MicroRNA expression in B-cell lymphomas

Tesis doctoral inédita, leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 04-06-201

miR-217 is an oncogene that enhances the germinal center reaction.

microRNAs are a class of regulators of gene expression that have been shown critical for a great number of biological processes; however, little is known of their role in germinal center (GC) B cells. Although the GC reaction is crucial to ensure a competent immune response, GC B cells are also the origin of most human lymphomas, presumably due to bystander effects of the immunoglobulin gene remodeling that takes place at these sites. Here we report that miR-217 is specifically upregulated in GC B cells. Gain- and loss-of-function mouse models reveal that miR-217 is a positive modulator of the GC response that increases the generation of class-switched antibodies and the frequency of somatic hypermutation. We find that miR-217 down-regulates the expression of a DNA damage response and repair gene network and in turn stabilizes Bcl-6 expression in GC B cells. Importantly, miR-217 overexpression also promotes mature B-cell lymphomagenesis; this is physiologically relevant as we find that miR-217 is overexpressed in aggressive human B-cell lymphomas. Therefore, miR-217 provides a novel molecular link between the normal GC response and B-cell transformation.S

Glutaminolysis is a metabolic dependency in FLT3ITD acute myeloid leukemia unmasked by FLT3 tyrosine kinase inhibition.

FLT3 internal tandem duplication (FLT3ITD) mutations are common in acute myeloid leukemia (AML) associated with poor patient prognosis. Although new-generation FLT3 tyrosine kinase inhibitors (TKI) have shown promising results, the outcome of FLT3ITD AML patients remains poor and demands the identification of novel, specific, and validated therapeutic targets for this highly aggressive AML subtype. Utilizing an unbiased genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 screen, we identify GLS, the first enzyme in glutamine metabolism, as synthetically lethal with FLT3-TKI treatment. Using complementary metabolomic and gene-expression analysis, we demonstrate that glutamine metabolism, through its ability to support both mitochondrial function and cellular redox metabolism, becomes a metabolic dependency of FLT3ITD AML, specifically unmasked by FLT3-TKI treatment. We extend these findings to AML subtypes driven by other tyrosine kinase (TK) activating mutations and validate the role of GLS as a clinically actionable therapeutic target in both primary AML and in vivo models. Our work highlights the role of metabolic adaptations as a resistance mechanism to several TKI and suggests glutaminolysis as a therapeutically targetable vulnerability when combined with specific TKI in FLT3ITD and other TK activating mutation-driven leukemias.P.G. is funded by the Wellcome Trust (109967/Z/15/Z) and was previously supported by the Academy of medical Sciences and Lady Tata Memorial Trust. The Huntly lab is funded by European Research Council, MRC, Bloodwise, the Kay Kendall Leukaemia Fund, the Cambridge NIHR Biomedical Research Centre, and core support grants to the Wellcome Trust - Medical Research Council Cambridge Stem Cell Institute. C.F. and A.S.H.C are funded by the Medical Research Council, Core Grant to the Cancer Unit. P.M-P. is supported by a grant from Cancer Research UK (C56179/A21617). D.S. is a Postdoctoral Fellow of the Mildred-Scheel Organisation, German Cancer Aid. This research was supported by the CIMR Flow Cytometry Core Facility. We would like to thank the Welcome Trust Sanger Institute facility for the MiSeq run

Clinical and biological markers predictive of treatment response associated with metastatic pancreatic adenocarcinoma.

Acknowledgements: The authors wish to acknowledge support from all Investigators and their research teams at sites which participated in the SIEGE trial. We thank all patients who consented to participate and supply research samples for the purpose of this study. This research was supported by the NIHR Cambridge Biomedical Research Centre (BRC-1215-20014). The views expressed are those of the authors and not necessarily those of the NIHR or the Department of Health and Social Care.BACKGROUND: Chemotherapy for metastatic pancreatic adenocarcinoma (PDAC) offers limited benefits, but survival outcomes vary. Reliable predictive response biomarkers to guide patient management are lacking. METHODS: Patient performance status, tumour burden (determined by the presence or absence of liver metastases), plasma protein biomarkers (CA19-9, albumin, C-reactive protein and neutrophils) and circulating tumour DNA (ctDNA) were assessed in 146 patients with metastatic PDAC prior to starting either concomitant or sequential nab-paclitaxel + gemcitabine chemotherapy in the SIEGE randomised prospective clinical trial, as well as during the first 8 weeks of treatment. Correlations were made with objective response, death within 1 year and overall survival (OS). RESULTS: Initial poor patient performance status, presence of liver metastases and detectable mutKRAS ctDNA all correlated with worse OS after adjusting for the different biomarkers of interest. Objective response at 8 weeks also correlated with OS (P = 0.026). Plasma biomarkers measured during treatment and prior to the first response assessment identified ≥10% decrease in albumin at 4 weeks predicted for worse OS (HR 4.75, 95% CI 1.43-16.94, P = 0.012), while any association of longitudinal evaluation of mutKRAS ctDNA with OS was unclear (β = 0.024, P = 0.057). CONCLUSIONS: Readily measurable patient variables can aid the prediction of outcomes from combination chemotherapy used to treat metastatic PDAC. The role of mutKRAS ctDNA as a tool to guide treatment warrants further exploration. CLINICAL TRIAL REGISTRATION: ISRCTN71070888; ClinialTrials.gov (NCT03529175)

