Abstract IA10: Targeting K-Ras for the treatment of pancreatic cancer.

Other risk factors for pancreatic cancer: Hormonal aspects

The effective treatment of pancreatic cancer relies on the diagnosis of the disease at an early stage, a difficult challenge. One major obstacle in the development of diagnostic biomarkers of early pancreatic cancer has been the dual expression of potential biomarkers in both chronic pancreatitis and cancer. To better understand the limitations of potential protein biomarkers, we used ICAT technology and tandem mass spectrometry-based proteomics to systematically study protein expression in chronic pancreatitis. Among the 116 differentially expressed proteins identified in chronic pancreatitis, most biological processes were responses to wounding and inflammation, a finding consistent with the underlining inflammation and tissue repair associated with chronic pancreatitis. Furthermore 40% of the differentially expressed proteins identified in chronic pancreatitis have been implicated previously in pancreatic cancer, suggesting some commonality in protein expression between these two diseases. Biological network analysis further identified c-MYC as a common prominent regulatory protein in pancreatic cancer and chronic pancreatitis. Lastly five proteins were selected for validation by Western blot and immunohistochemistry. Annexin A2 and insulin-like growth factor-binding protein 2 were overexpressed in cancer but not in chronic pancreatitis, making them promising biomarker candidates for pancreatic cancer. In addition, our study validated that cathepsin D, integrin beta1, and plasminogen were overexpressed in both pancreatic cancer and chronic pancreatitis. The positive involvement of these proteins in chronic pancreatitis and pancreatic cancer will potentially lower the specificity of these proteins as biomarker candidates for pancreatic cancer. Altogether our study provides some insights into the molecular events in chronic pancreatitis that may lead to diverse strategies for diagnosis and treatment of these diseases.

Introduction Pancreatic ductal adenocarcinoma (PDA) is a deadly cancer with limited treatment options. Oncogene KRAS mutations found in majority of PDA patients with KRAS(G12D) mutation being the most common. A new KRAS(G12D) specific inhibitor, MRTX1133 has been shown to induce rapid tumor regression in PDA models while the effect on stroma is only revealed by postmortem analyses of tumor specimens, making it hard to assess the dynamics of stroma remodeling. Furthermore, in clinical testing of KRAS(G12C) inhibitor, only ~50% of patients responded to the treatment despite the presence of correct genetic mutation in biopsy specimens, therefore, early and accurate assessment of the drug-target engagement is important in clinical setting. Our study was designed to test the hypothesis that multimetric MRI captures early responses to KRAS(G12D) inhibitor beyond the change of tumor size in a PDA model. Especially, changes in apparent diffusion coefficient (ADC) from diffusion weighted (DW) MRI, Ktrans from dynamic contrast enhanced (DCE) MRI, and magnetization transfer ratio (MTR). Methods A genetically engineered model of PDA bearing KRAS(G12D) and Trp53 mutation referred to as KPC was used. Male and female mice were enrolled to receive MRTX1133 at 30mg/kg BID or PBS via i.p injection. Murine PDA cells bearing KRAS(G12C) mutation were inoculated in the flank of C57BL/6 mice to grow subcutaneous tumors. In vivo DW- and DCE-MRI were obtained on a 9.4T Bruker system employing pulse sequences insensitive to respiratory motion. Results In KPC mice, striking increase of tumor ADC were detected as early as 48h after treatment initiation and was confirmed by statistical analyses at 48h and day7 accompanied by decrease of tumor size, in contrast to a trend of increased tumor size and decreased ADC in PBS-treated mice. H&E section revealed gross necrosis and extensive cell death. A clear increase of Ktrans suggesting an increase of capillary permeability and/or perfusion was detected at 48h, persisting to day7; while CD31 did not change significantly, capillary lumen area appeared enlarged at 48h, consistent with reduced interstitial fluid pressure resulted from clearance of stromal hyaluronic acid. A remarkable reduction of MTR at 48h clearly captured the stromal effect of MRTX1133 whereas no change observed in PBS-treated mice. Consistently, Sirius red (SR) staining for collagen revealed a high degree of heterogeneity where regions of depletion versus dense SR staining coexist. Lack of tumor regression and ADC change in KRAS(G12C) tumors confirmed the specificity of MRTX1133 to G12D however, a reduction of MTR was revealed, suggesting stromal impact as off-target effect of MRTX1133. Conclusion To our knowledge, this represents the first MRI study of the new KRAS inhibitor. The multimetric MRI approach is novel and significant because it captured MRTX1133-induced cancer cell death and stroma change over time. With further validation, these translatable imaging metrics can provide biological mechanism underneath tumor size change in the clinical setting. Citation Format: Mamta Gupta, Hoon Choi, Emma E. Furth, Miguel Joaquim, Stephen Pickup, Cynthia Clendenin, Margo Orlen, Thomas Karasic, Hee Kwon Song, Yong Fan, Peter O’Dwyer, Robert H. Vonderheide, Mark Rosen, Ben Z. Stanger, Rong Zhou. Quantitative MRI metrics capture pancreatic cancer and stroma responses to novel KRAS inhibitor [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 A098.

