Activation of pyruvate kinase by mitapivat potentially rescues ineffective erythropoiesis in models of diamond blackfan anemia. Free

Diamond-Blackfan anemia (DBA) is a rare congenital bone marrow failure syndrome, characterized by decreased red blood cell (RBC) production, congenital malformations and an increased susceptibility to cancer. It is predominantly caused by pathogenic variants in genes encoding ribosomal proteins and related factors, leading to imbalance in ribosome subunits, cellular stress, and premature cell death. Treatment options for DBA are limited to RBC transfusions and corticosteroids, both associated with significant side-effects.

We generated two conditional mouse models representing cardinal DBA features: macrocytic anemia with reticulocytopenia. Using Rps19 and Rpl5 haploinsufficient mice under a vav-iCre promoter—representing two commonly affected genes in DBA—we evaluated proliferation and RBC differentiation of cultured fetal liver cKit⁺cells. Flow cytometry revealed that Rpl5 haploinsufficient erythroid progenitors failed to progress to the late progenitor/early precursors stage, as evidenced by an accumulation of early erythroid progenitors EP1-EP2, and a reduction in late erythroid progenitors EP3-EP4 and pro- to polychromatic erythroblast populations. Erythroid progenitor differentiation of Rps19-haploinsufficient cells was comparable to controls, with a mild decrease in pro- to polychromatic erythroblast populations, albeit to a lesser extent than Rpl5-haploinsufficientcells. These haploinsufficient models were associated with distinct cell death mechanisms: apoptosis in Rps19-haploinsufficient cells and ferroptosis in Rpl5-haploinsufficient cells. Using a combination of polysome-sequencing and scRNAseq data, we found various metabolic pathways to be affected in the two models. In particular, there was differential expression of pyruvate kinase (PK) in Rps19- and Rpl5-haploinsufficient progenitors. Mitapivat, which activates PKM2 and PKR, two PK isoforms expressed in erythroid progenitors, has been shown to improve ineffective erythropoiesis in different models of anemia, notably β-thalassemia, by enhancing glycolysis, supporting antioxidant systems and preventing nuclear translocation of PKM2, ensuring effective erythroid maturation. Of note, dimeric PKM2 has been reported to induce ferroptosis in different cell models.

We hypothesized that mitapivat would improve erythropoiesis in Rpl5-haploinsufficient cells by metabolically supporting progenitor cells and reducing ferroptosis. We cultured 3.0x10⁵ cKit⁺ fetal liver cells for 48 hours and assessed cell count and RBC differentiation. Control and Rps19-haploinsufficient cells increased in cell count (median cell count after 24 hours of 6.1x10⁵ cells and 4.9x10⁵ cells, respectively). In contrast, the number of Rpl5-haploinsufficient cells decreased (median cell count 1.7x10⁵ cells). Concomitantly, RBC differentiation was impaired due to Rpl5 haploinsufficiency, with decreases in pro- to polychromatic erythroblasts (S3 population in control 48.8% (IQR 13.4) vs. Rpl5 20.9% (8.8), p<0.01). In cultured Rps19-haploinsufficient cells differentiation was similar to control cells, with the percentage of pro- to polychromatic erythroblasts being at the lower range of control cells (S3 population 40.3%). Upon culture with mitapivat (2 µM or 10 µM, DMSO 0.1% vehicle control), no changes in proliferation or differentiation were noted for control or Rps19-haploinsufficient cells. Rpl5-haploinsufficient cells treated with mitapivat showed a trend towards increased cell counts (1.7x10⁵ in vehicle-treated vs. 1.8x10⁵ in mitapivat 2 µM; 2.3x10⁵ cells in 10 µM). Also, there was a minor but non-significant increase in pro- to polychromatic erythroblasts (S3 population vehicle-treated 20.9% (8.8) vs. 2 µM 23.2% (4.7); 10 µM 23.6% (5.4)). While proliferation was similar after 48 hours, differentiation was marginally enhanced in mitapivat-treated Rpl5 haploinsufficient cells.

In CD34⁺-derived erythroid progenitors from RPL5 (n=4) and RPS19 (n=3) haploinsufficient DBA patients, we found similar levels of mRNA expression of PKLR and PKM2 when compared to controls (n=6). Cell culture with mitapivat (2 µM) improved the transition from EP1 to EP4 in RPL5, but not in RPS19 when compared to vehicle-treated cells.

In summary, Rps19- and Rpl5-haploinsufficient mice exhibit different mechanisms of cell death and activation of PKs may improve erythropoiesis in Rpl5-haploinsufficient progenitors by supporting proliferation and differentiation, with a possible beneficial effect on ferroptosis.