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Inside this Issue: November and December

Posted By Connections Editor, Wednesday, December 16, 2015



Newly identified mutations contributing to leukemogenesis and therapy resistance in AML
See Sykes et al., pages 989

Acute myeloid leukemia (AML) is the most common acute leukemia in adults. Unfortunately, even with aggressive treatment, including high-dose chemotherapy and bone marrow transplantation, most patients relapse and develop resistance to anti-leukemic therapies, resulting in 5-year overall survival rates between 30% and 40%. Increasing evidence supports a model for leukemia evolution whereby mutations accumulate in functionally normal hematopoietic stem cells during a prolonged “preleukemic” phase. Recent advances in whole-genome sequencing and global genomic approaches have provided a long list of newly identified mutations. However, even with these advances, how these molecular abnormalities contribute to the etiology, pathogenesis, and therapeutic responses of AML remains largely unknown. The International Society of Experimental Hematology invited Dr. Ross L. Levine and Dr. Ravindra Majeti, two authorities on AML, to conduct a webinar (available online at presenting their most recent research on this topic. In this perspective, Sykes et al. summarize the key points discussed in both presentations, which centered on the most recent published data concerning the pathogenesis of AML, including, but not limited to, clonal evolution, newly identified mutations in AML patients, and mutational cooperativity. In addition, they discuss the clinical and basic research implications of their work, as well as novel therapies that are currently emerging in AML treatment.


Multiple roads to genomic instability in Fanconi anemia
See Abdul-Sater et al. pages 1031

Inherited mutations within FANCA or other FA genes cause Fanconi anemia (FA), a bone marrow failure syndrome associated with myelodysplasia (MDS) and acute myeloid leukemia (AML). Acquired FA mutations drive AML in patients without FA. The FA pathway orchestrates both interphase DNA repair and mitosis; thus, FA mutations may unleash genomic instability via multiple mechanisms. However, the in vivo impact of interphase and mitotic errors on FA−/− hematopoiesis is unknown. To address this issue, Abdul-Sater et al. employed in this study a battery of quantitative assays and discovered increased frequency of abnormal mitoses during in vivo hematopoiesis in non-leukemic FANCA−/− patients and Fancc−/− mice, suggesting that these mitotic errors may ultimately cause leukemia. Mechanistically, live FANCA−/− cells failed to efficiently assemble mitotic spindles. Superresolution microscopy revealed that FANCA shuttles from centrioles to microtubule- attachment sites at the centrosome periphery in early mitosis, possibly to fine-tune microtubule nucleation.
In accordance with FA genes acting as mitotic housekeepers, loss of FANCA or FANCC rendered cells hypersensitive to antimitotic chemotherapeutics. Importantly, the authors illustrate that functionally different chemotherapeutics selectively damage FA−/− cells through distinct mechanisms. Although crosslinking drugs shatter DNA and activate the G2/M cell cycle checkpoint, low-dose antimitotic chemotherapy amplifies chromosome segregation errors. Accordingly, loss of FA signaling promotes genomic instability through complementary interphase and mitotic defects. Together, these findings not only provide insights into the complex mechanisms of genomic instability resulting from loss of the FANCA tumor suppressor, but also suggest that precision medicine therapeutics targeting erroneous mitosis may selectively kill FANCA−/− leukemia and other cancers.


VEGF may not be the most suitable target for anti-angiogenic therapy in primary myelofibrosis
See Dragoni et al., pages 1019

Increased angiogenesis is a major histopathologic feature of both bone marrow and spleen of patients with primary myelofibrosis (PMF). This has prompted the design of therapeutic protocols based on the use of anti-angiogenic drugs directed mainly against vascular endothelial growth factor (VEGF), the major regulator of such a process. However, despite the strong rationale for the use of anti-VEGF treatments, these trials have resulted in poor outcomes. In the recent past, it has been proposed that circulating endothelial progenitor cells have a role in angiogenesis in different diseases, including PMF. Dragoni et al. report that PMF patient-derived endothelial colony-forming cells, the only bona fide endothelial progenitor cells described so far, mount a defective proangiogenic response to VEGF in vitro, assessed in terms of Ca2+ oscillations, proliferation, and tubulogenesis. Importantly, this reported alteration in VEGF-mediated calcium flux in these endothelial colony-forming cells was previously unrecognized. These results, in addition to the previous report from the same group of an increased frequency of these cells in the peripheral blood of PMF patients, open new perspectives on the potential role of circulating endothelial progenitor cells in the pathogenesis of PMF. In particular, these data shed new light on the failure of anti-VEGF therapy in PMF patients and represent valuable information for the design and development of new anti-angiogenic drugs for the cure of this disease.


Innate and adaptive immune response: Help for curing CML?
See Caocci et al., pages 1015
Withdrawal of tyrosine kinase inhibitors (TKIs) in patients with chronic myeloid leukemia (CML) continues to be a much debated issue, and the search for clinical and/or biological patterns predictive of stable molecular remission (MR) and, perhaps, even definitive recovery is still underway. In a previous article, this group reported that homozygosity for killer cell immunoglobin-like receptor (KIR) A haplotype was significantly associated with achievement of deep MR. In this article, Caocci et al. report that after discontinuation of TKIs, MR was significantly higher in patients homozygous for KIR A haplotype and/or carrying specific combinations of KIRs and their HLA class I ligands. Although the study cohort is quite small and a bit heterogeneous, this article provides interesting novel findings on a hot topic in current CML treatment and suggests that natural killer cells and their KIRs cooperate with TKIs in the cure of CML.
There are many reasons why both physicians and their patients prefer to stop therapy. Some worth mentioning are the high cost, adverse events, poor compliance, the desire for pregnancy, and treatment-related poor quality of life. Ongoing clinical trials investigating TKI discontinuation should soon release more details on the safety of stopping treatment and the ideal candidates for this option. Meanwhile, on the basis of this and other studies, the important function of both innate and adaptive immune responses in controlling CML should be given attention. Further appreciation of immune regulation is a fundamental step toward improved treatment approaches and the development of anti-inhibitory KIR antibodies or vaccination strategies.





Reduced dose of cyclophosphamide post-HLA haploidentical peripheral blood stem cell transplantation as an option for high-risk leukemia and MDS patients
See Nakamae et al., pages 921–929

Nonmyeloablated T-cell–replete HLA haploidentical bone marrow transplantation with high-dose posttransplantation cyclophosphamide (PT/Cy) has shown encouraging outcomes with low transplantation-related mortality. High-dose Cy is capable of controlling graft versus host disease (GVHD) and preventing graft rejection; however, the high relapse rate remains a major issue of concern, particularly with high-risk hematological malignancies. In this prospective pilot study, Nakamae et al. investigate two reduced dosages of PT/Cy for patients with a poor prognosis, refractory leukemia, or myelodysplastic syndrome. The authors report a trend toward a lower incidence of severe acute GVHD at day 100 in the “25 mg/kg × 2 doses of PT/Cy” group compared with the “25 mg/kg of PT/Cy” group. Although they use peripheral blood as a stem cell source for all patients, the cumulative incidence of chronic GVHD was very low in both groups. BK virus hemorrhagic cystitis occurred more frequently in the double-dose PT/Cy group, particularly when busulfan was used for conditioning. The probability of overall survival at 1 year in the double dose group was better than that in the single dose group despite the absence of statistical significance. Overall, these results suggest that HLA haploidentical T-cell–replete peripheral blood stem cell transplantation with 25 mg/kg × 2 doses of PT/Cy might be a feasible option for high-risk leukemia and MDS patients. It thus might be worthwhile to compare the efficacy of a double dose of 25 mg/kg PT/Cy with the original PT/Cy double dose of 50 mg/kg in a large prospective study.


Osteoblast stimulating factor-5 (OSF-5); a novel tuner in B lymphopoiesis
See Fujita et al., pages 963–973

B lymphocytes are developed from hematopoietic stem cells (HSC) in their specialized microenvironment. In contrast to the recent progress in understanding the HSC niche, the mechanisms regulating the B lymphopoietic niche have remained unclear. To shed light on this issue, Fujita et al. identify several molecules secreted by the B lymphopoietic niche using signal sequence trap. First, the authors examined the proteins produced by MS5 stromal cells, which are B lymphopoiesis supportive cells commonly used in models of in vitro B lymphopoiesis. They identified pleiotrophin, placental proliferin-2, and osteoblast stimulating factor-5 (OSF-5). On the basis of experiments with transgenic mice and in vitro studies, they found that a secreted variant of OSF-5 inhibits proliferation at the pre–B cell stage. Furthermore, the authors show that human mesenchymal cells also express OSF-5 and, as in mice, this molecule suppresses the proliferation of human pre–B cells. These results identify OSF-5 as a new negative regulator of B lymphopoiesis and provide a framework to identify molecules that can be used to speed the regeneration of the humoral immune system after chemotherapy, and potentially suggesting ways to inhibit the growth of B lineage malignancies.


New biomarkers of AML dynamics
See Tzoran et al., pages 936–943

Acute myeloid leukemia (AML) is characterized by rapid growth of leukemic blasts. Cell exposure to chemo- or radiotherapy increases the shedding into the blood circulation of extracellular vesicles (EVs), which express antigens reflecting their cellular origin. Despite normalization of blood counts, AML patients in remission still harbor leukemic burden. This fact emphasizes the need to develop diagnostic tools capable of identifying minimal residual disease (MRD). The current study by Tzoran et al. explores the role of circulating EVs as potential biomarkers of AML activity and predictors of thrombogenicity in patients who are reported to be at increased risk for thrombotic events. The authors demonstrate that circulating EVs of AML patients express leukemic blast membrane proteins, with their levels significantly varying at diagnosis, nadir, and remission, thus reflecting the disease dynamics. Blast EV counts were found to be higher in patients at diagnosis compared with healthy controls. In remission, a statistically significant correlation between the percentage of CD117-labelled EVs in the peripheral blood and the amount of CD117-positive blasts in the bone marrow was revealed. Moreover, blast EV levels at remission were significantly lower in patients who were alive at 3 years of follow-up compared with those who succumbed to the disease, indicating the high predictive value of this biomarker. Additionally, EV procoagulant activity was increased in patients compared with controls and induced high endothelial cell thrombogenicity. This study provides a platform for the use of EVs as potential biomarkers of minimal residual disease and the procoagulant state in AML patients.


