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Macrophage PAK2 controls the SIRPα immune checkpoint via cytoskeletal dynamics and endocytic trafficking

ABSTRACT: Professional phagocytes are essential for immunomodulation, antimicrobial control, and initiation and resolution of inflammation. These cells rely on dynamic cytoskeletal rearrangements to respond to their environment. This plasticity requires a network of positive and negative regulators to control biomechanics and signaling circuits. P21-activated kinases (PAKs) regulate the actomyosin cytoskeleton, cell proliferation, and gene expression, yet their contributions to phagocyte functions remain unresolved. Analyzing PAK1/2-deficient phagocytes and utilizing a PAK inhibitor we show that PAK2 governs physio-mechanical macrophage properties, leading to membrane expansion, hyperphagic behavior and disruption of endocytic transport when absent, while PAKs cooperate to restrain oxidative neutrophil responses. In PAK1/mPAK2 ablated mice cytoskeletal remodeling combined with low-grade chronic inflammation prompts the spontaneous development of a macrophage-driven hematologic malignancy. Macrophage PAK2 ablation or inhibition markedly enhances cancer cell uptake by exerting biophysical control over the CD47-SIRPα checkpoint, indicating that biomechanical modification of macrophages holds promise in cancer immunotherapy.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Phagocyte

DISEASE(S): Cancer

SUBMITTER: Kieran Wynne

LAB HEAD: Prof Ulla Knaus

PROVIDER: PXD062325 | Pride | 2025-10-31

REPOSITORIES: Pride

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			Action	DRS
	240410kw_JDrieuASingh_1103_FX1_S2-B7_1_18898.d.zip	Other
	240410kw_JDrieuASingh_1103_FX1_S2-B8_1_18899.d.zip	Other
	240410kw_JDrieuASingh_1103_FX2_S2-B10_1_18901.d.zip	Other
	240410kw_JDrieuASingh_1103_FX2_S2-B9_1_18900.d.zip	Other
	240410kw_JDrieuASingh_1103_FX3_S2-B11_1_18902.d.zip	Other

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Project description:Introduction: Altered metabolism is a key hallmark of tumour cells (Pavlova & Thompson, 2016) and has long been associated with relapse and resistance to treatment (Gonçalves et al., 2021; Lv et al., 2022). Recently, we identified a new role for histone deacetylase 6 (HDAC6) in the regulation of glycolytic metabolism (Dowling et al., 2021). HDAC6 is an atypical member of the HDAC family as it is localised in the cytosol, has two deacetylase domains, and contains a ubiquitin-binding domain (Gallinari et al., 2007). HDAC6 has been shown to deacetylate proteins involved in processes including microtubule remodelling and stress responses (Hubbert et al., 2002; Kawaguchi et al., 2003). In a recent publication, we showed that HDAC6 knockdown (K/D) or inhibition, with a compound we discovered called BAS-2, altered the acetylation of glycolytic enzymes and reduced glycolysis in triple-negative breast cancer (TNBC) cells (Dowling et al., 2021). However, we did not investigate whether HDAC6 inhibition altered other metabolic pathways at the time. The mitochondrial tricarboxylic acid (TCA) cycle plays an important role in tumour biology, not alone for energy production through the respiratory chain, but also for metabolic intermediates that serve as building blocks for lipid and nucleic acid synthesis (Eniafe & Jiang, 2021). Some of these intermediates of the TCA cycle have dual roles as ‘oncometabolites’ (Frezza, 2017). Both fumarate hydratase (FH) and succinate dehydrogenase (SDH), two key enzymes in the TCA cycle, are known tumour suppressors (Rustin et al., 2002; Schmidt et al., 2020). Loss of FH is associated with an aggressive form of kidney cancer called hereditary leiomyomatosis and renal cell carcinoma (HLRCC) (Tomlinson et al., 2002). Deficiency of FH causes an increase in fumarate, which has various signalling properties including the non-enzymatic formation of 2-(succino)cysteine (2-SC) adducts that can alter protein activity, a process termed succination (Guberovic & Frezza, 2024; Kinch et al., 2011; Tyrakis et al., 2017). Two recent publications identified that macrophages with FH deficiency show an increase of intracellular fumarate and that this alters mitochondrial structure, causing the release of mitochondrial DNA or RNA to activate inflammatory responses (Hooftman et al., 2023; Zecchini et al., 2023). To date, the majority of studies on FH have been carried out using knockout mice to show the role of FH in tumourigenesis or macrophage inflammation (Valcarcel-Jimenez & Frezza, 2023; Zecchini et al., 2023). However, there are limited studies in tumour cells on the role of FH or on how FH activity is regulated. In this study, we identified that HDAC6 can alter mitochondrial structure, causing cristae damage and the release of mtDNA into the cytosol. We then show by mass spectrometry, immunoprecipitation, and super-resolution imaging that HDAC6 and FH directly interact. Inhibition of HDAC6 with BAS-2 reduces FH activity, resulting in an increase of fumarate, with a downstream increase in protein succination. Utilising stochastic optical reconstruction microscopy (STORM), we pinpoint sites of interaction to the mitochondria, thereby demonstrating a novel regulation of FH in tumour cells.

Project description:Macrophages play critical roles in immune function and are implicated as etiological agents in disease states such as cardiovascular disease and cancer. However, there is limited understanding of macrophage fitness molecular regulators, including survival, proliferation, and adhesion. This work identifies positive and negative regulators of macrophage fitness by harnessing the power of CRISPR/Cas9 whole genome screening technology. Here, we identified loss-of-function gene disruptions that caused mutant bone marrow-derived macrophages (BMDM) to decrease in culture abundance as supporters of macrophage fitness. Similarly, we identified genes that limit or suppress macrophage fitness by identifying those gene disruptions that caused BMDM to increase in abundance in cell culture. To do this, we analyzed the difference in frequency of sgRNA between the purified plasmid library used for lentiviral transfection versus the amplified sgRNA inserts from the DNA of BMDM following a 14-day post-viral transduction culture. With this approach, we identified gene disruptions to canonical essential genes, including previously described macrophage-specific essential genes such as Csf1r, as well as novel genes required for macrophage growth and survival. In addition, we also identified gene disruptions that increased macrophage abundance in culture, including canonical tumor suppressors such as p53 (Trp53) and cell cycle regulators (Cdkn1a/2a). Interestingly, targeting p21-activated kinase-2 (Pak2) led to those mutants becoming more productive, suggesting a novel role for Pak2 in suppressing macrophage fitness. Using targeted knockouts of PAK2, we show that PAK2 protein depletion affects NF2/Merlin-mediated downregulation of cyclin-d1. Further, this work indicates that PAK2 suppresses macropinocytic uptake in macrophages by modulating actin dynamics impacting pro-mitogenic signaling pathways downstream of macropinocytosis. Overall, this work shows how multiple pathways impact macrophage fitness and highlights the central role of PAK2 in macrophage fitness and macropinocytosis.

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Macrophage PAK2 controls the SIRPα immune checkpoint via cytoskeletal dynamics and endocytic trafficking

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