Background: Pelabresib (PELA) is an oral, small-molecule, investigational BET inhibitor that aims to decrease expression of genes involved in MF. MANIFEST-2 (NCT04603495), a global, randomized, double-blind, Phase 3 study, investigated the efficacy and safety of PELA + ruxolitinib (PELA+RUX) vs placebo + RUX (PBO+RUX) in JAKi treatment-naïve patients (pts) with MF. Methods: Eligible pts had DIPSS score ≥ INT-1, platelet count ≥100 × 10⁹/L, spleen volume ≥450 cm³, ≥2 symptoms with an average score ≥3 or total symptom score (TSS) ≥10 (MFSAF v4.0), peripheral blast count <5%, and ECOG PS ≤2. Pts were randomized 1:1. PELA or PBO was administered (QD for 14 consecutive days of 21) with RUX (BID for 21 days [1 cycle]). Primary endpoint was ≥35% spleen volume reduction from baseline (BL) (SVR35) at Week (Wk) 24. Secondary endpoints included absolute change in TSS and ≥50% reduction in TSS from BL (TSS50) at Wk 24, and safety. Other endpoints included hemoglobin (Hb) response (≥1.5 g/dL mean increase from BL without transfusions in the prior 12 wks), RBC transfusion number and bone marrow fibrosis (BMF). Results: As of Aug 31, 2023, 430 pts were randomized. At Wk 24, 65.9% (141/214) vs 35.2% (76/216) (p<0.001) of pts had an SVR35 response in the PELA+RUX vs PBO+RUX arms, respectively. SVR35 responders at any time were 80.4% (172/214) vs 50.0% (108/216); 80% (137/172) vs 63% (68/108) of responders reached SVR35 at Wk 12 scan; 83.7% (144/172) vs 79.6% (86/108) maintained response at cutoff. Mean change in absolute TSS was -15.99 (SE 1.028) vs -14.05 (SE 0.986) (p=0.0545), and TSS50 was 52.3% (112/214) vs 46.3% (100/216) (p=0.216) at Wk 24. There was a 2-fold difference in pts with both SVR35 and TSS50 with PELA+RUX (40.2% [86/214]) vs PBO+RUX (18.5% [40/216]). Hb response occurred in 10.7% (23/214) vs 6.0% (13/216) of pts, with differences in mean Hb levels maintained at 48 wks. In pts with anemia (Hb BL <10 g/dL), Hb response occurred in 16.4% (11/67) vs 14.1% (10/71). A total of 30.8% (66/214) vs 39.8% (86/216) of required RBC transfusion during the first 24 wks. BMF improvement ≥1 grade occurred in 38.5% (40/104) vs 24.2% (24/99) of pts (odds ratio 2.09; p=0.019). Of 426 pts evaluated for safety, the most common treatment-emergent AEs (≥20%) in the PELA+RUX vs PBO+RUX arms were anemia (43.9% vs 55.6% [Grade ≥3, 23.1% vs 36.4%]), thrombocytopenia (32.1% vs 23.4% [9% vs 5.6%]), platelet count decreased (20.8% vs 15.9% [4.2% vs 0.9%]), and diarrhea (23.1% vs 18.7% [0.5% vs 1.4%]). Updated results will be presented at the congress. Conclusions: PELA+RUX significantly and durably reduced splenomegaly, with a trend toward reduced TSS, and improved anemia and BMF at Wk 24 compared with PBO+RUX in JAKi treatment-naïve pts with MF, addressing key hallmarks of MF. Resultssupport a potential paradigm shift to combination therapy for MF. CH and JM contributed equally. Clinical trial information: NCT04603495.