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Inside this Issue: September and October

Posted By Connections Editor, Friday, November 6, 2015
Updated: Thursday, November 5, 2015


A new tool to identify the molecular and functional properties of individual stem cells at a single-cell resolution
Schulte et al., pages 803–811.


The recent discovery of significant heterogeneity in normal and malignant stem cell populations has driven the development of numerous tools to try and identify, at single-cell resolution, what the molecular and functional properties are of individual stem cells. Many groups have identified populations of hematopoietic stem cells that are based on a binary positive/negative gate and do not take into account the differing levels of proteins on the cell surface (i.e., how positive is a cell for marker X). To consider this, in this manuscript, Schulte et al. combine flow-cytometric index sorting (which quantifies the exact fluorescence of each cell surface marker form each single cell) with single-cell functional assays to determine whether or not different levels of cell surface molecules can enrich or deplete for functional activity. This technique is particularly useful when cell number is limited and can assess multiple marker intensities simultaneously in a single experiment. The technique has been already been usefully applied to uncover the molecular program of functional hematopoietic stem cells (HSCs; Wilson et al., Cell Stem Cell 2015) and can be applied to any cell-biological system with a defined single cell functional assay.

How the experimental setup of serial transplantations largely influences the interpretation of long-term HSC performance
Rundberg Nilsson et al., pages 812–817.

To study HSC self-renewal and longevity, serial transplantation has been considered the gold standard for many years. However, discrepancies in experimental procedures are widespread, including the choice to either transplant whole bone marrow (wBM) or fluorescence-activated cell sorting (FACS)-purified HSCs into secondary hosts. In this study, Rundberg Nilsson et al. show that this choice of experimental procedure significantly influences the readout following transplantation. The authors show that donor-derived HSCs can distribute highly unevenly among bones of the conditioned host, leading to serial transplantation of nonrepresentative HSC chimerism levels if wBM is isolated and transplanted from only one or a few bones. Moreover, by comparing mature donor cell output from transplanted wBM cells as opposed to FACS-purified HSCs, they found differences suggestive of cotransplantation of long-lived lymphoid progenitors and/or mature cells together with HSCs in the wBM, which could severely affect the interpretation of ongoing HSC activity. Furthermore, transplantation of different frequencies of HSCs using wBM transplantation between groups/recipients not only impeded the evaluation of the HSCs on a per cell basis, but also obscured the distinction between effects generated during the primary transplantation and those presenting during the secondary transplantation. Based on these data, the authors recommend serial transplantation of highly purified HSC populations using flow cytometry markers as opposed to unfractionated wBM. A standard experimental setup, such as the one suggested in this study, is highly important for the research community, both to aid comparison of results generated from different research groups and for optimal evaluation of HSC performance.

Targeting autophagy to promote differentiation in myeloid leukemia
Orfali et al., pages 781–793.

Acute myeloid leukemia (AML) affects predominantly an elderly population, who tend to tolerate intensive chemotherapy poorly. Differentiation therapy, a pharmacologic override of the differentiation block observed in this condition, is an attractive and potentially minimally toxic strategy. The use of all-trans-retinoic acid (ATRA) in the treatment of acute promyelocytic leukemia (APL) is the defining example of success in this field and a model for the study of differentiation pathways in myeloid cells. Recently published literature reports that ATRA treatment of APL cells induces autophagy, a lysosomal-dependent protein recycling pathway with defined roles in mammalian cell differentiation.
In this study, Orfali et al. investigate a functional role for autophagy in leukemic cell differentiation. The authors initially confirm the induction of autophagy by ATRA in
an APL cell line and in primary APL tissue cultured ex vivo. They proceed to demonstrate that autophagy blockade, using either pharmacologic agents (Chloroquine/3-methyladenine) or genetic inhibition of the critical autophagy regulator ATG7, impedes ATRA-mediated APL cell differentiation. Conversely, in an exciting experiment, the investigators show that promoting autophagy with the use of lithium chloride can enhance the differentiating effects of ATRA in AML cells previously resistant to differentiation.

In a time when the molecular heterogeneity of AML is increasingly understood, this work considers the endpoint at which these aberrancies converge: the differentiation block. If autophagy is of functional importance in leukemic cell differentiation, it is possible that defective autophagy could contribute to their malignant phenotype. Importantly, autophagy induction with readily available compounds may overcome ATRA-resistance in APL and potentiate the differentiation of non-APL leukemic subtypes.

Using cellular reprogramming to make blood
Singbrant et al., pages 756–759.

The current use of human HSCs to treat blood disorders is hampered by the need to find human leukocyte antigen (HLA)-matched donors and to obtain sufficient numbers of long-term engraftable HSCs. Despite the efforts of many researches, ex vivo expansion of HSCs to improve hematopoietic reconstitution and engraftment potential has been largely unsuccessful owing to the inability to generate sufficient HSC numbers and to excessive differentiation of the starting cell population. In search for an unlimited source of autologous HSCs, novel approaches, such as the reprogramming of somatic or pluripotent cells lines to HSCs, are now being pursued. Induced HSCs (iHSCs) hold much potential for regenerative medicine and, in the near future, could benefit patients through disease modeling and drug screening. The International Society of Experimental Hematology invited Drs. George Q. Daley and Derrick Rossi, two leaders in the field of cellular reprogramming, to present a webinar ith their most recent research on this topic. In this perspective, Singbrant et al. summarize the webinar (available online at and discuss the state of the field of hematopoietic specification.


Gene correction of a chronic granulomatous disease-causing mutation in iPS cells
Flynn et al., pages 838–848.

Chronic granulomatous disease (CGD) is a lifelong affliction causing serious morbidity and potential mortality, even in the present era of potent antimicrobials. Currently, the only curative treatment option involves allogenic bone marrow transplantation. However, a preferred option to avoid the inherent risks associated with this treatment, could be transplantation with genetically-modified, patient-derived cells (either from induced pluripotent stem [iPS] cell or the patient's bone marrow) would theoretically improve outcomes dramatically. To that end, in this study, Flynn et al. use the Cas9–clustered regularly interspaced short palindromic repeats (CRISPR) system to efficiently gene correct a CGD-causing point mutation in a patient-derived iPS cell line. This in situ modification of a single-point mutation within an intron of the main disease causing gene, CYBB, results in the correction of a splicing defect and restoration of the oxidative burst in macrophages derived from the iPS cells, a key antimicrobial response that is compromised in CGD sufferers. This work lays the foundation for future genetically clean (“footprintless”) gene therapy approaches to this, and other, life-threatening genetic diseases.

Encouraging advances in treating and curing globin disorders
Blobel et al., pages 821–837.

Throughout the era of molecular medicine, lessons learned from the globin gene disorders have led the way forward for research of many human genetic diseases. The pathway from understanding a human disease to developing an appropriate treatment or cure often takes decades. Thalassemia and sickle cell disease, which cause serious illness in millions of individuals worldwide, were arguably the first human genetic diseases to be understood at the molecular and cellular level, yet in more than 50 years of research, this increasingly detailed understanding has had only a modest impact on clinical treatment. However, recent, significant advances in our understanding of the developmental regulation of globin gene expression and in the technology for safely manipulating hematopoietic stem cells engender optimism that improved management for these disorders may be on the near horizon. Altering the normal developmental pattern of globin gene expression could be the most efficient way to treat the thalassemias, and over the past two decades, specific new pathways that regulate developmental switching have been identified, suggesting new ways to manipulate the activity of individual globin genes. Similarly, the promise of gene therapy to treat thalassemia, after three decades of meticulous study, has been achieved by lentiviral transduction of blood stem cells in affected patients. Genome editing of such cells may also soon be a reality. These advances, as well as many others, were presented at the 19th biennial Hemoglobin Switching Conference, held this past September in Oxford, United Kingdom, by the 160 participants who traveled from around the world to participate in this unique conference. This review by Blobel et al. highlights many of the most recent advances that were presented during the three days, and highlights why this meeting has become one of the most important international conferences examining the developmental biology, molecular genetics, cell biology, and epigenetics of human disease.

Interferon α promotes hematopoietic stem and progenitor division in humans
King et al., pages 912–918.

Named for their ability to interfere with viral transmission, interferons suppress the growth and survival of many cell types. Surprisingly, however, hematopoietic stem cells (HSCs) are activated to divide by the Type I interferon, interferon α, in murine in vivo studies. Similar effects have also been seen in response to Type II interferon. King et al. postulate in this study that interferon-mediated activation of HSC division could be a primitive mechanism for generating innate immune responses. To evaluate the effects of interferon α on human HSCs in vivo, serial bone marrow (BM) samples from patients with polycythemia vera or essential thrombocytosis were collected before and during treatment with pegylated interferon α. While the absolute number of CD34+CD38− hematopoietic stem and progenitor cells (HSPCs) did not change, the percentage of CD34+ cells undergoing cell division increased in most patients treated with pegylated interferon α. Furthermore, colony formation of whole BM cells in methylcellulose increased, indicative of enhanced differentiation. These findings were present even in patients with low or undetectable JAK2-mutant allele burden, suggesting that they reflect the activity of wild-type Jak2 in HSPCs. In contrast, patients treated with hydroxyurea, an antimetabolite, showed decreased cell division and decreased colony formation. This unique in vivo longitudinal study demonstrates for the first time in a human setting that interferon α can be used to promote division of quiescent HSPCs; this application may promote polyclonal hematopoiesis in patients with myeloproliferative neoplasms and improve chemosensitivity of hematologic malignancies.

MicroRNA-223 is a promoter of lineage commitment and differentiation and a modulator in AML
Gentner et al., pages 858–868.

