Integrative single-cell chromatin and transcriptome analysis of human plasma cell differentiation.

Plasma cells (PC) are highly specialized cells representing the end stage of B cell differentiation. We have shown that PC differentiation can be reproduced in vitro using elaborate culture systems. The molecular changes occurring during PC differentiation are recapitulated in this in vitro differentiation model. However, a major challenge exists to decipher the spatiotemporal epigenetic and transcriptional programs that drives the early stages of PC differentiation. We combined single cell (sc) RNA-seq and single cell ATAC-seq to decipher the trajectories involved in PC differentiation. ScRNA-seq experiments revealed a strong heterogeneity of the preplasmablastic and plasmablastic stages. Among genes that were commonly identified using scATAC-seq and scRNA-seq, we identified several transcription factors with significant stage specific potential importance in PC differentiation. Interestingly, differentially accessible peaks characterizing the preplasmablastic stage were enriched in motifs of BATF3, FOS and BATF, belonging to the AP-1 transcription factor family, that may represent key transcriptional nodes involved in PCD. Integration of transcriptomic and epigenetic data at the single cell level revealed that a population of preplasmablasts already undergone epigenetic remodeling related to PC profile together with UPR activation and are committed to differentiate in PC. These results and the supporting data generated with our in vitro PC differentiation model provide a unique resource for the identification of molecular circuits that are crucial for early and mature plasma cell maturation and biological functions. These data thus provide critical insights into epigenetic- and transcriptional-mediated reprogramming events that sustain PC differentiation.

PIM2 kinase has a pivotal role in plasmablast generation and plasma cell survival, opening up novel treatment options in myeloma.

The differentiation of B cells into plasmablasts (PBs) and then plasma cells (PCs) is associated with extensive cell reprogramming and new cell functions. By using specific inhibition strategies (including a novel morpholino RNA antisense approach), we found that early, sustained upregulation of the proviral integrations of Moloney virus 2 (PIM2) kinase is a pivotal event during human B-cell in vitro differentiation and then continues in mature normal and malignant PCs in the bone marrow. In particular, PIM2 sustained the G1/S transition by acting on CDC25A and p27Kip1 and hindering caspase 3-driven apoptosis through BAD phosphorylation and cytoplasmic stabilization of p21Cip1. In PCs, interleukin-6 triggered PIM2 expression, resulting in antiapoptotic effects on which malignant PCs were particularly dependent. In multiple myeloma, pan-PIM and myeloid cell leukemia-1 (MCL1) inhibitors displayed synergistic activity. Our results highlight a cell-autonomous function that links kinase activity to the newly acquired secretion ability of the PBs and the adaptability observed in both normal and malignant PCs. These findings should finally prompt the reconsideration of PIM2 as a therapeutic target in multiple myeloma.

Influence of manufacturing processes on cell surface properties of probiotic strain Lactobacillus rhamnosus Lcr35®.

The influence of the industrial process on the properties of probiotics, administered as complex manufactured products, has been poorly investigated. In the present study, we comparatively assessed the cell wall characteristics of the probiotic strain Lactobacillus rhamnosus Lcr35® together with three of its commercial formulations with intestinal applications. Putative secreted and transmembrane-protein-encoding genes were initially searched in silico in the genome of L. rhamnosus Lcr35®. A total of 369 candidate genes were identified which expressions were followed using a custom Lactobacillus DNA chip. Among them, 60 or 67 genes had their expression either upregulated or downregulated in the Lcr Restituo® packet or capsule formulations, compared to the native Lcr35® strain. Moreover, our data showed that the probiotic formulations (Lcr Lenio®, Lcr restituo® capsule and packet) showed a better capacity to adhere to intestinal epithelial Caco-2 cells than the native Lcr35® strain. Microbial (MATS) tests showed that the probiotic was an electron donor and that they were more hydrophilic than the native strain. The enhanced adhesion capacity of the active pharmaceutical ingredients (APIs) to epithelial Caco-2 cells and their antipathogen effect could be due to this greater surface hydrophilic character. These findings suggest that the manufacturing process influences the protein composition and the chemical properties of the cell wall. It is therefore likely that the antipathogen effect of the formulation is modulated by the industrial process. Screening of the manufactured products' properties would therefore represent an essential step in evaluating the effects of probiotic strains.

Staphylococcus aureus transcriptomic response to inhibition by H2O2-producing Lactococcus garvieae.

