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HOMERESEARCHPUBLICATIONSLAB MEMBERSCOURSES/LINKSPOSITIONSCONTACT
MRAZ LAB - MICROENVIRONMENT OF IMMUNE CELLS
We study the molecular pathways that regulate microenvironmental interactions in normal and malignant immune cells. The microenvironment of immune niches plays an important role in the onset, progression, and resistance of hematological malignancies. We largely focus on the role of non-coding RNAs (microRNAs [miRNAs] and long-noncoding RNAs [lncRNA]) in these pathways. The deregulation of non-coding RNAs can lead to aberrant regulation of pathways that are canonically associated with cancer biology like apoptosis, proliferation and differentiation. However, the role of non-coding RNAs in the microenvironmental interactions of normal and malignant cells is largely unknown.

MAIN PROJECTS:
i) The role of microRNAs in BCR-signalling and adhesion in B cell malignancies (Chronic Lymphocytic Leukemia, B-Cell Lymphomas)
ii) The regulation of BCR signalling in B cells, and the function of CD20
iii) The role of non-coding RNAs in DNA damage response in B cells
iv) Development of novel targeted therapies (miR-therapy, targeting of BCR-signalling/adhesion)
v) Prognostic and predictive markers in hematological malignancies

SELECTED PAPERS AND NEWS: 

ERC Starting Grant
ERC Starting Grant awarded to Marek Mraz (starting 2019). PHD AND POST-DOC POSITIONS FOR THE PROJECT ARE OPENED NOW...APPLY :-) [see "POSITIONS"].
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Marek Mraz
Blood. 2018 Sept (epub).
miR-150 downregulation contributes to the high-grade transformation of follicular lymphoma by upregulating FOXP1 levels.
Musilova K, Devan J, Cerna K, Seda V, Pavlasova G, Sharma S, Oppelt J, Pytlik R, Prochazka V, Prouzova Z, Trbusek M, Zlamalikova L, Liskova K, Kruzova L, Jarosova M, Mareckova A, Kornauth C, Simonitsch-Klupp I, Schiefer AI, Merkel O, Mocikova H, Burda P, Machova Polakova K, Kren L, Mayer J, Zent CS, Trneny M, Evans AG, Janikova A, Mraz M.
Follicular lymphoma (FL) is a common indolent B-cell malignancy with a variable clinical course. An unfavorable event in its course is histological transformation to a high-grade lymphoma, typically diffuse large B-cell lymphoma (DLBCL). Recent studies show that genetic aberrations of MYC or its overexpression are associated with FL transformation (tFL). However, the precise molecular mechanisms underlying FL transformation are unclear. Here we performed the first profiling of miRNAs' expression in paired samples of FL and tFL and identified five miRNAs as being differentially expressed. We focused on one of these miRNAs, namely miR-150, which was uniformly down-modulated in all examined tFL (~3.5-folds, n=13 pairs), and observed that high levels of MYC are responsible for repressing miR-150 in tFL by binding in its upstream region. This MYC-mediated repression of miR-150 in B cells is not dependent on LIN28A/B proteins which influence the maturation of miR-150 precursor (pri-miR-150) in myeloid cells. We also demonstrated that low miR-150 levels in tFL lead to upregulation of its target, namely FOXP1 protein, which is a known positive regulator of cell survival, as well as BCR and NF-kB signaling in malignant B cells. We have revealed that low levels of miR-150 and high levels of its target, FOXP1, are associated with shorter overall survival in FL, and suggest that miR-150 could serve as a good biomarker measurable in formalin-fixed paraffin-embedded (FFPE) tissue. Overall, our study demonstrates the role of the MYC/miR-150/FOXP1 axis in malignant B cells as a determinant of FL aggressiveness and its transformation.
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Marek Mraz
Leukemia. 2018 (in press).
MicroRNA miR-34a downregulates FOXP1 during DNA damage response to limit BCR signalling in chronic lymphocytic leukaemia B cells.
Cerna K, Oppelt J, Chochola V, Musilova K, Seda V, Pavlasova G, Radova L, Arigoni M, Calogero RA, Benes V, Trbusek M, Brychtova Y, Doubek M, Mayer J, Pospisilova S, Mraz M.
