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Purpose: Acute respiratory distress syndrome (ARDS) is a prevalent illness in intensive care units. Extracellular vesicles and particles released from activated alveolar macrophages (AMs) assist in ARDS lung injury and the inflammatory process through mechanisms that are unclear. This study investigated the role of AM-derived secretory autophagosomes (SAPs) in lung injury and microRNA (MiR)-199a-3p-regulated inflammation associated with ARDS in vitro and in a murine model., Methods: The ARDS model in mouse was established by intratracheal LPS lipopolysaccharide (LPS) injection. The agomirs or antagomirs of MiR-199a-3p were injected into the caudal vein to figure out whether MiR-199a-3p could influence ARDS inflammation and lung injury, whereas the mimics or inhibitors of MiR-199a-3p, siRNA of Rab8a, or PAK4 inhibitor were transfected or applied to RAW264.7 cells to evaluate the mechanism of SAP release. Culture supernatants of RAW264.7 cells treated with LPS or bronchoalveolar lavage fluid from mice were collected for the isolation of SAPs., Results: We found that MiR-199a-3p was over-expressed in the lungs of ARDS mice. The MiR-199a-3p antagomir alleviated, whereas the MiR-199a-3p agomir exacerbated LPS-induced inflammation in mice by promoting AM-derived SAP secretion. In addition, MiR-199a-3p over-expression exacerbated LPS-induced ARDS via activating Rab8a, and Rab8a silencing significantly suppressed the promoting influence of the MiR-199a-3p mimic on SAP secretion. Furthermore, MiR-199a-3p mimic activated Rab8a by directly inhibiting PAK4 expression., Conclusion: The novel finding of this study is that MiR-199a-3p participated in the regulation of SAP secretion and the inflammatory process via targeting of PAK4/Rab8a, and is a potential therapeutic candidate for ARDS treatment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Xu, Liu, Dong, Yang and Liu.)

Immunotherapy has delivered impressive outcomes in combating tumor malignancies. However, insufficient immune infiltration and poor immunogenicity within the tumor microenvironment (TME) greatly compromise patient response rates. Here, a photoactivatable silencing extracellular vesicle (PASEV) is developed for sensitized cancer immunotherapy. p21-Activated kinase 4 (PAK4) is a newly identified tumor-cell-intrinsic "guard" associated with immune exclusion. Small interfering RNA against PAK4 (siPAK4) is designed and assembled with a photoactivatable reactive-oxygen-species (ROS)-sensitive polymer to form the nanocomplex core, which is further camouflaged by extracellular vesicles from M1 macrophages. The PASEV not only serves as a vehicle for packaging, tumor accumulation, and ROS-responsive release of siPAK4 for potent PAK4 silencing, but also primes the TME through immunogenic phototherapy, thereby simultaneously boosting intratumoral infiltration and immune activation. The combined immunotherapy elicits robust anticancer immunity, thus showing great promise for fighting cancers. This work opens a new avenue to simultaneously boost intratumoral infiltration and immune activation for sensitized cancer immunotherapy., (© 2022 Wiley-VCH GmbH.)

The actin cytoskeleton reorganization during sperm capacitation is essential for the occurrence of acrosomal exocytosis (AR) in several mammalian species. Here, we demonstrate that in mouse sperm, within the first minutes of exposure upon capacitating conditions, the activity of RHOA/C and RAC1 is essential for LIMK1 and COFILIN phosphorylation. However, we observed that the signaling pathway involving RAC1 and PAK4 is the main player in controlling actin polymerization in the sperm head necessary for the occurrence of AR. Moreover, we show that the transient phosphorylation of COFILIN is also influenced by the Slingshot family of protein phosphatases (SSH1). The activity of SSH1 is regulated by the dual action of two pathways. On one hand, RHOA/C and RAC1 activity promotes SSH1 phosphorylation (inactivation). On the other hand, the activating dephosphorylation is driven by okadaic acid-sensitive phosphatases. This regulatory mechanism is independent of the commonly observed activating mechanisms involving PP2B and emerges as a new finely tuned modulation that is, so far, exclusively observed in mouse sperm. However, persistent phosphorylation of COFILIN by SSH1 inhibition or okadaic acid did not altered actin polymerization and the AR. Altogether, our results highlight the role of small GTPases in modulating actin dynamics required for AR., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Abstract: MicroRNAs (miRNAs) are regulators of numerous cellular events; accumulating evidence indicates that miRNAs play a key role in a wide range of biological functions, such as cellular proliferation, differentiation, and apoptosis in cancer. Down-regulated expression of miR-145 has been reported in colon cancer tissues and cell lines. The molecular mechanisms underlying miR-145 and the regulation of colon carcinogenesis remain unclear. In this study, we investigated the levels of miR-145 in human colon cancer cells using qRT-PCR and found markedly decreased levels compared to normal epithelial cells. We identified PAK4 as a novel target of miR-145 using informatics screening. Additionally, we demonstrated that miR-145 targets a putative binding site in the 3′UTR of PAK4 and that its abundance is inversely associated with miR-145 expression in colon cancer cells; we confirmed this relationship using the luciferase reporter assay. Furthermore, restoration of miR-145 by mimics in SW620 cells significantly attenuated cell growth in vitro, in accordance with the inhibitory effects induced by siRNA mediated knockdown of PAK4. Taken together, these findings demonstrate that miR-145 downregulates P-ERK expression by targeting PAK4 and leads to inhibition of tumor growth. [Copyright &y& Elsevier]

