Pathophysiology of Macrocytic Anemia in Diamond Blackfan Anemia and Del(5q) Myelodysplastic Syndrome

Twenty-five percent of Diamond Blackfan anemia (DBA) cases result from haploinsufficiency of RPS19 and ~30% from haploinsufficiencies of other ribosomal proteins. The macrocytic anemia of del(5q) MDS results from RPS14 haploinsufficiency.  When tested, these mutations disrupt ribosome assembly, yet how this causes macrocytic anemia remains uncertain. The dominant hypothesis is that P53 pathway activation leads to cell apoptosis, however, why erythropoiesis is severely impacted, while other hematopoietic as well as non-hematopoietic cells function appropriately, is difficult to explain. These observations led us to predict that DBA and del(5q) MDS share a pathogenesis that reflects the rapid kinetics of red cell and hemoglobin production, and specifically that poor ribosome assembly leads to an inability to quickly translate mRNA to protein, delayed globin synthesis in early erythroid precursors, and excess heme. Since free heme is toxic, cell death ensues. To explore this, we studied marrow from DBA (n=3) and del(5q) MDS (n=6) patients in cultures that optimize erythroid differentiation and showed that (a) cell numbers significantly decrease between stages I (lin-CD71+CD36-GlyA-)(BFU-E/early CFU-E) and II, (lin-CD71+CD36+GlyA-)(CFU-E/early proerythroblasts; Li et al. Blood 2014;124:3636);  and (b) the cells that survive this insult differentiate normally to stages III (lin-CD36+GlyA+) and IV (lin-CD36-GlyA+).  Using marrow from the index DBA patient, a 28-year old transfusion-dependent female with inherited RPS19 haploinsufficiency, and (as available) marrow from other DBA and del(5q) MDS patients (n=2-7 patients per study), we demonstrated that heme synthesis (assessed by ALAS2 mRNA and protein) initiates normally while globin production (α- and β-globin mRNA and protein) is slowed, and that stage I and II cells have excess heme. Next, to discern the cell-specific characteristics that allow a DBA or del(5q) MDS cell to survive and fully mature, we compared those cells able to advance to stages III and IV in patient and concurrent control cultures.  Stage III-IV GlyA⁺ DBA cells expressed 2.4-fold less ALAS2 mRNA and 2-fold more FLVCR mRNA than GlyA⁺ control cells (p values<0.05). Since the levels of ALAS2 and FLVCR mRNA were similar in stage I DBA and control cells, the time when heme synthesis initiates, these data imply that DBA cells capable of down-regulating their heme synthesis (have less ALAS2) or content (have more FLVCR and increased heme export) preferentially survive. Data from del(5q) MDS patients are similar - cells progressing to stages III and IV have 2.9-fold less ALAS2 mRNA (p<0.05) and 9.4-fold higher FLVCR mRNA (p<0.01) than control cells. That heme synthesis proceeds normally, while globin production initiates slowly, was confirmed by sequential sampling of marrow cultures at days 3, 7, 10, 13, and 17; and as expected, DBA and del(5q) MDS cells at day 7 had increased reactive oxygen species (ROS). Consistent with these observations, treatment with 10 μM succinylacetone, a specific inhibitor of heme synthesis, improved the erythroid cell output of DBA and del(5q) MDS cultures by 68-95% (p values<0.05), while the erythroid cell output of concurrent control cultures decreased by 4-13%. Cell death appears to result both from apoptosis (not shown) and ferroptosis, a ROS-induced process previously characterized in kidney epithelial cells and cancer cell lines (Dixon et al. Cell 2012;149:1060). Recent data showing P53 sensitizes cells to ferroptosis by impeding the transcription of SCL7A11, a cysteine-glutamate antiporter, leading to less cysteine uptake, less GSH synthesis, less GPX4, and less antioxidants (Jiang et al. Nature 2015;520:57) link our observations to work implicating P53 activation in DBA and del(5q) MDS anemia. In support of this connection, DBA marrow cells at culture day 10 have 57% less SLC7A11 mRNA than control cells in preliminary experiments. Together, our studies demonstrate that erythropoiesis fails when heme exceeds globin. The data also suggest that decreasing heme synthesis, either directly or by chelating iron, may improve these anemias. They further argue that lenalidomide (destabilizes P53) and therapies decreasing heme should be synergistic. Since diverse mutations could impede brisk protein transcription or translation, and hence brisk globin production, heme toxicity might also contribute to anemia in MDS patients without del(5q).