Abstract
Diamond-Blackfan anemia (DBA) is associated with hypomorphic mutations in at least 16 ribosomal proteins. Additionally, mutations in GATA1 that result in the preferential expression of variants lacking the transactivation domain cause a similar but not identical disease. Ribosomal haploinsufficiency can lead to imbalanced ribosome protein production resulting in the activation of p53 and additionally slows protein translation, including globin, resulting in excess heme during the early stages of erythropoiesis when heme synthesis (an enzymatic process) is brisk but globin levels are insufficient. Mice lacking the heme export protein FLVCR1 develop a severe macrocytic anemia similar to DBA resulting from a block in differentiation at the CFU-E/proerythroblast stage which is caused by excess heme and ROS (J Clin Invest 125:4681, 2015). This macrocytic anemia occurs independent of ribosomal haploinsufficiency or p53 activation, suggesting that elevated heme is a key factor in the pathophysiology of DBA. Indeed, erythroid cultures of marrow from individuals with DBA demonstrate delayed globin synthesis, excess heme, elevated ROS, and increased cell death of CFU-E/proerythroblasts (Sci Transl Med 8:338RA67, 2016). Rescue studies showed in vitro erythroid differentiation improved when heme synthesis was decreased. To further understand the role of heme and ribosomal haploinsufficiency in DBA, we are characterizing RPL11 haploinsufficient mice. RPL11 heterozygous mice develop a cell intrinsic macrocytic anemia with increased susceptibility to radiation-induced lymphomagenesis (Cell Reports 13:712, 2015). Our studies confirm RPL11 heterozygous mice have a chronic macrocytic anemia (HGB 12.0±1.7 vs 14.3±0.4 g/dL; MCV 58.0±2.2 vs 46.8±1.4 fL) concurrent with a block at the CFU-E/proerythroblasts stage (63% reduction in basophilic erythroblasts). Erythroblast heme content is 2-fold higher than control by the polychromatic erythroblast stage while ROS is elevated (15-75%) throughout terminal differentiation. To understand the pathophysiology leading to ineffective erythropoiesis in DBA we performed single cell RNA sequencing and cell surface protein quantification on erythroid precursors from control and Flvcr1 -deleted mice and are comparing these data to data from RPL11 haploinsufficient mice. Principal component analysis identified 4 transcriptionally unique clusters with negative, low, intermediate, and high Ter119 levels respectively. α- and β-globin transcription were highly correlated (r=0.975) and increased as Ter119 expression increased. Gene set enrichment analysis comparing control cells to Flvcr1 -deleted cells revealed significant upregulation of the ribosome pathway genes and downregulation of the hallmark heme metabolism pathway genes including GATA1 and GATA1-target genes. Quantitative PCR analysis of RPL11 haploinsufficient erythroid cells show 2-fold increases in Rps19, Rps14, Rpl4, and Rpl35 transcript levels during terminal erythroid differentiation, however, Rpl11 transcript levels are reduced 50% in precursor cells and fail to increase to comparable levels with other ribosomal protein genes. Cdkn1a was increased, consistent with activation of the p53 pathway. Comparable studies with Flvcr1 -deleted mice do not show any activation of the p53 pathway, indicating that p53 pathway activation is unique to ribosomal haploinsufficiency and not a result of excess heme. Both Flvcr1 -deleted and RPL11 haploinsufficient erythroid cells have reduced Gata1 expression. To resolve the role of heme from other driving factors, we tested the effect of ALA and iron treatment to induce endogenous heme synthesis in sorted human marrow cells. Within 15 minutes of treatment the early erythroid cells (Lin-CD36+GlyA-) upregulated ribosomal gene transcript levels while later erythroid cells (Lin-CD36+GlyA+) did not. Additionally, GATA1 protein levels were rapidly decreased by ALA and iron, but not by exogenous heme or iron treatment alone. Thus poor translation of globin in ribosomal haploinsufficiencies leads to excess heme. It is this excess heme which leads to premature termination of erythroid differentiation by reducing GATA1 levels, additionally, it exacerbates ribosomal protein imbalance, increasing p53 activation and cell death. Thus the key pathologies in DBA are a direct result of excess heme, suggesting new approaches for treatment.
Disclosures
No relevant conflicts of interest to declare.
Stability of alpha and beta globin messenger RNA during induced differentiation of mouse erythroleukemia cells