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The protein arginine methyltransferase PRMT5 confers therapeutic resistance to mTOR inhibition in glioblastoma

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Abstract

Introduction

Clinical trials directed at mechanistic target of rapamycin (mTOR) inhibition have yielded disappointing results in glioblastoma (GBM). A major mechanism of resistance involves the activation of a salvage pathway stimulating internal ribosome entry site (IRES)-mediated protein synthesis. PRMT5 activity has been implicated in the enhancement of IRES activity.

Methods

We analyzed the expression and activity of PRMT5 in response to mTOR inhibition in GBM cell lines and short-term patient cultures. To determine whether PRMT5 conferred resistance we used genetic and pharmacological approaches to ablate PRMT5 activity and assessed the effects on in vitro and in vivo sensitivity. Mutational analyses of the requisite IRES-trans-acting factor (ITAF), hnRNP A1 determined whether PRMT5-mediated methylation was necessary for ITAF RNA binding and IRES activity.

Results

PRMT5 activity is stimulated in response to mTOR inhibitors. Knockdown or treatment with a PRMT5 inhibitor blocked IRES activity and sensitizes GBM cells. Ectopic expression of non-methylatable hnRNP A1 mutants demonstrated that methylation of either arginine residues 218 or 225 was sufficient to maintain IRES binding and hnRNP A1-dependent cyclin D1 or c-MYC IRES activity, however a double R218K/R225K mutant was unable to do so. The PRMT5 inhibitor EPZ015666 displayed synergistic anti-GBM effects in vitro and in a xenograft mouse model in combination with PP242.

Conclusions

These results demonstrate that PRMT5 activity is stimulated upon mTOR inhibition in GBM. Our data further support a signaling cascade in which PRMT5-mediated methylation of hnRNP A1 promotes IRES RNA binding and activation of IRES-mediated protein synthesis and resultant mTOR inhibitor resistance.

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Acknowledgements

We thank Mark Schroeder and Drs. Jacob Fleischmann, Norimoto Yanagawa, Jann Sarkaria and Paul Mischel for cell lines and reagents. We also thank Dr. Alan Lichtenstein for comments on the manuscript.

Funding

This work was supported, in whole or in part, by Merit Review Award Number I01 BX002665 from the United States (U.S.) Department of Veterans Affairs Biomedical Laboratory R&D (BLRD) Service and NIH R01CA217820 Grants.

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Authors and Affiliations

  1. Department of Medicine, David Geffen School of Medicine At UCLA, Los Angeles, CA, USA

    Brent Holmes, Angelica Benavides-Serrato & Joseph Gera

  2. Department of Neurology, David Geffen School of Medicine At UCLA, Los Angeles, CA, USA

    Robert N. Nishimura

  3. Jonnson Comprehensive Cancer Center, Los Angeles, CA, USA

    Joseph Gera

  4. Molecular Biology Institute, University of California-Los Angeles, Los Angeles, CA, USA

    Joseph Gera

  5. Department of Research & Development, Greater Los Angeles Veterans Affairs Healthcare System, Los Angeles, CA, USA

    Brent Holmes, Angelica Benavides-Serrato, Jacquelyn T. Saunders, Kenna A. Landon, Adam J. Schreck, Robert N. Nishimura & Joseph Gera

  6. Greater Los Angeles VA Healthcare System, 16111 Plummer Street (151), Building 1, Room C111A, Los Angeles, CA, 91343, USA

    Joseph Gera

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  1. Brent Holmes
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Correspondence to Joseph Gera.

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All animal experiments were performed under an approved Institutional Animal Care and Use Committee protocol and conformed to the guidelines established by the Association for the Assessment and Accreditation of Laboratory Animal Care.

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Holmes, B., Benavides-Serrato, A., Saunders, J.T. et al. The protein arginine methyltransferase PRMT5 confers therapeutic resistance to mTOR inhibition in glioblastoma. J Neurooncol 145, 11–22 (2019). https://doi.org/10.1007/s11060-019-03274-0

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