ESTRO 2024 - Abstract Book

S5422

Radiobiology - Tumour biology

ESTRO 2024

Material/Methods:

In our study to investigate the role of Tri-Methyl-Histone H3 (Lys36) in response to ionizing radiation (IR), we conducted several experiments. We utilized clonogenic assays, γH2aX foci staining, and western blot analysis to assess DNA damage response proteins and histones. These experiments were performed on two well-established human rectal cancer cell lines, HCT116 and LoVo. We used two approaches: RNA inhibition (RNAi) and drug targeting, EPZ-719, to inhibit SETD2. Additionally, we employed CRISPR/Cas9 technology to disrupt the SETD2 gene in these targeted cell lines. Specifically, we designed guide RNAs (gRNAs) that targeted the SETD2 gene and introduced these gRNAs, along with Cas9 nuclease, into the cells to induce gene knockout. After successfully disrupting the SETD2 gene, we conducted an orthotopic rectal experiment. In this experiment, we implanted one million colorectal cells with the SETD2 knockout or control cells orthotopically into the rectum of athymic nude mice. After one week, we confirmed the formation of tumors using 7T small animal MRI. Subsequently, mice bearing tumors underwent image-guided radiation therapy (IGRT) on an Xstrahl small animal radiation research platform (SARRP), receiving a dose of 25 Gy delivered in 5 daily fractions. We assessed tumor response at 2 and 4 weeks after completing the radiation therapy. Following in vitro treatment with ionizing radiation (IR) at a dose of 5 Gy on two colorectal cancer cell lines, we noted an increase in the levels of Tri-Methyl-Histone H3 (Lys36). When we combined IR with RNA interference targeting SETD2 (SETD2 RNAi), we observed a reduction in Tri-Methyl-Histone H3 (Lys36), leading to decreased clonogenic survival and inhibited cell proliferation. Additionally, our in vitro experiments involved the use of a novel SETD2 inhibitor, EPZ-719, which resulted in increased DNA damage following IR (5 Gy), as evidenced by the quantification of γH2aX foci staining. The application of EPZ-719 showed a signficant enhancement in radiosensitization, with a 0.37 improvement in the dose enhancement ratio. In our in vivo investigation, a significant disparity in response rates was evident between tumors with SETD2 knockout and those with wild-type SETD2. Notably, SETD2 knockout tumors displayed a substantially higher complete response rate compared to SETD2 wildtype tumors, with frequencies of 75% and 25%, respectively (p=0.001). Results:

Conclusion:

Based on our preclinical findings, it appears that targeting SETD2 to diminish Tri-Methyl-Histone H3 (Lys36) guided DNA repair could potentially enhance the effectiveness of radiation therapy in patients with rectal cancer. These results underscore the potential clinical relevance of disrupting the SETD2 gene to elevate radiation response rates in rectal cancer cases.

Keywords: SETD2, Rectal Cancer, Radiosensitizer

References:

1. Chantalat S, Depaux A, Héry P, Barral S, Thuret JY, Dimitrov S, Gérard M. Histone H3 trimethylation at lysine 36 is associated with constitutive and facultative heterochromatin. Genome Res. 2011 Sep;21(9):1426-37. doi: 10.1101/gr.118091.110. Epub 2011 Jul 29. PMID: 21803857; PMCID: PMC3166828.