ESTRO 2024 - Abstract Book

S5401

Radiobiology - Tumour biology

ESTRO 2024

Purpose/Objective:

The role of High Mobility Group Box 2 (HMGB2) in radiation response has not been fully explored in gliomas. Here we sought to investigate the contributions of HMGB2 to cell proliferation and response to radiation in glioblastomas with and without TP53 mutations. Background: In our previous correlative studies 1 we described proteomic signatures associated with glioma evolution from lower grade (grades 2 and 3) to high grade (grade 4) using liquid chromatography tandem mass spectrometry (LC-MS/MS) and identified HMGB2 as a protein abundant in the early development stages of glioblastoma. This finding was further validated with multi-omics analyses [genomic characterization (Ion AmpliSeq targeted panel sequencing, Sanger sequencing, and Oncoscan), and mRNA array (ClarionD)]. High HMGB2 gene expression was associated to worse overall survival in diffuse gliomas (p=0.026). More importantly, HMGB2 expression increased in IDHwt lower grade gliomas preceding the development of the traditional histopathological features of glioblastoma (“molecular glioblastomas”). In histopathological analysis, HMGB2 protein expression by immunohistochemistry had strong positive association with proliferative activity (p=0.0091) in fully developed glioblastomas and grade 4 IDH mutant astrocytomas. Pathway analysis showed involvement of HMGB2 in the Retinoblastoma (RB) pathway, which along with p53 pathway, are canonical pathways in gliomas, inactivated in respectively 78% and 87% of glioblastomas (including astrocytomas grade 4 IDH mutant astrocytomas) 2 , forming the “p53-p21-RB Pathway” 3 . Previous studies have shown that HMGB2 modulates p53 phosphorylation during DNA damage response, and that high HMGB2 expression was related to increased resistance to temozolomide in gliomas 4 . In addition, HMGB2 downregulation increased radiation sensitivity in colon cancer cells with intact, but not mutant, TP53 5 . Nevertheless, the role of HMGB2 in the p53-p21-RB Pathway is not well explored in gliomas.

Material/Methods:

We utilized short hairpin RNA (shRNA) to achieve HMGB2 knockdown (HMGB2 KD) in established glioblastoma cell lines U87 MG and LN18 TP53-C238S ; primary 3359 cell line, and CRISPR-generated isogenic 3359 TP53-R248W . We then performed cell proliferation assays with MTS and methylene blue to determine, respectively, early, and late effects of the HMGB2 KD in glioblastoma cells. Clonogenic assay was performed to determine radiation dose effective to eliminate 50% of tumor cells (IC50). Finally, we defined the dynamics of HMGB2 expression in the cell cycle and compared the effects of HMGB2 KD in the cell lines using flow cytometry after cell synchronization with a double thymidine block.

Results:

1) The HMGB2 baseline protein expression was higher in the TP53-mutant cell lines (LN18 TP53-C238S and 3359 TP53 R248W ) compared with the TP53wt cell lines. 2) The decrease of HMGB2 expression in the HMGB2 KD cell lines compared with controls was: U87: 62%; LN18: 53%; 3359 TP53wt: 69%; 3359 TP53-R248W: 76%. 3) HMGB2 KD reduced cell proliferation in all cell lines (reduction compared with control: U87: p=0.001; LN18 TP53-C238S : p=0.06; 3359 TP53wt: p=0.05; 3359 TP53-R248W : p=0.01) 4) HMGB2 KD increased radiation sensitivity in all cell lines, independent of TP53 status (average colony formation reduction of 50% in all cell lines), and slightly reduced IC50 in all cell lines (maximum IC50 reduction observed in LN18- TP53-C238S : from 1.8Gy to 1.1 Gy and in 3359 TP53wt: from 1.4Gy to 1.0 Gy). 5) Maximum expression of HMGB2 protein occurred at the G2/M phase of the cell cycle in glioblastoma cells. 6) HMGB2 KD reduced the percentage of synchronized cells in G2/M up to 20% while increasing the percentage of cells in G0/G1 up to 13%, suggesting that HMGB2 KD alters the progression through the cell cycle.