f32f4367302afa4cd09a6548c1aae454 313049.pdf 72e625bc599a089560e132e8ef8ebf88424ba610 313049.pdf 751d8d7945f23865c1842ba63c7aa323fc85c632c56199df7488a8de235e7d11 313049.pdf Title: Multikinase Treatment of Glioblastoma: Evaluating the Rationale for Regorafenib Subject: We explored the rationale for treating glioblastoma (GBM) with regorafenib. In 103 newly diagnosed GBM patients, we assessed mutations, copy number variants (CNVs), fusions, and overexpression in 46 genes encoding protein kinases (PKs) potentially targeted by regorafenib or its metabolites and performed a functional enrichment analysis to assess their implications in angiogenesis. We analyzed regorafenib’s binding inhibitory activity and target affinity for these 46 PKs and focused on a subset of 18 genes inhibited by regorafenib at clinically achievable concentrations and on 19 genes involved in angiogenesis. Putative oncogenic alterations were defined as oncogenic/likely oncogenic mutations, oncogenic fusions, CNVs > 5, and/or gene overexpression. Regorafenib did not target all 46 PKs. For the 46-gene set, 40 genes (86.9%) and 73 patients (70.8%) harbored at least one alteration in genes encoding targetable PKs, but putative oncogenic alterations were present in only 34 patients (33%). In the 18-gene set, 18 genes (100%) and 48 patients (46.6%) harbored alterations, but putative oncogenic alterations were detected in only 26 patients (25.2%). Thirty patients (29.1%) had oncogenic alterations in the 18-gene set and/or in angiogenesis-related genes. Around 33% of patients had oncogenic alterations in any of the 46 potential targets. Additionally, the suboptimal dosing of regorafenib, due to its poor penetration of the blood–brain barrier, may reduce the likelihood of effectively targeting certain PKs. Future use of multi-target drugs must be guided by a thorough understanding of target presence, effective inhibition, and the drug’s ability to reach brain tumors at adequate concentrations. Keywords: glioblastoma; multikinase treatment; regorafenib; molecular target Author: Ana Maria Muñoz-Mármol, Bárbara Meléndez, Ainhoa Hernandez, Carolina Sanz, Marta Domenech, Oriol Arpí-Llucia, Marta Gut, Anna Esteve, Anna Esteve-Codina, Genis Parra, Cristina Carrato, Iban Aldecoa, Mar Mallo, Estela Pineda, Francesc Alameda, Nuria de la Iglesia, Eva Martinez-Balibrea, Anna Martinez-Cardús, Anna Estival-Gonzalez and Carmen Balana Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25 CreationDate: Thu Jan 23 16:48:32 2025 CET ModDate: Mon Jan 27 10:39:40 2025 CET Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 17 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 1726293 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- QKZKVH+VnURWPalladioL Type 1 Custom yes yes yes 10 0 VOWEQV+URWPalladioL-Roma Type 1 Custom yes yes yes 16 0 ZEYSOO+URWPalladioL-Bold Type 1 Custom yes yes yes 22 0 TAKLHZ+URWPalladioL-Ital Type 1 Custom yes yes yes 27 0 IKKWKP+CMSY10 Type 1 Builtin yes yes yes 59 0 DUNMPX+URWPalladioL-BoldItal Type 1 Custom yes yes yes 64 0 FKLPPG+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 85 0 FKLPPH+PalatinoLinotype CID TrueType Identity-H yes yes yes 88 0 FKLPPI+PalatinoLinotype TrueType WinAnsi yes yes no 94 0 FKLPPK+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 97 0 FKMALJ+PalatinoLinotype TrueType MacRoman yes yes no 100 0 FKMKMN+Calibri CID TrueType Identity-H yes yes yes 204 0 FKLPPG+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 210 0 FKLPPH+PalatinoLinotype CID TrueType Identity-H yes yes yes 213 0 FKLPPI+PalatinoLinotype TrueType WinAnsi yes yes no 219 0 FKLPPK+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 222 0 FKMALJ+PalatinoLinotype TrueType MacRoman yes yes no 225 0 FKLPPG+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 285 0 FKLPPH+PalatinoLinotype CID TrueType Identity-H yes yes yes 288 0 FKLPPI+PalatinoLinotype TrueType WinAnsi yes yes no 294 0 FKLPPK+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 297 0 FKMALJ+PalatinoLinotype TrueType MacRoman yes yes no 300 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2025-07-03 02:45:31 CEST RepresentationInformation: 313049.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2025-07-02 09:29:29 CEST Size: 1726293 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 556 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Materials and Methods Destination: section.2 Children: Item: Title: Patients Destination: subsection.2.1 Item: Title: Selection of Target PKs Destination: subsection.2.2 Item: Title: Molecular Analyses Destination: subsection.2.3 Item: Title: Statistical Analysis Destination: subsection.2.4 Item: Title: Results Destination: section.3 Children: Item: Title: Molecular Alterations in Genes Destination: subsection.3.1 Item: Title: Identification of Potential Candidates for Regorafenib Treatment Destination: subsection.3.2 Children: Item: Title: Alterations in Patient Samples Destination: subsubsection.3.2.1 Item: Title: Analysis of Potential Targets Destination: subsubsection.3.2.2 Item: Title: Discussion Destination: section.4 Item: Title: References Destination: appendix.A. Info: Title: Multikinase Treatment of Glioblastoma: Evaluating the Rationale for Regorafenib Author: Ana Maria Muñoz-Mármol, Bárbara Meléndez, Ainhoa Hernandez, Carolina Sanz, Marta Domenech, Oriol Arpí-Llucia, Marta Gut, Anna Esteve, Anna Esteve-Codina, Genis Parra, Cristina Carrato, Iban Aldecoa, Mar Mallo, Estela Pineda, Francesc Alameda, Nuria de la Iglesia, Eva Martinez-Balibrea, Anna Martinez-Cardús, Anna Estival-Gonzalez and Carmen Balana Subject: We explored the rationale for treating glioblastoma (GBM) with regorafenib. In 103 newly diagnosed GBM patients, we assessed mutations, copy number variants (CNVs), fusions, and overexpression in 46 genes encoding protein kinases (PKs) potentially targeted by regorafenib or its metabolites and performed a functional enrichment analysis to assess their implications in angiogenesis. We analyzed regorafenib’s binding inhibitory activity and target affinity for these 46 PKs and focused on a subset of 18 genes inhibited by regorafenib at clinically achievable concentrations and on 19 genes involved in angiogenesis. Putative oncogenic alterations were defined as oncogenic/likely oncogenic mutations, oncogenic fusions, CNVs > 5, and/or gene overexpression. Regorafenib did not target all 46 PKs. For the 46-gene set, 40 genes (86.9%) and 73 patients (70.8%) harbored at least one alteration in genes encoding targetable PKs, but putative oncogenic alterations were present in only 34 patients (33%). In the 18-gene set, 18 genes (100%) and 48 patients (46.6%) harbored alterations, but putative oncogenic alterations were detected in only 26 patients (25.2%). Thirty patients (29.1%) had oncogenic alterations in the 18-gene set and/or in angiogenesis-related genes. Around 33% of patients had oncogenic alterations in any of the 46 potential targets. Additionally, the suboptimal dosing of regorafenib, due to its poor penetration of the blood brain barrier, may reduce the likelihood of effectively targeting certain PKs. Future use of multi-target drugs must be guided by a thorough understanding of target presence, effective inhibition, and the drug’s ability to reach brain tumors at adequate concentrations. 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