Clinical and biological markers predictive of treatment response associated with metastatic pancreatic adenocarcinoma.

BACKGROUND: Chemotherapy for metastatic pancreatic adenocarcinoma (PDAC) offers limited benefits, but survival outcomes vary. Reliable predictive response biomarkers to guide patient management are lacking. METHODS: Patient performance status, tumour burden (determined by the presence or absence of liver metastases), plasma protein biomarkers (CA19-9, albumin, C-reactive protein and neutrophils) and circulating tumour DNA (ctDNA) were assessed in 146 patients with metastatic PDAC prior to starting either concomitant or sequential nab-paclitaxel + gemcitabine chemotherapy in the SIEGE randomised prospective clinical trial, as well as during the first 8 weeks of treatment. Correlations were made with objective response, death within 1 year and overall survival (OS). RESULTS: Initial poor patient performance status, presence of liver metastases and detectable mutKRAS ctDNA all correlated with worse OS after adjusting for the different biomarkers of interest. Objective response at 8 weeks also correlated with OS (P = 0.026). Plasma biomarkers measured during treatment and prior to the first response assessment identified ≥10% decrease in albumin at 4 weeks predicted for worse OS (HR 4.75, 95% CI 1.43-16.94, P = 0.012), while any association of longitudinal evaluation of mutKRAS ctDNA with OS was unclear (β = 0.024, P = 0.057). CONCLUSIONS: Readily measurable patient variables can aid the prediction of outcomes from combination chemotherapy used to treat metastatic PDAC. The role of mutKRAS ctDNA as a tool to guide treatment warrants further exploration. CLINICAL TRIAL REGISTRATION: ISRCTN71070888; ClinialTrials.gov (NCT03529175)

NF-κB directly mediates epigenetic deregulation of common microRNAs in Epstein-Barr virus-mediated transformation of B-cells and in lymphomas

MicroRNAs (miRNAs) have negative effects on gene expression and are major players in cell function in normal and pathological conditions. Epstein-Barr virus (EBV) infection of resting B lymphocytes results in their growth transformation and associates with different B cell lymphomas. EBV-mediated B cell transformation involves large changes in gene expression, including cellular miRNAs. We performed miRNA expression analysis in growth transformation of EBV-infected B cells. We observed predominant downregulation of miRNAs and upregulation of a few miRNAs. We observed similar profiles of miRNA expression in B cells stimulated with CD40L/IL-4, and those infected with EBNA-2- and LMP-1-deficient EBV particles, suggesting the implication of the NFkB pathway, common to all four situations. In fact, the NF-kappa B subunit p65 associates with the transcription start site (TSS) of both upregulated and downregulated miRNAs following EBV infection This occurs together with changes at histone H3K27me3 and histone H3K4me3. Inhibition of the NF-kappa B pathway impairs changes in miRNA expression, NF-kappa B binding and changes at the above histone modifications near the TSS of these miRNA genes. Changes in expression of these miRNAs also occurred in diffuse large B cell lymphomas (DLBCL), which are strongly NF-kappa B dependent. Our results highlight the relevance of the NF-kappa B pathway in epigenetically mediated miRNA control in B cell transformation and DLBCL

