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1817 High Dose Ascorbate Reduces Interleukin-1 Beta Secretion in TET2 Mutant Monocytes and Demonstrates Excellent Safety and Tolerability in CMML Patients in Combination with AzacitidineClinically Relevant Abstract

Program: Oral and Poster Abstracts
Session: 636. Myelodysplastic Syndromes: Basic and Translational: Poster I
Hematology Disease Topics & Pathways:
Chronic Myeloid Malignancies, CMML, Diseases, Myeloid Malignancies
Saturday, December 7, 2024, 5:30 PM-7:30 PM

Maha M Kamel, MBioChem, BScPharm1,2*, Nirmal Robinson, M.Pharm, PhD3*, Monika Kutyna, PhD4,5*, Kelly Lim, MSc, BSc1,2*, Chloe Thompson-Peach, PhD1,2*, Steven W Lane, MBBS, PhD, FRCPA, FRACP6, David T Yeung, MBBS, PhD, BSc, FRACP, FRCPA5, Agnes S. M. Yong, MD, PhD2,7, David M Ross, MD, PhD, FRACP, FRCPA2,8*, Devendra Hiwase, MD, MBBS, PhD, FRACP, FRCPA5,9 and Daniel Thomas, MD, PhD, FRACP, FRCPA5,8,10

1Adelaide Medical School, Faculty of Health and Medical Sciences, University of Adelaide, Adelaide, Australia
2South Australian Health and Medical Research Institute, Adelaide, Australia
3Centre for Cancer Biology, University of South Australia, Adelaide, Australia
4Adelaide Medical School, The University of Adelaide, Adelaide, SA, Australia
5South Australian Health and Medical Research Institute, Adelaide, SA, Australia
6Cancer Program, QIMR Berghofer Medical Research Institute, Royal Brisbane and Women’s Hospital, Brisbane, QLD, Australia
7Haematology Department, Royal Perth Hospital, Perth, Australia
8Adelaide Medical School, Faculty of Health and Medical Sciences, The University of Adelaide, Adelaide, Australia
9Department of Haematology, Central Adelaide Local Health Network, Adelaide, SA, Australia
10Central Adelaide Local Health Network, Adelaide, SA, Australia

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.

Disclosures: 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.

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

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