The CYCS gene is highly evolutionarily conserved, with only a few pathogenic variants that cause thrombocytopenia-4 (THC4). Here, we report a novel CYCS variant NM_018947.6: c.59C>T [NP_061820.1:p.(Thr20Ile)] segregating with thrombocytopenia in three generations of a Czech family. The phenotype of the patients corresponds to THC4 with platelets of normal size and morphology and dominant inheritance. Intriguingly, a gradual decline in platelet counts was observed across generations. CRISPR/Cas9-mediated gene editing was used to introduce the new CYCS gene variant into a megakaryoblast cell line (MEG-01). Subsequently, the adhesion, shape, size, ploidy, viability, mitochondrial respiration, cytochrome c protein (CYCS) expression, cell surface antigen expression and caspase activity were analysed in cells carrying the studied variant. Interestingly, the variant decreases the expression of CYCS while increasing mitochondrial respiration and the expression of CD9 cell surface antigen. Surprisingly, the variant abates caspase activation, contrasting with previously known effects of other CYCS variants. Some reports indicate that caspases may be involved in thrombopoiesis; thus, the observed dysregulation of caspase activity might contribute to thrombocytopenia. The findings significantly enhance our understanding of the molecular mechanisms underlying inherited thrombocytopenia and may have implications for diagnosis, prognosis and future targeted therapies.

• MeSH
• kaspasy * metabolismus   genetika   MeSH
• lidé MeSH
• rodokmen MeSH
• trombocytopenie * genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

Rituximab (RTX) plus chemotherapy (R-CHOP) applied as a first-line therapy for lymphoma leads to a relapse in approximately 40% of the patients. Therefore, novel approaches to treat aggressive lymphomas are being intensively investigated. Several RTX-resistant (RR) cell lines have been established as surrogate models to study resistance to R-CHOP. Our study reveals that RR cells are characterized by a major downregulation of CD37, a molecule currently explored as a target for immunotherapy. Using CD20 knockout (KO) cell lines, we demonstrate that CD20 and CD37 form a complex, and hypothesize that the presence of CD20 stabilizes CD37 in the cell membrane. Consequently, we observe a diminished cytotoxicity of anti-CD37 monoclonal antibody (mAb) in complement-dependent cytotoxicity in both RR and CD20 KO cells that can be partially restored upon lysosome inhibition. On the other hand, the internalization rate of anti-CD37 mAb in CD20 KO cells is increased when compared to controls, suggesting unhampered efficacy of antibody drug conjugates (ADCs). Importantly, even a major downregulation in CD37 levels does not hamper the efficacy of CD37-directed chimeric antigen receptor (CAR) T cells. In summary, we present here a novel mechanism of CD37 regulation with further implications for the use of anti-CD37 immunotherapies.

• MeSH
• antigeny CD20 * imunologie   metabolismus   genetika   MeSH
• antigeny nádorové imunologie   genetika   MeSH
• B-buněčný lymfom * imunologie   terapie   genetika   farmakoterapie   MeSH
• chemorezistence účinky léků   MeSH
• chimerické antigenní receptory imunologie   genetika   metabolismus   MeSH
• cyklofosfamid farmakologie   terapeutické užití   MeSH
• doxorubicin farmakologie   aplikace a dávkování   MeSH
• imunoterapie * metody   MeSH
• lidé MeSH
• monoklonální protilátky farmakologie   terapeutické užití   MeSH
• nádorové buněčné linie MeSH
• protokoly antitumorózní kombinované chemoterapie farmakologie   terapeutické užití   MeSH
• regulace genové exprese u nádorů MeSH
• rituximab * farmakologie   terapeutické užití   MeSH
• tetraspaniny * genetika   metabolismus   MeSH
• vinkristin farmakologie   terapeutické užití   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Bernard-Soulier syndrome (BSS) is a rare inherited disorder characterized by unusually large platelets, low platelet count, and prolonged bleeding time. BSS is usually inherited in an autosomal recessive (AR) mode of inheritance due to a deficiency of the GPIb-IX-V complex also known as the von Willebrand factor (VWF) receptor. We investigated a family with macrothrombocytopenia, a mild bleeding tendency, slightly lowered platelet aggregation tests, and suspected autosomal dominant (AD) inheritance. We have detected a heterozygous GP1BA likely pathogenic variant, causing monoallelic BSS. A germline GP1BA gene variant (NM_000173:c.98G > A:p.C33Y), segregating with the macrothrombocytopenia, was detected by whole-exome sequencing. In silico analysis of the protein structure of the novel GPIbα variant revealed a potential structural defect, which could impact proper protein folding and subsequent binding to VWF. Flow cytometry, immunoblot, and electron microscopy demonstrated further differences between p.C33Y GP1BA carriers and healthy controls. Here, we provide a detailed insight into its clinical presentation and phenotype. Moreover, the here described case first presents an mBSS patient with two previous ischemic strokes.

