Inhibition of DDR1 enhances in vivo chemosensitivity in KRAS-mutant lung adenocarcinoma

Platinum-based chemotherapy in combination with immune-checkpoint inhibitors is the current standard of care for patients with advanced lung adenocarcinoma (LUAD). However, tumor progression evolves in most cases. Therefore, predictive bioma ricers are needed for better patient stratification and for the identification of new therapeutic strategies, including enhancing the efficacy of chemotoxic agents. Here, we hypothesized that discoidin domain receptor 1 (DDR1) may be both a predictive factor for chemoresistance in patients with LUAD and a potential target positively selected in resistant cells. By using biopsies from patients with LUAD, KRAS-mutant LUAD cell lines, and in vivo genetically engineered KRAS-driven mouse models, we evaluated the role of DDR1 in the context of chemotherapy treatment. We found that DORT is upregulated during chemotherapy both in vitro and in viva. Moreover, analysis of a cohort of patients with LUAD suggested that high DOR1 levels in pretreatment biopsies correlated with poor response to chemotherapy. Additionally, we showed that combining DORI inhibition with chemotherapy prompted a synergistic therapeutic effect and enhanced cell death of KRAS-mutant tumors in vivo. Collectively, this study suggests a potential role for DDR1 as both a predictive and prognostic biomarker, potentially improving the chemotherapy response of patients with LUAD

The Expression of Myeloproliferative Neoplasm-Associated Calreticulin Variants Depends on the Functionality of ER-Associated Degradation

BACKGROUND: Mutations in CALR observed in myeloproliferative neoplasms (MPN) were recently shown to be pathogenic via their interaction with MPL and the subsequent activation of the Janus Kinase - Signal Transducer and Activator of Transcription (JAK-STAT) pathway. However, little is known on the impact of those variant CALR proteins on endoplasmic reticulum (ER) homeostasis. METHODS: The impact of the expression of Wild Type (WT) or mutant CALR on ER homeostasis was assessed by quantifying the expression level of Unfolded Protein Response (UPR) target genes, splicing of X-box Binding Protein 1 (XBP1), and the expression level of endogenous lectins. Pharmacological and molecular (siRNA) screens were used to identify mechanisms involved in CALR mutant proteins degradation. Coimmunoprecipitations were performed to define more precisely actors involved in CALR proteins disposal. RESULTS: We showed that the expression of CALR mutants alters neither ER homeostasis nor the sensitivity of hematopoietic cells towards ER stress-induced apoptosis. In contrast, the expression of CALR variants is generally low because of a combination of secretion and protein degradation mechanisms mostly mediated through the ER-Associated Degradation (ERAD)-proteasome pathway. Moreover, we identified a specific ERAD network involved in the degradation of CALR variants. CONCLUSIONS: We propose that this ERAD network could be considered as a potential therapeutic target for selectively inhibiting CALR mutant-dependent proliferation associated with MPN, and therefore attenuate the associated pathogenic outcomes

