Abstract
Background The progression of myelodysplastic syndromes (MDS) is driven by chronic sterile inflammation within the bone marrow microenviroment. Central to this process is activation of the NLRP3 inflammasome which triggers pyroptosis, a pro-inflammatory form of cell death. Pyroptosis leads to the release of damage-associated molecular patterns (DAMPs), including oxidized mitochondrial DNA (ox-mtDNA). Circulating cell-free ox-mtDNA serves as a potent endogenous ligand that further activates the inflammasome, creating a self-perpetuating cycle of inflammation. Prior studies have explored ox-mtDNA as a potential biomarker of pyroptosis in MDS.
Ofirnoflast (HT-6184), is a first-in-class oral allosteric inhibitor of NEK7, a scaffold protein that is required for NLRP3 inflammasome activation. It is currently under study in an ongoing two-stage Phase 2 clinical trial in patients with low-risk MDS and symptomatic anemia (NCT07052006). We hypothesize that by inhibiting NLRP3 inflammasome activation, ofirnoflast would reduce circulating ox-mtDNA levels and improve clinical outcomes for this patient population.
Methods In the ongoing Phase 2 trial, patients self-administered a single 2-mg capsule of ofirnoflast once daily for five consecutive days, followed by a two-day drug holiday, each week for an initial 16-week treatment period. Patients without disease progression after 16 weeks were allowed to continue treatment for an additional 16 weeks, for a maximum of 32 weeks. Hematologic improvement–erythroid (HI-E), per IWG 2018 criteria, was assessed at Week 16 and Week 32, and ox-mtDNA trends were summarized by erythroid response status.
Serum oxidized DNA (ox-DNA) was measured at three timepoints: baseline (pre-treatment), and of Week 16, and end of Week 32. Quantification of ox-DNA was performed using a commercially available high-sensitivity DNA/RNA oxidative damage ELISA. While this assay does not distinguish between mitochondrial and nuclear DNA, prior studies support that the majority of circulating cell-free DNA in MDS patients is mitochondrial in origin. Therefore, circulating ox-DNA was interpreted as primarily reflecting ox-mtDNA in this study.
Results Among the cohort of 18 patients evaluable for erythroid response enrolled, baseline serum ox-mtDNA levels ranged from 52.4 to 721.9 pg/mL, with a mean of 257.7 pg/mL (SD ±170.8). Treatment with ofirnoflast resulted in a reduction of circulating ox-mtDNA, with median percent changes from baseline of –53% at Week 16 and –29% at Week 32.
When stratified by hematologic improvement–erythroid (HI-E) response status, patients achieving HI-E (n=13) showed median reductions of 47% at Week 16 and 24% at Week 32. Non-responders demonstrated larger median reductions (64% at Week 16 [n=5] and 72% at Week 32 [n=2]), although this subgroup had higher median baseline levels (254 pg/mL vs. 231 pg/mL in responders) and a smaller sample size.
Patients with elevated baseline ox-mtDNA (>250 pg/mL) exhibited more consistent and durable declines, with sustained reductions exceeding 60% at both Week 16 and Week 32. Additionally, non-transfusion-dependent patients (n=6) demonstrated robust ox-mtDNA decreases, with median reductions of 51.8% at Week 16 and 62.4% at Week 32.
Conclusions Preliminary data suggest that ofirnoflast reduces circulating oxidized mitochondrial DNA in patients with low-risk MDS, with the most pronounced effects observed in those with elevated baseline levels of ox-mtDNA (>250 pg/mL). Notably, some patients with substantial reductions in ox-mtDNA did not meet hematologic response criteria, suggesting that absolute ox-mtDNA levels may be more clinically relevant than percent change alone. These findings support further evaluation of alternative dosing, schedules, and treatment durations to determine whether achieving lower circulating ox-mtDNA concentrations may enhance the likelihood of hematologic response in patients with low-risk MDS.