The serine-threonine kinase PAK4 plays a pivotal role in cell proliferation, survival, and control of the cytoskeleton. Mice that lack Pak4 die in midgestation prior to embryonic day E11 from unidentified causes. Analysis of PAK4 protein levels demonstrated that it was highly expressed in the whole embryo and in the developing heart but became low in the hearts of adult mice. In this study we analyzed development of the heart in conventional and conditional Pak4 knockout mice and embryos. We found that in conventional Pak4 knockout mice cardiogenesis is strongly affected from early developmental stages and by E9.5, hearts of Pak4 embryos developed multiple profound deficits. Conditional deletion of Pak4 in the progenitors of the secondary heart field led to abnormal development of the outflow tract, in which the pulmonary artery had a smaller diameter, and the aortal wall was thinner than in wildtype mice. The conditional knockout mice also displayed the characteristic enlargement of the right ventricles and right atria. Pak4 embryos and cardiomyocytes in which PAK4 was depleted exhibited low levels of LIMK1, a protein that plays key roles in cytoskeletal organization. Knock down of PAK4 in cultured cardiomyocytes led to severely compromised sarcomeric structure and deficits in contraction. These results indicate that PAK4 functions, including control of actin dynamics, are necessary for normal development of the heart. [ABSTRACT FROM AUTHOR]

PAKs 4, 5 and 6 are members of the group B family of p21- activated kinases. Among this group, PAK4 has been most extensively studied. While it has essential roles in embryonic development, in adults high levels of PAK4 are frequently associated with cancer. PAK4 is overexpressed in a variety of cancers, and the Pak4 gene is amplified in some cancers. PAK4 overexpression is sufficient to cause oncogenic transformation in cells and in mouse models. The tight connection between PAK4 and cancer make it a promising diagnostic tool as well as a potential drug target. The group B PAKs also have important developmental functions. PAK4 is important for many early developmental processes, while PAK5 and PAK6 play roles in learning and memory in mice. This chapter provides an overview of the roles of the group B PAKs in cancer as well as development, and includes a discussion of PAK mediated signaling pathways and cellular functions. [ABSTRACT FROM AUTHOR]

Abstract: P21-activated kinase 4 (PAK4), a serine/threonine protein kinase, has involved in the regulation of cytoskeletal reorganization, cell proliferation, gene transcription, oncogenic transformation and cell invasion. Moreover, PAK4 overexpression, genetic amplification and mutations were detected in a variety of human tumors, which make it potential therapeutic target. In this paper we found that LCH-7749944, a novel and potent PAK4 inhibitor, effectively suppressed the proliferation of human gastric cancer cells through downregulation of PAK4/c-Src/EGFR/cyclin D1 pathway. In addition, LCH-7749944 significantly inhibited the migration and invasion of human gastric cancer cells in conjunction with concomitant blockage of PAK4/LIMK1/cofilin and PAK4/MEK-1/ERK1/2/MMP2 pathways. Interestingly, LCH-7749944 also inhibited the formation of filopodia and induced cell elongation in SGC7901 cells. Importantly, LCH-7749944 caused successful inhibition of EGFR activity due to its inhibitory effect on PAK4. Taken together, these results provided novel insights into the development of PAK4 inhibitor and potential therapeutic strategies for gastric cancer. [Copyright &y& Elsevier]

Abstract: The canonical Wnt/β-catenin signaling pathway plays a central role in development and cancer. The p21-activated kinase 4 (PAK4) involves in a wide range of cellular processes, including cytoskeletal reorganization, cell proliferation, gene transcription and oncogenic transformation. However, the cross talk between the Wnt and PAK4 signaling pathways is poorly understood. Here, we show that PAK4 is a nucleo-cytoplasmic shuttling protein, containing three nuclear export signals (NESs) and two nuclear localization signals (NLSs). PAK4 is exported by the chromosome region maintenance-1 (CRM-1)-dependent pathway and is imported into the nucleus in an importin α5-dependent manner. PAK4 interacts with and phosphorylates β-catenin on Ser675, which promotes the TCF/LEF transcriptional activity and stabilizes β-catenin through inhibition of its degradation. Moreover, nuclear import of PAK4 accompanies with the nuclear import of β-catenin and increased TCF/LEF transcriptional activity. We further demonstrated that PAK4 associates with the TCF/LEF transcriptional complex by ChIP assays. These findings uncover a novel role for PAK4 in modulating intracellular translocation and signaling of β-catenin. [Copyright &y& Elsevier]