Although the effect of miR-223 on granulopoiesis was recognized over 10 years ago, its role in hematopoietic stem and progenitor cells (HSCPs) and in acute myeloid leukemia (AML) is still not completely understood. In this study, Gentner et al. explore the role of miR-223 in various murine AML models and human CD34+ cells. Considering that high miR-223 levels were associated with favorable prognosis and that the expression of miR-223 was low in leukemia-initiating cell (LIC) fractions of AML patients, it is surprising that genetic depletion of miR-223 decreased the LIC frequency in an AML mouse model, but was not mandatory for rapid onset AML, indicating only a minor role in AML development. This correlates with their observations in CD34+ cells, where miR-223 fine tunes myeloid progenitor cells with respect to myeloerythroid differentiation and expansion. The findings of Gentner et al. close the gap between in vitro findings and the actual function of miR-223 in vivo as a regulator of the expansion/differentiation equilibrium in HSPCs cells and in AML, highlighting its role as rheostat and not on-or-off switch in granulopoiesis.

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Inside Experimental Hematology: July 2015

Posted By Connections Editor, Monday, August 31, 2015
Updated: Tuesday, August 25, 2015

Current state of attempts to improve the efficacy of cord blood transplantation using stem cell-based technologies
Pineault et al., pages 498–513.

It is increasingly difficult to find suitablematched donors for hematopoietic stem cell transplant in developed countries due to a variety of factors including an aging population and decreased birth rates. Cord blood (CB) stem cells now represent an important source of hematopoietic stem and progenitor cells (HSPC) for patients with no suitable unrelated donor. However, the major limitation of CB transplantation is the limited number of total nucleated cells and HSPCs banked per unit. Transplantation of two partially matched units can overcome this shortcoming but fails to accelerate engraftment. Ex vivo expansion of CB HSPCs has made significant headway in recent years; accelerated neutrophil recovery, improved platelet engraftment, and superior therapeutic outcomes have now been reported in several clinical trials. In this review, Pineault et al. describe some of the most significant advances made toward improving CB engraftment through stem cell engineering. This review focuses on the development of culture strategies designed to expand human HSPCs ex vivo and the enhancement of engraftment through improved homing. The contributions of coculture, cytokines, developmental-related genes, and small molecules to advancements in CB transplantation are discussed. The major findings of studies that defined the short- and long-term engraftment properties of expanded HSPCs are presented in a series of supporting tables. Finally, the results and principal lessons learned from clinical trials conducted with expanded CB HSPCs are presented along with future perspectives of interest. In summary, this review provides experienced or entry-level readers of all fields with an up-to-date assessment of both fundamental and translational research on stem cell–based technologies developed to improve CB transplantation outcomes.


A widely applicable Kit and Sca-1 independent identification of hematopoietic stem cells
Vazquez et al., pages 578–585.

Hematopoietic stem cell (HSC) transplantation holds promise in many disease settings, including cancers, bone marrow failure, and autoimmune disease. The surface markers Kit and Sca-1 are commonly used to isolate HSCs in mice. However, some strains of mice, including the NOD mouse that is used to study autoimmune diabetes, do not express Sca-1 on their hematopoietic progenitors. Similarly, Kit is dynamically regulated in response to bone marrow injury and cannot be used to identify HSC following irradiation. It is therefore important to have an alternative identification approach to Kit and Sca-1 staining that is both simple and efficient. In this manuscript, Vazquez et al. found that antibodies to CD201 and CD27 enable HSC identification across a wide variety of mouse strains, and the expression of these markers is maintained following irradiation. These markers will facilitate the application of HSC transplantation to disease models such as the NOD mouse, as well as in settings where Kit and Sca-1 are dynamically regulated.


Proapoptotic function of hyperactivated AKT1 in AML
Tang et al., pages 554–564.

Elevated levels of phosphorylated AKT1 are frequently seen in leukemic blast cells. However, reoccurring mutations in the Akt1 gene are absent in patients with acute myeloid leukemia (AML), raising the question whether permanent AKT1 activation is incompatible with leukemogenesis. Previous reports have indicated that AKT1 activation leads to increased levels of reactive oxygen species (ROS), triggering apoptosis. Whether this is true in cells coexpressing other genetic events in leukemia is not known. To elucidate whether AKT1 causes apoptosis when coexpressed with additional oncogenes seen in AML, in this study, Tang et al. coexpressed hyperactivated AKT1 with STAT5, FLT3-ITD, or antiapoptotic Bcl-2 in hematopoietic progenitors. Hyperactivation of AKT1 was incompatible with both STAT5- and FLT3-ITD-driven proliferation, and triggered cell cycle block and apoptosis. Moreover, transplantable cells of Bcl-2 transgenic mice were impaired in their engraftment ability when expressing hyperactivated AKT1. Cells expressing hyperactivated AKT1 displayed higher levels of ROS, which could be counteracted by addition of the antioxidant N-acetyl-L-cysteine (NAC). These results indicate that constitutive AKT1 activity is incompatible with growth- and survival-promoting ability of other activated genes in AML. Because the harmful effects of hyperactivated AKT1 on hematopoietic cells was linked to an improper balance of ROS, this could be one reason why activating mutations of Akt1 are absent in AML. Considering that activating mutations of FLT3-ITD and other signaling proteins in leukemia activate the PI3K/AKT pathway, future drug development to enhance AKT1 activity could lead to novel ways of eradicating leukemic cells.


LSD-1 inhibitor RN-1 induces γ-globin expression in a sickle mouse model
Rivers et al., pages 546–553.

Increased fetal hemoglobin production is associated with decreased symptoms and increased life span in patients with sickle cell disease (SCD). Lysine demethylase-1 (LSD1), an enzyme that removes methyl groups from mono- and dimethylated lysine 4 amino acids of histone H3, is a repressor of γ-globin gene expression. LSD1 is also highly overexpressed in many cancers and is a target for anticancer drug therapies. Tranylcypromine (TCP), an FDA-approved LSD-1 inhibitor, increases γ-globin in cultured human erythroid progenitors and induces differentiation of AML cell lines in combination with retinoic acid. Using a novel approach, in this manuscript, Rivers et al. identified RN-1 as a biologically active LSD-1 inhibitor that induced differentiation of the U937 AML cell line with increased potency compared to tranylcypromine. The authors then performed in vivo studies in the SCD mouse model to test the ability of this drug to induce γ-globin messenger RNA. RN-1 treatment of SCD mice induced γ-globin messenger RNA, F cells, and F retics to higher levels than either TCP or hydroxyurea, and its effect was comparable to decitabine, currently in clinical trials for SCD. Because the level of γ-globin induction in the SCD mouse model is low, the authors intend to perform additional studies in the baboon nonhuman primate model that has reproducibly facilitated clinical translation of novel fetal hemoglobin–inducing therapies for SCD.

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Inside this Issue: May and June

Posted By Connections Editor, Wednesday, July 1, 2015
Updated: Monday, June 29, 2015


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.



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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Inside this Issue: March and April 2015

Posted By Connections Editor, Friday, May 1, 2015
Updated: Wednesday, April 29, 2015

March 2015

Mutant forms of c-Cbl and FLT3 cooperate to enhance myeloid leukemia development
Taylor et al., pages 191–206.

Fms-like tyrosine kinase (FLT) 3 mutations, especially internal tandem duplications (ITDs), are prevalent in acute myeloid leukemia (AML). Currently, there is conjecture over the negative regulation of the aberrantly localized FLT3-ITD protein, particularly in regard to E3 ubiquitin ligases such as c-Cbl. A greater understanding of these pathways could provide insight into novel targets for the treatment of AML. Hence, in this study, Taylor et al. investigated the role of c-Cbl in the regulation of the FLT3-ITD protein by generating of a double-mutant mouse containing both a FLT3-ITD and a c-Cbl RING finger domain mutation. The combination of the two mutations severely affected embryonic development, necessitating the generation of mice repopulated with E.14 fetal liver cells. These double-mutant transplanted mice developed an aggressive myeloid leukemia, with a phenotype markedly more severe than mice with either of the single mutations. The disease was characterized by a large increase in blast and myeloid cells and short survival. Interestingly, despite the severe phenotype, the c-Cbl mutation did not enhance FLT3-ITD protein levels or the downstream activity of signal transducer and activator of transcription 5. Rather, the severe leukemia was promoted by greater activity in additional signaling pathways that involved non-FLT3 c-Cbl targets. These included enhanced c-Kit signaling that promoted elevated activity of the phosphoinositide 3-kinase pathway. This study has highlighted the importance of wild-type signaling pathways contributing to leukemia development and suggests that pharmacological manipulation of these pathways may be of therapeutic benefit to control disease progression.


Potential targeting of B-cell lymphoma 2–associated AthanoGene-1 (BAG-1) in poor responding pediatric AML patients
Aveic et al., pages 180–190.

Although huge improvements have been made in the outcome of pediatric acute myeloid leukemia (AML), a significant number of patients still relapse, confirming that standard chemotherapy is not enough. There are several factors that may cause leukemic cells to escape the effects of cytotoxic drugs, and major among them is a failure in apoptosis activation. In the current work, Aveic et al. studied the B-cell lymphoma (BCL) 2–associated AthanoGene-1 (BAG-1), which is shown in the study to be a crucial antiapoptotic protein in AML. The authors demonstrate that the BAG-1 protein is overexpressed in a subgroup of AML patients that showed poor prognosis. The chemical inhibition of BAG-1 by Thioflavin S (which has been shown to interfere with BAG1 function) or BAG-1 silencing improved leukemia cell death by enhancing leukemic cells' sensitivity to conventional drugs. In addition, the authors report synergy between Thioflavin S and conventional chemotherapeutics and between the BCL2 inhibitor ABT-737 and Thioflavin S. In addition, the article reveals that two cellular pathways behind the activation of cell apoptosis, BCL-2 and endoplasmic reticulum stress and unfolded protein response, were both altered after BAG-1 depletion. These findings suggest that BAG-1 targeting could be evaluated as an innovative treatment that may decrease leukemia chemoresistance. The authors support the development and use of specific BAG-1 inhibitors in combination with standard chemotherapy drugs to reduce relapse and improve outcome in pediatric AML patients.


Autophagy regulates hematopoietic stem and progenitor cell cycle
Cao et al., pages 229–242.

