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Targeting PAK4 to reprogram the vascular microenvironment and improve CAR-T immunotherapy for glioblastoma

Nature Cancer volume 2, pages 83–97 (2021)Cite this article

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Abstract

Malignant solid tumors are characterized by aberrant vascularity that fuels the formation of an immune-hostile microenvironment and induces resistance to immunotherapy. Vascular abnormalities may be driven by pro-angiogenic pathway activation and genetic reprogramming in tumor endothelial cells (ECs). Here, our kinome-wide screening of mesenchymal-like transcriptional activation in human glioblastoma (GBM)-derived ECs identifies p21-activated kinase 4 (PAK4) as a selective regulator of genetic reprogramming and aberrant vascularization. PAK4 knockout induces adhesion protein re-expression in ECs, reduces vascular abnormalities, improves T cell infiltration and inhibits GBM growth in mice. Moreover, PAK4 inhibition normalizes the tumor vascular microenvironment and sensitizes GBM to chimeric antigen receptor–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 a unique opportunity to recondition the vascular microenvironment and strengthen cancer immunotherapy.

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Fig. 1: Identification of PAK4 as a critical regulator of mesenchymal-like transcriptional activation and functional abnormalities in GBM ECs.
Fig. 2: Endothelial-specific deletion of PAK4 inhibits vascular abnormalities and enhances T cell infiltration, leading to reduced tumor growth and increased mouse survival.
Fig. 3: Pharmacological PAK4 inhibition reduces proliferation selectively in GBM ECs and normalizes the tumor vasculature.
Fig. 4: PAK4 is critical for mesenchymal-like transcriptional reprogramming in GBM ECs.
Fig. 5: PAK4 suppresses adhesion protein expression via ZEB1 and SLUG, enhancing vessel permeability and reducing T cell adhesion to GBM ECs.
Fig. 6: PAK4 induces ZEB1 expression via MEF2D in GBM ECs.
Fig. 7: PAK4 inhibition sensitizes GBM to CAR-T immunotherapy.

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Data availability

RNA-seq data have been deposited in the National Center for Biotechnology Information’s Gene Expression Omnibus under accession code GSE154133. The MSigDB database used in the study is available at https://www.gsea-msigdb.org/gsea/msigdb. All of the other data supporting the findings of this study are available from the corresponding authors upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We are grateful to E. Holland (Fred Hutchinson Cancer Research Center) for providing the RCAS-PDGF GBM model. We thank S. Albelda and M. Leibowitz for help with CAR-T production and G. Linette for helpful discussions. This work was supported in part by the University of Pennsylvania Academic Development Fund (to Y.F.), an Abramson Cancer Center GBM-TCE Award (to Y.F., Z.A.B. and D.M.O.), a RadOnc-TCE Award (to Y.F.), an American Association for Cancer Research Judah Folkman Award (to Y.F.) and National Institutes of Health grants R01NS094533 and R01NS106108 (to Y.F.).

Author information

Author notes

  1. Wenjuan Ma & Rongxin Zhang

    Present address: State Key Laboratory of Oncology in South China and Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, China

  2. These authors contributed equally: Wenjuan Ma, Yanling Wang, Rongxin Zhang, Fan Yang.

  3. These authors jointly supervised this work: Yanqing Gong, Yi Fan.

Authors and Affiliations

  1. Department of Radiation Oncology, University of Pennsylvania, Philadelphia, PA, USA

    Wenjuan Ma, Yanling Wang, Rongxin Zhang, Fan Yang, Duo Zhang, Menggui Huang, Jay F. Dorsey & Yi Fan

  2. Department of Obstetrics and Gynecology, University of Pennsylvania, Philadelphia, PA, USA

    Lin Zhang

  3. Department of Neurosurgery, University of Pennsylvania, Philadelphia, PA, USA

    Zev A. Binder, Donald M. O’Rourke & Yi Fan

  4. Glioblastoma Translational Center of Excellence, Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA, USA

    Zev A. Binder, Donald M. O’Rourke & Yi Fan

  5. Department of Microbiology, University of Pennsylvania, Philadelphia, PA, USA

    Joseph A. Fraietta

  6. Center for Cellular Immunotherapies, Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA, USA

    Joseph A. Fraietta

  7. Division of Translational Medicine and Human Genetics, Department of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Yanqing Gong