Growth of the foodborne pathogen Staphylococcus aureus can be inhibited in milk and in cheese by the hydrogen peroxide-producing Lactococcus garvieae N201 dairy strain. Transcriptomic responses of two S. aureus strains, the S. aureus SA15 dairy strain and the MW2 human pathogenic strain, to this growth inhibition were investigated in Brain-Heart Infusion broth under a high or a low aeration level. We demonstrated that S. aureus MW2 had a higher resistance to L. garvieae inhibition under the high aeration level: this correlated to a higher survival under hydrogen peroxide exposure. Conversely, the two strains were similarly inhibited under the low aeration level. Expression of S. aureus genes involved in response to H2O2 or other stresses as well as in cell division was generally repressed by L. garvieae. However, differential expressions between the two S. aureus strains were observed, especially under the high aeration level. Additionally, expression of virulence-related genes (enterotoxins, regulatory genes) was modulated by L. garvieae depending on the aeration level and on the S. aureus strain. This study led to new insights into potential molecular mechanisms of S. aureus inhibition by Lactic Acid Bacteria via H2O2 production.

Var∣Decrypt: a novel and user-friendly tool to explore and prioritize variants in whole-exome sequencing data.

BACKGROUND: High-throughput sequencing (HTS) offers unprecedented opportunities for the discovery of causative gene variants in multiple human disorders including cancers, and has revolutionized clinical diagnostics. However, despite more than a decade of use of HTS-based assays, extracting relevant functional information from whole-exome sequencing (WES) data remains challenging, especially for non-specialists lacking in-depth bioinformatic skills. RESULTS: To address this limitation, we developed Var∣Decrypt, a web-based tool designed to greatly facilitate WES data browsing and analysis. Var∣Decrypt offers a wide range of gene and variant filtering possibilities, clustering and enrichment tools, providing an efficient way to derive patient-specific functional information and to prioritize gene variants for functional analyses. We applied Var∣Decrypt on WES datasets of 10 acute erythroid leukemia patients, a rare and aggressive form of leukemia, and recovered known disease oncogenes in addition to novel putative drivers. We additionally validated the performance of Var∣Decrypt using an independent dataset of ~ 90 multiple myeloma WES, recapitulating the identified deregulated genes and pathways, showing the general applicability and versatility of Var∣Decrypt for WES analysis. CONCLUSION: Despite years of use of WES in human health for diagnosis and discovery of disease drivers, WES data analysis still remains a complex task requiring advanced bioinformatic skills. In that context, there is a need for user-friendly all-in-one dedicated tools for data analysis, to allow biologists and clinicians to extract relevant biological information from patient datasets. Here, we provide Var∣Decrypt (trial version accessible here: https://vardecrypt.com/app/vardecrypt ), a simple and intuitive Rshiny application created to fill this gap. Source code and detailed user tutorial are available at https://gitlab.com/mohammadsalma/vardecrypt .

Comprehensive characterization of the epigenetic landscape in Multiple Myeloma.

Background: Human multiple myeloma (MM) cell lines (HMCLs) have been widely used to understand the molecular processes that drive MM biology. Epigenetic modifications are involved in MM development, progression, and drug resistance. A comprehensive characterization of the epigenetic landscape of MM would advance our understanding of MM pathophysiology and may attempt to identify new therapeutic targets. Methods: We performed chromatin immunoprecipitation sequencing to analyze histone mark changes (H3K4me1, H3K4me3, H3K9me3, H3K27ac, H3K27me3 and H3K36me3) on 16 HMCLs. Results: Differential analysis of histone modification profiles highlighted links between histone modifications and cytogenetic abnormalities or recurrent mutations. Using histone modifications associated to enhancer regions, we identified super-enhancers (SE) associated with genes involved in MM biology. We also identified promoters of genes enriched in H3K9me3 and H3K27me3 repressive marks associated to potential tumor suppressor functions. The prognostic value of genes associated with repressive domains and SE was used to build two distinct scores identifying high-risk MM patients in two independent cohorts (CoMMpass cohort; n = 674 and Montpellier cohort; n = 69). Finally, we explored H3K4me3 marks comparing drug-resistant and -sensitive HMCLs to identify regions involved in drug resistance. From these data, we developed epigenetic biomarkers based on the H3K4me3 modification predicting MM cell response to lenalidomide and histone deacetylase inhibitors (HDACi). Conclusions: The epigenetic landscape of MM cells represents a unique resource for future biological studies. Furthermore, risk-scores based on SE and repressive regions together with epigenetic biomarkers of drug response could represent new tools for precision medicine in MM.

miRNA profile at diagnosis predicts treatment outcome in patients with B-chronic lymphocytic leukemia: A FILO study.

