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A cell-penetrating MARCKS mimetic selectively triggers cytolytic death in glioblastoma

Oncogene volume 39, pages 6961–6974 (2020)Cite this article

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Abstract

Glioblastoma (GBM) is an aggressive malignancy with limited effectiveness of standard of care therapies including surgery, radiation, and temozolomide chemotherapy necessitating novel therapeutics. Unfortunately, GBMs also harbor several signaling alterations that protect them from traditional therapies that rely on apoptotic programmed cell death. Because almost all GBM tumors have dysregulated phosphoinositide signaling as part of that process, we hypothesized that peptide mimetics derived from the phospholipid binding domain of Myristoylated alanine-rich C-kinase substrate (MARCKS) could serve as a novel GBM therapeutic. Using molecularly classified patient-derived xenograft (PDX) lines, cultured in stem-cell conditions, we demonstrate that cell permeable MARCKS effector domain (ED) peptides potently target all GBM molecular classes while sparing normal human astrocytes. Cell death mechanistic testing revealed that these peptides produce rapid cytotoxicity in GBM that overcomes caspase inhibition. Moreover, we identify a GBM-selective cytolytic death mechanism involving plasma membrane targeting and intracellular calcium accumulation. Despite limited relative partitioning to the brain, tail-vein peptide injection revealed tumor targeting in intracranially implanted GBM PDX. These results indicate that MARCKS ED peptide therapeutics may overcome traditional GBM resistance mechanisms, supporting further development of similar agents.

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Fig. 1: MARCKS ED mimetic cytotoxicity in GBM.
Fig. 2: MED2 is similarly cytotoxic to adherent or suspension cells and induces cytoplasmic retraction and Annexin-V positive blebbing.
Fig. 3: MED2 cytotoxic effects are resistant to caspase inhibition.
Fig. 4: TAT-ED mimetic triggers rapid and sustained increases in intracellular calcium in GBM as compared to NHAs.
Fig. 5: MARCKS TAT-ED mimetic binds in a punctate manner near the plasma membrane and preferentially accumulates inside GBM in vitro.
Fig. 6: MED2 crosses the blood–brain barrier concentrating in periventricular brain regions of tumor-naïve mice or orthotopic-implanted tumors.

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Data presented in this manuscript are available from the corresponding author upon request.

References

  1. Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJ, et al. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med. 2005;352:987–96.

    Article  CAS  PubMed  Google Scholar 

  2. Fulda S. Tumor resistance to apoptosis. Int J Cancer. 2009;124:511–5.

    Article  CAS  PubMed  Google Scholar 

  3. Tchoghandjian A, Souberan A, Tabouret E, Colin C, Denicolai E, Jiguet-Jiglaire C, et al. Inhibitor of apoptosis protein expression in glioblastomas and their in vitro and in vivo targeting by SMAC mimetic GDC-0152. Cell Death Dis. 2016;7:e2325.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Krakstad C, Chekenya M. Survival signalling and apoptosis resistance in glioblastomas: opportunities for targeted therapeutics. Mol Cancer. 2010;9:135.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  5. Wijaya J, Fukuda Y, Schuetz JD. Obstacles to brain tumor therapy: key ABC transporters. Int J Mol Sci. 2017;18:2544.

    Article  CAS  PubMed Central  Google Scholar 

  6. Vadlakonda L, Pasupuleti M, Pallu R. Role of PI3K-AKT-mTOR and Wnt signaling pathways in transition of G1-S phase of cell cycle in cancer cells. Front Oncol. 2013;3:85.

    PubMed  PubMed Central  Google Scholar 

  7. Chang L, Graham PH, Hao J, Ni J, Bucci J, Cozzi PJ, et al. Acquisition of epithelial-mesenchymal transition and cancer stem cell phenotypes is associated with activation of the PI3K/Akt/mTOR pathway in prostate cancer radioresistance. Cell Death Dis. 2013;4:e875.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Toulany M, Rodemann HP. Phosphatidylinositol 3-kinase/Akt signaling as a key mediator of tumor cell responsiveness to radiation. Semin Cancer Biol. 2015;35:180–90.

