Background: Anti-PD-1 +/- anti-CTLA4 antibodies are the current standard of care immunotherapy for advanced melanoma. However, a significant proportion of patients do not achieve disease control. Epigenetic modulation, particularly histone deacetylase (HDAC) inhibition, can overcome tumor escape mechanisms and thus might increase the susceptibility to immunotherapy. Methods: Advanced unresectable/metastatic cutaneous melanoma patients primary refractory or non-responding to prior checkpoint inhibitor (CI) therapy were treated with domatinostat at 5 different dose levels (DL) (100 mg (QD), 200 (QD), and 200 mg (BID) using two different schedules (D1-14 and D1-21 q3w) in combination with pembrolizumab (2 mg/kg) q3w to evaluate safety and tolerability. Tumor assessments were performed every 12 weeks and assessed using irRECIST. Sequential tumor biopsies were taken for gene expression analysis and peripheral blood for pharmacokinetic (PK) analysis. Results: We report on preliminary results from the phase Ib part of the ongoing study, data cut-off Feb 1st, 2021 a total of 40 patients have been enrolled. Patient characteristics show that the median number of pretreatments at stage IV was 3, 65 % of patients stage M1c (AJCC 7 or 8) and 35 % with elevated LDH at trial inclusion. Treatment emergent adverse events (AEs) related to domatinostat reported in ≥ 10% of patients were: diarrhea (23%), nausea (20%), fatigue (20%), rash (15%), pyrexia (13%), blood alkaline phosphatase increased (13%), vomiting (10%), dyspnea (10%), all grade 1 and 2 - except one maculo-papular rash grade 3. In total, 8 patients (20 %) developed ≥ grade 3 AEs, with no treatment-related deaths. Patterns of AEs resembled the known safety profiles of domatinostat and pembrolizumab with no increase of immune related AEs for the combination. Maximum tolerated dose was not reached. Four patients discontinued treatment per protocol due to AEs grade 3. We observed clinical activity with 1 complete response, 2 confirmed partial responses and 9 stable diseases (6 confirmed), resulting in a disease control rate of 30% in highly pretreated patients throughout all DLs. Notably, 3 out of 7 patients achieved disease control in DL 3 (domatinostat 200 mg BID D1-14, q3w) and were on treatment ≥ 1.5 years, indicating a trend of dose-dependent clinical activity. Domatinostat treatment resulted in a trend to higher intra-tumoral expression of MHC/APM genes and a more inflamed tumor microenvironment reflecting enhanced T cell infiltration. Conclusions: The combination of domatinostat and pembrolizumab was safe and well tolerated. The observed clinical activity in advanced melanoma patients refractory to previous checkpoint inhibition and the favorable translational findings warrant further development of domatinostat in combination with CI in melanoma and beyond. Clinical trial information: NCT03278665