Gemcitabine In Pancreatic Cancer Research Articles

HENT1 Predicts Benefit from Gemcitabine in Pancreatic Cancer but Only with Low CDA mRNA.

Simple SummaryRecent clinical trials suggest that combination therapies that include either gemcitabine or 5-fluorouracil (5-FU) both give significant survival benefits for pancreatic cancer patients. The tumor level of the nucleoside transporter hENT1 is prognostic in patients treated with adjuvant gemcitabine but not adjuvant 5-FU. This work shows for the first time that hENT1 is only predictive of benefit from gemcitabine over 5-FU in patients with low levels of CDA transcript. A choice between adjuvant 5-FU based combination therapies (such as FOLFIRINOX) and gemcitabine-based therapy (e.g., GemCap) could be made based on a combination of hENT1 protein and CDA mRNA measured in a resected tumor.Gemcitabine or 5-fluorouracil (5-FU) based treatments can be selected for pancreatic cancer. Equilibrative nucleoside transporter 1 (hENT1) predicts adjuvant gemcitabine treatment benefit over 5-FU. Cytidine deaminase (CDA), inside or outside of the cancer cell, will deaminate gemcitabine, altering transporter affinity. ESPAC-3(v2) was a pancreatic cancer trial comparing adjuvant gemcitabine and 5-FU. Tissue microarray sections underwent in situ hybridization and immunohistochemistry. Analysis of both CDA and hENT1 was possible with 277 patients. The transcript did not correlate with protein levels for either marker. High hENT1 protein was prognostic with gemcitabine; median overall survival was 26.0 v 16.8 months (p = 0.006). Low CDA transcript was prognostic regardless of arm; 24.8 v 21.2 months with gemcitabine (p = 0.02) and 26.4 v 14.6 months with 5-FU (p = 0.02). Patients with low hENT1 protein did better with 5-FU, but only if the CDA transcript was low (median survival of 5-FU v gemcitabine; 29.3 v 18.3 months, compared with 14.2 v 14.6 with high CDA). CDA mRNA is an independent prognostic biomarker. When added to hENT1 protein status, it may also provide treatment-specific predictive information and, within the frame of a personalized treatment strategy, guide to either gemcitabine or 5FU for the individual patient.

Intraperitoneal siRNA Nanoparticles for Augmentation of Gemcitabine Efficacy in the Treatment of Pancreatic Cancer.

Pancreatic ductal adenocarcinoma is a deadly disease with limited treatment options due to late diagnosis and resistance to conventional chemotherapy. Among emerging therapeutic targets, the CXCR4 chemokine receptor and polo-like kinase 1 (PLK1) play critical roles in the progression, metastasis, and chemoresistance of pancreatic cancer. Here, we tested the hypothesis that combining CXCR4 inhibition by a polymeric CXCR4 antagonist PAMD-CHOL with PLK1 knockdown by siRNA will enhance the therapeutic effect of gemcitabine (GEM) in the orthotopic model of metastatic pancreatic cancer. We formulated nanoparticles with cholesterol-modified PAMD and siPLK1 and found strong synergism when combined with GEM treatment in vitro in both murine and human pancreatic cancer cell lines. The biodistribution of the nanoparticles in orthotopic pancreatic cancer models revealed strong accumulation in primary and metastatic tumors, with limited hepatic disposition. The cholesterol-containing nanoparticles showed not only increased tumor accumulation than the cholesterol-lacking control but also deeper penetration into the tumors. In a therapeutic study in vivo, the triple combination of PAMD-CHOL/siPLK1 and GEM showed superior anticancer activity when compared with single and dual combination controls. In conclusion, PAMD-CHOL/siPLK1 nanoparticles synergistically enhance anticancer activity of GEM in pancreatic cancer and represent a promising addition to the treatment arsenal.

Use of nab-paclitaxel and gemcitabine in pancreatic cancer without granulocyte colony-stimulating factor: A multicenter real-world experience.

