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A designer peptide against the EAG2–Kvβ2 potassium channel targets the interaction of cancer cells and neurons to treat glioblastoma

Nature Cancer volume 4, pages 1418–1436 (2023)Cite this article

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Abstract

Glioblastoma (GBM) is an incurable brain cancer that lacks effective therapies. Here we show that EAG2 and Kvβ2, which are predominantly expressed by GBM cells at the tumor–brain interface, physically interact to form a potassium channel complex due to a GBM-enriched Kvβ2 isoform. In GBM cells, EAG2 localizes at neuron-contacting regions in a Kvβ2-dependent manner. Genetic knockdown of the EAG2–Kvβ2 complex decreases calcium transients of GBM cells, suppresses tumor growth and invasion and extends the survival of tumor-bearing mice. We engineered a designer peptide to disrupt EAG2–Kvβ2 interaction, thereby mitigating tumor growth in patient-derived xenograft and syngeneic mouse models across GBM subtypes without overt toxicity. Neurons upregulate chemoresistant genes in GBM cells in an EAG2–Kvβ2-dependent manner. The designer peptide targets neuron-associated GBM cells and possesses robust efficacy in treating temozolomide-resistant GBM. Our findings may lead to the next-generation therapeutic agent to benefit patients with GBM.

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Fig. 1: EAG2 and Kvβ2 are upregulated at the GBM–brain interface.
Fig. 2: EAG2 localizes at GBM cell–neuron contact sites in a Kvβ2-dependent manner and regulates calcium transients of GBM cells.
Fig. 3: EAG2 and Kvβ2 promote GBM growth and invasion.
Fig. 4: Identification of Kvβ2 amino acid sequence that mediates EAG2–Kvβ2 interaction.
Fig. 5: K90-114TAT disrupts EAG2–Kvβ2 interaction and suppresses GBM growth.
Fig. 6: Cluster 7 GBM cells signify aggressive and TMZ-resistant GBM.
Fig. 7: K90-114TAT treatment is effective against TMZ-resistant GBM.
Fig. 8: A designer peptide to treat GBM by disruption of EAG2–Kvβ2 potassium channel complex-mediated cancer–neuron interaction.

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Data availability

The spatial expression of EAG2, Kvβ2 and other genes in GBM geometrical regions was derived from the Ivy Glioblastoma Atlas Project (https://glioblastoma.alleninstitute.org). The structure of Kvβ2 is available at the Protein Data Bank with accession code 3eau (https://www.ebi.ac.uk/pdbe/entry/pdb/3eau). IlluminaHiseq RNA-seq data of The Cancer Genome Atlas LGGs and GBMs are available at NCI’s Genomic Data Commons (https://gdc.cancer.gov/about-data/publications/lgggbm_2016). The clinical information and gene expression datasets of CGGA (mRNAseq_693, Illumina HiSeq) are available at the Chinese Glioma Genome Atlas (http://www.cgga.org.cn/index.jsp). Previously published microarray data of TMZ-resistant and -sensitive GBM clones that were reanalyzed here are available at ArrayExpress under accession code E-MTAB-2693 (https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-2693/). Preprocessed transcript data from the GLASS Consortium are available at Synapse (https://www.synapse.org/glass). Bulk RNA-seq data from GBM cell–neuron coculture, and scRNA-seq data from the PDX GBM model with peptide treatment reported in this manuscript, have been deposited in the NCBI Gene Expression Omnibus under accession code GSE231577. All unique materials such as patient-derived cell cultures are freely available and can be obtained by contacting the corresponding author and with a standard material transfer agreement with The Hospital for Sick Children. Data for all figures can be found within the manuscript, in the accompanying source data or from the corresponding author on reasonable request. Source data are provided with this paper.

Code availability

All codes used in this manuscript are freely available at GitHub (https://github.com/l-magnificence/Sequencing-Data-Analysis-Code-for-EAG2-Project).