• MeSH
• alely * MeSH
• Bernardův-Soulierův syndrom krev   diagnóza   genetika   MeSH
• fenotyp * MeSH
• genetická predispozice k nemoci * MeSH
• genetická variace * MeSH
• genetické asociační studie MeSH
• imunofenotypizace MeSH
• lidé MeSH
• mutační analýza DNA MeSH
• počet trombocytů MeSH
• rodokmen MeSH
• trombocytopenie krev   diagnóza   MeSH
• trombocytový glykoproteinový komplex Ib-IX genetika   metabolismus   MeSH
• trombocyty metabolismus   ultrastruktura   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• Geografické názvy
• Česká republika MeSH

Dědičné neutropenie (DN) patří mezi geneticky podmíněná onemocnění, která mohou být diagnostikována i v dospělém věku. DN se v dospělosti manifestuje spíše lehkými formami neutropenie a cytopenie. Případné další klinické příznaky bývají mírnější ve srovnání s DN diagnostikovanými u dětí. Molekulárně genetickou analýzou, zejména exomovým sekvenováním, bylo v různých genech identifikováno mnoho patogenních mutací zodpovídajících za fenotyp DN. Mutace některých těchto genů zároveň pacienty predisponuje ke zvýšenému riziku rozvoje myelodysplastického syndromu či akutní myeloidní leukemie. Správná strategie sledování pacientů, genetické poradenství a stanovení optimálního postupu léčby mohou poté významně ovlivnit prognózu tohoto onemocnění.

Congenital neutropenias (CNs) are a group of genetic disorders that may even be diagnosed in adulthood. In such cases, they manifest most often as mild neutropenia and cytopenia and other clinical symptoms tend to be less pronounced compared to CN diagnosed in childhood. Several gene variants responsible for the CN phenotype have been identified by molecular genetic approaches, especially by exome sequencing. Mutations of some of these genes also increase the risk of patients developing myelodysplastic syndrome or acute myeloid leukaemia. Proper patient monitoring strategies, genetic counselling and optimal treatment protocols can substantially affect the prognosis of these disorders.

• Klíčová slova
• varianty genů,
• MeSH
• genetické nemoci vrozené MeSH
• lidé MeSH
• neutropenie * genetika   MeSH
• sekvenování exomu MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• přehledy MeSH

Germline mutations in ETV6 gene cause inherited thrombocytopenia with leukemia predisposition. Here, we report on functional validation of ETV6 W380R mutation segregating with thrombocytopenia in a family where two family members also suffered from acute lymphoblastic leukemia (ALL) or essential thrombocythemia (ET). In-silico analysis predicted impaired DNA binding due to W380R mutation. Functional analysis showed that this mutation prevents the ETV6 protein from localizing into the cell nucleus and impairs the transcriptional repression activity of ETV6. Based on the germline ETV6 mutation, ET probably started with somatic JAK2 V617F mutation, whereas ALL could be caused by diverse mechanisms: high-hyperdiploidity; somatic deletion of exon 1 IKZF1 gene; or somatic mutations of other genes found by exome sequencing of the ALL sample taken at the diagnosis.

• MeSH
• akutní lymfatická leukemie genetika   MeSH
• esenciální trombocytemie genetika   MeSH
• lidé MeSH
• protoonkogenní proteiny c-ets metabolismus   MeSH
• represorové proteiny metabolismus   MeSH
• trombocytopenie metabolismus   MeSH
• zárodečné mutace genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Chimeric antigen receptor (CAR) T-cell therapy has already achieved remarkable remissions in some difficult-to-treat patients with B-cell malignancies. Although the clinical experience in chronic lymphocytic leukemia (CLL) patients is limited, the proportion of remissions reached in this disease is clearly the lowest from the spectrum of B-cell tumors. In this review, we discuss the antigenic targets exploited in CLL CAR-T therapy, the determinants of favorable responses, as well as the mechanisms of treatment failure specific to this disease.

