Nuplazid suppresses esophageal squamous cell carcinoma growth in vitro and in vivo by targeting PAK4

Background Due to the high recurrence and low 5-year survival rates of esophageal squamous cell carcinoma (ESCC) after treatment, the discovery of novel drugs for recurrence chemoprevention is of particular importance. Methods We screened the FDA-approved drug library and found that Nuplazid, an atypical antipsychotic that acts as an effective 5-HT 2 A receptor inverse agonist, could potentially exert anticancer effects in vitro and in vivo on ESCC. Results Pull-down results indicated that Nuplazid binds with p21-activated kinase 4 (PAK4), and a kinase assay showed that Nuplazid strongly suppressed PAK4 kinase activity. Moreover, Nuplazid exhibited inhibitory effects on ESCC in vivo. Conclusions Our findings indicate that Nuplazid can suppress ESCC progression through targeting PAK4.

Effect of Gastrodin on Cognitive Dysfunction in Diabetes by Inhibiting PAK2 Phosphorylation

Diabetes and cognitive dysfunction are highly prevalent disorders, while the underlying mechanism is still elusive. The effects of Gastrodin on central nervous system have been emphasized recently. In this study, we aim to explore the potential mechanism leading to cognitive dysfunction in diabetes and the therapeutic effect of Gastrodin. Diabetes was induced by a single injection of streptozotocin. RNA sequencing technique was used to identify the potential factors involved. Western blot and immunofluorescence were applied to detect the protein expression. Our results have shown that spatial learning was impaired and hippocampal pyramidal neurons were damaged in diabetic rats, which could be ameliorated by Gastrodin intervention. Transcriptional analysis identified differential expression genes, which were confirmed by qPCR and western blot. Furthermore, p21 activated kinase 2 (PAK2) was selected and its inhibitor could promote the survival of primary hippocampal neurons. It suggested that PAK2 pathway may be involved in cognitive dysfunction in diabetes and a therapeutic target for Gastrodin intervention.

EXTH-37. PAK4 INHIBITION REPROGRAMS VASCULAR MICROENVIRONMENT AND IMPROVES CAR T IMMUNOTHERAPY IN GLIOBLASTOMA

Malignant solid tumors are characterized by aberrant vascularity that inhibits T cell adhesion to vasculature and impedes T cell delivery into the tumors, contributing to glioblastoma (GBM) resistance to T cell-based immunotherapy such as adoptive chimeric antigen receptor (CAR)–T transfer. Vascular abnormality is driven by pro-angiogenic pathway activation and genetic reprogramming in tumor endothelial cells (ECs). Here, our kinome-wide functional screening of mesenchymal-like transcriptional activation in human GBM-derived ECs identifies p21-activated kinase 4 (PAK4) as a selective regulator of genetic programming and aberrant vascularization in tumor. Genetic ablation of PAK4 reprograms EC transcriptome and inhibits mesenchymal-like transformation. Interestingly, deficiency of PAK4 induces adhesion protein re-expression in tumor ECs, reduces vascular abnormalities in tumors, improves T cell infiltration into the tumors, and inhibits GBM growth in mice. Moreover, pharmacological PAK4 inhibition normalizes the tumor vascular microenvironment and sensitizes GBM to Egfrviii CAR–T cell immunotherapy. Finally, we reveal a MEF2D/ZEB1- and SLUG-mediated mechanism by which PAK4 reprograms the EC transcriptome and downregulates claudin-14 and VCAM-1 expression, enhancing vessel permeability and reducing T cell adhesion to the endothelium. Thus, targeting PAK4-mediated EC plasticity may offer exciting opportunities to recondition the vascular microenvironment and strengthen cancer immunotherapy.

737 Inhibition of P21-activated kinase 4 (PAK4) reverts immune exclusion and restores anti-tumor immunity in the tumor microenvironment