During many years, chemo-immunotherapy fludarabine-cyclophosphamide-rituximab (FCR) was the gold standard for first line treatment of medically fit patients with symptomatic B-chronic lymphocytic leukemia (CLL). Over the last decade, targeted biotherapies have revolutionized the treatment of B-CLL patients and almost entirely supplanted FCR. However, no biomarker still exists to predict the complete remission (CR) with undetectable minimal residual disease (uMRD) in bone marrow (BM), which remains the best predictive factor for survival. MicroRNAs represent a class of molecular biomarkers which expression is altered in B-CLL. Our study aimed at identifying before treatment blood miRNAs that predict treatment outcome in previously untreated B-CLL patients (NCT01370772, https://clinicaltrials.gov/ct2/show/NCT01370772). Using hierarchical clustering of miRNA expression profiles discriminating 8 patients who achieved CR with BM uMRD from 8 patients who did not achieve CR and displayed detectable BM MRD, we identified 25 miRNAs differentially expressed before treatment. The expression of 11 miRNAs was further validated on a larger cohort (n=123). Based on the dosage of 5 miRNAs at diagnosis, a decision tree was constructed to predict treatment outcome. We identified 6 groups of patients with a distinct probability of being CR with BM uMRD to FCR treatment, ranging from 72% (miR-125b, miR-15b and miR-181c high) to 4% (miR-125b and miR-193b low). None of the patients displaying high expression levels of miR-125b, miR-15b and miR-181c relapsed during study follow-up. In contrast, patients with low miR-15b and high miR-412, or with low miR-125b and miR-193b, demonstrated significant low PFS. RNA sequencing of blood at diagnosis identified that patients relapsing after treatment are characterized by significant enrichment of gene signatures related to cell cycle, MYC target genes, metabolism and translation regulation. Conversely, patients achieving CR with BM uMRD displayed significant enrichment in genes related to communication between CLL cells and the microenvironment, immune system activation and upregulation of polycomb PRC2 complex target genes. Our results suggest that blood miRNAs are potent predictive biomarkers for FCR treatment efficacy and might be implicated in the FCR efficacy in B-CLL patients, providing new insight into unmet need for the treatment of B-CLL patients and identifying pathways predictive of patients' remission. Clinical trial registration: ClinicalTrials.gov, identifier NCT01370772.

The BLM helicase is a new therapeutic target in multiple myeloma involved in replication stress survival and drug resistance.

Multiple myeloma (MM) is a hematologic cancer characterized by accumulation of malignant plasma cells in the bone marrow. To date, no definitive cure exists for MM and resistance to current treatments is one of the major challenges of this disease. The DNA helicase BLM, whose depletion or mutation causes the cancer-prone Bloom's syndrome (BS), is a central factor of DNA damage repair by homologous recombination (HR) and genomic stability maintenance. Using independent cohorts of MM patients, we identified that high expression of BLM is associated with a poor outcome with a significant enrichment in replication stress signature. We provide evidence that chemical inhibition of BLM by the small molecule ML216 in HMCLs (human myeloma cell lines) leads to cell cycle arrest and increases apoptosis, likely by accumulation of DNA damage. BLM inhibition synergizes with the alkylating agent melphalan to efficiently inhibit growth and promote cell death in HMCLs. Moreover, ML216 treatment re-sensitizes melphalan-resistant cell lines to this conventional therapeutic agent. Altogether, these data suggest that inhibition of BLM in combination with DNA damaging agents could be of therapeutic interest in the treatment of MM, especially in those patients with high BLM expression and/or resistance to melphalan.

Angiogenic factors could help us to define patients obtaining complete response with undetectable minimal residual disease in untreated CLL patients treated by FCR: results from the CLL2010FMP, a FILO study.

Angiogenesis is in a constant balance between pro and anti-angiogenic factors. Neoangiogenesis, implicated in metastatic spreading is characterized in solid cancers, but fairly new in chronic lymphocytic leukemia (CLL). We hypothesize that secretion of angiogenic factors could be correlated to the pathogenesis of CLL, and therefore predict the outcome of patients. We investigated concentrations of 22 cytokines and chemokines in 137 non-del 17p B-CLL patients, treated with a fludarabine-cyclophosphamide-rituximab (FCR)-based regimen. We constructed a biomarker index defining different risk groups based on lymphocyte count, the intensity of CD20 antigen on CD19+ cells, Ang-2, and PDGF-BB plasma concentrations at diagnosis. Four groups were defined, exhibiting specific molecular signatures and correlated with progression-free survival of patients. Our results suggest that we can determine at diagnosis of non-del 17p B-CLL patients, those with a very high probability of progression-free survival, independently of IGVH mutational status and residual disease at the end of treatment.

