Biology subjects

Obr, A.

Publications and source records attributed to Obr, A..

4 recordsLinked to original sources

bioRxiv2021

Inhibition of Casein Kinase 2 induces Cell Death in Chronic Myelogenous Leukemia Cells with Different Mechanisms of Resistance to Tyrosine Kinase Inhibitors

Chronic myelogenous leukemia (CML) is a myeloproliferative disease characterized by the presence of a BCR-ABL oncogene. Despite the high performance of treatment with tyrosine kinase inhibitors (TKI), about 30 % of patients develop resistance to the therapy. To improve the outcomes, identification of new targets of treatment is needed. Here, we explored the Casein Kinase 2 (CK2) as a potential target for CML therapy. Previously, we detected increased phosphorylation of HSP90{beta} Serine 226 in patients non-responding to TKIs imatinib and dasatinib. This site is known to be phosphorylated by CK2, which was also linked to CML resistance to imatinib. In the present work, we established six novel imatinib- and dasatinib-resistant CML cell lines, all of which had increased CK2 activation. A CK2 inhibitor, CX-4945, induced cell death of CML cells in both parental and resistant cell lines. In some cases, CK2 inhibition also potentiated the effects of TKI on the cell metabolic activity. No effects of CK2 inhibition were observed in normal mononuclear blood cells from healthy donors and BCR-ABL negative HL60 cell line. Our data indicate that CK2 kinase supports CML cell viability even in cells with different mechanisms of resistance to TKI, and thus represents a potential target for treatment.

cancer biology↗

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↗

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↗

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↗