abstract Mutant K-RAS addiction of pancreatic ductal adenocarcinoma cells (PDAC) coordinates their transformation, proliferation, and survival. Therefore, initially, it had an impression that targeting mutant K-RAS could be an ideal strategy to treat PDAC. However, after decades of studies, no effective RAS inhibitors reached the clinic, indicating that RAS-oncoprotein is an undruggable target in PDAC. Thus, identifying a new target linked with K-RAS signaling was most needed. Using genetically engineered cell lines and K-RAS-driven genetically engineered mouse models (KC or KPC) with or without pancreas-specific CCN1 conditional knockout background, we identified a positive feedback circuit between CCN1 and mutant K-RAS expression. Moreover, blocking this feedback via conditional knockout of CCN1 in the pancreas significantly reduced invasive phenotypes, PDAC growth, and metastasis in the lung in KC and KPC mice. Mechanistically, we found that the K-RAS-CCN1 feedback circuit is regulated by microRNA miR-145 via a feedforward mechanism. Collectively, these studies highlight the clinical potential of targeting CCN1 in PDAC. (VA Merit Grant supports this work) Citation Format: Anjali Kambhampati, Archana De, Inamul Haque, Snigdha Banerjee, Sushanta K. Banerjee. The role of CCN1 in mutant K-RAS addiction in 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 1667.

Abstract 5957: Development of a selective, oral ATP-competitive CDK9 inhibitor, BLX-3030, for treatment of pancreatic cancer

Abstract Introduction Transcription regulators, such as cyclin-dependent kinase 9 (CDK9), have been implicated in the super-enhancer driven transcriptional control of oncogenes in multiple cancers including pancreatic cancer. CDK9 has been shown to be employed by cancer cells for the constant production of short-lived oncoproteins such as c-Myc to maintain their survival. Due to its critical role in regulating transcription in cancer cells, CDK9 has emerged as a potential therapeutic target. Here, we report the antitumor activity of a selective, oral ATP-competitive CDK9 inhibitor, BLX-3030, in preclinical models for pancreatic cancer. Experimental Procedures Fragment-based MolecuLern driven AI workflow methods were used to design a series of ATP-competitive CDK9 specific kinase inhibitors. BLX-3030 was selected as one of the most potent leads with an IC50 of 1.1 nM in a cell-free CDK9 kinase assay. A Sulforhodamine B (SRB) assay was used to evaluate the cell growth inhibitory activity of BLX-3030 in a panel of 10 pancreatic ductal adenocarcinoma (PDAC) and 2 pancreatic neuroendocrine tumor (pNET) cell lines. Western blotting was used to determine the effects of BLX-3030 treatment on c-Myc expression in cancer cells. The combination effects between BLX-3030 and standard of care regimen, gemcitabine (GEM) and nab-paclitaxel (NP), was assessed using the SRB assay. Finally, the in vivo antitumor activity of BLX-3030 alone or in combination with GEM and NP was evaluated in a mouse cell line xenograft model for pancreatic cancer. Results BLX-3030 exhibited potent cell growth inhibitory activity in the panel of pancreatic cancer cell lines with IC50 values ranging from 133.5 nM to 508.1 nM. The potency of BLX-3030 in the cell lines appears to correlate with the expression level of c-Myc in the cell lines. BLX-3030 showed similar activities in PDAC and pNET cell lines with mean IC50 values of 244.5 nM and 194.6 nM, respectively. However, treatment with BLX-3030 for 48 hours did not seem to significantly reduce c-Myc expression in PSN-1 PDAC cells. When combined with GEM and NP, BLX-3030 demonstrated mostly additive effects in PSN-1 cells. Preliminary data from the in vivo study show that BLX-3030 improves the antitumor activity of GEM and NP. Conclusion In summary, BLX-3030 potently inhibited the growth of both PDAC and pNET cells in vitro. The growth inhibitory activity of BLX-3030 in PDAC cell lines correlates with their c-Myc expression levels. BLX-3030 showed an additive effect when combined with standard of care drugs (GEM and NP) in PDAC cell lines. Overall, the novel CDK9 inhibitor, BLX-3030, demonstrated potent activity in pancreatic cancer cell line models and presents a promising preclinical lead for pancreatic cancer. (This work was supported in part by Biolexis Therapeutics and the Seena Magowitz Foundation). Citation Format: Yesenia Barrera-Millan, Zhaoliang Li, Hariprasad Vankayalapati, David J. Bearss, Daniel D. Von Hoff, Haiyong Han. Development of a selective, oral ATP-competitive CDK9 inhibitor, BLX-3030, for treatment 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 5957.

