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Grant Projects (ongoing in the year 2023)
View ongoing grants in the year: 2009, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2018, 2019, 2020, 2021, 2022, 2023, 2024, 2025, 2026, 2027, 2028.
Study of molecular pathogenesis of bone marrow failure and identification of clinically relevant biomarkers in hypoplastic myelodysplastic syndrome and acquired aplastic anemia
AZV NU21-03-00565 [2021 – 2024]
Hypoplastic myelodysplastic syndrome (hMDS) and acquired aplastic anemia (AA) are severe hematopoietic disorders whose clinicopathological features overlap that makes diagnosis complicated. In both disorders, bone marrow (BM) failure occurs due to damage to hematopoietic cells by cytotoxic T-lymphocytes. However, the molecular mechanisms involved in this process are still unclear. In this context, a comprehensive genome analysis in hMDS and AA will be performed in the proposed project to characterize the molecular basis of BM failure and to define clinically relevant biomarkers for easier differential diagnosis, prediction of disease progression, and evaluation of the effect of immunosuppressive therapy on pathological clone evolution. Whole-exome sequencing will be used for characterization of genomic landscape, the transcriptome will be analyzed by RNASeq at the level of CD34+ BM cells and CD3+ lymphocytes, and mRNA and lnRNA expression profiles will be determined. Due to the etiology of the diseases, we will focus on the factors mediating the immune response and the T-cell population that will be studied in detail by flow cytometry. Knowledge of the molecular background of hMDS/AA will provide us an understanding of the disease development and will also enable to define new biomarkers which may strengthen the personalized approach to patients.
Multifunctional nanostructures for plasmonic sensing of biomolecules related to onco-hematological diseases
GA ČR 22-27329S [2022 – 2024]
prof. Ing. Jiří Homola CSc., DSc., Ústav fotoniky a elektrotechniky AV ČR
Ing. Jiří Suttnar, CSc., Ing. Andrés de los Santos Pereira Ph.D., Ústav makromolekulární chemie AV ČR
This is a multidisciplinary project that aims to advance research in plasmonics, functional coatings and onco-hematological diseases with the goal of developing a new generation of optical biosensors that enable simultaneous and sensitive detection of microRNA and protein biomarkers of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). This goal will be achieved by the development of new multi-functional plasmonic nanostructures that support surface plasmons in VIS/NIR and UV spectral regions, and functionalization methods using UV-triggered chemical reactions, and their combination for site-specific receptor immobilization in plasmonic hotspot regions. The biosensor technology to be developed in this project will provide a new tool for the research of onco-hematological diseases, open up new opportunities for monitoring MDS/AML patients and pave the way to personalized therapies.
Role of acute myeloid leukemia-associated nucleophosmin mutations in the NPM-p53-Mdm2 regulatory network
GA ČR 22-03875S [2022 – 2024]
prof. RNDr. Petr Heřman CSc., MFF UK Praha
Mgr. Barbora Brodská, Ph.D.
Nucleophosmin (NPM) mutations characteristic for the acute myeloid leukemia (AML) cause aberrant cytoplasmic localization of NPM and many of its interaction partners, e.g. of tumor suppressor p53. The p53 level is regulated at several stages by mdm2, which is also known to interact with NPM. Impact of the mutation on the dynamic equilibria within the NPM-p53-mdm2 network, mutual protein interactions, and potential formation of the ternary complex in live cells will be examined by microscopic and immunochemical methods. We will focus on the role of NPM in the regulation of p53 activity with the possibility to increase sensitivity and response of cells with mutated NPM to cytostatics. We will evaluate impact of the NPM mutations on relations between specific phosphorylations and oligomerization of p53 and NPM. Experiments will be done with cell lines transfected with fluorescent proteins and verified on primary cells from AML patients. Results will contribute to understanding of the role of NPM mutation in leukemogenesis and will open new strategies for AML treatment.
Genomic characterization of adult B-precursor acute lymphoblastic leukemia for prediction of treatment response to targeted therapy
AZV NU22-03-00210 [2022 – 2025]
doc. MUDr. Mgr. Cyril Šálek, Ph.D.
