scholarly journals Ruxolitinib and Polycation Combination Treatment Overcomes Multiple Mechanisms of Resistance of Pancreatic Cancer Cells to Oncolytic Vesicular Stomatitis Virus

2017 ◽  
Vol 91 (16) ◽  
Author(s):  
Sébastien A. Felt ◽  
Gaith N. Droby ◽  
Valery Z. Grdzelishvili
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Vesicular Stomatitis Virus ◽  
Oncolytic Virus ◽  
Ductal Adenocarcinoma ◽  
Mechanisms Of Resistance ◽  
Pdac Cell ◽  
Ldl Uptake ◽  
Vesicular Stomatitis ◽  
Pdac Cell Line

ABSTRACT Vesicular stomatitis virus (VSV) is a promising oncolytic virus (OV). Although VSV is effective against a majority of pancreatic ductal adenocarcinoma cell (PDAC) cell lines, some PDAC cell lines are highly resistant to VSV, and the mechanisms of resistance are still unclear. JAK1/2 inhibitors (such as ruxolitinib and JAK inhibitor I) strongly stimulate VSV replication and oncolysis in all resistant cell lines but only partially improve the susceptibility of resistant PDACs to VSV. VSV tumor tropism is generally dependent on the permissiveness of malignant cells to viral replication rather than on receptor specificity, with several ubiquitously expressed cell surface molecules playing a role in VSV attachment to host cells. However, as VSV attachment to PDAC cells has never been tested before, here we examined if it was possibly inhibited in resistant PDAC cells. Our data show a dramatically weaker attachment of VSV to HPAF-II cells, the most resistant human PDAC cell line. Although sequence analysis of low-density lipoprotein (LDL) receptor (LDLR) mRNA did not reveal any amino acid substitutions in this cell line, HPAF-II cells displayed the lowest level of LDLR expression and dramatically lower LDL uptake. Treatment of cells with various statins strongly increased LDLR expression levels but did not improve VSV attachment or LDL uptake in HPAF-II cells. However, LDLR-independent attachment of VSV to HPAF-II cells was dramatically improved by treating cells with Polybrene or DEAE-dextran. Moreover, combining VSV with ruxolitinib and Polybrene or DEAE-dextran successfully broke the resistance of HPAF-II cells to VSV by simultaneously improving VSV attachment and replication. IMPORTANCE Oncolytic virus (OV) therapy is an anticancer approach that uses viruses that selectively infect and kill cancer cells. This study focuses on oncolytic vesicular stomatitis virus (VSV) against pancreatic ductal adenocarcinoma (PDAC) cells. Although VSV is effective against most PDAC cells, some are highly resistant to VSV, and the mechanisms are still unclear. Here we examined if VSV attachment to cells was inhibited in resistant PDAC cells. Our data show very inefficient attachment of VSV to the most resistant human PDAC cell line, HPAF-II. However, VSV attachment to HPAF-II cells was dramatically improved by treating cells with polycations. Moreover, combining VSV with polycations and ruxolitinib (which inhibits antiviral signaling) successfully broke the resistance of HPAF-II cells to VSV by simultaneously improving VSV attachment and replication. We envision that this novel triple-combination approach could be used in the future to treat PDAC tumors that are highly resistant to OV therapy.

scholarly journals Expanding the Spectrum of Pancreatic Cancers Responsive to Vesicular Stomatitis Virus-Based Oncolytic Virotherapy: Challenges and Solutions

Cancers ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 1171
Author(s):  
Molly C. Holbrook ◽  
Dakota W. Goad ◽  
Valery Z. Grdzelishvili
Keyword(s):  
Cell Lines ◽  
Vesicular Stomatitis Virus ◽  
Constitutive Expression ◽  
The United States ◽  
Ductal Adenocarcinoma ◽  
Type I ◽  
Phase I Clinical Trials ◽  
Pdac Cell ◽  
Viral Attachment ◽  
Vesicular Stomatitis