Abstract: Phagocytosis of immunoglobulin G-opsonised particles takes place via Fcγ receptor ligation, leading to uptake through an actin-dependent mechanism. Myosin regulatory light chains have previously been reported to control contractility during uptake through the Fcγ receptor. In this study, we show that p21-activated kinase 4 contributes to Fcγ receptor-mediated uptake downstream of actin cup formation by regulating phosphorylation of myosin regulatory light chain. siRNA-mediated knockdown of p21-activated kinase 4 leads to reduced myosin regulatory light chain phosphorylation at Serine 19, with a corresponding reduction in phospho-myosin regulatory right chain localised to bound immunoglobulin G-opsonised red blood cells. p21-activated kinase 4 phosphorylates myosin light chain 9 at Serine 19 in vitro and RNA interference against myosin light chain 9 implicates this isoform, but not myosin light chain 12A or 12B, in Fcγ receptor-mediated uptake. Taken together, these data indicate that p21-activated kinase 4 regulates regulatory myosin light chain phosphorylation and myosin contractility during FcγR-mediated phagocytosis. [Copyright &y& Elsevier]

Abstract: The Pak4 serine/threonine kinase regulates cytoskeletal organization, and controls cell growth, proliferation, and survival. Deletion of Pak4 in mice results in embryonic lethality prior to embryonic day 11.5. Pak4 knockout embryos exhibit abnormalities in the nervous system, the heart, and other tissues. In this study a conditional deletion of Pak4 was generated in order to study the function of Pak4 in the development of the brain. Nervous system-specific conditional deletion of Pak4 was accomplished by crossing mice with a floxed allele of Pak4 with transgenic mice expressing Cre recombinase under the control of the nestin promoter. The conditional Pak4 knockout mice were born normally, but displayed growth retardation and died prematurely. The brains showed a dramatic decrease in proliferation of cortical and striatal neuronal progenitor cells. In vitro analyses revealed a reduced proliferation and self-renewing capacity of neural progenitor cells isolated from Pak4 knockout brains. The mice also exhibited cortical thinning, impaired neurogenesis and loss of neuroepithelial adherens junctions. By the time the mice died, by 4weeks after birth, severe hydrocephalus could also be seen. These results suggest that Pak4 plays a critical role in the regulation of neural progenitor cell proliferation and in establishing the foundation for development of the adult brain. [Copyright &y& Elsevier]

Abstract: Overexpression, genetic amplification and mutations of p21-activated kinase 4 (PAK4) were found in a variety of human cancers. PAK4 regulated actin cytoskeleton reorganization by phosphorylating LIMK1 and promoted cancer cells migration. Using yeast two-hybrid screen, we identified a novel PAK4 binding protein, DGCR6L, which was associated with cancer cell metastasis. We confirmed PAK4 binding to the DGCR6L specifically by GST pull-down assay, and found an association between endogenous PAK4 and DGCR6L by immunoprecipitation in mammalian cells. Furthermore, L115 of DGCR6L was the critical amino acid to bind 466–572aa in the very C-terminus of PAK4. Importantly, DGCR6L was required for the formation of PAK4-DGCR6L-β-actin complex. Overexpressed DGCR6L promoted migration of AGS cells mediated by PAK4, whereas knock-down of DGCR6L markedly inhibited the migration of those cells. Moreover, DGCR6L (L115V), which did not bind to PAK4, lost the ability to promote AGS cells migration. DGCR6L colocalized with PAK4 or F-actin and enhanced the phosphorylation level of LIMK1 and cofilin in a dose dependent manner. Taken together, our results demonstrated that DGCR6L, a novel PAK4 interacting protein, regulated PAK4-mediated migration of human gastric cancer cells via LIMK1. [Copyright &y& Elsevier]

Abstract: Pak4 is a member of the group B family of Pak serine/threonine kinases, originally identified as an effector protein for the Rho GTPase Cdc42. Pak4 knockout mice are embryonic lethal and do not survive past embryonic day 11.5. Previous work on Pak4 knockout mice has focused on studying the phenotype of the embryo. Abnormalities in the extraembryonic tissue, however, are common causes of early embryonic death in knockout mice. Extraembryonic tissue associated with the Pak4-null embryos was therefore examined. Abnormalities in both yolk sacs and placentas resulted when Pak4 was deleted. These included a lack of vasculature throughout the extraembryonic tissue, as well as an abnormally formed labyrinthine layer of the placenta. Interestingly, epiblast-specific deletion of Pak4 using a conditional knockout system, did not rescue the embryonic lethality. In fact, it did not even rescue the extraembryonic tissue defects. Our results suggest that the extraembryonic tissue abnormalities are secondary to defects that occur in response to epiblast abnormalities. More detailed analysis suggests that abnormalities in vasculature throughout the extraembryonic tissue and the epiblast may contribute to the death of the Pak4-null embryos. [Copyright &y& Elsevier]

