Background: EZH2 is the catalytic subunit of PRC2, which catalyzes H3K27me3 of target gene promotors and silences transcription of several cancer-related genes. EZH2 overexpression is common in HNSCC and correlated with methylation of several tumor suppressor genes. EZH2 siRNA downregulated EZH2 mRNA and protein and reduced colony formation in an EZH2-expressing HNSCC cell line. EZH2 inhibitors decreased H3K27me3 in HPV + and - cell lines. Tazemetostat, an EZH2 inhibitor, upregulated expression of HLA class I molecules in anti-PD-1-resistant HNSCC cell lines and mouse models, and increased antigen-specific CD8+ T cell proliferation, IFN-γ production, CXCL10 expression, and tumor cytotoxicity. In an anti-PD-1-resistant HNSCC model, tazemetostat and an anti-PD-1 suppressed tumor growth. The primary aims of this phase 1-2 trial were to establish 1) the recommended phase 2 dose (RP2D) of tazemetostat given with pembrolizumab and 2) the objective response rate of this regimen among patients (pts) with anti-PD-1 resistant, PD-L1 positive HNSCC. We report the results of Phase 1. Methods: In phase 1, eligible pts had recurrent/metastatic (RM) HNSCC, adequate performance status and organ function, and ability to swallow study drug. A 3+3 dose-escalation phase 1 design was used to assess 3 dose-levels of tazemetostat (400, 600, and 800 mg orally twice daily) with pembrolizumab (200 mg IV). Cycle 1 was 35 days, with tazemetostat given on days 1-35 and pembrolizumab on day 15. Subsequent cycles were 21 days, with tazemetostat given on days 1-21 and pembrolizumab on day 1. Dose-limiting toxicity (DLT), assessed during cycle 1, was defined as grade 4 neutropenia/thrombocytopenia, grade 3 febrile neutropenia, or study drug-related grades 3-4 non-hematologic adverse events (AEs). Pts must have completed cycle 1 to be evaluable for DLT assessment; otherwise, an equal number of additional pts were enrolled. The RP2D was defined as the highest dose level in which 0 of the first 3 or <1 of 6 pts experienced a DLT. Results: 12 pts enrolled to phase 1: 3 each on the 400 and 600 mg dose-levels of tazemetostat and 6 on the 800 mg dose-level. Key tumor characteristics included primary site (larynx: 4, oral cavity: 3, oropharynx: 3, nasopharynx: 1, cutaneous: 1), HPV status (+: 1, -: 11), and number of lines of prior systemic treatment for RM disease (1-2: 6, >3: 6). Nine pts were evaluable for DLT; 3 pts on the 800 mg dose-level did not complete cycle 1 and were not evaluable for DLT. DLT events and grade 4-5 AEs did not occur. Grade 3 AEs that occurred after cycle 1 included anemia (2 pts, both at 800 mg dose) and fatigue (1, at 400 mg dose). Across all cycles, the most common AEs included anemia (10 pts), fatigue (9), rash (7), and myalgia (4). Conclusions: Among pts with RM-HNSCC, the RP2D of tazemetostat was 800 mg twice daily when given with pembrolizumab. Enrollment to phase 2 is ongoing. Clinical trial information: NCT04624113.