Autophagy (or autophagocytosis) is the basic catabolic mechanism that involves cell degradation of unnecessary or dysfunctional cellular components through the actions of lysosomes. The breakdown of cellular components promotes cellular survival during starvation by maintaining cellular energy levels. In this manuscript, Cao et al. explored the role of autophagy in regulating the cell cycle of mouse hematopoietic stem and progenitor cells (HSPCs) using in vivo and ex vivo experimental systems as well as genetically modified mouse models. They show that autophagy regulates the cell cycle of HSPCs in a nutrient-dependent manner. Autophagy signaling under nutrient rich conditions were not upregulated in HSPCs, and cyclin D3 maintained a high level, which promoted cell-cycle entry and cell cycling of HSPCs; however, upon nutrient stress, autophagy of early signaling was activated, which in turn triggered ubiquitinational degradation of cyclin D3 to slow down the cell-cycle entry and G1/S transition of HSPCs, maintaining a proper cell cycling rate. Therefore, autophagy signaling accelerates or decelerates the cell cycle of HSPCs by recruiting ubiquitination machinery to upregulate or downregulate cyclin D3, depending on the nutrient supply. This study nicely demonstrates that autophagy responds to the nutrient supply to adjust the cell cycle and maintain the appropriate quiescence and self-renewal of hematopoietic stem cells as well as cell cycling of HSPCs, which is essential for a functioning hematopoietic system during adult life.

Melatonin overcomes resistance to clofarabine in two leukemic cell lines by increased expression of deoxycytidine kinase
Yamanishi et al., pages 207–214.

Drug resistance remains a serious problem in leukemia therapy. In this study, Yamanishi et al. investigated the drug resistance mechanism from the viewpoint of epigenetics. Among newly developed nucleoside antimetabolites, clofarabine has broad cytotoxic activity showing therapeutic promise and is currently approved for relapsed acute lymphoblastic leukemia. To investigate the mechanisms responsible for clofarabine resistance, the authors established two clofarabine-resistant lymphoblastic leukemia cell lines from parental lines. They found that expression of deoxycytidine kinase (dCK), which phosphorylates clofarabine to exert cytotoxicity, in clofarabine-sensitive and -resistant cells, showed significant decreased expression of dCK RNA in clofarabine-resistant cells compared with sensitive cells. Interestingly, there was no difference between clofarabine-sensitive and -resistant cells in the methylation status of CpG islands of the dCK promoter and expression of MDR1, MRP1, and ABCG2. Total histone, histone H3, and histone H4 acetylation on ChIP assay were significantly decreased in resistant cells. The authors then examined the effects of melatonin in clofarabine-resistant cells. Melatonin is an indolamine that functions in the regulation of chronobiological rhythms to exert cytotoxic effects. Melatonin treatment led to increased cytotoxicity with clofarabine in resistant cells via increased acetylation, which is another histone deacetylase inhibitor. Melatonin is readily available in the clinic and may be a useful candidate for overcoming clofarabine resistance. Based on the data presented, combination therapy with cytotoxic drugs and histone deacetylase inhibitor might be useful in the clinic for enhancement of therapeutic efficacy in relapsed or refractory leukemia.

April 2015
Csf1r collaborates with a C-terminal mutant of C/EBPα to develop aggressive AML
Togami et al., pages 300–308.

CCAAT-enhancer-binding protein (C/EBPα) is a transcription factor that regulates proliferation and differentiation of myeloid cells. Two types of C/EBPα mutations are found in patients with acute myeloid leukemia (AML). It has been previously demonstrated, by using either a mouse bone marrow transplantation model or a knock-in mouse, that a C-terminal mutant of C/EBPα(C/EBPα-Cm) alone induces AML and that it collaborates with an N-terminal mutant of C/EBPα(C/EBPα-Nm) in the efficient induction of AML. In this study, Togami et al. investigate the molecular mechanisms underlying C/EBPα-Cm–induced leukemogenesis. By analyzing gene expression profiles of C/EBPα-Cm– and mock-transduced c-Kit+Sca-1+Lin− cells, they identified Csf1r as a gene downregulated by C/EBPα-Cm. In addition, leukemic cells expressing C/EBPα-Cm exhibited low levels of Csf1r in mice. The authors then tested the possibility that the downregulated expression of Csf1r plays critical roles in leukemogenesis, given that Csf1r is required for myeloid differentiation. However, in contrast to their expectation, Csf1r overexpression collaborated with C/EBPα-Cm in inducing fulminant AML with leukocytosis with shorter latencies compared to those of AML induced by C/EBPα-Cm alone. These results suggest that C/EBPα-Cm–mediated downregulation of Csf1r negatively regulates the progression of myeloid malignancies involving C/EBPα-Cm, and may relate to the fact that Csfr1 is a maker of leukemic stem cell in the mouse leukemia model induced by MOZ-TIF2. Further experiments using the C/EBPα-Cm–induced leukemia model will help us understand the molecular mechanisms by which C/EBPα-Cm–induced leukemia in mice and identify novel therapeutic targets.


BIRB796 stimulates hematopoietic progenitor stem cell growth in FANCA
Svahn et al., pages 295–299.

Bone marrow failure, the main cause of mortality and morbidity in Fanconi anemia (FA), has been largely related to overproduction of tumor necrosis factor α (TNF-α), to which FA stem- and progenitor-cells are hypersensitive. It is known that TNF-α suppression improves the growth of patients' hematopoietic progenitor cells and, in nonhuman experimental settings, it was shown that TNF-α overproduction involves the Toll-like receptor (TLR) 4/8 and p38 mitogen-activiated protein kinase (MAPK) pathway. In this study, Svahn et al. attempted to ameliorate the FA pathway by perturbation of p38 MAPK. The work was conducted on primary human FA complementation group A (FANCA)-deficient monocytes from nine patients and shows that: (i) inhibition of p38 MAPK reduces TLR4 and 7/8-mediated TNF-α production; (ii) inhibitors of p38MAPK improve in vitro erythropoiesis; and (iii) this effect occurs on auxiliary cells (monocytes) and not directly on CD34+ cells. The two latter findings are novel and important for two reasons. The first is that translation to patients' primary cells confirms the same findings obtained on cell lines and mice. This may not always be true; FANCA mice, for example, do not have detectable developmental abnormalities. Therefore, “real ground” confirmation supports continuation of this research. The second reason is that, not only did the authors show the positive effect of inhibition of p38 MAPK on FA erythropoiesis, but they also identified the cellular target of this inhibition. This is important to direct the effect of future compounds counteracting marrow failure on a selective cell target preserving CD34+ cells, which is a severely hampered cell population in FA. At large, these findings might be also useful in other marrow failure disorders like acquired aplastic anemia, in which TNF-α is known to play an important pathogenic role.


The antimalarial drug artemisinin depletes erythrocytes
Yang et al., pages 331–341.

Artemisinin is a major antimalarial drug due to its extraordinary efficacy. However, hemolytic anemia is an evident adverse effect for which underlying mechanisms are unclear. Through a small-molecule screen in zebrafish embryos, Yang et al. found in this study that artemisinin treatments led to diminishing red blood cells but had little to no effect on hematopoietic stem cells and myeloid cells. RNA-Seq revealed that artemisinin suppressed a cluster of genes in the heme biosynthesis and globin synthesis pathways, including the gata 1 regulated genes alas2, urod, fech, and ppox. Artimisinin treatment resulted in erythroid cell apoptosis in both zebrafish and human K562 cell-differentiated red blood cells. Importantly, artemisinin suppressed the ectopic expression of erythroid genes in a zebrafish model of polycythemia vera, a blood disorder in which the bone marrow produces too many erythrocytes. This study provides not only a novel insight into the pharmacologic action of artemisinin on differentiated erythroids but also its potential utility in the intervention and therapy on polycythemia vera.

Bone marrow failure in C57BL/6 mice
Chen et al. pages 256–267.

Murine models of immune-mediated bone marrow (BM) failure have been previously developed with proven utility in the study of disease pathophysiology. However, important elements, such as inciting antigens that initiate autoimmune responses and key molecules that direct T cell lodging, remain uncharacterized. In this study, Chen et al developed a new model of immune-mediated BM failure in C57BL/6 (B6) mice through sublethal irradiation and infusion of allogeneic lymphocytes from major histocompatibility complex–mismatched Friend leukemia virus B/N (FVB) donors. Recipient animals underwent oligoclonal cytotoxic T cell expansion and activation along with significant elevations in inflammatory cytokines and chemokines, leading to severe marrow hypoplasia and fatal pancytopenia. This new model establishes a useful platform that could be extended to a large array of gene knockout and transgenic stocks readily available on the B6 strain background. As proof, B6 mice carrying the lpr mutation deficient in the apoptotic receptor Fas (B6-Fas−/−) were tested in the study. B6-Fas−/− mice showed significant attenuation in BM destruction when treated with the same levels of irradiation and allogeneic FVB lymphocyte infusion. Extension of this model to other B6-based knockout and transgenic mutants will help define the roles of individual genes and their products in the development of immune-mediated BM failure. This is needed to understand the mechanisms behind aplastic anemia and other forms of BM failure and treat patients with these devastating diseases.

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Inside Experimental Hematology: January and February 2015

Posted By Connections Editor, Friday, February 27, 2015
Updated: Monday, February 23, 2015


An ALL xenograft model that simulates the process of clonal selection of chemoresistant clones
Nowak et al., pages 32-93.

The combination of high throughput molecular techniques such as next generation sequencing and copy number micro arrays in combination with xenotransplantation models have revealed that the leukemic cell pool of patients suffering from acute lymphoblastic leukemia (ALL) is not only composed of a sequentially developed dominant clone, but also of a disturbingly large collection of additional highly variable subclonal populations with a complex branching architecture of ancestry. This subclonal diversity, which is formed by a high genetic plasticity of ALL cells, is thought to constitute a reservoir for disease relapse and for cell populations resistant to therapy. This study by Nowak et al tested the feasibility of simulating the process of clonal selection of chemoresistant ALL clones in an in vivo xenograft model. Exposing ALL cells to the selective pressure of chemotherapy led to a rapid outgrowth of clones harboring genomic lesions conferring resistance. Even by next-generation deep sequencing, an emerging chemoresistant clone could not be detected in the samples before drug selection. This finding shows a need to further investigate the genetic plasticity of ALL cells under treatment with chemotherapy to elucidate the dynamics by which resistant subclones emerge. The presented model for induction of chemoresistance provides a platform for performing such studies.

