MYCN reprograms cysteine metabolism to drive immune-suppression and neuroblastoma progression
Introduction
Neuroblastoma (NB) is the most common pediatric malignancy diagnosed in the first year of life and accounts for 15% of pediatric cancer mortalities. High-risk NB is particularly lethal; 50% of patients will relapse despite aggressive therapy regimens and the post-relapse survival rate is only 20%. Amplification of the MYCN oncogene drives high-risk NB and regulates many oncogenic processes. However, strategies directly targeting MYCN have so far proved ineffective, thus requiring further efforts to understand downstream MYCN processes for therapeutic targeting. Our lab and others have identified a critical role for MYCN in reprogramming NB tumor cell metabolism to promote nutrient synthesis and uptake to support tumor progression. Further, our lab has identified that the TME in advanced-stage MYCN-driven TH-MYCN GEM tumors (TH-MYCN+/+) is highly immune-suppressive and that MYCN represses immune-related transcripts in both murine and human NB. Both tumor cells and immune cells rely on nutrient synthesis and uptake for survival and effector functions. Therefore, we aim to elucidate how MYCN, through metabolic reprogramming of NB cells, promotes an immune-suppressive TME.
Methods
To understand how MYCN reprograms tumor metabolism and immune-suppressive phenotypes, TH-MYCN+/+ tumors and healthy kidneys or adrenal glands were subjected to untargeted metabolomics (HD4 platform, Metabolon) and mRNA-Sequencing. To investigate the nutrient composition of the TME, MYCN-activated SK-N-AS MYCN-ER™ cell supernatant (4-OHT for 72h) was analyzed by targeted LC-MS. To define the TME of MYCN-driven NB, TH-MYCN+/+ tumors were phenotyped using high-parameter flow cytometry. To characterize T cells following tumor or modified-nutrient exposure, tumor-infiltrating and splenic T cells, isolated by CD4/CD8 Miltenyi Biotec MicroBeads, were stimulated ex vivo (anti-CD3/anti-CD28/IL-2 for 24-96h) to measure proliferation (CellTrace™ Violet), apoptosis (Annexin V), cytokine production (intracellular antibody staining), and gene/protein expression (RT-qPCR and Western Blot). To investigate tumor cell metabolic dependencies, NB cells were cultured in cyst(e)ine- or methionine-free medias or treated with Erastin (cystine uptake inhibitor) or Propargylglycine (transsulfuration inhibitor) and assessed for cell viability.
Results
Compared to healthy tissue, THMYCN+/+ tumors have reduced intratumoral pools of cyst(e)ine and precursor metabolites (methionine and serine) with a corresponding upregulation of GSH, suggesting that MYCN drives tumor consumption of cyst(e)ine to meet redox balance needs. MNA cells are more sensitive to Erastin treatment and exogenous cyst(e)ine deprivation than non-MNA cells, suggesting a MYCN-driven tumor dependency on exogenous cyst(e)ine. Moreover, MYCN-activated SK-N-AS MYCN-ER™ cells consume exogenous cyst(e)ine, thus depriving it from the TME. Murine and human T cells activated ex vivo in cyst(e)ine-deplete media are unable to proliferate. When activated without exogenous cyst(e)ine, murine T cells produce reduced levels of effector cytokines (IFNγ, TNFα, GZMB) and undergo apoptosis. Ex vivo activated T cells upregulate their expression of xCT (cystine transporter) and lack the required machinery (CBS enzyme) for de novo cysteine synthesis, suggesting that activated T cells are heavily dependent on exogenous cyst(e)ine.
Conclusion
Our results suggest that MYCN reprograms NB cysteine metabolism to promote a cyst(e)ine poor, immune-suppressive microenvironment. Microenvironmental cells, like T cells, depend on exogenous cyst(e)ine during activation for cell viability, proliferation, and effector functions. These data suggest that MYCN-driven NB tumor cells and T cells directly compete for exogenous cyst(e)ine and that developing therapies to shift this competition to favor T cell consumption of cyst(e)ine would be an effective strategy for MNA high-risk NB patients.