Diffuse midline gliomas (DMG), including diffuse intrinsic pontine gliomas (DIPG), are the most lethal of childhood cancers. Palliative radiotherapy is the only established treatment, with median patient survival of 9 to 11 months. ONC201 is a DRD2 antagonist and ClpP agonist that has shown preclinical and emerging clinical efficacy in DMG. However, further work is needed to identify the mechanisms of response of DIPGs to ONC201 treatment and to determine whether recurring genomic features influence response. Using a systems-biological approach, we showed that ONC201 elicits potent agonism of the mitochondrial protease ClpP to drive proteolysis of electron transport chain and tricarboxylic acid cycle proteins. DIPGs harboring PIK3CA mutations showed increased sensitivity to ONC201, whereas those harboring TP53 mutations were more resistant. Metabolic adaptation and reduced sensitivity to ONC201 was promoted by redox-activated PI3K/Akt signaling, which could be counteracted using the brain penetrant PI3K/Akt inhibitor, paxalisib. Together, these discoveries coupled with the powerful anti-DIPG/DMG pharmacokinetic and pharmacodynamic properties of ONC201 and paxalisib have provided the rationale for the ongoing DIPG/DMG phase II combination clinical trial NCT05009992. Significance: PI3K/Akt signaling promotes metabolic adaptation to ONC201-mediated disruption of mitochondrial energy homeostasis in diffuse intrinsic pontine glioma, highlighting the utility of a combination treatment strategy using ONC201 and the PI3K/Akt inhibitor paxalisib.

The concept of polypharmacology involves the interaction of drug molecules with multiple molecular targets. It provides a unique opportunity for the repurposing of already‐approved drugs to target key factors involved in human diseases. Herein, we used an in silico target prediction algorithm to investigate the mechanism of action of mebendazole, an antihelminthic drug, currently repurposed in the treatment of brain tumors. First, we confirmed that mebendazole decreased the viability of glioblastoma cells in vitro (IC₅₀ values ranging from 288 nm to 2.1 µm). Our in silico approach unveiled 21 putative molecular targets for mebendazole, including 12 proteins significantly upregulated at the gene level in glioblastoma as compared to normal brain tissue (fold change > 1.5; P < 0.0001). Validation experiments were performed on three major kinases involved in cancer biology: ABL1, MAPK1/ERK2, and MAPK14/p38α. Mebendazole could inhibit the activity of these kinases in vitro in a dose‐dependent manner, with a high potency against MAPK14 (IC₅₀ = 104 ± 46 nm). Its direct binding to MAPK14 was further validated in vitro, and inhibition of MAPK14 kinase activity was confirmed in live glioblastoma cells. Consistent with biophysical data, molecular modeling suggested that mebendazole was able to bind to the catalytic site of MAPK14. Finally, gene silencing demonstrated that MAPK14 is involved in glioblastoma tumor spheroid growth and response to mebendazole treatment. This study thus highlighted the role of MAPK14 in the anticancer mechanism of action of mebendazole and provides further rationale for the pharmacological targeting of MAPK14 in brain tumors. It also opens new avenues for the development of novel MAPK14/p38α inhibitors to treat human diseases.

Despite recent advances in deciphering cancer drug resistance mechanisms, relapse is a widely observed phenomenon in advanced cancers, mainly due to intratumor clonal heterogeneity. How tumor clones progress and impact each other remains elusive. In this study, we developed 2D and 3D non-small cell lung cancer co-culture systems and defined a phenomenological mathematical model to better understand clone dynamics. Our results demonstrated that the drug-sensitive clones inhibit the proliferation of the drug-resistant ones under untreated conditions. Model predictions and their experimental in vitro and in vivo validations indicated that a metronomic schedule leads to a better regulation of tumor cell heterogeneity over time than a maximum-tolerated dose schedule, while achieving control of tumor progression. We finally showed that drug-sensitive and -resistant clones exhibited different metabolic statuses that could be involved in controlling the intratumor heterogeneity dynamics. Our data suggested that the glycolytic activity of drug-sensitive clones could play a major role in inhibiting the drug-resistant clone proliferation. Altogether, these computational and experimental approaches provide foundations for using metronomic therapy to control drug-sensitive and -resistant clone balance and highlight the potential of targeting cell metabolism to manage intratumor heterogeneity.

