Mraz lab

We are hiring a Postdoc and PhD students. See "positions" and apply ASAP! Before end of january 2025 or better sooner

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Eva Hoferkova succesfully defended her PhD thesis (12/2024) on novel co-culture model mimicking CLL-T cell interactions. CONGRATULATIONS !!!

Marek Mraz has presented an invited talk during American Society of Hematology (ASH) Annual Meeting (San Diego, 12/2024) and discuss non-genetic adaptation to BTK inhibitors in CLL .

EXPRO GACR Grant
GACR EXPRO grant of 2 million Euros from Czech Science Foundation awarded to Marek Mraz to study the Role of non-coding RNAs in the microenviromental interactions of chronic lymphocytic leukemia (2025-2029).

Veronika Sandova succesfully defended her PhD thesis (10/2024) on CD20 regulation by IL4. CONGRATULATIONS !!!

J CLIN INVESTIGATION. 2024 Oct (epub). IF=13,3
FoxO1/Rictor axis induces a non-genetic adaptation to Ibrutinib via Akt activation in chronic lymphocytic leukemia
video Laura Ondrisova summarized the results in a short video.
Ondrisova L, Seda V, Hlavac K, Pavelkova P, Hoferkova E, Chiodin G, Kostalova L, Mladonicka Pavlasova G, Filip D, Vecera J, Zeni PF, Oppelt J, Kahounova Z, Vichova R, Soucek K, Panovska A, Plevova K, Pospisilova S, Simkovic M, Vrbacky F, Lysak D, Fernandes SM, Davids MS, Maiques-Diaz A, Charalampopoulou S, Martin-Subero JI, Brown JR, Doubek M, Forconi F, Mayer J, Mraz M
BTK inhibitor therapy induces peripheral blood lymphocytosis in chronic lymphocytic leukemia (CLL) lasting for several months. It remains unclear whether non-genetic adaptation mechanisms exist, allowing CLL cells' survival during BTK inhibitor-induced lymphocytosis and/or playing a role in therapy resistance. We show that in approximately 70 % of CLL cases, ibrutinib treatment in vivo increases Akt activity above pre-therapy levels within several weeks, leading to compensatory CLL cell survival and a more prominent lymphocytosis on therapy. Ibrutinib-induced Akt phosphorylation (pAktS473) is caused by the upregulation of FoxO1 transcription factor, which induces expression of Rictor, an assembly protein for mTORC2 protein complex that directly phosphorylates Akt at serine 473 (S473). Knock-out or inhibition of FoxO1 or Rictor led to a dramatic decrease in Akt phosphorylation and growth disadvantage for malignant B cells in the presence of ibrutinib (or PI3K inhibitor idelalisib) in vitro and in vivo. FoxO1/Rictor/pAktS473 axis represents an early non-genetic adaptation to BCR inhibitor therapy not requiring PI3K? or BTK kinase activity. We further demonstrate that FoxO1 can be targeted therapeutically, and its inhibition induces CLL cells' apoptosis alone or in combination with BTK inhibitors (ibrutinib, acalabrutinib, pirtobrutinib) and blocks their proliferation triggered by T-cell factors (CD40L, IL-4, and IL-21)

LEUKEMIA. 2024 June (epub). IF=12,8
Stromal cells engineered to express T cell factors induce robust CLL cell proliferation in vitro and in PDX co-transplantations allowing the identification of RAF inhibitors as anti-proliferative drugs
Hoferkova E, Seda V, Kadakova S, Verner J, Loja T, Matulova K, Skuhrova Francova H, Ondrouskova E, Filip D, Blavet N, Boudny M, Mladonicka Pavlasova G, Vecera J, Ondrisova L, Pavelkova P, Hlavac K, Kostalova L, Michaelou A, Pospisilova S, Dorazilova J, Chochola V, Jaros J, Doubek M, Jarosova M, Hampl A, Vojtova L, Kren L, Mayer J, Mraz M
Several in vitro models have been developed to mimic chronic lymphocytic leukemia (CLL) proliferation in immune niches; however, they typically do not induce robust proliferation. We prepared a novel model based on mimicking T-cell signals in vitro and in patient-derived xenografts (PDXs). Six supportive cell lines were prepared by engineering HS5 stromal cells with stable expression of human CD40L, IL4, IL21, and their combinations. Co-culture with HS5 expressing CD40L and IL4 in combination led to mild CLL cell proliferation (median 7% at day 7), while the HS5 expressing CD40L, IL4, and IL21 led to unprecedented proliferation rate (median 44%). The co-cultures mimicked the gene expression fingerprint of lymph node CLL cells (MYC, NFkB, and E2F signatures) and revealed novel vulnerabilities in CLL-T-cell-induced proliferation. Drug testing in co-cultures revealed for the first time that pan-RAF inhibitors fully block CLL proliferation. The co-culture model can be downscaled to five microliter volume for large drug screening purposes or upscaled to CLL PDXs by HS5-CD40L-IL4 +/- IL21 co-transplantation. Co-transplanting NSG mice with purified CLL cells and HS5-CD40L-IL4 or HS5-CD40L-IL4-IL21 cells on collagen-based scaffold led to 47% or 82% engraftment efficacy, respectively, with ~20% of PDXs being clonally related to CLL, potentially overcoming the need to co-transplant autologous T-cells in PDXs.

