NFE2L2 mutations enhance radioresistance in head and neck cancer by modulating intratumoral myeloid cells

To identify the mechanism by which Nrf2E79Q mediates RT resistance and CB-839 overcomes RT resistance in Nrf2E79Q, we performed RNA sequencing of SAS cells stably expressing either Nrf2WT or Nrf2E79Q treated with RT±CB-839 and analyze the differentially expressed genes in Nrf2E79Q vs. Nrf2WT cells. Nrf2WT or Nrf2E79Q SAS cells were pretreated with 0.1μM CB-839 or vehicle for 24 hours, then irradiated (4 Gy). RT was performed as previously described(14) using a 225kVp cabinet x-ray irradiator filtered with 0.5 mm Cu (IC-250, Kimtron Inc.). Thus 4 groups were created, including Nrf2WT+RT, Nrf2E79Q+RT, Nrf2WT+RT+CB-839, Nrf2E79Q+RT+CB-839. All the experiments were performed in triplicate, generating a total of 12 samples. Next, RNA was isolated using Trizol (Catalog# 15596026, Invitrogen) 24 hours after RT. Library preparation and RNA sequencing were performed by Novogene via Illumina platforms based on mechanism of SBS (Sequencing by synthesis).