BACKGROUND: The mechanistic target of rapamycin (mTOR) is a crucial regulator of cell metabolic activity. It forms part of several distinct protein complexes, particularly mTORC1 and mTORC2. The lack of specific inhibitors still hampers the attribution of mTOR functions to these complexes. JR-AB2-011 has been reported as a specific mTORC2 inhibitor preventing mTOR binding to RICTOR, a unique component of mTORC2. We aimed to describe the effects of JR-AB2-011 in leukemia/lymphoma cells, where the mTOR pathway is often aberrantly activated. METHODS: The impact of JR-AB2-011 on leukemia/lymphoma cell metabolism was analyzed using the Seahorse platform. AKT phosphorylation at Ser473 was used as a marker of mTORC2 activity. mTOR binding to RICTOR was assessed by co-immunoprecipitation. RICTOR-null cells were derived from the Karpas-299 cell line using CRISPR/Cas9 gene editing. RESULTS: In leukemia/lymphoma cell lines, JR-AB2-011 induced a rapid drop in the cell respiration rate, which was variably compensated by an increased glycolytic rate. In contrast, an increase in the respiration rate due to JR-AB2-011 treatment was observed in primary leukemia cells. Unexpectedly, JR-AB2-011 did not affect AKT Ser473 phosphorylation. In addition, mTOR did not dissociate from RICTOR in cells treated with JR-AB2-011 under the experimental conditions used in this study. The effect of JR-AB2-011 on cell respiration was retained in RICTOR-null cells. CONCLUSION: JR-AB2-011 affects leukemia/lymphoma cell metabolism via a mechanism independent of mTORC2.

• MeSH
• fosforylace účinky léků   MeSH
• leukemie * farmakoterapie   metabolismus   MeSH
• lidé MeSH
• mTOR inhibitory farmakologie   MeSH
• mTORC2 * metabolismus   MeSH
• nádorové buněčné linie MeSH
• protein RICTOR * metabolismus   MeSH
• protoonkogenní proteiny c-akt metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The p21-activated kinase (PAK) family of proteins regulates various processes requiring dynamic cytoskeleton organization such as cell adhesion, migration, proliferation, and apoptosis. Among the six members of the protein family, PAK2 is specifically involved in apoptosis, angiogenesis, or the development of endothelial cells. We report a novel de novo heterozygous missense PAK2 variant, p.(Thr406Met), found in a newborn with clinical manifestations of Knobloch syndrome. In vitro experiments indicated that this and another reported variant, p.(Asp425Asn), result in substantially impaired protein kinase activity. Similar findings were described previously for the PAK2 p.(Glu435Lys) variant found in two siblings with proposed Knobloch syndrome type 2 (KNO2). These new variants support the association of PAK2 kinase deficiency with a second, autosomal dominant form of Knobloch syndrome: KNO2.

• MeSH
• degenerace retiny genetika   patologie   MeSH
• encefalokéla MeSH
• lidé MeSH
• missense mutace genetika   MeSH
• novorozenec MeSH
• odchlípení sítnice genetika   patologie   vrozené   MeSH
• p21 aktivované kinasy * genetika   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• novorozenec MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH

Somatic JAK2 mutations are the main molecular cause of the vast majority of polycythemia vera (PV) cases. According to a recent structural model, the prevalent acquired V617F mutation improves the stability of the JAK2 dimer, thereby enhancing the constitutive JAK2 kinase activity. Germline JAK2 mutations usually do not largely alter JAK2 signaling, although they may modulate the impact of V617F. We found an unusual germline JAK2 mutation L604F in homozygous form in a young PV patient, along with a low allele burden JAK2 V617F mutation, and in her apparently healthy sister. Their father with a PV-like disease had L604F in a heterozygous state, without V617F. The functional consequences of JAK2 L604Fmutation were compared with those induced by V617F in two different in vitro model systems: (i) HEK293T cells were transfected with plasmids for exogenous JAK2-GFP expression, and (ii) endogenous JAK2 modifications were introduced into HeLa cells using CRISPR/Cas9. Both mutations significantly increased JAK2 constitutive activity in transfected HEK293T cells. In the second model, JAK2 modification resulted in reduced total JAK2 protein levels. An important difference was also detected: as described previously, the effect of V617F on JAK2 kinase activity was abrogated in the absence of the aromatic residue F595. In contrast, JAK2 hyperactivation by L604F was only partially inhibited by the F595 change to alanine. We propose that the L604F mutation increases the probability of spontaneous JAK2 dimer formation, which is physiologically mediated by F595. In addition, L604F may contribute to dimer stabilization similarly to V617F.