Simple Summary: Non-Hodgkin's lymphomas (NHL) are cancers of the white blood cells. While some NHL subtypes, such as Diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL), grow and spread aggressively, others, like follicular lymphoma (FL), are indolent in nature. Irrespective of how fast they grow, all NHL subtypes can spread to other organs in the body if not treated. In this study, we have demonstrated that the targeted inhibition of p21-activated kinase 4 (PAK4) and nicotinamide phosphoribosyl transferase (NAMPT) in different NHL subtypes by a novel, orally bioavailable, dual inhibitor KPT-9274 can lead to energy depletion, inhibition of cell proliferation, and ultimately apoptosis. KPT-9274 treatment shows potent anti-tumor effects in DLBCL and MCL subcutaneous xenograft models and enhances mice survival in a systemic FL model. Therefore, this study demonstrates the potential of targeting PAK4 and NAMPT by a small molecule inhibitor KPT-9274 for NHL therapy. Diffuse large B-cell lymphoma (DLBCL), grade 3b follicular lymphoma (FL), and mantle cell lymphoma (MCL) are aggressive non-Hodgkin's lymphomas (NHL). Cure rates are suboptimal and novel treatment strategies are needed to improve outcomes. Here, we show that p21-activated kinase 4 (PAK4) and nicotinamide phosphoribosyl transferase (NAMPT) is critical for lymphoma subsistence. Dual targeting of PAK4-NAMPT by the Phase I small molecule KPT-9274 suppressed cell proliferation in DLBCL, FL, and MCL. Growth inhibition was concurrent with apoptosis induction alongside activation of pro-apoptotic proteins and reduced pro-survival markers. We observed NAD suppression, ATP reduction, and consequent cellular metabolic collapse in lymphoma cells due to KPT-9274 treatment. KPT-9274 in combination with standard-of-care chemotherapeutics led to superior inhibition of cell proliferation. In vivo, KPT-9274 could markedly suppress the growth of WSU-DLCL2 (DLBCL), Z-138, and JeKo-1 (MCL) sub-cutaneous xenografts, and a remarkable increase in host life span was shown, with a 50% cure of a systemic WSU-FSCCL (FL) model. Residual tumor analysis confirmed a reduction in total and phosphorylated PAK4 and activation of the pro-apoptotic cascade. This study, using various preclinical experimental models, demonstrates the therapeutic potential of targeting PAK4-NAMPT in DLBCL, FL, and MCL. The orally bioavailable, safe, and efficacious PAK4-NAMPT dual inhibitor KPT-9274 warrants further clinical investigation. [ABSTRACT FROM AUTHOR]

Pancreas ductal adenocarcinoma (PDAC) is a highly lethal cancer that typically presents as advanced. unresectable disease. This invasive tendency, coupled with intrinsic resistance to standard therapies and genome instability, are major contributors to poor long-term survival. The genetic elements governing the invasive propensity of PDAC have not been well elucidated. Here, in the course of validating resident genes in highly recurrent and focal amplifications in PDAC, we have identified Rio Kinase 3 (RIOK3) as an amplified gene that alters cytoskeletal architecture as well as promotes pancreatic ductal cell migration and invasion. We determined that RIOK3 promotes its invasive activities through activation of the small G protein, Rac. This genomic and functional link to Rac signaling prompted a genome wide survey of other components of the Rho family network, revealing p21 Activated Kinase 4 (PAK4) as another amplified gene in PDAC tumors and cell lines. Like RIOK3, PAK4 promotes pancreas ductal cell motility and invasion. Together, the genomic and functional profiles establish the Rho family GTP-binding proteins as integral to the hallmark invasive nature of this lethal disease. [ABSTRACT FROM AUTHOR]

Abstract: Hepatocyte growth factor (HGF) is associated with tumour progression and increases the invasiveness of prostate carcinoma cells. Cell migration and invasion requires reorganisation of the actin cytoskeleton; processes mediated by the Rho family GTPases. p21 activated kinase 4 (PAK4), an effector of the Rho family protein Cdc42, is activated downstream of HGF. We report here the novel finding that in prostate cancer cells PAK4 binds to and phosphorylates LIM kinase 1 (LIMK1) in an HGF-dependent manner. We show for the first time that variations in the level of PAK4 expression change the level of cofilin phosphorylation in cells, a change we correlate with LIMK1 activity, cell morphology and migratory behaviour. We identify for the first time a direct and localised interaction between PAK4 and LIMK1 within cells using FRET: FLIM. Moreover we show here that HGF mediates this interaction which is concentrated in small foci at the cell periphery. PAK4 and LIMK1 act synergistically to increase cell migration speed, whilst a reduction in PAK4 expression decreases cell speed. It is well established that unphosphorylated (active) cofilin is a required to drive cell migration. Our results support a model whereby HGF-stimulated cell migration also requires a cofilin phosphorylation step that is mediated by PAK4. [Copyright &y& Elsevier]

Abstract: Mucin, a major component of mucus, plays a critical role in host mucosal defense response by participating in mucociliary clearance. However, if overproduced, overproduced mucus leads to airway mucus obstruction and conductive hearing loss. Despite extensive studies that focus on investigating how MUC5AC mucin is regulated by one inducer at a time, how MUC5AC is synergistically regulated by multiple factors remains unknown. Here we provide direct evidence for the first time that bacterial pathogen NTHi and human growth factor EGF synergize with each other to potently up-regulate MUC5AC mucin transcription. Moreover, activation of both p38 and ERK is required for synergistic induction of MUC5AC by NTHi and EGF. Finally, PAK2 and PAK4 are differentially involved in this synergistic induction of MUC5AC by acting upstream of p38 and ERK. Our studies bring novel insights into our understanding of synergistic regulation of MUC5AC mucin by both pathological and physiological inducers. [Copyright &y& Elsevier]