• MeSH
• antigeny CD19 imunologie   MeSH
• B-lymfocyty imunologie   MeSH
• chimerické antigenní receptory imunologie   MeSH
• chronická lymfatická leukemie imunologie   terapie   MeSH
• imunoterapie adoptivní metody   MeSH
• indukce remise MeSH
• lidé MeSH
• T-lymfocyty imunologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

BACKGROUND: Anti-CD19 chimeric antigen receptor T cells (CART-19) frequently induce remissions in hemato-oncological patients with recurred and/or refractory B-cell tumors. However, malignant cells sometimes escape the immunotherapeutic targeting by CD19 gene mutations, alternative splicing or lineage switch, commonly causing lack of CD19 expression on the surface of neoplastic cells. We assumed that, in addition to the known mechanisms, other means could act on CD19 to drive antigen-negative relapse. METHODS: Herein, we studied the mechanism of antigen loss in an in vivo CD19-negative recurrence model of chronic lymphocytic leukemia (CLL) to CART-19, established using NOD-scid IL2Rgnull mice and HG3 cell line. We validated our findings in vitro in immortalized B-cell lines and primary CLL cells. RESULTS: In our in vivo CLL recurrence model, up to 70% of CART-19-treated mice eventually recurred with CD19-negative disease weeks after initial positive response. We found that the lack of CD19 expression was caused by promoter DNA hypermethylation. Importantly, the expression loss was partially reversible by treatment with a demethylating agent. Moreover, this escape mechanism was common for 3 B-cell immortalized lines as well as primary CLL cells, as assessed by in vitro coculture experiments. CONCLUSIONS: Epigenetically driven antigen escape could represent a novel, yet at least partially reversible, means of CD19 loss to CART-19 in B-cell tumors.

• MeSH
• antigeny CD19 imunologie   MeSH
• lidé MeSH
• metylace DNA imunologie   MeSH
• myši MeSH
• receptory antigenů T-buněk imunologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: While achieving prolonged remissions in other B cell-derived malignancies, chimeric antigen receptor (CAR) T cells still underperform when injected into patients with chronic lymphocytic leukemia (CLL). We studied the influence of genetics on CLL response to anti-CD19 CAR T-cell therapy. METHODS: First, we studied 32 primary CLL samples composed of 26 immunoglobulin heavy-chain gene variable (IGHV)-unmutated (9 ATM-mutated, 8 TP53-mutated, and 9 without mutations in ATM, TP53, NOTCH1 or SF3B1) and 6 IGHV-mutated samples without mutations in the above-mentioned genes. Then, we mimicked the leukemic microenvironment in the primary cells by '2S stimulation' through interleukin-2 and nuclear factor kappa B. Finally, CRISPR/Cas9-generated ATM-knockout and TP53-knockout clones (four and seven, respectively) from CLL-derived cell lines MEC1 and HG3 were used. All these samples were exposed to CAR T cells. In vivo survival study in NSG mice using HG3 wild-type (WT), ATM-knockout or TP53-knockout cells was also performed. RESULTS: Primary unstimulated CLL cells were specifically eliminated after >24 hours of coculture with CAR T cells. '2S' stimulated cells showed increased survival when exposed to CAR T cells compared with unstimulated ones, confirming the positive effect of this stimulation on CLL cells' in vitro fitness. After 96 hours of coculture, there was no difference in survival among the genetic classes. Finally, CAR T cells were specifically activated in vitro in the presence of target knockout cell lines as shown by the production of interferon-γ when compared with control (CTRL) T cells (p=0.0020), but there was no difference in knockout cells' survival. In vivo, CAR T cells prolonged the survival of mice injected with WT, TP53-knockout and ATM-knockout HG3 tumor cells as compared with CTRL T cells (p=0.0485, 0.0204 and <0.0001, respectively). When compared with ATM-knockout, TP53-knockout disease was associated with an earlier time of onset (p<0.0001), higher tumor burden (p=0.0002) and inefficient T-cell engraftment (p=0.0012). CONCLUSIONS: While in vitro no differences in survival of CLL cells of various genetic backgrounds were observed, CAR T cells showed a different effectiveness at eradicating tumor cells in vivo depending on the driver mutation. Early disease onset, high-tumor burden and inefficient T-cell engraftment, associated with TP53-knockout tumors in our experimental setting, ultimately led to inferior performance of CAR T cells.