• MeSH
• HEK293 buňky MeSH
• HeLa buňky MeSH
• Janus kinasa 2 genetika   MeSH
• lidé MeSH
• mutace MeSH
• zárodečné buňky * MeSH
• zárodečné mutace * MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

P21-activated kinases (PAKs) regulate processes associated with cytoskeletal rearrangements, such as cell division, adhesion, and migration. The possible regulatory role of PAKs in cell metabolism has not been well explored, but increasing evidence suggests that a cell metabolic phenotype is related to cell interactions with the microenvironment. We analyzed the impact of PAK inhibition by small molecule inhibitors, small interfering RNA, or gene knockout on the rates of mitochondrial respiration and aerobic glycolysis. Pharmacological inhibition of PAK group I by IPA-3 induced a strong decrease in metabolic rates in human adherent cancer cell lines, leukemia/lymphoma cell lines, and primary leukemia cells. The immediate effect of FRAX597, which inhibits PAK kinase activity, was moderate, indicating that PAK nonkinase functions are essential for cell metabolism. Selective downregulation or deletion of PAK2 was associated with a shift toward oxidative phosphorylation. In contrast, PAK1 knockout resulted in increased glycolysis. However, the overall metabolic capacity was not substantially reduced by PAK1 or PAK2 deletion, possibly due to partial redundancy in PAK1/PAK2 regulatory roles or to activation of other compensatory mechanisms.

• MeSH
• HEK293 buňky MeSH
• HeLa buňky MeSH
• lidé MeSH
• mitochondrie enzymologie   genetika   MeSH
• nádorové mikroprostředí * MeSH
• nádorové proteiny genetika   metabolismus   MeSH
• nádory enzymologie   genetika   MeSH
• p21 aktivované kinasy genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The immune system is important for elimination of residual leukemic cells during acute myeloid leukemia (AML) therapy. Anti-leukemia immune response can be inhibited by various mechanisms leading to immune evasion and disease relapse. Selected markers of immune escape were analyzed on AML cells from leukapheresis at diagnosis (N = 53). Hierarchical clustering of AML immunophenotypes yielded distinct genetic clusters. In the absence of DNMT3A mutation, NPM1 mutation was associated with decreased HLA expression and low levels of other markers (CLIP, PD-L1, TIM-3). Analysis of an independent cohort confirmed decreased levels of HLA transcripts in patients with NPM1 mutation. Samples with combined NPM1 and DNMT3A mutations had high CLIP surface amount suggesting reduced antigen presentation. TIM-3 transcript correlated not only with TIM-3 surface protein but also with CLIP and PD-L1. In our cohort, high levels of TIM-3/PD-L1/CLIP were associated with lower survival. Our results suggest that AML genotype is related to blast immunophenotype, and that high TIM-3 transcript levels in AML blasts could be a marker of immune escape. Cellular pathways regulating resistance to the immune system might contribute to the predicted response to standard therapy of patients in specific AML subgroups and should be targeted to improve AML treatment.

• MeSH
• akutní myeloidní leukemie * diagnóza   genetika   MeSH
• antigeny CD274 genetika   MeSH
• biologické markery MeSH
• buněčný receptor 2 viru hepatitidy A genetika   MeSH
• DNA methyltransferasa 3A * genetika   MeSH
• lidé MeSH
• mutace MeSH
• nukleofosmin * genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• Publikační typ
• abstrakt z konference MeSH

Knobloch syndrome is an autosomal recessive phenotype mainly characterized by retinal detachment and encephalocele caused by biallelic pathogenic variants in the COL18A1 gene. However, there are patients clinically diagnosed as Knobloch syndrome with unknown molecular etiology not linked to COL18A1. We studied an historical pedigree (published in 1998) designated as KNO2 (Knobloch type 2 syndrome with intellectual disability, autistic behavior, retinal degeneration, encephalocele). Whole exome sequencing of the two affected siblings and the normal parents resulted in the identification of a PAK2 non-synonymous substitution p.(Glu435Lys) as a causative variant. The variant was monoallelic and apparently de novo in both siblings indicating a likely germ-line mosaicism in one of the parents; the mosaicism, however, could not be observed after deep sequencing of blood parental DNA. PAK2 encodes a member of a small group of serine/threonine kinases; these P21-activating kinases (PAKs) are essential in signal transduction and cellular regulation (cytoskeletal dynamics, cell motility, death and survival signaling and cell cycle progression). Structural analysis of the PAK2 p.(Glu435Lys) variant that is located in the kinase domain of the protein predicts a possible compromise in the kinase activity. Functional analysis of the p.(Glu435Lys) PAK2 variant in transfected HEK293T cells results in a partial loss of the kinase activity. PAK2 has been previously suggested as an autism-related gene. Our results show that PAK2-induced phenotypic spectrum is broad and not fully understood. We conclude that the KNO2 syndrome in the studied family is dominant and caused by a deleterious variant in the PAK2 gene.

