EXTH-31. EXPLOITING THE GENETIC DEPENDENCY ON PI3K/MTOR SIGNALING FOR THE TREATMENT OF H3-ALTERED DIFFUSE MIDLINE GLIOMA
Authors: Duchatel Ryan , Jackson Evangeline , Parackal Sarah , Sun Claire , Daniel Paul , Mannan Abdul , Findlay Izac , Staudt Dilana , Germon Zacary , Laternser Sandra , Kiltschewskij Dylan , Kearney Padraic , Jamaluddin M Fairuz B , Douglas Alicia , Beitaki Tyrone , Perrson Mika , Thomas Bryce , Manning Elizabeth , Murray Heather , Verrills Nikki , Vilain Ricardo , Skerrett-byrne David , Nixon Brett , Hua Susan , DeBock Charles , Valdes-Mora Fatima , Tsoli Maria , Ziegler David , Cairns Murray , Raabe Eric , Vitanza Nicholas , Phoenix Timothy , Koschmann Carl , Alvaro Frank , Dayas Christopher , Tinkle Christopher , Eisenstat David , Firestein Ron , Mueller Sabine , Nazarian Javad , Cain Jason , Dun Matthew 
Published: November 2023

Abstract
Recurring activating mutations, amplifications and allelic loss of the negative repressors of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) signaling genes are overarching contributors to the poor survival of patients with H3K27-altered diffuse midline gliomas (DMG) diagnosed in the pons (diffuse intrinsic pontine glioma – DIPG), highlighting the clinical potential of therapies that target this axis. Employing a loss-of-function CRISPR-Cas9 gene deletion screen, we identified PIK3CA and MTOR as survival dependences in DMG/DIPG patient derived cell line models (n=36), highlighting the clinical potential of targeting PI3K/Akt/mTOR. The brain-penetrant, PI3K/mTOR inhibitor paxalisib is currently under clinical investigation in DIPG (NCT03696355 and NCT05009992), but dosing limiting toxicities including hyperglycaemia, mucositis and skin irritation limit patient compliance. To exploit this therapeutic vulnerability, we optimized the preclinical use of paxalisib, identifying both precision medicine combination strategies and systemic interventions that reduced side effects and elicited profound tumor control in vivo. Mimicking the patient experience, paxalisib elevated blood glucose levels, promoting the phosphorylation of the insulin receptor (p-INSR) in patient derived DIPG xenograft models. Combining paxalisib with the anti-glycemic drug metformin, synergistically extended the survival of two DIPG xenograft models, decreasing p-Akt, p-mTOR and p-INSR in vivo. Phosphoproteomic profiling of DIPG cells identified paxalisib induced intracellular calcium-regulated PKC signaling. The brain penetrant PKC inhibitor enzastaurin extended the survival of DIPG xenograft models alone, and synergistically combined with paxalisib, enhanced using metformin in three DIPG xenograft models, as well as the immunocompetent H3f3aK27M, PdgfraD842Vmut, Trp53-/- murine brainstem glioma model. Mechanisms of adaptation/plasticity were assessed by ATAC-Seq and quantitative proteomic profiling xenograft tumors refractory to treatment. Here we address the intrinsic neoplastic sequela of DIPG, by combined targeting of PI3K/Akt/mTOR using paxalisib, compensatory PKC signaling using enzastaurin, coupled with strategies to manage treatment-related side-effects and reduced efficacy using metformin; providing the preclinical rationale for the addition of metformin to NCT05009992.