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Inside this Issue: May and June

Posted By Connections Editor, Wednesday, July 01, 2015
Updated: Monday, June 29, 2015

MAY

RPL11 and RDH11 induce erythroid proliferation without Epo
Kummalue et al., pages 414–423.


Understanding the essential genes involved in erythropoiesis would help to clarify the pathobiology and improve the treatment of patients with anemia resulting from the abnormal production of erythroid cells. In this study, using lentivirally transduced UT-7/Epo erythroleukemic cells, Kummalue et al. identified 2 pivotal genes, i.e., ribosomal protein L11 (RPL11) and retinol dehydrogenase 11 (RDH11). Both RPL11- and RDH11-transduced cells could proliferate without erythropoietin with RPL11-transduced cells showing high proliferation rate. The antiapoptotic protein BCL-XL was highly detected in both transduced cells, and STAT5 signaling pathway was activated by Lyn and CREB phosphorylation. Moreover, cell-cycle profiles of both transduced cells revealed G0/G1 prolongation and low percentages of apoptosis. Interestingly, the hemoglobins in both transduced cells were switched from adult to fetal. These findings provide important insights into the mechanisms underlying induction of erythroid proliferation and may lead to improved therapeutic approaches for patients with diseases such as myelodysplastic syndrome, Diamond–Blackfan anemia, and acute erythroleukemia, which are still too difficult to manage, and specific treatments remain to be developed.

 

Components of the microRNA-induced silencing complex in red blood cells
Azzouzi et al., pages 382–392.


Erythropoietic cells extrude their nucleus but retain their ability to respond to stimuli by regulating protein translation, a process that can be regulated by microRNAs (miRNAs). In this study, Azzousi et al. identified both the miRNA and the protein components forming the miRNA-inducing silencing complex in circulating red blood cells (RBCs) by using a combination of deep sequencing of small RNA species and endogenous Argonaute 2-immunoprecipitation followed by mass spectrometry. Almost 200 different miRNAs and 26 different proteins that interact with Argonaute 2 were identified, giving a comprehensive overview of the miRNA-inducing silencing complex components in circulating RBCs. MicroRNA-451a was found to be the dominant species, with over 60% of total reads, and was shown to interact with its known target, 14-3-3zeta, in an Argonaute2-dependent manner. This study points at the possibility of miRNAs in circulating RBCs as biologically active molecules, and provides a starting point for in depth analysis of miRNA-regulated processes in these enucleated cells. This study not only gives novel insight into erythrocyte homeostasis, but will lead to a better understanding of erythroid diseases as well.

 

TXNIP, a novel player in erythroid differentiation
Gasiorek et al., pages 393–403.


Besides a well-characterized role in redox control and cell cycle, there are only a few reports that have implicated Thioredoxin-interacting protein (TXNIP) in the function of hematopoietic cells, namely natural killer, dendritic, and hematopoietic stem cells. In this study, Gasiorek et al. present the first report of a role for TXNIP in terminal erythropoiesis. TXNIP was robustly upregulated upon induction of erythroid differentiation in two different cellular models. Furthermore, inhibiting the MAP kinases p38 and JNK induced the levels of TXNIP. Flow cytometry experiments revealed that, in vivo, TXNIP knockout mice displayed impaired splenic erythropoiesis, characterized by a partial block between basophilic and late basophilic/polychromatic erythroblasts. It will be of interest to identify the pathways targeted by TXNIP at this transition step. In this report, the authors suggest that one of the mechanisms may be the effect of TXNIP on cellular iron levels, since they observed increased iron uptake upon TXNIP overexpression. This study introduces TXNIP as a new player in the field of erythropoiesis and opens the door to research on its implication in an important process for red blood cell differentiation, namely iron homeostasis.

 

Potential side effects of dasatanib on host immunity
Oksvold et al., pages 352–363.


Dasatinib is a widely used broad-spectrum tyrosine kinase inhibitor with potential for its clinical application to be expanded beyond chronic myeloid leukemia. Recently, this group found that dasatinib significantly reduced B cell numbers when used as a treatment for myeloproliferative disease in c-Cbl RING finger mutant mice. These findings highlighted the lack of studies investigating the effects of dasatinib on B cells from wild-type mice and humans. In this study, Oksvold et al. show that dasatinib has an inhibitory impact on human and mouse B-lymphocytes. They found that dasatinib markedly reduced the number of human CD19+ peripheral B cells in culture through the induction of apoptosis, yet it had no impact on T cell viability despite broad inhibition of early signaling events in both B and T cells. Sensitivity to dasatinib was also shown in human pre-B cells in vitro and in mouse pre-B cells in vivo. Furthermore, dasatinib targeted actively cycling mouse B-lineage cells in the spleen and caused a marked loss of thymocytes. In contrast, no impact was seen in myeloid lineage cells or hematopoietic progenitors. These findings are important and have potential implications for infection prevention strategies for patients receiving dasatinib. In this regard, Rodriguez et al. reported in August 2012 in Leukemia & Lymphoma a single institute cohort study in which 69 patients received dasatinib treatment for chronic myeloid leukemia or Ph+ ALL. Bacteria were the most common offending microorganisms and pneumonia was the most common type of infection associated with dasatinib. A more recent study published by Chang et al. last August in the International Journal of Infectious Diseases reported Pneumocystis jiroveci pneumonia in patients receiving dasatinib treatment. Both studies have raised attention to the possible effects of dasatinib on cellular immunity. Hence, studies such as the one performed by Oksvold et al. are important to further our understanding of the potential effects of this drug on cellular immunity, especially in view of the fact that dasatinib is increasingly used as a treatment modality for hematopoietic malignancies. Overall, these results underline the importance of thorough investigation of the effects of kinase inhibitors on immunity and suggest that dasatinib may adversely impact patients' immune responses, especially those receiving long-term treatment.