The variable clinical course in chronic lymphocytic leukaemia (CLL) largely depends on p53 functionality and B-cell receptor (BCR) signalling propensity; however, it is unclear if there is any crosstalk between these pathways. We show that DNA damage response (DDR) activation leads to down-modulating the transcriptional factor FOXP1, which functions as a positive BCR signalling regulator and its high levels are associated with worse CLL prognosis. We identified microRNA (miRNA) miR-34a as the most prominently upregulated miRNA during DDR in CLL cells in vitro and in vivo during FCR therapy (fludarabine, cyclophosphamide, rituximab). MiR-34a induced by DDR activation and p53 stabilization potently represses FOXP1 expression by binding in its 3'-UTR. The low FOXP1 levels limit BCR signalling partially via derepressing BCR-inhibitory molecule CD22. We also show that low miR-34a levels can be used as a biomarker for worse response or shorter progression free survival in CLL patients treated with FCR chemoimmunotherapy, and shorter overall survival, irrespective of TP53 status. Additionally, we have developed a method for the absolute quantification of miR-34a copies and defined precise prognostic/predictive cutoffs. Overall, herein, we reveal for the first time that B cells limit their BCR signalling during DDR by down-modulating FOXP1 via DDR-p53/miR-34a axis.
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Marek Mraz
Leukemia. 2018 (in press).
Rituximab primarily targets an intra-clonal BCR signaling proficient CLL subpopulation characterized by high CD20 levels.
Pavlasova G, Borsky M, Svobodova V, Oppelt J, Cerna K, Novotna J, Seda V, Fojtova M, Fajkus J, Brychtova Y, Doubek M, Pospisilova S, Mayer J, Mraz M.
The key feature of chronic lymphocytic leukemia (CLL) cells is their re-circulation between peripheral blood and immune niches to obtain pro-proliferative and pro-survival signals. The CLL cells that have recently exited the immune niches to the peripheral blood are characterized by weak cell-surface chemokine receptor CXCR4 levels and high levels of activation molecule CD5. These CXCR4dimCD5bright CLL cells have a ~2-fold higher CD20 expression due to the activation of the CXCR4/SDF-1 axis, and ~2-fold higher cell-surface IgM levels. Here we describe that higher CD20 expression supports B-cell receptor (BCR) signaling fitness in CLL cells, and contributes to the activated phenotype and aggressiveness of an intra-clonal subpopulation of CXCR4dimCD5brightCD20bright cells. CD20 levels directly affect BCR-induced calcium flux and the phosphorylation of BCR/PI3K-associated molecules (LYN, SYK, ERK, GAB1) after BCR-crosslinking. The CXCR4dimCD5brightCD20bright subpopulation contains more proliferative (Ki67+) cells, higher levels of pAKT/pERK/pCD79a, and their gene expression signature is significantly enriched for genes involved in BCR and MAPK signaling, migration, and actin cytoskeleton organization. CD20 up-regulation on these cells leads also to their more efficient (~9-fold) elimination by rituximab during FCR therapy in vivo, which might partially account for the success of anti-CD20 antibodies.
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Marek Mraz
Semin Oncol. 2018 Oct 22 (in press).
New concepts in follicular lymphoma biology: From BCL2 to epigenetic regulators and non-coding RNAs.
Devan J, Janikova A, Mraz M.
The molecular pathogenesis of follicular lymphoma (FL) was partially revealed 3 decades ago, with the discovery of the translocation that brings BCL2 under the influence of immunoglobulin heavy chain enhancers in a vast majority of cases. Despite the importance of this seminal observation, it has become increasingly clear that additional genetic alterations need to occur to trigger neoplastic transformation and disease progression. In this review, we summarize recent knowledge of molecular pathways and microenvironmental components involved in FL biology, and discuss them in the context of physiological B-cell development, FL evolution, and targeted therapies.
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Marek Mraz
Oncotarget. 2018 (in press).
p53 limits B cell receptor (BCR) signalling: a new role for miR-34a and FOXP1.
Cerna K, Mraz M.
The role of p53-miR-34a-FOXP1 axis described in Cerna et al. (Leukemia, 2018) is discussed in this editorial
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NEURON Award
Marek Mraz received the prestigious NEURON Award for his contribution to the studies of B cell malignancies. .
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Discovery Award
Gabriela Pavlasova received the prestigious Discovery Award in Biomedicine from Novartis for her work on the regulation and function of CD20 in B cell malignancies. Marek Mraz received this award in 2014..
