Combination drug screen identifies synergistic drug interaction of BCL-XL and class I histone deacetylase inhibitors in MYC-amplified medulloblastoma cells.

PURPOSE: Patients with MYC-amplified Group 3 medulloblastoma (MB) (subtype II) show poor progression-free survival rates. Class I histone deacetylase inhibitors (HDACi) are highly effective for the treatment of MYC-amplified MB in vitro and in vivo. Drug combination regimens including class I HDACi may represent an urgently needed novel treatment approach for this high risk disease. METHODS: A medium-throughput in vitro combination drug screen was performed in three MYC-amplified and one non-MYC-amplified MB cell line testing 75 clinically relevant drugs alone and in combination with entinostat. The drug sensitivity score (DSS) was calculated based on metabolic inhibition quantified by CellTiter-Glo. The six top synergistic combination hits were evaluated in a 5 × 5 combination matrix and a seven-ray design. Synergy was validated and characterized by cell counts, caspase-3-like-activity and poly-(ADP-ribose)-polymerase-(PARP)-cleavage. On-target activity of drugs was validated by immunoprecipitation and western blot. BCL-XL dependency of the observed effect was explored with siRNA mediated knockdown of BCL2L1, and selective inhibition with targeted compounds (A-1331852, A-1155463). RESULTS: 20/75 drugs effectively reduced metabolic activity in combination with entinostat in all three MYC-amplified cell lines (DSS ≥ 10). The combination entinostat and navitoclax showed the strongest synergistic interaction across all MYC-amplified cell lines. siRNA mediated knockdown of BCL2L1, as well as targeted inhibition with selective inhibitors showed BCL-XL dependency of the observed effect. Increased cell death was associated with increased caspase-3-like-activity. CONCLUSION: Our study identifies the combination of class I HDACi and BCL-XL inhibitors as a potential new approach for the treatment of MYC-amplified MB cells.

Class I HDAC inhibition reduces DNA damage repair capacity of MYC-amplified medulloblastoma cells.

PURPOSE: MYC-driven Group 3 medulloblastoma (MB) (subtype II) is a highly aggressive childhood brain tumor. Sensitivity of MYC-driven MB to class I histone deacetylase inhibitors (HDACi) has been previously demonstrated in vitro and in vivo. In this study we characterize the transcriptional effects of class I HDACi in MYC-driven MB and explore beneficial drug combinations. METHODS: MYC-amplified Group 3 MB cells (HD-MB03) were treated with class I HDACi entinostat. Changes in the gene expression profile were quantified on a microarray. Bioinformatic assessment led to the identification of pathways affected by entinostat treatment. Five drugs interfering with these pathways (olaparib, idasanutlin, ribociclib, selinexor, vinblastine) were tested for synergy with entinostat in WST-8 metabolic activity assays in a 5 × 5 combination matrix design. Synergy was validated in cell count and flow cytometry experiments. The effect of entinostat and olaparib on DNA damage was evaluated by γH2A.X quantification in immunoblotting, fluorescence microscopy and flow cytometry. RESULTS: Entinostat treatment changed the expression of genes involved in 22 pathways, including downregulation of DNA damage response. The PARP1 inhibitors olaparib and pamiparib showed synergy with entinostat selectively in MYC-amplified MB cells, leading to increased cell death, decreased viability and increased formation of double strand breaks, as well as increased sensitivity to additional induction of DNA damage by doxorubicin. Non-MYC-amplified MB cells and normal human fibroblasts were not susceptible to this triple treatment. CONCLUSION: Our study identifies the combination of entinostat with olaparib as a new potential therapeutic approach for MYC-driven Group 3 MB.

A Cell-Based MAPK Reporter Assay Reveals Synergistic MAPK Pathway Activity Suppression by MAPK Inhibitor Combination in BRAF-Driven Pediatric Low-Grade Glioma Cells.

Pilocytic astrocytomas as well as other pediatric low-grade gliomas (pLGG) exhibit genetic events leading to aberrant activation of the MAPK pathway. The most common alterations are KIAA1549:BRAF fusions and BRAFV600E and NF1 mutations. Novel drugs targeting the MAPK pathway (MAPKi) are prime candidates for the treatment of these single-pathway diseases. We aimed to develop an assay suitable for preclinical testing of MAPKi in pLGGs with the goal to identify novel MAPK pathway-suppressing synergistic drug combinations. A reporter plasmid (pDIPZ) with a MAPK-responsive ELK-1-binding element driving the expression of destabilized firefly luciferase was generated and packaged using a lentiviral vector system. Pediatric glioma cell lines with a BRAF fusion (DKFZ-BT66) and a BRAFV600E mutation (BT-40) background, respectively, were stably transfected. Modulation of the MAPK pathway activity by MAPKi was measured using the luciferase reporter and validated by detection of phosphorylated protein levels. A screening of a MAPKi library was performed, and synergy of selected combinations was calculated. Screening of a MAPKi library revealed MEK inhibitors as the class inhibiting the pathway with the lowest IC50s, followed by ERK and next-generation RAF inhibitors. Combination treatments with different MAPKi classes showed synergistic effects in BRAF fusion as well as BRAFV600E mutation backgrounds. Here, we report a novel reporter assay for medium- to high-throughput preclinical drug testing in pLGG cell lines. The assay confirmed MEK, ERK, and next-generation RAF inhibitors as potential treatment approaches for KIAA1549:BRAF and BRAFV600E-mutated pLGGs. In addition, the assay revealed that combination treatments synergistically suppressed MAPK pathway activity.