Prof. MUDr. Tomáš Stopka, Ph.D., 1. LF UK Praha, Priv.-Doz. Dr. rer. nat. habil. Radislav Sedláček, ÚMG AV ČR Praha
Subtypes of B-precursor acute lymphoblastic leukemias (BCP-ALL) with distinct genomic or gene expression profile have been recently revealed using high-throughput sequencing technology, including BCR-ABL1-like ALL, ALL with DUX4, ZNF384, MEF2D and NUTM1 rearrangements, ALL with PAX5 alterations or IKZFplus ALL. They are mostly associated with poor response to standard chemotherapy and impaired progression-free survival. This can be overcome with targeted pharmaceuticals including inhibitors of distinct kinases, monoclonal antibodies (including bispecific agents and immunoconjugates), and chimeric antigen receptor T-cells (CAR-T). These drugs have shown unprecedent therapeutic efficacy in relapsed/refractory cohorts. Czech Leukemia Study Group is running three academic trials with targeted agents in frontline settings: 1) Blina-CELL Study with anti-CD19/anti-CD3 bispecific monoclonal antibody in the induction phase for BCR-ABL1-negative ALL); 2) EWALL-INO Study with anti-CD22 immunoconjugate in the induction phase for BCR-ABL1-negative ALL; and 3) Pona-CELL Study combining third generation tyrosine kinase inhibitor ponatinib with reduced-intensity chemotherapy in BCR-ABL1-positive ALL. These trials are aiming to accelerate treatment response and induce negativity of minimal residual disease (MRD) at early phases of the therapy. The aim of proposed project is to perform detailed genomic characterization and search for specific genomic patterns in subjects enrolled to above mentioned academic studies and to compare their genomic profile with the treatment response. Prospective cohort treated with targeted agents will be compared to a consecutive retrospective cohort treated with standard chemotherapy. The goal is to identify specific genomic signatures that confer a high risk of treatment failure on standard chemotherapy protocols and to prove that high-risk genomic features can be overcome with modern targeted agents if administered early in the front-line therapy.
Gene engineered killer T cells for the therapy of acute myeloid leukemia
AZV NU22-05-00374 [2022 – 2025]
doc.MUDr. Pavel Otáhal, Ph.D.
Prof. MUDr. Tomáš Stopka, Ph.D., 1. LF UK Praha, Priv.-Doz. Dr. rer. nat. habil. Radislav Sedláček, ÚMG AV ČR Praha
The aim of the project is to develop new applied methods of cell therapy of AML based on genetically modified autologous T lymphocytes. This procedure is based on a genetic modification by a recombinant T-cell receptor (TCR), which redirects the specificity of T lymphocytes to the selected antigen, and their subsequent in vitro expansion. One of the targets studied in this way is Wilms Tumor Antigen 1 (WT1), which is expressed by AML cells and is at the same time effectively recognized by the immune system and has therefore been used as a target in a number of clinical trials. This identical TCR reacting with the RMFNAPYL antigen antigen WT1 in the HLA A2 complex was prepared and tested at the same time as a number of other TCR control TCR reacting with other relevant antigens such as NY-ESO-1. The main goal of the project will be to develop a GMP-certified method of preparing WT1-specific TCR-T cells using the non-viral gene transduction method using piggyBac transposon, in a similar way as CD19-specific CAR-T cells are currently being prepared in UHKT. The function of prepared cells will be studied using in vitro and in vivo using mouse models. The anticancer effect will be monitored by in-vivo bio-luminescent methods using standard cell lines of acute myeloid leukaemia and primary AML cells. In addition, humanized mouse models will be introduced to allow a more detailed study of a number of immunological aspects of the treatment of acute myeloid leukemia via TCR-T cells. In the next objective of the project, we will prepare new types of TCR vectors that will allow to target additional epitopes from antigens WT1, or PRAME and we will develop new types of TCR vectors with enhanced activity via co-expression of cytokines such as IL-15 and IL-21. The applied output of the project will be the GMP production of TCR-T lymphocytes specific to the WT1 antigen, ands the submission of an application for a clinical trial (Phase I) in patients with AML.
