(F-227) Downregulation of TFAM enhances the sensitivity of Human Papillomavirus-negative head and neck cancer to radiation and immunotherapy

Introduction:: In the United States, it is estimated that over 65,000 people will be diagnosed with and over 15,000 people will die from head and neck cancer (HNC) in 20231. HNC is associated with risk factors such as human papillomavirus (HPV) infections and carcinogen exposure, and it is treated using surgery, radiotherapy, chemotherapy, or immunotherapy^2,3. Patients with HPV-negative HNC exhibit significantly lower survival rates than HPV-positive patients, so new treatment strategies must be explored to treat HPV-negative HNC resisting traditional therapies⁴_.

Mitochondria are cellular organelles responsible for generating energy and maintaining homeostasis in the cell. Cancer cells alter mitochondrial activity to increase proliferation, so mitochondria play a pivotal role in cancer cell viability5. Mitochondrial transcription factor A (TFAM) is a transcription factor required for the replication of mitochondrial DNA and transcription within the mitochondria. TFAM is essential for cellular functions such as metabolism, calcium homeostasis, and apoptosis. TFAM has been shown to be upregulated in cancer cells and is being investigated as a potential target to alter the sensitivity of HNC cells to radiotherapy and immunotherapy^6,7.

This study assesses the hypothesis that reducing the expression of TFAM increases radiosensitivity and immunosensitivity, which reduces the survivability of HNC cells treated with ionizing radiation (IR) and immune checkpoint inhibitors (ICIs). Cells with lowered TFAM expression experience greater oxidative stress, which could increase the cell’s likelihood of dying following radiation or immunotherapy treatment^8,9. In practice, this could translate to reduced tumor sizes and better outcomes in HPV-negative HNC patients.

Materials and Methods:: TFAM’s effect on HNC cells was examined using mouse oral squamous cell carcinoma (MOC2) cells. TFAM expression was reduced by shRNA. TFAM expression in TFAM knockdown (TFAM KD) cells and TFAM wild-type (parental) cells was analyzed using RT-qPCR.

To examine the effects of altering TFAM expression on cell survival in response to IR, a clonogenic assay was performed¹⁰. TFAM KD and parental cell lines were subjected to differing amounts of radiation, and the colonies were counted to calculate survival fractions for each cell line.

The cGAS-STING pathway is a part of the innate immune system that detects the presence of cytosolic DNA and activates the expression of inflammatory genes and genes involved in Type I interferon gene expression. The effects of the STING pathway were studied by comparing the expression of IFNβ, OAS2, and PDL1 in parental and TFAM KD cells after a 10 Gy dose of IR by RT-qPCR.

A mouse tumor model showed how TFAM expression impacts the effectiveness of treatments in-vivo. Female C57Bl/6 mice were inoculated with TFAM KD or parental cells via right flank subcutaneous injection. Tumor sizes were measured using calipers until tumor burden was observed. Survival was compared in mice who received no treatment, radiation therapy (RT), ICIs, or a combination of RT and ICI treatments. Mice in the RT groups received a 10 Gy dose of RT on day 0, and mice in the ICI groups received PDL1 and CTLA4 ICIs via intraperitoneal injections on days 1 and 7.

Results, Conclusions, and Discussions:: RT-qPCR analysis showed that TFAM KD cells have approximately 15 percent TFAM expression relative to parental cells (p < 0.001, t-test). Despite this, TFAM KD and parental cells had comparable growth rates and viability, as observed in cell culture.

A clonogenic assay examined the ability of TFAM KD and parental cells to survive and proliferate after radiation treatment. At lower doses of radiation (0, 2 Gy), there is no difference between the surviving fractions in parental and TFAM KD cells. However, there is a significantly lower surviving fraction of TFAM KD cells at doses of 4 (p = 0.010, t-test), 6 (p < 0.001, test), and 8 Gy (p < 0.001, t-test) in comparison to parental cells (Figure 1). Down-regulation of TFAM raises oxidative stress via the elevated production of ROS¹¹. When combined with IR treatment, TFAM KD cells experience more DNA damage and pro-apoptotic signaling than parental cells due to the increased ROS level, which prevents the survival of MOC2 cells⁶.

The cGAS-STING pathway detects dsDNA in the cytosol of cells and initiates immune responses¹². Activation of the cGAS-STING pathway upregulates the transcription of interferon response genes such as IFNβ, OAS2, and PDL1^13-15. RT-qPCR results demonstrate that TFAM KD cells experience a greater increase in IFNβ, OAS2, and PDL1 expression after radiation treatment than parental cells (Figure 2). Mitochondrial stress due to radiation or TFAM reduction causes the release of mtDNA into the cytosol, so TFAM knockdown and IR increase immunosensitivity due to greater cGAS-STING activation^16,17.

Tumor growth in mice bearing MOC2 parental or TFAM KD cells was monitored over time. The combination of RT and ICIs in TFAM KD-bearing animals resulted in the most significant difference (p = 0.005, log-rank test), enabling greater survival of the mice over 60 days and 80% tumor reversion (Figure 3). This suggests that partial TFAM knockdown could improve the effectiveness of the other therapeutic treatments.

A combined treatment consisting of reduced TFAM expression, RT, and ICIs could translate to increased survival in clinical applications. HPV-negative HNC often resists traditional oncology treatments, so using TFAM as a sensitizing target could benefit patients.

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