High Dose Ascorbate Reduces Interleukin-1 Beta Secretion in TET2 Mutant Monocytes and Demonstrates Excellent Safety and Tolerability in CMML Patients in Combination with Azacitidine

Introduction

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 partially restored by high dose ascorbate, presumably by preserving intracellular iron (II) in a reduced state. 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 inflammasome function has not been studied rigorously in human TET2 mutant monocytes nor the effects of ascorbate. Here we report the effect of ascorbate in CRISPR-engineered human model of TET2 mutant monocyte differentiation and report safety data from TET2 mutant CMML patients treated with high dose ascorbate.

Methods: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. IL-1β was quantified by Western blot +/- 250 uM sodium ascorbate. Total 5hmC levels were determined by dot blot using polyclonal antibody (Active Motif). 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.

Results: “Tracking of Indels by Decomposition” analysis confirmed 90% targeting of at least one TET2 allele with >90% loss of TET2 protein. Differentiation to Mono/Mac was confirmed by CD14 (87.3%), CD11b (85.7%), CD206 (86.2%), CD68 (73.8%), and CD80 (38.2% that increased to 59.6% after LPS stimulation). Interestingly, we noted a 2 fold increase in NLRP3 inflammasome protein and 2.8 fold increase in active IL-1β excretion after LPS/ATP stimulation in TET2-KO cells compared to safe harbor. Strikingly, ascorbate reduced IL-1β secretion in TET2-KO monocytes by 82% and increased total 5hmC levels.

We then 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 2024, 7 patients had completed at least 3 cycles of ascorbate and azacitidine treatment (27 enrolled in the total; 20 subjects in Lenzilumab arm). IL-1β showed a trend towards increased levels in plasma compared with healthy subjects (2.6-fold increase, P=0.25). Vitamin C deficiency (< 11 umol/L) was observed in 29% of subjects in the ascorbate arm and 27% overall; 14% were “at risk” of deficiency (11-23 umol/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 umol/L after cycle 1, 80.7±6.5 umol/L at cycle 3 and 68.2±12.9 umol/L at cycle 7 (P=0.05, 0.001 and 0.035 respectively). Overall, high dose ascorbate was well tolerated with 22 grade 3 or 4 adverse effects reported as probably secondary to azacitidine and none attributed to ascorbate. A trending improvement in mean blood monocyte counts were noted from 2.7 to 1.0 x10⁹/L after cycle 3 and 1.2 x10⁹/L after 6 cycles (P<0.1, 0.3 respectively).

Conclusion: 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 umol/L) was detected in 27% of CMML patients, a higher rate than reported for the general population (8.7%), which warrants further investigation. In CMML patients, high dose ascorbate, given with azacitidine, demonstrates adequate safety and tolerability and restores vitamin C blood levels.

Lane:GSK: Consultancy; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Other: Drugs, Research Funding. Yeung:Pfizer: Honoraria; Takeda: Honoraria; BMS: Research Funding; Amgen: Honoraria; Novartis: Honoraria, Research Funding; Ascentage: Honoraria. Yong:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Research Funding; Celgene: Research Funding. Ross:Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Keros: Membership on an entity's Board of Directors or advisory committees; Menarini: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees. Hiwase:Abbvie: Honoraria; Astella Pharma: Honoraria; Otsuka: Honoraria.

High Dose Sodium Ascorbate given to patients with chronic myelomonocytic leukaemia that have mutation in TET2 as an intravenous infusion in combination with azacitidine