Chloroquine plays a cell-dependent role in the response to treatment of pancreatic adenocarcinoma

In this study, our aim is to assess the role played by autophagy and its inhibition in the different PDAC cellular compartments, and its involvement in chemo-resistance using primary human pancreatic cancer-derived cells (PCC) and Cancer Associated Fibroblasts (CAF). Autophagy flux, as measured by LC3-I and -II in the presence of Chloroquine, showed a variable level in PCC and CAFs. We found no correlation between autophagy level and degree of tumor differentiation. Association of Chloroquine with gemcitabine, 5FU, oxaliplatin, irinotecan and docetaxel revealed that its effect on survival is cell- and drug-dependent in vitro and in vivo. In addition, we demonstrated that autophagy in CAFs can play an important role in sensitizing PDAC to anticancer treatments since its inhibition increased the resistance of PCCs to gemcitabine. In conclusion, this work clearly shows a heterogeneity in the effect of Chloroquine and highlights a role of CAFs autophagy in sensitizing tumors to treatments. It also reveals that the role of autophagy is more complex than expected in PDAC as well as its sensitivity to treatments.Fil: Molejon, Maria Ines. Institut National de la Santé et de la Recherche Médicale; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Ciencias de la Tierra y Ambientales de La Pampa. Universidad Nacional de La Pampa. Facultad de Ciencias Exactas y Naturales. Instituto de Ciencias de la Tierra y Ambientales de La Pampa; ArgentinaFil: Swayden, Mirna. Institut National de la Santé et de la Recherche Médicale; FranciaFil: Fanale, Daniele. 3 Department of Surgical, Oncological and Oral Sciences, Section of Medical Oncology, University of Palermo, 90127 Palermo, Italy.; Argentina. University of Palermo. Department of Surgical, Oncological and Oral Sciences; ItaliaFil: Bintz, Jennifer. Institut National de la Santé et de la Recherche Médicale; FranciaFil: Gayet, Odile. Institut National de la Santé et de la Recherche Médicale; FranciaFil: Soubeyran, Philippe. Institut National de la Santé et de la Recherche Médicale; FranciaFil: Iovanna, Juan Lucio. Institut National de la Santé et de la Recherche Médicale; Franci

Inhibition of glucuronidation in pancreatic cancer improves gemcitabine anticancer activity

Pancreatic ductal adenocarcinoma (PDAC) treatmentis focused on two regimens. The polychemotherapy, FOLFIRINOX (folinic acid, fluorouracil, irinotecan, oxali-platin), is used in patients with good health conditions, while gemcitabine, as monotherapy, in patients withpoor health conditions. Gemcitabine resistance-associated pathways have been targeted to sensitize cancercells, but the results were disappointing. Using a transcrip-tomic bioinformatics analysis combined with biologicalvalidation, we showed that glucuronidation was associated with the gemcitabine resistance in PDAC, and its inhibition could switch tumors from resistant to sensitive.To unravel the biological drivers of gemcitabineresponse in PDAC, we determined the transcriptomic dissimilarity between two preclinical models with definedgemcitabine sensitivity.Fil: Fraunhoffer Navarro, Nicolas Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Meilerman Abuelafia, Analía. Inserm; FranciaFil: Chanez, Brice. Inserm; FranciaFil: Bigonnet, Martin. Inserm; FranciaFil: Gayet, Odile. Inserm; FranciaFil: Roques, Julie. Inserm; FranciaFil: Chuluyan, Hector Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Dusetti, Nelson. Inserm; FranciaFil: Iovanna, Juan Lucio. Inserm; Franci

Evidencing a pancreatic ductal adenocarcinoma subpopulation sensitive to the proteasome inhibitor Carfilzomib

