Abstract 6935: Combinatorial treatment with small molecule inhibitors C26-A6 and MYCi975 in hepatocellular carcinoma (HCC)

Abstract Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer. The median overall survival of advanced HCC patients is only 6.5-10.7 months. Identifying novel therapeutic targets and developing effective combinatorial treatment is highly relevant to HCC. Two oncogenes, Astrocyte elevated gene-1(AEG-1) and MYC (coding for c-Myc) are co-amplified in HCC patients and cooperate to promote aggressive, metastatic HCC. MYC is a driver oncogene for HCC and overexpression of MYC alone can initiate HCC. AEG-1 overexpression itself cannot induce HCC but it can significantly accelerate tumor progression once initiated. The objective is to evaluate the therapeutic efficacy of the combination of C26-A6 and MYCi975, small molecule inhibitors of AEG-1 and MYC, respectively, for HCC. Human HCC cell lines SNU-449, SNU-182, HuH-7, and Hep3B were treated with C26-A6 and MYCi975, alone or in combination. Optimum concentration of the inhibitors, to be used in combination, was determined by titration experiments. Cell viability was measured by MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] and colony formation assays. Data was analyzed by a two-tailed T-test. Cell invasion was analyzed by Matrigel invasion assay. Differential gene expression was analyzed by RNA-sequencing (RNA-seq) and Ingenuity Pathway Analysis (IPA). In all four cell lines, MYCi975 was more potent than C26-A6 to inhibit cell viability. For MYCi975, MTT assay over 5 days identified the IC50 ranging between 2-4 μM, while for C26-A6 IC50 was more than 100 μM. A combination of C26-A6 and MYCi975 showed significant and synergistic inhibition in viability. Similarly, the combination treatment showed significant inhibition in cell invasion compared to single agent treatment. SNU-449 cells were treated with C26-A6, MYCi975, or C26-A6+MYCi975 combination for 24 h and RNA-seq was performed. This time point was chosen to identify differentially expressed genes (DEGs) by inhibitor treatment before cell death starts. IPA of DEGs revealed that MYCi975 inhibited pathways associated with cell proliferation, C26-A6 inhibited pathways regulating cell migration, invasion, and metastasis, and the combination inhibited both. Inhibition of several oncogenes, such as STAT3, E2F2, and FOXM1 which are associated with poor prognosis and aggressive HCC, was identified by the combination treatment. MYCi975 inhibited cell proliferation, while C26-A6 inhibited cell invasion indicating that in an in vivo setting C26-A6 and MYCi975 combination might inhibit both primary and metastatic tumors and the combination might exert a synergistic inhibitory effect compared to either agent alone. Studies are ongoing to evaluate in vivo therapeutic efficacy of the treatment and elucidate the underlying mechanism mediating the effects of the combination treatment. Citation Format: Eva Davis, Ali G. Ermi, Devanand Sarkar. Combinatorial treatment with small molecule inhibitors C26-A6 and MYCi975 in hepatocellular carcinoma (HCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6935.