Assessing BRCA1 activity in DNA damage repair using human induced pluripotent stem cells as an approach to assist classification of BRCA1 variants of uncertain significance.

Funder: King’s College LondonFunder: The European Union Scholarship ProgrammeFunder: National Institute for Health Research (NIHR)Establishing a universally applicable protocol to assess the impact of BRCA1 variants of uncertain significance (VUS) expression is a problem which has yet to be resolved despite major progresses have been made. The numerous difficulties which must be overcome include the choices of cellular models and functional assays. We hypothesised that the use of induced pluripotent stem (iPS) cells might facilitate the standardisation of protocols for classification, and could better model the disease process. We generated eight iPS cell lines from patient samples expressing either BRCA1 pathogenic variants, non-pathogenic variants, or BRCA1 VUSs. The impact of these variants on DNA damage repair was examined using a ɣH2AX foci formation assay, a Homologous Repair (HR) reporter assay, and a chromosome abnormality assay. Finally, all lines were tested for their ability to differentiate into mammary lineages in vitro. While the results obtained from the two BRCA1 pathogenic variants were consistent with published data, some other variants exhibited differences. The most striking of these was the BRCA1 variant Y856H (classified as benign), which was unexpectedly found to present a faulty HR repair pathway, a finding linked to the presence of an additional variant in the ATM gene. Finally, all lines were able to differentiate first into mammospheres, and then into more advanced mammary lineages expressing luminal- or basal-specific markers. This study stresses that BRCA1 genetic analysis alone is insufficient to establish a reliable and functional classification for assessment of clinical risk, and that it cannot be performed without considering the other genetic aberrations which may be present in patients. The study also provides promising opportunities for elucidating the physiopathology and clinical evolution of breast cancer, by using iPS cells

Masitinib (AB1010), a Potent and Selective Tyrosine Kinase Inhibitor Targeting KIT

International audienceBackground: The stem cell factor receptor, KIT, is a target for the treatment of cancer, mastocytosis, and inflammatory diseases. Here, we characterise the in vitro and in vivo profiles of masitinib (AB1010), a novel phenylaminothiazole-type tyrosine kinase inhibitor that targets KIT. Methodology/Principal Findings: In vitro, masitinib had greater activity and selectivity against KIT than imatinib, inhibiting recombinant human wild-type KIT with an half inhibitory concentration (IC50) of 200 ± 40 nM and blocking stem cell factor-induced proliferation and KIT tyrosine phosphorylation with an IC50 of 150 ± 80 nM in Ba/F3 cells expressing human or mouse wild-type KIT. Masitinib also potently inhibited recombinant PDGFR and the intracellular kinase Lyn, and to a lesser extent, fibroblast growth factor receptor 3. In contrast, masitinib demonstrated weak inhibition of ABL and c-Fms and was inactive against a variety of other tyrosine and serine/threonine kinases. This highly selective nature of masitinib suggests that it will exhibit a better safety profile than other tyrosine kinase inhibitors; indeed, masitinib-induced cardiotoxicity or genotoxicity has not been observed in animal studies. Molecular modelling and kinetic analysis suggest a different mode of binding than imatinib, and masitinib more strongly inhibited degranulation, cytokine production, and bone marrow mast cell migration than imatinib. Furthermore, masitinib potently inhibited human and murine KIT with activating mutations in the juxtamembrane domain. In vivo, masitinib blocked tumour growth in mice with subcutaneous grafts of Ba/F3 cells expressing a juxtamembrane KIT mutant. Conclusions: Masitinib is a potent and selective tyrosine kinase inhibitor targeting KIT that is active, orally bioavailable in vivo, and has low toxicit

Etude de l'implication des protéines fes et fer dans la signalisation des recepteurs à activité tyrosine kinase kit et FLT3

La recherche de nouveaux acteurs de la signalisation du récepteur tyrosine kinase KIT, nous a conduit à étudier les protéines tyrosine kinase cytoplasmiques FES et FER. Trois projets ont émané de cette identification. Une première étude a porté sur le récepteur oncogénique KITD816V (mutant retrouvé chez la majorité des patients atteints de mastocytose). Dans ce contexte, FES est phosphorylée sur tyrosines, reflétant son activation constitutive. De plus, en aval de ce récepteur, FES est un régulateur positif de la prolifération cellulaire et plus pre cisément, cette kinase est impliquée dans la transition des phases G1/S du cycle cellulaire. Au plan moléculaire, FES n est pas nécessaire à l activation de la MAP Kinase p38, mais pour celles des protéines STAT et p70S6K. Alors que l action de FES sur la phosphorylation des STAT semble dépendante du modèle d étude, son action sur la p70S6K s avère toujours être une régulation positive. De plus, en aval de KITD816V, FER n est pas impliquée dans la prolifération cellulaire. Un deuxième sujet tente de déterminer la ou les fonction(s) de FES dans le contexte du récepteur KITWT. Ainsi, nous avons déjà pu montrer que FES interagissait avec KIT stimulé par son ligand et que selon la même cinétique, FES était activée. Ces deux événements sont d une part, relativement tardifs et d autre part, transitoires. D un point de vue fonctionnel, FES et FER interviennent dans le chimiotactisme en réponse au SCF mais pas dans la prolifération cellulaire. Un troisième travail a été mené sur les rôles des protéines FES et FER en aval du récepteur oncogénique FLT3ITD (mutation majoritaire chez les patients atteints de LAM). Pour ces deux kinases, leurs activations sont dépendantes du contexte oncogénique induit par FLT3ITD. L absence de FES ou de FER dans le système FLT3ITD provoque une diminution de la prolifération cellulaire. Dans le cas de FER, cet effet est consécutif à des défauts de transition entre les phases G1/S et G2/M du cycle cellulaire. Dans le cas de FES, sa présence semble nécessaire à la survie cellulaire. Au niveau moléculaire, ces deux protéines sont des régulateurs positifs de l activation de STAT5 et des protéines en aval de la PI3Kinase. Ces études ont donc conduit à révéler les protéines FES et FER comme des effecteurs indispensables en aval des récepteurs KIT et FLT3.AIX-MARSEILLE2-BU Sci.Luminy (130552106) / SudocSudocFranceF

