ATRT-02. THE DUAL MTORC1/2 INHIBITOR, TAK-228 COMBINES SYNERGISTICALLY WITH THE BH3 MIMETIC, OBATOCLAX TO IMPROVE SURVIVAL IN MICE BEARING ORTHOTOPIC XENOGRAFTS OF AT/RT release_ucza7b42mnduhm5gkbtamrr7ge

Abstract

<jats:title>Abstract</jats:title> mTOR activation drives tumorigenicity by regulating transcription factor expression and downstream growth and survival pathways. We have previously shown that mTORC1 and mTORC2 are highly activated in AT/RT and the dual mTORC1/2 inhibitor, TAK-228 (Sapanisertib) improves survival in mice bearing orthotopic xenografts of AT/RT. To design a rational combination therapy that enhances TAK-228's efficacy and durability, we performed RNASeq 4 hours after TAK-228 treatment of AT/RT cell models. Pathway analysis revealed disruption of the NRF2-mediated stress response. NRF2 is a cap'n'collar leucine zipper transcription factor that regulates expression of genes involved in redox homeostasis, energy metabolism, cell proliferation, and survival. Analysis of publicly available RNASeq data on 32 human tumors identified elevated expression of NRF2 in AT/RT (median expression 40.78, normal brain 18.81). Short-hairpin knockdown of NRF2 decreased the expression of NRF2 as well as the anti-apoptotic proteins MCL-1, BCL-xL, and BCL-2 (western blot), and intracellular concentrations of reduced glutathione (p&amp;lt;0.005, t-test). TAK-228 similarly decreased expression of NRF2, MCL-1, and glutathione (p&amp;lt;0.005, t-test) demonstrating that TAK-228 compromises AT/RT defenses against oxidative stress and cell death. The brain-penetrant BH3 mimetic, Obatoclax increases oxidative stress and induces apoptosis in AT/RT (MUSE oxidative stress, cPARP western blot, t-test p&amp;lt;0.05). These complementary mechanisms of action synergize to slow AT/RT cell growth (MUSE Cell viability assay, ANOVA p&amp;lt;0.05) and induce high rates of cell death (MUSE ANNEXIN V assay, ANOVA p&amp;lt;0.05, Western blot for cPARP, Compusyn Synergy analysis CI&amp;lt;1.0). Once-weekly treatments of TAK-228 combined with Obatoclax in orthotopic mouse models of AT/RT is well tolerated, slows tumor growth (bioluminescence imaging, ANOVA p&amp;lt;0.05) and significantly extends median survival from 35 to 55 days (Log-rank p&amp;lt;0.05). These findings support a new clinical trial aimed at improving AT/RT survival.
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