Abstract 2087: Targeting lipid metabolism in pancreatic cancer

Abstract Background PIKfyve is a lipid kinase that serves as the sole source of cellular PI(3,5)P2 and PI5P, critical phosphatidylinositols for autophagy and lysosome function. Pancreatic ductal adenocarcinoma (PDAC) is known to upregulate and depend on lysosomal functions such as autophagy to exist in its harsh, nutrient-disrupted microenvironment. Thus, we aimed to establish PIKfyve as a therapeutic target in PDAC. Using a PIKfyve-knockout genetically engineered mouse model of pancreatic cancer (KC and KPC), we determined that PIKfyve was necessary for PDAC development. Given these results, we hypothesized that PIKfyve was critical for maintaining metabolic homeostasis in PDAC and that inhibiting PIKfyve would be an efficacious therapeutic strategy for PDAC. Methods and results We first validated the known role of PIKfyve in regulating autophagy using CRISPRi mediated knockdown, PIKfyve inhibitors Apilimod and ESK981, and a PIKfyve degrader, which all demonstrated that PIKfyve perturbation disrupted autophagic flux and suppressed PDAC cell growth. Little is known about the metabolic impacts of PIKfyve inhibition in PDAC beyond disruption of autophagy. Thus, we paired RNA-seq and a metabolic CRISPR screen to discover that PIKfyve inhibition obligated PDAC cells to upregulate de novo lipid synthesis. Specifically, PDAC cells upregulated fatty acid synthesis genes ACACA and FASN upon PIKfyve inhibition. Additionally, these genes were synthetically critical in the context of PIKfyve inhibition. We further performed metabolomics and lipidomics which revealed accumulation of sphingolipids upon PIKfyve inhibition. Given this, we concluded that PIKfyve regulates lipid homeostasis in PDAC. To further establish PIKfyve as a target in PDAC, we investigated the role of PIKfyve upon inhibition of KRAS, which drives PDAC through MAPK pathway signaling. First, KRAS/MAPK inhibition diminished expression of FASN and ACACA, genes synthetically essential upon PIKfyve inhibition. Additionally, KRAS/MAPK inhibition upregulated autophagy which was attenuated upon concurrent PIKfyve inhibition. Given this metabolic conflict between KRAS/MAPK and PIKfyve, we assessed the effects of dual inhibiting KRAS/MAPK and PIKfyve in a syngeneic orthotopic model. Encouragingly, combination therapy of trametinib (MEK inhibitor) and ESK981 eliminated tumor burden in most of mice, while single treatments only had modest effects. Further, we established a patient-derived xenograft model and showed that while the treatment with individual compounds trametinib, MRTX-1133 (KRASG12D inhibitor), and ESK981 all suppressed tumor growth, the combination of ESK981 and trametinib or ESK981 and MRTX-1133 caused substantial tumor regression in nearly all mice. Taken together, targeting lipid metabolism through inhibition of PIKfyve is a promising therapeutic strategy for PDAC, particularly in combination with the numerous KRAS/MAPK inhibitors in clinical development. Citation Format: Caleb Cheng, Jasmine P. Wisniewski, Ahmet Korkaya, Bailey Jackson, Rahul Mannan, Somnath Mahapatra, Nicholas Rossiter, Rupam Bhattacharyya, Pietro Morlacchi, Sydney Peters, Yuanyuan Qiao, Costas A. Lyssiotis, Arul M. Chinnaiyan. Targeting lipid metabolism in 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 2087.

Abstract 2904: Small molecule inhibitor targeting DDR represents a novel therapeutic strategy for the treatment of pancreatic ductal adenocarcinoma (PDAC)

