However, in view of the promising clinical results seen with immunotherapy in HNSCC, understanding the immune related mechanisms of drug resistance is also important

However, in view of the promising clinical results seen with immunotherapy in HNSCC, understanding the immune related mechanisms of drug resistance is also important. PD-L1. We sought to determine Mouse monoclonal to COX4I1 whether PD-L1 expression is elevated in head and neck squamous cell carcinoma (HNSCC) models of acquired cetuximab resistance and whether the expression is regulated by bromodomain and extraterminal domain (BET) proteins. Methods: Expression of PD-L1 was assessed in HNSCC cell line models of acquired cetuximab resistance. Proteolysis targeting chimera (PROTAC)- and RNAi-mediated targeting were used to assess the role of BET proteins. Results: Cetuximab resistant HNSCC cells expressed elevated PD-L1 compared to cetuximab sensitive controls. Treatment with the BET inhibitor JQ1, the BET PROTAC MZ1, or RNAi-mediated knockdown of BRD2 decreased PD-L1 expression. Knockdown of BRD2 also reduced the elevated levels XMD8-87 of PD-L1 seen in a model of acquired cisplatin resistance. Conclusions: PD-L1 is significantly XMD8-87 elevated in HNSCC models of acquired cetuximab and cisplatin resistance where BRD2 is the primary regulator. and role for BET proteins in mediating cetuximab resistance by highlighting the importance of BRD4, but the potential role of BRD3 and BRD2 in driving cetuximab resistance remains unclear. Emerging evidence suggests BET proteins regulate the expression of immune checkpoint proteins.7,8 Zhu and colleagues reported that BRD4 directly binds to the promoter region of studies and MZ1 was purchased from Tocris Bioscience (Bristol, UK). Crystal violet viability assay Cell Lines were seeded in 96 well plates followed by cetuximab or cisplatin exposure for 96 hours. Viability was assessed after staining the cells with crystal violet for 30 minutes. The crystal violet solution was rinsed off with tap water, the plate was dried overnight. Crystal violet was dissolved with 5% SDS solution and the absorbance was determined using a colorimetric plate reader as previously described.4 Flow Cytometry Parental and CTXR clones were seeded in 6 cm dishes and treated in the absence or presence of inhibitors for 72 hours. Cells were detached with 0.05% trypsin, counted and incubated with PD-L1/PE-Cy7 antibody or PE-Cy7 isotype control (1:300; Biolegend) for 15 minutes in the dark on ice. Cells were washed and resuspended in FACS buffer and analyzed on a FACS Calibur Dx instrument. Data was analyzed using FlowJo Software. Immunoblotting Cells were washed with cold PBS and lysed with RIPA lysis buffer. Protein was quantified and lysates were resolved by SDS-PAGE, transferred to XMD8-87 PVDF membranes, before an overnight incubation with primary antibodies. Membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (BioRad) for 1 hour followed by visualization of immunoreactive bands by chemiluminescence (Santa Cruz Biotechnology). Antibodies used for immunoblotting included: PD-L1 (CST;#13684; 1:1000), -actin (Abcam; ab6276. 1:5000), BRD4 (Abcam; 128874. 1:1000), BRD2 ( CST; #5848. 1:1000). To measure PD-L1 glycosylation, PE/CA-PJ49 and FaDu parental and CTXR lysates were treated with or without PNGaseF (New England BioLabs) according to the manufacturers instructions. The lysates were then resolved by SDS-PAGE as described earlier. RT-PCR Messenger RNA was isolated from cells using the RNAeasy Kit (Qiagen), followed by cDNA synthesis using the iSCRIPT cDNA Synthesis Kit (Bio-Rad). PCR reactions were executed using the SYBR Green Master Mix (Bio-Rad) and the Bio-Rad CFX96 cycler. Relative mRNA levels were quantified following normalization to the housekeeping gene, (were: forward: 5- TGGCATTTGCTGAACGCATTT-3 and reverse: 5- TGCAGCCAGGTCTAATTGTTTT-3. RNA interference Parental and cetuximab-resistant clones were transfected with 50 pmol of XMD8-87 pooled siRNA oligonucleotides and Lipofectamine RNAiMAX (Life Technologies). BRD4- and BRD2-specific siRNAs were ordered through Sigma-Aldrich. Sequences for BRD4 siRNAs have been described previously.8 Sequences for BRD2 siRNA were: BRD2 siRNA#1: 5-CAAGAAAGCGAAUGAGAAA-3, and BRD2 siRNA#2: 5-CAGAAGAGAUUGAGAUUGA-3. Chromatin Immunoprecipitation FaDu parental and the isogenic cetuximab-resistant subline CTXR#3 cells were cultured to 70 C 80% confluency, and 24 hours after seeding the cells, freshly prepared complete cell fixation solution (CFS) was used to fix the cells according to the manufacturers instruction (Active Motif #53040). The Active Motif Kit was used to perform chromatin immunoprecipitation (ChIP). Following cell lysis, chromatin was sonicated and prepared for immunoprecipitation. Sheared chromatin was immunoprecipitated with normal IgG (1:200; CST, #3900), BRD2 (1:200;.