Mechanisms of AXL overexpression and function in Imatinib-resistant chronic myeloid leukemia cells

AXL is a receptor tyrosine kinase of the TAM family, the function of which is poorly understood. We previously identified AXL overexpression in Imatinib (IM)-resistant CML cell lines and patients. The present study was conducted to investigate the role of AXL and the mechanisms underlying AXL overexpression in Tyrosine Kinase Inhibitor (TKI)-resistant CML cells. We present evidence that high AXL expression level is a feature of TKI-resistant CML cells and knockdown of AXL sensitized TKI-resistant cells to IM. In addition, expression of wild-type AXL but not a dominant negative form of AXL confers IM-sensitive CML cells the capacity to resist IM effect. AXL overexpression required PKCα and β and constitutive activation of ERK1/2. Accordingly, GF109203X a PKC inhibitor, U0126 a MEK1 inhibitor and PKCα/β knockdown restore sensitivity to IM while PKCα or PKCβ overexpression in CML cells promotes protection against IM-induced cell death. Finally, using luciferase promoter activity assays we established that AXL is regulated transcriptionally through the AP1 transcription factor. Our findings reveal an unexpected role of AXL in resistance to TKI in CML cells, identify the molecular mechanisms involved in its overexpression and support the notion that AXL is a new marker of resistance to TKI in CML

Effects of proton versus photon irradiation on (lymph) angiogenic, inflammatory, proliferative and anti-tumor immune responses in head and neck squamous cell carcinoma

International audienceThe proximity of organs at risk makes the treatment of head and neck squamous cell carcinoma (HNSCC) challenging by standard radiotherapy. The higher precision in tumor targeting of proton (P) therapy could promote it as the treatment of choice for HNSCC. Besides the physical advantage in dose deposition, few is known about the biological impact of P versus photons (X) in this setting. To investigate the comparative biological effects of P versus X radiation in HNSCC cells, we assessed the relative biological effectiveness (RBE), viability, proliferation and mRNA levels for genes involved in (lymph)angiogenesis, inflammation, proliferation and anti-tumor immunity. These parameters, particularly VEGF-C protein levels and regulations, were documented in freshly irradiated and/or long-term surviving cells receiving low/high-dose, single (SI)/multiple (MI) irradiations with P/X. The RBE was found to be 1.1 Key (lymph)angiogenesis and inflammation genes were downregulated (except for vegf-c) after P and upregulated after X irradiation in MI surviving cells, demonstrating a more favorable profile after P irradiation. Both irradiation types stimulated vegf-c promoter activity in a NF-κB-dependent transcriptional regulation manner, but at a lesser extent after P, as compared to X irradiation, which correlated with mRNA and protein levels. The cells surviving to MI by P or X generated tumors with higher volume, anarchic architecture and increased density of blood vessels. Increased lymphangiogenesis and a transcriptomic analysis in favor of a more aggressive phenotype were observed in tumors generated with X-irradiated cells. Increased detection of lymphatic vessels in relapsed tumors from patients receiving X radiotherapy was consistent with these findings. This study provides new data about the biological advantage of P, as compared to X irradiation. In addition to its physical advantage in dose deposition, P irradiation may help to improve treatment approaches for HNSCC

Anti-tumor Activity of Novel Compounds Targeting BCR-ABL, c-SRC and BCR-ABLT315I in Chronic Myelogenous Leukemia

Chronic myelogenous leukemia (CML) is a hematological stem-cell disorder characterized by the expression of the BCR-ABL fusion protein, a constitutively active tyrosine kinase that causes pathogenesis. The development of tyrosine kinase inhibitors (TKIs) targeting the BCR-ABL oncogene has proven an effective approach to treat CML, but a non-negligible proportion of patients develop a resistance to this class of drugs. Of note, the T315I mutant of BCR-ABL is resistant to all known TKIs, with the noticeable exception of ponatinib. To address this unmet medical need, a new series of compounds was designed and tested for anti-tumor effects against BCR-ABLT315I CML. The effects of three OriBase Pharma compounds (OR1001, OR1002 and OR1003) on the kinase activity of wild-type and mutant BCR-ABL proteins, on cell proliferation and on the growth of subcutaneous xenografts of CML cells in athymic mice were investigated. In vitro, the three compounds were potent inhibitors of both ABL and c-SRC kinases and of the main mutants of ABL, including T315I. The three compounds inhibited the proliferation of cell lines expressing the wild-type and several mutated forms of BCR-ABL, including T315I. Finally, in a mouse xenograft model, OR1001, was found to significantly reduce tumor growth. These data support the potential of OR1001 as an effective therapy for the treatment of de novo and TKI-resistant patients