Precise spatial and temporal regulation of Rho GTPases is required in controlling F-actin-based changes in cell morphology. The molecular mechanisms through which microtubules (MTs) modulate the activity of RhoGTPases and regulate the actin cytoskeleton are unclear. Here we show that p21-activated-kinase 4 (PAK4) mediates morphological changes through its association with the Rho-family guanine nucleotide exchange factor (GEF), GEF-H1. We show that this association is dependent upon a novel GEF-H1 interaction domain (GID) within PAK4. Further, we show that PAK4-mediated phosphorylation of Ser810 acts as a switch to block GEF-H1-dependent stress fiber formation while promoting the formation of lamellipodia in NIH-3T3 cells. We found that the endogenous PAK4-GEF-H1 complex associates with MTs and that PAK4 phosphorylation of MT-bound GEF-H1 releases it into the cytoplasm of NIH-3T3 cells, which coincides with the dissolution of stress fibers. Our observations propose a novel role for PAK4 in GEF-H1-dependent crosstalk between MTs and the actin cytoskeleton. [ABSTRACT FROM AUTHOR]

Slingshot (SSH) phosphatases and LIM kinases (LIMK) regulate actin dynamics via a reversible phosphorylation (inactivation) of serine 3 in actin-depolymerizing factor (ADF) and cofilin. Here we demonstrate that a multi-protein complex consisting of SSH-1L, LIMK1, actin, and the scaffolding protein, 14-3-3?, is involved, along with the kinase, PAK4, in the regulation of ADF/cofilin activity. Endogenous LIMK1 and SSH-1L interact in vitro and co-localize in vivo, and this interaction results in dephosphorylation and downregulation of LIMK1 activity. We also show that the phosphatase activity of purified SSH-1L is F-actin dependent and is negatively regulated via phosphorylation by PAK4. 14-3-3?binds to phosphorylated slingshot, decreases the amount of slingshot that co-sediments with F-actin, but does not alter slingshot activity. Here we define a novel ADF/cofilin phosphoregulatory complex and suggest a new mechanism for the regulation of ADF/cofilin activity in mediating changes to the actin cytoskeleton. [ABSTRACT FROM AUTHOR]

• PAK4 overexpression is a predictive biomarker for tumor development and resistance to cancer treatments. • PAK4 is also a regulator of the anti-tumor immune response. • PAK4 and PD-L1 over-expression is mediated by the same signalling pathways (mainly PI3K/AKT, and Wnt/β-catenin) • Targeting PAK4 would regulate immune cells response to anti-PD-1/anti-PD-L1 immune checkpoint inhibition therapy. The p21 activated kinases (PAKs) are known to play a role in the regulation of cell morphology and functions. Among the various members of PAKs family, only the PAK4 protein has been shown to be overexpressed in cancer cells and its upregulation was associated with tumor development. Indeed, several studies have shown that PAK4 overexpression is implicated in carcinogenesis by different mechanisms including promotion of cell proliferation, invasion and migration, protection of cells from apoptosis, stimulation of the tumor-specific anchorage-independent cell growth and regulation of the cytoskeletal organisation and adhesion. Moreover, high PAK4 protein levels have been observed in several solid tumors and have been shown able to enhance cancer cell resistance to many treatments especially chemotherapy. Interestingly, it has been recently demonstrated that PAK4 downregulation can inhibit the PD-1/PD-L1 immune regulatory pathway. Taken together, these findings not only implicate PAK4 in oncogenic transformation and in prediction of tumor response to treatment but also suggest its role as an attractive target for immunotherapy. In the current review we will summarize the different mechanisms of PAK4 implication in tumor development, describe its role as a regulator of the immune response and as a potential novel target for cancer immunotherapy. [ABSTRACT FROM AUTHOR]

Over 95% of Pancreatic ductal adenocarcinomas (PDA) carry mutations in the oncogene KRas which has been proven to be a difficult drug target. P21-activated kinase 4 (PAK4), acts downstream of KRas, and is overexpressed in PDA contributing to its growth and chemoresistance, and thus becomes an attractive therapeutic target. We have developed a new PAK4 inhibitor, PAKib and tested its effect on pancreatic cancer (PC) cell growth in vitro and in a syngeneic mouse model of PC. PAKib suppressed PC cell growth by inducing cell death and cycle arrest. PAKib inhibited PC growth and enhanced the inhibition by gemcitabine of PC in cell culture and in PC mouse model. PAKib acted through multiple signaling pathways involved in cell cycle checkpoints, apoptosis, cell junction, and focal adhesion. These proof-of-concept studies demonstrated the anti-cancer effect of PAKib alone and in combination with gemcitabine and warrant a further clinical investigation., (Copyright © 2021. Published by Elsevier Inc.)