Mixed chimerism in C3a-deficient mice
Baskiewicz-Ha1asa et al., pages 14-22

Transplantation tolerance is an important goal in the effort to reduce long-term morbidity and mortality in organ transplant recipients. Mixed chimerism promises transplant tolerance because
donor-derived antigen-presenting cells migrate to the thymus, present donor antigens to developing recipient thymocytes, and promote the elimination of donor reactive cells through negative selection. Following up on recent studies showing that the C3a-complement component plays a major role in hematopoietic cell circulation and attraction to hematopoietic niches after
transplantation, Ba_skiewicz-Ha1asa et al. investigated in this study the role of C3a in immune tolerance induction in a chimeric mice model. The analysis of mixed chimerism rate in peripheral blood leukocytes during the 20-week experiment revealed that stable multilineage mixed chimerism was not effectively established in C3a_/_ mice, in contrast to wild-type animals. The tolerance to donor antigens (Balb/c) related to mixed chimerism was also not achieved in C3a_/_ mice, as it was observed employing lymphocytes expressing Vb5 and Vb11 TCRs measurement and skin graft acceptance analysis. The present study demonstrates that C3a is essential for achieving stable mixed chimerism and resulting transplant tolerance. Although the levels of hematopoietic stem and progenitor cells were comparable in C3a_/_ and wild-type mice, the hematopoietic environment in C3a-deficient animals was defective for engraftment of transplanted cells. Because blocking C3a anaphylatoxin has been considered as a potential technique to avoid graft rejection, the authors suggest that blocking C3a might not be applicable in transplantation procedures that are aimed at mixed chimerism.

Donor chimerism in CD25D-activated leukocytes as a predictor of GvHD after stem cell transplantation
Martínez -Laperche et al., pages 4-13

Development of severe graftvs-host disease (GvHD) remains the main complication of hematopoietic stem cell transplantation.CD3þ T lymphocytes (TL) chimerism is currently the cornerstone of leukocyte lineage chimerism analysis for the evaluation of events related to the development of GvHD. However, the TL subset may include both alloreactive and naive cells, what underscores the need to further dissect the TL subset, targeting activated leukocytes (AL) for chimerism studies, since only alloreactive cells would be considered in the analysis. In this study, Martínez -Laperche et al. evaluated the impact of the status of ALs (CD25þ) chimerism on the incidence and clinical course of GvHD in allogeneic transplant recipients after myeloablative conditioning. This retrospective study shows that the analysis of chimerism in AL at day 30 and day 90 after stem cell tranplantation adds to the study of TL and could be useful for the improved anticipation of acute GvHD and chronic GvHD, respectively. The association between
chimerism in AL and GvHD would aid in clinical decision-making by allowing early post-transplant modification of immunomodulatory therapies.


Increase level of intact CXCL12 in the spleen of patients with myelofibrosis: a mechanism underlying the development of extramedullary hematopoiesis
Wang et al., pages 100-109

Myelofibrosis (MF) is characterized by the constitutive mobilization of hematopoietic stem and progenitor cells (HSCs/HPCs) and the establishment of extramedullary hematopoiesis (EMH). The mechanisms underlying this abnormal HSC/HPC trafficking pattern remain poorly understood. In this study, Wang and coworkers identified a possible mechanism by which MF CD34+ cells lodge in the spleens of MF patients, eventually leading to the development of EMH. The authors observed a dramatically higher number of splenic MF CD34+ cells that migrated in response to splenic homogenate supernatant (plasma) of MF patients, as compared with peripheral blood (PB) MF plasma. They documented that the concentration of the intact HSC/HPC chemoattractant CXCL12 was greater in splenic MF plasma than PB MF plasma, as quantified using mass spectrometry. By contrast, comparable concentrations of the four functionally inactive truncated products of CXCL12, which are due to proteolytic degradation by serine proteases, were detected in both splenic and PB MF plasmas. The authors further showed that the treatment with an anti-CXCL12 neutralizing antibody resulted in a reduction in the degree of migration of splenic MF CD34+ cells toward both PB and splenic MF plasma, validating the role of CXCL12 as a functional chemoattractant. Their data indicate that the MF splenic microenvironment is characterized by increased levels of intact, functional CXCL12, which contributes to the homing of MF CD34+ cells to the spleens, rather than the marrows, of MF patients, ultimately leading to EMH in the spleen. Therapeutic strategies that would be capable of reversing such pathological chemokine gradients might represent a possible means of correcting the abnormal cellular trafficking characteristic of MF-HSC/HPC and delaying disease progression. 


A novel anti-HLA monoclonal antibody with anti-GVHD activity
Nakauchi et al., pages 79-88

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) using cord blood or haploidentical donor cells is a promising alternative option for patients who cannot find a human leukocyte antigen (HLA)-matched donor. However, HLA-mismatched allo-HSCT may be complicated by graft-versus-host disease (GVHD), a major cause of nonrelapse mortality mediated by alloreactive T cells. Although several agents have been developed for effective control of GVHD, serious side effects are the major issues to be addressed. In this paper, Nakauchi et al. established a novel therapeutic approach to GVHD using allele-specific anti-HLA monoclonal antibodies (ASHmAbs) that specifically recognize donor-derived cells and cause their death. One such ASHmAb against HLA-A*02:01 (A2-kASHmAb) was examined for its effectiveness in preventing disease in a xenogeneic GVHD mouse model. To induce fatal GVHD, nonirradiated immunodeficient mice were injected with healthy-donor human peripheral blood mononuclear cells with or without expression of HLA-A*02:01. Besides the induction of complement-dependent cell death on their target cells in vitro, the administration of the allele-specific anti-HLA antibody effectively ameliorated the GVHD symptoms of the xenogeneic nonirradiated immunodeficient mice and markedly improved their survival. Furthermore, the authors observed the recovery of human blood-cell chimerism after the antibody treatment, indicating that human hematopoietic stem cells were preserved. This study is the first reported instance of the effectiveness of a cytotoxic anti-HLA antibody against GVHD. This novel therapeutic approach may offer hope for effective treatment of GVHD, favorably influencing the outcome of allo-HSCT.


Do osteoclasts have a role in HSC trafficking?
Rao et al., pages 110-114

The role of osteoclasts in regulating hematopoietic stem/progenitor cell (HSPC) trafficking in the bone marrow is controversial. Initial reports suggested that osteoclasts promote HSPC egress from the bone marrow. On the other hand, genetic or pharmacologic inhibition of osteoclast activity is associated with accentuated granulocyte colony–stimulating factor (G-CSF)-induced HSPC mobilization. This question has potential clinical relevance, since pharmacologic agents that inhibit osteoclasts are widely used in the clinic to treat osteoporosis. Definitive studies addressing this issue have been limited by the osteopetrosis and extramedullary hematopoiesis associated with transgenic mouse models of osteoclast deficiency. To overcome this limitation, Rao et al. developed in this study two complementary nonosteopetrotic mouse models to assess the impact of osteoclast deficiency on HSPC trafficking. In the first model, they generated Rank−/− fetal liver chimeras in Csf3r−/− recipient mice. These mice have normal osteoclast numbers but the osteoclasts lack G-CSF receptor expression. In the second model, they acutely depleted osteoclasts by treating wild-type mice with osteoprotegrin-Fc (OPG-Fc, Figure). In both models, HPSC mobilization at baseline and after G-CSF treatment was comparable to control mice. These data show that osteoclasts are not required for the efficient retention of HSPCs in the murine bone marrow and are dispensable for HSPC mobilization by G-CSF. According to these data, pharmacologic strategies to augment HSPC mobilization by disrupting osteoclast function are unlikely to be effective.


Mature adipocytes as potential cell source for therapeutic angiogenesis
Poloni et al., pages 137-146.

Mature adipocytes are generally considered terminally differentiated because they have lost their proliferative abilities. However, this group has previously reported that adipocytes can dedifferentiate to mesenchymal stem cells. In this study, Poloni et al. show that human adipocytes can also differentiate toward the endothelial lineage without change in karyotype. The cells formed cord- or tube-like structures when cultured in specific medium and expressed several endothelial marker genes and proteins, such as vascular endothelial growth factor receptor 2, vascular endothelial cadherin, von Willebrand factor, and CD133. The characteristics displayed by the cells may reflect an intrinsic plasticity of mature adipocytes to change into different cell types and, moreover, to revert into an immature phase without any chromosomal alterations. These results also highlight the concept that adipose lineage cells may represent a suitable new cell source for therapeutic angiogenesis in ischemic disease.

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Inside Experimental Hematology: November/December 2014

Posted By Connections Editor, Monday, January 5, 2015
Updated: Tuesday, December 30, 2014


An ALL xenograft model that simulates the process of clonal selection of chemoresistant clones

The combination of high throughput molecular techniques such as next generation sequencing and copy number micro arrays in combination with xenotransplantation models have revealed that the leukemic cell pool of patients suffering from acute lymphoblastic leukemia (ALL) is not only composed of a sequentially developed dominant clone, but also of a disturbingly large collection of additional highly variable subclonal populations with a complex branching architecture of ancestry. This subclonal diversity, which is formed by a high genetic plasticity of ALL cells, is thought to constitute a reservoir for disease relapse and for cell populations resistant to therapy. This study by Nowak et al tested the feasibility of simulating the process of clonal selection of chemoresistant ALL clones in an in vivo xenograft model. Exposing ALL cells to the selective pressure of chemotherapy led to a rapid outgrowth of clones harboring genomic lesions conferring resistance. Even by next-generation deep sequencing, an emerging chemoresistant clone could not be detected in the samples before drug selection. This finding shows a need to further investigate the genetic plasticity of ALL cells under treatment with chemotherapy to elucidate the dynamics by which resistant subclones emerge. The presented model for induction of chemoresistance provides a platform for performing such studies.

Mixed chimerism in C3a-deficient mice

See Baśkiewicz-Hałasa et al., pages 14-22.

