Oncogene-Induced Senescence in RTK-fused Infant High Grade Gliomas

Background: While the association between histologic grade and patient outcomes in pediatric and adult glioma diagnoses is well established, this correlation is less predictable in infants under the age of two years. Typically, infant low-grade glioma (LGG) express a more aggressive course and infant high-grade gliomas (iHGG) exhibit superior outcomes. Recent findings have highlighted the unique genomic landscape of iHGG as a potential explanation, characterized by an enrichment in receptor tyrosine kinase (RTK) fusions – thus these tumors may display a more pediatric “LGG-like” and favorable methylation pattern. However, this biology neither explains this tumor behavior nor provides sufficient evidence to avoid the classic, and potentially excessive, treatment approach for iHGG. 

Oncogene-induced senescence (OIS) is a process by which aberrant activation of an oncogene triggers a stable form of cell cycle arrest in normal cells. OIS in MAPK-driven pLGG seems to explain the variable behavior of pediatric LGG, a subset of tumors that behave and display methylation patterns quite similar to iHGG. Whether RTK-fused iHGG is similarly mediated by OIS, and whether this is identifiable from a prognostic standpoint or targetable from a therapeutic standpoint is currently unknown and under investigation.

Hypothesis: We expect that the percentage of cells positive for the senescence markers in RTK-fused iHGG will be similar to MAPK-activated pLGG, and higher compared to pediatric/adult HGG specimens. These results will suggest that senescence contributes to the different behaviors and outcomes of infant and pediatric/adult glioma. 

Methods: Infant and pediatric/adult HGG and LGG tissues have been obtained and experiments will be performed in triplicates and with 2 biological repeats. RNA was extracted from the tumor tissues and reversed transcribed. Quantitative real-time PCR was then performed to determine the expression levels several senescence associated secretory phenotype (SASP) factors using GAPDH signal as an internal control. Immunohistochemical staining was performed on sections of these samples to detect expression of senescence markers. Sections of the frozen tissue samples were then stained for senescence-associated β-galactosidase (SA-β-gal), a lysosomal enzyme found in high concentrations in senescent cells, and compared to aHGG controls to assess the tissue’s degree of senescence.

Results: RTK+ iHGG tissue samples displayed a mean of over 40% SA-β-gal positive cells, a significant increase compared to less than 20% in aHGG samples (p=0.021). Further, when comparing both RTK- and RTK+ iHGG samples to aHGG tissue, there was an increase of over 20% SA-β-gal positive cells (p=0.037). These results suggest that senescence is one differentiating factor between iHGG and aHGG.

Conclusions: Senescent profiles in iHGG tissue, compared to aHGG, suggest that iHGG harboring RTK-fusions may be a distinct subset of potentially favorable tumors. The significance of this finding lies in the potential for the modification of the current, extremely aggressive treatment plan in infants with this tumor. This will be further explored through analysis of the effects of manipulations of RTK fusions on senescence and tumorigenesis in primary human gliocytes and cell lines derived from iHGG tissues without RTK fusions. By overexpressing RTK fusion genes in non-cancerous gliocytes and iHGG cell lines without endogenous RTK fusion, we can isolate the impact of RTK fusions on tumorigenesis and OIS initiation. This may also provide insight into whether OIS can be induced as a therapeutic approach to potentially render a mass more benign and/or more amenable to resection.