3b9c3bdb988fe26fe038342592d3da1f 317416.pdf 40eceb0d6f7881bc01a48ada25d2bc6d6ff67532 317416.pdf 7ef41fa7a07cf62b578c0a8949455e6856b68707e6498e502d1c3cc9895605ec 317416.pdf Title: Tinostamustine (EDO-S101), an Alkylating Deacetylase Inhibitor, Enhances the Efficacy of Daratumumab in Multiple Myeloma by Upregulation of CD38 and NKG2D Ligands Subject: Multiple myeloma is a malignancy characterized by the accumulation of malignant plasma cells in bone marrow and the production of monoclonal immunoglobulin. A hallmark of cancer is the evasion of immune surveillance. Histone deacetylase inhibitors have been shown to promote the expression of silenced molecules and hold potential to increase the anti-MM efficacy of immunotherapy. The aim of the present work was to assess the potential effect of tinostamustine (EDO-S101), a first-in-class alkylating deacetylase inhibitor, in combination with daratumumab, an anti-CD38 monoclonal antibody (mAb), through different preclinical studies. Tinostamustine increases CD38 expression in myeloma cell lines, an effect that occurs in parallel with an increment in CD38 histone H3 acetylation levels. Also, the expression of MICA and MICB, ligands for the NK cell activating receptor NKG2D, augments after tinostamustine treatment in myeloma cell lines and primary myeloma cells. Pretreatment of myeloma cell lines with tinostamustine increased the sensitivity of these cells to daratumumab through its different cytotoxic mechanisms, and the combination of these two drugs showed a higher anti-myeloma effect than individual treatments in ex vivo cultures of myeloma patients’ samples. In vivo data confirmed that tinostamustine pretreatment followed by daratumumab administration significantly delayed tumor growth and improved the survival of mice compared to individual treatments. In summary, our results suggest that tinostamustine could be a potential candidate to improve the efficacy of anti-CD38 mAbs. Keywords: multiple myeloma; daratumumab; tinostamustine; immunotherapy Author: Andrea Díaz-Tejedor, Javier Rodríguez-Ubreva, Laura Ciudad, Mauro Lorenzo-Mohamed, Marta González-Rodríguez, Bárbara Castellanos, Janet Sotolongo-Ravelo, Laura San-Segundo, Luis A. Corchete, Lorena González-Méndez, Montserrat Martín-Sánchez, María-Victoria Mateos, Enrique M. Ocio, Mercedes Garayoa and Teresa Paíno Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25 CreationDate: Mon Apr 29 11:18:13 2024 CEST ModDate: Mon Apr 29 11:25:21 2024 CEST Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 15 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 4075600 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- IOJCNK+VnURWPalladioL-Bold Type 1 Custom yes yes yes 10 0 MNLGEK+URWPalladioL-Bold Type 1 Custom yes yes yes 16 0 NXWQJX+URWPalladioL-Roma Type 1 Custom yes yes yes 22 0 HWQDMO+URWPalladioL-Ital Type 1 Custom yes yes yes 27 0 PSKSBY+VnURWPalladioL Type 1 Custom yes yes yes 32 0 IRWHZX+TimesNewRomanPSMT Type 1C WinAnsi yes yes no 48 0 CKUPDE+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 51 0 HPTLXK+EURM10 Type 1 Builtin yes yes yes 68 0 JYUNWZ+CMSY10 Type 1 Builtin yes yes yes 76 0 FJIAJL+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 93 0 FJIAJM+PalatinoLinotype CID TrueType Identity-H yes yes yes 96 0 FJIAJN+PalatinoLinotype TrueType WinAnsi yes yes no 102 0 FJIAKO+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 105 0 FJIBEL+PalatinoLinotype TrueType MacRoman yes yes no 108 0 FJIAJL+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 131 0 FJIAJM+PalatinoLinotype CID TrueType Identity-H yes yes yes 134 0 FJIAJN+PalatinoLinotype TrueType WinAnsi yes yes no 140 0 FJIAKO+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 143 0 FJIBEL+PalatinoLinotype TrueType MacRoman yes yes no 146 0 FJIAJL+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 173 0 FJIAJM+PalatinoLinotype CID TrueType Identity-H yes yes yes 176 0 FJIAJN+PalatinoLinotype TrueType WinAnsi yes yes no 182 0 FJIAKO+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 185 0 FJIBEL+PalatinoLinotype TrueType MacRoman yes yes no 188 0 FJIAJL+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 216 0 FJIAJM+PalatinoLinotype CID TrueType Identity-H yes yes yes 219 0 FJIAJN+PalatinoLinotype TrueType WinAnsi yes yes no 225 0 FJIAKO+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 228 0 FJIBEL+PalatinoLinotype TrueType MacRoman yes yes no 231 0 FJIAJL+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 251 0 FJIAJM+PalatinoLinotype CID TrueType Identity-H yes yes yes 254 0 FJIAJN+PalatinoLinotype TrueType WinAnsi yes yes no 260 0 FJIAKO+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 263 0 FJIBEL+PalatinoLinotype TrueType MacRoman yes yes no 266 0 WABLSD+SFRM1000 Type 1 Custom yes yes yes 284 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2025-09-09 03:49:19 CEST RepresentationInformation: 317416.