ID #998 Precision Targeting of PI3K/mTOR in Diffuse Midline Glioma: GCT007 as a Novel Brain-Penetrant PIK3CA Inhibitor

Diffuse midline glioma (DMG) is a universally fatal pediatric brainstem cancer. Outside of clinical trials, palliative radiotherapy (RT) remains the only approved treatment, with a median overall survival (OS) of < 12 months. Through CRISPR-Cas9 loss-of-function screens across 38 patient-derived models, we identified consistent, mutation-independent dependencies on PIK3CA and MTOR, confirming PI3K/mTOR signaling as a core therapeutic vulnerability in DMG. However, in Phase I studies (NCT01547546), the systemic pan-PI3K inhibitor, paxalisib, induces adverse events in patients (e.g., hyperglycemia), undermining treatment efficacy. This is mirrored in the Phase II clinical trial, PNOC022/NCT050009992, where approximately 20% of patients experienced immune-related toxicities-mucositis, colitis, and skin reactions, likely driven by paxalisib’s pan-PI3K inhibition, as PIK3CG (p110γ) and PIK3CD (p110δ) are enriched in hematopoietic cells. We hypothesized that the brain-penetrant, p110α-selective inhibitor, GCT-007, could maintain therapeutic efficacy while reducing immune toxicity. DMG patient-derived cell lines showed comparable sensitivity to paxalisib and GCT-007 (mean IC50: 0.76 μM vs. 0.97 μM, respectively). Unbiased proteomic profiling in two DMG lines treated with GCT-007 identified robust downregulation of PI3K/AKT/mTOR signaling, confirmed via immunoblotting. In aggressive immunocompetent syngeneic models, GCT-007 significantly extended overall survival (median 53 vs. 40 days, p = 0.0001). Phosphoproteomic profiling following PI3K/AKT/mTOR inhibition showed activated calcium-dependent PKC signaling, therefore, we tested GCT-007 in combination with the brain-penetrant PKC inhibitors: enzastaurin, and JLD14 (novel therapy in development in our laboratory) and with immune checkpoint inhibitor anti-PD1 therapies. GCT-007 increased survival in both syngeneic and humanized immune-competent DMG models by an average of 53% to 77% compared to vehicle (p = 0.001), with 25% of mice in the GCT-007+enzastaurin+anti-PD1 and GCT-007+JLD14+anti-PD1 cohorts achieving long-term survival. Together these findings define a precision treatment strategy which targets DMG genetic dependencies, circumvents therapeutic resistance and primes the tumor for immune engagement. Ongoing spatial profiling of the tumor immune microenvironment and organ-toxicity is underway to support translation.