Conditional Synthetic Lethality between the Initial Steps of Heme Biosynthesis and Pyridoxine Metabolism during Erythropoiesis

Heme biosynthesis depends upon pyridoxal 5' phosphate (PLP), the active form of pyridoxine or vitamin B6. PLP is the cofactor of ALAS, the first enzyme of heme biosynthesis that condenses glycine and succinyl-CoA to form 5-ALA, the sole precursor of porphyrins and heme. PLP is also the cofactor for multiple other enzymes relevant to heme synthesis, notably several involved in glycine metabolism. ALAS2 is the erythroid-specific isoform of ALAS. Loss-of-function and gain-of-function mutations in ALAS2 cause two rare diseases: X-linked Sideroblastic Anemia (XLSA) and X-linked Protoporphyria (XLPP), respectively. We recently generated mouse models of XLSA and XLPP harboring knock-in mutations found in patients; these mouse models recapitulate their respective human diseases. Using diets with defined amounts of pyridoxine, we demonstrated that XLSA mutant animals of three different genotypes (R170H, R411H, and R452H), are dependent upon pyridoxine to ensure their erythropoiesis and red blood cells (RBC) formation, independent of the initial severity of the anemia under baseline conditions. Control littermates or XLPP animals only develop moderate sideroblastic anemia (hemoglobin >8g/dL) after eight weeks of a pyridoxine-deficient diet, which corresponds roughly to RBC lifespans. Whereas pyridoxine-deficient XLSA animals develop massive splenomegaly and a progressively profound anemia in the same period.

Methodology. To determine if this phenomenon is specific to XLSA, we assessed the effect of pyridoxine deficiency in other mouse models of erythroid heme or globin synthesis. We used an erythroid protoporphyria (EPP) model (Fech^m1Pas/m1Pas), a beta-thalassemia model (HBB^Th3/+), and a new model of congenital sideroblastic anemia (SLC25A38^c/-vav-cre+). SLC25A38 is the only known mitochondrial importer of glycine, and the anemia of SLC25A38-CSA patients is typically pyridoxine non-responsive. At baseline, SLC25A38^c/-vav-cre+ animals have a moderate microcytic hypochromic anemia with abundant siderocytes. For each of these strains, we fed male animals diets containing 0, 10, or 100 ppm pyridoxine at weaning. Complete blood counts (2, 5, and 8 weeks of diet) and steady-state and stress erythropoiesis (8 weeks of diet) were assessed.

Results. Pyridoxine-depleted EPP animals developed an anemia similar to the control and XLPP animals but continued to accumulate free protoporphyrin in RBCs. The anemia in beta-thalassemia animals was not substantially impacted by pyridoxine deficiency. In contrast, the response of SLC25A38^{c/- vav-cre+} mice to pyridoxine deficiency was dramatic. After only three weeks of depletion, the anemia was already severe (HGB <2g/dL). In pyridoxine-depleted animals, the remaining RBCs did not present with siderocytes. Notably, the spleen size was normal, and no blockage of erythropoiesis was found by flow cytometry. Instead, both the marrow and spleen contained <15% ter119+ cells, half of them being RBCs, suggesting a profound and early disruption of erythropoiesis.

Conclusion. Heme-dependent Congenital Sideroblastic Anemia (heme-CSA), specifically XLSA and SLC25A38-CSA, and pyridoxine deficiency are non-lethal in mouse models. Nevertheless, heme-CSA mouse models are not viable in the absence of pyridoxine due to impaired erythropoiesis. This phenomenon can be considered conditional synthetic lethality between early heme biosynthesis and pyridoxine metabolism in erythroid cells. Furthermore, an additional PLP-dependent mechanism in both diseases supports the impaired erythropoiesis induced by the genetic mutation. As PLP is not only ALAS2 cofactor, including many enzymes involved in glycine metabolism, which contribute to heme synthesis, identifying the underlying PLP pathway(s) that compensate for other enzymatic defects could offer new therapeutic approaches for heme-CSAs.

Fleming:DISC medicine: Current holder of stock options in a privately-held company, Honoraria.