Project Title

Production of NRF2 and p62 in EBV Latency

Authors' Affiliations

Nicholas Roberts is the first author and the person completing registration Nicholas Roberts, Quillen College of Medicine, East Tennessee State University, Johnson City, TN Marry Howell is the second author Marry Howell, Center for Inflammation, Infectious Disease and Immunity, Department of Internal Medicine, Quillen College of Medicine, East Tennessee State University Dr. Shunbin Ning is the third author Shunbin Ning, Center for Inflammation, Infectious Disease and Immunity, Department of Internal Medicine, Quillen College of Medicine, East Tennessee State University

Location

Culp Ballroom

Start Date

4-7-2022 9:00 AM

End Date

4-7-2022 12:00 PM

Poster Number

5

Faculty Sponsor’s Department

Internal Medicine

Name of Project's Faculty Sponsor

Shunbin Ning

Classification of First Author

Medical Student

Competition Type

Competitive

Type

Poster Presentation

Project's Category

Cytopathology

Abstract or Artist's Statement

Abstract

Introduction: The Epstein Barr Virus (EBV) is one of the most common viruses infecting around 95% of adults worldwide. The virus is responsible for 1% of all cancers including Burkitt’s lymphoma, gastric carcinoma, Hodgkin’s lymphoma, and nasopharyngeal carcinoma. EBV-associated tumor cells display radiation and chemotherapy resistance despite the accumulation of intracellular reactive oxygen species (ROS). Our previous study showed that EBV infected lymphocytes release products (LMP1, EBNA1/2, and EBERs) that spontaneously activated the Keap1-NRF2 pathway. When this pathway is activated, the transcription factor NRF2 dissociated from Keap1 and entered the nucleus where it increased transcription for the protein p62. As intracellular p62 levels rose as a result of ROS, this protein competed with NRF2 for binding on Keap1 further increasing the rate of p62 transcription. This created a positive feedback loop that caused a high level of p62 and ROS within the cell. At such high levels, p62 was no longer capable of DNA damage repair through selective autophagy leading to uncontrolled cellular proliferation while escaping DNA damage-induced cell death. The mechanism by which EBV activated the Keap1-NRF2 antioxidant defense pathway is not well understood. In this preliminary stage of the study, the goal is to identify drug treatments affecting different proteins within the keap1-NRF2 pathway that alters the production of NRF2 and p62. In the future, this data will be utilized in a mouse model to identify and therapeutically suppress tumor growth in EBV-positive patients.

Methods: The cells used in this study were human B and T cells derived from EBV-positive Burkitt’s lymphoma patients. The cell lines utilized were Sav3, SLCL7, IB4 (B cells), and MT4 (T cell). The drugs tested included GSK8612 (TBK1 inhibitor), PP2 (SRC inhibitor), GSK2606414 (Perk inhibitor), LY294002 (PI3K/AKT inhibitor), and MRT68291 (ulk1 inhibitor). All cell lines were tested in concentration/time studies to determine the survivability of each cell line and the optimal concentration of each drug. At the end of the incubation period, the cells were collected, and a western blot assay was performed for the proteins GAPDH (standard), pNRF2, NRF2, p62, phosphorylated p62, and Keap1. The optimal concentration was determined for each drug. After preliminary data was collected, cell lines were treated with each drug at the appropriate concentration and incubation time. The cells were then collected, and their protein concentration was determined by spectrophotometry. Eight samples of equal protein concentration underwent gel electrophoresis utilizing a 10% agarose gel. The protein was transferred to a nitrocellulose membrane paper and rinsed with TBS-Tween. Mouse and rabbit human antibodies specific to each protein of interest were used as the primary antibodies. Goat antibodies specific for mouse or rabbit were used as secondary antibodies. Enhanced chemiluminescence (ECL) specific for each secondary antibody was added to anti-body bound protein and exposed to film paper.

Results: The Perk inhibitor (GSK2606414) did not show a significant change in p62 but the IB4 cell line showed an increase in NRF2. The PI3K/AKT inhibitor (LY294002) displayed a decrease in p62 across all cell lines with no change in NRF2 or Keap1. The SRC inhibitor (PP2) showed an increase in p62 concentration across all cell lines with no noticeable change in NRF2 or pNRF2. The ulk1 inhibitor (MRT68291) showed a decrease in p62 concentration in Sav3, SLCL7, and IB4 with no significant difference in NRF2 or Keap1. The TBK1 inhibitor (GSK8612) showed a slight decrease in p62 concentration in SLCL7 and IB4. NRF2 stayed the same or decreased with drug exposure.

