PI3K/mTOR is a therapeutically targetable genetic dependency in diffuse intrinsic pontine glioma
Ryan J. Duchatel, … , Jason E. Cain, Matthew D. Dun
Ryan J. Duchatel, … , Jason E. Cain, Matthew D. Dun
Published February 6, 2024
Citation Information: J Clin Invest. 2024;134(6):e170329. https://doi.org/10.1172/JCI170329.
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Research Article Oncology

PI3K/mTOR is a therapeutically targetable genetic dependency in diffuse intrinsic pontine glioma

Abstract

Diffuse midline glioma (DMG), including tumors diagnosed in the brainstem (diffuse intrinsic pontine glioma; DIPG), are uniformly fatal brain tumors that lack effective treatment. Analysis of CRISPR/Cas9 loss-of-function gene deletion screens identified PIK3CA and MTOR as targetable molecular dependencies across patient derived models of DIPG, highlighting the therapeutic potential of the blood-brain barrier–penetrant PI3K/Akt/mTOR inhibitor, paxalisib. At the human-equivalent maximum tolerated dose, mice treated with paxalisib experienced systemic glucose feedback and increased insulin levels commensurate with patients using PI3K inhibitors. To exploit genetic dependence and overcome resistance while maintaining compliance and therapeutic benefit, we combined paxalisib with the antihyperglycemic drug metformin. Metformin restored glucose homeostasis and decreased phosphorylation of the insulin receptor in vivo, a common mechanism of PI3K-inhibitor resistance, extending survival of orthotopic models. DIPG models treated with paxalisib increased calcium-activated PKC signaling. The brain penetrant PKC inhibitor enzastaurin, in combination with paxalisib, synergistically extended the survival of multiple orthotopic patient-derived and immunocompetent syngeneic allograft models; benefits potentiated in combination with metformin and standard-of-care radiotherapy. Therapeutic adaptation was assessed using spatial transcriptomics and ATAC-Seq, identifying changes in myelination and tumor immune microenvironment crosstalk. Collectively, this study has identified what we believe to be a clinically relevant DIPG therapeutic combinational strategy.

Authors

Ryan J. Duchatel, Evangeline R. Jackson, Sarah G. Parackal, Dylan Kiltschewskij, Izac J. Findlay, Abdul Mannan, Dilana E. Staudt, Bryce C. Thomas, Zacary P. Germon, Sandra Laternser, Padraic S. Kearney, M. Fairuz B. Jamaluddin, Alicia M. Douglas, Tyrone Beitaki, Holly P. McEwen, Mika L. Persson, Emily A. Hocke, Vaibhav Jain, Michael Aksu, Elizabeth E. Manning, Heather C. Murray, Nicole M. Verrills, Claire Xin Sun, Paul Daniel, Ricardo E. Vilain, David A. Skerrett-Byrne, Brett Nixon, Susan Hua, Charles E. de Bock, Yolanda Colino-Sanguino, Fatima Valdes-Mora, Maria Tsoli, David S. Ziegler, Murray J. Cairns, Eric H. Raabe, Nicholas A. Vitanza, Esther Hulleman, Timothy N. Phoenix, Carl Koschmann, Frank Alvaro, Christopher V. Dayas, Christopher L. Tinkle, Helen Wheeler, James R. Whittle, David D. Eisenstat, Ron Firestein, Sabine Mueller, Santosh Valvi, Jordan R. Hansford, David M. Ashley, Simon G. Gregory, Lindsay B. Kilburn, Javad Nazarian, Jason E. Cain, Matthew D. Dun

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Figure 1

Patient-derived DMG cell lines are sensitive to paxalisib in vitro.

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Patient-derived DMG cell lines are sensitive to paxalisib in vitro. (A) ...
(A) CRISPR/Cas9 loss-of-function screening across H3K27-altered subtypes of DMG; WT-H3 (EZHIP) (n = 3), H3.1K27M (n = 8), H3.3K27M (n = 27). (B) Sensitivity of DMG WT-H3 (circles), H3.1K27M (squares), H3.3K27M (triangles), (n = 18), GBM (diamonds) (n = 4) and HGG (hexagons) (n = 2) patient-derived cell lines and normal (upsidedown triangle) (n = 3) to 72 hours paxalisib treatment. (C) Comparison of DMG to HGG/GBM and normal cell lines (AUC) to 72 hours paxalisib treatment (DMG versus HGG/GBM, P = 0.0023 and normal P = 0.0008, 1-way ANOVA). (D) Oncoprint of aberrations (TSO500) in DIPG cell lines (n = 16). (E) Comparison of paxalisib sensitivity (AUC) mutant versus WT PIK3CA DMG cell lines and H3K27M mutation subgroups (1-way ANOVA). (F) Analysis of paxalisib sensitivity versus PIK3CA AUC z-score in PIK3CA mutant versus WT DIPG cell lines and H3K27M mutation subgroups (PIK3CA mut versus PIK3CA WT; H3.1K27M versus H3.3K27M; 1-way ANOVA, *P < 0.05). (G–J) Phosphorylation of PI3K/Akt/mTOR signaling proteins after 1 μM paxalisib treatment for 3, 6, 12, and 24 hours, SU-DIPG-VI, SU-DIPG-XIII and SU-DIPG-XVII (n = 3, 1-way ANOVA, treated versus untreated; #P < 0.05, ##P < 0.01, ###P < 0.001). Analysis of altered gene expression of SU-DIPG-VI following 6 and 12 hours 1 μM paxalisib treatment identifying (K) activated canonical pathways (red) and inactivated pathways (blue); (L) upregulated upstream regulators, and (M) transcriptional regulators; (N) decreased upstream regulators, and (O) transcriptional regulators (activation z-score, P value, size correlating to number of target molecules in data set).