Pancreatic ductal adenocarcinoma (PDAC) is a devastating malignancy with poor prognosis and a dismal survival rate, expected to become the second leading cause of cancer-related deaths in the United States. Oncolytic virus (OV) is an anticancer approach that utilizes replication-competent viruses to preferentially infect and kill tumor cells. Vesicular stomatitis virus (VSV), one such OV, is already in several phase I clinical trials against different malignancies. VSV-based recombinant viruses are effective OVs against a majority of tested PDAC cell lines. However, some PDAC cell lines are resistant to VSV. Upregulated type I IFN signaling and constitutive expression of a subset of interferon-simulated genes (ISGs) play a major role in such resistance, while other mechanisms, such as inefficient viral attachment and resistance to VSV-mediated apoptosis, also play a role in some PDACs. Several alternative approaches have been shown to break the resistance of PDACs to VSV without compromising VSV oncoselectivity, including (i) combinations of VSV with JAK1/2 inhibitors (such as ruxolitinib); (ii) triple combinations of VSV with ruxolitinib and polycations improving both VSV replication and attachment; (iii) combinations of VSV with chemotherapeutic drugs (such as paclitaxel) arresting cells in the G2/M phase; (iv) arming VSV with p53 transgenes; (v) directed evolution approach producing more effective OVs. The latter study demonstrated impressive long-term genomic stability of complex VSV recombinants encoding large transgenes, supporting further clinical development of VSV as safe therapeutics for PDAC.

scholarly journals Experimental Evolution Generates Novel Oncolytic Vesicular Stomatitis Viruses with Improved Replication in Virus-Resistant Pancreatic Cancer Cells

2019 ◽  
Vol 94 (3) ◽  
Author(s):  
Sara L. Seegers ◽  
Connor Frasier ◽  
Sarah Greene ◽  
Irina V. Nesmelova ◽  
Valery Z. Grdzelishvili
Keyword(s):  
Cell Lines ◽  
Genomic Stability ◽  
Oncolytic Viruses ◽  
Ductal Adenocarcinoma ◽  
Pdac Cell ◽  
Virus Attachment ◽  
Vesicular Stomatitis ◽  
Moderately Resistant ◽  
Evolution Approach

ABSTRACT Vesicular stomatitis virus (VSV) based oncolytic viruses are promising agents against various cancers. We have shown that pancreatic ductal adenocarcinoma (PDAC) cell lines exhibit great diversity in susceptibility and permissibility to VSV. Here, using a directed evolution approach with our two previously described oncolytic VSV recombinants, VSV-p53wt and VSV-p53-CC, we generated novel oncolytic VSVs with an improved ability to replicate in virus-resistant PDAC cell lines. VSV-p53wt and VSV-p53-CC encode a VSV matrix protein (M) with a ΔM51 mutation (M-ΔM51) and one of two versions of a functional human tumor suppressor, p53, fused to a far-red fluorescent protein, eqFP650. Each virus was serially passaged 32 times (which accounts for more than 60 viral replication cycles) on either the SUIT-2 (moderately resistant to VSV) or MIA PaCa-2 (highly permissive to VSV) human PDAC cell lines. While no phenotypic changes were observed for MIA PaCa-2-passaged viruses, both SUIT-2-passaged VSV-p53wt and VSV-p53-CC showed improved replication in SUIT-2 and AsPC-1, another human PDAC cell line also moderately resistant to VSV, while remaining highly attenuated in nonmalignant cells. Surprisingly, two identical VSV glycoprotein (VSV-G) mutations, K174E and E238K, were identified in both SUIT-2-passaged viruses. Additional experiments indicated that the acquired G mutations improved VSV replication, at least in part due to improved virus attachment to SUIT-2 cells. Importantly, no mutations were found in the M-ΔM51 protein, and no deletions or mutations were found in the p53 or eqFP650 portions of virus-carried transgenes in any of the passaged viruses, demonstrating long-term genomic stability of complex VSV recombinants carrying large transgenes. IMPORTANCE Vesicular stomatitis virus (VSV)-based oncolytic viruses are promising agents against pancreatic ductal adenocarcinoma (PDAC). However, some PDAC cell lines are resistant to VSV. Here, using a directed viral evolution approach, we generated novel oncolytic VSVs with an improved ability to replicate in virus-resistant PDAC cell lines, while remaining highly attenuated in nonmalignant cells. Two independently evolved VSVs obtained 2 identical VSV glycoprotein mutations, K174E and E238K. Additional experiments indicated that these acquired G mutations improved VSV replication, at least in part due to improved virus attachment to SUIT-2 cells. Importantly, no deletions or mutations were found in the virus-carried transgenes in any of the passaged viruses. Our findings demonstrate long-term genomic stability of complex VSV recombinants carrying large transgenes and support further clinical development of oncolytic VSV recombinants as safe therapeutics for cancer.

