FLVCR Deletion Does Not Impact Normal Hematopoietic Stem Cell Function but Is Necessary for Erythroid and T-Cell Maturation and Enhances Platelet Formation

Abstract 2363

Hematopoietic stem cells (HSCs) are mainly in a quiescent state, but activate and proliferate in response to stress. To maintain quiescence, as well as respond to activation signals, HSCs must coordinately regulate cell cycle status versus metabolic demand. Mismatch in these pathways can lead to HSC exhaustion, failure or malignant transformation. One metabolic pathway that is essential to all cells is heme biosynthesis. Heme can act directly as a signaling molecule by modulating the function of transcriptional (Bach1) or translational (eIFa2 kinase) regulators. In addition, heme can act indirectly through the action of hemoproteins (hemoglobin, oxidases, peroxidases, cytochromes). However, excess free intracellular heme is toxic, therefore organisms have developed “safety valve” pathways to regulate intracellular heme concentrations. The most well studied mechanism is free heme degradation by the heme-oxygenase pathway. We have recently described a second pathway to prevent intracellular heme excess, namely heme export by the cytoplasmic transporter FLVCR. Previously we have shown that FLVCR is essential for erythropoiesis, as FLVCR−/− mice die of anemia (Keel et. al., Science 319: 825, 2008). FLVCR is expressed in many other tissues and blood cells, including HSCs (Lucas et. al., Blood 106:51, 2005). This suggests that FLVCR may be important in HSC function or the development of other lineages, besides red cells. We used a competitive transplantation model to determine the effect of FLVCR deletion on HSC function and lineage maturation. FLVCR−/− or wild type (WT) donor cells and equal numbers of competitor cells were transplanted into lethally irradiated, syngeneic recipients. FLVCR−/− marrow cells reconstitute the granulocyte compartment equally as well as WT cells in serial transplantation (up to tertiary transplants). Equal contributions also remain after myelotoxic stress (radiation or arsenic trioxide). Therefore, FLVCR may be dispensable for normal HSC function under steady-state and stress conditions. As anticipated, FLVCR is required for proerythroblast survival and maturation. Eight weeks after competitive transplantation with FLVCR−/− donor cells, 100% of red cells are derived from competitor cells. This is also seen when the ratio of FLVCR−/−:competitor cells is increased to 95:5 from 50:50. FLVCR−/− cells fail to mature past the proerythroblast stage (CD71^pos lin-Ter119^{neg or lo}) and undergo significantly more apoptosis than WT donor cells (88% vs. 36%, p<0.001). FLVCR deletion also blocks T-cell development. Due to biological variability in our experimental model, T-cell numbers were normalized to granulocytes in each determination, in each mouse and expressed as a percentage of the expected value. At 5 weeks post-transplant, the percentage of FLVCR−/− derived T-cells was 65.8% of the percentage of FLVCR−/− derived granulocytes. The percentage of WT derived T-cells was 93.4% of the percentage of WT derived granulocytes (p<0.02). Over time, FLVCR−/− derived T-cells steadily decreased, dropping to 22% of expected values by week 24, and remaining low, whereas T-cells derived from control, WT donor cells remained stable (74–98% of expected values). FLVCR−/− T-cells are arrested at the double positive (CD4+CD8+) stage (Philip et. al., Blood (ASH Abstracts) 114:913, 2009). Surprisingly, loss of FLVCR improves platelet production. FLVCR−/− donor cells make up ∼60% of all platelets (avg. 1.71 x expected, when normalized to granulocytes). In the separate control cohort competitively transplanted with WT donor cells, ∼40% of the platelet population was derived from WT cells (avg. 0.91 x expected, when normalized to granulocytes). Our studies suggest that the increased platelet production likely results from increased megakaryocyte ploidy (e.g. avg. 14.2N in FLVCR−/− vs 5.2N in control) and not from increased megakaryocyte progenitors. Of note, FLVCR−/− deleted mice have thrombocytosis (2221/μL vs 1008/μL, p<0.001 in littermate controls), a finding we initially dismissed as a stress response to severe anemia. Together, these findings reveal stage specific roles for FLVCR in discrete hematopoietic lineages and support the broader hypothesis that heme excess might impair cell division, leading to decreased red and T-cell production, but improved endomitosis and increased platelet production.