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Figure 3 from Coordinated Targeting of S6K1/2 and AXL Disrupts Pyrimidine Biosynthesis in PTEN-Deficient Glioblastoma

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posted on 2024-08-23, 10:20 authored by Catherine A. Behrmann, Kelli N. Ennis, Pranjal Sarma, Collin Wetzel, Nicholas A. Clark, Kate M. Von Handorf, Subrahmanya Vallabhapurapu, Cristina Andreani, James Reigle, Pier Paolo Scaglioni, Jarek Meller, Maria F. Czyzyk-Krzeska, Ady Kendler, Xiaoyang Qi, Jann N. Sarkaria, Mario Medvedovic, Soma Sengupta, Biplab Dasgupta, David R. Plas

S6K1 and AXL inhibitors counteract pyrimidine biosynthesis in PTEN-deficient GBM. A, Steady state metabolite abundance in LN229 GBM transfected with siPTEN and then treated with vehicle control or combination S6K1 (LY-2584702, 10 μmol/L) and AXL (BMS-777607, 10 μmol/L) inhibitors for 5 hours (n = 4). B, Detail of nucleotide and their precursor metabolites from A. C, log₂ fold change of [U]-¹³C glucose labeled metabolites in U87MG GBM pretreated for 3 hours with 10 μmol/L LY-2584702 and 10 μmol/L BMS-777607 vs. vehicle control at 60 and 300 minutes after addition of ¹³C-glucose (n = 4). Statistically significant (>1.5 fold) metabolites are highlighted. D, JHH136 spheres were treated with inhibitors (10 μmol/L each) for 72 hours for western blot analysis. LY-2584702, S6K1 inhibitor, reduces phosphorylation of rpS6 and CAD leading to sustained impairment of pyrimidine synthesis and cell growth. Treatment with BMS-777607 increases H2A.X phosphorylation at Serine 139, indicating an increase in double-stranded DNA breaks. E, Mayo59 spheres were treated as in D. S6K1 inhibition reduces phosphorylation of rpS6 and CAD, impairing pyrimidine synthesis. DNA damage is evident in combination S6K1 and AXL inhibition.

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ARTICLE ABSTRACT

Intrinsic resistance to targeted therapeutics in PTEN-deficient glioblastoma (GBM) is mediated by redundant signaling networks that sustain critical metabolic functions. Here, we demonstrate that coordinated inhibition of the ribosomal protein S6 kinase 1 (S6K1) and the receptor tyrosine kinase AXL using LY-2584702 and BMS-777607 can overcome network redundancy to reduce GBM tumor growth. This combination of S6K1 and AXL inhibition suppressed glucose flux to pyrimidine biosynthesis. Genetic inactivation studies to map the signaling network indicated that both S6K1 and S6K2 transmit growth signals in PTEN-deficient GBM. Kinome-wide ATP binding analysis in inhibitor-treated cells revealed that LY-2584702 directly inhibited S6K1, and substrate phosphorylation studies showed that BMS-777607 inactivation of upstream AXL collaborated to reduce S6K2-mediated signal transduction. Thus, combination targeting of S6K1 and AXL provides a kinase-directed therapeutic approach that circumvents signal transduction redundancy to interrupt metabolic function and reduce growth of PTEN-deficient GBM. Therapy for glioblastoma would be advanced by incorporating molecularly targeted kinase-directed agents, similar to standard of care strategies in other tumor types. Here, we identify a kinase targeting approach to inhibit the metabolism and growth of glioblastoma.