SRC-Family Kinases in Acute Myeloid Leukaemia and Mastocytosis

International audienc

The tyrosine kinase FES is an essential effector of KITD816V proliferation signal

International audienceKIT is a tyrosine kinase receptor that is aberrantly activated in several neoplasms. In human pathologies, the most frequent mutation of KIT occurs at codon 816. The resulting KIT mutant protein is activated in the absence of ligand and is resistant to the clinically available inhibitors of KIT. In this report, we provide evidence for an essential function of the cytoplasmic tyrosine kinase FES downstream of KITD816V. FES is phosphorylated on tyrosine residues in cells that carry KITD816V mutation, and this phosphorylation is KIT dependent. Reduction of FES expression using RNA interference results in decreased cell proliferation in human or murine cells harboring KITD816V or the homologous mouse mutation KITD814Y. The reduced cell growth can be rescued using another cytokine (granulocyte-macrophage colony-stimulating factor [GM-CSF]) and is not observed when the closely related fer gene is targeted. Finally, signaling downstream of KITD816V is altered in cells lacking FES expression. This study shows a major function of FES downstream of activated KIT receptor and thereby points to FES as a novel target in KIT-related pathologies

p38 alpha Negatively Regulates Survival and Malignant Selection of Transformed Bronchioalveolar Stem Cells

Lung cancer is the cause of most cancer-related deaths in the Western world. Non-small cell lung cancer accounts for almost 80% of all lung cancers, and 50% of this type are adenocarcinomas. The cellular and molecular origin of this type of lung cancer remains elusive and the mechanisms are poorly known. It is known that K-Ras mutations appear in 25-30% of lung adenocarcinomas and it is the best known single mutation that can be related to lung cancers. Recently, it has been suggested that a putative population of mouse bronchioalveolar stem cells could be considered as the cell of origin of adenocarcinomas. These cells are expanded in the early stages of lung tumorigenesis. We have isolated a population of mouse bronchioalveolar stem cells and induced their transformation by oncogenic K-RasG12. Different approaches have shown that an intracellular network linking the p38α MAPK and the PI3K-Pdk1 pathways is involved in regulating the survival and malignant progression of the transformed cells. Absence of p38α catalytic activity leads to further Pdk1 activation (independent of Akt and Erk activity), enhancing the survival and proliferation of the more malignant lung cancer cells. This specifically selects high Sca-1/Sox9 cells that harbour a stronger colonizing potential, as they maintain their capacity to produce secondary tumors after serial transplantations.status: publishe

p38α negatively regulates survival and malignant selection of transformed bronchioalveolar stem cells.

Lung cancer is the cause of most cancer-related deaths in the Western world. Non-small cell lung cancer accounts for almost 80% of all lung cancers, and 50% of this type are adenocarcinomas. The cellular and molecular origin of this type of lung cancer remains elusive and the mechanisms are poorly known. It is known that K-Ras mutations appear in 25-30% of lung adenocarcinomas and it is the best known single mutation that can be related to lung cancers. Recently, it has been suggested that a putative population of mouse bronchioalveolar stem cells could be considered as the cell of origin of adenocarcinomas. These cells are expanded in the early stages of lung tumorigenesis. We have isolated a population of mouse bronchioalveolar stem cells and induced their transformation by oncogenic K-RasG12. Different approaches have shown that an intracellular network linking the p38α MAPK and the PI3K-Pdk1 pathways is involved in regulating the survival and malignant progression of the transformed cells. Absence of p38α catalytic activity leads to further Pdk1 activation (independent of Akt and Erk activity), enhancing the survival and proliferation of the more malignant lung cancer cells. This specifically selects high Sca-1/Sox9 cells that harbour a stronger colonizing potential, as they maintain their capacity to produce secondary tumors after serial transplantations

An essential pathway links FLT3-ITD, HCK and CDK6 in acute myeloid leukemia

CDK4/CDK6 and RB proteins drive the progression through the G1 phase of the cell cycle. In acute myeloid leukemia (AML), the activity of the CDK/Cyclin D complex is increased. The mechanism involved is unknown, as are the respective roles played by CDK4 or CDK6 in this process. Here, we report that AML cells carrying FLT3-ITD mutations are dependent on CDK6 for cell proliferation while CDK4 is not essential. We showed that FLT3-ITD signaling is responsible for CDK6 overexpression, through a pathway involving the SRC-family kinase HCK. Accordingly, FLT3-ITD failed to transform primary hematopoietic progenitor cells from Cdk6-/- mice. Our results demonstrate that CDK6 is the primary target of CDK4/CDK6 inhibitors in FLT3-ITD positive AML. Furthermore, we delineate an essential protein kinase pathway -FLT3/HCK/CDK6- in the context of AML with FLT3-ITD mutations.This project was supported by La Ligue Contre le Cancer (Equipe labelisee) and a grant from the Fondation ARC pour la Recherche sur le Cancer