Dr. Vaclav Seda was awarded the EHA JUNIOR RESEARCH GRANT (2024). There are only two recipients of this grant in the Czech Republic so far; both from this laboratory (M. Mraz received the grant in 2014).
news report
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Marek Mraz presented an invited talk during ESH 3rd Translational Research Conference: CLL (Vienna, 3/2024) discussing non-genetic adaptation to BCR signalling inhibitors in CLL .

Lab Christmas party 2023
Mraz lab
Lab Christmas party was quadruple festive: i) we celebrated the return of many lab members from an Indian wedding of our former Ph.D. student Sonali ii) Peter Kacz has been awarded the Ph.D. talent scholarship iii) Vasek Seda, Laura Ondrisova, and Marek Mraz received the CEITEC Award for scientific achievement iv) MM has been recently promoted to Full professor. The picture has all students, including undergrads and also some partners :-)

Blood. 2023 Nov (epub), IF 25,5.
5'-UTR mRNA splicing determines CD20 levels
Mraz M
In this issue of Blood, Ang et al show that human CD20 messenger RNA (mRNA) undergoes alternative splicing to generate distinct 5' untranslated region (5'-UTR) variants, which determine the cell-surface CD20 levels in malignant B cells and the interpatient variability in the expression of this therapeutic target. These variants can also be used as an escape mechanism from anti-CD20 therapies.

We have obtained a US patent for use of GAB1 inhibitors in hematological malignancies

Patent
PATENT
INHIBITORS FOR TREATMENT OF HEMATOLOGICAL MALIGNANCIES (US 11648255B2).
In this patent, we describe the use of novel GAB1 protein inhibitors as therapeutics for a number of hematological malignancies. In the context of B-cell leukemias and lymphomas, we have described that GAB1-001 and GAB1-004 compounds inhibit malignant cell migration and induce cell death by blocking the BCR signaling pathway. Other BCR inhibitors have been approved for clinical use in B malignancies (e.g., ibrutinib, acalabrutinib, zanubrutinib, idelalisib), and the development of this class of drugs continues to be very intensive. We have also described that GAB1 inhibition is toxic to acute myeloid leukemia cells (AML), the most aggressive adult leukemia, where there is a clear unmet medical need for new therapies.

RNA Biology. 2021 May (epub).
Our review on lncRNAs in adaptive immunity featured on the cover of RNA Biology Journal
Zeni P,Mraz M
The adaptive immune system is responsible for generating immunological response and immunological memory. Regulation of adaptive immunity including B cell and T cell biology was mainly understood from the protein and microRNA perspective. However, long non-coding RNAs (lncRNAs) are an emerging class of non-coding RNAs (ncRNAs) that influence key factors in lymphocyte biology such as NOTCH, PAX5, MYC and EZH2. LncRNAs were described to modulate lymphocyte activation by regulating pathways such as NFAT, NF?B, MYC, interferon and TCR/BCR signalling (NRON, NKILA, BCALM, GAS5, PVT1), and cell effector functions (IFNG-AS1, TH2-LCR). Here we review lncRNA involvement in adaptive immunity and the implications for autoimmune diseases (multiple sclerosis, inflammatory bowel disease, rheumatoid arthritis) and T/B cell leukaemias and lymphomas (CLL, MCL, DLBCL, T-ALL). It is becoming clear that lncRNAs are important in adaptive immune response and provide new insights into its orchestration.

