Biology subjects

Kuzelova, K.

Publications and source records attributed to Kuzelova, K..

7 recordsLinked to original sources

bioRxiv2021

PAK1 and PAK2 in the cell metabolism regulation

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. Alternatively, 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.

cell biology↗

bioRxiv2021

Associations between recurrent mutations and blast immunophenotype in acute myeloid leukemia

The immune system undoubtly plays an important role in final elimination of residual leukemic cells during acute myeloid leukemia (AML) therapy. However, the anti-leukemia immune response can be inhibited by a variety of mechanisms enabling immune escape and eventual disease relapse. We analyzed selected markers of immune escape on AML cells at diagnosis (N = 53) and used them for hierarchical clustering analysis, which yielded distinct clusters with different incidence of mutations in nucleophosmin 1 (NPM1) and in the methyltransferase DNMT3A. More detailed analysis showed that in the absence of DNMT3A mutation, NPM1 mutation is associated with decreased HLA expression and also with low levels of other markers (CLIP, PD-L1, TIM-3). On the other hand, samples with concomitant DNMT3A mutation had high CLIP surface amount suggesting reduced antigen presentation. Higher CLIP exposition was also found in patients with internal tandem duplications in FLT3 (FLT3-ITD). TIM-3 transcript correlated not only with TIM-3 protein surface amount, but also with CLIP and PD-L1, suggesting acquisition of a complex immunoresistant phenotype. Our results indicate that AML genotype is to some extent related to the blast immunophenotype, and the established predictive values of particular mutations might also reflect an inherent cell resistance to the immune system.

cancer biology↗

bioRxiv2020

Dominant monoallelic variant in the PAK2 gene causes Knobloch syndrome type 2

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 germline 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 which 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.

genetics↗

bioRxiv2020

Group I p21-activated kinases in leukemia cell adhesion to fibronectin

P21-activated kinases (PAK) regulate many processes associated with cytoskeleton dynamics, including cell adhesion, migration, and apoptosis. PAK function is frequently altered in cancer, and PAK were proposed as therapy targets both in solid tumors and in hematological malignancies. However, current knowledge about PAK function in cell adhesion is mainly based on adherent cell models. Moreover, existing functional differences among the individual PAK family members are unsufficiently characterized. We measured expression of PAK group I members in leukemia cell lines and in primary leukemia cells, both on protein and mRNA levels. In functional assays, we analyzed the effect of two PAK inhibitors with different mechanisms of action, IPA-3 and FRAX597. Changes in cell interaction with fibronectin were monitored through impedance measurement and by interference reflection microscopy. Cytotoxic effects of inhibitors were assessed by Annexin V/propidium iodide test. PAK intracellular localization was analyzed by confocal microscopy. PAK2 transcript was dominant in cell lines, whereas primary leukemia cells also expressed comparable amount of PAK1, which was detected as two transcription isoforms: PAK1-full and PAK1{Delta}15. PAK1{Delta}15 and PAK2 transcript levels correlated with surface density of integrins {beta}1 and V{beta}3. PAK1-full, but not PAK2, was present in membrane protrusions. The inhibitors had partly opposed effects: 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. Although many cells accumulated high FRAX597 amounts, low doses were sufficient to kill sensitive cells. FRAX597-induced cell death was fast in the MV4-11 cell line and in primary AML cells. Although PAK group I seem to be essential for leukemia cell adhesion and survival, and might thus serve as therapy targets, many PAK functions still remain to be attributed to individual isoforms and to their functional domains.

cell biology↗

bioRxiv2020

NSC348884 cytotoxicity is not mediated by inhibition of nucleophosmin oligomerization

Oligomerization of the nucleolar phosphoprotein nucleophosmin (NPM) is mediated by its N-terminal domain. In acute myeloid leukemia, a frequent NPM mutation occurring at the C-terminus causes NPM delocalization to the cytoplasm. Due to formation of NPM heterooligomers, the wild-type NPM as well as many of NPM interaction partners are also delocalized. Proper localization and function of mislocalized proteins in the cells with mutated NPM may be restored by targeting NPM oligomerization. We introduce a reliable set of complementary methods for monitoring NPM oligomerization in both cell lysates and live cells. Using this methodological background we show that a putative inhibitor of NPM oligomerization, NSC348884, does not prevent formation of NPM oligomers in leukemia cells. Instead, we reveal that the observed cytotoxic effect of NSC348884 is associated with changes in cell adhesion signaling.

cell biology↗

bioRxiv2019

PAK1, PAK1Δ15, and PAK2: similarities, differences and mutual interactions

P21-activated kinases (PAK) are key effectors of the small GTPases Rac1 and Cdc42, as well as of Src family kinases. In particular, PAK1 has several well-documented roles, both kinase-dependent and kinase-independent, in cancer-related processes, such as cell proliferation, adhesion, and migration. However, PAK1 properties and functions have not been attributed to individual PAK1 isoforms: besides the full-length kinase (PAK1-full), a splicing variant lacking the exon 15 (PAK1{Delta}15) is annotated in protein databases. In addition, it is not clear if PAK1 and PAK2 are functionally overlapping. Using fluorescently tagged forms of human PAK1-full, PAK1{Delta}15, and PAK2, we analyzed their intracellular localization and mutual interactions. Effects of PAK inhibition or depletion on cell-surface adhesion were monitored by real-time microimpedance measurement. We show that PAK1{Delta}15 is in many aspects similar to PAK2, rather than to PAK1-full. Both PAK1{Delta}15 and PAK2, but not PAK1-full, were enriched in focal adhesions, indicating that the C-terminus might be important for PAK intracellular localization. Using immunoprecipitation, we documented direct interactions among the studied PAK group I members: PAK1 and PAK2 form homodimers, but all possible heterodimers were also detected. Our results indicate that PAK1 and PAK2 have distinct roles in cell adhesion and mutually affect their function. PAK1-full is required for formation of membrane protrusions, whereas PAK2 is involved in focal adhesion assembly. We have also noted that PAK inhibition was associated with a large reduction of the cell glycolytic rate. Altogether, our data suggest a complex interplay among different PAK group I members, which have largely non-redundant functions.

cell biology↗

bioRxiv2018

Association of HLA class I type with prevalence and outcome of patients with acute myeloid leukemia and mutated nucleophosmin

Acute myeloid leukemia with mutated nucleophosmin (NPMc+ AML) forms a distinct AML subgroup with better prognosis which can potentially be associated with immune response against the mutated nucleophosmin (NPM). As the T-cell-mediated immunity involves antigen presentation on HLA class I molecules, we hypothesized that individuals with suitable HLA type could be less prone to develop NPMc+ AML. We compared HLA class I distribution in NPMc+ AML patient cohort (398 patients from 5 centers) with the HLA allele frequencies of the caucasian population and found HLA-A*02, B*07, B*40 and C*07 underrepresented in the NPMc+ AML group. Presence of B*07 or C*07:01 antigen was associated with better survival in patients without concomitant FLT3 internal tandem duplication. Candidate NPM-derived immunopeptides were found for B*40 and B*07 using prediction software tools. Our findings suggest that a T-cell-mediated immune response could actually explain better prognosis of NPMc+ patients and provide a rationale for attempts to explore the importance of immunosuppressive mechanisms in this AML subgroup.

cancer biology↗