Significance of T cell response to vaccination against SARS-CoV2 for leukemic patients with weakend immune system
AZV NU22-E-138 [2022 – 2023]
The project is aimed on choosing optimal methods for measurement of total and SARS-CoV-2 specific immunity through quantification of specific effectors of adaptive immunity of haemato-oncologic patients treated with cellular therapy, that will be suitable for optimizing of vaccination against COVID-19 and for evaluation of its immunogenicity. The state of immune system of patients will be characterized and the impact of its deterioration on immune response to vaccination will be assessed. Immune responses to vaccination of recipients of various types of cellular therapy and of healthy individuals will be compared. Impact of vaccination on the course of therapy of underlying disease will be determined. The goal of the project includes proposing a recommendation of a suitable method for examination of specific anti-SARS-CoV-2 cellular immunity, acceptable for clinical praxis.
Advanced genomic characterization of secondary myeloid malignancies: implications for diagnostics and prognostication
AZV NU23-03-00401 [2023 – 2026]
RNDr. Monika Beličková, Ph.D.
Mgr. Jana Kotašková, Ph.D., Fakultní nemocnice Brno
Therapy-related myeloid neoplasms (t-MNs) are secondary malignancies that occur after chemotherapy and/or radiation therapy of a previous cancer. The t-MN category accounts for approximately 10-20% of newly diagnosed patients with myeloid neoplasms (MNs) and the latency between treatment and development of t-MN ranges from a few months to years. The prognosis of these patients is generally poor, and unlike primary MNs, there are no specific prognostic scoring systems for this group nor a defined complex genomic background. We hypothesized that the combined effect of acquired somatic mutations and numerous germline variants, including common and rare variants, which underlie the multistep molecular pathogenesis, contributes to t-MN development. Thus, we propose an advanced analysis of the t-MN genome, which will include the examination of somatic and germline mutations using whole-genome and targeted sequencing. Furthermore, transcriptome analysis of protein-coding and noncoding RNAs at the level of bone marrow CD34+ cells will be performed to determine the signaling pathways involved in t-MN development. Genomic data along with cytogenetic findings will be correlated with clinical parameters. Relevant molecular markers will be incorporated into the diagnostic and prognostic evaluation of t-MN patients. Knowledge of the molecular background of secondary malignancies will lead to a more detailed subclassification of patients, determination of a more accurate prognosis and selection of an appropriate treatment, ultimately strengthening personalized approaches for patients.
Next generation CAR-T cells for the therapy of hematological malignancies
AZV NU23-03-00188 [2023 – 2026]
doc.MUDr. Pavel Otáhal, Ph.D.
Prof. MUDr. Pavel Klener, Ph.D., 1. LF UK Praha
The proposed project deals with new methods of cell therapy of hematological malignancies such as AML, B-ALL and B-NHL using CAR-T T lymphocytes. The aim of the project is to build on the ongoing clinical study with CD19-specific CAR-T lymphocytes, which are produced in IHBT for therapeutic purposes (UHKT CAR19) and are tested in a clinical trial in patients with refractory/relapsing B-ALL/B-NHL. Treatment with commercial CD19 CAR-T products cures about 20-30% of patients indicated for this treatment, the remaining patients either do not respond to treatment or relapse even when complete remission is achieved. The causes of CAR-T treatment failure are being intensively studied. One of the ways to prevent lies in the development of so-called next-generation CAR-T cells that have enhanced antitumor activity, e.g. by co-expression of cytokines (IL-12, IL-15, IL-18, IL-21) or—by secreted bi-specific antibodies. The first goal of this project is to develop such modified CD19-specific CAR-T lymphocytes. The second goal is the development of CD123-specific CAR-T lymphocytes, for the treatment of acute myeloid leukemia. These new types of CAR-T products will be analogically to the UHKT CAR19 T cells that are now being produced at UHKT. During the solution of the project, we will establish GMP-certified production of both CAR-T products in the Clean Rooms of the IHBT, prepare pharmaceutical documentation and submit an application for two clinical trials with these products - one for AML and the other for refractory/relapsing B-ALL/B-NHL.