    Article  CAS  PubMed  Google Scholar 

  9. Birge RB, Boeltz S, Kumar S, Carlson J, Wanderley J, Calianese D, et al. Phosphatidylserine is a global immunosuppressive signal in efferocytosis, infectious disease, and cancer. Cell Death Differ. 2016;23:962–78.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Cancer Genome Atlas Research N. Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature. 2008;455:1061–8.

    Article  CAS  Google Scholar 

  11. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144:646–74.

    Article  CAS  PubMed  Google Scholar 

  12. Fruman DA, Chiu H, Hopkins BD, Bagrodia S, Cantley LC, Abraham RT. The PI3K pathway in human disease. Cell. 2017;170:605–35.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Langhans J, Schneele L, Trenkler N, von Bandemer H, Nonnenmacher L, Karpel-Massler G, et al. The effects of PI3K-mediated signalling on glioblastoma cell behaviour. Oncogenesis. 2017;6:398.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  14. Castellano E, Downward J. RAS interaction with PI3K: more than just another effector pathway. Genes Cancer. 2011;2:261–74.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Denley A, Gymnopoulos M, Kang S, Mitchell C, Vogt PK. Requirement of phosphatidylinositol(3,4,5)trisphosphate in phosphatidylinositol 3-kinase-induced oncogenic transformation. Mol Cancer Res. 2009;7:1132–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Nichol D, Mellinghoff IK. PI3K pathway inhibition in GBM-is there a signal? Neuro Oncol. 2015;17:1183–4.

    Article  PubMed  PubMed Central  Google Scholar 

  17. Li X, Wu C, Chen N, Gu H, Yen A, Cao L, et al. PI3K/Akt/mTOR signaling pathway and targeted therapy for glioblastoma. Oncotarget. 2016;7:33440–50.

    Article  PubMed  PubMed Central  Google Scholar 

  18. Wen PY, Lee EQ, Reardon DA, Ligon KL, Alfred Yung WK. Current clinical development of PI3K pathway inhibitors in glioblastoma. Neuro Oncol. 2012;14:819–29.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Morton LA, Yang H, Saludes JP, Fiorini Z, Beninson L, Chapman ER, et al. MARCKS-ED peptide as a curvature and lipid sensor. ACS Chem Biol. 2013;8:218–25.

    Article  CAS  PubMed  Google Scholar 

  20. Arbuzova A, Schmitz AA, Vergeres G. Cross-talk unfolded: MARCKS proteins. Biochem J. 2002;362:1–12.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Tanabe A, Kamisuki Y, Hidaka H, Suzuki M, Negishi M, Takuwa Y. PKC phosphorylates MARCKS Ser159 not only directly but also through RhoA/ROCK. Biochem Biophys Res Commun. 2006;345:156–61.

    Article  CAS  PubMed  Google Scholar 

  22. Brudvig JJ, Weimer JM. X MARCKS the spot: myristoylated alanine-rich C kinase substrate in neuronal function and disease. Front Cell Neurosci. 2015;9:407.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  23. Fong LWR, Yang DC, Chen CH. Myristoylated alanine-rich C kinase substrate (MARCKS): a multirole signaling protein in cancers. Cancer Metastasis Rev. 2017;36:737–47.

    Article  CAS  PubMed  Google Scholar 

  24. Rohrbach TD, Shah N, Jackson WP, Feeney EV, Scanlon S, Gish R, et al. The effector domain of MARCKS is a nuclear localization signal that regulates cellular PIP2 levels and nuclear PIP2 localization. PLoS ONE. 2015;10:e0140870.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  25. Iqbal S, Walsh TR, Rodger A, Packer NH. Interaction between polysialic acid and MARCKS-ED peptide at the molecular level. ACS Chem Neurosci. 2020;11:1944–54.

    Article  CAS  PubMed  Google Scholar 

  26. Kastelowitz N, Tamura R, Onasoga A, Stalker TJ, White OR, Brown PN, et al. Peptides derived from MARCKS block coagulation complex assembly on phosphatidylserine. Sci Rep. 2017;7:4275.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  27. Chen CH, Statt S, Chiu CL, Thai P, Arif M, Adler KB, et al. Targeting myristoylated alanine-rich C kinase substrate phosphorylation site domain in lung cancer. Mechanisms and therapeutic implications. Am J Respiratory Crit Care Med. 2014;190:1127–38.