Transplantation tolerance is an important goal in the effort to reduce long-term morbidity and mortality in organ transplant recipients. Mixed chimerism promises transplant tolerance because donor-derived antigen-presenting cells migrate to the thymus, present donor antigens to developing recipient thymocytes, and promote the elimination of donor reactive cells through negative selection. Following up on recent studies showing that the C3a-complement component plays a major role in hematopoietic cell circulation and attraction to hematopoietic niches after transplantation, Baśkiewicz-Hałasa et al. investigated in this study the role of C3a in immune tolerance induction in a chimeric mice model. The analysis of mixed chimerism rate in peripheral blood leukocytes during the 20-week experiment revealed that stable multilineage mixed chimerism was not effectively established in C3a−/− mice, in contrast to wild-type animals. The tolerance to donor antigens (Balb/c) related to mixed chimerism was also not achieved in C3a−/− mice, as it was observed employing lymphocytes expressing Vβ5 and Vβ11 TCRs measurement and skin graft acceptance analysis. The present study demonstrates that C3a is essential for achieving stable mixed chimerism and resulting transplant tolerance. Although the levels of hematopoietic stem and progenitor cells were comparable in C3a−/− and wild-type mice, the hematopoietic environment in C3a-deficient animals was defective for engraftment of transplanted cells. Because blocking C3a anaphylatoxin has been considered as a potential technique to avoid graft rejection, the authors suggest that blocking C3a might not be applicable in transplantation procedures that are aimed at mixed chimerism.

Donor chimerism in CD25+-activated leukocytes as a predictor of GvHD after stem cell transplantation

See Martínez-Laperche et al., pages 4-13.

Development of severe graft-vs-host disease (GvHD) remains the main complication of hematopoietic stem cell transplantation. CD3+ T lymphocytes (TL) chimerism is currently the cornerstone of leukocyte lineage chimerism analysis for the evaluation of events related to the development of GvHD. However, the TL subset may include both alloreactive and naive cells, what underscores the need to further dissect the TL subset, targeting activated leukocytes (AL) for chimerism studies, since only alloreactive cells would be considered in the analysis. In this study, Martínez-Laperche et al. evaluated the impact of the status of ALs (CD25+) chimerism on the incidence and clinical course of GvHD in allogeneic transplant recipients after myeloablative conditioning. This retrospective study shows that the analysis of chimerism in AL at day 30 and day 90 after stem cell tranplantation adds to the study of TL and could be useful for the improved anticipation of acute GvHD and chronic GvHD, respectively. The association between chimerism in AL and GvHD would aid in clinical decision-making by allowing early posttransplant modification of immunomodulatory therapies.



Murine CALM-AF10 cells are sensitive to iron depletion in vitro but not in vivo

See Heath et al., pages 1022-1030.

Iron is known to be an essential element for the growth and proliferation of neoplastic cells, and iron depletion as treatment for malignancy has been studied in the context of several hematopoietic and solid tumors, with mixed results. One possible reason for these inconsistent results is that normal cells also require iron, rendering the potential therapeutic index too narrow to avoid undue host toxicity. Iron (complexed with transferrin [Tf]) enters cells primarily via clathrin-mediated endocytosis of the Tf-Tf Receptor (TfR) complex. This endocytic process is altered in leukemias bearing a CALM-AF10 translocation, which demonstrate haploinsufficiency of the normal CALM protein as well as a dominant negative effect of the abnormal CALM-AF10 fusion protein. In this manuscript, Heath et al. hypothesize that CALM-AF10 leukemia cells are iron deficient and, therefore, particularly sensitive to the cytotoxic effects of iron depletion. The authors confirmed that CALM-AF10 leukemia cells are indeed iron deficient and are sensitive to the cytotoxic effects of iron depletion in vitro—an effect which was additive when combined with traditional chemotherapy. However, in a murine model of CALM-AF10 leukemia, no survival benefit was seen with iron deprivation alone or in combination with traditional chemotherapy. It is possible that, even in the face of a pre-existing sensitivity of neoplastic cells to iron depletion, the therapeutic index remains too narrow to provide benefit. Future studies involving more potent iron chelators could show benefit if the organism can be rescued from the toxic effects of severe iron deprivation without disrupting its antineoplastic effect.


The mixed-lineage leukemia gene (MLL1), a paradigm to understand leukemogenesis through aberrant epigenetic gene regulation

See Li et al., pages 995-1012.

Chromosomal translocations in the human mixed-lineage leukemia (MLL) gene were among the first molecularly characterized lesions resulting in deregulated gene expression in leukemia. In this review, Li et al. discuss the evolution of animal models to understand how deregulating MLL activity results in leukemia and discuss results obtained over the intervening years using these animal models to understand the genetic pathways regulated by MLL fusion oncoproteins. Significant progress has been made recently in understanding the molecular basis by which this mysterious histone methyltransferase is deregulated to result in leukemia. Studies focused on the importance of both direct and indirect downstream targets of MLL oncoproteins are numerous in the literature and have recently included several shRNA-based high-throughput screens to identify such targets. Epistasis experiments performed to determine the relative significance of MLL-regulated genes or direct target genes have then suggested pathways that could be targeted in leukemia driven by MLL fusion oncoproteins. Furthermore, data from primary leukemia samples have been integrated with mouse-model genetic data to distill the critical pathways upon which MLL fusion proteins selectively depend. These studies have already led to several new molecularly targeted therapeutics to treat leukemia harboring MLL gene rearrangements. Furthermore, this body of work will continue to inform the targeting of other cancers driven by transcriptional and epigenetic regulators. For those clinical or basic science-oriented investigators interested in how MLL fusion proteins lead to leukemia, or those interested in following a paradigm in applying basic science to translate targeted therapeutics, this review covers this topic in significant enough detail to appreciate the progress made using this particular paradigmatic leukemia pathway.

STAT3 and PRL-3: two prominent oncogenic molecules are now connected

See Zhou et al., pages 1041-1052.

PRL-3 (encoded by PTP4A3) is a VH1-like protein tyrosine phosphatase with dual-specificity and plays a critical role in cancer cell metastasis, invasion, migration and tumor angiogenesis. It is reported that the PRL-3 protein is overexpressed in a subset of acute myeloid leukemia (AML) patients, and high PRL-3 level is associated with poor survival. However, the mechanism by which PRL-3 is regulated in AML is not fully elucidated. Constitutive activation of the STAT3 pathway has been demonstrated in a variety of solid tumors and hematologic malignancies. Particularly in AML, aberrant STAT3 signaling has been found in about 50% of cases and has been associated with adverse disease-free survival. In this study, Zhou et al. revealed for the first time the interaction between these two prominent oncogenic molecules, STAT3 and PRL-3. STAT3 specifically bound to the -201 to -210 conserved region of the PRL-3 promoter. Ectopic expression of STAT3 in mouse STAT3-/- liver cells could rescue the reduced expression of the PRL-3 protein. Furthermore, the authors generated a core STAT3 signature, which was derived from the largest datasets in the literature. This STAT3 core signature is enriched in AML with high PRL-3 expression. Importantly, Zhou et al. provide strong evidence supporting the conclusion that the STAT3/PRL-3 regulatory loop contributes to the pathogenesis of AML and propose that intervention of the STAT3-PRL-3 regulatory loop is therefore of potential benefit in AML patients with high PRL-3AML patients with high PRL-3.


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Inside this Issue: September/October 2014

Posted By Connections Editor , Thursday, October 30, 2014
Updated: Tuesday, October 28, 2014


Furthering our understanding of the role of miRNAs in hematopoiesis

See Wojtowicz et al., pages 909-918.; Oh et al., pages 919-923., and Byon et al., pages 852-856.

MicroRNAs (miRNAs) are short, endogenous, noncoding RNA molecules that regulate the expression of crucial genes involved in regulatory functions related to cell growth, development, and differentiation, and they are associated with a wide variety of human diseases. MicroRNAs are transcribed by RNA polymerase II as large RNA precursors, which are processed in the nucleus by the microprocessor complex, consisting of the RNase III enzyme Drosha and the double-stranded-RNA-binding protein Pasha/DGCR8. The resulting pre-miRNAs are approximately 70 nucleotides in length and are transported into the cytoplasm, where they undergo an additional processing step by the RNAse III enzyme Dicer, generating the mature miRNA, a double-stranded RNA approximately 22 nucleotides in length. Dicer also initiates the formation of the RNA-induced silencing complex, which pairs the miRNAs with the 3' untranslated region of their target mRNA, leading to its destabilization and resulting in degradation or translational suppression.

A growing body of evidence indicates that miRNAs are vital for the proper functioning of hematopoietic stem and progenitor cells (HSPCs) and that they can influence erythroid lineage commitment and differentiation. The article by Wojtowicz et al. compares, side by side, the role in hematopoiesis of miR-125a, miR-125b1, and miR-125b2, which share the same seed sequence and are highly expressed in HSPCs. The authors could not detect any functional difference between them, showing that overexpression of each of the three miR-125 family members preserves HSPCs in a primitive state in vitro, results in a competitive advantage upon serial transplantation, and promotes skewing toward the myeloid lineage. Mechanistically, the study suggests that a seed-mutated version of miR-125 can rescue the effects of miR-125 overexpression, indicating that they likely operate in a seed-sequence-dependent manner.

In the brief communication by Byon et al., the authors describe a mouse model of conditional Dicer deletion limited to late erythroid cells (beyond proerythroblast). Under normal conditions, Dicer deletion did not affect any hematopoietic parameters; however, following stress with phenylhydrazine or 5-fluorouracil, the mice showed impaired recovery from anemia, suggesting that miRNAs primarily regulate stress erythropoiesis. This novel mouse model can be exploited to delineate miRNA function in late erythropoiesis, specifically under stress conditions, either by combining this model with other genetic models of stress erythropoiesis or by performing miRNA/shRNA rescue screens to identify specific miRNAs/mRNAs that restore the normal response to stress erythropoiesis.

Anti-T-cell globulin (ATG) mediates antitumor activity toward a variety of hematologic malignancies

See Westphal et al., pages 875-882.

Graft-versus-host disease (GVHD) is a major complication after allogeneic stem cell transplantation (allo-HSCT), leading to considerable morbidity and mortality. In vivo or in vitro depletion of T cells is effective to reduce the incidence of GVHD but has been demonstrated to be associated with increased relapse rates of malignancies after allo-HSCT.