The metastatic pancreatic adenocarcinoma clinical trial (MPACT) trial established gemcitabine (gem) and nab-paclitaxel (nab) as a standard treatment for pancreatic cancer utilizing granulocyte colony-stimulating factors to manage neutropenia. This was a challenge for jurisdictions that do not use granulocyte colony-stimulating factors in palliative settings. We developed dosage guidelines to dose modify gem and nab without granulocyte colony-stimulating factors. We undertook a retrospective review to determine the efficacy and safety of these dose adjustment guidelines in the real world. A multi-centered, retrospective chart review was performed on pancreatic patients between December 1, 2014, and August 21, 2018. Provincial electronic medical health records were reviewed. Using Log-rank statistics we determined the patient's progression-free survival and overall survival. Of 248 patients, 209 met patient selection criteria. Patients were excluded if they were lost to follow-up, on gem alone prior to nab/gem combination therapy or did not receive nab or gem. Patients who received nab/gem as first-line therapy had a median progression-free survival of 6.3 months (95% CI, 5.1-7.4), and median overall survival of 11.1 months (95% CI, 9.5-12.8). Those who received gem/nab in the second line had a median progression-free survival of 4.6 months (95% CI, 2.8-6.5), and median overall survival of 19.3 months (95% CI, 12.6-26.0). The patient's progression-free survival and overall survival taking nab/gem using our dose modification algorithm were equivalent or superior to the MPACT trial's progression-free survival and overall survival. Gem/nab can be given by our dose modification scheme without granulocyte colony-stimulating factor.

Retraction Note to: S-Adenosylmethionine synergistically enhances the antitumor effect of gemcitabine against pancreatic cancer through JAK2/STAT3 pathway.

Gemcitabine (GEM) has been widely used for pancreatic cancer (PC) treatment but limited by the development of drug resistance. The agents that reverse its resistance and improve the chemo-sensitivity are urgently needed. S-Adenosylmethionine (SAM) is a precursor for polyamine biosynthesis in mammalian cells and plays a key role in biological transmethylation events. It is reported that SAM could be used as a therapeutic reagent for cancer treatments. In this study, we investigated the chemo-sensitization of SAM to potentiate the antitumor effect of GEM in PC. After treating PC cells with GEM and/or SAM, different subsequent experiments were performed. Results showed that SAM plus GEM could significantly inhibit the growth and proliferation of PC cells, and SAM acts synergistically with GEM. The combinative treatment could induce cell apoptosis and inhibit invasion and migration through JAK2/STAT3 inactivation. Inhibition of JAK2/STAT3 pathway significantly enhanced the pro-apoptotic effect of SAM, suggesting the key roles of JAK2/STAT3 in the process. Moreover, co-treatment with GEM and SAM exhibited more efficient inhibition of tumor weight and volume on PANC-1 xenograft mouse model compared to GEM or SAM alone and has no significant effect on the function of the liver and kidney. In general, this study indicated that SAM synergistically enhanced the antitumor effect of GEM against PC through suppressed JAK2/STAT3 pathway, and SAM is applicable as a promising agent to improve the sensitivity of PC to GEM.

The Antitumor Activity of Sodium Selenite Alone and in Combination with Gemcitabine in Pancreatic Cancer: An In Vitro and In Vivo Study.

Simple SummaryThe treatment of advanced pancreatic cancer represents a major challenge because of its chemoresistance and low survival rate. The aim of this research is to test a new antitumor agent (sodium selenite) in pancreatic cancer, both in vitro and in vivo, focusing on the molecular mechanisms involved and its combination with traditional chemotherapy (gemcitabine). Our results show that selenite has a strong antitumor effect against pancreatic cancer, via parthanatos cell death, and enhances the effect of gemcitabine via the p38 pathway in both in vitro and in vivo assays. These results could improve the outcome of pancreatic cancer patients and could be the start of future clinical trials.Sodium selenite acts by depleting enzymes that protect against cellular oxidative stress. To determine its effect alone or in combination with gemcitabine (GMZ) in pancreatic cancer, we used PANC-1 and Pan02 cell lines and C57BL mice bearing a Pan02-generated tumor. Our results demonstrated a significant inhibition of pancreatic cancer cell viability with the use of sodium selenite alone and a synergistic effect when associated with GMZ. The molecular mechanisms of the antitumor effect of sodium selenite alone involved apoptosis-inducing factor (AIF) and the expression of phospho-p38 in the combined therapy. In addition, sodium selenite alone and in association with GMZ significantly decreased the migration capacity and colony-forming ability, reduced tumor activity in multicellular tumor spheroids (MTS) and decreased sphere formation of cancer stem cells. In vivo studies demonstrated that combined therapy not only inhibited tumor growth (65%) compared to the untreated group but also relative to sodium selenite or GMZ used as monotherapy (up to 40%), increasing mice survival. These results were supported by the analysis of C57BL/6 albino mice bearing a Pan02-generated tumor, using the IVIS system. In conclusion, our results showed that sodium selenite is a potential agent for the improvement in the treatment of pancreatic cancer and should be considered for future human clinical trials.

Girdin Knockdown Increases Gemcitabine Chemosensitivity to Pancreatic Cancer by Modulating Autophagy.

