CTPS1 inhibition synergizes with replication stress signaling inhibition in MYC-amplified Group 3 medulloblastoma

Abstract

MYC-driven medulloblastomas (MBs) represent the most aggressive and deadly subgroup of MB, the most common malignant pediatric brain tumor. Direct targeting of MYC itself remains an unmet clinical need, therefore focusing on vulnerabilities driven by MYC may be a viable option for novel therapeutic interventions. Using whole-genome CRISPR screening, we identified the de novo pyrimidine synthesis enzyme CTP synthase (CTPS1) as a strong dependency in MYC-driven MB. CTPS1 is the final and rate-limiting step in the de novo pyrimidine synthesis pathway. Targeted inhibition of CTPS1 leads to decreased tumor cell proliferation and markedly reduces MYC expression in G3 MB models. Mechanistically, we demonstrate that single agent CTPS1 inhibition activates the replication stress signaling pathway mediated by ATM-CHK2 and ATR-CHK1. Blockade of CHK1 kinase activity increases sensitivity to CTPS1 inhibition and significantly impedes heterotopic MB tumor growth. CTPS1 enzymatic activity requires the amino acid glutamine, therefore we inhibited CTPS1 using the glutamine antagonists, JHU083 and JHU395. These compounds are prodrugs of 6-diazo-5-oxo-L-norleucine (DON) which were developed to exhibit better tumor targeting and enhanced blood-brain barrier penetrability. Combining JHU083 and CHK1 inhibition demonstrates potent synergy against patient-derived MB xenografts in vivo. Our findings strongly suggest that combining de novo pyrimidine synthesis and ATR-CHK1 inhibitors is a promising treatment for MYC-driven MBs. Key PointsO_LICTPS1 is a unique vulnerability in MYC-driven medulloblastoma C_LIO_LICTPS1 inhibition activates the ATR-CHK1 replication stress response pathway for cell survival C_LIO_LIBlockade of CTPS1 enzymatic activity synergizes with CHK1 inhibition in vitro and in vivo C_LI Importance of the StudyMYC hyperactivation in tumors drives multiple anabolic processes which contribute to tumor proliferation and aggressiveness in patients. We show that targeting de novo pyrimidine synthesis (via CTPS1) limits tumor growth and targets MYC itself through a feedback mechanism. CTPS1 inhibition potently combines with CHK1 blockade and enhances disease control in both heterotopic and orthotopic models of medulloblastoma (MB). Our results support the clinical evaluation of combined CTPS1 and CHK1 inhibition in patients with MYC-driven MB.