DIPG-04. TUMOR CELL-INTRINSIC TIM-3 PROMOTES DIFFUSE INTRINSIC PONTINE GLIOMA TUMORIGENESIS

Diffuse intrinsic pontine glioma (DIPG) is an aggressive brain tumor and the leading cause of pediatric death caused by cancer. Despite great strides in the understanding of this disease, the median overall survival is still dismal. TIM-3 is emerging as an alternative to the classic immune checkpoints CTLA-4 and PD-1, and its expression regulates by various mechanisms the function of innate and adaptative immunity. In our work, we demonstrated that TIM-3 is expressed in murine and human DIPG tumor cell lines in vitro and in vivo. TIM-3 knockout using CRISPR/Cas9 was lethal in all human DIPG cell lines evaluated. However, a doxycycline-inducible shRNA-TIM-3 significantly reduced the proliferation of DIPG human cell lines (100% control vs. 25% shRNA TIM-3). Additionally, TIM-3KO murine cell lines displayed a significant decrease in clonogenicity, proliferation, and migration in vitro compared with parental DIPG murine cell lines. Interestingly, loss of TIM-3 expression did not result in cell death, as shown by the evaluation of apoptosis. In vivo evaluation of TIM-3KO cell lines tumorigenicity demonstrated that mice bearing TIM3-KO cells had an overall median survival (35 days) significantly higher than the ones bearing the control guide cells (16 days). RNAseq data indicated that TIM-3 plays a critical role in DIPG murine cells tumorigenesis and survival through the MAPK signaling pathway. Finally, single-cell sequencing and immunochemistry of human tumor samples uncovered TIM-3 expression in patients. TIM-3 expression in DIPG tumor cells correlated with genes positively involved in regulating ion transport, IL-6 production, and response to IFN-γ pathways. We also observed enrichment in genes regulating MAPK and ERK1/ERK2 cascades in these patients. Collectively, these data suggest that TIM-3 expression might have a role in the survival and tumorigenesis of DIPG cells.