More recently, clinical studies found that in vivo T-cell depletion with rabbit anti-T-cell globulin (ATG-F) lowered the incidence of GVHD without increasing tumor relapse rates. In this article, Westphal et al. examine the antitumor effect of ATG-F. They show that ATG-F binds to a variety of hematologic tumor cells, including acute myeloid leukemia and B-cell lymphoma. They demonstrate that ATG-F mediates antitumor effects in vitro by inducing antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, and apoptosis. This is an interesting study that contributes to our understanding of antitumor mechanisms in the early phase after allo-HSCT. The findings described in the study may have clinical implications during allo-HSCT in situations when T-cell depletion is required but tumor relapse is a concern.

Understanding stem cell heterogeneity in disease

See Prick et al., pages 841-851.

Recent descriptions of heterogeneity in stem cells and cancers have emphasized the need to understand how single stem cells are subverted to cause tumors. Human myeloproliferative neoplasms arise from a transformed hematopoietic stem cell and provide a paradigm for studying the early stages of tumor establishment and progression.

This review postulates that the selective pressure placed on the first progeny in the initial clonal outgrowth is the source of substantial heterogeneity, which eventually manifests as distinct disease subtypes. It underscores the need to think about oncogenesis as a constantly evolving and highly interactive process, both with a tumor’s own clonal progeny and the tumor microenvironment. This review also details recent advances in our understanding of clonal evolution of the myeloproliferative neoplasms, including the molecular cataloguing of the genomic landscape and the current theories for how a single point mutation in JAK2 can be responsible for three distinct disease subtypes.



MSC-derived osteoblasts, a new source of feeder cells for the expansion of HSPC with enhanced thrombopoietic activity

See Dumont et al., pages 741-752.

The delay in platelet and neutrophil recovery following single and double umbilical cord blood transplantations limits their widespread utilization. Ex vivo expansion of hematopoietic stem and progenitor cells (HSPC) provides a mean to raise the dose of transplantable progenitors in order to promote early engraftment and prevent graft failure. Moreover, such procedure may one day allow for single cord blood unit transplant in patients that currently require two independent units. Multiple strategies have been reported to expand HSPC ex vivo, including co-culture of hematopoietic cells with mesenchymal stromal cells (MSC). Just recently, da Lima et al. reported that patients transplanted with 2 cord-blood units, 1 of which was expanded ex vivo in co-culture with MSC, had accelerated neutrophil recovery and also a significant improvement in platelet engraftment. In this issue, Dumont and colleagues compared the growth- and differentiation-modulatory activities of osteoblasts derived in vitro from human MSCs (i.e. M-OST) to that of the parental MSCs and found that M-OST supported greater expansion of HSPCs. More importantly, HSPCs expanded in M-OST conditioned medium yielded superior platelet engraftment than HSPCs expanded in MSC conditioned medium or in control cultures. Taken together, these data suggest that M-OST represent a new underappreciated source of feeder cells for the expansion of HSPC with enhanced thrombopoietic activity.

Bone marrow Th2 cells promote erythropoiesis at high altitude

See, Li et al., pages 804-815.

High-altitude hypoxia can lead to moderate or excessive increase of red blood cells in different individuals. The excessive erythropoiesis is termed high-altitude polycythemia (HAPC) and its mechanism remains largely obscure. The hypoxia-induced erythropoietin (EPO) is considered an underlying cause of HAPC; however, EPO levels usually do not correlate well with the severity of HAPC among individuals. A growing body of evidence has suggested that T lymphocytes, particularly bone marrow (BM) T cells, are involved in hematopoietic regulation. In this study, Li et al reported an association between altered BM Th2 cells and accelerated BM erythropoiesis at high altitude. Using a mouse model, they found that CXCR4-dependent Th2 cells trafficking to the BM during hypoxic exposure and their production of activin A and interleukin-9 contribute to erythropoiesis at high altitude. These findings provide a new insight into the EPO-independent mechanism underlying erythroid regulation at high altitude. More research is needed to reveal whether this pathway actually contributes to the pathology of excessive erythropoiesis at high altitude in humans. If this is the case, strategies to inhibit BM Th2 lymphocytes may be a new approach to cure HAPC patients.

CD45 regulatory elements facilitate efficient lentiviral tracking of transplanted cells

See Duong et al., pages 761-772.

Cell transplantation for the treatment of hematologic disease remains challenging due to a lack of suitably matched donors, and efforts are underway to use cellular reprogramming as a platform for attaining autologous blood cells for therapy. All studies to date employing defined factors to reprogram fibroblasts into induced pluripotent stem (iPS) cells or to an alternate cell lineage have utilized a reporter to document lineage respecification; however, a genetically tractable reporter system does not currently exist for marking the production of blood cells following differentiation or reprogramming. To construct a widely applicable hematopoietic delineation system, in this paper, Duang et al used transcription factor chromatin occupancy (ChIP-seq), promoter nuclease sensitivity (DNase-seq) and evolutionary conservation to define regulatory elements within the mouse and human blood surface gene CD45. The resulting lentiviral reporter enabled highly efficient and stable marking of lymphoid, myeloid and nucleated erythroid progenitor cells following long-term reconstitution in vivo. The CD45 reporter is hematopoietic restricted, and therefore not activated in fibroblasts or pluripotent cells. This specificity makes the system well-suited for following blood cell transplantation kinetics and persistence, isolating hematopoietic lineages from embryonic stem (ES) or induced pluripotent stem (iPS) cells, and for the development and facile monitoring of direct reprogramming strategies.

Induced pluripotent stem cells from myelofibrosis patients-a novel source of research material

See Hosoi et al., pages 816-825.

Induced pluripotent stem cells (iPSCs) derived from disease cells are expected to provide new experimental material, especially for the diseases for which the sample is difficult to obtain. Myelofibrosis (MF) is a rare and serious hematologic malignancy classified as a Philadelphia chromosome-negative myeloproliferative neoplasm (MPN). The disease is more common in males and in older individuals. Of the MPNs, MF presents with the most severe morbidity and greatest mortality. Although the cause of MF is unknown, it is thought to occur from acquired mutations that target the hematopoietic stem cell. The only curable treatment option is stem cell transplantation; however, it is warranted only to young patients without challenging complications. Although novel therapeutics to improve the outcome of MF patients are clearly needed, research material for this disease is often difficult to obtain from patients because of progressive scarring of the bone marrow. To overcome this problem, in this manuscript, Hosoi et al. established induced pluripotent stem cells (iPSCs) derived from primary and secondary MF patient samples. The authors confirmed that the disease specific genomic markers were sustained in those iPSCs, and also that those iPSCs were capable of differentiating into hematopoietic cells, such as megakaryocyte, erythrocyte and myelocyte. Megakaryocytes are considered to be responsible for generating effectors to myelofibrotic transformation in MF. These megakaryocytes are extremely difficult to harvest from MF patients, and thus this alternative source, through the differentiation of iPSCs, could potentially be a valuable tool to learn more about MF. Indeed, the authors used whole MF-iPSC derived megakaryocytes to study the expression level of IL8, a cytokine known to stimulate fibroblasts to produce collagen and extracellular matrix and which is highly elevated in patients with MF, especially in those with a poor prognosis. They showed that expression of IL8 in MF-iPSC was largely increased compared to normal iPSC derived megakaryocytes. Based on the data presented here, MF-iPSC provide a novel platform to investigate MF pathogenesis on the basis of patient-derived samples and should proved useful to accelerate the development of novel therapies which are urgently needed to help patients with this devastating disease.

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Inside Experimental Hematology July and August 2014

Posted By Connections Editor, Friday, September 5, 2014
Updated: Wednesday, September 3, 2014

Inside this issue July 2014

Phenotypic characterization of the erythroid island macrophages that mediate the block of medullary erytrhropoiesis by G-CSF

See Jacobsen et al., pages 547561.

In the bone marrow (BM), as well as in the spleen and liver, macrophages are the central component of erythroblastic islands, which are specialized niches within which definitive mammalian erythroblasts proliferate and differentiate. In these islands, the central macrophage extends cytoplasmic protrusions to a ring of surrounding erythroblasts. It has been proposed that early in erythroid maturation, the macrophages provide nutrients and proliferative and survival signals to the erythroblasts, and phagocytose extrudes erythroblast nuclei at the conclusion of erythroid maturation. This group and others have previously demonstrated that G-CSF mediates the mobilization of hematopoietic stem cells (HSCs) from the BM via the suppression of BM macrophages. Due to the key role of macrophages in erythropoiesis, in this study, Jacobsen et al. investigated the effect of G-CSF in red blood cell formation and characterized the phenotype of the macrophages involved. They report that G-CSF blocks erythropoiesis in mouse BM but not in the spleen or liver, via the depletion of medullar erythroid island macrophages. These macrophages express F4/80, VCAM-1, ER-HR3, CD169, and Ly-6G antigens. This precise identification of the phenotype of the erythropoietic island macrophages should facilitate future studies of these cells in steady-state and disease, which until now have been hampered by the lack of specific markers.

Signaling via TLR9 augments production of human NK-like cells

See Vadillo et al., pages 562573.

It is well known that the hematopoietic system can respond to different types of insults by favoring the production of particular cell types. In keeping with this, it has been shown that pathogens and their products can influence blood cell production in a Toll-like receptor (TLR)-dependent manner. For example, lymphopoiesis is suppressed in herpes simplex–infected mice, whereas dendritic cells are preferentially made from progenitor cells that reside within bone marrow. However, the vast majority of such studies focused on the myeloid arm of hematopoiesis, and they were conducted with experimental animals. Thus, much remains to be learned about lymphoid, and particularly natural killer (NK) cell production in humans. In this issue, Vadillo et al. demonstrate that ligation of the TLR9 stimulates adult human lymphoid progenitors to divide and generate NK-like cells. Interestingly, this phenomenon—marked by elevated expression of IL-15Rβ—was observed in adult, but not neonatal cells. These findings accord with previous reports concerning ontogeny-related changes in human lymphocyte production and raise interesting questions. Are neonates threatened by infections because they lack the ability to boost innate immunity? Alternatively, NK progenitors may have already been exposed to endogenous TLR ligands and sterile inflammatory processes in utero. Related to that issue, do the neonatal versus adult differences represent residual maternal/fetal tolerance processes? It is clear that we need to better understand immune system development within the context of endogenous and pathogen-derived TLR ligands.

