Meechan, M. | bioRxiv Science

bioRxiv2025

Preclinical efficacy of combinatorial B7-H3 CAR T cells and ONC206 against diffuse intrinsic pontine glioma

BackgroundDiffuse intrinsic pontine glioma (DIPG) is a fatal pediatric brain tumor affecting over 300 children annually in the United States. Chimeric antigen receptor (CAR) T cells are a targeted immune effector cell therapy with substantial clinical benefit against hematologic cancers. Against CNS tumors, CAR T cells targeting B7-H3, a protein highly expressed on DIPG, have rapidly advanced from preclinical studies to clinical trials. BrainChild-03 (NCT04185038), a phase 1 trial of repeatedly delivered intracerebroventricular (ICV) B7-H3-targeting CAR T cells (B7-H3 CAR T cells), demonstrated tolerability and potential efficacy for children and young adults with DIPG. However, clinical benefits were not uniformly seen, and multi-agent treatment strategies may be required against such an aggressive disease. Here, we combined B7-H3 CAR T cells with ONC206, an imipridone molecule also under clinical investigation. MethodsWe tested B7-H3 CAR T cells combined with ONC206 across multiple DIPG cell cultures and orthotopic xenograft mouse models. ResultsB7-H3 CAR T cell monotherapy induced robust cytotoxicity while ONC206 treatment resulted in significant mitochondrial dysfunction against DMG/DIPG cells. The combination of low effector-to-target ratios of B7-H3 CAR T cells and IC50 concentrations of ONC206 led to significantly enhanced cytotoxicity in vitro (p<0.003) and increased IL-2, IL-29, VEGF-A, and Granzyme B levels. In vivo combinatorial studies of ONC206 and a single ICV dose of B7-H3 CAR T cells significantly extended survival in multiple DIPG xenograft mouse models (p<0.05). ConclusionsB7-H3 CAR T cells combined with ONC206 is a feasible and efficacious multi-agent approach against multiple DIPG models. Importance of the studyDiffuse intrinsic pontine glioma (DIPG) is a fatal pediatric brain tumor. While B7-H3 CAR T cells have shown tolerability and potential benefit in early trials, combinatorial regimens may be required for consistent cures against this aggressive disease. This study demonstrates that a preclinical therapeutic regimen of B7-H3 CAR T cells with ONC206, a second-generation imipridone, increases anti-tumor efficacy in vitro and in orthotopic DIPG mouse models. To our knowledge, this is the first study to evaluate ONC206 in combination with CAR T cells. Our findings provide a preclinical roadmap for evaluating small molecules with CAR T cells to interrogate both their combined benefit and the effect of small molecules on T cells themselves. This work offers a biologically-informed, clinically translatable strategy integrating small molecule therapeutics with CAR T cell therapy and support the development of multi-agent immunotherapy trials for children with DIPG and other high-grade brain and spinal cord tumors. Key PointsO_LIB7-H3 CAR T cells are cytotoxic against preclinical DMG models. C_LIO_LIONC206 causes metabolic apoptosis in preclinical DMG models. C_LIO_LIB7-H3 CAR T cells and ONC206 have combinatorial efficacy against DMG. C_LI

cancer biology↗

bioRxiv2024

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

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.

cancer biology↗