Conclusion: If the positive feedback loop between NRF2 and p62 is disrupted, we would expect to see an increase in NRF2 and Keap1 as well as a decrease in p62 concentration. The results showed a decrease in p62 concentration in cells treated with the drugs GSK8612, LY294002, and MRT68291. The same drug testing will be replicated in an asplenic EBV-positive mouse model in an attempt to achieve similar results. This successful drug study is an important step in further understanding the complex relationship between the Keap1-NRF2 pathway and p62.

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Apr 7th, 9:00 AM Apr 7th, 12:00 PM

Production of NRF2 and p62 in EBV Latency

Culp Ballroom

Abstract

Introduction: The Epstein Barr Virus (EBV) is one of the most common viruses infecting around 95% of adults worldwide. The virus is responsible for 1% of all cancers including Burkitt’s lymphoma, gastric carcinoma, Hodgkin’s lymphoma, and nasopharyngeal carcinoma. EBV-associated tumor cells display radiation and chemotherapy resistance despite the accumulation of intracellular reactive oxygen species (ROS). Our previous study showed that EBV infected lymphocytes release products (LMP1, EBNA1/2, and EBERs) that spontaneously activated the Keap1-NRF2 pathway. When this pathway is activated, the transcription factor NRF2 dissociated from Keap1 and entered the nucleus where it increased transcription for the protein p62. As intracellular p62 levels rose as a result of ROS, this protein competed with NRF2 for binding on Keap1 further increasing the rate of p62 transcription. This created a positive feedback loop that caused a high level of p62 and ROS within the cell. At such high levels, p62 was no longer capable of DNA damage repair through selective autophagy leading to uncontrolled cellular proliferation while escaping DNA damage-induced cell death. The mechanism by which EBV activated the Keap1-NRF2 antioxidant defense pathway is not well understood. In this preliminary stage of the study, the goal is to identify drug treatments affecting different proteins within the keap1-NRF2 pathway that alters the production of NRF2 and p62. In the future, this data will be utilized in a mouse model to identify and therapeutically suppress tumor growth in EBV-positive patients.

Methods: The cells used in this study were human B and T cells derived from EBV-positive Burkitt’s lymphoma patients. The cell lines utilized were Sav3, SLCL7, IB4 (B cells), and MT4 (T cell). The drugs tested included GSK8612 (TBK1 inhibitor), PP2 (SRC inhibitor), GSK2606414 (Perk inhibitor), LY294002 (PI3K/AKT inhibitor), and MRT68291 (ulk1 inhibitor). All cell lines were tested in concentration/time studies to determine the survivability of each cell line and the optimal concentration of each drug. At the end of the incubation period, the cells were collected, and a western blot assay was performed for the proteins GAPDH (standard), pNRF2, NRF2, p62, phosphorylated p62, and Keap1. The optimal concentration was determined for each drug. After preliminary data was collected, cell lines were treated with each drug at the appropriate concentration and incubation time. The cells were then collected, and their protein concentration was determined by spectrophotometry. Eight samples of equal protein concentration underwent gel electrophoresis utilizing a 10% agarose gel. The protein was transferred to a nitrocellulose membrane paper and rinsed with TBS-Tween. Mouse and rabbit human antibodies specific to each protein of interest were used as the primary antibodies. Goat antibodies specific for mouse or rabbit were used as secondary antibodies. Enhanced chemiluminescence (ECL) specific for each secondary antibody was added to anti-body bound protein and exposed to film paper.

Results: The Perk inhibitor (GSK2606414) did not show a significant change in p62 but the IB4 cell line showed an increase in NRF2. The PI3K/AKT inhibitor (LY294002) displayed a decrease in p62 across all cell lines with no change in NRF2 or Keap1. The SRC inhibitor (PP2) showed an increase in p62 concentration across all cell lines with no noticeable change in NRF2 or pNRF2. The ulk1 inhibitor (MRT68291) showed a decrease in p62 concentration in Sav3, SLCL7, and IB4 with no significant difference in NRF2 or Keap1. The TBK1 inhibitor (GSK8612) showed a slight decrease in p62 concentration in SLCL7 and IB4. NRF2 stayed the same or decreased with drug exposure.

Conclusion: If the positive feedback loop between NRF2 and p62 is disrupted, we would expect to see an increase in NRF2 and Keap1 as well as a decrease in p62 concentration. The results showed a decrease in p62 concentration in cells treated with the drugs GSK8612, LY294002, and MRT68291. The same drug testing will be replicated in an asplenic EBV-positive mouse model in an attempt to achieve similar results. This successful drug study is an important step in further understanding the complex relationship between the Keap1-NRF2 pathway and p62.