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Contributions

W.M. performed the kinome screening analysis, RNA-seq and animal studies with transgenic mice. Y.W. and F.Y. conducted the mechanistic studies and CAR-T therapy work. R.Z. performed the cell function experiments. D.Z. conducted the three-dimensional vasculature imaging. M.H. generated the screening constructs. D.Z. and L.Z. contributed to the bioinformatics analysis. J.F.D. contributed to the experimental design. Z.A.B. and D.M.O. contributed to preparation of the cells derived from patients with GBM. J.A.F. helped to design the CAR-T therapy. Y.G. and Y.F. designed and co-supervised the experiments. Y.F. conceived of the ideas and wrote the manuscript. All of the authors commented on the manuscript.

Corresponding authors

Correspondence to Yanqing Gong or Yi Fan.

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Competing interests

Y.F. is an inventor on a patent application covering the use of PAK4 inhibitors for vessel normalization therapy. L.Z. received research funding from AstraZeneca, Bristol-Myers Squibb/Celgene and Prelude Therapeutics.

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Extended data

Extended Data Fig. 1 Effects of siRNA-mediated PAK4 knockdown on EC functions.

a,b, Human GBM-derived ECs from patient #5377 were lentivirally transduced to express SMA-fLuc and CMV-rLuc, followed by transfection with an siRNA targeting PAK4 or a random sequence. a, Cell lysates were immunoblotted. This experiment was repeated independently twice with similar results. b, Four days after transfection, fLuc and rLuc bioluminescence was analyzed (n = 3 EC samples each derived from a distinct GBM tumor, mean ± SEM). Statistical analysis by two-way ANOVA. c-f, ECs isolated from human GBM tumors or normal brains were transfection with an siRNA targeting PAK4 or a random sequence. c, ECs isolated from normal human brain were subjected to proliferation analysis (n = 3 independent experiments, mean ± SEM). Statistical analysis by two-tailed Student’s t test. d, GBM ECs isolated from patient #5465 were subjected to proliferation analysis (n = 3 independent experiments, mean ± SEM). Statistical analysis by two-tailed Student’s t test. e, ECs isolated from normal human brain were subjected to migration analysis (n = 3 EC samples each derived from a distinct GBM tumor, mean ± SEM). Statistical analysis by two-tailed Students’ t-test. f, GBM ECs isolated from three patients were subjected to migration analysis (n = 3 independent assays, mean ± SEM). Statistical analysis by one-way ANOVA.

Source data

Extended Data Fig. 2 Effects of PAK4 knockout on EC proliferation and migration and mouse growth. Ten-day-old Pak4fl/fl (WT) and Cdh5-Cre;Pak4fl/fl (PAK4-∆EC) mice were treated with tamoxifen for three days.

a,b, Aortic ECs were isolated from 21-day-old mice. a, Cell proliferation was determined using a MTT-based assay (n = 5 EC samples each derived from a distinct mouse, mean ± SEM). Statistical analysis by two-tailed Student’s t test. b, Cell migration in response to FBS was measured using a transwell assay (n = 3 EC samples each derived from a distinct mouse, mean ± SEM). Statistical analysis by two-tailed Students’ t-tests. c, Animal body weight was monitored (mean ± SEM; WT group, n = 4 mice; PAK4-ΔEC group, n = 6 mice).

Source data

Extended Data Fig. 3 PAK knockout in ECs inhibits FSP-1 expression in GBM-associated ECs.

GBM was genetically induced, followed by implantation into WT or PAK4-ΔEC mice. Tumor sections were immunostained using anti-CD31 and anti-FSP-1 antibodies, and subjected to immunofluorescence analysis. Representative images are shown (n = 4 mice). Arrows indicated FSP-1 expression in CD31+ cells. Scale bar: 100 μm.

Extended Data Fig. 4 PAK4 knockout in ECs restores VCAM-1 expression in GBM-associated ECs.