Chemotherapy is crucial for the treatment of pancreatic cancer (PC). Gemcitabine (GEM) as the first-line chemotherapy drug has a high resistance rate. Increasing the sensitivity of gemcitabine is currently the objectives and challenges of this study. Our previous study showed Girdin was closely related to the progression and prognosis of PC, indicating that Girdin may be associated with chemosensitivity. In the current study, we use recombinant adenovirus to specifically knockdown Girdin in PC cell lines to determine the effect of Girdin in the process of gemcitabine chemosensitivity. Autophagy is one of the pathways affecting the gemcitabine chemosensitivity in PC. Further research validated that Girdin may activate autophagy by interacting with autophagy protein p62/SQSTM1, which could enhance chemotherapy resistance to gemcitabine in PC. Down-regulation of Girdin may therefore increase gemcitabine chemosensitivity in PC. Our results reveal that Girdin acted as a negative regulator of gemcitabine chemosensitivity in PC. Increased autophagy activity caused by abnormally high Girdin expression may be one of the main factors for the reduction in chemosensitivity, which may provide new perspectives on understanding chemosensitization in PC.

Intracellular KRAS-specific antibody enhances the anti-tumor efficacy of gemcitabine in pancreatic cancer by inducing endosomal escape.

KRAS mutation is associated with the progression and growth of pancreatic cancer and contributes to chemo-resistance, which poses a significant clinical challenge in pancreatic cancer. Here, we developed a RT22-ep59 antibody (Ab) that directly targets the intracellularly activated GTP-bound form of oncogenic KRAS mutants after it is internalized into cytosol by endocytosis through tumor-associated receptor of extracellular epithelial cell adhesion molecule (EpCAM) and investigated its synergistic anticancer effects in the presence of gemcitabine in pancreatic cancer. We first observed that RT22-ep59 specifically recognized tumor-associated EpCAM and reached the cytosol by endosomal escape. In addition, the anticancer effect of RT22-ep59 was observed in the high-EpCAM-expressing pancreatic cancer cells and gemcitabine-resistant pancreatic cancer cells, but it had little effect on the low-EpCAM-expressing pancreatic cancer cells. Additionally, co-treatment with RT22-ep59 and gemcitabine synergistically inhibited cell viability, migration, and invasion in 3D-cultures and exhibited synergistic anticancer activity by inhibiting the RAF/ERK or PI3K/AKT pathways in cells with high-EpCAM expression. In an orthotopic mouse model, combined administration of RT22-ep59 and gemcitabine significantly inhibited tumor growth. Furthermore, the co-treatment suppressed cancer metastasis by blocking EMT signaling in vitro and in vivo. Our results demonstrated that RT22-ep59 synergistically increased the antitumor activity of gemcitabine by inhibiting RAS signaling by specifically targeting KRAS. This indicates that co-treatment with RT22-ep59 and gemcitabine might be considered a potential therapeutic strategy for pancreatic cancer patients harboring KRAS mutation.

Huaier increases the antitumor effect of gemcitabine on pancreatic cancer in vitro and in vivo.

BackgroundPancreatic cancer has a high degree of malignancy and poor prognosis. As the first-line chemotherapy drug for pancreatic cancer, gemcitabine is widely used but is limited in its efficacy due to the development of chemoresistance. Huaier is a traditional Chinese medicine with anticancer effects. This present study explored the antitumor effect of gemcitabine combined with Huaier on pancreatic cancer in vitro and in vivo.MethodsAfter treatment with gemcitabine combined with Huaier in PaTu8988 pancreatic cancer cells, including 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, wound healing, and Transwell invasion in vitro assays were performed to investigate the proliferation, migration and invasion of cells, respectively. The apoptotic rate of cells was detected by propidium iodide—annexin V staining and flow cytometry. In vivo PaTu8988 pancreatic cancer xenograft and tail vein injection into lung metastasis nude mice models were used to determine the tumor growth and lung metastasis efficiency.ResultsHuaier could not only inhibit the proliferation, migration, and invasion of cancer cells, but could also induce the apoptosis of pancreatic cancer in vitro and suppress tumor growth and lung metastasis in vivo. It further significantly increased the tumor suppressing effects of gemcitabine, and combined use of the two drugs exhibited a synergistic effect.ConclusionsOur present study concluded that Huaier was capable of enhancing the antitumor effect of gemcitabine in pancreatic cancer in vitro and in vivo. Therefore, Huaier may be a potential drug to increase the therapy sensitivity of gemcitabine and improve the prognosis of pancreatic cancer patients.