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2025-09-08 13:49:46 CEST Size: 4075600 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 508 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Results Destination: section.2 Children: Item: Title: Tinostamustine Increases CD38 Expression in Myeloma Cells Destination: subsection.2.1 Item: Title: Tinostamustine Increases the Expression of MICA and MICB, Ligands for the Activating NK Cell Receptor NKG2D, in Myeloma Cell Lines and Malignant Plasma Cells from Myeloma Patients Destination: subsection.2.2 Item: Title: Tinostamustine Enhances Daratumumab-Mediated Anti-Myeloma Activity In Vitro and Ex Vivo Destination: subsection.2.3 Item: Title: Tinostamustine Enhances Daratumumab-Mediated Anti-Myeloma Activity In Vivo Destination: subsection.2.4 Item: Title: Discussion Destination: section.3 Item: Title: Materials and Methods Destination: section.4 Children: Item: Title: Drugs and Reagents Destination: subsection.4.1 Item: Title: MM Cell Lines, Patient Samples and Cultures Destination: subsection.4.2 Item: Title: Flow Cytometry (FCM) Destination: subsection.4.3 Item: Title: Real-Time Quantitative PCR (RT-qPCR) Destination: subsection.4.4 Item: Title: Chromatin Immunoprecipitation (ChIP) Destination: subsection.4.5 Item: Title: Evaluation of Daratumumab Binding to Myeloma Cells Destination: subsection.4.6 Item: Title: ADCC, CDC and Apoptosis via Crosslinking Destination: subsection.4.7 Item: Title: Ex Vivo Analysis of Apoptosis in BM Samples from Myeloma Patients Destination: subsection.4.8 Item: Title: In Vivo Evaluation of the Efficacy of the Combination of Tinostamustine + Daratumumab in Subcutaneous Plasmacytoma Models Destination: subsection.4.9 Item: Title: Statistical Analyses Destination: subsection.4.10 Item: Title: References Destination: appendix.A. Info: Title: Tinostamustine (EDO-S101), an Alkylating Deacetylase Inhibitor, Enhances the Efficacy of Daratumumab in Multiple Myeloma by Upregulation of CD38 and NKG2D Ligands Author: Andrea Díaz-Tejedor, Javier Rodríguez-Ubreva, Laura Ciudad, Mauro Lorenzo-Mohamed, Marta González-Rodríguez, Bárbara Castellanos, Janet Sotolongo-Ravelo, Laura San-Segundo, Luis A. Corchete, Lorena González-Méndez, Montserrat Martín-Sánchez, María-Victoria Mateos, Enrique M. Ocio, Mercedes Garayoa and Teresa Paíno Subject: Multiple myeloma is a malignancy characterized by the accumulation of malignant plasma cells in bone marrow and the production of monoclonal immunoglobulin. A hallmark of cancer is the evasion of immune surveillance. Histone deacetylase inhibitors have been shown to promote the expression of silenced molecules and hold potential to increase the anti-MM efficacy of immunotherapy. The aim of the present work was to assess the potential effect of tinostamustine (EDO-S101), a first-in-class alkylating deacetylase inhibitor, in combination with daratumumab, an anti-CD38 monoclonal antibody (mAb), through different preclinical studies. Tinostamustine increases CD38 expression in myeloma cell lines, an effect that occurs in parallel with an increment in CD38 histone H3 acetylation levels. Also, the expression of MICA and MICB, ligands for the NK cell activating receptor NKG2D, augments after tinostamustine treatment in myeloma cell lines and primary myeloma cells. Pretreatment of myeloma cell lines with tinostamustine increased the sensitivity of these cells to daratumumab through its different cytotoxic mechanisms, and the combination of these two drugs showed a higher anti-myeloma effect than individual treatments in ex vivo cultures of myeloma patients’ samples. In vivo data confirmed that tinostamustine pretreatment followed by daratumumab administration significantly delayed tumor growth and improved the survival of mice compared to individual treatments. In summary, our results suggest that tinostamustine could be a potential candidate to improve the efficacy of anti-CD38 mAbs. 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