MYC ASO Impedes Tumorigenesis and Elicits Oncogene Addiction in Autochthonous Transgenic Mouse Models of HCC and RCC

Abstract

The MYC oncogene is dysregulated in most human cancers and hence is an attractive target for cancer therapy. We and others have shown experimentally in conditional transgenic mouse models that suppression of the MYC oncogene is sufficient to induce rapid and sustained tumor regression, a phenomenon known as oncogene addiction. However, it is unclear whether a therapy that targets the MYC oncogene could similarly elicit oncogene addiction. In this study, we report that using antisense oligonucleotides (ASOs) to target and reduce the expression of MYC impedes tumor progression and phenotypically elicits oncogene addiction in transgenic mouse models of MYC-driven primary hepatocellular carcinoma (HCC) and renal cell carcinoma (RCC). Quantitative image analysis of MRI was used to demonstrate the inhibition of HCC and RCC progression. After 4 weeks of drug treatment, tumors had regressed histologically. ASOs depleted MYC mRNA and protein expression in primary tumors in vivo, as demonstrated by real-time PCR and immunohistochemistry. Treatment with MYC ASO in vivo, but not with a control ASO, decreased proliferation, induced apoptosis, increased senescence, and remodeled the tumor microenvironment by recruitment of CD4⁺ T cells. Importantly, although MYC ASO reduced both mouse Myc and transgenic human MYC, the ASO was not associated with significant toxicity. Lastly, we demonstrate that MYC ASO inhibits the growth of human liver cancer xenografts in vivo. Our results illustrate that targeting MYC expression in vivo using ASO can suppress tumorigenesis by phenotypically eliciting both tumor-intrinsic and microenvironment hallmarks of oncogene addiction. Hence, MYC ASO therapy is a promising strategy to treat MYC-driven human cancers.

Keywords: MYC oncogene; antisense oligonucleotide; hepatocellular carcinoma; renal cell carcinoma; targeted therapy.

Graphical abstract

Figure 1

MYC ASO Inhibits MYC Expression in Murine Cancer (A) Schematic of treatment of MYC-HCC and MYC-RCC with PBS or control ASO or MYC ASO. (B) Representative immunohistochemistry (IHC) images (×10 and ×40) and quantification show that treatment with MYC ASO inhibits MYC protein expression in liver cancer. (C) Representative IHC images (×10 and ×40) and quantification show that treatment with MYC ASO inhibits MYC protein expression in renal cell cancer. (D) Quantitative PCR for transgenic human MYC and endogenous mouse MYC in primary small and large liver tumors treated with PBS or control ASO or MYC ASO. ∗p < 0.05, ∗p < 0.01, ∗∗∗p < 0.001. Error bars represent standard error of mean (SEM).

Figure 2

MYC ASO Delays Tumor Progression in a Transgenic Mouse Model of Hepatocellular Carcinoma (A) Schematic of treatment of MYC transgenic mice with PBS or control ASO or MYC ASO. (B) Representative MRI images of liver tumors at week 0 and at week 4 of treatment. (C) Quantification to fold change in tumor volume between week 0 and week 4 in the three treatment groups. (D) Gross images and histopathology of liver tumors treated with PBS or control ASO or MYC ASO. ∗p < 0.05, ∗∗∗p < 0.001. Error bars represent standard error of mean (SEM).

Figure 3

MYC ASO Delays Tumor Progression in a Transgenic Mouse Model of Renal Cell Carcinoma (A) Representative MRI images of mice with MYC-driven kidney cancers at week 0 and at week 4 of treatment. (B) Quantification to fold change in kidney size between week 0 and week 4 in the three treatment groups. (C) Gross images and histopathology of kidney tumors treated with PBS or control ASO or MYC ASO. (D) Comparison of kidney weights at the time of euthanasia between the three treatment groups. ∗p < 0.05, ∗∗∗p < 0.001. Error bars represent standard error of mean (SEM).

Figure 4

Mechanism of Action of MYC ASO (A) Liver tumors treated with MYC ASO demonstrate a lower proliferative index as measured by phospho-histone H3 staining. Quantification of CC3 staining in primary liver tumors. (B) Cleaved caspase-3 staining is higher in liver tumors treated with MYC ASO than PBS or control ASO treated. Quantification of CC3 staining in primary liver tumors. (C) IHC shows increased infiltration of CD4⁺ T cells in MYC ASO-treated tumors compared to PBS- or control ASO-treated tumors. Treatment with MYC ASO is associated with increased expression of the senescence marker β-galactosidase. ∗p < 0.05, ∗p < 0.01, ∗∗∗p < 0.001. Error bars represent standard error of mean (SEM).

Figure 5

ASO-Mediated MYC Knockdown Is Well Tolerated in Mice (A) Body weight of MYC-driven HCC and RCC treated with PBS, control ASO. or MYC ASO. (B) Quantitative PCR for endogenous mouse MYC expression in non-target organs such as the adrenal glands, testes, spleen, and normal liver in transgenic kidney cancer mice treated with PBS or control ASO or MYC ASO. (C–F) Histopathology and immunohistochemical staining for cleaved caspase-3 in mouse liver (C), kidney (D), spleen (E), and testis (F) of transgenic mice treated with PBS or control ASO or MYC ASO. (G) IHC for neutrophils and macrophages (F4/80) in three treatment groups. (H) Graphs show quantification of IHC staining. Error bars represent standard error of mean (SEM).

Figure 6

MYC ASO Inhibits Growth of Human Liver Cancer Xenograft (A) Immunoblotting shows that treatment of HepG2 cells with MYC ASO leads to decreased MYC expression compared to PBS treatment or control ASO treatment. Quantification of immunoblots is shown. (B) MTT assay to measure cell growth of HepG2 upon in vitro treatment with MYC ASO or control at day 4, day 6, or day 8. (C) Experimental scheme for treatment of subcutaneous xenografts of HepG2 in immunocompromised mice. ∗∗∗p < 0.001. (D) In vivo growth of HepG2 xenografts upon treatment with PBS or control ASO or MYC ASO. ∗p < 0.05, ∗p < 0.01, ∗∗∗p < 0.001. Error bars represent standard error of mean (SEM).