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Targeting destabilized DNA G-quadruplexes and aberrant splicing in drug-resistant glioblastoma

Deanna M Tiek, Roham Razaghi, Lu Jin, View ORCID ProfileNorah Sadowski, View ORCID ProfileCarla Alamillo-Ferrer, View ORCID ProfileJ Robert Hogg, Bassem R Haddad, View ORCID ProfileDavid H Drewry, View ORCID ProfileCarrow I Wells, Julie E. Pickett, View ORCID ProfileWilliam J Zuercher, View ORCID ProfileWinston Timp, View ORCID ProfileRebecca B Riggins
doi: https://doi.org/10.1101/661660
Deanna M Tiek
1Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC, USA
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  • For correspondence: dmt53@georgetown.edu rbr7@georgetown.edu
Roham Razaghi
2Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD, USA
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Lu Jin
1Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC, USA
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Norah Sadowski
2Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD, USA
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  • ORCID record for Norah Sadowski
Carla Alamillo-Ferrer
3Structural Genomics Consortium, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
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J Robert Hogg
4Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA
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Bassem R Haddad
1Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC, USA
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David H Drewry
3Structural Genomics Consortium, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
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Carrow I Wells
3Structural Genomics Consortium, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
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Julie E. Pickett
3Structural Genomics Consortium, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
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William J Zuercher
3Structural Genomics Consortium, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
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Winston Timp
2Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD, USA
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Rebecca B Riggins
1Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC, USA
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  • ORCID record for Rebecca B Riggins
  • For correspondence: dmt53@georgetown.edu rbr7@georgetown.edu
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Abstract

Temozolomide (TMZ) is a chemotherapy agent that adds mutagenic adducts to guanine, and is first-line standard of care for the aggressive brain cancer glioblastoma (GBM). Methyl guanine methyl transferase (MGMT) is a DNA repair enzyme that can remove O6-methyl guanine adducts prior to the development of catastrophic mutations, and is associated with TMZ resistance. However, inhibition of MGMT fails to reverse TMZ resistance. Guanines are essential nucleotides in many DNA and RNA secondary structures. In several neurodegenerative diseases (NDs), disruption of these secondary structures is pathogenic. We therefore took a structural view of TMZ resistance, seeking to establish the role of guanine mutations in disrupting critical nucleotide secondary structures. To test whether these have functional impacts on TMZ-resistant GBM, we focused on two specific guanine-rich regions: G-quadruplexes (G4s) and splice sites. Here we report broad sequence- and conformation-based changes in G4s in acquired or intrinsic TMZ resistant vs. sensitive GBM cells, accompanied by nucleolar stress and enrichment of nucleolar RNA:DNA hybrids (r-loops). We further show widespread splice-altering mutations, exon skipping, and deregulation of splicing-regulatory serine/arginine rich (SR) protein phosphorylation in TMZ-resistant GBM cells. The G4-stabilizing ligand TMPyP4 and a novel inhibitor of cdc2-like kinases (CLKs) partially normalize G4 structure and SR protein phosphorylation, respectively, and are preferentially growth-inhibitory in TMZ-resistant cells. Lastly, we report that the G4- and RNA-binding protein EWSR1 forms aberrant cytoplasmic aggregates in response to acute TMZ treatment, and these aggregates are abundant in TMZ resistant cells. Preliminary evidence suggests these cytoplasmic EWSR1 aggregates are also present in GBM clinical samples. This work supports altered nucleotide secondary structure and splicing deregulation as pathogenic features of TMZ-resistant GBM. It further positions cytoplasmic aggregation of EWSR1 as a potential indicator for TMZ resistance, establishes the possibility of successful intervention with splicing modulatory or G4-targeting agents, and provides a new context in which to study aggregating RNA binding proteins.

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Posted June 05, 2019.
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Targeting destabilized DNA G-quadruplexes and aberrant splicing in drug-resistant glioblastoma
Deanna M Tiek, Roham Razaghi, Lu Jin, Norah Sadowski, Carla Alamillo-Ferrer, J Robert Hogg, Bassem R Haddad, David H Drewry, Carrow I Wells, Julie E. Pickett, William J Zuercher, Winston Timp, Rebecca B Riggins
bioRxiv 661660; doi: https://doi.org/10.1101/661660
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Targeting destabilized DNA G-quadruplexes and aberrant splicing in drug-resistant glioblastoma
Deanna M Tiek, Roham Razaghi, Lu Jin, Norah Sadowski, Carla Alamillo-Ferrer, J Robert Hogg, Bassem R Haddad, David H Drewry, Carrow I Wells, Julie E. Pickett, William J Zuercher, Winston Timp, Rebecca B Riggins
bioRxiv 661660; doi: https://doi.org/10.1101/661660

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