RNA-Sequencing-Based Transcriptomic Score with Prognostic and Theranostic Values in Multiple Myeloma.

Multiple myeloma (MM) is the second most frequent hematological cancer and is characterized by the clonal proliferation of malignant plasma cells. Genome-wide expression profiling (GEP) analysis with DNA microarrays has emerged as a powerful tool for biomedical research, generating a huge amount of data. Microarray analyses have improved our understanding of MM disease and have led to important clinical applications. In MM, GEP has been used to stratify patients, define risk, identify therapeutic targets, predict treatment response, and understand drug resistance. In this study, we built a gene risk score for 267 genes using RNA-seq data that demonstrated a prognostic value in two independent cohorts (n = 674 and n = 76) of newly diagnosed MM patients treated with high-dose Melphalan and autologous stem cell transplantation. High-risk patients were associated with the expression of genes involved in several major pathways implicated in MM pathophysiology, including interferon response, cell proliferation, hypoxia, IL-6 signaling pathway, stem cell genes, MYC, and epigenetic deregulation. The RNA-seq-based risk score was correlated with specific MM somatic mutation profiles and responses to targeted treatment including EZH2, MELK, TOPK/PBK, and Aurora kinase inhibitors, outlining potential utility for precision medicine strategies in MM.

High Content Screening Using New U2OS Reporter Cell Models Identifies Harmol Hydrochloride as a Selective and Competitive Antagonist of the Androgen Receptor.

Prostate cancer is the most commonly diagnosed malignancy in men. Its growth mainly relies on the activity of the androgen receptor (AR), justifying the use of androgen deprivation therapy as a gold standard treatment for the metastatic disease. Inhibition of the androgen axis using second generation antagonists has improved patients' survival, but is systematically confronted to resistance mechanisms, leading to a median survival that does not exceed 5 years. Counteracting this resistance has been the object of a large number of investigations, with a particular emphasis towards the identification of new AR inhibitors, whether they antagonize the receptor by a competitive or a non-competitive binding. To this end, many high content screens have been performed, to identify new non-steroidal AR antagonists, using a variety of approaches, but reported somewhat controversial results, depending on the approach and on the cell model that was used for screening. In our study, we used the U2OS osteosarcoma cells stably transfected with AR or ARv7 and a luciferase reporter as a previously validated model to screen the Prestwick Phytochemical library. The results of our screen identified ellipticine, harmol, and harmine hydrochloride as confirmed hits. Surprisingly, we could demonstrate that harmol hydrochloride, previously identified as a non-competitive inhibitor of AR or a weak inhibitor of androgen signaling, was actually a competitive antagonist of AR, which inhibits the growth of VCaP prostate cancer line, at concentrations for which it did not affect the growth of the AR negative DU145 and PC3 cells. Interestingly, we also report for the first time that harmol hydrochloride was selective for AR, as it could not alter the activity of other nuclear receptors, such as the glucocorticoid receptor (GR), the progesterone receptor (PR), or the mineralocorticoid receptor (MR). Additionally, we demonstrate that, conversely to enzalutamide, harmol hydrochloride did not show any agonistic activity towards the pregnane X receptor (PXR), a master regulator of drug metabolism. Together, our results shed light on the importance of the cellular context for the screening of new AR antagonists. They further indicate that some of the potential hits that were previously identified may have been overlooked.

The Anti-Cancer Drug Dabrafenib Is a Potent Activator of the Human Pregnane X Receptor.

The human pregnane X receptor (hPXR) is activated by a large set of endogenous and exogenous compounds and plays a critical role in the control of detoxifying enzymes and transporters regulating liver and gastrointestinal drug metabolism and clearance. hPXR is also involved in both the development of multidrug resistance and enhanced cancer cells aggressiveness. Moreover, its unintentional activation by pharmaceutical drugs can mediate drug-drug interactions and cause severe adverse events. In that context, the potential of the anticancer BRAF inhibitor dabrafenib suspected to activate hPXR and the human constitutive androstane receptor (hCAR) has not been thoroughly investigated yet. Using different reporter cellular assays, we demonstrate that dabrafenib can activate hPXR as efficiently as its reference agonist SR12813, whereas it does not activate mouse or zebrafish PXR nor hCAR. We also showed that dabrafenib binds to recombinant hPXR, induces the expression of hPXR responsive genes in colon LS174T-hPXR cancer cells and human hepatocytes and finally increases the proliferation in LS174T-hPXR cells. Our study reveals that by using a panel of different cellular techniques it is possible to improve the assessment of hPXR agonist activity for new developed drugs.