scholarly journals Proteome-Wide Alterations of Asymmetric Arginine Dimethylation Associated With Pancreatic Ductal Adenocarcinoma Pathogenesis

2020 ◽  
Vol 8 ◽  
Author(s):  
Meijin Wei ◽  
Chaochao Tan ◽  
Zhouqin Tang ◽  
Yingying Lian ◽  
Ying Huang ◽  
...  
Keyword(s):  
Pancreatic Ductal Adenocarcinoma ◽  
Cell Line ◽  
Cell Lines ◽  
Affinity Purification ◽  
Arginine Methylation ◽  
Mapk Signaling ◽  
Mitogen Activated Protein Kinase ◽  
Ductal Adenocarcinoma ◽  
Pdac Cell ◽  
Protein Levels

Arginine methylation catalyzed by protein arginine methyltransferases (PRMTs) performs essential roles in regulating cancer initiation and progression, but its implication in pancreatic ductal adenocarcinoma (PDAC) requires further elucidation. In this study, asymmetric dimethylarginine (ADMA)-containing peptides in PDAC cell line PANC-1 were identified by label-free quantitative proteomics combined with affinity purification, using human non-cancerous pancreatic ductal epithelium cell line HPDE6c7 as the control. In total, 289 ADMA sites in 201 proteins were identified in HPDE6c7 and PANC-1 cells, including 82 sites with lower dimethylation and 37 sites with higher dimethylation in PANC-1 cells compared with HPDE6c7 cells. These ADMA-containing peptides demonstrated significant enrichment of glycine and proline residues in both cell lines. Importantly, leucine residues were significantly enriched in ADMA-containing peptides identified only in HPDE6c7 cells or showing lower dimethylation in PANC-1 cells. ADMA-containing proteins were significantly enriched in multiple biological processes and signaling cascades associated with cancer development, such as spliceosome machinery, the Wnt/β-catenin, Hedgehog, tumor growth factor beta (TGF-β), and mitogen-activated protein kinase (MAPK) signaling pathways. Moreover, PDAC cell lines with enhanced cell viability showed lower PRMT4 protein abundance and global ADMA-containing protein levels compared with HPDE6c7. PRMT4 overexpression partially recovered ADMA-containing protein levels and repressed viability in PANC-1 cells. These results revealed significantly altered ADMA-containing protein profiles in human pancreatic carcinoma cells, which provided a basis for elucidating the pathogenic roles of PRMT-mediated protein methylation in pancreatic cancer.

scholarly journals P03.29 Characterization of treatment-induced adaptive immune responses in pancreatic ductal adenocarcinoma

2020 ◽  
Vol 8 (Suppl 2) ◽  
pp. A34.2-A34
Author(s):  
J Heetmeyer ◽  
C Falcomatà ◽  
S Bärthel ◽  
C Schneeweis ◽  
A Coluccio ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Line ◽  
Poor Prognosis ◽  
Ductal Adenocarcinoma ◽  
Pdac Cell ◽  
Adaptive Immune ◽  
Epithelial Morphology ◽  
Pdac Cell Line ◽  
Combinatorial Treatment

BackgroundPancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy marked by poor prognosis and profound drug resistance characterized in more than 90% of cases by KRAS mutations. To recapitulate central aspects of PDAC, we employed genetically engineered mouse models presenting KrasG12D pancreas specific expression. Through a high-throughput combination drug screen with trametinib as backbone we identified a high synergism with the multikinase inhibitor nintedanib, preferentially in mesenchymal PDAC, a subtype of this disease characterized by poor prognosis and therapeutic resistance. This combinatorial treatment, that led to the induction of apoptosis in vitro and disease regression in vivo, was accompanied by a strong tumor infiltration of CD8 positive T cells.Materials and MethodsTo characterize the treatment-induced adaptive immune cell infiltration in vivo, we performed orthotopic transplantations of KRAS-driven murine PDAC cell lines presenting mesenchymal and epithelial morphology. The derived control and nintedanib + trametinib treated PDAC tumors were analyzed by multi-color immunofluorescence stainings. We compared the findings to high parameter flow cytometry results.ResultsConfocal microscopy of the immunofluorescence stainings revealed an overall increase of tumor-infiltrating lymphocytes (TIL) in the tumors upon combinatorial treatment with substantial differences in quantity and spatial distribution. Tumors derived from a PDAC cell line of epithelial morphology were characterized by few TIL mainly located at the invasive margins of the tumors, while tumors derived from a mesenchymal PDAC cell line showed a strong increase of TIL even in the center of the tumor mass. Furthermore, an increased ratio of CD8 positive cytotoxic T cells to CD4 positive helper T cells as well as a decrease of Foxp3 and CD4 positive regulatory T cells could be observed for tumors derived from the mesenchymal PDAC cell line under combinatorial treatment. To investigate if the observed recruitment of T cells was indispensable for treatment efficacy of the combinatorial therapy, we orthotopically transplanted the mesenchymal PDAC cell line in immunodeficient CD3-Knockout (CD3ko) mice and applied an analogous combinatorial treatment scheme. In the CD3ko mice, the combinatorial treatment did not lead to an increased survival or tumor regression as observed in immunocompetent mice. However, flow cytometry and immunofluorescence stainings revealed an increase of B cells upon nintedanib + trametinib treatment.ConclusionsOur findings indicate a reduced efficacy of the combinatorial treatment in T cell deficient mice, underlining the importance of T cells in treatment-induced anti-tumor responses and enlarging the understanding of the role of TIL in PDAC.Disclosure InformationJ. Heetmeyer: None. C. Falcomatà: None. S. Bärthel: None. C. Schneeweis: None. A. Coluccio: None. C. Veltkamp: None. G. Schneider: None. D. Saur: None.

Effect of Liposome Encapsulated Vesicular Stomatitis Virus Matrix Protein in Hematological Malignancy

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4634-4634
Author(s):  
Ting Niu ◽  
Wei Liu ◽  
Hua Gao ◽  
Wentong Meng ◽  
Ting Liu
Keyword(s):  
Growth Inhibition ◽  
Cell Line ◽  
Cell Lines ◽  
Vesicular Stomatitis Virus ◽  
Matrix Protein ◽  
Hematological Malignancy ◽  
K562 Cells ◽  
M Protein ◽  
Time Points ◽  
Vesicular Stomatitis

Abstract Objective: Vesicular Stomatitis Virus (VSV) has shown antitumor effects in many studies, however as an active virus,it has biosafety problems when being used in human body. Matrix protein (M protein) of VSV also has antitumor effects without the existence of other portions of VSV.Recently liposome is widely used as a gene delivery vector. So the present study was to investigate the effect of liposome as a gene delivery vector in hematological malignant cell lines and to investigate the effect of liposome encapsulated Vesicular Stomatitis Virus Matrix protein in hematological malignancy in vitro. Methods: First, Lipofectamine 2000 was selected as a kind of gene delivery vector and its efficacy in different cell lines was identified by transfecting the plasmid DNA encoding GFP gene into suspension cell lines, including K562 cells (human chronic myeloid leukemia), Jurkat cells (human T cell lymphoma), Raji cells (human B cell lymphoma) and both attachment and suspension cell line CZ-1 cells (human multiple myeloma) according to the instruction provided by the Invitrogen company when the ratios of DNA to Lipofectamine2000 were 1:2,1:2.5,1:3,1:4 and 1:5, respectively. Selecting those cell lines was based on that the transfection efficiency of liposome was above 20% and the ratios which could help to receive the greatest efficiency. The human ovarian cancer cell line A2780 was the positive control and the ratio of DNA to Lipofectamine varied from 1:2.5 to 1:3. Secondly, in vitro study, the P-M group was the one in which the plasmid DNA encoding M protein of Vesicular Stomatitis Virus (VSV-M) was transfected into the selected cell lines by the best ratios of DNA to Lipofectamine2000.The groups of Lipofectamine2000- treated (LIPO) , Lipofectamine2000 encapsulated empty plasmid pVAX -treated (P) and untreated one (BLK) were set as the control groups. Morphological changes were observed and the cell growth inhibition rates in different groups were measured by MTT (Methylthiazolyldiphenyl-tetrazolium bromide) method at 24h, 48h and 72h after transfection. The cell apoptosis rates of K562 cells were tested by Flow Cytometry at 48h after transfection. Results The Lipofectamine2000 transfection efficiency in K562 cells could reach up to 50%, while in different conditions the efficiency in CZ-1 was only 20% and that in Raji and jurkat cells was below 10%. The efficiency could also reach 50% in A2780 cells when the ratio was1:3. Study of the effect of liposome encapsulated Vesicular Stomatitis Virus Matrix protein in hematological malignancy: 2.1 K562 and CZ-1 cells: The morphology of K562 and CZ-1 cells in P-M groups was nearly not changed at 24h,48h and 72h after transfection. Futhermore, MTT method showed that, compared with the P and LIPO groups, there were no growth inhibition in K562 and CZ-1 cells at different time points(P>0.05). 2.2 A2780 cells: In the P-M groups, the morphology of A2780 cells was changed ,and a lot of suspension cells were observed in the medium after 48h. Compared with the P and LIPO groups, there was obvious growth inhibition in A2780 cells at different time points (P<0.05).In the P groups, the morphology of A2780 cells was also changed little. Compared with the LIPO group, there was some growth inhibition in A2780 cells at different time points (P<0.05). In the LIPO and BLK groups, these cells had no morphological changes and no growth inhibition. Flow Cytometry showed that there was no difference between the P-M group and P,LIPO groups in K562 cell line in terms of cell apoptosis rates after 48 hours of transfection with VSV-M by means of- liposome(P>0.05). Conclusions Liposome was not a good choice for the gene transfection in hematological cell lines, including Raji,CZ-1 and Jurkat; however it had good efficacy in K562 and A2780 cell lines. M protein of Vesicular Stomatitis Virus had no obvious inhibition effect in several hematological malignancy cell lines in the current transfection conditions in vitro, while it had good inhibition effect in A2780 cell line. Futhermore, M protein could not induce cell apoptosis in K562 cells in this study.