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Marek Mraz
Blood. 2016 Aug 1 (epub).
Ibrutinib inhibits CD20 up-regulation on CLL B cells mediated by the CXCR4/SDF-1 axis.
Pavlasova G, Borsky M, Seda V, Cerna K, Osickova J, Doubek M, Mayer J, Calogero R, Trbusek M, Pospisilova S, Davids MS, Kipps TJ, Brown JR,Mraz M.
Agents targeting B cell receptor (BCR) signaling-associated kinases such as BTK or PI3K can induce mobilization of neoplastic B cells from the lymphoid tissues into the blood, which makes them potentially ideal to combine with anti-CD20 monoclonal antibodies (such as rituximab or ofatumumab) for treatment of B cell lymphomas and chronic lymphocytic leukemia (CLL). Here we show that interactions between leukemia cells and stromal cells (HS-5) up-regulate CD20 on CLL cells and that administering ibrutinib down-modulates CD20 expression in vivo. We observed that CLL cells that have recently exited the lymph node microenvironment and moved into the peripheral blood (CXCR4dimCD5bright subpopulation) have higher cell surface levels of CD20 than the cells circulating in the bloodstream for a longer time (CXCR4brightCD5dim cells). We found that CD20 is directly up-regulated by CXCR4 ligand SDF-1? (CXCL12) produced by stromal cells, and BTK-inhibitor ibrutinib and CXCR4-inhibitor plerixafor block SDF-1? mediated CD20 up-regulation. Ibrutinib also down-modulated Mcl1 levels in CLL cells in vivo and in co-culture with stromal cells. Overall, our study provides a detailed mechanistic explanation of CD20 expression regulation in the context of microenvironmental interactions and that it may have important implications for microenvironment-targeting therapies.
This publication was rewarded by the Czech Society of Hematology Award for best publication in 2016.
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Werner von Siemens Award
Eva Vojackova received the Werner von Siemens Award for her diploma thesis. She is now a PhD student in the group..
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UK CLL forum Dr Marek Mraz presented an invited talk at the UK CLL Forum Annual Scientific Day (March 2017) on MicroRNAs in BCR signalling in CLL


Award from Bone Marrow Transplant Foundation
Marek Mraz received the "Prof. Vladimir Koza Award" (2017) from Bone Marrow Transplant Foundation to support his research in B cell malignancies .
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Josef V. Kostir Award
Marek Mraz received the Josef V. Kostir Award (2016) for excellence in science from Czech Society of Biochemistry and Molecular Biology .
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 We have obtained a patent:
Patent
PATENT
Method for absolute quantification of miRNA expression, particularly that of miR-34a and/or miR-150 and their use in prognostics and predictive biomarkers in B-cell malignancies. .
The invention provides an absolute quantification method of miRNA expression, namely from miR-34a and/or miR-150, along with the expression of endogenous control gene. It is based on the use of synthetic standards for miRNA and endogenous control which provides absolute number of miRNA copies, and precise cut-offs. This quantification method can be used for determining prognosis of patients with B-cell leukemias/lymphomas, and prediction of therapeutic response of patients with B-cell lymphomas, especially chronic lymphocytic leukemia, and identification of p53 abnormalities.
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European Hematology Association Congress, 2016 Copenhagen
EHA 2016 Copenhagen
Marek Mraz talk at European Hematology Association (EHA) Congress.
The biology of microRNAs/ncRNAs and how to move this into clinical trials in hematological malignancies.
Translational research from the basic science perspective
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Marek Mraz
Leukemia. 2015.
MicroRNAs in B cell lymphomas: How a complex biology gets more complex.
Musilova K, Mraz M.