Spectrum of NGS detected somatic mutations and their association with prognosis and outcome of adolescent and young adult patients with Ph-positive leukemias
AZV NU21-07-00225 [2021 – 2024]
doc. Mgr. Kateřina Machová Poláková, Ph.D.
Prof. MUDr. Jan Trka,, Ph.D., Fakultní nemocnice v Motole, Praha
Adolescent and young adult (AYA) patients with Ph+ leukemias comprise a unique subgroup of patients with hematologic malignancies. The disease demands the careful monitoring and good compliance. Up to now, except for few clinical trials, the specific aspects and biology of Ph+ leukemias of AYA patients have not been studied in detail. Currently, most patients with chronic myeloid leukemia in chronic phase (CML-CP) have a normal life expectancy. Before tyrosine kinase inhibitors (TKIs) introduction into the clinical practice, the higher age was a negative prognostic factor. In the current era of TKIs it seems that younger patients with CML-CP at age 15-39, defined by National Comprehensive Cancer Network Guidelines as AYAs, have worse prognosis and response to TKIs. In recent years, somatic mutations in hematologic malignancies are of importance and have been highly reported. However, very little is known about the genomic landscape of somatic mutations outside the oncogene BCR-ABL1 in Ph positive leukemias - CML and Ph positive acute lymphoblastic leukemia (Ph+ALL) - of AYAs and their relation to the resistance to TKI treatment and relapses. We expect that the complex NGS-based screening of somatic mutations proposed in this project will allow us to determine whether the clonal hematopoiesis represents a worse prognostic factor for AYA patients with Ph+ leukemias. We anticipate that the similar spectrum of mutations can be identified in CML and Ph+ALL AYA patients, but with different patterns of clonal evolution in association with the different treatment protocols. This project may identify high risk AYA CML patients, who may profit from more intensive treatment protocol, similar to protocol of the Ph+ALL.
Changes in expression of adhesion molecules: a tool for prediction of extramedullary relapse of B cell acute lymphoblastic leukemia following blinatumomab treatment
AZV NU23J-03-00033 [2023 – 2026]
Blinatumomab (Blincyto ®) is a bi-specific therapeutic antibody mediating the connection of the CD19 receptor on malignant B-cells to CD3 on effector T-lymphocytes. This enables the formation of a cytolytic synapse and effective elimination of the tumor. According to current knowledge, however, the relapse into extramedullary disease (EMD) is unusually common in blinatumomab-treated patients with B-cell acute lymphoblast leukemia (B-ALL). The main hypothesis of the project assumes that blinatumomab affects leukemia cell adhesion properties and thereby homing of B-ALL cells to tissues where blinatumomab is not effective. This may eventually lead to EMD. The sole interaction of blinatumomab with CD19 can affect the signaling of several adhesion-related molecules (integrins, Lyn). Furthermore, immune synapse formation can lead to changes in the expression of surface cell adhesion molecules (CAM) on both involved cells. Additionally, cytoskeleton changes induced by B-cell receptor (BCR), a vital component of the immune synapse, are dependent on Lyn kinase signaling. The project aims to analyze the effect of blinatumomab on B-cell adhesion processes. Mechanisms of blinatumomab-induced changes in adhesion signaling will be studied on B-cell leukemia (B-ALL) cell lines, both in the absence and in the presence of model T-lymphocytes. We will assess changes in the cell affinity to proteins of the extracellular matrix and to model stromal cells and, on a molecular level, changes in the activation status of selected proteins to find a clinically useful marker of the changes induced by blinatumomab. In primary samples (B-ALL, ~60 patients and up to 250 samples during the project course), changes in expression and activation of relevant CAMs (integrins, CD44, CD62L), as well as selected intracellular markers (Lyn) after blinatumomab treatment, will be evaluated, and correlated with the treatment results.