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer with a survival rate less than 5%. Multiple chemotherapeutic drugs have been tested to improve patient prognosis; however, the clinical efficacy of these treatments is low. One of the most controversial family of drugs are the proteasome inhibitors, which have displayed promising effects in preclinical studies, but low clinical performance. Here, we unravel a specific transcriptomic signature that discriminates a subgroup of patients sensitive to the proteasome inhibitor carfilzomib. Experimental Design: First, we identified a subpopulation of PDAC-derived primary cells cultures (PDPCC) sensitive to the proteasome inhibitor carfilzomib. Then, we selected a transcriptomic signature that predicts carfilzomib chemosensitivity using independent component analysis on the transcriptome of PDPCC. Finally, we validated the signature in an independent cohort of PDAC biopsy-derived pancreatic organoids. Results: Sensitive phenotype was characterized by a high expression of genes related with a cornified/squamous pathway and a downregulation of epithelial-mesenchymal transition genes. Interestingly, carfilzomib-sensitive transcriptomic profile did not show any association with the proteasome activity but strongly correlates with ATF4 and CHOP expression, which are key markers of the unfolded protein response and critical to trigger the cell death program. Concordantly, sensitive phenotype showed a high level of the de novo RNA and protein synthesis compared with the resistant one and, most important, cell death induced by carfilzomib is dependent of the translational activity. Conclusions: We demonstrate the existence of a carfilzomib-sensitive PDAC subgroup with a specific transcriptomic phenotype that could explain the biological reason for this responsiveness.Fil: Fraunhoffer Navarro, Nicolas Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentina. Inserm; FranciaFil: Meilerman Abuelafia, Miriam Analia. Inserm; FranciaFil: Bigonnet, Martin. Inserm; FranciaFil: Gayet, Odile. Inserm; FranciaFil: Roque, Julie. Inserm; FranciaFil: Telle, Emmanuel. Inserm; FranciaFil: Santofimia-Castaño, Patricia. Inserm; FranciaFil: Borrello, Maria Teresa. Inserm; FranciaFil: Chuluyan, Hector Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Dusetti, Nelson. Inserm; FranciaFil: Iovanna, Juan Lucio. Inserm; Franci

Exploring the complementarity of pancreatic ductal adenocarcinoma preclinical models

Purpose: Compare pancreatic ductal adenocarcinoma (PDAC), preclinical models, by their transcriptome and drug response landscapes to evaluate their complementarity. Experimental De-sign: Three paired PDAC preclinical models—patient‐derived xenografts (PDX), xenograft‐derived pancreatic organoids (XDPO) and xenograft‐derived primary cell cultures (XDPCC)—were derived from 20 patients and analyzed at the transcriptomic and chemosensitivity level. Transcriptomic characterization was performed using the basal‐like/classical subtyping and the PDAC molecular gradient (PAMG). Chemosensitivity for gemcitabine, irinotecan, 5‐fluorouracil and oxaliplatin was established and the associated biological pathways were determined using independent component analysis (ICA) on the transcriptome of each model. The selection criteria used to identify the different components was the chemosensitivity score (CSS) found for each drug in each model. Results: PDX was the most dispersed model whereas XDPO and XDPCC were mainly classical and basal-like, respectively. Chemosensitivity scoring determines that PDX and XDPO display a positive correlation for three out of four drugs tested, whereas PDX and XDPCC did not correlate. No match was observed for each tumor chemosensitivity in the different models. Finally, pathway analysis shows a significant association between PDX and XDPO for the chemosensitivity‐associated pathways and PDX and XDPCC for the chemoresistance‐associated pathways. Conclusions: Each PDAC preclinical model possesses a unique basal‐like/classical transcriptomic phenotype that strongly in-fluences their global chemosensitivity. Each preclinical model is imperfect but complementary, sug-gesting that a more representative approach of the clinical reality could be obtained by combining them. Translational Relevance: The identification of molecular signatures that underpin drug sensitivity to chemotherapy in PDAC remains clinically challenging. Importantly, the vast majority of studies using preclinical in vivo and in vitro models fail when transferred to patients in a clinical setting despite initially promising results. This study presents for the first time a comparison between three preclinical models directly derived from the same patients. We show that their applica-bility to preclinical studies should be considered with a complementary focus, avoiding tumor-based direct extrapolations, which might generate misleading conclusions and consequently the overlook of clinically relevant features.Fil: Hoare, Owen. Centre National de la Recherche Scientifique; FranciaFil: Fraunhoffer Navarro, Nicolas Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Elkaoutari, Abdessamad. Centre National de la Recherche Scientifique; FranciaFil: Gayet, Odile. Centre National de la Recherche Scientifique; FranciaFil: Bigonnet, Martin. Centre National de la Recherche Scientifique; FranciaFil: Roques, Julie. Centre National de la Recherche Scientifique; FranciaFil: Nicolle, Rémy. No especifíca;Fil: McGuckin, Colin. Cell Therapy Research Institute; FranciaFil: Forraz, Nico. Cell Therapy Research Institute; FranciaFil: Sohier, Emilie. Le Centre Régional de Lutte Contre Le Cancer Léon Bérard; FranciaFil: Tonon, Laurie. Le Centre Régional de Lutte Contre Le Cancer Léon Bérard; FranciaFil: Wajda, Pauline. Le Centre Régional de Lutte Contre Le Cancer Léon Bérard; FranciaFil: Boyault, Sandrine. Le Centre Régional de Lutte Contre Le Cancer Léon Bérard; FranciaFil: Attignon, Valéry. Le Centre Régional de Lutte Contre Le Cancer Léon Bérard; FranciaFil: Tabone, Luciana Belen. Le Centre Régional de Lutte Contre Le Cancer Léon Bérard; FranciaFil: Barbier, Sandrine. No especifíca;Fil: Mignard, Caroline. No especifíca;Fil: Duchamp, Olivier. No especifíca;Fil: Iovanna, Juan. Centre National de la Recherche Scientifique; FranciaFil: Dusetti, Nelson J.. Centre National de la Recherche Scientifique; Franci