Desmoplasia of Pancreatic Ductal Adenocarcinoma

Pancreatic cancers respond poorly to chemotherapy, more effective and less toxic treatment is a crucial need. We have employed a biological assay (MTS assay) to identify small molecule compounds that induce cytotoxicity specifically in pancreatic cancer cells and not in normal cells by screening large number of compounds from combinatorial chemistry library. One of the compounds, LWKS22 (MW: 547.94) kills pancreatic cancer cells and not the normal pancreatic cells (h-tert HPNE). LWKS22 kills pancreatic cancer cells isolated from all the four stages and the EC50 is in the range of 2-10 μM, whereas for normal cells is 70 μM. Maximum tolerated dose is 80 mg/kg of body weight. At 20 mg/Kg body weight, it showed significant tumor shrinkage in human PDAC xenografts mice model when compared to PBS controls. Hypothesis and Specific Aims: The structure activity relationship studies of LWKS22 revealed that the parent compound structure is very important for its killing activity. LWKS22 was submitted to National drug therapeutics center, NIH, to screen for 60 different cancer cells derived from various human cancers. LWKS22 showed significant killing in human colon cancer cells and lymphoma cells, suggesting that this compound specifically targets some oncoproteins that are expressed in these cancer types. Now, the therapeutic potential of LWKS22 is confirmed. Identification of LWKS22 binding partner and its signaling mechanism to kill PDAC cancer cells will certainly add more benefits for the pancreatic cancer therapy. We hypothesize that LWKS22 can be developed as a therapeutic agent against pancreatic cancer along with other chemotherapy. To make it is a therapeutic agent, its mechanism of action, targeting receptors, signaling pathway, biodistribution and pharmacokinetics studies need to be conducted. Experimental Design: Therapeutic efficacy of LWKS22 was evaluated with mice bearing PDAC primary tumors. Photoaffinity labeling, chemoaffinity labeling methods and molecular biology techniques will be used to identify LWKS22’s binding partners. Impact: We have identified a compound that will kill cancer cells and not the normal cell. Targeting more than one pathway is one of the best ways to control this lethal disease. Functional characterization of this LWKS22 will certainly add more benefits for pancreatic cancer therapy. Citation Format: Yan Wang, Angela Echaverri, Anastasia Warde, Thiru Arumugham, Kit Lam, Pappanaicken Kumaresan. Small-molecule (LWKS22) drug discovery for pancreatic cancer therapy. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr A20.

Therapeutic Advances in Pancreatic Cancer

KRAS mutations occur in 95% of pancreatic ductal adenocarcinomas (PDAC) and are a well-validated driver of PDAC growth. Therefore, anti-KRAS therapies are expected to make a significant impact on the treatment of this deadly cancer, where there are currently no effective targeted therapies. Supporting this premise, early clinical trial results with KRASG12C inhibitors have shown promising disease control rates (84-100%) in KRASG12C-mutant PDAC. Despite these observations, two key issues limit the impact of KRASG12C inhibitors in PDAC. First, KRASG12C(OFF) mutations comprise less than 2% of KRAS mutations in PDAC. Second, patients initially responsive to KRASG12C inhibitors invariably relapse due to treatment-induced resistance. To begin qualifying KRAS inhibitors that target KRAS mutations more frequently found in PDAC, we characterized a RAS inhibitor that targets the multiple KRAS mutations as well as wild-type RAS proteins. We evaluated the impact of this inhibitor on RAS signaling and anti-proliferative activity in KRAS-mutant pancreatic cancer human cell lines and in a panel of RASless MEFs (ras-null mouse embryo fibroblasts) stably expressing exogenous RAS mutant proteins that are commonly found in PDAC. To identify genetic mechanisms of resistance to KRAS inhibition in pancreatic cancer, we applied CRISPR-Cas9 loss-of-function screens to KRASG12C-, KRASG12D-, KRASG12R-, and KRASQ61H-mutant PDAC cells treated with KRAS inhibitors to identify genes that modulate KRAS inhibitor anti-proliferative activity. We identified expected and novel mechanisms of resistance, including those that have been observed in patients treated with KRASG12C inhibitors. Citation Format: Andrew Michael Waters, Wen-Hsuan Chang, Clint Stalnecker, Cole Edwards, Runying Yang, Craig M. Goodwin, Adrienne D. Cox, Channing J. Der. Identification of resistance mechanisms to direct KRAS inhibition in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1075.