    Article  CAS  Google Scholar 

  28. Chen CH, Fong LWR, Yu E, Wu R, Trott JF, Weiss RH. Upregulation of MARCKS in kidney cancer and its potential as a therapeutic target. Oncogene. 2017;36:3588–98.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Rohrbach TD, Jones RB, Hicks PH, Weaver AN, Cooper TS, Eustace NJ, et al. MARCKS phosphorylation is modulated by a peptide mimetic of MARCKS effector domain leading to increased radiation sensitivity in lung cancer cell lines. Oncol Lett. 2017;13:1216–22.

    Article  CAS  PubMed  Google Scholar 

  30. Dinca A, Chien WM, Chin MT. Intracellular delivery of proteins with cell-penetrating peptides for therapeutic uses in human disease. Int J Mol Sci. 2016;17:263.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  31. Panzarini E, Inguscio V, Dini L. Timing the multiple cell death pathways initiated by Rose Bengal acetate photodynamic therapy. Cell Death Dis. 2011;2:e169.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Seguin L, Desgrosellier JS, Weis SM, Cheresh DA. Integrins and cancer: regulators of cancer stemness, metastasis, and drug resistance. Trends Cell Biol. 2015;25:234–40.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Babiychuk EB, Monastyrskaya K, Potez S, Draeger A. Blebbing confers resistance against cell lysis. Cell Death Differ. 2011;18:80–9.

    Article  CAS  PubMed  Google Scholar 

  34. Wlodkowic D, Telford W, Skommer J, Darzynkiewicz Z. Apoptosis and beyond: cytometry in studies of programmed cell death. Methods Cell Biol. 2011;103:55–98.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Chaitanya GV, Steven AJ, Babu PP. PARP-1 cleavage fragments: signatures of cell-death proteases in neurodegeneration. Cell Commun Signal. 2010;8:31.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Elmore S. Apoptosis: a review of programmed cell death. Toxicol Pathol. 2007;35:495–516.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Dasari S, Tchounwou PB. Cisplatin in cancer therapy: molecular mechanisms of action. Eur J Pharm. 2014;740:364–78.

    Article  CAS  Google Scholar 

  38. Keoni CL, Brown TL. Inhibition of apoptosis and efficacy of pan caspase inhibitor, Q-VD-OPh, in models of human disease. J Cell Death. 2015;8:1–7.

    Article  PubMed  PubMed Central  Google Scholar 

  39. Zhivotovsky B, Orrenius S. Calcium and cell death mechanisms: a perspective from the cell death community. Cell Calcium. 2011;50:211–21.

    Article  CAS  PubMed  Google Scholar 

  40. Collatz MB, Rudel R, Brinkmeier H. Intracellular calcium chelator BAPTA protects cells against toxic calcium overload but also alters physiological calcium responses. Cell Calcium. 1997;21:453–9.

    Article  CAS  PubMed  Google Scholar 

  41. Haar CP, Hebbar P, Wallace GCT, Das A, Vandergrift WA 3rd, Smith JA, et al. Drug resistance in glioblastoma: a mini review. Neurochem Res. 2012;37:1192–200.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Groothuis DR. The blood-brain and blood-tumor barriers: a review of strategies for increasing drug delivery. Neuro Oncol. 2000;2:45–59.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Micallef J, Taccone M, Mukherjee J, Croul S, Busby J, Moran MF, et al. Epidermal growth factor receptor variant III-induced glioma invasion is mediated through myristoylated alanine-rich protein kinase C substrate overexpression. Cancer Res. 2009;69:7548–56.

    Article  CAS  PubMed  Google Scholar 

  44. Johnson DE. Noncaspase proteases in apoptosis. Leukemia. 2000;14:1695–703.

    Article  CAS  PubMed  Google Scholar 

  45. Galluzzi L, Vitale I, Aaronson SA, Abrams JM, Adam D, Agostinis P, et al. Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018. Cell Death Differ. 2018;25:486–541.

    Article  PubMed  PubMed Central  Google Scholar 

  46. Boohaker RJ, Lee MW, Vishnubhotla P, Perez JM, Khaled AR. The use of therapeutic peptides to target and to kill cancer cells. Curr Med Chem. 2012;19:3794–804.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Kepp O, Tesniere A, Schlemmer F, Michaud M, Senovilla L, Zitvogel L, et al. Immunogenic cell death modalities and their impact on cancer treatment. Apoptosis. 2009;14:364–75.