FBW7-NRA41-SCD1 axis synchronously regulates apoptosis and ferroptosis in pancreatic cancer cells

FBW7 functions as a tumor suppressor by targeting oncoproteins for degradation. Our previous study found FBW7 was low expressed in pancreatic cancer due to sustained activation of Ras-Raf-MEK-ERK pathway, which destabilized FBW7 by phosphorylating at Thr205. MicroPET/CT imaging results revealed that FBW7 substantially decreased 18F-fluorodeoxyglucose uptake in xenograft tumors. Mechanistically, FBW7 inhibited glucose metabolism via c-Myc/TXNIP axis. But in these studies, we observed FBW7 down-regulated genes were widely involved in redox reaction and lipid metabolism. Here we reanalyzed previous gene expression profiling and conducted targeted cell metabolites analysis. Results revealed that FBW7 regulated lipid peroxidation and promoted ferroptosis, a non-apoptotic form of cell death. Mechanistically, we found FBW7 inhibited the expression of stearoyl-CoA desaturase (SCD1) via inhibiting nuclear receptor subfamily 4 group A member 1 (NR4A1). SCD1 was reported to inhibit both ferroptosis and apoptosis, which was consistent with the function of FBW7 and NR4A1, another FBW7 down-regulated gene in the gene expression profiling. Moreover, FBW7 potentiated cytotoxic effect of gemcitabine via activating ferroptosis and apoptosis. Combination ferroptosis inducers and apoptosis activators could also significantly potentiated cytotoxic effect of gemcitabine in pancreatic cancer. Therefore, our findings might provide new strategies for the comprehensive treatment of pancreatic cancer.

Abstract PO-007: PRMT5 inhibition sensitizes pancreatic cancer to gemcitabine in orthotopic and metastatic murine models

Abstract Purpose: The purpose of this study was to investigate protein arginine methyltransferase 5 (PRMT5) as a therapeutic target in combination with gemcitabine for pancreatic cancer in preclinical models. Procedures: In an orthotopic xenograft model, PRMT5 wild-type (WT) or knockout (KO) mPanc96 cells were injected into the pancreata of athymic nude mice. Mice were randomized to control or gemcitabine (100 mg/kg) arms. Tumor volume was measured with magnetic resonance imaging (MRI) at 21, 28, and 35 days post-injection. In a liver metastasis model, PRMT5 WT or KO mPanc96 cells were injected into the spleens of athymic nude mice followed by splenectomy after 10 minutes. Mice were randomized to control or gemcitabine (100 mg/kg) arms. Metastatic tumor burden within the liver was measured with in vivo bioluminescence imaging twice weekly for 19 days post-injection. Relative bioluminescence was calculated relative to post-injection day 2. Results: In the orthotopic model, untreated PRMT5 KO tumors exhibited a 46% reduction in tumor volume compared to untreated PRMT5 WT tumors (959 mm−3 vs. 1766 mm−3, p=0.009). Furthermore, gemcitabine-treated PRMT5 KO tumors exhibited a 30% reduction in tumor volume compared to gemcitabine-treated PRMT5 WT tumors (405 mm−3 vs. 579 mm−3, p=0.046). Compared to untreated PRMT5 WT tumors, gemcitabine-treated PRMT5 KO tumors exhibited a 77% reduction in tumor volume (405 mm−3 vs. 1766 mm−3, p=<0.001). In the liver metastasis model, untreated PRMT5 KO cells exhibited a 45% reduction in relative bioluminescence compared to untreated PRMT5 WT metastases (83.3 vs. 152.8, p=0.02). Additionally, gemcitabine-treated PRMT5 KO metastases exhibited a 52% reduction in relative bioluminescence compared to gemcitabine-treated PRMT5 WT metastases (17.2 vs. 36.0, p=0.02). Compared to untreated PRMT5 WT metastases, gemcitabine-treated PRMT5 KO metastases exhibited an 89% reduction in liver tumor burden (17.2 vs. 152.8, p=<0.001). Conclusions: PRMT5 genetic depletion significantly reduces pancreatic cancer growth in murine models of orthotopic growth and liver metastasis. Additionally, PRMT5 depletion improves gemcitabine response in these models. PRMT5 is a possible therapeutic target in pancreatic cancer treatment that necessitates further investigation. Citation Format: William J. Kane, Sara J. Adair, Sarbajeet Nagdas, Denis Liu, Xiaolong Wei, Mazhar Adli, Todd W. Bauer. PRMT5 inhibition sensitizes pancreatic cancer to gemcitabine in orthotopic and metastatic murine models [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-007.