ABC transporters are the active transport systems of the cell involved in the export or import of a wide variety of molecules. We discovered that a member of the ABC transporter family called ABCC3 has a critical role in pancreatic cancer. ABCC3 blockade using genetic knockdown inhibits pancreatic cancer growth in vitro and in vivo. In addition, we demonstrate that knockdown of ABCC3 reduces cell proliferation by inhibition of STAT3 and HIF1α signalling pathways, which are key regulators of pancreatic cancer progression. A focused chemical library of indenes was screened for ABCC3 inhibition using ABCC3 expressing pancreatic tumour cells. A drug development candidate, designated as S3, emerged following extensive chemical modification to optimize target selectivity and oral bioavailability. Oral administration of S3 significantly inhibited tumour growth and increased survival in several mouse models of pancreatic cancer without discernible toxicity. Interestingly, using the KPC transgenic mouse model that closely mimics human pancreatic cancer, we identified a dual activity of S3 to inhibit the growth of the primary tumour and impact the surrounding stroma. Strikingly, a significant increase in survival was achieved with S3 treatment compared to vehicle treated KPC mice. A two-fold increase in lifespan was observed from 72.5 days (median survival) in the control group to 146.5 days in the treatment group. Importantly, we observed no overt toxicity from S3 treatment at a dosage of 50 mg/kg, which generated plasma levels exceeding growth inhibitory IC50 values. Furthermore, we show that stromal cells in pancreatic tumours, which actively participate in cancer progression, are enriched for ABCC3, and that its inhibition may contribute to stroma reprogramming. In other studies, we found that S3 inhibits the closely related transporter, ABCC1, and that pharmacological inhibition of ABCC1 reduced prostate cancer cell growth in vitro and potentiated the effects of Docetaxel in vitro and in mouse models of prostate cancer in vivo. Mechanistically, we have shown that ABCC3, is overexpressed in pancreatic cancer cells and can efflux the bioactive lipid lysophosphatidylinositol (LPI) which, in turn, activates its receptor G protein-coupled receptor 55 in an autocrine mitogenic loop. Similarly, ABCC1 mediates LPI efflux in prostate cancer cells. The fact that both ABCC1 and ABCC3 transport LPI and are inhibited by S3 is not surprising considering that they share a high primary sequence identity and are known to have overlapping substrate specificity. Interestingly, unlike known ABC inhibitors, S3 has anticancer activity as a single agent. Our goal is to further study the antitumor activity of S3 alone and in combination with conventional chemotherapy or molecular targeted drugs used for the treatment of pancreatic and prostate cancer. Citation Format: Marco Falasca, Xi Chen, Gary Piazza. Novel ABC transport inhibitor as a treatment for pancreatic and prostate 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 1814.

Pancreatic Cancer: Novel Approaches to Diagnosis and Therapy

Within the last few years the Helmholtz Zentrum München has established several initiatives enabling the translation of basic research results into discovery of novel small molecules that affect pathomechanisms of chronic and complex diseases. Here, one of the main operations is the Assay Development and Screening Platform (ADSP) that has state-of-the-art equipment for compound screening and provides knowledge in a variety of biochemical or cell-based phenotypic assays. In particular, ADSP has a strong focus on complex assays such as high-content screening in stem cells that are likely to provide an innovative approach complementary to biochemical assays for the discovery of novel small molecules modulating key biological processes.

ASGE guideline on screening for pancreatic cancer in individuals with genetic susceptibility: summary and recommendations

From the *Department of Surgery, University of Texas Medical Branch, Galveston, TX; and †Department of Surgery, Indiana University School of Medicine, Indianapolis, IN. Reprints: Taylor S. Riall, MD, Assistant Professor of Surgery, Department of Surgery, University of Texas Medical Branch, 301 University Boulevard, Johns Sealy Annex 6.312b, Galveston, TX 77555-0542. E-mail: [email protected].

Origin of cancer is strongly related to the unusual epigenetic regulation of gene function as indicated by recent reports. The covalent modifications to DNA or histones without affecting genomes that finally lead to phenotypical changes in cells or organisms are referred as "Epigenetics." The possibility to reprogram the epigenetics in the cancer epigenome is the most important target for cancer treatment and drug resistance. The development of epigenetic drugs holds a great potential for the current cancer therapeutic approaches. Nevertheless, targeting cancer epigenetic pathways is still exciting due to the lack of selective and effective small molecule compounds or drug molecules. Therefore, the current book chapter highlights epigenetic pathways for cancer and potential small molecule inhibitors and epidrugs targeting DNA methyltransferase, histone modification, and more new therapies with nanomaterials and imaging to improve the effectiveness of cancer treatment. The structural aspects on discovery of novel small molecules or drugs targeting epigenetic pathways in cancer exploration as promising strategies will be also discussed.