BackgroundP21-activated kinase 4 (PAK4) is a serine/threonine protein kinase that is mostly expressed in tumor and stroma cells. PAK4 activates tumor WNT/β-catenin pathway and regulates cellular morphology, motility, EMT, cell proliferation and survival. Recent studies also showed that PAK4 can actively exclude T cells from tumors, suggesting that therapeutic inhibition of PAK4 can increase T cell infiltration in tumor microenvironment and overcome resistance to checkpoint inhibitor immunotherapy.1MethodsWe generated PAK4 knockout (KO) clones in human and mouse tumor cells to validate its biology in vitro and in vivo. We also performed pharmacological evaluation of PAK4 inhibition using Pfizer compounds (referred to as 'PAK4i compounds' below) for their potential tumor-intrinsic and immune-regulatory roles.ResultsNanostring, qPCR and RNASeq analysis showed that PAK4 depletion led to increase of cytokine expression in tumor, including conventional dendritic cell (cDC)- recruiting chemokine CCL4, and type I IFN / ISG pathway genes that are associated with MHC upregulation such as CXCL10. In addition, PAK4 KO sensitizes B16F10 tumors to anti-PD-1 treatment and increases infiltration of cDC and T cells in the tumor microenvironment.We also showed that small molecule PAK4i compounds induced more potent cancer cell growth inhibition over treated normal PBMCs. PAK4i compounds also increased immune-activating and decreased immune exclusion genes in B16F10 cells and tumor explants in vitro. Although PAK4 target engagement is demonstrated by CETSA assay, the compound potency on modulating PAK4 downstream Wnt/ β-catenin pathway is low, suggesting that the aforementioned phenotypic changes induced by PAK4i compounds may be partially attributed to other off-target effects.ConclusionsCollectively, our data suggests that genetic depletion or pharmacological inhibition of PAK4 may induce immune-activating cytokine production in tumor cells, revert immune cell exclusion in tumor microenvironment, and synergize with checkpoint blockade therapies. However, further optimization on these PAK4i compounds is needed to improve its specificity on modulating PAK4 enzyme activities.ReferenceAbril-Rodriguez G, Torrejon DY, Liu W, Zaretsky JM, Nowicki TS, Tsoi J, Puig-Saus C, Baselga-Carretero I, Medina E, Quist MJ, Garcia AJ, Senapedis W, Baloglu E, Kalbasi A, Cheung-Lau G, Berent-Maoz B, Comin-Anduix B, Hu-Lieskovan S, CWang CY, Grasso CS & Ribas A. PAK4 inhibition improves PD-1 blockade immunotherapy. Nat Cancer 2020;1:46–58.Ethics ApprovalAll animal studies were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee of Pfizer. Approved protocol # LAJ-2019-01347

Proximity proteomics identifies PAK4 as a component of Afadin–Nectin junctions

Human PAK4 is an ubiquitously expressed p21-activated kinase which acts downstream of Cdc42. Since PAK4 is enriched in cell-cell junctions, we probed the local protein environment around the kinase with a view to understanding its location and substrates. We report that U2OS cells expressing PAK4-BirA-GFP identify a subset of 27 PAK4-proximal proteins that are primarily cell-cell junction components. Afadin/AF6 showed the highest relative biotin labelling and links to the nectin family of homophilic junctional proteins. Reciprocally >50% of the PAK4-proximal proteins were identified by Afadin BioID. Co-precipitation experiments failed to identify junctional proteins, emphasizing the advantage of the BioID method. Mechanistically PAK4 depended on Afadin for its junctional localization, which is similar to the situation in Drosophila. A highly ranked PAK4-proximal protein LZTS2 was immuno-localized with Afadin at cell-cell junctions. Though PAK4 and Cdc42 are junctional, BioID analysis did not yield conventional cadherins, indicating their spatial segregation. To identify cellular PAK4 substrates we then assessed rapid changes (12’) in phospho-proteome after treatment with two PAK inhibitors. Among the PAK4-proximal junctional proteins seventeen PAK4 sites were identified. We anticipate mammalian group II PAKs are selective for the Afadin/nectin sub-compartment, with a demonstrably distinct localization from tight and cadherin junctions.

A kinase-independent function of PAK is crucial for pathogen-mediated actin remodelling

The p21-activated kinase (PAK) family regulate a multitude of cellular processes, including actin cytoskeleton remodelling. Numerous bacterial pathogens usurp host signalling pathways that regulate actin reorganisation in order to promote Infection. Salmonella and pathogenic Escherichia coli drive actin-dependent forced uptake and intimate attachment respectively. We demonstrate that the pathogen-driven generation of both these distinct actin structures relies on the recruitment and activation of PAK. We show that the PAK kinase domain is dispensable for this actin remodelling, which instead requires the GTPase-binding CRIB and the central poly-proline rich region. PAK interacts with and inhibits the guanine nucleotide exchange factor β-PIX, preventing it from exerting a negative effect on cytoskeleton reorganisation. This kinase-independent function of PAK may be usurped by other pathogens that modify host cytoskeleton signalling and helps us better understand how PAK functions in normal and diseased eukaryotic cells.