Prof. Klaus Rajewsky arrived (13.10.22) to give an invited talk during the Mendels Lecture Series, and to discuss research with us. He was awarded the "Mendel Medal" by Masaryk University for his tremendous contribution to understanding B cell biology

Vasek Seda succesfully defended his PhD thesis (9/2021). CONGRATULATIONS !!! He received an award from YIM iwCLL 2021 for the best presentation (see below)

Blood. 2021 March (epub), IF 25,5.
FoxO1-GAB1 Axis Regulates Homing Capacity and Tonic AKT Activity in Chronic Lymphocytic Leukemia
video presenting the research at iwCLL 2021 [Awarded the best presentation at YIM iwCLL].
Seda V,Eva Vojackova, Laura Ondrisova, Lenka Kostalova, Sonali Sharma, Tomas Loja, Gabriela Mladonicka Pavlasova, Daniel Zicha, Marie Kudlickova Peskova, Jan Krivanek, Kvetoslava Liskova, Leos Kren, Vladimir Benes, Katerina Musilova Litzmanova, Marek Borsky, Jan Oppelt, Jan Verner, Sarka Pospisilova, Yvona Brychtova, Anna Panovska, Zhi Tan, Shuxing Zhang, Michael Doubek, Katerina Amruz Cerna, Jiri Mayer, Mraz M.
Recirculation of chronic lymphocytic leukemia (CLL) cells between the peripheral blood and lymphoid niches plays a critical role in disease pathophysiology, and inhibiting this process is one of the major mechanisms of action for BCR inhibitors such as ibrutinib or idelalisib. Migration is a complex process guided by chemokine receptors and integrins. However, it remains largely unknown how precisely CLL cells regulate their homingintegrate multiple migratory signals while balancing survival in peripheral blood and the decision to return to immune niches. Here we provide evidence for the use of CXCR4/CD5 intraclonal subpopulations to study CLL cells migration regulation. We performed RNA profiling of CXCR4dimCD5bright versus CXCR4brightCD5dim cells and identified differential expression of dozens of molecules with a putative function cell migration. We have shown that GRB2 associated binding protein 1 (GAB1) positively regulates CLL cell homing capacity of CXCR4brightCD5dim cells. Gradual GAB1 accumulation in CLL cells outside immune niches is mediated by FoxO1-based transcriptional GAB1 activation. We also describe that upregulation of GAB1 plays an important role in maintaining basal PI3K activity and "tonic" AKT phosphorylation required to sustain survival of resting CLL cells. This is important during ibrutinib therapy since CLL cells induce the FoxO1-GAB1-pAKT axis, which represents an adaptation mechanism to the inability to home to immune niches. We have demonstrated that GAB1 can be targeted therapeutically by novel GAB1 inhibitors alone or in combination with BTK inhibition. GAB1 inhibitors induce CLL cell apoptosis, impair cell migration, inhibit "tonic" or BCR-induced AKT phosphorylation, and block compensatory AKT activity during ibrutinib therapy.

