The treatment regimen developed by Duchatel, Jackson, and colleagues exemplifies this approach. Their strategy included targeting the PI3K/mTOR pathway with paxalisib, mitigating paxalisib-associated toxicity (particularly hyperglycemia) with the antidiabetic drug metformin, and tackling one potential resistance mechanism, namely upregulation of PKC signaling, with the PKC inhibitor enzastaurin (Figure 1) (2).

Combinatorial treatment with paxalisib, metformin, and enzastaurin prolongs survival in models of DIPG. Signaling through PDGFRA activates the PI3K/mTOR axis, which promotes tumor growth. Paxalisib effectively targets PI3K, a therapeutic vulnerability in DIPG cells; however, it induces hyperglycemia, which is mitigated by combining it with metformin. DIPG cells escape this dual therapy by upregulation of PKC signaling, which is counteracted by adding enzastaurin as a triple combinatorial strategy. Adapted from a graphic created in BioRender.com (2024).

The authors began by demonstrating the critical importance of the PI3K/mammalian target of rapamycin (mTOR) pathway in DIPG. The role of this pathway has been suggested based on recurrent mutations in PI3K genes and based on frequent activation of PDGF receptors, whose downstream effects are mediated in part by PI3K. The authors confirmed the PI3K/mTOR signaling pathway as a therapeutic vulnerability in various DIPG-derived models by performing CRISPR-Cas9 screens and high-throughput drug screens and pinpointed paxalisib as a potential drug for DIPG irrespective of PI3K mutational status. This last finding is especially interesting, since it highlights a PI3K-pathway dependency across a broad range of patients with DIPG.

To define an optimal dose for paxalisib, Duchatel and Jackson et al. performed pharmacokinetic and pharmacodynamic studies and observed that treating mice twice daily with 5 mg/kg — a dose used in a previous study by the same group (5) — was relatively well-tolerated and had good brainstem penetration, which is critical for efficacy against DIPG. However, Duchatel and Jackson et al. (2) observed that the upregulation of insulin receptor signaling resulted in hyperglycemia and associated symptoms (9). To overcome this problem, they coadministered metformin, an antidiabetic drug with a known and favorable safety profile and showed that it counteracted the activation of the insulin pathway and thereby increased the survival benefit for mice treated with 5 mg/kg paxalisib twice daily (2).

Notably, however, even mice treated with the combination therapy succumbed to the disease, suggesting that this therapy evokes resistance. To gain insight into the mechanisms of resistance, the authors performed phosphoproteomic analysis of treatment-resistant DIPG cells and showed upregulation of the PKC pathway. To counteract this response, they combined paxalisib plus metformin with PKC inhibitors. Specifically, they used enzastaurin, a brain-penetrant, FDA-approved PKC inhibitor that has already been tested in patients with DIPG as a monotherapy. Importantly, they showed additive effects of the combinatorial treatment in vitro and in vivo. There was a survival benefit from the triple therapy (paxalisib-metformin-enzastaurin) compared with either monotherapy with enzastaurin or paxalisib plus metformin (Figure 1); remarkably, the combination of triple therapy with radiation was a safe strategy and prolonged survival in an immunocompetent DIPG model. To understand the mechanism underlying the effects of their therapy, the authors performed spatial transcriptomic and ATAC-Seq analysis of a treated patient-derived xenograft model. This study showed that triple treatment resulted in downregulation of key myelination-associated genes. The authors speculated that this combination strategy targeted a key cell population of these tumors, since DIPG arises from cells of the oligodendroglial lineage that specialize in making myelin (2, 10). Moreover, they observed upregulation of the PDGFRA, JAK-STAT, and TGF-β signaling pathways, identifying additional potential mechanisms of resistance to their treatment regimen (2). These findings pave the way for further improvements in DIPG therapy.