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

Abstract

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 PAK1Δ15. PAK1Δ15 and PAK2 transcript levels correlated with surface density of integrins β1 and αVβ3. 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.

PAK1 and PAK2 in cell metabolism regulation

“…Although FRAX597 is less specific and might inhibit other kinases in addition to PAK group I (Licciulli et al, 2013), it had only a limited effect on the metabolic rates within 1 h (Figs 2B and 3B). PAK kinase activity is rapidly and largely inhibited by FRAX597 at the doses used (Grebenova et al, 2019;Kuželová et al, 2021), and we thus suggest that a nonkinase PAK function is essential for cell metabolism. Possible synergy between targeting PAK kinase activity and nonspecific effects, which might have explained the observed strong effect of IPA-3, was ruled out by analysis of combined treatment with FRAX597 and PIR3.5 (Supplementary Fig.…”

“…Accordingly, both the OCR and ECAR were markedly reduced by 20 µM IPA-3 in all leukemia cell lines tested (Fig. 3A), except for MOLM-7, which has a very low amount of PAK1 and a high PAK2 level (Kuželová et al, 2021). Increasing the IPA-3 dose to 50 µM was sufficient to achieve a marked decrease in the OCR and ECAR also in the latter cell line (data not shown).…”

“…Leukemia cells were reported to be very sensitive to PAK inhibition (Kuzelova et al, 2014;Kuželová et al, 2021;Pandolfi et al, 2015), and treatment with IPA-3 induces cell death in the majority of leukemia cell lines. Accordingly, both OCR and ECAR were markedly reduced by 20 µM IPA-3 in all leukemia cell lines tested (Fig.…”

“…The adhesion structures of leukemia cells are more dynamic than those of adherent cells and they are usually not tightly connected to the actin cytoskeleton, but PAKs are still involved in the regulation of leukemia cell adhesion to the ECM (Kuželová et al, 2021). In addition, PAKs are indispensable for leukemia cell survival, as their inhibition leads to rapid cell death (Kuzelova, Grebenova, Holoubek, Roselova, & Obr, 2014;Kuželová et al, 2021;Pandolfi et al, 2015). Similar to solid tumors, leukemia cells often display a preference for aerobic glycolysis (Jiang & Nakada, 2016), and the metabolic phenotype has been proposed as a prognostic parameter for clinical outcome in acute myeloid leukemia (Chen, W. L. et al, 2014;Herst, Howman, Neeson, Berridge, & Ritchie, 2011).…”

“…Nevertheless, they interact with stromal cells, as well as with the extracellular matrix (ECM) of the bone marrow, and these interactions have a profound impact on their behavior. The adhesion structures of leukemia cells are more dynamic than those of adherent cells and they are usually not tightly connected to the actin cytoskeleton, but PAKs are still involved in the regulation of leukemia cell adhesion to the ECM (Kuželová et al, 2021). In addition, PAKs are indispensable for leukemia cell survival, as their inhibition leads to rapid cell death (Kuzelova, Grebenova, Holoubek, Roselova, & Obr, 2014;Kuželová et al, 2021;Pandolfi et al, 2015).…”