Blood. 2021, IF 25,5.
miR-29 Modulates CD40 Signaling in Chronic Lymphocytic Leukemia by Targeting TRAF4: an Axis Affected by BCR inhibitors
video presenting the research at EHA
awarded as the most influential publication by Czech Hematology Society (2021), and by Masaryk University (MUNI Scientists, Vice-rector award and Deans award for S.Sharma) and CEITEC MU (Award for best publication).
Sharma S,Pavlasova G, Seda V, Cerna K, Vojackova E, Filip D, Ondrisova L, Sandova V, Kostalova L, Zeni PF, Borsky M, Oppelt J, Liskova K, Kren L, Janikova A, Pospisilova S, Fernandes SM, Shehata M , Rassenti LZ, Jaeger U, Doubek M, Davids MS, Brown JR, Mayer J, Kipps TJ, Mraz M.
B cell receptor (BCR) signaling and T cell interactions play a pivotal role in chronic lymphocytic leukemia (CLL) pathogenesis and disease aggressiveness. CLL cells can utilize microRNAs (miRNAs) and their targets to modulate microenvironmental interactions in the lymph node niches. To identify miRNA expression changes in the CLL microenvironment, we performed complex profiling of short non-coding RNAs in this context by comparing CXCR4/CD5 intraclonal cell subpopulations (CXCR4dimCD5bright vs. CXCR4brightCD5dim cells). This identified dozens of differentially expressed miRNAs including several that have previously been shown to modulate BCR signaling (miR-155, miR-150, and miR-22), but also other candidates for a role in microenvironmental interactions. Notably, all three miR-29 family members (miR-29a, miR-29b, miR-29c) were consistently down-modulated in the immune niches, and lower miR-29(a/b/c) levels associated with an increased relative responsiveness of CLL cells to BCR ligation, and significantly shorter overall survival of CLL patients. We identified Tumor-Necrosis Factor Receptor-Associated Factor 4 (TRAF4) as a novel direct target of miR-29s and revealed that higher TRAF4 levels increase CLL responsiveness to CD40 activation and downstream NFkB signaling. In CLL, BCR-represses miR-29 expression via MYC, allowing for concurrent TRAF4 upregulation and stronger CD40-NFkB signaling. This regulatory loop is disrupted by "BCR inhibitors" (BTK inhibitor ibrutinib or PI3K inhibitor idelalisib). In summary, we showed for the first time that a miRNA-dependent mechanism acts to activate CD40 signaling/T-cell interactions in a CLL microenvironment and described a novel miR-29-TRAF4-CD40 signaling axis modulated by the BCR activity.

Haematologica. 2021 July (epub), IF 9,9.
IL4-STAT6 signaling induces CD20 in chronic lymphocytic leukemia and this axis is repressed by PI3K? inhibitor idelalisib
video presenting the research at iwCLL 2021
Sandova V,Pavlasova GM, Seda V, Cerna KA, Sharma S, Palusova V, Brychtova Y, Pospisilova S, Fernandes SM, Panovska A, Doubek M, Davids MS, Brown JR, Mayer J, Mraz M.
In order to effectively combine anti-CD20 antibodies (such as rituximab or obinutuzumab) with BTK/PI3K inhibitors, a better understanding is required of the mechanisms of CD20 regulation and its function(s) in this context. It has been shown that the BTK inhibitor ibrutinib down-modulates CD20 levels in chronic lymphocytic leukemia (CLL) by interfering with its regulators in the lymph node microenvironment, namely the CXCR4/SDF1 axis, FoxO1 levels, and NF?B signaling. Here we show that interleukin 4 (IL4; IL-4) produced by T-cells is a potent inducer of CD20 (MS4A1) transcription via STAT6 activation and its binding to the CD20 promotor. CD20 itself is involved in IL4 signaling propensity, creating a feed-forward positive loop where IL4 induces CD20 and further increases IL4 responsiveness. The IL4-STAT6 axis includes PI3K?, and single agent idelalisib therapy in CLL leads to CD20 down-modulation in vivo. Moreover, the BTK inhibitor ibrutinib also interferes with the IL4 signaling pathway, resulting in decreased STAT6 phosphorylation and reduced CD20 levels. Altogether, here we describe a novel mechanism of CD20 regulation in CLL B cells, and this has important implications in the context of combinatorial targeted therapy with BCR inhibitors.

Haematologica. 2020 June (epub).
The regulation and function of CD20: an enigma of B-cell biology and targeted therapy.Review
Pavlasova G, Mraz M.
The introduction of anti-CD20 monoclonal antibodies such as rituximab, ofatumumab, or obinutuzumab improved the therapy of B-cell malignancies even though the precise physiological role and regulation of CD20 remains unclear. Furthermore, CD20 expression is highly variable between different B-cell malignancies, patients with the same malignancy, and even between intraclonal subpopulations in an individual patient. Several epigenetic (EZH2, HDAC1/2, HDAC1/4, HDAC6, complex Sin3A-HDAC1) and transcription factors (USF, OCT1/2, PU.1, PiP, ELK1, ETS1, SP1, NFkB, FOXO1, CREM, SMAD2/3) regulating CD20 expression (encoded by MS4A1) have been characterized. CD20 is induced in the context of microenvironmental interactions by CXCR4/SDF1 (CXCL12) chemokine signaling and the molecular function of CD20 has been linked to the signaling propensity of B-cell receptor (BCR). CD20 has also been shown to interact with multiple other surface proteins on B cells (such as CD40, MHCII, CD53, CD81, CD82, and CBP). Current efforts to combine anti-CD20 monoclonal antibodies with BCR signaling inhibitors targeting BTK or PI3K (ibrutinib, acalabrutinib, idelalisib, duvelisib) or BH3-mimetics (venetoclax) lead to the necessity to better understand both the mechanisms of regulation and the biological functions of CD20. This is underscored by the observation that CD20 is decreased in response to the BCR inhibitor ibrutinib which largely prevents its successful combination with rituximab. Several small molecules (such as histone deacetylase inhibitors, DNA methyl-transferase inhibitors, aurora kinase A/B inhibitors, farnesyltransferase inhibitors, FOXO1 inhibitors, and bryostatin-1) are being tested to upregulate cell-surface CD20 levels and increase the efficacy of anti-CD20 monoclonal antibodies. Herein, we review the current understanding of CD20 function, and the mechanisms of its regulation in normal and malignant B cells, highlighting the therapeutic implications.

