CRISPR/Cas9 Editing of the Polyomavirus Tumor Antigens Inhibits Merkel Cell Carcinoma Growth In Vitro

Abstract

Merkel cell carcinoma (MCC) is an aggressive type of skin cancer whose main causative agent is Merkel cell polyomavirus (MCPyV). MCPyV is integrated into the genome of the tumor cells in most MCCs. Virus-positive tumor cells constitutively express two viral oncoproteins that promote cell growth: the small (sT) and the large (LT) tumor antigens (TAs). Despite the success of immunotherapies in patients with MCC, not all individuals respond to these treatments. Therefore, new therapeutic options continue to be investigated. Herein, we used CRISPR/Cas9 to target the viral oncogenes in two virus-positive MCC cell lines: MS-1 and WAGA. Frameshift mutations introduced in the target sequence upon repair of the Cas9-induced DNA break resulted in decreased LT protein levels, which subsequently impaired cell proliferation, caused cell cycle arrest, and led to increased apoptosis. Importantly, a virus-negative non-MCC cell line (HEK293T) remained unaffected, as well as those cells expressing a non-targeting single-guide RNA (sgRNA). Thus, we presumed that the noted effects were not due to the off-target activity of the TAs-targeting sgRNAs. Additionally, WAGA cells had altered levels of cellular proteins involved in cell cycle regulation, supporting the observed cell cycle. Taken together, our findings provide evidence for the development of a CRISPR/Cas9-based therapeutic option for virus-positive MCC.

Merkel Cell Polyomavirus and Merkel Cell Carcinoma

“…CRISPR/Cas9 targeting of LT/57kT impaired MS-1 and WaGa cell proliferation, decreased G1/S cell cycle progression and increased apoptosis. Additional targeting of sT did not enhance the effect in LT/57kT mutated cells [ 112 ].…”

“…Depletion of T antigens expression by the CRISPR/Cas9 technology was shown to impair proliferation and induce apoptosis of VP-MCC cell lines [ 112 ]. Development of a CRISPR/Cas9-based therapeutic tool against T antigens in VP-MCC is a possibility.…”

Synergistic targeting of the PI3K/mTOR and MAPK/ERK pathways in Merkel cell carcinoma

“…Merkel cell polyomavirus (MCPyV), was identified clonally integrated in ∼80% of MCCs [ 1 ]. Tumorigenesis of MCPyV-positive (MCPyV + ) MCC is driven by the constitutive expression of two viral oncoproteins, the small (sT) and large (LT) tumor antigens (TAs), which maintain cell growth by blocking retinoblastoma (Rb) function [ [2] , [3] , [4] ], among other mechanisms [ 5 ]. Conversely, MCPyV-negative (MCPyV − ) MCCs arise upon accumulation of mutations with an UV-light signature [ 6 ].…”

Applications of CRISPR‐Cas9 in gynecological cancer research

“…It was used to knock out the beta‐globin gene, the pathogenic gene of β‐hemoglobinopathy, and it also employed rAAV6, as a donor template, to effectively correct the disease‐causing mutation of SCD through homologous recombination . In addition, The CRISPR‐Cas9 gene editing technology has been widely used treatment‐related researches and in the establishment of disease model of many cancers, including pancreatic, lung, gastric, colon, kidney, liver, head and neck, and skin cancers …”