Pharmacological synergisms are an attractive anticancer strategy. However, with more than 5000 approved-drugs and compounds in clinical development, identifying synergistic treatments represents a major challenge. High-throughput screening was combined with target deconvolution and functional genomics to reveal targetable vulnerabilities in glioblastoma. The role of the top gene hit was investigated by RNA interference, transcriptomics and immunohistochemistry in glioblastoma patient samples. Drug combination screen using a custom-made library of 88 compounds in association with six inhibitors of the identified glioblastoma vulnerabilities was performed to unveil pharmacological synergisms. Glioblastoma 3D spheroid, organotypic ex vivo and syngeneic orthotopic mouse models were used to validate synergistic treatments. Nine targetable vulnerabilities were identified in glioblastoma and the top gene hit RRM1 was validated as an independent prognostic factor. The associations of CHK1/MEK and AURKA/BET inhibitors were identified as the most potent amongst 528 tested pairwise drug combinations and their efficacy was validated in 3D spheroid models. The high synergism of AURKA/BET dual inhibition was confirmed in ex vivo and in vivo glioblastoma models, without detectable toxicity. Our work provides strong pre-clinical evidence of the efficacy of AURKA/BET inhibitor combination in glioblastoma and opens new therapeutic avenues for this unmet medical need. Besides, we established the proof-of-concept of a stepwise approach aiming at exploiting drug poly-pharmacology to unveil druggable cancer vulnerabilities and to fast-track the identification of synergistic combinations against refractory cancers. This study was funded by institutional grants and charities.

Medulloblastoma is the most frequent brain malignancy of childhood. The current multimodal treatment comes at the expense of serious and often long-lasting side effects. Drug repurposing is a strategy to fast-track anti-cancer therapy with low toxicity. Here, we showed the ability of β-blockers to potentiate radiotherapy in medulloblastoma with bad prognosis. Medulloblastoma cell lines, patient-derived xenograft cells, 3D spheroids and an innovative cerebellar organotypic model were used to identify synergistic interactions between β-blockers and ionising radiations. Gene expression profiles of β-adrenergic receptors were analysed in medulloblastoma samples from 240 patients. Signaling pathways were explored by RT-qPCR, RNA interference, western blotting and RNA sequencing. Medulloblastoma cell bioenergetics were evaluated by measuring the oxygen consumption rate, the extracellular acidification rate and superoxide production. Low concentrations of β-blockers significantly potentiated clinically relevant radiation protocols. Although patient biopsies showed detectable expression of β-adrenergic receptors, the ability of the repurposed drugs to potentiate ionising radiations did not result from the inhibition of the canonical signaling pathway. We highlighted that the efficacy of the combinatorial treatment relied on a metabolic catastrophe that deprives medulloblastoma cells of their adaptive bioenergetics capacities. This led to an overproduction of superoxide radicals and ultimately to an increase in ionising radiations-mediated DNA damages. These data provide the evidence of the efficacy of β-blockers as potentiators of radiotherapy in medulloblastoma, which may help improve the treatment and quality of life of children with high-risk brain tumours. This study was funded by institutional grants and charities.

Drug development in oncology is highly challenging, with less than 5% success rate in clinical trials. This alarming figure points out the need to study in more details the multiple biological effects of drugs in specific contexts. Indeed, the comprehensive assessment of drug poly-pharmacology can provide insights into their therapeutic and adverse effects, to optimize their utilization and maximize the success rate of clinical trials. Recent technological advances have made possible in-depth investigation of drug poly-pharmacology. This review first highlights high-throughput methodologies that have been used to unveil new mechanisms of action of existing drugs. Then, we discuss how emerging chemo-proteomics strategies allow effectively dissecting the poly-pharmacology of drugs in an unsupervised manner.