BCL2L10 is a predictive factor for resistance to Azacitidine in MDS and AML patients

Azacitidine is the leading compound to treat patients suffering myelodysplastic syndrome (MDS) or AML with less than 30% of blasts, but a majority of patients is primary refractory or rapidly relapses under treatment. These patients have a drastically reduced life expectancy as compared to sensitive patients. Therefore identifying predictive factors for AZA resistance is of great interest to propose alternative therapeutic strategies for non-responsive patients. We generated AZA-resistant myeloid cell line (SKM1-R) that exhibited increased expression of BCL2L10 an anti-apoptotic Bcl-2 family member. Importantly, BCL2L10 knockdown sensitized SKM1-R cells to AZA effect suggesting that increased BCL2L10 expression is linked to AZA resistance in SKM1-R. We next established in 77 MDS patients that resistance to AZA is significantly correlated with the percentage of MDS or AML cells expressing BCL2L10. In addition, we showed that the proportion of BCL2L10 positive bone marrow cells can predict overall survival in MDS or AML patients. We propose a convenient assay in which the percentage of BCL2L10 expressing cells as assessed by flow cytometry is predictive of whether or not a patient will become resistant to AZA. Therefore, systematic determination of BCL2L10 expression could be of great interest in newly diagnosed and AZA-treated MDS patients

A New Hydroxylated Nonaprenylhydroquinone from the Mediterranean Marine Sponge Sarcotragus spinosulus

Chemical investigation of the Mediterranean sponge Sarcotragus spinosulus led to the isolation of a new hydroxylated nonaprenylhydroquinone, along with two known metabolites, hepta- and octaprenylhydroquinones. The structure of the new metabolite was assigned by extensive 1D and 2D NMR analyses and MS studies. The antileukemic effect of the three compounds towards the chronic myelogenous leukemia (CML) cells line K562 was also evaluated

DIFFERENTIAL EFFECTS OF PROTONTHERAPY AND PHOTONTHERAPY ON HEAD AND NECK SQUAMOUS CELL CARCINOMA (HNSCC) POST-TREATMENT AGGRESSIVENESS

International audienceHead and neck cancers, the 7 th cause of death worldwide, are currently treated with a combination of surgical resection of the primary tumor, chemotherapy and radiotherapy, depending on the disease stage. Conventional photontherapy nevertheless remains difficult to apply to tumors such as head and neck squamous cell carcinomas (HNSCC), due to the proximity of numerous organs at risk (i.e. salivary glands, esophagus, larynx). Protontherapy has been proposed to treat such sensitive tumors, due to its high precision in tumor targeting. Despite the current therapeutic strategies, the five-year overall survival rate of HNSCC patients is only 53%, with a high percentage of poor response to therapy and a high recurrence rate. Lymph node metastasis, the first sign of tumor progression, has been directly correlated to Vascular Endothelial Growth Factor-C (VEGF-C) expression levels in HNSCC and to VEGF-C-dependent tumoral lymphatic vessel development. In the present study, we investigated the hypothesis that, beside the advantage in dose deposition, protontherapy may show distinct biological properties than photontherapy (at similar doses). We thus examined several in vitro biological behaviors of HNSCC-derived cells when exposed to photons or protons, focusing on molecules with key roles in the progression and prognosis of HNSCC, such as genes/proteins involved in (lymph)angiogenesis/metastasis, inflammation, tumor cell proliferation and anti-tumor immunity, tumorigenic potential. We showed that cell proliferation decreased with the irradiation dose, both in proton and photon irradiated cells. Proton and photon irradiations increased VEGF-C and PD-L1 expression in HNSCC cells. In cells surviving multiple irradiation, key (lymph)angiogenesis and inflammation genes were down-regulated (except for VEGF-C) after protontherapy and up-regulated after photontherapy. Both irradiation types stimulated VEGF-C promoter activity via NF-kB-dependent transcriptional regulation. We conclude that cell resistance, tumor progression and lymphangiogenesis induction is less pronounced after proton irradiation than after photon irradiation. We validated these results by in vivo experiments: Photon-or proton-irradiated HNSCC-derived cells were xenografted subcutaneously into immunodeficient mice. Cells surviving to multiple irradiations by protons or photons generated tumors with higher volume, anarchic architecture and increased density of blood vessels than non-irradiated cells. Increased lymphangiogenesis and a transcriptomic analysis in favor of a more aggressive phenotype were observed in tumors generated with X irradiated cells. Detection of a denser lymphatic vessel network in relapsed tumors from patients receiving conventional X radiotherapy is consistent with these results

Activation of HER3 Interferes with Antitumor Effects of Axl Receptor Tyrosine Kinase Inhibitors: Suggestion of Combination Therapy.

