High dose ascorbate downregulates innate immune cytokines in the bone marrow of TET2 mutant chronic myelomonocytic leukemia

Abstract

Abstract The most frequent mutation in CMML is a loss of function missense or frameshift variant in the gene encoding the epigenetic dioxygenase, ten eleven translocation 2 (TET2). Interestingly, deleterious TET2 function on 5-hydroxymethylation can be restored by high dose ascorbate. Macrophages from TET2-deficient mice have been shown to produce increased IL-1β in response to lipopolysaccharide (LPS) challenge (Fuster, et al. 2017, Science; Sano, et al. 2018, J Am Coll Cardiol) but the inflammatory cytokines in TET2 mutant CMML have not been described in detail or the anti-inflammatory responses to ascorbate. Here we report the serial cytokine profiles of bone marrow plasma in a cohort of TET2 mutant CMML patients treated with high dose ascorbate plus azacitidine and show TET2 mutation results in LPS-CD14-NLRP3 inflammasome activation rather than interferon/STING activation in a CRISPR-engineered human model of TET2 mutant monocyte differentiation. Methods: PREACH-M is a phase 2/3 non-randomized, open-label trial in 54 subjects with newly diagnosed CMML in which patients with TET2 mutations are stratified to be treated with high dose sodium ascorbate (30g IV for 7 days then 1.1g oral powder every 28 days) in combination with azacitidine. Vitamin C levels were measured at baseline, cycle 1, 4 and 7. Cytokine profiling was performed at baseline, cycle 4 day 1, cycle 7 day 1, cycle 13 day 1 using Milliplex Human Cytokine/Chemokine Magnetic bead panel. TET2 knockout human cord blood stem progenitor cells versus safe harbor AAVS1 targeting were engineered using dual CRISPR CAS9 sgRNA “hit & run” methods (Nakauchi, et al. 2022, Blood Cancer Discovery) and then differentiated into monocytes/macrophages over 21 days. Results: We analysed the molecular and clinical profiles of TET2 mutant CMML subjects treated with high dose ascorbate. TET2 mutations were detected in 78% overall (n=27) with 76% of these having at least two TET2 variants with variant allele frequency > 3%. Three subjects had TET2 loss of heterozygosity on chromosome 4q24, while one subject had loss of one copy. As of 1st August 2025, 8 patients had completed at least 3 cycles of ascorbate and azacitidine treatment (27 enrolled in the total; 20 subjects in Lenzilumab arm). Vitamin C deficiency (<11 µmol/L) was observed in 29% of subjects in the ascorbate arm and 27% overall; 14% were “at risk” of deficiency (11-23 µmol/L). As expected, treatment with high dose ascorbate significantly increased vitamin C from a mean of 27.9±8.7 at screening to 147.7±52 µmol/L after cycle 1, 80.7±6.5 µmol/L at cycle 3 and 68.2±12.9 µmol/L at cycle 7 (P=0.05, 0.001 and 0.035 respectively). We noted multiple fold change reductions in cytokine levels noted to be produce by innate immune activation including IL-1β (baseline vs cycle 13, P=0.001), IL-8 (baseline vs cycle 7, P=0.04, and baseline vs cycle 13, P=0.001), IL-12p70 (baseline vs cycle 13, P=0.0005), G-CSF (baseline vs cycle 13, P=0.05), GM-CSF (baseline vs cycle 13, P=0.002), IL-1RA (baseline vs cycle 4, P=0.001, baseline vs cycle 7, P=0.03, and baseline vs cycle 13, P=0.007), IL-13 (baseline vs cycle 4, P=0.003, baseline vs cycle 7, P<0.0001, and baseline vs cycle 13, P=0.005), IL-15 (baseline vs cycle 4, P=0.002, baseline vs cycle 7, P=0.07, and baseline vs cycle 13, P<0.0001). Significantly we did not observed changes in IFNγ following ascorbate treatment. Consistent with the plasma results we noted a 2-fold increase in NLRP3 infammasome protein (P=0.0076 and 2.8-fold increase in active IL-1β excretion after LPS/ATP stimulation (P=0.015) in TET2-KO cells compared to safe harbor but no increase in STING activation, tank-binding kinase (P=n.s.) that is associated with interferon signalling.Conclusion: Our preliminary data demonstrates a role for high dose ascorbate in down regulating multiple innate immune cytokines including IL-1β and GM-CSF but not IFNγ in the bone marrow of TET2 mutant CMML patients. This is consistent with our in vitro data showing engineered human TET2-mutated monocytes exhibit hyper-activated innate immune responses through CD14-NLRP3-IL-1β axis reversible with high dose ascorbate. Ascorbate deficiency (< 11 µmol/L) was detected in 27% of CMML patients, a higher rate than reported for the general population (8.7%). In contrast we did not observed hypersensitivity to interferon signalling, tank-binding kinase or STING activation in TET2 mutant monocytes.