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is ranked as the second leading cause of cancer-related mortality with a 5-year survival rate of merely 12%. The complexity of PDAC lies in its intrinsic resistance to treatment, attributable to the desmoplastic tumor microenvironment, characterized by an abundance of tumor-promoting extracellular matrix (ECM) and stromal cells. We hypothesize that Discoidin Domain Receptors (DDR), members of the receptor tyrosine kinase family known for their affinity to fibrillar collagens within the ECM, play an important role in promoting PDAC growth and progression. While several drugs targeting DDR proteins have been developed, no inhibitors to DDR proteins are specific or approved for PDAC treatment, thus necessitating the development of novel therapeutic interventions. Methods. A comprehensive high-throughput screening approach employing small molecule inhibitors was executed to identify authentic inhibitors targeting DDR. Subsequently, a rigorous evaluation of specificity of these small molecules was conducted through structural activity relationship (SAR) analyses and reference-based assessments. The efficacy of candidate inhibitors was assessed through functional assays, while mechanistic insights were gleaned using a multifaceted approach, including RNA-seq, real-time PCR, western blot analysis, in vivo experimentation, and Phospho-Receptor Tyrosine Kinase (RTK) profiling to discern any unintended off-target effects. Results: Using an unbiased screening approach, we identified 13 inhibitors that specifically target DDR. SAR studies and cellular thermal shift assay validated CIDD-0108633 (8633) as a bona fide DDR2 inhibitor. Functional assays and RNA-seq analyses demonstrated 8633's capacity to inhibit PDAC growth, metastatic potential, and expression of DNA replication, cell-cycle, and DNA damage repair genes. Combination therapy of 8633 with gemcitabine synergistically reduced PDAC cell viability and metastatic potential. Mechanistic studies revealed the role of ABC transporters in mediating gemcitabine resistance. RTK profiling shows moderate kinase selectivity. In vivo studies affirmed its antitumor effects. Conclusions: The results indicate that the selective inhibition of DDR with small molecule inhibitors suppresses PDAC cell growth and progression, implying that DDR inhibitors have the potential to be developed as safe and efficacious therapeutics for the treatment of pancreatic cancers. Citation Format: Trong Phat Do, Jack Prochnau, Prabhakar Pitta Venkata, Sajid Khan, Santosh Timilsina, Deepika Singh, Daisy Medina, Radhika Amaradhi, Daohong Zhou, Matthew Hart, Stanton McHardy, Manjeet Rao. Small molecule inhibitor targeting DDR represents a novel therapeutic strategy for the treatment of pancreatic ductal adenocarcinoma (PDAC) [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 2904.

Structural Basis for Multivalent MUC16 Recognition and Robust Anti-Pancreatic Cancer Activity of Humanized Antibody AR9.6.

Mucin-16 (MUC16) is a target for antibody-mediated immunotherapy in pancreatic ductal adenocarcinoma (PDAC) among other malignancies. The MUC16-specific monoclonal antibody AR9.6 has shown promise for PDAC immunotherapy and imaging. Here, we report the structural and biological characterization of the humanized AR9.6 antibody (huAR9.6). The structure of huAR9.6 was determined in complex with a MUC16 SEA (Sea urchin sperm, Enterokinase, Agrin) domain. Binding of huAR9.6 to recombinant, shed, and cell-surface MUC16 was characterized, and anti-PDAC activity was evaluated in vitro and in vivo. HuAR9.6 bound a discontinuous, SEA domain epitope with an overall affinity of 88 nmol/L. Binding affinity depended on the specific SEA domain(s) present, and glycosylation modestly enhanced affinity driven by favorable entropy and enthalpy and via distinct transition state thermodynamic pathways. Treatment with huAR9.6 reduced the in vitro growth, migration, invasion, and clonogenicity of MUC16-positive PDAC cells and patient-derived organoids (PDO). HuAR9.6 blocked MUC16-mediated ErbB and AKT activation in PDAC cells, PDOs, and patient-derived xenografts and induced antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. More importantly, huAR9.6 treatment caused substantial PDAC regression in subcutaneous and orthotopic tumor models. The mechanism of action of huAR9.6 may depend on dense avid binding to homologous SEA domains on MUC16. The results of this study validate the translational therapeutic potential of huAR9.6 against MUC16-positive PDACs.

DDX3X interacts with SIRT7 to promote PD-L1 expression to facilitate PDAC progression