MicroRNAs (miRNAs) represent important regulators of gene expression besides transcriptional control. miRNA regulation can be involved in the cell developmental fate decisions, but can also have more subtle roles in buffering stochastic fluctuations in gene expression. They participate in pathways fundamental to B-cell development like B-cell receptor (BCR) signalling, B-cell migration/adhesion, cell-cell interactions in immune niches, and the production and class-switching of immunoglobulins. miRNAs influence B-cell maturation, generation of pre-, marginal zone, follicular, B1, plasma and memory B cells. In this review, we discuss miRNAs with essential functions in malignant B-cell development (such as miR-150, miR-155, miR-21, miR-34a, miR-17-92 and miR-15-16). We also put these miRNAs in the context of normal B-cell differentiation, as this is intimately connected to neoplastic B-cell development. We review miRNAs' role in the most common B-cell malignancies, including chronic lymphocytic leukaemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL) and mantle cell lymphoma (MCL). We focus on miR-contribution to the regulation of important signalling pathways (such as NF-?B, PI3K/AKT and TGF-), BCR signalling and its modulators (such as PTEN, SHIP-1, ZAP-70, GAB1 and BTK), anti- and pro-apoptotic proteins (such as BCL2, MCL1, TCL1, BIM, p53 and SIRT1) and transcription factors (such as MYC, MYB, PU.1, FOXP1 and BCL6). We also discuss the association of miRNAs' expression levels with the patients' survival and response to therapy, summarizing their potential use as predictive and prognostic markers. Importantly, the targeting of miRNAs (like use of anti-miR-155 or miR-34a mimic) could provide a novel therapeutic approach as evidenced by tumour regression in xenograft mouse models and initial promising data from clinical trials.
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Marek Mraz
Blood. 2014 Jul 3;124(1):84-95.
miR-150 influences B-cell receptor signaling in chronic lymphocytic leukemia by regulating expression of GAB1 and FOXP1.
Mraz M, Chen L, Rassenti LZ, Ghia EM, Li H, Jepsen K, Smith EN, Messer K, Frazer KA, Kipps TJ.
We examined the microRNAs (miRNAs) expressed in chronic lymphocytic leukemia (CLL) and identified miR-150 as the most abundant, but with leukemia cell expression levels that varied among patients. CLL cells that expressed zeta-chain-associated protein of 70 kDa (ZAP-70) or that used unmutated immunoglobulin heavy chain variable (IGHV) genes, each had a median expression level of miR-150 that was significantly lower than that of ZAP-70-negative CLL cells or those that used mutated IGHV genes. In samples stratified for expression of miR-150, CLL cells with low-level miR-150 expressed relatively higher levels of forkhead box P1 (FOXP1) and GRB2-associated binding protein 1 (GAB1), genes with 3' untranslated regions having evolutionary-conserved binding sites for miR-150. High-level expression of miR-150 could repress expression of these genes, which encode proteins that enhance B-cell receptor signaling, a putative CLL-growth/survival signal. Also, high-level expression of miR-150 was a significant independent predictor of longer treatment-free survival or overall survival, whereas an inverse association was observed for high-level expression of GAB1 or FOXP1 for overall survival. This study demonstrates that expression of miR-150 can influence the relative expression of GAB1 and FOXP1 and the signaling potential of the B-cell receptor, thereby possibly accounting for the noted association of expression of miR-150 and disease outcome.
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Marek Mraz
Michal Jez was awarded the PhD Talent Fellowship (2014)
JCMM.
.
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Marek Mraz
Dr. Marek Mraz was awarded the EHA RESEARCH GRANT (2014)
EHA
.
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Marek Mraz
Chapter in an Elsevier book "MicroRNA in Regenerative Medicine"
Elsevier
.
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Marek Mraz
Blood. 2014 Jul 24;124(4):546-54
MicroRNA-155 influences B-cell receptor signaling and associates with aggressive disease in chronic lymphocytic leukemia
Cui B, Chen L, Zhang S, Mraz M, Fecteau JF, Yu J, Ghia EM, Zhang L, Bao L, Rassenti LZ, Messer K, Calin GA, Croce CM, Kipps TJ.
High-level leukemia cell expression of micro-RNA 155 (miR-155) is associated with more aggressive disease in patients with chronic lymphocytic leukemia (CLL), including those cases with a low-level expression of ?-chain-associated protein of 70 kD. CLL with high-level miR-155 expressed lower levels of Src homology-2 domain-containing inositol 5-phosphatase 1 and were more responsive to B-cell receptor (BCR) ligation than CLL with low-level miR-155. Transfection with miR-155 enhanced responsiveness to BCR ligation, whereas transfection with a miR-155 inhibitor had the opposite effect. CLL in lymphoid tissue expressed higher levels of miR155HG than CLL in the blood of the same patient. Also, isolated CD5(bright)CXCR4(dim) cells, representing CLL that had been newly released from the microenvironment, expressed higher levels of miR-155 and were more responsive to BCR ligation than isolated CD5(dim)CXCR4(bright) cells of the same patient. Treatment of CLL or normal B cells with CD40-ligand or B-cell-activating factor upregulated miR-155 and enhanced sensitivity to BCR ligation, effects that could be blocked by inhibitors to miR-155. This study demonstrates that the sensitivity to BCR ligation can be enhanced by high-level expression of miR-155, which in turn can be induced by crosstalk within the tissue microenvironment, potentially contributing to its association with adverse clinical outcome in patients with CLL.