PO-1534: Tumour response modelling to radiochemotherapy with gemcitabine for pancreatic cancer

PO-1534: Tumour response modelling to radiochemotherapy with gemcitabine for pancreatic cancer

"Open Sesame?": Biomarker Status of the Human Equilibrative Nucleoside Transporter-1 and Molecular Mechanisms Influencing its Expression and Activity in the Uptake and Cytotoxicity of Gemcitabine in Pancreatic Cancer.

Simple SummaryDespite the enormous advance in biomarker discovery, many potential biomarkers of drug activity are unable to satisfy the clinical need due to inadequate sensitivity and specificity. The nucleoside transporter hENT-1 has been studied as a potential biomarker to predict the effect of the widely used anticancer drug gemcitabine in pancreatic cancer. However, several studies showed controversial results regarding the predictive value of hENT-1, prompting new analyses with larger cohorts of patients and standardized methodologies. Improved insights on molecular mechanisms underlying hENT-1 expression and activity should also help in the identification of subsets of patients who are more likely to benefit from specific treatments and improve their clinical outcome. The establishment of such biomarker is especially valuable in pancreatic cancer, which is frequently characterized by complex disease biology and high mortality.Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive tumor characterized by early invasiveness, rapid progression and resistance to treatment. For more than twenty years, gemcitabine has been the main therapy for PDAC both in the palliative and adjuvant setting. After the introduction of FOLFIRINOX as an upfront treatment for metastatic disease, gemcitabine is still commonly used in combination with nab-paclitaxel as an alternative first-line regimen, as well as a monotherapy in elderly patients unfit for combination chemotherapy. As a hydrophilic nucleoside analogue, gemcitabine requires nucleoside transporters to permeate the plasma membrane, and a major role in the uptake of this drug is played by human equilibrative nucleoside transporter 1 (hENT-1). Several studies have proposed hENT-1 as a biomarker for gemcitabine efficacy in PDAC. A recent comprehensive multimodal analysis of hENT-1 status evaluated its predictive role by both immunohistochemistry (with five different antibodies), and quantitative-PCR, supporting the use of the 10D7G2 antibody. High hENT-1 levels observed with this antibody were associated with prolonged disease-free status and overall-survival in patients receiving gemcitabine adjuvant chemotherapy. This commentary aims to critically discuss this analysis and lists molecular factors influencing hENT-1 expression. Improved knowledge on these factors should help the identification of subgroups of patients who may benefit from specific therapies and overcome the limitations of traditional biomarker studies.

Glycogen synthase kinase-3β participates in acquired resistance to gemcitabine in pancreatic cancer.

Acquisition of resistance to gemcitabine is a challenging clinical and biological hallmark property of refractory pancreatic cancer. Here, we investigated whether glycogen synthase kinase (GSK)‐3β, an emerging therapeutic target in various cancer types, is mechanistically involved in acquired resistance to gemcitabine in human pancreatic cancer. This study included 3 gemcitabine‐sensitive BxPC‐3 cell‐derived clones (BxG30, BxG140, BxG400) that acquired stepwise resistance to gemcitabine and overexpressed ribonucleotide reductase (RR)M1. Treatment with GSK3β‐specific inhibitor alone attenuated the viability and proliferation of the gemcitabine‐resistant clones, while synergistically enhancing the efficacy of gemcitabine against these clones and their xenograft tumors in rodents. The gemcitabine‐resensitizing effect of GSK3β inhibition was associated with decreased expression of RRM1, reduced phosphorylation of Rb protein, and restored binding of Rb to the E2 transcription factor (E2F)1. This was followed by decreased E2F1 transcriptional activity, which ultimately suppressed the expression of E2F1 transcriptional targets including RRM1, CCND1 encoding cyclin D1, thymidylate synthase, and thymidine kinase 1. These results suggested that GSK3β participates in the acquisition of gemcitabine resistance by pancreatic cancer cells via impairment of the functional interaction between Rb tumor suppressor protein and E2F1 pro‐oncogenic transcription factor, thereby highlighting GSK3β as a promising target in refractory pancreatic cancer. By providing insight into the molecular mechanism of gemcitabine resistance, this study identified a potentially novel strategy for pancreatic cancer chemotherapy.