Pancreatic ductal adenocarcinoma (PDAC) is recognized as the most aggressive and fatal malignancy. A previous study reported that PDAC patients who exhibit elevated levels of DDX3X have a poor prognosis and low overall survival rate. However, the underlying molecular mechanism remains unclear. This study aimed to investigate the specific roles of DDX3X in PDAC. Multiple bioinformatics analyses were used to evaluate DDX3X expression and its potential role in PDAC. In vitro and in vivo studies were performed to assess the effects of DDX3X on PDAC cell growth. Furthermore, Western blotting, quantitative PCR, immunohistochemistry, immunofluorescence, mass spectrometry, coimmunoprecipitation and multiplexed immunohistochemical staining were conducted to identify the specific regulatory mechanism in PDAC. The results verified that DDX3X expression is notably upregulated in the tumor tissue vs. normal tissue of PDAC patients. DDX3X knockdown markedly suppressed the proliferation, invasion and migration of PDAC cells in vitro and inhibited tumor growth in vivo. Conversely, overexpression of DDX3X induced the opposite effect. Further studies supported that the DDX3X protein can associate with sirtuin 7 (SIRT7) to stimulate PDAC carcinogenesis and progression. Furthermore, SIRT7 inhibition significantly impeded DDX3X-mediated tumor growth both ex vivo and in vivo. The results also revealed that programmed death ligand 1 (PD-L1) expression is positively correlated with DDX3X expression. These results reveal significant involvement of the DDX3X-SIRT7 axis in the initiation and advancement of PDAC and offer previously undiscovered therapeutic options for PDAC management.

Abstract C031: Differential growth of pancreatic ductal adenocarcinoma cells in serum amyloid A (SAA) deficient mouse model through immune modulation

Abstract Objective: Pancreatic ductal adenocarcinoma (PDAC) is a formidable challenge to cure and expected to be the second leading cause of cancer-related deaths in the US by 2030. Its aggressive nature stems from late diagnosis and early metastasis. PDAC cells interact with the host system and impair the immune response. This communication leads to the production of mediators by host cells, creating an environment that supports PDAC cells growth and the formation of premetastatic niches. Despite progress, the specific factors and mechanisms underlying immune modulation in PDAC progression are not fully understood. Methods: PDAC mice models (WT-KPCL and KOSAA-KPCL) were developed by administering luciferase-expressing KPC cells (KPCL) intraperitoneally into eight weeks old male C57BL/6 mice. The control groups (WT and KOSAA) received PBS. Plasma proteomics was analyzed by using Mass Spectrometry (MS). PDAC cells growth was visualized by bioluminescence using Lago X in-vivo imaging system. Results: During tumor progression, KPCL mice exhibited significant weight loss, with the loss of adipose tissue and skeletal muscle, indicating a hallmark of pancreatic cancer. To identify potential circulating molecules involved in tumor progression, we conducted proteomics analysis on plasma samples using MS. Comparative analysis revealed that out of 734 circulating proteins, 86 were upregulated and 35 were downregulated in tumor mice compared to controls. Serum Amyloid A 1 and 2 were found to be the most abundantly expressed proteins in the plasma of tumor mice, with a remarkable 1024-fold increase compared to controls. To determine the major source of SAA in tumor mice, we measured SAA1, 2, and 3 expressions in various tissues, including adipose tissue, liver, muscle, and tumor. Interestingly, adipose tissue displayed a substantial contribution to SAA levels in WT-KPCL. However, we were unable to detect SAA expression in the tumor tissue from KOSAA-KPCL indicating negligible source of SAA from KPCL cells. Intrigued by these findings, we studied the role of SAA in PDAC cell proliferation in-vivo. Luciferase-expressing KPC cells were injected into SAA-KO mice (KOSAA-KPCL) and controls (WT-KPCL). Unexpectedly, bioluminescence imaging revealed a dramatic growth of KPC cells in the pancreas of SAA-deficient animals. This was supported by a significantly higher rate of metastasis to the liver, increased by 62.5% in KOSAA-KPCL compared to WT-KPCL. SAA is retinol-binding protein and retinoic acid plays a vital role in the immune system. We found a decreased concentration of retinoic acid in the plasma of KOSAA-KPCL mice, with a significant decrease in CD8 expression in tumor tissue isolated from KOSAA-KPCL. These results suggest that SAA modulates immune response during PDAC cells growth. Conclusion: Our study demonstrates that adipose tissue is likely a major source of SAA, where the absence of SAA leads to enhanced PDAC cells growth and metastasis through immune modulation. These findings suggest the therapeutic potential of SAA for pancreatic cancer. Citation Format: Yuki Takamuku, Mengistu Lemecha, Keiichi Itakura. Differential growth of pancreatic ductal adenocarcinoma cells in serum amyloid A (SAA) deficient mouse model through immune modulation [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C031.

