Oncogene MYCN regulates localization of NKT cells to the site of disease in neuroblastoma
Liping Song, Tasnim Ara, Hong-Wei Wu, Chan-Wook Woo, C. Patrick Reynolds, Robert C. Seeger, Yves A. DeClerck, Carol J. Thiele, Richard Sposto, Leonid S. Metelitsa
Liping Song, Tasnim Ara, Hong-Wei Wu, Chan-Wook Woo, C. Patrick Reynolds, Robert C. Seeger, Yves A. DeClerck, Carol J. Thiele, Richard Sposto, Leonid S. Metelitsa
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Research Article Oncology

Oncogene MYCN regulates localization of NKT cells to the site of disease in neuroblastoma

Abstract

Vα24-invariant natural killer T (NKT) cells are potentially important for antitumor immunity. We and others have previously demonstrated positive associations between NKT cell presence in primary tumors and long-term survival in distinct human cancers. However, the mechanism by which aggressive tumors avoid infiltration with NKT and other T cells remains poorly understood. Here, we report that the v-myc myelocytomatosis viral related oncogene, neuroblastoma derived (MYCN), the hallmark of aggressive neuroblastoma, repressed expression of monocyte chemoattractant protein–1/CC chemokine ligand 2 (MCP-1/CCL2), a chemokine required for NKT cell chemoattraction. MYCN knockdown in MYCN-amplified neuroblastoma cell lines restored CCL2 production and NKT cell chemoattraction. Unlike other oncogenes, MYCN repressed chemokine expression in a STAT3-independent manner, requiring an E-box element in the CCL2 promoter to mediate transcriptional repression. MYCN overexpression in neuroblastoma xenografts in NOD/SCID mice severely inhibited their ability to attract human NKT cells, T cells, and monocytes. Patients with MYCN-amplified neuroblastoma metastatic to bone marrow had 4-fold fewer NKT cells in their bone marrow than did their nonamplified counterparts, indicating that the MYCN-mediated immune escape mechanism, which we believe to be novel, is operative in metastatic cancer and should be considered in tumor immunobiology and for the development of new therapeutic strategies.

Authors

Liping Song, Tasnim Ara, Hong-Wei Wu, Chan-Wook Woo, C. Patrick Reynolds, Robert C. Seeger, Yves A. DeClerck, Carol J. Thiele, Richard Sposto, Leonid S. Metelitsa

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Figure 5

MYCN regulates NKT cell localization to neuroblastoma xenografts in BM of NOD/SCID mice.

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MYCN regulates NKT cell localization to neuroblastoma xenografts in BM o...
(A) CHLA-255 and CHLA-255/MYCN neuroblastoma cells were injected into the bone cavities of left and right femurs in NOD/SCID mice, and 4 weeks later in vitro–expanded human NKT cells were injected i.v. together with human PBMCs (1:10 ratio, 5 × 107 total cells). Mice were sacrificed after 24 hours, and BM from the indicated sources was analyzed by 5-color flow cytometry. After exclusion of DAPI+ dead cells, the neuroblastoma (NB) cells and leukocytes (Leuk) were identified as CD56+CD45– and CD45+ events, respectively. T cells (CD3+6B11–) and NKT cells (CD3+6B11+) were analyzed after gating on CD45+ region. BM of tumor-free tibias served as control. Numbers within parentheses and brackets represent percentages of all viable cells (DAPI–) and of leukocytes, respectively. Data are from a representative individual mouse. ND, not detected. (B) Scatter plot of T, NKT cell, and monocyte (Mono) frequency among all non-neuroblastoma cells in BM from individual mice as indicated in 3 different experiments. Data are mean ± SEM from 16 animals.