Antiretroviral Drug Repositioning for Glioblastoma

Abstract

Outcomes for glioblastoma (GBM) remain poor despite standard-of-care treatments including surgical resection, radiation, and chemotherapy. Intratumoral heterogeneity contributes to treatment resistance and poor prognosis, thus demanding novel therapeutic approaches. Drug repositioning studies on antiretroviral therapy (ART) have shown promising potent antineoplastic effects in multiple cancers; however, its efficacy in GBM remains unclear. To better understand the pleiotropic anticancer effects of ART on GBM, we conducted a comprehensive drug repurposing analysis of ART in GBM to highlight its utility in translational neuro-oncology. To uncover the anticancer role of ART in GBM, we conducted a comprehensive bioinformatic and in vitro screen of antiretrovirals against glioblastoma. Using the DepMap repository and reversal of gene expression score, we conducted an unbiased screen of 16 antiretrovirals in 40 glioma cell lines to identify promising candidates for GBM drug repositioning. We utilized patient-derived neurospheres and glioma cell lines to assess neurosphere viability, proliferation, and stemness. Our in silico screen revealed that several ART drugs including reverse transcriptase inhibitors (RTIs) and protease inhibitors (PIs) demonstrated marked anti-glioma activity with the capability of reversing the GBM disease signature. RTIs effectively decreased cell viability, GBM stem cell markers, and proliferation. Our study provides mechanistic and functional insight into the utility of ART repurposing for malignant gliomas, which supports the current literature. Given their safety profile, preclinical efficacy, and neuropenetrance, ARTs may be a promising adjuvant treatment for GBM.

Keywords: abacavir; antiretroviral; drug repurposing; glioblastoma; lamivudine; reverse transcriptase inhibitors.

Figure 1

Unbiased screen of ART identifies candidate drugs with potent anti-glioma activity. (A) Heatmap illustrating the impact of antiretroviral drugs on the survival of individual glioma cell lines in terms of log₂fold change (range = −2.13, 1.92, SD= 0.4692). Data sourced from the PRISM Repurposing Screen and analyzed using DepMap, with warmer colors indicating improved survival and cooler colors indicating reduced survival. (B) Heatmap depicting the capacity of individual antiretroviral drugs to reverse the genetic signature of various primary cancers based on sRGESs calculated using drug expression profiles accessed from the LINCS L1000 assay (range = −0.37, 0.22, SD = 0.124). (C) Heatmap illustrating the impact of select antiretroviral drugs on the expression of 9 genes critically associated with reversal of genetic signature in glioblastoma. (D) Table supplying sRGES and CNS-MPO values for each antiretroviral drug to inform both the effect of each drug on genetic signature reversal and the drug’s optimal pharmacokinetic profile [sRGES range: −0.0235, −0.630, SD: 0.282], [CNS-MPO range: 1.53, 5.83, SD: 1.78].

Figure 2

In vitro validation of candidate ARTs. (A) XTT assay of patient-derived glioblastoma cell lines (GBM 28, 43) and a pure glioma cell line (A172) showing the effect of select antiretrovirals on cell viability relative to increased dosing. (B) Violin plots depicting the impact of select antiretroviral drugs on the survival of established cell lines stratified based on primary cancers in the Cancer Dependency Map database with greater than 10 established cell lines. This demonstrates the relative effect of each antiretroviral drug on the log₂fold change in survival for each primary cancer. Highlighted plots represent GBM and demonstrate a median log₂fold change of less than 0 when treated with abacavir, lamivudine, and raltegravir.

Figure 3

Reverse transcriptase inhibitors decrease stemness and self-renewal capacity. (A,B) Identified foci of stemness markers using IF of A172 and U87 cells treated with ABC 20 μM and LMV 20 μM for 48 h. OCT-4 (green), vimentin (red), and DAPI. (C,D) Quantification of fold change of foci shows a significant decrease in expression after treatment. (E,F) Cell proliferation assessed by xCelligence assay of A172 and U87 cell lines while treated with ABC 20 μM and LMV 20 μM: RTIs significantly decreased proliferation compared to control. (ANOVA, r * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Figure 4

Synergistic effects of ART against the genetic landscape in GBM. (A) Heatmap depicting a correlation matrix that represents the comprehensive evaluation of antiretroviral drug efficacy against glioma cell lines using the Cancer Dependency Map (DepMap) database. In total, 16 antiretroviral drugs were identified from 4518 drugs in the PRISM Repurposing Screen. Each cell indicates the strengths of the Pearson correlation coefficient (r) between two antiretrovirals, highlighting their comparative effectiveness. More positive values signify a strong positive correlation, suggesting similar effectiveness, while more negative values indicate more distinct efficacy profiles of the compared drugs. (B) Schematic of ART capacity to decrease proliferation, stemness, migration, and invasion in CNS tumors. Figure depicts RTI involvement in decreased proliferation and promoting differentiation. Additionally, PIs are involved in mitigating migration and invasion. (C) Data obtained from SynergySeq used to evaluate drugs from the LINCS 1000 small molecules database for disease transcriptional response signatures. Scatterplot of all ARTs (blue) and drugs from recent and current NCI-supported GBM clinical trials (red) that were identified in the database depicting similarity to the transcriptional response of GBM to TMZ (x-axis) and degree of glioblastoma (GBM) transcriptional signature reversal (y-axis) derived from an independent cohort of 71 patients and matched normal brain tissue.

Figure 5

Systematic review flow chart for preclinical trials utilizing antiretroviral medications for the treatment of established glioma cell lines and clinical trials utilizing antiretroviral medications for the treatment of various cancers.