Etude des rétrotransposons LINE-1 dans la leucémie myéloïde chronique

Le gène hybride BCR-ABL1, responsable de la leucémie myéloïde chronique (LMC), code une protéine à activité tyrosine kinase constitutive. Lors d une étude transcriptomique menée au laboratoire sur des patients résistants secondaires à l imatinib, les deux gènes codant les protéines des rétrotransposons LINE-1 ont été trouvés sous exprimés d environ 20 fois lorsque les patients rechutent. Le rôle des transposons n a jamais été clairement défini, ils assurent certainement une fonction importante puisqu ils sont conservés au cours de l évolution et présents chez tous les organismes. Le but de ce travail a été d étudier l implication de LINE-1 dans la LMC. La sous-expression de LINE-1 est-elle une conséquence de la présence de BCR-ABL1 ou une cause de son apparition ? Différents groupes ont montré que les rétrotransposons LINE-1 possédent la capacité de réparation des cassures double-brin de l ADN. Nous avons fait l hypothèse qu une diminution de l expression des gènes codés par les rétrotransposons LINE-1 entraînerait l instabilité génétique observée dans la LMC. Une étude réalisée chez des patients atteints de LMC et des sujets contrôles a montré une correlation inverse entre l expression de LINE-1 et celle de l oncogène BCR-ABL1. Parallèlement, une étude sur des lignées cellulaires leucémiques humaines BCR-ABL positives et négatives a été réalisée. Nous avons recherché le lien qui existe entre l expression de LINE 1, de BCR-ABL1 et la réparation des cassures double-brin de l ADN. Nous avons montré d une part qu une inhibition de l expression de BCR-ABL1 induit une augmentation de l expression des transposons LINE-1 D autre part, une diminution de l expression de LINE-1 entraîne une apparition du transcrit BCR-ABL1 dans les cellules BCR-ABL negatives.BCR-ABL1 fusion gene, responsible of the chronic myeloid leukemia (CML) encodes a constitutively activated tyrosine kinase protein. Expression of both LINE-1 retrotransposon ORFs were found decreased at the time of imatinib resistance in a comparative transcriptional study focused on secondary resistant patients. The role of retrotransposons is unclear. They are conserved through evolution. This project focuses on the involvement of LINE-1 in CML. Is LINE-1 under expression a result of BCR-ABL1 expression or is it at the origin of BCR ABL1? Different groups have shown that LINE-1 retrotransposons were able to repair DNA double strands breaks. We suggest that LINE-1 under expression could be responsible of genetic instability observed in CML. We show in a study on CML patients and healthy subjects that LINE-1 expression is inverse correlated to BCR-ABL1 expression. Moreover, study on BCR-ABL+ and BCR-ABL- human leukemic cell lines was carried on. First, we show that decrease of BCR-ABL1 expression induces increase of LINE-1 expression. Then that decrease of LINE-1 expression generates BCR-ABL1 transcript in BCR-ABL negatives cell lines.BORDEAUX2-Bib. électronique (335229905) / SudocSudocFranceF

The ura5 gene of the filamentous fungus Podospora anserina: nucleotide sequence and expression in transformed strains

International audienceWe have sequenced the ura5 gene of the filamentous fungus Podospora anserina. The deduced sequence for the orotidylic acid pyrophosphorylase (OMPppase) has been compared with the Escherichia coli enzyme which is the only known sequence for this enzyme. This comparison shows extensive blocks of homology. The expression of the ura5 gene has been studied in a ura5 mutant which has been transformed by a recombinant plasmid carrying the ura5 gene. We observed that strains carrying integrated multicopies of the transforming vector exhibit higher specific activity for OMPppase than wild type (wt). By recombination we have constructed a strain in which the level of this enzyme is 32 times higher than in the wt strain

A gene responsible for vegetative incompatibility in the fungus Podospora anserina encodes a protein with a GTP-binding motif and Gβ homologous domain

International audienceThe het-e-1 gene of the fungus Podospora anserina is responsible for vegetative incompatibility through specific interactions with different alleles of the unlinked gene, het-c. Coexpression of two incompatible genes triggers a cell death reaction that prevents heterokaryon formation. The het-e1 allele has been cloned to get information on the function of the locus. It encodes a putative 1356-amino-acid polypeptide that displays two sequence motifs that have not yet been reported to be present on a single polypeptide. They are a GTP-binding domain and a repeated region that shares similarity with that of the beta-transducin. Contrary to other members of the beta-transducin family, sequence conservation between the repeated units is very strong and the number of repeats is different in wild-type het-e alleles

Use of a Linear Plasmid Containing Telomeres as an Efficient Vector for Direct Cloning in The Filamentous FungusPodospora anserina

