Epigenetic immune reprogramming overcomes PD-1 resistance in metastatic melanoma patients: The phase II NIBIT-ML1 study

Background: In the NIBIT Foundation-sponsored phase Ib NIBIT-M4 study, we firstly reported that the hypomethylating agent (HMA) guadecitabine (G), a prodrug of decitabine (D), followed by ipilimumab (I), was safe and had promising clinical and tumor-immunomodulatory activity in metastatic melanoma (MM) patients (pts) ( CCR 2019; Nature Commun 2023 ). Thus, we designed the NIBIT-ML1 trial to investigate the efficacy of G plus I+nivolumab (I+N) in MM or non-small cell lung cancer (NSCLC) pts, progressing to PD-1/PDL-1 inhibitors. Clinical results, integrated analyses of transcriptomic and methylome profiles, and immune contextures of serial tumor biopsies are being investigated in the MM Cohort. 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 W12. Results: Run-in phase : 6 Stage IV MM pts (2 male; median age 71y) received G (2 pts) or ASTX727 (4 pts) plus I+N. No DLT occurred. One CR, 2 PR, 1 SD, and 2 PD were observed. Stage I : 36 Stage III (3)/IV (33) MM pts (22 male; median age 62y), were randomized to Arm A or B. 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. In Arm A+Run-in (A/R) the number of hypermethylated probes at W0 was higher in tumor biopsies from pts with iDCR (R) compared to non 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 in 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 in intra-tumoral T-cell infiltration was observed at W0 between Arm A/R and Arm B; 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.