Diffuse large B-cell lymphoma (DLBCL), grade 3b follicular lymphoma (FL), and mantle cell lymphoma (MCL) are aggressive non-Hodgkin's lymphomas (NHL). Cure rates are suboptimal and novel treatment strategies are needed to improve outcomes. Here, we show that p21-activated kinase 4 (PAK4) and nicotinamide phosphoribosyl transferase (NAMPT) is critical for lymphoma subsistence. Dual targeting of PAK4-NAMPT by the Phase I small molecule KPT-9274 suppressed cell proliferation in DLBCL, FL, and MCL. Growth inhibition was concurrent with apoptosis induction alongside activation of pro-apoptotic proteins and reduced pro-survival markers. We observed NAD suppression, ATP reduction, and consequent cellular metabolic collapse in lymphoma cells due to KPT-9274 treatment. KPT-9274 in combination with standard-of-care chemotherapeutics led to superior inhibition of cell proliferation. In vivo, KPT-9274 could markedly suppress the growth of WSU-DLCL2 (DLBCL), Z-138, and JeKo-1 (MCL) sub-cutaneous xenografts, and a remarkable increase in host life span was shown, with a 50% cure of a systemic WSU-FSCCL (FL) model. Residual tumor analysis confirmed a reduction in total and phosphorylated PAK4 and activation of the pro-apoptotic cascade. This study, using various preclinical experimental models, demonstrates the therapeutic potential of targeting PAK4-NAMPT in DLBCL, FL, and MCL. The orally bioavailable, safe, and efficacious PAK4-NAMPT dual inhibitor KPT-9274 warrants further clinical investigation.

With a dismal survival rate, pancreatic cancer (PC) remains one of the most aggressive and devastating malignancies, predominantly due to the absence of a valid biomarker for diagnosis and limited therapeutic options for advanced diseases. Exosomes (Exo) as cell-derived vesicles, are widely used as natural nanocarriers for drug delivery. P21-activated kinase 4 (PAK4) is oncogenic when overexpressed, promoting cell survival, migration and anchorage-independent growth. Herein we validated PAK4 as a therapeutic target in an in vivo PC tumour mouse model using Exo-mediated RNAi following intra-tumoural administration. PC derived Exo were firstly isolated by ultracentrifugation on sucrose cushion and characterised for their surface marker expression, size, number, purity and morphology. SiRNA was encapsulated into Exo via electroporation and dual uptake of Exo and siRNA was investigated by flow cytometry and confocal microscopy. In vitro siPAK4 silencing in PC cells following uptake was assessed by flow cytometry, western blotting, and in vitro scratch assay. In vivo efficacy (tumour growth delay and mouse survival) of siPAK4 was evaluated in PC bearing NSG mouse model. Ex vivo tumours were examined using Haematoxylin and eosin (H&E) staining and immunohistochemistry. Results showed high quality PC-derived PANC-1 Exo were obtained. SiRNA was incorporated in Exo with 16.5% encapsulation efficiency. In vitro imaging confirmed Exo and siRNA co-localisation in cells. PAK4 knockdown was successful with 30 nM Exo-siPAK4 at 24 h post incubation in vitro. Intra-tumoural administration of Exo-siPAK4 (0.03 mg/kg siPAK4 and 6.1 × 1011 Exo, each dose, two doses) reduced PC tumour growth in vivo and enhanced mice survival (p < 0.001), with minimal toxicity observed compared to polyethylenimine (PEI) used as a commercial transfection reagent. H&E staining of tumours showed significant tissue apoptosis in siPAK4 treated groups. PAK4 knockdown prolongs survival of PC-bearing mice suggesting its potential as a new therapeutic target for PC. PANC-1 Exo demonstrated comparable efficacy but safer profile than PEI as in vivo RNAi transfection reagent. Image 1 [ABSTRACT FROM AUTHOR]

Parkinson's disease (PD) is characterized by a progressive loss of dopamine-producing neurons in the midbrain, which results in decreased dopamine levels accompanied by movement symptoms. Oral administration of l-3,4-dihydroxyphenylalanine (L-dopa), the precursor of dopamine, provides initial symptomatic relief, but abnormal involuntary movements develop later. A deeper understanding of the regulatory mechanisms underlying dopamine homeostasis is thus critically needed for the development of a successful treatment. Here, we show that p21-activated kinase 4 (PAK4) controls dopamine levels. Constitutively active PAK4 (caPAK4) stimulated transcription of tyrosine hydroxylase (TH) via the cAMP response element-binding protein (CREB) transcription factor. Moreover, caPAK4 increased the catalytic activity of TH through its phosphorylation of S 40 , which is essential for TH activation. Consistent with this result, in human midbrain tissues, we observed a strong correlation between phosphorylated PAK4 S474 , which represents PAK4 activity, and phosphorylated TH S40 , which reflects their enzymatic activity. Our findings suggest that targeting the PAK4 signaling pathways to restore dopamine levels may provide a new therapeutic approach in PD.

