Abstract LB-C004: Epigenetic immune reprogramming overcomes PD-1 resistance in metastatic melanoma patients: the phase II NIBIT-ML1 study

Abstract Background: Our phase Ib NIBIT-M4 study firstly reported that the hypomethylating agent guadecitabine (G), a prodrug of decitabine (D), followed by ipilimumab (I) is safe, with clinical and tumor-immunomodulatory activity in metastatic melanoma (MM) pts (CCR 2019; Nat Commun 2023). Thus, we designed the NIBIT-ML1 trial investigating the efficacy of G plus I+nivolumab (I+N) in PD-1/PDL-1 resistant MM and NSCLC pts. The Stage I MM Cohort results are reported. Methods: The NIBIT-ML1 is a multicenter, run-in, phase II randomized, non-comparative study, in Stage III/IV MM (Cohort A) or NSCLC (Cohort B) pts progressing on PD-1/PDL-1 therapy. An amendment replaced G with ASTX727 (oral D combined with cedazuridine). After a safety Run-in (6 pts/Cohort), 36 MM pts were randomized (1:1) to ASTX727 plus I+N (Arm A) or to I+N (Arm B) in the Stage I. Immune(i)-ORR and safety, iDCR and PFS were primary and secondary endpoints, respectively; exploratory analysis integrated RNA-seq and DNA methylation profiling, and multiplex immunofluorescence (mIF) for CD3, CD4, CD8, CD20, CD163 of tumor biopsies at week (W)0 and at W12. Results: 36 Stage III (3)/IV (33) MM pts (22 male; median age 62y), were randomized in Arm A or B, in Stage I. As of December 15, 2025, at a median follow-up of 19 months (IQR: 11-20), the iORR was 33% (3 CR, 3 PR) (95% CI: 13.3-59.0) and 17% (1 CR, 2 PR) (95% CI: 3.6-41.4) in Arm A and B, respectively; both Arms met the primary endpoint. The iDCR and the median PFS were 56% (95% CI: 30.7-78.5) and 9.4 (CI 95%: 5.0-13.8) months in Arm A and were 39% (95% CI: 17.3-64.2) and 5.8 (95% CI: 5.0-6.6) months in Arm B. The number of hypermethylated probes at W0 was higher in tumor biopsies from pts with iDCR (R) compared to NR,in Arm A+Run-in (A+R). Comparative analysis of differentially methylated probes identified 35,319 probes hypermethylated specifically in R from Arm A+R at screening; among those, 43 were hypomethylated by treatment only in R, and were associated with genes involved viral mimicry and antitumor immunity. Integrated DNA methylation and transcriptomic tumor analyses revealed an epigenetic reactivation driven by treatment-induced promoter hypomethylation of 166 immune-related genes in R from Arm A+R but not in Arm B. No significant difference was observed at W0 between Arm A+R and Arm B in intra-tumoral T-cell infiltration; CD8+and CD3+ enriched on-therapy in over 50% of R from Arm A+R. Conclusions: ASTX727 plus I+N induces clinically meaningful objective responses that correlate with epigenetic immune reprogramming in PD-1 refractory MM pts. Baseline tumor methylation profiling may identify MM pts who will benefit from the addition of a DNA hypomethylating agent to ICI therapy. Citation Format: Anna Maria Di Giacomo, Alessia Covre, Maria Fortunata Lofiego, Francesca Pia Caruso, Maura Colucci, Vincenzo D'Alonzo, Raffaella Grifoni, Roberta Depenni, Laura Solmonese, Francesco Marzani, Emma Bello, Antonio De Falco, Monica Valente, Ramiz Rana, Eleonora Carbonari, Giovanni Amato, Elena Manenti, Ilenia Vizzari, Sandra Coral, Harold Keer, Aram Oganesian, Danna Chan, Roberta Mortarini, Maresa Altomonte, Diana Giannarelli, Andrea Anichini, Teresa Maria Rosaria Noviello, Michele Ceccarelli, Michele Maio. Epigenetic immune reprogramming overcomes PD-1 resistance in metastatic melanoma patients: the phase II NIBIT-ML1 study [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr LB-C004.