    Article  PubMed  Google Scholar 

  48. Gimple RC, Kidwell RL, Kim LJY, Sun T, Gromovsky AD, Wu Q, et al. Glioma stem cell-specific superenhancer promotes polyunsaturated fatty-acid synthesis to support EGFR signaling. Cancer Discov. 2019;9:1248–67.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Willey CD, Gilbert AN, Anderson JC, Gillespie GY. Patient-derived xenografts as a model system for radiation research. Semin Radiat Oncol. 2015;25:273–80.

    Article  PubMed  PubMed Central  Google Scholar 

  50. Sarkaria JN, Carlson BL, Schroeder MA, Grogan P, Brown PD, Giannini C, et al. Use of an orthotopic xenograft model for assessing the effect of epidermal growth factor receptor amplification on glioblastoma radiation response. Clin Cancer Res. 2006;12:2264–71.

    Article  CAS  PubMed  Google Scholar 

  51. Verhaak RG, Hoadley KA, Purdom E, Wang V, Qi Y, Wilkerson MD, et al. Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1. Cancer Cell. 2010;17:98–110.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. de Boer E, Samuel S, French DN, Warram JM, Schoeb TR, Rosenthal EL, et al. Biodistribution study of intravenously injected cetuximab-IRDye700DX in cynomolgus macaques. Mol Imaging Biol. 2016;18:232–42.

    Article  PubMed  CAS  Google Scholar 

  53. Jarboe JS, Anderson JC, Duarte CW, Mehta T, Nowsheen S, Hicks PH, et al. MARCKS regulates growth, radiation sensitivity and is a novel prognostic factor for glioma. Clin Cancer Res. 2012. https://doi.org/10.1158/1078-0432.CCR-11-3091.

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Acknowledgements

A special thanks to Neil A. Durso, Ph.D. from ThermoFisher for assistance with time-lapse and calcium imaging using CellInsight Cx7 instrument. ChemoMetec loaned us the Xcyto10 image cytometer for initial work and provided microscope slides, BlueMask-1, and DAPI reagents but no financial compensation. We would also like to acknowledge the UAB Nanostring Laboratory in the Department of Radiation Oncology, directed by Eddy S. Yang, MD, PhD and managed by Debbie Della Manna, as well as the UAB Kinome Core for the molecular analyses services performed in the Supplementary data.

Funding

This work was supported by funding from the National Institutes of Health (the UAB MSTP training Grant: T32GM008361 and the UAB Training Program in Brain Tumor Biology: T32NS048039), the American Cancer Society RSG-14-071-01-TBG, the UAB Physician Scientist Training Program Medical Student Summer Research Training Program, and an intramural research grant from the UAB Department of Radiation Oncology.

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Authors and Affiliations

  1. Department of Radiation Oncology, The University of Alabama at Birmingham, Birmingham, AL, USA

    Nicholas J. Eustace, Joshua C. Anderson, Hasan Alrefai, Zeel Patel, Patricia H. Hicks & Christopher D. Willey

  2. Department of Otolaryngology, The University of Alabama at Birmingham, Birmingham, AL, USA

    Jason M. Warram

  3. Department of Biochemistry and Molecular Genetics, The University of Alabama at Birmingham, Birmingham, AL, USA

    Hayley N. Widden & William J. Placzek

  4. ChemoMetec, DK-3450, Allerod, Denmark

    Rune T. Pedersen

  5. Department of Neurosurgery, The University of Alabama at Birmingham, Birmingham, AL, USA

    G. Yancey Gillespie

  6. Department of Cell, Developmental and Integrative Biology, The University of Alabama at Birmingham, Birmingham, AL, USA

    Anita B. Hjelmeland

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Correspondence to Christopher D. Willey.

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RTP is an employee at ChemoMetec and assisted in the use of Xcyto10, aided in Xcyto10 data analysis, and reviewed the manuscript. Unrelated to this work, but for full disclosure, CDW is a part time consultant with LifeNet Health and Varian Medical Systems.

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Eustace, N.J., Anderson, J.C., Warram, J.M. et al. A cell-penetrating MARCKS mimetic selectively triggers cytolytic death in glioblastoma. Oncogene 39, 6961–6974 (2020). https://doi.org/10.1038/s41388-020-01511-9

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