Pb1990: glycolytic activity and effect of ex-vivo treatment with the pyruvate kinase (pk) activator ag-946 in red blood cells from low-risk myelodysplastic syndromes patients: a proof-of-concept study

Topic: 9. Myelodysplastic syndromes - Biology & Translational Research Background: Anemia is the most common cytopenia occurring in 80%–85% of patients (pts) with low-risk myelodysplastic syndromes (LR-MDS) and therapeutic strategies remain limited. Beyond defective maturation, preliminary data has shown decreased glycolytic activity in MDS-RBCs. Glycolytic pathway is the prominent metabolic cascade in RBCs and is currently targeted with PK activators in clinical trials of congenital anaemias which are also marked by ineffective erythropoiesis like MDS. AG946 is an investigational, potent activator of PK with potential to enhance red blood cells (RBC) functionality and survival by increasing glycolysis. Aims: Evaluate enzymatic activity of the glycolytic pathway in pts with anemia due to LR-MDS and effect of ex-vivo treatment with PK activator (AG946) in RBCs of LR-MDS pts Methods: In part A, 66 anemic pts (i.e., Hb<11 g/dL) diagnosed with MDS according to WHO 2022 were enrolled in the study and sampled for peripheral blood ≥4 weeks after the last RBC unit to reduce blood donor contamination. The determination of activities of RBC enzymes hexokinase (HK) and PK (the initial and terminal steps of glycolytic pathway), and of ATP levels was performed by standard spectrophotometric method according to Beutler et al, 1984. 2,3DPG levels were tested by (2,3-BPG) ELISA kit (ElisaGenie, IE). In part B, purified RBC or whole blood of 10 LRMDS pts and 5 healthy controls (HC) were incubated for 6 and 24 h at 37°C in presence or absence of AG946 in phosphate-buffered saline containing 1% glucose, 170 mg/L adenine, and 5.25 g/L mannitol (AGAM, pH 7.40). RBC were incubated with 1, 5 and 50 uM of AG946 for up to 24 h, at 37°C. After 6 and 24 h, enzymatic activities (HK, PK) and ATP levels were measured. Results: Part A: Pts were mainly elderly males (median age 80yr, 58-92), and mostly belonged to IPSS-R low (78%) and very low/low groups (85%). PK activity (median 13.7 IU/gHb, 7.5-35.9) was reduced in 35% of cases and HK activity (median 2.3 IU/gHb, 0.8-8.1) was increased in 91%, resulting in an abnormally reduced PK/HK ratio in majority of pts (95%). Notably, ATP levels (median 3.4 umol/gHb, 1.3-6.5) were also reduced in 33/56 tested patients (59%), whilst 2,3DPG levels were close to the upper limit of the normal range (median 389 nmol/gHb, 318-639). Part B: 10 MDS pts (All IPSS-R low, 6 MDS-RS/SF3B1, 3 MDS-MLD/LB, and 1 MDS-del5q) were included, 9 male/1 female, median age 82 years (58-87). Baseline (BL) PK activity, PK/HK ratio, and ATP levels were confirmed reduced in pts vs HC (Fig 1A). After 24h incubation with AG946 ex-vivo (Fig 1B), 5/10 pts (3 MDS-RS/SF3B1, 1 MDS-MLD/LB, 1 MDS-del5q) had a significant increase in mean PK activity from BL across all AG946 concentrations evaluated (BL: mean 9.68 IU/gHb [SD 1.4], 1uM: 11.68 IU/gHb [SD 0.9], 5uM: 11.2 IU/gHb [SD 0.89], 50uM: 11.54 IU/gHb [SD 0.8]; p<0.01). Pts who demonstrated an increase in PK activity also had higher HK levels at BL. 4/5 pts who demonstrated an increase in PK activity, (1 excluded for experiment failure) maintained stable ATP levels across all evaluated concentrations of AG946. Summary/Conclusion: Our data demonstrates decreased glycolytic activity in a large cohort of anemic pts with LR-MDS versus HC, with reduced PK activity in 1/3 of cases, decreased PK/HK ratio in nearly all subjects, and reduced ATP in 2/3 of pts. Furthermore, ex-vivo treatment with AG946 led to an increase in PK activity and PK/HK ratio and stable ATP levels in RBCs of LR-MDS pts across all WHO classifications. Combined, these data further support clinical investigation of potent PK activator AG946 in LR-MDS.Keywords: Pyruvate kinase, Anemia, Ex vivo, Myelodysplastic syndrome