Abstract B077: KRASG12D inhibitor MRTX1133 synergizes with the next generation nuclear transport protein inhibitor eltanexor resulting in enhanced antitumor activity against pancreatic ductal adenocarcinoma

Abstract Introduction: The novel KRAS inhibitor MRTX1133 that targets the G12D isoform, a major KRAS mutation in pancreatic ductal adenocarcinoma (PDAC) patients, has been recently developed and expected to benefit a large PDAC patient population. However, based on clinical outcomes of sotorasib (KRASG12C inhibitor) in the lung cancer setting, it is likely that the KRASG12D inhibitor will also show modest increase in disease-free survival in PDAC patients because of emergence of drug resistance. Hence, there is a pressing need to identify strategies to enhance the efficacy of KRAS inhibitors in PDAC. There is evidence that nuclear transport protein exportin 1 (XPO1) is a druggable vulnerability in KRAS mutant cancers. XPO1 guides the movement of several key tumor suppressors, genome surveillance proteins and transcription factors out of the nucleus and is known to be overexpressed in many cancers including PDAC. In this study, using in vitro and in vivo models, we have tested eltanexor/KPT-8602 (an investigational, next generation selective inhibitor of XPO1) for its ability to synergize with the KRASG12D inhibitor and to sensitize KRASG12D inhibitor resistant PDAC cells. Methods: We examined the cytotoxicity and molecular profile of KRASG12D inhibitor MRTX1133 in combination with XPO1 inhibitor KPT-8602 in KRASG12D mutant 2D and 3D cellular/spheroid models. The antitumor activity of the combination was further evaluated in a KRASG12D mutant cell line-derived xenograft model. Results: KPT-8602 synergized with MRTX1133 yielding suppressed growth of KRASG12D mutant PDAC cells, but not in KRASwt, KRASG12V, and KRASG12C cell lines (NCI RASless MEFs). Similar synergistic effects were also seen in 3D culture models where the combination synergistically enhanced the disintegration of PDAC spheroids. In addition, the combination reduced the clonogenic potential of KRASG12D mutant PDAC cells. Furthermore, we developed a PDAC cell line resistant to MRTX1133 and observed that such MRTX1133-resistant cell line was acutely sensitive to treatment with KPT-8602. Moreover, KPT-8602 and MRTX1133 combination resulted in enhanced inhibition of KRAS downstream effector pathways. In xenograft studies, oral administration of KPT-8602 and intraperitoneal administration of MRTX1133 at sub-optimal doses demonstrated remarkable suppression of KRASG12D mutant HPAC tumor burden in ICR-SCID mice. Conclusion: This is the first study showing that XPO1 inhibitor KPT-8602 can synergize with KRASG12D inhibitor MRTX1133. This combination can simultaneously target direct KRAS downstream effectors and the nuclear transport machinery warranting further clinical investigations. Citation Format: Husain Y. Khan, Amro Aboukameel, Md Hafiz Uddin, Mohammad Najeeb Al-Hallak, Eliza W. Beal, Ibrahim Azar, Erkan Baloglu, Rafic Beydoun, Ammar Sukari, Misako Nagasaka, Amr Mohamed, Ahmed Kaseb, Philip A. Philip, Basel El-Rayes, Herbert Chen, Anthony Shields, Asfar Azmi. KRASG12D inhibitor MRTX1133 synergizes with the next generation nuclear transport protein inhibitor eltanexor resulting in enhanced antitumor activity against pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B077.

Epigenetic silencing ZSCAN23 promotes pancreatic cancer growth by activating Wnt signaling

ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) is the most malignant tumor. Zinc finger and SCAN domain-containing protein 23 (ZSCAN23) is a new member of the SCAN domain family. The expression regulation and biological function remain to be elucidated. In this study, we explored the epigenetic regulation and the function of ZSCAN23 in PDAC. ZSCAN23 was methylated in 60.21% (171/284) of PDAC and its expression was regulated by promoter region methylation. The expression of ZSCAN23 inhibited cell proliferation, colony formation, migration, invasion, and induced apoptosis and G1/S phase arrest. ZSCAN23 suppressed Panc10.05 cell xenograft growth in mice. Mechanistically, ZSCAN23 inhibited Wnt signaling by interacting with myosin heavy chain 9 (MYH9) in pancreatic cancer cells. ZSCAN23 is frequently methylated in PDAC and may serve as a detective marker. ZSCAN23 suppresses PDAC cell growth both in vitro and in vivo.