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Acknowledgements

This work is supported by the SickKids Foundation, Arthur and Sonia Labatt Brain Tumour Research Centre, Garron Family Cancer Centre, Canadian Cancer Society Challenge Grant, Concern Foundation Conquer Cancer Now Award, b.r.a.i.n.child, Sontag Foundation Distinguished Scientist Award, Meagan’s HUG, Natural Sciences and Engineering Research Council Discovery Grant, Ontario Institute for Cancer Research Translational Research Initiative, Early Researcher Award and Canadian Institute of Health Research Project Grants (to X.H.), a Garron Family Cancer Centre Pitblado Discovery Grant (to L.-Y.W. and X.H.), Canadian Institute of Health Research Project Grants PJT-156034 and PJT-156439 and a Natural Sciences and Engineering Research Council grant RGPIN-2017-06665 (to L.-Y.W.). Xin C. is supported by SickKids Restracomp Scholarship. S.B. is supported by an International Postdoc Grant from the Swedish Research Council (Vetenskapsrådet) and Powered by Pablove Research Grant from Pablove Foundation. We thank J. Lathia for sharing GL261 cells, S. O. Kelley for sharing PC9 and H1975 cells and J. Ellis for sharing HEK293T cells, human astrocytes and lentivirus packaging plasmids. We thank P. Paroutis and K. Lau at SickKids Imaging Facility for help with confocal imaging and image analysis. We thank C. Simpson at SickKids SPARC BioCentre Molecular Analysis for assistance with mass spectrometry experiments. We thank the staff at The Centre for Phenogenomic for their help on animal works. We thank W. Wang for technical assistance and Huang laboratory members for comments on the manuscript. Schematics in Figs. 2a, 5c,e, 7a and 8 created with BioRender.com. X.H. is a Catalyst Scholar at The Hospital for Sick Children and Canada Research Chair in Cancer Biophysics.

Author information

Authors and Affiliations

  1. Developmental and Stem Cell Biology Program, The Hospital for Sick Children, Toronto, Ontario, Canada

    Weifan Dong, Xiaodi Chen, Xin Chen, Shahrzad Bahrampour, Yi Xiong, Hongyu Zhao, Ji-Eun Kim, Peter B. Dirks, Tae-Hee Kim, Siyi Wanggou & Xi Huang

  2. Arthur and Sonia Labatt Brain Tumour Research Centre, The Hospital for Sick Children, Toronto, Ontario, Canada

    Weifan Dong, Xiaodi Chen, Xin Chen, Shahrzad Bahrampour, Yi Xiong, Hongyu Zhao, Peter B. Dirks, Siyi Wanggou & Xi Huang

  3. Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada

    Weifan Dong, Xiaodi Chen, Daniel Schramek, Peter B. Dirks, Tae-Hee Kim & Xi Huang

  4. Neurosciences and Mental Health Program, The Hospital for Sick Children, Toronto, Ontario, Canada

    Adam Fekete, Tian Kong & Lu-Yang Wang

  5. Department of Neurosurgery, Xiangya Hospital, Central South University, Changsha, China

    Hongwei Liu, Yi Xiong, Qi Yang, Hongyu Zhao, Siyi Wanggou & Xuejun Li

  6. Hunan International Scientific and Technological Cooperation Base of Brain Tumor Research, Xiangya Hospital, Central South University, Changsha, China

    Hongwei Liu, Yi Xiong, Qi Yang, Hongyu Zhao, Siyi Wanggou & Xuejun Li

  7. Molecular Medicine Program, The Hospital for Sick Children, Toronto, Ontario, Canada

    Greg L. Beilhartz & Roman A. Melnyk

  8. Department of Physiology, University of Toronto, Toronto, Ontario, Canada

    Tian Kong & Lu-Yang Wang

  9. Macaulay Honors College, City College of New York, New York, NY, USA

    Malia S. Morioka & Ye He

  10. Advanced Science Research Center at the Graduate Center, City University of New York, New York, NY, USA

    Malia S. Morioka & Ye He

  11. The Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA

    Geena Jung & Yuanquan Song

  12. Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Geena Jung & Yuanquan Song

  13. Centre for Molecular and Systems Biology, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada

    Daniel Schramek

  14. Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada

    Roman A. Melnyk

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  1. Weifan Dong
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Contributions

X.H. directed the study. Experimental design, data acquisition and data analysis were carried out by W.D., A.F., Xiaodi C., H.L., G.L.B., Xin C., S.B., Y.X., Q.Y., H.Z., T.K., M.S.M., G.J., J.-E.K., G.J., Y.S., T.-H.K., Y.H., S.W., X.L., R.A.M. and L.-Y.W. Reagent contributions were provided by D.S. and P.B.D. W.D. and X.H. wrote the manuscript.

Corresponding author

Correspondence to Xi Huang.

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Competing interests

W.D. and X.H. have filed a PCT patent for the composition of matter and use of designer peptides to treat cancer. The remaining authors declare no competing interests.