GBM was genetically induced, followed by transplantation into WT or PAK4-ΔEC mice. Tumor sections were immunostained using anti-CD31 and anti-VCAM-1 antibodies, followed by immunofluorescence analysis. Representative images are shown (n = 4 mice). Scale bar: 100 μm.

Extended Data Fig. 5 PAK4 knockdown did not affect claudin-5 or CD31 expression in GBM ECs and normal ECs.

ECs isolated from human GBM tumor (patient #5377) or normal brain were transfected with siRNA targeting PAK4 or control sequence. Cell lysates were analyzed by immunoblot. This experiment was repeated independently twice with similar results.

Source data

Extended Data Fig. 6 PAK4 kinase activity is critical for mesenchymal-like transcriptional reprogramming and cell proliferation and migration in GBM ECs.

ECs isolated from human GBM tumors were transduced to express CRISPR targeting PAK4 or a random sequence, followed by transfection with plasmids expressing WT PAK4 or kinase-dead K350M mutant PAK4 or empty vector (EV). a, Cell lysates were analyzed by immunoblot. This experiment was repeated independently twice with similar results. b, Cells were subjected to cell proliferation analysis (n = 6 EC samples derived from different tumors, mean ± SD). Statistical analysis by two-way ANOVA. c, Cells were subjected to transwell-based cell migration analysis (mean ± SEM, n = 3 EC samples derived from different tumors for control group, and n = 6 EC samples derived from different tumors for other groups). Statistical analysis by one-way ANOVA.

Source data

Extended Data Fig. 7 PAK4 induces MEF2D phosphorylation at Ser180.

a, ECs isolated from human GBM tumor (patient #5377) were transduced to express CRISPR targeting PAK4 or a random sequence, followed by transfection with plasmids expressing WT PAK4 or kinase-dead K350M mutant PAK4 or empty vector (EV). Cell lysates were analyzed by immunoblot. b, Purified MEF2D and PAK4 proteins were incubated in kinase buffer, followed by immunoblot analysis. These experiments were repeated independently twice with similar results.

Source data

Extended Data Fig. 8 A murine Egfrviii CAR T system.

a, Schematic diagram of the mouse Egfrviii CAR T construct. b, Mouse spleen-derived T cells were transduced with MSGV retrovirus that encodes Egfrviii 139 CAR or with an empty vector, followed by flow cytometry analysis of 139 CAR expression. Representative cell sortings are shown. c, Mouse T cells expressing 139 CAR were incubated with mouse GL261 glioma cells expressing mouse Egfrviii or control WT Egfr. Cell lysis was determined by europium cytotoxicity assay (mean ± SEM, n = 3 T cell samples derived from different mice). Statistical analysis by two-way ANOVA.

Source data

Extended Data Fig. 9 Expression of Egfrviii by retroviral transduction in mouse GBM cells.

GBM was genetically engineered induced in mice. Tumor-derived spheres were transduced with retrovirus that expresses mouse Egfrviii, followed by flow cytometry analysis for Egfrviii expression. Representative cell sortings are shown.

Supplementary information

Reporting Summary

Supplementary Video 1

GBM was induced in Rosa-LSL-tdTomato;Cdh5-CreETR2 mice, followed by treatment with saline or KPT9274. Tumors were excised and imaged by light sheet microscopy (n = 3 mice). Three-dimensional images of tumor vasculature were reconvoluted.

Supplementary Video 2

GBM was induced in Rosa-LSL-tdTomato;Cdh5-CreETR2 mice, followed by treatment with saline or KPT9274. Mice were injected with Hypoxyprobe-1 (pimonidazole HCl), and brain tissues were probed with antibodies against pimonidazole adducts, followed by light sheet microscopy imaging (n = 3 mice). Three-dimensional images of the vasculature (red) and hypoxic areas (green) in tumors and normal brains were reconvoluted.

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Ma, W., Wang, Y., Zhang, R. et al. Targeting PAK4 to reprogram the vascular microenvironment and improve CAR-T immunotherapy for glioblastoma. Nat Cancer 2, 83–97 (2021). https://doi.org/10.1038/s43018-020-00147-8

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