International audienceIn Podospora anserina a linear plasmid with telomeric ends behaves as an artificial acentric minichromosome. Transformation is at least 100 times more efficient than with integrative vectors. Genomic DNA was inserted in this plasmid in vitro and the mixture used to transform a leu1-1 strain. Many fungal clones containing the leu1 gene as a genomic insert in the linear plasmid were identified. The leu1 gene was rescued as a circular plasmid in Escherichia coli demonstrating that a direct cloning procedure can be applied for the fungus P. anserina. The conservation of telomeric sequences among filamentous fungi suggests that a telomere-based linear plasmid could provide a general cloning vector for filamentous fungi

A new mechanism of resistance to ABL1 tyrosine kinase inhibitors in a BCR-ABL1-positive cell line

International audienceTyrosine kinase inhibitors (TKI) constitute the frontline treatment for chronic myeloid leukemia patients. Dasatinib, a second-generation TKI, was developed to overcome TKI resistances. However, dasatinib resistances are also described but remain less characterized. To mimic in vivo acquired dasatinib resistance, the BCR-ABL1-positive cell line K562 was transiently treated with a pharmacological concentration of dasatinib, for a short time in the presence of stem cell factor. A dasatinib resistant counterpart (K562 RES) was developed. Investigation of resistance mechanisms using kinase substrate arrays revealed that FYN was overactivated in K562 RES. The FYN inhibitor KX2-391 cooperated with dasatinib to block K562 RES proliferation. Cell tracking experiments showed that activated FYN support cell proliferation independently of BCR-ABL1 in K562 RES cells. Moreover, the MEK-ERK pathway was found hyper-phosphorylated in K562 RES cells even in the presence of dasatinib. Actually, ERK1/2 activity supported viability in K562 RES only in the absence of BCR-ABL1 activity. Finally, BCR-ABL1 and MEK inhibitor combination was sufficient to induce cell death even in non-proliferating resistant cells. Considering the conditions used to generate this dasatinib resistant cell line, such a resistance mechanism could be found in dasatinib treated patients. Consequently, it is valuable to know that inhibition of the MEK-ERK1/2 axis can overcome this resistance

Combined use of foraminifera Mg/Ca and Sr/Ca concentrations to strengthen temperature reconstructions over geological timescales.

International audienceTo overcome the lack of long-term instrumental records, paleo-reconstructions using geochemical signatures preserved in the carbonate shells of foraminifera, provide a unique opportunity to reconstruct changes in seawater environmental parameters over time and evaluate the validity of climate change scenarios. High resolution paleoceanographic studies can provide new insights about Paleoclimatic perturbations allowing better prediction of biotic responses to modern warming and acidification. In this study, we will present calibrations using modern foraminifera sampled from surface seawater of the Atlantic Ocean, where all environmental parameters were recorded. Mg and Sr concentrations were determined using LA-ICP-MS, while the oxygen isotope composition of the same tests as used for the elemental analyses was subsequently measured by IRMS. Our results show that combining Mg and Sr data to compute temperature improve temperature reconstruction most probably by accounting for the impact of salinity and/or carbonate chemistry.To further test this hypothesis a new set of temperature data covering the Paleogene was extracted from δ18O, porosity, and combined Mg/Ca and Sr/Ca data, measured on Subbotina linaperta, from three localities: South and North Atlantic, and North Pacific. This foraminifera multi-proxies approach allowed to determine a continuous Paleogene sub-surface temperature. For the first time, the results of the three proxies are consistent with each other and highlight the existence of a latitudinal gradient over the Paleogene, punctuated by short term variations. Hence, we emphasize here: 1) the combined use of Mg/Ca and Sr/Ca concentrations within temperature equation reconstruction to strengthen paleoenvironmental reconstructions on geological timescales, and 2) the utility of porosity as a complementary ecological proxy, to either discard or complement geochemical data

Combined use of foraminifera Mg/Ca and Sr/Ca concentrations to strengthen temperature reconstructions over geological timescales.

International audienc