• MeSH
• antigeny CD19 terapeutické užití   MeSH
• chimerické antigenní receptory imunologie   MeSH
• chronická lymfatická leukemie genetika   MeSH
• lidé MeSH
• myši MeSH
• T-lymfocyty imunologie   MeSH
• zdraví dobrovolníci pro lékařské studie MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Surface protein CD20 serves as the critical target of immunotherapy in various B-cell malignancies for decades, however its biological function and regulation remain largely elusive. Better understanding of CD20 function may help to design improved rational therapies to prevent development of resistance. Using CRISPR/Cas9 technique, we have abrogated CD20 expression in five different malignant B-cell lines. We show that CD20 deletion has no effect upon B-cell receptor signaling or calcium flux. Also B-cell survival and proliferation is unaffected in the absence of CD20. On the contrary, we found a strong defect in actin cytoskeleton polymerization and, consequently, defective cell adhesion and migration in response to homeostatic chemokines SDF1α, CCL19 and CCL21. Mechanistically, we could identify a reduction in chemokine-triggered PYK2 activation, a calcium-activated signaling protein involved in activation of MAP kinases and cytoskeleton regulation. These cellular defects in consequence result in a severely disturbed homing of B cells in vivo.

• MeSH
• aktiny metabolismus   MeSH
• antigeny CD20 genetika   metabolismus   fyziologie   MeSH
• B-buněčný lymfom metabolismus   patologie   MeSH
• B-lymfocyty patologie   fyziologie   MeSH
• buněčná adheze fyziologie   MeSH
• genový knockdown MeSH
• leukemie B-buněčná metabolismus   patologie   MeSH
• lidé MeSH
• multimerizace proteinu fyziologie   MeSH
• myši inbrední NOD MeSH
• myši SCID MeSH
• myši transgenní MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• pohyb buněk fyziologie   MeSH
• polymerizace MeSH
• receptory antigenů B-buněk metabolismus   MeSH
• signální transdukce imunologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Monoclonal antibody (mAb) therapy often encounters resistance due to downmodulation of the target antigens. The exact underlying mechanisms remain unknown as well as the efficacy of newly developed mAbs. We propose to investigate the impact of several clinically advanced mAbs on the expression level of their target antigen and determine functional consequences of lower target expression on the potency of mAbs in cell killing. We will characterize cellular mechanisms regulating the surface expression of antigens that are downregulated in response to any of the mAbs. We shall investigate how are these antigens degraded and explore changes on the transcriptional and translational level, changes in promoter activity and epigenetic alterations. Finally, we will perform rescue screen with inhibitors of epigenetic modifying enzymes and a whole genome loss-of-function screen using novel state-of-the-art technique. This will identify proteins involved in the regulation of surface expression and suggest intervention strategies that may lead to upregulation of the antigens on the cell surface.

Terapie monoklonálními protilátkami (mAbs) se často potýká s rezistencí způsobenou snížením exprese cílových antigenů. Podstata zapříčiňujících mechanismů zůstává neznámá, stejně jako účinnost nově vyvinutých mAbs. V tomto projektu budeme testovat vliv několika klinicky nejvíce pokročilých mAbs na úroveň exprese jejich cílového antigenu a určíme funkční důsledky snížené exprese na efektivitu protilátek v usmrcování buněk. Charakterizujeme buněčné mechanismy regulující povrchovou expresi antigenů, které jsou downregulovány v reakci na některou z mAbs. Prozkoumáme, jakými mechanismy jsou tyto antigeny degradovány a budeme sledovat změny na úrovni transkripce a translace, změny v aktivitě promotoru a epigenetické modifikace. Na závěr provedeme screening s inhibitory epigenetických enzymů a celogenomový “loss-of-function” screening s využitím nové technologie na úrovni dnešní doby. Tento přístup povede k identifikaci proteinů podílejících se na regulaci povrchové exprese a navrhne intervenční strategie, které mohou vést k upregulaci antigenů na buněčném povrchu.

• MeSH
• antigeny povrchové MeSH
• chronická lymfatická leukemie terapie   MeSH
• exprese genu MeSH
• inhibitory enzymů MeSH
• léková rezistence MeSH
• monoklonální protilátky terapeutické užití   MeSH
• protein Cas9 MeSH
• regulace genové exprese MeSH

• NLK Obory
• onkologie
• biochemie
• genetika, lékařská genetika
• NLK Publikační typ
• závěrečné zprávy o řešení grantu AZV MZ ČR