Interferon-beta enhances sensitivity to gemcitabine in pancreatic cancer

BackgroundAdjuvant gemcitabine for pancreatic cancer has limited efficacy in the clinical setting. Impaired drug metabolism is associated with treatment resistance. We aimed to evaluate the chemosensitising effect of interferon-beta (IFN-β).MethodsBxPC-3, CFPAC-1, and Panc-1 cells were pre-treated with IFN-β followed by gemcitabine monotherapy. The effect on cell growth, colony formation, and cell cycle was determined. RT-qPCR was used to measure gene expression. BxPC-3 cells were used in a heterotopic subcutaneous mouse model.ResultsIFN-β increased sensitivity to gemcitabine (4-, 7.7-, and 1.7-fold EC50 decrease in BxPC-3, CFPAC-1, and Panc-1, respectively; all P < 0.001). Findings were confirmed when assessing colony formation. The percentage of cells in the S-phase was significantly increased after IFN-β treatment only in BxPC-3 and CFPAC-1 by 12 and 7%, respectively (p < 0.001 and p < 0.05, respectively). Thereby, IFN-β upregulated expression of the drug transporters SLC28A1 in BxPC-3 (252%) and SLC28A3 in BxPC-3 (127%) and CFPAC-1 (223%) (all p < 0.001). In vivo, combination therapy reduced tumor volume with 45% (P = 0.01). Both ex vivo and in vivo data demonstrate a significant reduction in the number of proliferating cells, whereas apoptosis was increased.ConclusionsFor the first time, we validated the chemosensitising effects of IFN-β when combined with gemcitabine in vitro, ex vivo, and in vivo. This was driven by cell cycle modulation and associated with an upregulation of genes involving intracellular uptake of gemcitabine. The use of IFN-β in combination with gemcitabine seems promising in patients with pancreatic cancer and needs to be further explored.

Abstract B32: High-throughput chemical screening reveals YAP-mediated alterations in drug sensitivities

Abstract The Hippo pathway is evolutionarily conserved and plays a critical role in determining organ size and in tumorigenesis. Aberrant genetic alterations in the Hippo pathway genes including LATS1/2, NF2, and STK11, as well as increased expression and activity of YAP have been found in some human cancers. Loss of function of Hippo genes results in increased nuclear localization of YAP, which promotes cell proliferation, migration, and invasion. Despite our increasing understanding of molecular phenotypes associated with Hippo aberration and excess YAP activity, there are as yet no FDA-approved therapies targeting this pathway, representing a large, unmet patient need. Previous work from our lab has established an additional role for the pathway in altering cellular response to gemcitabine in pancreatic cancer (PNAS 2017). To uncover therapeutic vulnerabilities caused by increased YAP activity, we performed a quantitative, high-throughput screen using the Mechanism Interrogation PlatE (MIPE) library of compounds. All the drugs in this library are relevant for clinical use in cancer and have known mechanisms of action. Annotated gene families represented in the MIPE library include kinases, GPCRs, and ion channels as well as broad cellular mechanisms including modulators of nuclear receptor transcription factors, lipid metabolism, histone epigenetic machinery, and apoptosis. The screen was carried out in twenty 1,536-well plates consisting of 2,154 compounds at nine different doses on both Panc02.13 cells expressing GFP or constitutively active YAP (YAPS6A). Preliminary data have confirmed our previous work in YAP’s role in increasing sensitivity to gemcitabine and several other chemotherapeutics. Pathway enrichment analysis of drugs based on similar mechanism of action allowed us to identify specific signaling nodes and cellular processes that present points of vulnerability or resistance caused by excess YAP activity. We discovered a profound increase in cellular resistance to MEK inhibitors. AC50 values for seventeen out of eighteen MEK inhibitors tested were on average more than four-fold higher in YAPS6A-expressing cells. We have validated these results in several other pancreatic cell lines in both 2D and 3D spheroid assays. Molecular analysis revealed that nuclear YAP causes sustained PI3K/AKT activation, providing a plausible compensatory pathway for cell survival. Moreover, treatment with PI3K/AKT inhibitors can abrogate this YAP-induced resistance to MEK inhibitors. Overall our data are consistent with previous findings that increases in PI3K and AKT signaling function as an escape mechanism to MEK inhibition. Our data suggest that increased YAP activity may represent a distinct molecular mechanism for how this escape is accomplished in refractory cancers. Cotargeting PI3K/AKT may thus provide therapeutic options for patients carrying mutations in Hippo pathway genes. Citation Format: Andrew J. Bondesson, Aya Miyaki, Stella Shin, Michele Ceribelli, Craig J. Thomas, Taran S. Gujral. High-throughput chemical screening reveals YAP-mediated alterations in drug sensitivities [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr B32.

Noninvasive Preclinical Evaluation of Targeted Nanoparticles for the Delivery of Curcumin in Treating Pancreatic Cancer.

