EACR25-2204

Functional impact of histone methyltransferase KMT2B in pediatric astrocytomas

A. Giannopoulou1, A. Klonou1, P. Korkolopoulou2, A. Pambalou2, D. Kanakoglou2, A. Mitsios3, S. Sgouros3, A. Papavassiliou1, C. Piperi1, C. Troungos1
1Medical School, National and Kapodistrian University of Athens, Department of Biological Chemistry, Athens, Greece
2Medical School, National and Kapodistrian University of Athens, Department of Pathology, Athens, Greece
3IASO Children's Hospital, National and Kapodistrian University of Athens, Department of Pediatric Neurosurgery, Athens, Greece
Introduction:

Histone modifications coordinate gene expression by regulating transcription factor binding. Although the role of the epigenome is clear in a variety of cancers, in pediatric astrocytomas still remains ambiguous. The Mixed Lineage Leukemia 2 (MLL2/KMT2B) protein, responsible for H3K4 trimethylation (H3K4me3), was shown to mediate pro-oncogenic effects in several cancers. Here, we aimed to investigate the functional role of histone KMT2B and KMT2B-regulated signaling pathways in pediatric gliomas.

Material and method:

We first performed in silico analysis of a publicly available microarray dataset of 49 pediatric astrocytoma samples using the R2: Genomic Analysis. Then, we evaluated the mRNA and protein expression levels of KMT2B by qRT-PCR, immunohistochemistry, and Western immunoblotting in 38 archival pediatric astrocytoma tissues (Grade 2-4) and normal brain samples. We proceeded to evaluate the functional role of KMT2B silencing (using siRNA) in pediatric glioblastoma cell lines (SJGBM2, CHLA-200) proliferation by XTT, as well as in cell migration by scratch assay.

Result and discussion:

In silico analysis revealed that pediatric astrocytomas exhibit significantly increased KMT2B expression levels compared to normal brain tissues, which was further confirmed by qRT-PCR, and Western immunoblotting in tissue samples. Immunohistochemical analysis showed significantly lower KMT2B immunoreactivity in pilocytic astrocytomas compared to grade 3-4 diffusely infiltrating tumors. Accordingly, H3K4me3 protein levels were detected significantly lower in normal brain compared to astrocytomas grade 1 and grade 2-4. The univariate survival analysis of the entire cohort showed correlation of reduced patient’s survival with increased KMT2B expression, indicating a significant clinical impact. To this end, silencing of KMT2B in pediatric astrocytoma cell lines showed a significant reduction in cell proliferation and p53 expression, as well as in cell migration and at mesenchymal marker vimentin levels. Moreover, exposure of these pediatric cell lines to MM-102, a potent inhibitor of the WDR5/MLL interaction, led to a reduction in cell proliferation and migration.

Conclusion:

Taken together, our data show a potential oncogenic role of KMT2B in pediatric astrocytomas, correlating to tumor progression and inferior patients’ survival that needs further investigation.