Combinatorial effects of microRNAs to suppress the Myc oncogenic pathway

Many mammalian transcripts contain target sites for multiple miRNAs although it is not clear to what extent miRNAs may coordinately regulate single genes. We have mapped the interactions between downregulated miRNAs and overexpressed target protein-coding genes in murine and human lymphomas. Myc, one of the hallmark oncogenes in these lymphomas, stands out as the upregulated gene with the highest number of genetic interactions with downregulated miRNAs in mouse lymphomas. The regulation of Myc by several of these miRNAs is confirmed by cellular and reporter assays. The same approach indentifies MYC and multiple Myc targets as a preferential target of downregulated miRNAs in human Burkitt's lymphoma, a pathology characterized by translocated MYC oncogenes. These results indicate that several miRNAs must be coordinately downregulated in order to enhance critical oncogenes such as Myc. Some of these Myc-targeting miRNAs are repressed by Myc, suggesting that these tumors are a consequence of the unbalanced activity of Myc versus miRNAs.This work was supported by grants from the Association for International Cancer Research (AICR #08-0188), Fundación Ramón Areces, Fundación Mutua Madrileña Automovilista (to M.M.), and the MICINN (SAF2009-11426 to J.F.P.; SAF2007-64571 to I.P.C.; and SAF2009-07973 to M.M.). The CBM/UAM group is supported by the Centro de Investigaciones Biomédicas en Red de Enfermedades Raras (CIBERER) network from the MICINN. The Bioinformatics Unit of the CNIO is supported by the Spanish National Bioinformatics Institute (Genoma España). The Cell Division and Cancer Group of the CNIO is supported by the OncoCycle Programme (Comunidad de Madrid, S-BIO-0283-2006), the OncoBIO Consolider-Ingenio 2010 Programme (MICINN, CSD2007-00017) and the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement n° [HEALTH-F5-2010-241548]Peer reviewe

NF-κB directly mediates epigenetic deregulation of common microRNAs in Epstein-Barr virus-mediated transformation of B-cells and in lymphomas

MicroRNAs (miRNAs) have negative effects on gene expression and are major players in cell function in normal and pathological conditions. Epstein-Barr virus (EBV) infection of resting B lymphocytes results in their growth transformation and associates with different B cell lymphomas. EBV-mediated B cell transformation involves large changes in gene expression, including cellular miRNAs. We performed miRNA expression analysis in growth transformation of EBV-infected B cells. We observed predominant downregulation of miRNAs and upregulation of a few miRNAs. We observed similar profiles of miRNA expression in B cells stimulated with CD40L/IL-4, and those infected with EBNA-2- and LMP-1-deficient EBV particles, suggesting the implication of the NF-kB pathway, common to all four situations. In fact, the NF-kB subunit p65 associates with the transcription start site (TSS) of both upregulated and downregulated miRNAs following EBV infection This occurs together with changes at histone H3K27me3 and histone H3K4me3. Inhibition of the NF-kB pathway impairs changes in miRNA expression, NF-kB binding and changes at the above histone modifications near the TSS of these miRNA genes. Changes in expression of these miRNAs also occurred in diffuse large B cell lymphomas (DLBCL), which are strongly NF-kB dependent. Our results highlight the relevance of the NF-kB pathway in epigenetically mediated miRNA control in B cell transformation and DLBCL

NF-κB directly mediates epigenetic deregulation of common microRNAs in Epstein-Barr virus-mediated transformation of B-cells and in lymphomas

MicroRNAs (miRNAs) have negative effects on gene expression and are major players in cell function in normal and pathological conditions. Epstein-Barr virus (EBV) infection of resting B lymphocytes results in their growth transformation and associates with different B cell lymphomas. EBV-mediated B cell transformation involves large changes in gene expression, including cellular miRNAs. We performed miRNA expression analysis in growth transformation of EBV-infected B cells. We observed predominant downregulation of miRNAs and upregulation of a few miRNAs. We observed similar profiles of miRNA expression in B cells stimulated with CD40L/IL-4, and those infected with EBNA-2- and LMP-1-deficient EBV particles, suggesting the implication of the NF-kB pathway, common to all four situations. In fact, the NF-kB subunit p65 associates with the transcription start site (TSS) of both upregulated and downregulated miRNAs following EBV infection This occurs together with changes at histone H3K27me3 and histone H3K4me3. Inhibition of the NF-kB pathway impairs changes in miRNA expression, NF-kB binding and changes at the above histone modifications near the TSS of these miRNA genes. Changes in expression of these miRNAs also occurred in diffuse large B cell lymphomas (DLBCL), which are strongly NF-kB dependent. Our results highlight the relevance of the NF-kB pathway in epigenetically mediated miRNA control in B cell transformation and DLBCL