• MeSH
• degenerace retiny * genetika   patologie   MeSH
• encefalokéla diagnóza   genetika   patologie   MeSH
• HEK293 buňky MeSH
• lidé MeSH
• mutace MeSH
• odchlípení sítnice * vrozené   genetika   MeSH
• p21 aktivované kinasy genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

P21-activated kinases (PAK) regulate processes associated with cytoskeleton dynamics. PAK expression in leukemia cells was measured on protein and mRNA levels. In functional assays, we analyzed the effect of PAK inhibitors IPA-3 and FRAX597 on cell adhesivity and viability. PAK2 was dominant in cell lines, whereas primary cells also expressed comparable amount of PAK1 transcription isoforms: PAK1-full and PAK1Δ15. PAK1Δ15 and PAK2 levels correlated with surface density of integrins β1 and αVβ3. PAK1-full, but not PAK2, was present in membrane protrusions. IPA-3, which prevents PAK activation, induced cell contraction in semi-adherent HEL cells only. FRAX597, which inhibits PAK kinase activity, increased cell-surface contact area in all leukemia cells. Both inhibitors reduced the stability of cell attachment and induced cell death.

• MeSH
• buněčná adheze MeSH
• buněčné linie MeSH
• fibronektiny genetika   MeSH
• leukemie * genetika   MeSH
• lidé MeSH
• p21 aktivované kinasy * genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Nucleophosmin (NPM) mutations causing its export from the nucleoli to the cytoplasm are frequent in acute myeloid leukemia (AML). Due to heterooligomerization of wild type NPM with the AML-related mutant, the wild-type becomes misplaced from the nucleoli and its functions are significantly altered. Dissociation of NPM heterooligomers may thus restore the proper localization and function of wild-type NPM. NSC348884 is supposed to act as a potent inhibitor of NPM oligomerization. The effect of NSC348884 on the NPM oligomerization was thoroughly examined by fluorescence lifetime imaging with utilization of FRET and by a set of immunoprecipitation and electrophoretic methods. Leukemia-derived cell lines and primary AML cells as well as cells transfected with fluorescently labeled NPM forms were investigated. Our results clearly demonstrate that NSC348884 does not inhibit formation of NPM oligomers neither in vivo nor in vitro. Instead, we document that NSC348884 cytotoxicity is rather associated with modified cell adhesion signaling. The cytotoxic mechanism of NSC348884 has therefore to be reconsidered.

• MeSH
• apoptóza účinky léků   MeSH
• HEK293 buňky MeSH
• indoly farmakologie   MeSH
• jaderné proteiny genetika   metabolismus   MeSH
• leukemie farmakoterapie   genetika   metabolismus   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• protinádorové látky farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Nucleophosmin (NPM) C-terminal mutations are detected in about 30% of patients with acute myeloid leukemia (AML). Our pilot study indicated that individuals with appropriate HLA alleles (about 85% of population) are able to raise spontaneous immune response against mutated NPM which can prevent AML development. Anti-NPM immune response is also active during therapy and essentially contributes to better outcome of patients having NPM mutations. The objectives of the project are: (i) to confirm and to extend these findings on a larger patient cohort, (ii) to obtain experimental evidence of the existence of NPM-specific T-cells, (iii) to establish diagnostic methods for the detection of reasons for transient or complete failure of the immune response which allows for AML development. The practical aim of this applied research is to prepare conditions for implementation of individualized immunotherapy into the treatment regimen of AML patients in order to achieve the disease eradication.

Mutace v C-koncové části nukleofosminu (NPM) se vyskytují u zhruba 30% pacientů s akutní myeloidní leukémií (AML). Naše pilotní studie naznačila, že jedinci s vhodnými HLA alelami (tvořící asi 85% populace) jsou schopni vyvinout spontánní imunitní reakci proti mutovanému NPM, která může zabránit rozvoji AML. Imunitní reakce proti NPM působí i během léčby a zásadně přispívá k lepší prognóze u pacientů s mutovaným NPM. Úkolem projektu je: (i) potvrdit a rozšířit naše předběžná zjištění na větším souboru pacientů, (ii) získat experimentální důkazy existence T-buněk specifických pro NPM a (iii) zavést diagnostické metody pro analýzu důvodů přechodného nebo úplného selhání imunitní odpovědi, které vede k rozvoji AML. Praktickým cílem tohoto aplikovaného výzkumu je připravit podmínky pro zavedení individualizované imunoterapie do léčebného protokolu AML tak, aby bylo dosaženo trvalého vyléčení u co největšího počtu pacientů.

• MeSH
• akutní myeloidní leukemie terapie   MeSH
• HLA antigeny MeSH
• imunita MeSH
• imunoterapie metody   MeSH
• lidé MeSH
• mutace MeSH
• nukleofosmin škodlivé účinky   MeSH
• pilotní projekty MeSH
• Check Tag
• lidé MeSH

• Konspekt
• Patologie. Klinická medicína
• NLK Obory
• onkologie
• alergologie a imunologie
• genetika, lékařská genetika
• NLK Publikační typ
• závěrečné zprávy o řešení grantu AZV MZ ČR