The Axl receptor tyrosine kinase (RTK) has been established as a strong candidate for targeted therapy of cancer. However, the benefits of targeted therapies are limited due to acquired resistance and activation of alternative RTKs. Therefore, we asked if cancer cells are able to overcome targeted Axl therapies. Here, we demonstrate that inhibition of Axl by short interfering RNA or the tyrosine kinase inhibitor (TKI) BMS777607 induces the expression of human epidermal growth factor receptor 3 (HER3) and the neuregulin 1(NRG1)-dependent phosphorylation of HER3 in MDA-MB231 and Ovcar8 cells. Moreover, analysis of 20 Axl-expressing cancer cell lines of different tissue origin indicates a low basal phosphorylation of RAC-alpha serine/threonine-protein kinase (AKT) as a general requirement for HER3 activation on Axl inhibition. Consequently, phosphorylation of AKT arises as an independent biomarker for Axl treatment. Additionally, we introduce phosphorylation of HER3 as an independent pharmacodynamic biomarker for monitoring of anti-Axl therapy response. Inhibition of cell viability by BMS777607 could be rescued by NRG1-dependent activation of HER3, suggesting an escape mechanism by tumor microenvironment. The Axl-TKI MPCD84111 simultaneously blocked Axl and HER2/3 signaling and thereby prohibited HER3 feedback activation. Furthermore, dual inhibition of Axl and HER2/3 using BMS777607 and lapatinib led to a significant inhibition of cell viability in Axl-expressing MDA-MB231 and Ovcar8 cells. Therefore, we conclude that, in patient cohorts with expression of Axl and low basal activity of AKT, a combined inhibition of Axl and HER2/3 kinase would be beneficial to overcome acquired resistance to Axl-targeted therapies

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency–Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Implication de l’inflammasome NLRP3 dans la détection des toxines bactériennes et dans l’évolution du COVID-19