Parkinson's disease (PD) provokes bradykinesia, resting tremor, rigidity and postural instability, and also non-motor symptoms such as depression, anxiety, sleep and cognitive impairments. Similar phenotypes can be induced in Drosophila melanogaster through modification of PD-relevant genes or the administration of PD-inducing toxins. Recent studies correlated deregulation of human p21-activated kinase 4 (PAK4) with PD, leaving open the question of a causative relationship of mutations in this gene for manifestation of PD symptoms. To determine whether flies lacking the PAK4 homolog Mushroom bodies tiny (Mbt) show PD-like phenotypes, we tested for a variety of PD criteria. Here, we demonstrate that mbt mutant flies show PD-like phenotypes including age-dependent movement deficits, reduced life expectancy and fragmented sleep. They also react to a stressful situation with higher immobility, indicating an influence of Mbt on emotional behavior. Loss of Mbt function has a negative effect on the number of dopaminergic protocerebral anterior medial (PAM) neurons, most likely caused by a proliferation defect of neural progenitors. The age-dependent movement deficits are not accompanied by a corresponding further loss of PAM neurons. Previous studies highlighted the importance of a small PAM subgroup for age-dependent PD motor impairments. We show that impaired motor skills are caused by a lack of Mbt in this PAM subgroup. In addition, a broader re-expression of Mbt in PAM neurons improves life expectancy. Conversely, selective Mbt knockout in the same cells shortens lifespan. We conclude that mutations in Mbt/PAK4 can play a causative role in the development of PD phenotypes., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

FPR1, FPR2, and FPR3 are members of Formyl Peptides Receptors (FPRs) family belonging to the GPCR superfamily. FPR2 is a low affinity receptor for formyl peptides and it is considered the most promiscuous member of this family. Intracellular signaling cascades triggered by FPRs include the activation of different protein kinases and phosphatase, as well as tyrosine kinase receptors transactivation. Protein kinases and phosphatases act coordinately and any impairment of their activation or regulation represents one of the most common causes of several human diseases. Several phospho-sites has been identified in protein kinases and phosphatases, whose role may be to expand the repertoire of molecular mechanisms of regulation or may be necessary for fine-tuning of switch properties. We previously performed a phospho-proteomic analysis in FPR2-stimulated cells that revealed, among other things, not yet identified phospho-sites on six protein kinases and one protein phosphatase. Herein, we discuss on the selective phosphorylation of Serine/Threonine-protein kinase N2, Serine/Threonine-protein kinase PRP4 homolog, Serine/Threonine-protein kinase MARK2, Serine/Threonine-protein kinase PAK4, Serine/Threonine-protein kinase 10, Dual specificity mitogen-activated protein kinase kinase 2, and Protein phosphatase 1 regulatory subunit 14A, triggered by FPR2 stimulation. We also describe the putative FPR2-dependent signaling cascades upstream to these specific phospho-sites. [ABSTRACT FROM AUTHOR]

p21-Activated kinase 4 (PAK4), a serine/threonine kinase, is purported to localize to podosomes: transient adhesive structures that degrade the extracellular matrix to facilitate rapid myeloid cell migration. We find that treatment of transforming growth factor β (TGF-β)-differentiated monocytic (THP-1) cells with a PAK4-targeted inhibitor significantly reduces podosome formation and induces the formation of focal adhesions. This switch in adhesions confers a diminution of matrix degradation and reduced cell migration. Furthermore, reduced PAK4 expression causes a significant reduction in podosome number that cannot be rescued by kinase-dead PAK4, supporting a kinase-dependent role. Concomitant with PAK4 depletion, phosphorylation of Akt is perturbed, whereas a specific phospho-Akt signal is detected within the podosomes. Using superresolution analysis, we find that PAK4 specifically localizes in the podosome ring, nearer to the actin core than other ring proteins. We propose PAK4 kinase activity intersects with the Akt pathway at the podosome ring:core interface to drive regulation of macrophage podosome turnover. • PAK4 kinase activity is crucial to macrophage podosome dynamics • Podosome loss due to PAK4 suppression reduces migration speed and degradation • PAK4 localizes to the podosome ring where it interacts with vinculin and paxillin The differential role of PAK family proteins in podosome function has not been previously evaluated. Foxall et al. demonstrate that PAK4 rather than PAK1 plays a pivotal role and use super resolution microscopy to position PAK4 at the podosome ring:core interface. [ABSTRACT FROM AUTHOR]

Pancreatic neuroendocrine tumors (PNET) remain an unmet clinical need. In this study, we show that targeting both nicotinamide phosphoribosyltransferase (NAMPT) and p21-activated kinase 4 (PAK4) could become a synthetic lethal strategy for PNET. The expression of PAK4 and NAMPT was found to be higher in PNET tissue compared to normal cells. PAK4-NAMPT dual RNAi suppressed proliferation of PNET cell lines. Treatment with KPT-9274 (currently in a Phase I trial or analogs, PF3758309 (the PAK4 selective inhibitor) or FK866 (the NAMPT inhibitor)) suppressed the growth of PNET cell lines and synergized with the mammalian target of rapamycin (mTOR) inhibitors everolimus and INK-128. Molecular analysis of the combination treatment showed down-regulation of known everolimus resistance drivers. KPT-9274 suppressed NAD pool and ATP levels in PNET cell lines. Metabolomic profiling showed a statistically significant alteration in cellular energetic pathways. KPT-9274 given orally at 150 mg/kg 5 days/week for 4 weeks dramatically reduced PNET sub-cutaneous tumor growth. Residual tumor analysis demonstrated target engagement in vivo and recapitulated in vitro results. Our investigations demonstrate that PAK4 and NAMPT are two viable therapeutic targets in the difficult to treat PNET that warrant further clinical investigation. [ABSTRACT FROM AUTHOR]

