Abstract 6021: Genetic and epigenetic features OF KMT2D, CREBBP, ATM, TSC2 and GNAS in uterine leyomiosarcomas
Authors: dos Anjos Laura G. , Bizinelli Daniela , Carvalho Katia C. 
Published: April 2023

Background: Uterine leiomyosarcoma (LMS) comprises 60% to 70% of the uterine sarcoma (US). These tumors present high rates of recurrence and metastasis, and the patients’ survival rates are very low (20% in five years). It is known that somatic mutations and modifications in the DNA methylation levels are associated with several types of neoplasms. Previously, our group identified missense and loss of function mutations in KMT2D, CREBBP, ATM, TSC2, and GNAS, using a genetic screening method in LMS samples. Based on these results, we decided to investigate whether a genetic and epigenetic crosstalk could help to understand the biological processes involved in the LMS origin, development, and pathogenesis. The present study aimed to evaluate the KMT2D, CREBBP, ATM, TSC2 and GNAS gene expression profile in LMS cell line, and their methylation levels in formalin-fixed and paraffin-embedded (FFPE) patients´ samples.

Methods: We selected 15 LMS - FFPE samples obtained via surgical procedures performed between 2012 and 2019 at the Instituto do Cancer do Estado de São Paulo (ICESP). Genomic DNA was extracted using the QIAamp DNA FFPE Tissue Kit and we evaluated methylation levels using the Illumina Infinium Methylation EPIC BeadChip system 850k, including samples treated with bisulfite-treated DNA. Leiomyoma (LM) (THESCs CRL-4003) and LMS (SK-UT-1) cell lines were cultivated for gene expression analysis. Cells were growth for 24, 48, 72, and 96 hours, and the total RNA was extracted by TRIzol. High-Capacity cDNA Reverse Transcription Kit was used for cDNA synthesis and for gene expression evaluation, real-time PCR reactions were performed using TaqMan Universal PCR Master Mix and inventoried TaqMan probes.

Results: KMT2D and TSC2 showed increased expression in LMS compared to LM (cut-off ≤ -2 and ≥ 2 for down and upregulation, respectively), with higher expression in 24 hours of KMT2D [Fold Regulation (FR): 2.77] and TSC2 (FR: 2.58). In contrast, CREBBP was downregulated, with lowest expression in 72 hours (FR: -6.57). ATM was upregulated at 24, 48 with higher expression in 72 hours (FR: 2.32), and GNAS was downregulated, with lowest expression in 96 hours (FR: -4.31). Moreover, the methylation analyzes showed an expressive hypomethylation in LMS samples, compared to myometrium, for KMT2D (β value: 0.45; p= 0.004); CREBBP (β value: 0.51 p &lt; 0.0001); ATM (β value: 0.47 p &lt; 0.0001); TSC2 (β value: 0.53 p &lt; 0.0001) and GNAS (β value: 0.41 p &lt; 0.0001). In conclusion, our study showed that potentially pathogenic mutations may be associated with an aberrant hypomethylation profile, as well as increased expression of KMT2D, TSC2, ATM and loss expression of CREBBP and GNAS in LMS. The interconnection of these genetic and epigenetic events is essential for understanding the complex biology of these tumors, in addition to enabling identification of biomarkers.

Citation Format: Laura G. dos Anjos, Daniela Bizinelli, Katia C. Carvalho. Genetic and epigenetic features OF KMT2D, CREBBP, ATM, TSC2 and GNAS in uterine leyomiosarcomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6021.