 

JUNE

Autophagy regulates platelet production
Cao et al., pages 488–494.

Megakaryopoiesis, megakaryocyte differentiation, and thrombopoiesis are regulated at multiple stages during hematopoiesis. These involve successive lineage commitment followed by polyploidization, maturation, and development of an extensive internal demarcation membrane system, leading to the release of platelets in circulation. The cellular mechanisms by which megakaryocytes derive from their progenitors and megakaryocytes differentiate into platelets are not fully understood. In this paper, Cao et al. set out to study the role of autophagy, a metabolic process essential in homeostasis and cellular remodeling, on megakaryopoiesis and platelet function. Using an autophagy-related gene (Atg7) hematopoietic conditional knockout mouse model, they report that loss of autophagy caused mitochondrial and cell cycle dysfunction, impeding megakaryopoiesis and megakaryocyte differentiation, as well as thrombopoiesis. In addition, abnormal platelets with larger size and smaller number were produced in peripheral blood, ultimately leading to severely impaired platelet production and failed hemostasis. These data suggest that autophagy is essential for megakaryopoiesis, megakaryocyte differentiation, thrombopoiesis, and platelet production. Given these results, autophagy may serve as a suitable target for megakaryocyte/platelet disorders in clinical conditions.

 

You can count (on) this: Chimerism analysis going digital
Stahl et al., pages 462–468.


Hematopoietic chimerism, i.e., the ratio between donor and recipient blood cells after allogeneic stem cell transplantation, is a crucial diagnostic parameter in the posttransplant period. To assess chimerism, donor-/patient-specific genetic markers in polymorphic genome regions are investigated. The current gold standard of chimerism analysis employs polymerase chain reaction (PCR)-based amplification of repetitive DNA sequences, such as short-tandem repeats (STRs), and quantification of donor-/recipient-specific PCR products by capillary electrophoresis. This method is robust, but its detection limit of approximately 1% is unsatisfactory. Newer techniques measuring the presence of insertion/deletion polymorphisms by real-time quantitative PCR (qPCR) are much more sensitive, but are hampered by comparatively low accuracy in the state of mixed chimerism (10%–90% donor cells). To overcome the inherent limitations of classical qPCR in chimerism analysis, Stahl et al. propose in this study the use of digital PCR. Digital PCR (dPCR) is based on the compartmentalization of single DNA molecules, allowing the parallel but separate amplification of large numbers of individual target sequences. Digital PCR represents an end-point analysis and is, in contrast to qPCR, not influenced by the efficiency of individual PCR reactions. Taking advantage thereof, the authors simultaneously quantified both donor- and patient-specific alleles in single tubes (duplex). They show that dPCR combines the excellent sensitivity of qPCR with the high accuracy and reproducibility of STR-PCR. Since dPCR is easy to perform and interpret, it has the potential to become the new standard in chimerism analysis. To achieve this, a broad panel of assays covering multiple insertion/deletion polymorphisms will be required.

 

Transferrin receptor 1 (TfR1) signals erythron iron need
Keel et al., pages 469–478.


When there is systemic iron deficiency, marrow erythroid cells upregulate TfR1 to assure that residual transferrin-bound iron is preferentially imported for hemoglobin synthesis and red cell production. Similarly, TfR1 is upregulated whenever erythropoiesis expands and additional iron is needed. In both settings, the liver also synthesizes less hepcidin, which acts by binding the iron export protein, ferroportin, leading to its degradation. This inhibits dietary iron absorption and macrophage iron recycling so that iron is more rapidly absorbed form the gastrointestinal tract, more rapidly released from macrophages, and thus more available to the erythron. How an erythroid precursor in the bone marrow communicates its iron need to hepatocytes is a major unresolved question in the field of iron homeostasis. In this manuscript, Keel et al. ask whether TfR1, besides assuring adequate marrow iron import, is also responsible for alerting the liver to suppress hepcidin. The authors studied a unique pure red cell aplasia patient with erythroid arrest at the proerythroblast stage, presenting with excess marrow proerythroblasts (highly expressing TfR1) but no hemoglobinized cells. The patient's serum hepcidin was low, while pure red cell aplasia patients who lack all erythroid precursors had high serum hepcidins, suggesting that TfR1 is a proximal mediator of the erythroid regulator of hepcidin expression. Their additional studies in mice genetically engineered to be deficient in TfR1 further imply that TfR1 either regulates the release of a yet-to-be-defined mediator of hepcidin expression or regulates at the post transcriptional level erythroferrone, a tumor necrosis factor α superfamily member that regulates hepcidin production after phlebotomy and after erythropoietin administration. The data presented in this study also help explain prior findings from ferrokinetic studies in humans, studies in hypotransferrinemic mice, and studies in mice lacking signal transducer and activator of transcription 5 in hematopoietic cells. The data also imply that iron homeostasis and red cell production are intricately connected through multiple, and likely independent, links.

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