Gene expression profiling of patient‐derived pancreatic cancer xenografts predicts sensitivity to the BET bromodomain inhibitor JQ1: implications for individualized medicine efforts

Abstract c‐MYC controls more than 15% of genes responsible for proliferation, differentiation, and cellular metabolism in pancreatic as well as other cancers making this transcription factor a prime target for treating patients. The transcriptome of 55 patient‐derived xenografts show that 30% of them share an exacerbated expression profile of MYC transcriptional targets (MYC‐high). This cohort is characterized by a high level of Ki67 staining, a lower differentiation state, and a shorter survival time compared to the MYC‐low subgroup. To define classifier expression signature, we selected a group of 10 MYC target transcripts which expression is increased in the MYC‐high group and six transcripts increased in the MYC‐low group. We validated the ability of these markers panel to identify MYC‐high patient‐derived xenografts from both: discovery and validation cohorts as well as primary cell cultures from the same patients. We then showed that cells from MYC‐high patients are more sensitive to JQ1 treatment compared to MYC‐low cells, in monolayer, 3D cultured spheroids and in vivo xenografted tumors, due to cell cycle arrest followed by apoptosis. Therefore, these results provide new markers and potentially novel therapeutic modalities for distinct subgroups of pancreatic tumors and may find application to the future management of these patients within the setting of individualized medicine clinics

Induction of Apoptosis in Human Pancreatic Cancer Stem Cells by the Endoplasmic Reticulum-Targeted Alkylphospholipid Analog Edelfosine and Potentiation by Autophagy Inhibition

© 2021 by the authors.Pancreatic cancer is one of the most lethal malignancies with a poor and gloomy prognosis and the highest mortality-to-incidence ratio. Pancreatic cancer remains an incurable malignancy, and current therapies are ineffective. We isolated cancer stem cells (CSCs) from the human PANC-1 pancreatic cancer cell line as CD44+CD24+EpCAM+ cells. These CSCs form pancreatic cancer spheres or spheroids and develop tumors in SCID mice after subcutaneous injection of as few as 100 cells per mouse. Here, we found that the alkylphospholipid analog edelfosine inhibited CSC pancreatic cancer spheroid formation and induced cell death, as assessed by an increase in the percentage of cells in the sub-G0/G1 region by means of flow cytometry, indicative of DNA breakdown and apoptosis. This correlated with an increase in caspase-3 activity and PARP breakdown, as a major substrate of caspase-3, following PANC-1 CSC treatment with edelfosine. The antitumor ether lipid edelfosine colocalized with the endoplasmic reticulum in both PANC-1 cells as well as PANC-1 CSCs by using a fluorescent edelfosine analog, and induced an endoplasmic reticulum stress response in both PANC-1 cells and PANC-1 CSCs, with a potent CHOP/GADD153 upregulation. Edelfosine elicited a strong autophagy response in both PANC-1 cells and PANC-1 CSCs, and preincubation of CSCs with autophagy inhibitors, chloroquine or bafilomycin A1, enhanced edelfosine-induced apoptosis. Primary cultures from pancreatic cancer patients were sensitive to edelfosine, as well as their respective isolated CSCs. Nontumorigenic pancreatic human cell line HPNE and normal human fibroblasts were largely spared. These data suggest that pancreatic CSCs isolated from established cell lines and pancreatic cancer patients are sensitive to edelfosine through its accumulation in the endoplasmic reticulum and induction of endoplasmic reticulum stress.This work was supported by grants from Ministerio de Ciencia e Innovación (SAF2017-89672-R and PID2020-119656RB-I00); INCa (2018-078 and 2018-079); Canceropole PACA; and Amidex Foundation. This study is part of the national program Cartes d’Identité des Tumeurs (CIT) funded and developed by Ligue Nationale Contre le Cancer

