Abstract Glioblastoma (GBM), the most common malignant primary brain cancer in adults, has an average survival of one year. Treatment includes maximal safe resection, followed by chemoradiation and adjuvant temozolomide (TMZ), the latter only increasing median survival by 2.5 months. Moreover, most targeted therapy trials have been unsuccessful due to activation of tyrosine kinase receptors and relative blood-brain barrier (BBB) impermeability. Thus, a pressing need remains to find a more effective therapy. This study builds on preliminary data generated using Connectivity Map (CMap), developed by the BROAD institute to identify drugs for repurposing based on cancer’s genetic profile. By analyzing 99 GBM and 38 adjacent normal samples from 4 datasets, CMap identified histone deacetylase (HDAC) inhibitors as top candidates. Database analysis using GEPIA identified HDAC1 and HDAC2 as the most upregulated and HDAC11 as the most downregulated HDACs. Thus, we selected the BBB permeant PCI-24781/abexinostat due to its specificity for GBM gene signature- specific HDACs (inhibiting HDAC1 and HDAC2, but not HDAC11, or class IIa HDACs) to evaluate. In preliminary studies, as compared to other pan HDAC inhibitors, PCI-24781 induced significantly greater apoptosis and downregulated DNA repair machinery (CHK1, RAD51, and MGMT) in GBM cell lines in vitro. Further, PCI-24781 efficiently decreased the tumor burden in orthotopic murine models in combination with TMZ compared to vorinostat with TMZ and enhanced survival. From this, we hypothesize that inhibiting class 1 HDACs with PCI- 24781 will enhance the efficacy of TMZ in GBM by targeting DNA repair machinery. To test this hypothesis, we propose two specific aims: In Aim 1, we will evaluate the efficacy of PCI-24781 with TMZ in in vivo GBM models. Murine GBM organoids, genetically engineered GBM mouse models, and patient-derived xenografts will be used to evaluate BBB permeability and to measure toxicity. Therapeutic efficacy and survival will be recorded for each group of mice treated with PCI-24781 and TMZ singly and in combination. We will analyze the effect of combination therapy on DNA repair machinery proteins by IHC in resected tumors and will also perform RNA- seq and Omni-ATAC-seq analyses to identify additional pathways and chromatin accessibility, respectively, impacted by PCI-24781. In Aim 2, a phase 1 clinical trial will evaluate toxicity and determine the MTD of PCI- 24781 with TMZ in recurrent high-grade glioma patients. We will analyze patient exosomes to demonstrate protein acetylation and BBB permeability. Together, these aims will elucidate mechanisms for synergy between and tolerance of PCI-24781 with TMZ in GBM. Combining PCI-24781 with TMZ will successfully overcome TMZ resistance, negatively impacting tumor growth and recurrence and effectively improving the survival of GBM patients.