Abstract A046: nSMase2-generated ceramide promotes PDA aggression through exosome reprogramming of the stroma

Abstract The pleiotropic roles of nSMase2-generated ceramide include regulation of intracellular ceramide signaling and exosome biogenesis. Packaging of specific cellular constituents, such as prion protein and specific miRNAs involved in cancer progression, into exosomes are dependent on nSMase2. Employing the KPC mouse model of pancreatic ductal adenocarcinoma (PDA), we demonstrate that nSMase2 regulates exosome secretion and in vivo growth of PDA cells. PDA cell exosomes generated through nSMase2 accelerate PDA progression, and PDA cell exosomes generated independently of nSMase2 are anti-tumorigenic during PDA progression. Homozygous loss of Smpd3 (gene which encodes nSMase2) in KPC mouse pancreatic epithelial cells during development extends survival of KPC mice. KPC; Smpd3f/f mice display less PanIN and tumor burden compared to KPC; Smpd3wt/wt controls. In addition, nSMase2 loss in KPC PDA cells promoted a PDA subtype switch from aggressive basal-like to classical. RNA-sequencing of PDA cell lines generated from pancreatic tumors of end-stage KPC; Smpd3wt/wt, KPC; Smpd3f/wt, and KPC; Smpd3f/f mice demonstrated that nSMase2-generated ceramide is a key regulator of intracellular pathways regulating tumor microenvironment dynamics in PDA cells including fibrosis and integrins. We observed a decrease in extracellular matrix collagen abundance and fewer activated stellate cells and fibroblasts in KPC; Smpd3f/f compared to control KPC; Smpd3wt/wt mouse pancreata. Exosomes isolated from KPC; Smpd3wt/wt PDA cell lines significantly stimulate proliferation of primary murine stellate cells in vitro, while exosomes isolated from KPC; Smpd3f/f PDA cell lines do not. Abrogation of Smpd3 expression also affected immune cell infiltration, as demonstrated by a significant increase in iNOS+ F4/80+ double positive proinflammatory macrophages in KPC; Smpd3f/f pancreata when compared to KPC; Smpd3wt/wt pancreata. Lipidomics analysis of epithelial cell lines generated from end-stage pancreatic tumors of KPC; Smpd3f/f and KPC; Smpd3wt/wt mice demonstrated an alteration in hundreds of lipid species including ceramides, triacylglycerides, sphingomyelins, and phosphatidylcholines. Proteomics and small RNA-seq analysis of exosomes secreted from KPC; Smpd3wt/wt and KPC; Smpd3f/f PDA cell lines demonstrated significant alteration of >700 proteins and 12 miRNAs. Our patient data demonstrate that low nSMase2 expression in treatment naïve primary resected pancreatic tumors significantly correlates with fewer CD31+ endothelial cells. In parallel, loss of Smpd3 resulted in a reduction in CD31+ endothelial cells in pancreatic tumors of KPC; Smpd3f/f mice when compared to KPC; Smpd3wt/wt controls. Thus, nSMase2 is a critical mediator of angiogenesis during PDA progression, which is integral to chemotherapeutic response. Collectively, our data show that nSMase2-generated exosomes promote PDA tumorigenesis, specifically proliferation of stellate cells, which may in turn induce a fibrotic tumor microenvironment that affects vasculature development. Citation Format: Audrey M. Hendley, Atsushi Urano, Xianlu L. Peng, Sudipta Ashe, Tuan A. Phu, Martin Ng, Natanya R. Kerper, David I. Berrios, Jonghyun Lee, David Jin, Gun H. Jang, Oghenekevwe M. Gbenedio, Jeroen P. Roose, Jen J. Yeh, Steven Gallinger, Andrew V. Biankin, Vasilis Ntranos, David K. Chang, David W. Dawson, Grace E. Kim, Valerie M. Weaver, Robert L. Raffai, Matthias Hebrok. nSMase2-generated ceramide promotes PDA aggression through exosome reprogramming of the stroma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A046.

Antitumor Activity of the Xanthonoside XGAc in Triple-Negative Breast, Ovarian and Pancreatic Cancer by Inhibiting DNA Repair.

