Preclinical Studies of the GlyT1 Inhibitor Bitopertin in Diamond-Blackfan Anemia

Diamond-Blackfan anemia (DBA) results from the haploinsufficiency of 1 of 20 different ribosomal proteins. While DBA patients have hematopoietic stem/progenitor cell defects which broadly impact hematopoiesis, the dominant phenotype is ineffective erythropoiesis and severe anemia. In ~60% of patients, the anemia responds to corticosteroid therapy, leaving an unmet need for safe and effective treatment. In DBA, the synthesis of heme (a chemical chelate) proceeds at a near normal rate while the ribosomal haploinsufficiency leads to slowed translation and delayed globin protein synthesis, resulting in a heme-globin imbalance. DBA patient colony forming unit-erythroid (CFU-E)/proerythroblasts and murine Rpl11 haploinsufficient CFU-E/proerythroblasts show increased heme- and reactive oxygen species (ROS)-mediated cell damage, apoptosis, and ferroptosis. Single cell RNAseq analyses also suggest that excessive heme is a major factor in DBA erythroid marrow failure. When patient marrow cells are cultured in the presence of succinylacetone to reduce heme synthesis or hemopexin to facilitate heme export, erythroid output significantly increases. Succinylacetone competitively inhibits the 2 ^nd step in heme synthesis, while hemopexin enhances heme export 100-fold and scavenges the extracellular heme. Neither would be a pragmatic and safe therapy for DBA patients.

In the first step of heme biosynthesis, glycine and succinyl-CoA combine to form aminolevulinic acid. Erythroid precursors are unable to synthesize enough glycine to support their robust heme synthesis and must import glycine via glycine transporter 1 (GlyT1) (PMID 28495915). Bitopertin is an investigational oral small-molecule inhibitor of GlyT1. In Phase 3 clinical studies of schizophrenia, it was found to be ineffective but exhibited a favorable safety profile (PMID 27816567).

To determine whether bitopertin could rebalance heme and globin synthesis, reduce ineffective erythropoiesis, and improve red cell production in DBA, we cultured marrow cells from patients with DBA without and with 10 nM bitopertin (n=3). Following 7 days of culture, bitopertin increased the mean number of cells (21%) and the number of CD71+ erythroid precursors (31%) without impacting erythroid maturation. We next made human cord blood CD34+ deficient for RPS19 (44±2% reduction) with RPS19 shRNA transduction (or control luciferase shRNA) to model RPS19 haploinsufficient DBA. Treatment with bitopertin improved erythroid expansion of RPS19-deficient CD34+ cells 60% (P<0.01) but not control CD34+ cells. Additionally, to assess erythroid differentiation in vitro in the context of an erythroblastic island (EBI) niche, we cultured the CD34+ cell-derived erythroblasts with cord blood-derived macrophages. Erythroid expansion is 7.9±1.3-fold higher (P<0.02) in EBI cultures compared to cultures without macrophages. Treatment of the EBI cultures with bitopertin increased the expansion of RPS19-deficient CD34+ cell-derived erythroblasts 3.9±0.3-fold (P<0.002) but did not increase the expansion of control CD34+ cell-derived erythroblasts.

As a final study, Rpl11 haploinsufficient mice were treated with bitopertin (10 to 150 ppm in chow, 2-30 mg/kg/day) for 8 weeks. Bitopertin treatment resulted in a dose-dependent improvement in anemia. After treatment with an optimal dose of bitopertin (100 ppm in chow, 20 mg/kg/day), there was an 8.8% increase in the hemoglobin values, and there was significant improvement in red cell numbers (7.5±1.1 M/µL to 8.9±1.3 M/µL, p=0.005), hematocrit (41.6±4.9% to 45.5±4.2%, p<0.05), and mean corpuscular volume (55.7±3.5 fL to 51.8±4.2 fL, p=0.01). Additionally, bitopertin treatment diminished the developmental block at the CFU-E/proerythroblast stage and reduced erythroblast ROS.

The improved in vitro erythroid differentiation of DBA patient marrow and RPS19-deficient CD34+ cells and the improved anemia of RPL11 haploinsufficient mice suggest that bitopertin might mitigate anemia in DBA patients. These encouraging preclinical data supported the initiation of a Phase 1/2 clinical trial (NCT05828108: Young et al, abstract this meeting).