Pharmacological inhibition of polrmt mimicking polrmt rare variants associated with sideroblastic anemia, strongly inhibits proliferation and differentiation of human erythroblasts Free

Introduction Congenital sideroblastic anemia (CSA) result from abnormal erythroid differentiation related to mutations in genes involved in one of four key mitochondrial pathways: i) heme biosynthesis; ii) iron-sulfur cluster biosynthesis and transport; iii) tRNA synthesis and maturation and iv) mitochondrial respiratory chain synthesis. However, genetic analyses reveal a monogenic cause in only 50% of cases in French cohort. To identify new variants in genetically uncharacterized patients, we used an exome sequencing approach with an analysis focused on mitochondrial pathways. We identified two unrelated families with probands P1 and P2 carrying bi-allelic variants in POLRMT, a nuclear gene encoding a mitochondrial RNA polymerase. So far, POLRMT variants were associated with mitochondriopathies with neurological symptoms but no erythroid defect was reported. Though controlling mitochondrial gene expression, POLRMT is involved in oxidative phosphorylation (OXPHOS), known to provide energy required for early steps of erythropoiesis. In this report, using an ex vivo system of erythroid differentiation, we studied extensively whether POLRMT inhibition alters human erythropoiesis and whether POLRMT bi-allelic variants could represent a new cause of CSA.

Methods P1 (14 y.o.) and P2 (21 y.o.) referred to Hematology for non-regenerative chronic anemia. For the respectively past 3 and 10 years, they received transfusions every 1 to 3 months, hemoglobin level dropping as low as 4-6 g/dl. Bone marrow aspiration showed in both cases erythroblastopenia and respectively presence of 58% and 74% of ring sideroblasts.

CD34+ cells obtained from P1 were sorted from PBMC and cultured using a 3 step-protocol allowing a strong erythroid commitment. Normal CD34+cells from PBMC were cultured using a 3 step-protocol after synchronisation without EPO with or without the POLRMT chemical inhibitor IMT1. Ex vivo erythroid differentiation was monitored by flow cytometry, mitochondrial function by seahorse, mitochondrial morphology using electronic microscopy. Gene expression at RNA and protein levels was assessed by RNA-Seq, RQ-PCR and WB.

Results Exposure of primary CD34+cells to 0.2 µM IMT1 from D7 inhibited cell proliferation and altered drastically early erythropoiesis as shown by a decreased GPA expression and the nearly total loss of erythroid clonogenic potential. IMT1 increased the proportion of cells in G0/G1 at D11 and apoptosis rate at D14. RNAseq analysis confirmed the decreased expression of genes associated with erythroid maturation and heme synthesis. IMT1 exposure strongly decreased the transcriptional expression of mitochondrial-encoded components of the respiratory chains (MT-ND1, MT-CYB and MT-CO1), whereas no change was observed for nucleus-encoded ones (NDUFB8). At protein level, the decreased expression concerned the mitochondrial respiratory chain complexes encoded by nuclear and mitochondrial genes (C1) or mitochondrial genes only (C4). Expression of proteins belonging to C2, encoded by nuclear genes remained unchanged. POLRMT inhibition increased the mitochondrial biomass in erythroid cells assessed by TOM20 quantification and Mitotracker Probe, together with a decrease in the mitochondrial membrane potential. Seahorse experiments revealed that POLRMT inhibition altered the mitochondrial energy metabolism in erythroblasts, as shown by decreased oxygen consumption rates and ATP levels. Moreover, IMT1 exposure altered deeply the mitochondrial morphology. Ex vivo erythroid differentiation from CD34+ cells obtained from P1 confirmed the delay in GPA acquisition at D9, a lower proliferation rate, an increased mitochondrial mass at D15 and a decreased expression of transcripts of the respiratory chain complexes encoded by mitochondrial genes at D11.

Conclusion These findings demonstrate a role of POLRMT in mitochondrial gene expression required for an optimal oxidative phosphorylation during erythropoiesis. We observed a strong impact of POLRMT chemical inhibition in the proliferation and differentiation of erythroid cells in response to EPO. Same defects, although milder, were also observed using ex vivo culture of P1-derived primary CD34+ cells, arguing for a role of the identified POLRMT bi-allelic variants in the pathophysiology of the anemia. Thus, POLRMT should be analysed in rare inherited anemia, particularly in erythroid hypoplasia associated with ring sideroblasts.