Loss of Protein Kinase R increases cell-intrinsic radiosensitivity of B16 melanoma cells

Committee/Faculty

Presenting Author(s)

SC

Scott C. Chin, n/a

Microbiology, Immunology, and Molecular Genetics, BS
University of California, Los Angeles
Los Angeles, California, United States

Disclosure: I do not have any relevant financial / non-financial relationships with any proprietary interests.

Co-Author(s)

JD

Jie Deng

MD, PhD
University of California, Los Angeles
Los Angeles, California, United States

Disclosure: Disclosure information not submitted.
SK

Sarah Kremer, n/a

Microbiology, Immunology, and Molecular Genetics, BS
University of California, Los Angeles
Los Angeles, California, United States

Disclosure: Disclosure information not submitted.
MT

Mito Tariveranmoshabad, n/a

Molecular, Cellular, and Developmental Biology, BS
University of California, Los Angeles
Los Angeles, California, United States

Disclosure: Disclosure information not submitted.
DS

Dorthe Schaue, n/a

PhD
University of California, Los Angeles
Los Angeles, California, United States

Disclosure: Disclosure information not submitted.
AK

Anusha Kalbasi, n/a

MD
University of California, Los Angeles
Los Angeles, California, United States

Disclosure: Disclosure information not submitted.

Purpose: Radiation therapy (RT)-induced activation of pattern recognition receptors (PRRs) is implicated in the direct cytotoxicity and the indirect immunologic effects of RT. Understanding the contribution of PRR pathways to RT responses may inform therapeutic opportunities that leverage both cytotoxic and immunologic effects of RT. For example, double-stranded RNA (dsRNA) sensor RIG-I mediates cell-intrinsic radiosensitivity and RT-induced type I interferon (1). In contrast, Protein Kinase R (PKR) is a dsRNA sensor that has been shown to mediate cell-intrinsic radio-resistance (2). We hypothesized that loss of PKR will increase cell-intrinsic radiosensitivity in vitro, but these effects will be diminished in vivo due to reduced RT-induced immunogenicity.

Methodology: PKR-knockout (PKR-KO) B16 murine melanoma cells were generated using CRISPR/Cas9 and verified by sequencing and protein expression. We quantified colony frormation at 0 Gy, 4 Gy, and 8 Gy and assessed radiosensitvity in the radiation-treated groups through a clonogenic assay. We determined the surviving fraction (SF) of WT and PKR-KO B16 cells compared to their respective untreated. Next, we implanted tumor cells subcutaneously in C57BL/6 mice to evaluate tumor growth and RT response.

Results: Untreated PKR-KO cells formed larger and more numerous colonies than WT cells in vitro. The mean surviving fraction of PKR-KO cells in response to 4 Gy (mean SF  standard deviation, 0.107  0.046) was significantly lower (p < 0.0001) than that of the WT cells (0.511 0.013). Furthermore, at 8 Gy, the mean SF of PKR-KO (0.007  0.008) remains significantly lower (p < 0.0001) than the mean SF of WT cells (0.039  0.001). Overall PKR-KO cells formed fewer colonies than WT cells after RT indicating conferred radiosensitivity with the loss of PKR. Ongoing preliminary in vivo data indicate no difference in growth or radiosensitivity of WT and PKR-KO B16 melanoma cells.

Conclusions: Our results demonstrate that PKR may play a role in sensitizing B16 cells to radiotherapy in vitro, but these effects may be diminished in vivo due to loss of RT-induced immunogenicity. Ongoing studies will fully characterize the effect of PKR on tumor growth and radiosensitivity in vivo, including immunogenic effects of RT.

References: [1] Ranoa, D. R. E., Parekh, A.D., Pitroda, S.P., et al. (2016). Cancer therapies activate RIG-I-like receptor pathway through endogenous non-coding RNAs. Oncotarget, 7(18), 26496.
[2] Hao, C., Shao, R., Raju, U., et al. (2016). Accumulation of RNA-dependent protein kinase in the nuclei of lung cancer cells mediates radiation resistance. Oncotarget, 7(25), 38235.