Aim: To investigate whether kaempferol exhibits a protective effect on high glucose-induced epithelial-mesenchymal transition (EMT) by mediating the  PVT1 / miR-214 and PAK4/β-catenin pathways in SRA01/04 cells. Methods & methods: qRT-PCR and western blot assays were used for gene and protein determination, and migration and invasion assays were conducted. A coimmunoprecipitation assay was used for determining protein interactions. Results: High glucose effectively upregulated PVT1 expression, downregulated miR-214 expression and promoted cell migration and invasion. Kaempferol attenuated high glucose-induced EMT by increasing PVT1 expression and decreasing miR-214 expression. PAK4 was identified as a direct target of miR-214 . PAK4 overexpression could rescue the effects of PVT1 deficiency on SRA01/04 cells. Conclusion: Kaempferol ameliorated the regulatory effects of PVT1 / miR-214 on high glucose-induced EMT through PAK4/β-catenin in SRA01/04 cells.

Pulmonary arterial hypertension (PAH) is a progressive pulmonary vascular disease associated with dysfunction of pulmonary artery endothelial cells and pulmonary artery smooth muscle cells (PASMCs). To explore the potential mechanism of miR-193-3p in pulmonary arterial hypertension, human PASMCs and rats were respectively stimulated by hypoxia and monocrotaline to establish PAH model in vivo and in vitro. The expressions of miR-193-3p and p21-activated protein kinase 4 (PAK4) in the lung samples of PAH patients and paired healthy samples from the healthy subjects in PHA cells and rats were detected by quantitative reverse transcriptase-PCR. Morphological changes in lung tissues were determined using hematoxylin and eosin staining. Right ventricular systolic pressure (RVSP) and ratio of right ventricle to left ventricle plus septum (RV/LV p S) were measured. The binding relationship between miR-193-3p and PAK4 was analyzed by TargetScan and verified by luciferase reporter assay. Cell viability, apoptosis, and migration were detected by 3-(4, 5-Dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide (MTT) flow cytometry, and wound-healing assays, respectively. The protein expressions of PAK4, proliferating cell nuclear antigen (PCNA), P21, p-AKT, and AKT in vivo or in vitro were determined by Western blot. In this study, we found that in pulmonary arterial hypertension, miR-193-3p expression was downregulated and PAK4 expression was up-regulated. MiR-193-3p directly targeted PAK4 and negatively regulated its expression. Hypoxia condition promoted cell proliferation, migration, and inhibited apoptosis accompanied with increased expressions of PCNA and p-AKT/AKT and decreased expression of P21 in PASMCs. MiR-193-3p overexpression attenuated the effects of hypoxia on PASMCs via downregulating PAK4. Monocrotaline treatment increased p-AKT/AKT and decreased P21 expression and caused pulmonary vascular remodeling in the model rats. MiR-193-3p overexpression attenuated pulmonary vascular remodeling, decreased p-AKT/AKT, and increased P21 levels via downregulating PAK4 in monocrotaline-induced rats. The results in this study demonstrated that upregulation of miR-193-3p reduced cell proliferation, migration, and apoptosis of PAH in vitro and pulmonary vascular remodeling in PAH in vivo through downregulating PAK4., Competing Interests: Conflict of interest: The author(s) declare that there is no conflict of interest., (© The Author(s) 2020.)

Esophageal squamous cell carcinoma (ESCC) is one of the most common malignant tumors of the digestive tract in humans. Several studies have indicated that PAK4 is associated with the risk of ESCC and may be a potential druggable kinase for ESCC treatment. However, the underlying mechanism remains largely unknown. The aim of our study is to identify the functional role of PAK4 in ESCC. To determine the expression of PAK4 in ESCC, Western blot analysis and immunohistochemistry were performed, and the results showed that PAK4 is significantly upregulated in ESCC tissues and cell lines compared with normal controls and normal esophageal epithelial cell line. To further investigate the role of PAK4 in ESCC, cell viability assays, anchorage-independent cell growth assays, wound healing assays, cellular invasion assays, in vivo xenograft mouse models, and metastasis assays were conducted, and the results showed that PAK4 can significantly facilitate ESCC proliferation and metastasis in vitro and in vivo. To determine the potential target of PAK4 in ESCC progression, a pull-down assay was performed, and the results showed that LASP1 may be a potential target of PAK4. An immunoprecipitation assay and confocal microscopy analysis confirmed that PAK4 can bind to and colocalize with LASP1 in vitro and in cells. Notably, rescue experiments further illustrated the mechanistic network of PAK4/LASP1. Our research reveals the oncogenic roles of PAK4 in ESCC and preliminarily elucidates the mechanistic network of PAK4/LASP1 in ESCC., (© 2020 The Authors. Molecular Carcinogenesis published by Wiley Periodicals LLC.)