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Nature Cancer thanks Justin Lathia, Stephen Robbins and Harald Sontheimer for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 EAG2 and Kvβ2 expression in xenograft GBM tumors, EAG2 localization in GBM cells, EAG2 and Kvβ2 knockdown validation, and tumor growth comparison between control tumor and tumor with knockdown of EAG2, Kvβ2, or both.

a. EAG2, Kvβ2, GFP, and DAPI expression in G411 xenograft tumor. EAG2, Tau, and GFAP expression in G489, G523, and G532 xenograft tumors. b. Representative images and quantification of EAG2 localization in GBM cells with or without contact of astrocytes. n = 24 cells examined over 3 independent experiments. P values, two-sided unpaired t-test. Error bars, mean ± s.e.m. c. Validation of Dox-induced knockdown of EAG2 and Kvβ2. d. Bioluminescence images show tumor burdens of mice bearing G411 tumors before and after Dox treatment. Kaplan-Meier survival curves are shown. P values, log-rank test. All experiments were performed 3 times using biologically independent samples.

Source data

Extended Data Fig. 2 Characterization of peptide treatment on GBM cell growth in vitro and tumor growth in vivo.

a. Co-IP shows EAG2-Kvβ2 interaction in G411 GBM cells treated with TAT, K59-78TAT, K90-114, or K90-114TAT. 3 biologically independent experiments were performed. b. Cell number, microtube number per cell, and microtube length of G489 GBM cells, which are co-cultured with neurons, after TAT, K59-78TAT, K90-114, or K90-114TAT treatment. Sample size (from left to right): n = 31, 42, 31, 43, 50, 52, 33, 41, 78, 146, 116, 49 cells examined over 3 independent experiments. Adjusted P value was calculated by Tukey’s multiple comparisons test (n.s. means not significant, P > 0.05). Error bars, mean ± s.e.m. Cell number and microtube length of K59-78TAT and K90-114TAT treated cells are also shown in Fig. 5b for easier data interpretation. c. Representative images and quantification of tumor areas of G411 xenograft tumors treated for 7 days with K59-78TAT or K90-114TAT. n = 15 samples examined from 5 biologically independent animals. P values, two-sided unpaired t-test. Error bars, mean ± s.e.m. d. Bioluminescence images and survival comparison of G411 GBM-bearing mice treated with K59-78TAT or K90-114TAT at various dosages. P values, log-rank test. Each P value was generated individually by comparing to K59-78TAT. e. Bioluminescence images and survival comparison of G411 GBM-bearing mice treated with TAT, K90-114, or randomized K90-114 TAT. K59-78TAT survival curve is derived from Fig. 5d. P values, log-rank test.

Source data

Extended Data Fig. 3 K90-114TAT treatment alters proliferation and apoptosis of GBM tumors.

Representative images showing proliferation and apoptosis of G489 xenograft tumors (pHis3: n = 54, cleaved caspase 3: n = 53) or GL261 syngeneic tumors (pHis3: n = 53 and 48, cleaved caspase 3: n = 54) treated with K59-78TAT or K90-114TAT. STEM121 labels G489 tumor cells. GFP labels GL261 tumor cells. pHis3 labels mitotic cells. cleaved caspase 3 labels apoptotic cells. Samples were evenly and independently collected from 3 animals. P values, two-sided unpaired t-test. Error bars, mean ± s.e.m.

Source data

Extended Data Fig. 4 K90-114TAT treatment does not affect mouse body weight, survival, and internal organs.

a. Body weight of K59-78TAT- and K90-114TAT-treated tumor-free NSG mice (n = 9 mice in each group). b. Survival of K59-78TAT- and K90-114TAT-treated tumor-free NSG mice (n = 9 mice in each group). c. Pathological analysis of hearts, kidneys, livers, and lungs harvested from control, K59-78TAT and K90-114TAT-treated tumor-bearing NSG mice (n = 3 mice in each group).

Source data

Extended Data Fig. 5 K90-114TAT treatment decreases proliferation and increases apoptosis of TMZ-resistant GBM.

Representative images showing STEM121+ tumor cells, pHis3+ mitotic cells, and cleaved caspase 3+ apoptotic cells in TMZ-resistant xenograft tumors treated with TMZ, K59-78TAT and K90-114TAT. Sample size (from left to right): G411-TMZr: n = 24, 25, 21, 24, 24, 24; G532-TMZr: n = 7, 15, 21, 17, 17, 21. Samples were evenly and independently collected from 3 animals. P values, two-sided unpaired t-test. Error bars, mean ± s.e.m.

Source data

Supplementary information

Reporting Summary

Supplementary Table 1

Sequences of peptides, shRNAs and PCR primers.

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Unprocessed immunoblots and gels.

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Dong, W., Fekete, A., Chen, X. et al. A designer peptide against the EAG2–Kvβ2 potassium channel targets the interaction of cancer cells and neurons to treat glioblastoma. Nat Cancer 4, 1418–1436 (2023). https://doi.org/10.1038/s43018-023-00626-8

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