In this issue, Pine and colleagues compared single-cell RNA-sequencing data across four distinct types of glioblastoma stem cell-derived tumor models, reinforcing the importance of a three-dimensional microenvironment for accurate recapitulation of cellular states.See related article by Pine et al., p. 964.

On-target-off-tissue drug engagement is an important source of adverse effects that constrains the therapeutic window of drug candidates^1,2. In diseases of the central nervous system, drugs with brain-restricted pharmacology are highly desirable. Here we report a strategy to achieve inhibition of mammalian target of rapamycin (mTOR) while sparing mTOR activity elsewhere through the use of the brain-permeable mTOR inhibitor RapaLink-1 and the brain-impermeable FKBP12 ligand RapaBlock. We show that this drug combination mitigates the systemic effects of mTOR inhibitors but retains the efficacy of RapaLink-1 in glioblastoma xenografts. We further present a general method to design cell-permeable, FKBP12-dependent kinase inhibitors from known drug scaffolds. These inhibitors are sensitive to deactivation by RapaBlock, enabling the brain-restricted inhibition of their respective kinase targets.

The mTOR signaling is dysregulated prominently in human cancers including glioblastoma, suggesting mTOR as a robust target for therapy. Inhibitors of mTOR have had limited success clinically, however, in part because their mechanism of action is cytostatic rather than cytotoxic. Here, we tested three distinct mTOR kinase inhibitors (TORKi) PP242, KU-0063794, and sapanisertib against glioblastoma cells. All agents similarly decreased proliferation of glioblastoma cells, whereas PP242 uniquely induced apoptosis. Apoptosis induced by PP242 resulted from off-target cooperative inhibition of JAK2 and protein kinase C alpha (PKCα). Induction of apoptosis was also decreased by additional on-target inhibition of mTOR, due to induction of autophagy. As EGFR inhibitors can block PKCα, EGFR inhibitors erlotinib and osimertinib were tested separately in combination with the JAK2 inhibitor AZD1480. Combination therapy induced apoptosis of glioblastoma tumors in both flank and in patient-derived orthotopic xenograft models, providing a preclinical rationale to test analogous combinations in patients. SIGNIFICANCE: These findings identify PKCα and JAK2 as targets that drive apoptosis in glioblastoma, potentially representing a clinically translatable approach for glioblastoma.

Background

The transcription factor signal transducer and activator of transcription 3 (STAT3) drives progression in glioblastoma (GBM), suggesting STAT3 as a therapeutic target. Surprisingly however, GBM cells generally show primary resistance to STAT3 blockade.

Methods

Human glioblastoma cell lines LN229, U87, SF767, and U373, and patient-derived xenografts (PDXs) GBM8 and GBM43 were used to evaluate epidermal growth factor receptor (EGFR) activation during STAT3 inhibition. Protein and gene expression experiments, protein stability assays, cytokine arrays, phospho-tyrosine arrays and EGFR-ligand protein arrays were performed on STAT3 inhibitor-treated cells. To evaluate antitumor activity, we administered a betacellulin (BTC)-neutralizing antibody alone and in combination with STAT3 inhibition. BTC is an EGFR ligand. We therefore treated mice with orthotopic xenografts using the third-generation EGFR inhibitor osimertinib, with or without STAT3 knockdown.

Results

We demonstrate that both small-molecule inhibitors and knockdown of STAT3 led to expression and secretion of the EGFR ligand BTC, resulting in activation of EGFR and subsequent downstream phosphorylation of nuclear factor-kappaB (NF-κB). Neutralizing antibody against BTC abrogated activation of both EGFR and NF-κB in response to inhibition of STAT3; with combinatorial blockade of STAT3 and BTC inducing apoptosis in GBM cells. Blocking EGFR and STAT3 together inhibited tumor growth, improving survival in mice bearing orthotopic GBM PDXs in vivo.

Conclusion

These data reveal a feedback loop among STAT3, EGFR, and NF-κB that mediates primary resistance to STAT3 blockade and suggest strategies for therapeutic intervention.