Identification of sulfur components enhancing the anti-Candida effect of Lactobacillus rhamnosus Lcr35.

GYNOPHILUS (Lcr REGENERANS) is a live biotherapeutic product (LBP) aimed at restoring the vaginal microbiome and contains the live biotherapeutic microorganism Lactobacillus rhamnosus Lcr35. In this study, the LBP formulation and manufacturing process significantly enhanced the anti-Candida activity of L. rhamnosus Lcr35, with a complete loss of viability of the yeast after 48 h of coincubation. Sodium thiosulfate (STS), one excipient of the product, was used as a potentiator of the anti-Candida spp. activity of Lactobacilli. This contact-independent phenomenon induced fungal cell disturbances, as observed by electron microscopy observations. Nonverbal sensory experiments showed clear odor dissimilarities between cocultures of L. rhamnosus Lcr35 and C. albicans in the presence and absence of STS, suggesting an impact of odor-active metabolites. A volatolomic approach allowed the identification of six odor-active compounds, including one sulfur compound that was identified as S-methyl thioacetate (MTA). MTA was associated with the antifungal effect of Lcr35, and its functional link was established in vitro. We show for the first time that the LBP GYNOPHILUS, which is a highly active product in the reduction of vulvovaginal candidiasis, requires the presence of a sulfur compound to fully achieve its antifungal effect.

Automated and simplified identification of normal and abnormal plasma cells in Multiple Myeloma by flow cytometry.

BACKGROUND: Multiple myeloma (MM) is an incurable disease characterized by clonal plasma cell (PC) proliferation within the bone marrow (BM). Next-generation flow cytometry has become the reference tool to follow minimal residual disease (MRD). We developed a new simpler and cheaper flow cytometry method to analyze bone marrow samples in patients with MM. METHODS: To identify and characterize abnormal PCs, we designed a simple panel composed of anti-CD38, antikappa, and antilambda light chain antibodies, combined with two antibody pools with the same fluorophore (anti-CD19 and anti-CD27 for the negative pool and anti-CD56, anti-CD117, and anti-CD200 antibodies for the positive pool). We also developed dedicated software for the automated identification of malignant PCs and MRD assessment. We then compared PC identification with our simple antibody panel and with the larger antibody panel routinely used at Montpellier University Hospital Center in 52 patients with MM (M-CHU cohort). RESULTS: Results for total PC detection (r2  = 0.9965; P < 0.001; n = 52) and malignant PC detection (r2  = 0.9486; P < 0.001; n = 38) obtained with the two panels were significantly correlated. Moreover, comparison of the results obtained by automated detection with our software and by manual gating analysis in 80 BM samples (38 from the M-CHU cohort and 42 patients from another MM cohort) showed strong correlation for both total and malignant PC selection (respectively, r2  = 0.936; P < 0.001 and r2  = 0.9505; P < 0.001). CONCLUSIONS: Our simple and automated strategy for MRD assessment in MM could help increasing reproducibility and productivity without compromising sensitivity and specificity, while decreasing the test cost. © 2017 International Clinical Cytometry Society.

CD24, CD27, CD36 and CD302 gene expression for outcome prediction in patients with multiple myeloma.

Multiple myeloma (MM) is a B cell neoplasia characterized by clonal plasma cell (PC) proliferation. Minimal residual disease monitoring by multi-parameter flow cytometry is a powerful tool for predicting treatment efficacy and MM outcome. In this study, we compared CD antigens expression between normal and malignant plasma cells to identify new potential markers to discriminate normal from malignant plasma cells, new potential therapeutic targets for monoclonal-based treatments and new prognostic factors. Nine genes were significantly overexpressed and 16 were significantly downregulated in MMC compared with BMPC (ratio ≥2; FDR CD24, CD27, CD36 and CD302) was associated with a prognostic value in two independent cohorts of patients with MM (HM cohort and TT2 cohort, n=345). The expression level of these four genes was then used to develop a CD gene risk score that classified patients in two groups with different survival (P = 2.06E-6) in the HM training cohort. The prognostic value of the CD gene risk score was validated in two independent cohorts of patients with MM (TT2 cohort and HOVON65/GMMGHD4 cohort, n=282 patients). The CD gene risk score remained a prognostic factor that separated patients in two groups with significantly different overall survival also when using publicly available data from a cohort of relapsing patients treated with bortezomib (n=188). In conclusion, the CD gene risk score allows identifying high risk patients with MM based on CD24, CD27, CD36 and CD302 expression and could represent a powerful tool for simple outcome prediction in MM.