Conventional therapy regimens for pancreatic cancer (PC) are surgical resection and systemic gemcitabine based chemotherapy. Recent studies showed that curcumin could potentiate the anticancer effect of gemcitabine in PC. However, due to its poor water solubility, effective bioavailability of curcumin is insufficient, resulting in poor efficacy. To address this issue, mesoporous silica nanoparticles (MSN) were prepared by the sol-gel method, then loaded with curcumin (Cur), coated with polyethylene glycol (PEG), and finally conjugated with the targeting moiety transferrin (Tf) to target human PC cells. TEM analysis revealed that uniform sized spherical MSN formed with an average size of 100 nm, which increased to 120 nm after PEG coating on MSN surface. Confocal microscopy proved that curcumin uptake being seven-times higher for MSN-NH2-Cur-PEG-Tf, when compared to free curcumin. The in vitro cytotoxicity study on MIA PaCa-2 cells showed that MSN-NH2-Cur-PEG-Tf exhibited three-fold higher cytotoxicity than free curcumin. On the basis of the encouraging in vitro cytotoxicity results obtained, preclinical assessment of antitumor efficacy in MIA PaCa-2 subcutaneous xenograft model proves that both MSN-NH2-Cur-PEG and MSN-NH2-Cur-PEG-Tf inhibit tumor growth and minimize distant metastasis to major organ sites. The in vitro studies also proved that nanoparticles can enhance the sensitization effect, caused by curcumin on cancer cells, which help the gemcitabine to kill a higher percentage of cancer cells. Hence, we propose that transferrin targeted, PEGylated, mesoporous silica nanoparticles can be used as a carrier to deliver curcumin, and used in addition to gemcitabine to reduce disease burden significantly for pancreatic cancer patients.

Gas Plasma-Conditioned Ringer's Lactate Enhances the Cytotoxic Activity of Cisplatin and Gemcitabine in Pancreatic Cancer In Vitro and In Ovo.

Pancreatic cancer is one of the most aggressive tumor entities. Diffuse metastatic infiltration of vessels and the peritoneum restricts curative surgery. Standard chemotherapy protocols include the cytostatic drug gemcitabine with limited efficacy at considerable toxicity. In search of a more effective and less toxic treatment modality, we tested in human pancreatic cancer cells (MiaPaca and PaTuS) a novel combination therapy consisting of cytostatic drugs (gemcitabine or cisplatin) and gas plasma-conditioned Ringer’s lactate that acts via reactive oxygen species. A decrease in metabolic activity and viability, change in morphology, and cell cycle arrest was observed in vitro. The combination treatment was found to be additively toxic. The findings were validated utilizing an in ovo tumor model of solid pancreatic tumors growing on the chorion-allantois membrane of fertilized chicken eggs (TUM-CAM). The combination of the drugs (especially cisplatin) with the plasma-conditioned liquid significantly enhanced the anti-cancer effects, resulting in the induction of cell death, cell cycle arrest, and inhibition of cell growth with both of the cell lines tested. In conclusion, our novel combination approach may be a promising new avenue to increase the tolerability and efficacy of locally applied chemotherapeutic in diffuse metastatic peritoneal carcinomatosis of the pancreas.

The Benzimidazole-Based Anthelmintic Parbendazole: A Repurposed Drug Candidate That Synergizes with Gemcitabine in Pancreatic Cancer.

Pancreatic cancer (PC) is one of the most lethal, chemoresistant malignancies and it is of paramount importance to find more effective therapeutic agents. Repurposing of non-anticancer drugs may expand the repertoire of effective molecules. Studies on repurposing of benzimidazole-based anthelmintics in PC and on their interaction with agents approved for PC therapy are lacking. We analyzed the effects of four Food and Drug Administration (FDA)-approved benzimidazoles on AsPC-1 and Capan-2 pancreatic cancer cell line viability. Notably, parbendazole was the most potent benzimidazole affecting PC cell viability, with half maximal inhibitory concentration (IC50) values in the nanomolar range. The drug markedly inhibited proliferation, clonogenicity and migration of PC cell lines through mechanisms involving alteration of microtubule organization and formation of irregular mitotic spindles. Moreover, parbendazole interfered with cell cycle progression promoting G2/M arrest, followed by the emergence of enlarged, polyploid cells. These abnormalities, suggesting a mitotic catastrophe, culminated in PC cell apoptosis, are also associated with DNA damage in PC cell lines. Remarkably, combinations of parbendazole with gemcitabine, a drug employed as first-line treatment in PC, synergistically decreased PC cell viability. In conclusion, this is the first study providing evidence that parbendazole as a single agent, or in combination with gemcitabine, is a repurposing candidate in the currently dismal PC therapy.