ERC Starting Grant
ERC Starting Grant awarded to Marek Mraz to study the Propensity of microenviromental interactions in chronic lymphocytic leukemia(5/2019-5/2024). In relation to this, Dr. Mraz received the Award of the Rector of Masaryk University .

Fron Oncology. 2020 Oct 26;10:591577. (epub).
Genetic and Non-Genetic Mechanisms of Resistance to BCR Signaling Inhibitors in B Cell Malignancies.Review
Ondrisova L, Mraz M.
The review describes genetic and non-genetic mechanisms of resistance to first generation of BTK or PI3K inhibitors.

iwCLL 2019,
short video about the talk
Marek Mraz presented an invited talk during iwCLL 2019 discussing the role of Regulatory RNAs in CLL Biology .

Marek Mraz received the award for the best finished research project from the Czech Science Foundation (GACR). The project was focused on studying the role of microRNAs in the DNA damage response in malignant B cells.
Article about the award,
short video about the awarded project

Marek Mraz received an award from the committee of the League Against Cancer.
Article about the award,

Katka Cerna and Katka Musilova succesfully defended their PhD thesis (6/2019). CONGRATULATIONS !!! Katka M. also received an award from Purkyne Foundation for the best publication in 2018

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.

Leukemia. 2019.
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.

Leukemia. 2018.
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.

Semin Oncol. 2018
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.

Leuk Lymphoma. 2019 Aug;54(8):1836-9.
The role of MYC in the transformation and aggressiveness of 'indolent' B-cell malignancies
Filip D, Mraz M
MYC was found to be involved in many germinal center derived lymphomas, and more recently in the histological transformation of indolent mature B-cell malignancies, such as follicular lymphoma (FL), chronic lymphocytic leukemia (CLL) and mucosa-associated lymphoid tissue lymphoma (MALT) to aggressive diffuse large B-cell lymphoma (DLBCL). Pathological MYC activity gain in lymphomas is able to overcome its regulation by repressors, which leads to bypassing the affinity-based selection of B-cells. Arguably the MYC activity gain is the most constantly observed phenomenon (>70% of cases) in transformed FL/MALT/CLL (Richter's transformation) and co-occurs with specific aberrations such as the loss of p53, CDKN2A/B, or gain of BCL2/BCL6. Here we summarize recent progress in the understanding of MYC regulatory network in lymphoma B-cells and highlight its involvement in lymphomas' histological transformation by regulating cyclins, CDKs, p21, p27, BCL2, E2F, FOXP1, BCR signaling components, and non-coding microRNA (miRNA) genes such as miR-150, miR-29, miR-17-92, and miR-34a.

Oncotarget. 2018
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

NEURON Award
Marek Mraz received the prestigious NEURON Award for his contribution to the studies of B cell malignancies. .

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..

Blood. 2016.
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.

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..

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 .

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 .

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.

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

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.

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.

Michal Jez was awarded the PhD Talent Fellowship (2014)
JCMM.
.

Dr. Marek Mraz was awarded the EHA RESEARCH GRANT (2014)
EHA
.

Chapter in an Elsevier book "MicroRNA in Regenerative Medicine"
Elsevier
.

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.

Dr. Marek Mraz was awarded the SoMoPro Fellowship (2014)
JCMM.
.

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.

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.

Eur J Haematol. 2014
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.

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.