Optimization of a Bioluminescence Resonance Energy Transfer-Based Assay for Screening of Trypanosoma cruzi Protein/Protein Interaction Inhibitors

Chagas disease, a parasitic disease caused by Trypanosoma cruzi, is a major public health burden in poor rural populations of Central and South America and a serious emerging threat outside the endemic region, since the number of infections in non-endemic countries continues to rise. In order to develop more efficient anti-trypanosomal treatments to replace the outdated therapies, new molecular targets need to be explored and new drugs discovered. Trypanosoma cruzi has distinctive structural and functional characteristics with respect to the human host. These exclusive features could emerge as interesting drug targets. In this work, essential and differential protein–protein interactions for the parasite, including the ribosomal P proteins and proteins involved in mRNA processing, were evaluated in a bioluminescence resonance energy transfer-based assay as a starting point for drug screening. Suitable conditions to consider using this simple and robust methodology to screening compounds and natural extracts able to inhibit protein–protein interactions were set in living cells and lysates.Fil: Mild, Jesica Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Fernandez, Lucia Raquel. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Gayet, Odile. Centre de Recherche en Cancérologie de Marseille; FranciaFil: Iovanna, Juan. Centre de Recherche en Cancérologie de Marseille; FranciaFil: Dusetti, Nelson. Centre de Recherche en Cancérologie de Marseille; FranciaFil: Edreira, Martin Miguel. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentin

Basal‐like and classical cells coexist in pancreatic cancer revealed by single‐cell analysis on biopsy‐derived pancreatic cancer organoids from the classical subtype

International audiencePancreatic ductal adenocarcinoma (PDAC) is composed of stromal, immune, and cancerous epithelial cells. Transcriptomic analysis of the epithelial compartment allows classification into different phenotypic subtypes as classical and basal-like. However, little is known about the intra-tumor heterogeneity particularly in the epithelial compartment. Growing evidences suggest that this phenotypic segregation is not so precise and different cancerous cell types may coexist in a single tumor. To test this hypothesis, we performed single-cell transcriptomic analyses using combinational barcoding exclusively on epithelial cells from six different classical PDAC patients obtained by Endoscopic Ultrasound (EUS) with Fine Needle Aspiration (FNA). To purify the epithelial compartment, PDAC were grown as biopsy-derived pancreatic cancer organoids. Single-cell transcriptomic analysis allowed the identification of four main cell clusters present in different proportions in all tumors. Remarkably, although all these tumors were classified as classical, one cluster present in all corresponded to a basal-like phenotype. These results reveal an unanticipated high heterogeneity of pancreatic cancers and demonstrate that basal-like cells, which have a highly aggressive phenotype, are more widespread than expected

Selection of Intracellular Single-Domain Antibodies Targeting the HIV-1 Vpr Protein by Cytoplasmic Yeast Two-Hybrid System

International audienceThe targeting of HIV-1 using antibodies is of high interest as molecular tools to better understand the biology of the virus or as a first step toward the design of new inhibitors targeting critical viral intracellular proteins. Small and highly stable llama-derived single-domain antibodies can often be functionally expressed as intracellular antibodies in the cytoplasm of eukaryotic cells. Using a selection method based on the Sos Recruitment System, a cytoplasmic yeast two-hybrid approach, we have isolated single-domain antibodies able to bind HIV-1 Vpr and Capside proteins in the yeast cytoplasm. One anti-Vpr single domain antibody was able to bind the HIV-1 regulatory Vpr protein in the cytoplasm of eukaryotic cells, leading to its delocalization from the nucleus to the cytoplasm. To our knowledge, this is the first description of a functional single-domain intrabody targeting HIV-1 Vpr, isolated using an in vivo cytoplasmic selection method that alleviates some limitations of the conventional yeast two-hybrid system