scholarly journals Neutralizing activity of Sputnik V vaccine sera against SARS-CoV-2 variants

2021 ◽  
Author(s):  
Satoshi Ikegame ◽  
Mohammed Siddiquey ◽  
Chuan-Tien Hung ◽  
Griffin Haas ◽  
Luca Brambilla ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Cell Line ◽  
Vesicular Stomatitis Virus ◽  
Neutralizing Antibodies ◽  
Vaccine Development ◽  
The Novel ◽  
Serum Samples ◽  
Egfp Reporter ◽  
Vesicular Stomatitis ◽  
Reduced Activity

Abstract The novel pandemic betacoronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected at least 120 million people since its identification as the cause of a December 2019 viral pneumonia outbreak in Wuhan, China1,2. Despite the unprecedented pace of vaccine development, with six vaccines already in use worldwide, the emergence of SARS-CoV-2 ‘variants of concern’ (VOC) across diverse geographic locales have prompted re-evaluation of strategies to achieve universal vaccination3. All three officially designated VOC carry Spike (S) polymorphisms thought to enable escape from neutralizing antibodies elicited during initial waves of the pandemic4–8. Here, we characterize the biological consequences of the ensemble of S mutations present in VOC lineages B.1.1.7 (501Y.V1) and B.1.351 (501Y.V2). Using a replication-competent EGFP-reporter vesicular stomatitis virus (VSV) system, rcVSV-CoV2-S, which encodes S from SARS coronavirus 2 in place of VSV-G, and coupled with a clonal HEK-293T ACE2 TMPRSS2 cell line optimized for highly efficient S-mediated infection, we determined that only 1 out of 12 serum samples from a cohort of recipients of the Gamaleya Sputnik V Ad26 / Ad5 vaccine showed effective neutralization (IC90) of rcVSV-CoV2-S: B.1.351 at full serum strength. The same set of sera efficiently neutralized S from B.1.1.7 and showed only moderately reduced activity against S carrying the E484K substitution alone. Taken together, our data suggest that control of some emergent SARS-CoV-2 variants may benefit from updated vaccines.

scholarly journals Vesicular Stomatitis Virus Oncolysis of T Lymphocytes Requires Cell Cycle Entry and Translation Initiation

2008 ◽  
Vol 82 (12) ◽  
pp. 5735-5749 ◽  
Author(s):  
Stephanie Oliere ◽  
Meztli Arguello ◽  
Thibault Mesplede ◽  
Vanessa Tumilasci ◽  
Peyman Nakhaei ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
T Lymphocytes ◽  
Vesicular Stomatitis Virus ◽  
Oncolytic Virus ◽  
Protein Translation ◽  
Lymphocytic Leukemia ◽  
Activated T Cells ◽  
Cell Cycle Entry ◽  
Vesicular Stomatitis