The orphan nuclear receptor NR4A1 (TR3/Nur77) is overexpressed as a nuclear protein in multiple human tumors and this receptor exhibits pro-oncogenic activity in many tumor types. A recent study in this laboratory has shown that inactivation of TR3 decreases human pancreatic cancer cell and tumor growth through inhibition of Sp1-dependent expression of antiapoptotic genes such as bcl-2 and survivin. In this study, we performed 2-D gel electrophoresis and MALDI-TOF-TOF mass spectrometry to further characterize the mechanisms of TR3-mediated cell growth inhibition and apoptosis in Panc-1 human pancreatic cancer cells transfected with TR3 siRNA (siTR3) or treated with a TR3 deactivator 1,1-bis(3’-indolyl)-1-(p-hydroxyphenyl)methane (DIM-C-pPhOH) and found 40 proteins that were differentially expressed. Many proteins showing increased expression in cells treated with siTR3 or DIM-C-pPhOH were associated with the endoplasmic reticulum (ER) and ER stress-related pathways, including glucose-regulated protein 78 (GRP78/BiP) and C/EBP homologous protein (CHOP/GADD153) which mediates ER stress-induced apoptosis. It was confirmed by transmission electron microscopy (TEM) and Western blot analysis that TR3 inactivation by siTR3 or DIM-C-pPhOH induces ER stress and ER stress/CHOP-mediated apoptosis. Furthermore, we also observed that ER stress-mediated apoptosis by siTR3 or the TR3 inactivator is primarily due to increase of reactive oxygen species (ROS) production and is abrogated after cotreatment with antioxidants. These results demonstrate that endogenous nuclear TR3 regulates intracellular ROS homeostasis and ER stress-mediated apoptosis, and TR3 deactivation again represents a novel approach for treatment of pancreatic and other cancers that overexpress this nuclear receptor. Grant support: National Institutes of Health (R01CA124998) Citation Format: Syng-Ook Lee, Jeong Han Kang, Un-Ho Jin, Aaron S. Guthrie, Sandeep Sreevalsan, Stephen H. Safe. Inactivation of the orphan nuclear receptor TR3 induces ER stress-mediated apoptosis by increasing ROS production in pancreatic cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr B101.

Soil microbial communities are pivotal to plant health and nutrient acquisition. It is becoming increasingly clear that many interactions, both among and between microbes and plants, are governed by small bioactive molecules or "secondary metabolites" that can aid in communication, competition, and nutrient uptake. Yet, secondary metabolite biogeography - who makes what, where, and why-is in its infancy. Further, secondary metabolite biosynthesis genes are often silent or weakly expressed under standard laboratory conditions, making it incredibly difficult to study these small molecules. To begin to address these dual challenges, we focused on redox-active metabolites (RAMs), a specific class of small molecules, and took advantage of recent findings that many RAMs aid in acquiring phosphorus and that their production is frequently stimulated by stress for this macronutrient. We developed a screen for RAM-producing bacteria that leverages phosphorus limitation to stimulate metabolite biosynthesis and uses a colorimetric (ferrozine) iron-reduction assay to identify redox activity. We isolated 557 root-associated bacteria from grasses collected at sites across the United States (Santa Rita Experimental Range [AZ], Konza Prairie Biological Station [KS], and Harvard Forest [MA]) and from commercial tomato plants and screened them for RAM production. We identified 128 soil isolates of at least 19 genera across Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes that produced RAMs under phosphorus stress. Our work reveals that the production of RAMs under phosphorus stress is common across diverse soil bacteria and provides an approach to screen for these small molecules rapidly.IMPORTANCEBy secreting secondary metabolites, bacteria at the plant root can defend against diseases and help acquire essential nutrients. However, the genes that synthesize secondary metabolites are typically inactive or are weakly expressed under standard laboratory conditions. This fact makes it difficult to study these small molecules and hinders the discovery of novel small molecules that may play crucial roles in agricultural and biomedical settings. Here, we focus on redox-active metabolites (RAMs), a class of secondary metabolites that can help bacteria solubilize phosphorus and are often produced when phosphorus is limited. We developed a screen that rapidly identifies RAM-producing bacteria by utilizing a colorimetric iron-reduction assay in combination with phosphorus limitation to stimulate biosynthesis. The screen reveals that RAM-producing bacteria are far more prevalent in soil than previously appreciated and that this approach can be used to identify RAM producers.