The innate immune detection of pathogenic microbes is crucial to enhance the immune response. During an infection, the activation of the innate immune system is the first step to establish an adaptative immune response. A physiological immune response is defined as adapted to the microbial environment and as an effectively resolved inflammation. To do so, the host must be able to qualitatively and quantitatively detect microbes. The detection of conserved MAMPs allows the host to evaluate the number of microbes. On the other hand, the detection of virulence factors’ activity – pathogen-specific – give a qualitative view of microbes.The discovery of inflammasomes has allowed a major advance in the understanding of the mechanisms of detection of pathogen- and danger associated molecular patterns. Inflammasomes are macromolecular complexes responsible for the activation of Caspase-1 and the maturation of IL-1ß and IL-18. Initially discovered for their involvement in autoinflammatory pathologies, there is growing evidence for the role of inflammasomes in detecting the activity of toxins and microbial effectors including those targeting Rho GTPases.Rho GTPases are located at the crossroads of major cell signaling pathways; by regulating cell migration, phagocytosis or even gene transcription, these are major players in immunity. This makes them preferential targets for pathogenic microorganisms. Indeed, more than 30 bacterial virulence factors manipulate Rho GTPases using various strategies leading to their inhibition or activation.The uropathogenic Escherichia coli (E. coli) is the primary cause of cystitis and pyelonephritis and in the most severe cases of bacteremia. More than a third of uropathogenic E. coli possess the CNF1 toxin, a de-amidase that activates Rho GTPases. This modification destroys the intrinsic GTPase activity stimulated by the GAPs (GTPase-activating protein) of Rho, Rac and Cdc42.Before my arrival at the laboratory, Dr. Laurent Boyer's team showed that the CNF1 toxin induces an immune response. Indeed, during bacteremia in mice or systemic infection in drosophila, E. coli expressing CNF1 are eliminated more quickly than E. coli deleted for CNF1. At the cellular level, the activation of Rho GTPases by CNF1 is responsible for the production of pro-inflammatory cytokines. Alongside this transcriptional response, the CNF1 toxin causes IL-1β to mature in a Caspase-1 dependent fashion, suggesting the involvement of an inflammasome.My thesis project was to identify the inflammasome involved in the detection of the CNF1 toxin and to characterize the signaling pathway leading to this activation.We have identified the NLRP3 inflammasome as being responsible for the detection of GTPase Rac2 activation by the CNF1 toxin. We then studied the role of the kinase Pak1, a major effector of Rac2, in this signaling pathway and we were able to show that Pak1 plays a crucial role in the activation of the inflammasome NLRP3 by CNF1. Pak1 phosphorylates Thr659 of the NLRP3 receptor and this phosphorylation is critical for the recruitment of the regulatory protein Nek7 and the activation of the NLRP3 inflammasome downstream of the detection of CNF1. Finally, we were able to show the major role of the Pak1-NLRP3 pathway in the establishment of an anti-virulence immune response during mice bacteremia.In addition, we used our expertise to study the activation of the NLRP3 inflammasome in patients infected with SARS-CoV-2. This study allowed us to establish the signature of the NLRP3 response in the circulating myeloid cells of COVID-19 patients and to use these parameters to define a score to predict the patients’ outcome.My work on the involvement of the NLRP3 inflammasome in the detection of virulence has provided a better understanding of the mechanisms involved in an anti-infective response.Lors d’une infection, la détection des microorganismes pathogènes est indispensable à la réponse immunitaire innée et à l’initiation de l’immunité adaptative. Une réponse immunitaire physiologique est définie comme une réponse proportionnelle et adaptée à la stimulation microbienne et doit conduire à une résolution de l’inflammation. Pour cela, l’hôte doit être capable de détecter quantitativement et qualitativement les micro-organismes. Du point de vue quantitatif, l’hôte détecte des motifs structuraux conservés au sein d’une classe de micro-organismes. D’autre part, la détection de l’activité des facteurs de virulence – spécifiques aux pathogènes – permet une détection qualitative.La découverte des PRR et plus précisément des inflammasomes a permis une avancée majeure dans la compréhension des mécanismes de détection des pathogènes et des signaux de danger liés aux dommages cellulaires produits lors de l’infections. Les inflammasomes sont des complexes responsables de l’activation de la Caspase-1 et de la maturation de l’IL-1ß et IL-18. Initialement découverts pour leur implication dans des maladies auto-inflammatoires, les inflammasomes sont également impliqués dans la détection de l’activité des facteurs de virulence notamment ceux ciblant les Rho-GTPases.Les Rho-GTPases sont situées au carrefour de grandes voies de signalisation cellulaire ; en régulant la migration cellulaire, la phagocytose ou encore la transcription des gènes, ceux sont des acteurs majeurs de l’immunité. Cela en fait des cibles préférentielles pour les bactéries pathogènes. En effet, plus de 30 facteurs de virulence bactériens manipulent les Rho GTPases en utilisant diverses stratégies menant à leur inhibition ou leur activation.La bactérie E. coli uropathogène est la première cause de cystites, pyélonéphrites et dans les cas les plus graves de bactériémies. Plus de 30% des E. coli uropathogènes possèdent la toxine CNF1, une dé-amidase activant les Rho-GTPases.Avant mon arrivée au laboratoire, l’équipe du Dr Laurent Boyer a montré que la toxine CNF1 induit une réponse immunitaire. En effet, au cours de la bactériémie chez la souris ou de l’infection systémique chez la drosophile, la détection de la toxine CNF1 induit la clairance bactérienne. Au niveau cellulaire, l’activation des Rho-GTPases par CNF1 est responsable de la production de cytokines pro-inflammatoires. En parallèle de cette réponse transcriptionnelle, la toxine CNF1 provoque la maturation de l’IL-1ß de façon dépendante de la Caspase-1, suggérant l’implication d’un inflammasome.Mon projet de thèse était d’identifier l’inflammasome impliqué dans la détection de la toxine CNF1 et de caractériser la voie de signalisation menant à cette activation.Nous avons identifié l’inflammasome NLRP3 comme étant responsable de la détection de l’activation de la GTPase Rac2 par la toxine CNF1. Nous avons ensuite étudié le rôle de la kinase Pak1, effecteur de Rac2, dans cette voie de signalisation et nous avons pu montrer que Pak1 joue un rôle crucial dans l’activation de l’inflammasome NLRP3 par CNF1. Pak1 phosphoryle la Thr659 du récepteur NLRP3 et cette phosphorylation est déterminante pour le recrutement de la protéine régulatrice Nek7 et l’activation de l’inflammasome NLRP3. Finalement, nous avons pu montrer le rôle majeur de la voie Pak1-NLRP3 dans la mise en place d’une réponse immunitaire anti-virulence au cours de la bactériémie.De plus, nous avons utilisé notre expertise pour étudier l’activation de l’inflammasome NLRP3 chez les patients infectés par le SARS-CoV-2. Cette étude nous a permis d’établir la signature de la réponse NLRP3 dans les cellules myéloïdes circulantes des patients COVID-19 et d’utiliser ces paramètres pour définir un score permettant de prédire l’évolution des patients.Mon travail sur l’implication de l’inflammasome NLRP3 dans la détection de la virulence a permis une meilleure compréhension des mécanismes impliqués lors d’une réponse immunitaire anti-infectieuse