Efficacy and safety of tyrosine kinase inhibitors’ discontinuation after two-step dose reduction in patients with chronic myeloid leukemia (A prospective multi-centre phase II clinical trial - HALF)
AZV NU22-03-00136 [2022 – 2025]
Prof. MUDr. Jiří Mayer, CSc., LF MU Brno
MUDr. Daniela Žáčková, Ph.D., FN Brno, doc. Mgr. Kateřina Machová Poláková, Ph.D., MUDr. Lukáš Stejskal, FN Ostrava, Prof. MUDr. Edgar Faber, CSc., FN Olomouc, MUDr. Michal Karas, Ph.D., RN Plzeň, MUDr. Petra Bělohlávková, Ph.D., FN Hradec Králové
Chronic myeloid leukemia (CML) patients’ treatment and prognosis improved significantly with tyrosine kinase inhibitors (TKI) introduction into clinical practice. However, long-lasting or even lifelong therapy can cause even serious adverse events and represents a substantial economic burden for health care systems. Treatment-free remission (TFR) has therefore become a new goal of CML therapy, since its feasibility and safety was confirmed by multiple clinical trials as well as clinical practice experience. However, usually abrupt therapy discontinuation has been successful only in about half of eligible patients and even if so, it can cause burdening symptoms known as a TKI withdrawal syndrome (TWS) in about 30% of patients. On top of that, sustainable deep molecular response as a main prerequisite for TKI discontinuation has been achieved in only 20-40% of patients. Thus, majority of CML patients need to be treated with lifelong therapy with all its drawbacks. Moreover, any robust clinical nor biological factor predictive for successful TFR has not been identified yet. We propose the project of multi-centre investigator-initiated phase II clinical trial (HALF) representing an original concept of the two-step TKI dose reduction (i.e. to half of standard dose during the first 6 months and to the same dose given alternatively during the next 6 months) before complete TKI withdrawal. We suppose that this approach will both induce higher rate of patients achieving successful TFR with less frequent/pronounce TWS and proof feasibility and safety of sequential TKI dose reduction as an alternative to the complete therapy interruption versus its lifelong administration. Besides our focus on efficacy and safety improvement, we plan to provide many analyses in order to identify both clinical and biological factors predictive for successful TFR. We also aim to evaluate and eventually reduce financial burden of the long-term therapy as well as improve patients’ quality of life
VEXAS syndrome; characterization and clinical consequences of individual UBA1 genetic variants among patients with rheumatic and hematologic disorders
AZV NU23-10-00160 [2023 – 2026]
MUDr. Heřman Mann, Ph.D., Revmatologický ústav, Praha
RNDr. Monika Beličková, Ph.D.
VEXAS (vacuoles, E1 activating enzyme, X-linked, auto-inflammatory, somatic) syndrome is a clinically serious and potentially fatal adult-onset disease caused by somatic mutations in the UBA1 (Ubiquitin-like modifier activating enzyme 1) gene in hematopoietic progenitor cells. It was first described in 2020 therefore data regarding its clinical manifestations, genetic associations, prognosis and treatment efficacy are limited. UBA1 missense mutations in VEXAS syndrome patients cause activation of inflammatory pathways, which clinically present in older patients with systemic inflammation and a potential to progress into myelodysplastic, myeloproliferative, or lymphoproliferative disease. Patients may meet diagnostic criteria for several rheumatic and/or hematological disorders. Given its heterogeneous clinical manifestations a high degree of clinical suspicion is required for pursuing the diagnosis of VEXAS syndrome. Demonstration of known pathogenic variants in hot spot sites of p.Met41 in exon 3 in UBA1 sequencing analysis of hematopoietic cells is currently required to establish the diagnosis of VEXAS syndrome. However, this approach may not be sufficient since an extended analysis of all coding regions of the UBA1 gene may uncover other mutations with putative functional consequences as we have shown. VEXAS syndrome is associated with high morbidity and mortality. There is currently no information regarding the impact of VEXAS syndrome on patients’ quality of life. Many patients experience partial response to treatment with glucocorticoids and/or immunosuppressive therapy. Limited evidence suggests that hematopoietic stem cell transplantation may be curative in some cases. More data from careful clinical observations is clearly needed to provide much evidence regarding the optimal diagnostic approach, impact of the disease on patients’ quality of life, prognosis and value of individual treatments.
