Use of DNA–Damaging Agents and RNA Pooling to Assess Expression Profiles Associated with BRCA1 and BRCA2 Mutation Status in Familial Breast Cancer Patients

A large number of rare sequence variants of unknown clinical significance have been identified in the breast cancer susceptibility genes, BRCA1 and BRCA2. Laboratory-based methods that can distinguish between carriers of pathogenic mutations and non-carriers are likely to have utility for the classification of these sequence variants. To identify predictors of pathogenic mutation status in familial breast cancer patients, we explored the use of gene expression arrays to assess the effect of two DNA–damaging agents (irradiation and mitomycin C) on cellular response in relation to BRCA1 and BRCA2 mutation status. A range of regimes was used to treat 27 lymphoblastoid cell-lines (LCLs) derived from affected women in high-risk breast cancer families (nine BRCA1, nine BRCA2, and nine non-BRCA1/2 or BRCAX individuals) and nine LCLs from healthy individuals. Using an RNA–pooling strategy, we found that treating LCLs with 1.2 µM mitomycin C and measuring the gene expression profiles 1 hour post-treatment had the greatest potential to discriminate BRCA1, BRCA2, and BRCAX mutation status. A classifier was built using the expression profile of nine QRT–PCR validated genes that were associated with BRCA1, BRCA2, and BRCAX status in RNA pools. These nine genes could distinguish BRCA1 from BRCA2 carriers with 83% accuracy in individual samples, but three-way analysis for BRCA1, BRCA2, and BRCAX had a maximum of 59% prediction accuracy. Our results suggest that, compared to BRCA1 and BRCA2 mutation carriers, non-BRCA1/2 (BRCAX) individuals are genetically heterogeneous. This study also demonstrates the effectiveness of RNA pools to compare the expression profiles of cell-lines from BRCA1, BRCA2, and BRCAX cases after treatment with irradiation and mitomycin C as a method to prioritize treatment regimes for detailed downstream expression analysis

Caractérisation des fonctions de l'oncogenèse Spi-1/PU.1 dans la transformation érythroïde

L érythroleucémie développée dans les souris transgéniques pour l oncogène spi-1 est un processus multi-étapes de la transformation du proérythroblaste. Deux évènements principaux ont été caractérisés : la surexpression de Spi-1 dans les proérythroblastes qui entraîne le blocage de la différenciation érythroïde et des mutations activatrices du récepteur au SCF, Kit, qui confèrent aux progéniteurs érythroïdes une croissance indépendante de l Epo et un caractère tumorigène in vivo. Ce modèle représente le paradigme du développement leucémique impliquant une coopération entre deux évènements : une mutation bloquant la différenciation de la cellule et une mutation lui conférant un avantage prolifératif. Pour caractériser le rôle de Spi-1 dans la transformation érythroïde, une analyse des différences phénotypiques et moléculaires entre les proérythroblastes qui surexpriment ou n expriment pas Spi-1 a été effectuée. Pour cela, un modèle cellulaire utilisant des siRNAs anti-Spi-1 dont les expressions sont stables et inductibles par la doxycycline, a été établi dans les proérythroblastes préleucémiques transgéniques spi-1. La diminution de Spi-1 permet une reprogrammation des cellules dans un processus de différenciation, révélant que Spi-1 est le facteur limitant permettant le blocage de la différenciation. Parallèlement, Spi-1 empêche la mort des proérythroblastes par apoptose et altère le mécanisme de la réplication de l ADN. Nous montrons que la surexpression de Spi-1 entraîne une augmentation de la vitesse des fourches de réplication et une instabilité génétique accrue, suggérant que la dérégulation de l élongation de la réplication est impliquée dans la leucémogenèse.PARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Rôle du facteur de transcription Spi-1/PU.1 dans la progression leucémique (dérégulation des réseaux transcriptionnnels et inhibition de l'apoptose)

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Oncogenic kit triggers Shp2/Erk1/2 pathway to down-regulate the pro-apoptotic protein Bim and to promote apoptosis resistance in leukemic cells.

Oncogenic mutations leading to persistent kinase activities are implicated in various human malignancies. Thereby, signaling pathway-targeted therapies are powerful customized treatment to eradicate cancer cells. In murine and human leukemia cells harboring mutations in Kit, we previously showed that distinct and independent pathways controlled resistance to apoptosis or cell cycle. A treatment with PI3Kinase inhibitors to reduce cell proliferation combined with inhibitors of Erk1/2 activity to promote apoptosis had synergistic effects allowing eradication of leukemia cell growth. We reported here that Bim(EL), a pro-apoptotic member of the Bcl2 family proteins, is the target of Erk1/2 signaling and that its down-regulation is responsible for the apoptosis resistance of murine and human leukemic cells. Downstream of Kit mutant, the tyrosine phosphatase Shp2 maintains Bim(EL) expression at a low level, through Erk/2 activation and proteosomal Bim(EL) degradation. This process is controlled by Shp2 independently of other signaling pathways activated downstream of oncogenic Kit, demonstrating that Shp2 is a key regulator of Bim expression in the context of an oncogenic signaling. The increase in Bim(EL) expression is associated to an increased apoptosis. Moreover, the depletion of Bim overcomes apoptosis associated with Erk1/2 inactivation in UO126-treated leukemic cells, thereby establishing the contribution of Bim to drug-induced apoptosis. These data provide a molecular rationale for using BH3 mimetics in combination with PI3K inhibitors to treat leukemia, especially in the case of an oncogenic signaling refractory to Tyrosine Kinase inhibitors

CHIPIN: ChIP-seq inter-sample normalization based on signal invariance across transcriptionally constant genes

Background Multiple studies rely on ChIP-seq experiments to assess the effect of gene modulation and drug treatments on protein binding and chromatin structure. However, most methods commonly used for the normalization of ChIP-seq binding intensity signals across conditions, e.g., the normalization to the same number of reads, either assume a constant signal-to-noise ratio across conditions or base the estimates of correction factors on genomic regions with intrinsically different signals between conditions. Inaccurate normalization of ChIP-seq signal may, in turn, lead to erroneous biological conclusions. Results We developed a new R package, CHIPIN, that allows normalizing ChIP-seq signals across different conditions/samples when spike-in information is not available, but gene expression data are at hand. Our normalization technique is based on the assumption that, on average, no differences in ChIP-seq signals should be observed in the regulatory regions of genes whose expression levels are constant across samples/conditions. In addition to normalizing ChIP-seq signals, CHIPIN provides as output a number of graphs and calculates statistics allowing the user to assess the efficiency of the normalization and qualify the specificity of the antibody used. In addition to ChIP-seq, CHIPIN can be used without restriction on open chromatin ATAC-seq or DNase hypersensitivity data. We validated the CHIPIN method on several ChIP-seq data sets and documented its superior performance in comparison to several commonly used normalization techniques. Conclusions The CHIPIN method provides a new way for ChIP-seq signal normalization across conditions when spike-in experiments are not available. The method is implemented in a user-friendly R package available on GitHub: https://github.com/BoevaLab/CHIPINISSN:1471-210

From normal hematopoiesis to malignancies: Highlights from the 2021 Meeting of the Club Hematopoiesis and Oncogenesis

International audienc