Dysregulation of the DNA damage response may contribute to the sensitization of cancer cells to DNA-targeting agents by impelling cell death. In fact, the inhibition of the DNA repair pathway is considered a promising anticancer therapeutic strategy, particularly in combination with standard-of-care agents. The xanthonoside XGAc was previously described as a potent inhibitor of cancer cell growth. Herein, we explored its antitumor activity against triple-negative breast cancer (TNBC), ovarian cancer and pancreatic ductal adenocarcinoma (PDAC) cells as a single agent and in combination with the poly(ADP-ribose) polymerase inhibitor (PARPi) olaparib. We demonstrated that XGAc inhibited the growth of TNBC, ovarian and PDAC cells by inducing cell cycle arrest and apoptosis. XGAc also induced genotoxicity, inhibiting the expression of DNA repair proteins particularly involved in homologous recombination, including BRCA1, BRCA2 and RAD51. Moreover, it displayed potent synergistic effects with olaparib in TNBC, ovarian cancer and PDAC cells. Importantly, this growth inhibitory activity of XGAc was further reinforced in a TNBC spheroid model and in patient-derived ovarian cancer cells. Also, drug-resistant cancer cells showed no cross-resistance to XGAc. Additionally, the ability of XGAc to prevent cancer cell migration was evidenced in TNBC, ovarian cancer and PDAC cells. Altogether, these results highlight the great potential of acetylated xanthonosides such as XGAc as promising anticancer agents against hard-to-treat cancers.

Coordinate transcriptional regulation of ErbB2/3 by C-terminal binding protein 2 signals sensitivity to ErbB2 inhibition in pancreatic adenocarcinoma

There is a critical need to identify new therapeutic vulnerabilities in pancreatic ductal adenocarcinoma (PDAC). Transcriptional co-regulators C-terminal binding proteins (CtBP) 1 and 2 are highly overexpressed in human PDAC, and CRISPR-based homozygous deletion of Ctbp2 in a mouse PDAC cell line (CKP) dramatically decreased tumor growth, reduced metastasis, and prolonged survival in orthotopic mouse allografts. Transcriptomic profiling of tumors derived from CKP vs. Ctbp2-deleted CKP cells (CKP/KO) revealed significant downregulation of the EGFR-superfamily receptor Erbb3, the heterodimeric signaling partner for both EGFR and ErbB2. Compared with CKP cells, CKP/KO cells also demonstrated reduced Erbb2 expression and did not activate downstream Akt signaling after stimulation of Erbb3 by its ligand neuregulin-1. ErbB3 expression in human PDAC cell lines was similarly dependent on CtBP2 and depletion of ErbB3 in a human PDAC cell line severely attenuated growth, demonstrating the critical role of ErbB3 signaling in maintaining PDAC cell growth. Sensitivity to the ErbB2-targeted tyrosine kinase inhibitor lapatinib, but not the EGFR-targeted agent erlotinib, varied in proportion to the level of ErbB3 expression in mouse and human PDAC cells, suggesting that an ErBb2 inhibitor can effectively leverage CtBP2-driven transcriptional activation of physiologic ErbB2/3 expression and signaling in PDAC cells for therapeutic benefit.

AUM302, a novel triple kinase PIM/PI3K/mTOR inhibitor, is a potent in vitro pancreatic cancer growth inhibitor.

Pancreatic cancer is one of the leading causes of cancer deaths, with pancreatic ductal adenocarcinoma (PDAC) being the most common subtype. Advanced stage diagnosis of PDAC is common, causing limited treatment opportunities. Gemcitabine is a frequently used chemotherapeutic agent which can be used as a monotherapy or in combination. However, tumors often develop resistance to gemcitabine. Previous studies show that the proto-oncogene PIM kinases (PIM1 and PIM3) are upregulated in PDAC compared to matched normal tissue and are related to chemoresistance and PDAC cell growth. The PIM kinases are also involved in the PI3K/AKT/mTOR pathway to promote cell survival. In this study, we evaluate the effect of the novel multikinase PIM/PI3K/mTOR inhibitor, AUM302, and commercially available PIM inhibitor, TP-3654. Using five human PDAC cell lines, we found AUM302 to be a potent inhibitor of cell proliferation, cell viability, cell cycle progression, and phosphoprotein expression, while TP-3654 was less effective. Significantly, AUM302 had a strong impact on the viability of gemcitabine-resistant PDAC cells. Taken together, these results demonstrate that AUM302 exhibits antitumor activity in human PDAC cells and thus has the potential to be an effective drug for PDAC therapy.