Targeting of FLT3 using new dual inhibitors as a therapeutic approach in MLL-rearranged acute myeloid leukemia
GAČR 23-05462S [2023 – 2025]
Doc. RNDr. Vladimír Kryštof, Ph.D., PřF UP Olomouc
RNDr. Hana Votavová , Ph.D.
Translocations of MLL are associated with aggressive acute leukemias. MLL fusions deregulate gene expression through aberrantly recruited epigenetic and transcription regulators and also often activate FLT3 signaling. Unfortunately, FLT3 inhibitors induce acquired drug resistance and therefore new compounds with rational dual activity are sought for. We recently identified new FLT3 inhibitors with high selectivity towards myeloid cells expressing oncogenic MLL and FLT3. Besides FLT3, the compounds target few other kinases and in this project we intend to explore dual FLT3/CDK9 and FLT3/SFK inhibition as a therapeutic approach for MLL. Activity of compounds will be studied in leukemic cell lines, primary cells from leukemic patients and in in patient-derived xenograft models. Finally, using our murine model of MLL-ENL leukemogenesis we will test the hypothesis that ATR suppression activates FLT3-dependent signalling in pre-leukemic progenitor cells and that ATR inhibition results in a hyperproliferative MLL-ENL phenotype vulnerable to inhibition of the FLT3 pathway.
Role of transposable elements and PIWI-interacting RNAs in myelodysplastic syndrome and their potential clinical applications
AZV NU20-03-00412 [2020 – 2024]
Ing. Michaela Dostálová Merkerová, Ph.D.
Doc. Ing. Jiří Kléma, Ph.D., FEL ČVUT Praha
Myelodysplastic syndrome (MDS) is a malignant hematopoietic disorder characterized by aberrant differentiation of hematopoietic stem cells (HSCs). Genome instability is one of the key features of MDS HSCs and mobilization of transposable elements (TEs) is a known destabilizing factor of the genome integrity. PIWI-interacting RNAs (piRNAs) inhibit TE mobilization, functioning as central players in stem cell mechanisms to preserve genome integrity. Despite recent advances in the understanding of MDS pathogenesis, the role of TEs and piRNAs in development and progression of the disease has not been studied yet. We will analyze transcription of TEs and piRNAs by next generation sequencing and examine their activity in MDS. Besides the implication in the MDS pathogenesis, utilization of TEs and piRNAs as potential molecular markers of the disease progression will be tested. Moreover, possible effect of azacitidine treatment on TE and piRNA transcriptions will be addressed to prove their applicability as novel predictive markers for the drug responsiveness and relapse prevention.
Natural Killer cell based adoptive cellular immunotherapy of hemato-oncology disorders
AZV NU22-08-00287 [2022 – 2025]
The use of natural killer (NK) cells as a cellular immunotherapy has increased over the past decade, specifically in patients with hematologic malignancies. NK cell adoptive therapy is a promising cancer therapeutic approach, but there are significant challenges that are limiting its feasibility and clinical efficacy. Acute myeloid leukemia (AML) is a clinically and genetically heterogeneous disease with remaining unsatisfactory outcomes. In this project, we propose to develop methods for expansion and activation of NK cells from human peripheral blood mononuclear cells as well as clinical-scale method to produce NK cells for immunotherapy under Current Good Manufacturing Practices (cGMP) conditions. We plan to generate the data correlating the NK cells phenotype with the expansion and killing capacity allowing to stratify the NK cells donors in order to achieve reproducible and efficient product of cellular immunotherapy. In this study, we will use irradiated autologous peripheral blood mononuclear cells as cell-based feeder cells for large-scale expansion of highly purified cytotoxic NK cells. Along with those steps, we will investigate the possibilities of NK cell donor stratification based on the correlation of immunophenotypic markers and expansion and cytotoxic potential of NK cell product. The generated knowledge will be used for further improvements of the therapeutic strategy and more personalized donor selection. The ultimate goal of the project is to develop an immunotherapeutic protocol using adoptively transferred NK cells to achieve full remission of AML and myelodysplastic syndrome patients.