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Marek Mraz
Dr. Marek Mraz was awarded the SoMoPro Fellowship (2014)
JCMM.
.
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Marek Mraz
Dr. Marek Mraz was awarded the Discovery Award 2014
Discovery Award.
Discovery Award.
The award was established to promote research and development in medicine and pharmacy. The company wants to support and encourage brave and innovative achievements that will benefit patients, improve their quality of life and also contribute to the innovative potential of the Czech healthcare and pharmacy. Novartis wants to encourage especially the young generation of researchers and motivate them to be more interested in working in the Czech Republic.

Marek Mraz
COMMENT ON OUR PAPER "miR in CLL: more than mere markers of prognosis?" Blood. 2014 Jul 3;124(1):84-95.
miR-150 influences B-cell receptor signaling in chronic lymphocytic leukemia by regulating expression of GAB1 and FOXP1.
Mraz M, Chen L, Rassenti LZ, Ghia EM, Li H, Jepsen K, Smith EN, Messer K, Frazer KA, Kipps TJ.
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Marek Mraz
Eur J Haematol. 2014 Aug 1
B-cell receptor signalling and its crosstalk with other pathways in normal and malignant cells
Seda V Mraz M.
The physiology of B cells is intimately connected with the function of their B-cell receptor (BCR). B-cell lymphomas frequently (dys)regulate BCR signalling and thus take advantage of this pre-existing pathway for B-cell proliferation and survival. This has recently been underscored by clinical trials demonstrating that small molecules (fosfamatinib, ibrutinib, idelalisib) inhibiting BCR-associated kinases (SYK, BTK, PI3K) have an encouraging clinical effect. Here we describe the current knowledge of the specific aspects of BCR signalling in diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukaemia (CLL) and normal B cells. Multiple factors can contribute to BCR pathway (dys)regulation in these malignancies and the activation of 'chronic' or 'tonic' BCR signalling. In lymphoma B cells, the balance of initiation, amplitude and duration of BCR activation can be influenced by a specific immunoglobulin structure, the expression and mutations of adaptor molecules (like GAB1, BLNK, GRB2, CARD11), the activity of kinases (like LYN, SYK, PI3K) or phosphatases (like SHIP-1, SHP-1 and PTEN) and levels of microRNAs. We also discuss the crosstalk of BCR with other signalling pathways (NF-?B, adhesion through integrins, migration and chemokine signalling) to emphasise that the 'BCR inhibitors' target multiple pathways interconnected with BCR, which might explain some of their clinical activity.
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Marek Mraz
Leuk Lymphoma. 2013 Aug;54(8):1836-9.
MicroRNAs and B cell receptor signaling in chronic lymphocytic leukemia
Mraz M Kipps TJ.
The relative expression levels of certain microRNAs (miRNAs) correlate with known prognostic markers in chronic lymphocytic leukemia (CLL), such as leukemia-cell expression of zeta-associated protein of 70 kDa (ZAP-70), use of unmutated immunoglobulin heavy-chain variable region genes (IGHV), chromosomal abnormalities or dysfunctional p53. Here we review studies that provide evidence suggesting that certain miRNAs (e.g. miR-155, miR-17-92, miR-181, miR-29) can regulate the activated phenotype of CLL cells and/or fitness of the surface-immunoglobulin (sIg) B cell receptor (BCR) complex expressed by CLL cells, thereby accounting for the differential leukemia-cell expression of these miRNAs in different CLL prognostic subgroups. How these miRNAs influence cellular activation and/or BCR signaling through the post-transcriptional regulation of critical signaling molecules (e.g. Lyn, Syk, BTK, SHIP-1, SHP1) is a topic of current research.
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