Hexokinase 2 dimerization and interaction with voltage-dependent anion channel promoted resistance to cell apoptosis induced by gemcitabine in pancreatic cancer.

Gemcitabine (GEM) is the standard chemotherapy drug for pancreatic cancer. Because of widespread drug resistance, the effect is limited. Therefore, it is urgent to reveal the underlying mechanism. Glycolysis is the most remarkable character of tumor aberrant metabolism, which plays vital roles on tumor drug resistance. Hexokinase 2 (HK2), as the key enzyme regulating the first‐step reaction of glycolysis, is overexpressed in many kinds of tumors. The putative role of HK2 resisting GEM therapy was investigated in this study. We found that HK2 was overexpressed in pancreatic cancer and associated with poor prognosis. HK2 knockdown decreased pancreatic cancer cell proliferation, migration viability, and promoted cell apoptosis in vitro. HK2 high expression in pancreatic cancer showed GEM resistance. HK2 knockdown increased the sensitivity of pancreatic cancer cell to GEM, the growth of xenograft tumor with HK2 knockdown was also further decreased with the GEM treatment compared with control in vivo. GEM‐resistant pancreatic cancer showed the increase of HK2 dimer rather than HK2 mRNA or protein. Our study revealed that the ROS derived from GEM promoted HK2 dimerization combining with voltage‐dependent anion channel, which resulted in the resistance to GEM. Meanwhile, our study established a new sight for GEM resistance in pancreatic cancer.

Abstract 3625: Nanodelivery platform for targeting mutant-KRAS and improving response to gemcitabine therapy

Abstract Background: Although, surgical resection and chemotherapy are the gold standard for treating pancreatic cancer (PanCa), poor patient survival remains the problem. Despite being one of the most common oncogenes in human cancer, to date, no success has been achieved to inhibit KRAS. Targeting KRAS has been shown to synergize anti-cancer activity of gemcitabine in pancreatic cancer. Herein, we have developed a supermagnetic iron oxide (SP) nanoparticles for the sustained delivery of KRAS siRNA to the tumor and simultaneous sensitization of gemcitabine in PanCa. Methods: PanCa cells, AsPC1 and Panc-1 were used in the study. A precipitation approach was employed to develop the SP formulation. These particles were further conjugated with siKRAS (12D; most common KRAS mutation) and investigated for its anticancer efficacy alone and in combination with gemcitabine. Particles were investigated for size, physico-chemical characterization (Dynamic light scattering), hemocompatiblity (hemolysis assay) and the complexation of siKRAS (gel retardation assay). Cellular internalization and uptake of the particles were investigated using FAM labelled siRNA and Prussian blue assay. kRAS silencing was confirmed at both mRNA and protein levels using quantitative reverse-transcription PCR and Western blotting, respectively. Anti-cancer efficacy of SP-siKRAS particles alone or in combination with gemcitabine treatment was determined using in vitro functional assays for cell viability (MTT), migration (Boyden chambers), invasion (Matrigel), colonogenicity and tumor spheroid formation. Results: Our results demonstrate optimal particle size (190.137 nm) and zeta potential (18.73mV) of SP-siKRAS formulation. SP-siKRAS efficiently internalized in PanCa cells and suppressed KRAS G12D expression as well as its downstream targets, YAP and PDL-1. SP-siKRAS improved gemcitabine response as observed through enhanced inhibition of cell proliferation, clonogenicity, migration, and invasion of pancreatic cancer cells. Additionally, SP-siKRAS together with gemcitabine resulted in the activation of death related mechanisms in PanCa cells, such as Bax, bcl-2, PARP cleavage. Interestingly, SP-siKRAS inhibited the secondary tumorsphere formation in combined PanCa and cancer associated fibroblast (CAFs) cells, alone as well as in combination with gemcitabine. At day 14, analysis of secondary tumorspheres, treated with SP-siKRAS revealed diminished levels of KRAS G12D through PCR. This further provides a clinical validation demonstrating potential of SP-siKRAS particles to efficiently silence KRAS expression. SP-siKRAS also exhibited haemocompatibility, suggesting its potential of silencing KRAS without being toxic to the body. Conclusion: Therefore, SP-siKRAS provide a highly efficient and safe platform for silencing KRAS and improving gemcitabine therapy in pancreatic cancer. Citation Format: Nirnoy Dan, Sheema Khan, Saini Setua, Sonam Kumari, Pallabita Chowdhury, Kamalika Samanta, Meena Jaggi, Murali Yallappu, Subhash Chauhan. Nanodelivery platform for targeting mutant-KRAS and improving response to gemcitabine therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3625.