Critical Role of Novel O-GlcNAcylation of S550 and S551 on the p65 Subunit of NF-κB in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies, with a mere 5-year survival of ~10%. This highlights the urgent need for innovative treatment options for PDAC patients. The nuclear factor κB (NF-κB) is a crucial transcription factor that is constitutively activated in PDAC. It mediates the transcription of oncogenic and inflammatory genes that facilitate multiple PDAC phenotypes. Thus, a better understanding of the mechanistic underpinnings of NF-κB activation holds great promise for PDAC diagnosis and effective therapeutics. Here, we report a novel finding that the p65 subunit of NF-κB is O-GlcNAcylated at serine 550 and 551 upon NF-κB activation. Importantly, the overexpression of either serine-to-alanine (S-A) single mutant (S550A or S551A) or double mutant (S550A/S551A) of p65 in PDAC cells impaired NF-κB nuclear translocation, p65 phosphorylation, and transcriptional activity, independent of IκBα degradation. Moreover, the p65 mutants downregulate a category of NF-κB-target genes, which play a role in perpetuating major cancer hallmarks. We further show that overexpression of the p65 mutants inhibited cellular proliferation, migration, and anchorage-independent growth of PDAC cells compared to WT-p65. Collectively, we discovered novel serine sites of p65 O-GlcNAcylation that drive NF-κB activation and PDAC phenotypes, thus opening new avenues by inhibiting the NF-κB O-GlcNAcylation enzyme, O-GlcNAc transferase (OGT), for PDAC treatment in the future.

Dinaciclib exerts a tumor-suppressing effect via β-catenin/YAP axis in pancreatic ductal adenocarcinoma.

Dinaciclib, a cyclin-dependent kinase-5 (CDK5) inhibitor, has significant anti-tumor properties. However, the precise mechanism of dinaciclib requires further investigation. Herein, we investigated the anti-tumor functions and molecular basis of dinaciclib in pancreatic ductal adenocarcinoma (PDAC). PDAC and matched para-carcinoma specimens were collected from the patients who underwent radical resection. Immunohistochemistry was performed to assess CDK5 expression. Cell proliferation ability, migration, and invasion were measured using Cell Counting Kit-8, wound healing, and transwell assay, respectively. The cell cycle and apoptosis were assessed using flow cytometry. Gene expression was examined using RNA-seq and quantitative real-time PCR. Protein expression of proteins was measured by western blot analysis and immunofluorescence microscopy. Tumor-bearing mice were intraperitoneally injected with dinaciclib. CDK5 is highly expressed in PDAC. The expression level of CDK5 was significantly related to tumor size, T stage, and the American Joint Committee on Cancer stage. High CDK5 expression can predict poor survival in PDAC patients. In addition, the expression level of CDK5 might be an independent prognostic factor for PDAC patients. Dinaciclib inhibits the growth and motility of PDAC cells and induces apoptosis and cell cycle arrest in the G2/M phase. Mechanistically, dinaciclib down-regulated yes-associated protein (YAP) mRNA and protein expression by reducing β-catenin expression. Moreover, dinaciclib significantly inhibited PDAC cell growth in vivo . Our findings reveal a novel anti-tumor mechanism of dinaciclib in which it decreases YAP expression by down-regulating β-catenin at the transcriptional level rather than by activating Hippo pathway-mediated phosphorylation-dependent degradation.

Osteopontin secreted from obese adipocytes enhances angiogenesis and promotes progression of pancreatic ductal adenocarcinoma in obesity.

Obesity is a risk factor and poor prognostic factor for pancreatic ductal adenocarcinoma (PDAC), but the underlying mechanisms remain unclear. PDAC cells and obese visceral adipocytes (O-Ad) derived from mice and humans were used to analyze interactions between the two cell types, and human microvascular endothelial cells were used for angiogenesis assay. A xenograft mouse model with subcutaneously injected PDAC cells was used for animal studies. The relationship between visceral fat and prognosis was analyzed using resected tissues from PDAC patients with and without obesity. Conditioned media (CM) from O-Ad significantly increased PDAC cell growth and migration and angiogenic capacity in both human and mice cells, and blocking osteopontin (OPN) in O-Ad canceled O-Ad-induced effects in both mouse and human cells. In addition, O-Ad directly increased the migratory and tube-forming capacities of endothelial cells, while blocking OPN canceled these effects. O-Ad increased AKT phosphorylation and VEGFA expression in both PDAC and endothelial cells, and OPN inhibition in O-Ad canceled those O-Ad-induced effects. In the xenograft model, PDAC tumor volume was significantly increased in obese mice compared with lean mice, whereas blocking OPN significantly inhibited obesity-accelerated tumor growth. OPN expression in adipose tissues adjacent to human PDAC tumor was significantly higher in obese patients than in non-obese patients. In PDAC patients with obesity, high OPN expression in adipose tissues was significantly associated with poor prognosis. Obese adipocytes trigger aggressive transformation in PDAC cells to induce PDAC progression and accelerate angiogenesis via OPN secretion.