ABSTRACT Vesicular stomatitis virus (VSV) is a candidate oncolytic virus that replicates and induces cell death in cancer cells while sparing normal cells. Although defects in the interferon antiviral response facilitate VSV oncolysis, other host factors, including translational and growth regulatory mechanisms, also appear to influence oncolytic virus activity. We previously demonstrated that VSV infection induces apoptosis in proliferating CD4+ T lymphocytes from adult T-cell leukemia samples but not in resting T lymphocytes or primary chronic lymphocytic leukemia cells that remain arrested in G0. Activation of primary CD4+ T lymphocytes with anti-CD3/CD28 is sufficient to induce VSV replication and cell death in a manner dependent on activation of the MEK1/2, c-Jun NH2-terminal kinase, or phosphatidylinositol 3-kinase pathway but not p38. VSV replication is specifically impaired by the cell cycle inhibitor olomoucine or rapamycin, which induces early G1 arrest, but not by aphidicolin or Taxol, which blocks at the G11S or G21M phase, respectively; this result suggests a requirement for cell cycle entry for efficient VSV replication. The relationship between increased protein translation following G0/G1 transition and VSV permissiveness is highlighted by the absence of mTOR and/or eIF4E phosphorylation whenever VSV replication is impaired. Furthermore, VSV protein production in activated T cells is diminished by small interfering RNA-mediated eIF4E knockdown. These results demonstrate that VSV replication in primary T lymphocytes relies on cell cycle transition from the G0 phase to the G1 phase, which is characterized by a sharp increase in ribogenesis and protein synthesis.

Investigating autophagy and glutamine metabolism as therapeutic targets for pancreatic cancer.

2015 ◽  
Vol 33 (3_suppl) ◽  
pp. 381-381
Author(s):  
Mario Jardon ◽  
Steve Kalloger ◽  
Christina Iggulden ◽  
Nancy Erro Go ◽  
Paalini Sathiyaseelan ◽  
...  
Keyword(s):  
Cell Lines ◽  
Therapeutic Targets ◽  
Glutamine Metabolism ◽  
Ductal Adenocarcinoma ◽  
Pdac Cell ◽  
Multiple Cancer ◽  
Tissue Micro Array ◽  
Cancer Hallmarks ◽  
Functional Studies ◽  
New Therapeutic Approaches

381 Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest types of cancer, for which new therapeutic approaches are urgently needed. We are developing novel combination therapy approaches based on the inhibition of glutamine metabolism and autophagy, to improve current treatments for PDAC. Since both processes are key mediators of multiple cancer hallmarks, and have inter-related but non-redundant roles, this combination may result in a more efficient disruption of cancer cell resistance to treatments. Methods: We interrogated the Pancreas Centre BC tissue micro-array, containing the epithelial component of 252 PDAC samples, for expression of three key glutamine-metabolizing proteins and two autophagy-related proteins: glutamine synthetase (GLUL), asparagine synthetase (ASNS), glutaminase C (GLS-GAC), microtubule-associated protein 1 light chain 3 beta (MAP1-LC3B or LC3B) and autophagy-related protein 4B (ATG4B). While previous efforts by other groups have focused on GLS-GAC, the role of GLUL in cancer has remained less well understood. We thus investigated the functional relevance of GLUL using a panel of PDAC cell lines. We are also investigating interactions between glutamine metabolism and autophagy, including the exploration of strategies to target GLUL and the evaluation of a new class of ATG4B inhibitors for the treatment of pancreatic cancers. Results: We found that GLUL, ASNS, GLS-GAC, LC3B and ATG4B were expressed in 31%, 58%, 99%, 51% and 73%, respectively, of PDAC samples. Furthermore, higher LC3B expression correlated with poor outcome. Our functional studies revealed that various PDAC cells express GLUL, which can be upregulated upon glutamine deprivation. In all cell lines tested, GLUL knockdown sensitized them to gemcitabine, as assessed by a long-term recovery assay. We also found that the candidate ATG4B inhibitors, shown to inhibit this target in cell-free assays, effectively inhibit PDAC cell proliferation at micromolar concentrations. Conclusions: Our study reveals that glutamine metabolism and autophagy are clinically relevant in PDAC and may have potential as therapeutic targets. Supported by Pancreas Centre BC, BC Cancer Foundation and VGH Foundation.

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