Lmo2 has epigenetic effects on CD4 in T-cell leukemia

See Cleveland et al., pages 581–593.

LIM domain Only-2 (Lmo2) is one of the most frequently deregulated oncogenes in human T-cell acute lymphoblastic leukemia (T-ALL). This group and others have found that prior to the onset of leukemia, Lmo2 overexpression causes an arrest in the differentiation sequence of T-cell progenitor cells before the onset of expression of CD4 and CD8. In this paper, Cleveland et al. discovered a mouse T-ALL cell line derived from CD2-Lmo2 transgenic mice that has an unusual differentiation block. The cell line, 32080, is clonal but has cells with negative, intermediate, and high CD4 expression, whereas CD8 expression is homogeneous. Pure CD4− or CD4+ populations were sorted but the expression of CD4 was unstable since the sorted cells acquired CD4 or lost CD4 after replication. The CD4+CD8+ population resembled the double positive (DP) cells of normal T-cell differentiation and the CD4−CD8+ population resembled the intermediate single positive (ISP) T-cell progenitors. Similar to their normal counterparts, the DP-like population had a higher baseline rate of apoptosis and increased sensitivity to dexamethasone. This variegated pattern of CD4 expression in 32080 cells was regulated by Lmo2, Notch1, Runx1, and histone deacetylation. The 32080 cell line provides a unique model of ISP to DP developmental stage transition, where the CD4 gene is known to be epigenetically regulated, and also suggests unique epigenetic effects of the Lmo2 oncogene.

Inside this Issue August 2014

Editorial for the Special Issue on Genomics and Model Organisms: New Horizons for Experimental Hematology

A comment from the Editor-in-Chief, Dr. Keith Humphries, pages 595-597

On behalf of the journal, I am very pleased to introduce a special issue devoted to two exciting research topics of growing importance to our field—Genomics, guest edited by Dr. Bertie Gottgens, and Model Organisms, guest edited by Dr. David Traver. This combined series of reviews highlights recent advances across the wide range of model organisms and genome-scale approaches with a particular emphasis on strategies that are showing promise for extracting new biological insights relevant to normal hematopoiesis and disease. In the following, Drs. Gottgens and Traver provide a glimpse of the topics covered in their respective review series that I hope will encourage a deep read and discussion.

A special note of thanks to the many authors who so enthusiastically and kindly agreed to contribute to this review series.

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Inside Experimental Hematology: May and June 2014

Posted By Connections Editor, Monday, June 30, 2014

Inside this issue May

Immunogenicity of ESC-derived hematopoietic progenitors

See Thompson et al., pages 347359.

Successful transplantation of embryonic stem cell (ESC) derivatives into adult recipients could facilitate their clinical translation as cellular therapies. However, a significant knowledge gap exists in how ESC-derivatives are recognized and rejected by the adult host immune system. To address this, Thompson et al. examined the potential immunogenicity of ESC-derived hematopoietic progenitors (ESHPs). ESHPs were analyzed for the expression of immunogenicity markers and their ability to stimulate T cell proliferation. During T cell-mediated immune rejection of allogeneic transplants, donor peptides can be presented by the donor antigen-presenting cells, via a process termed “direct presentation.” In contrast, phagocytosis of donor cells by host antigen-presenting cells, such as macrophages, results in presentation of donor peptides on host MHC II molecules to host T cells via indirect presentation. Thompson et al. found that adult macrophages preferentially phagocytosed donor ESHPs compared to adult lineage-negative bone marrow progenitors. Furthermore, macrophages presenting ESHP-derived peptides stimulated proliferation of host CD4+ T cells. These results demonstrate that ESHPs can stimulate allogeneic responses in vitro and suggest that determination of ESHP immunogenicity profiles, as well as identification of embryonic antigens that are recognized by adult macrophages, may improve the success of ESHP survival and function after transplantation in vivo.

Parsing out the Integrins' work on erythropoiesis

See Ulyanova et al., pages 404409.

By studying mice with conditional deletion of all β1-integrins or only α4β1 at the stem/progenitor cell level, this group has previously uncovered their distinct influences on erythropoiesis at homeostasis and after stress. However, it was unclear at what stage these effects were exerted and whether only combinatorial effects were in place. In this study, Ulyanova et al. have created novel mice with deletion of the erythroid integrins, α5 or α4, only in erythroid cells. Studying these mice yielded two important conclusions: a) α5β1 when ablated in erythroid cells was dispensable for completion of erythroid maturation; this was in contrast to their previous data and to certain previous conclusions in vitro; b) by contrast, α4β1 has a dominant effect on erythroblast retention and on enhancement of terminal erythroid maturation, especially after stress, regardless of being ablated early in hematopoiesis or only in erythroid cells. These conclusions advance the current state of knowledge on the role of integrins in erythropoiesis. Furthermore, by exploiting the use of a surface antigen deletion using the EpoR-cre mice, the authors have uncovered the intricate details of EpoR-directed ablation during erythroid differentiation at homeostasis and the surprisingly altered profile of ablation under stress.

Is bio-manufacture of human platelets for transfusion possible?

See Haylock et al., pages 332–346.       

Can the production of mature blood cells from hematopoietic stem cells via large-scale manufacture provide an alternative source of cells for transfusion? This review by Haylock et al. provides a detailed outline of both the challenges involved with the bio-manufacture of platelets and the opportunities available to move away from the reliance on blood donations. The authors examine the scale requirements for platelet bio-manufacture to deliver sufficient cells for transfusion. In order to achieve the numbers required, a three stage linked process may be required, with the first involving the expansion of hematopoietic stem cells and progenitors, and the second focusing on megakaryocyte differentiation and maturation. The third stage calls for optimal conversion of megakaryocytes into platelets. In addition, the authors present a brief outline of the current understanding of megakaryopoiesis and thrombogenesis and highlight how this impacts on the design of culture systems and bioreactors for producing megakaryocytes and platelets. The review conveys the message that ex vivo culture conditions need to be carefully optimised for the distinct stages of expansion, differentiation and maturation as well as platelet release. The authors highlight several key issues that must be addressed to ensure that the bio-manufacture of platelets becomes a reality and stress the importance of a multidisciplinary approach in achieving this. A major challenge to be overcome is how to optimally induce megakaryocyte proplatelet formation and platelet release. While the ex vivo bio-manufacture of platelets is an emerging area of experimental hematopoiesis, currently involving only a relatively small number of research groups worldwide, the issues associated with scaling small-scale research devices to large-scale platform technologies remain across the board for all cellular therapies.

Hes1 contributes to leukemic transformation of FIP1L1-PDGFRA-positive leukemia

See Uchida et al., pages 369379.

Hairy enhancer of split 1 (Hes1) is a transcriptional repressor that regulates cellular differentiation and tissue morphogenesis. It also immortalizes committed progenitors and inhibits myeloid differentiation. This laboratory previously reported that overexpression of Hes1 contributes to blast crisis of chronic myelogenous leukemia through inhibition of myeloid differentiation. In the present article, Uchida et al. examined Hes1 expression in patients with hematologic malignancies and found that Hes1 expression was observed only in a fraction of patients with AML, MDS, and MDS/AML. Interestingly, Hes1 was overexpressed in 2 out of 5 patients with eosinophilia-associated leukemia harboring the Fip1-like1-platelet-derived growth factor receptor alpha (FIP1L1-PDGFRA) fusion gene. Clinically, FIP1L1-PDGFRA is identified in patients with chronic eosinophilic leukemia, eosinophilia-associated AML and T cell acute lymphoblastic leukemia. In a mouse bone marrow transplantation model, FIP1L1-PDGFRA was reported to induce myeloproliferative neoplasm or T cell acute lymphoblastic leukemia. On the other hand, combination of FIP1L1-PDGFRA and Hes1 induced AML in the transplanted mice. The leukemic cells are morphologically immature cells without eosinophilic granules, but express eosinophil markers such as IL-5 receptor, indicating some commitment of the leukemic cells to the eosinophilic lineage. The authors hypothesize that overexpression of Hes1 might have inhibited differentiation of leukemic cells. Alternatively, it is possible that IL-5 expression is required for further commitment of the blasts to eosinophil lineage as previously reported. This article, together with the previous report on chronic myelogenous leukemia in blast crisis, implicates Hes1 in the leukemic transformation of myeloproliferative neoplasm, and helps understand its etiology, although the molecular mechanisms for Hes1-upregulation remain elusive.


Inside this issue June

Overlapping roles of Snail proteins in hematopoiesis and strategies to assay transcription factor families for intramember compensation

See Pioli and Weis, pages 425430.

Transcriptional activation pathways have been closely scrutinized for their roles in the development of bone marrow lineages. Less emphasis has been placed upon transcriptional repressors and how they could function to repress gene activation during hematopoiesis. This review by Pioli and Weiss presents the existing knowledge on the Snail family of transcriptional repressors in hematopoiesis. The Snail proteins consists of three members (Snai1, Snai2, and Snai3) that possess nearly identical DNA-binding domains (recognizing the canonical E box motif) and N-terminal repressor domains. These three proteins are expressed in a variety of hematopoietic lineages and mature end stage cells. While a mouse deficient in Snai1 does not survive embryogenesis, those lacking Snai2 or Snai3 have few, if any, anatomical or hematological defects. Mice lacking both Snai2 and Snai3, however, do show dramatic differences in hematopoietic cell--derived populations as well as in organizations of tissues (thymus and spleen) that are occupied by mature end stage cells. These findings suggest that, in cells co-expressing Snai2 and Snai3, single Snai2 or Snai3 protein deficiencies are complemented by the other Snail protein and that a true representation of the role of the Snail proteins in hematopoietic lineage will require elimination of all three genes in such lineage cells. These studies parallel those of other transcriptional regulatory systems in which highly homologous family members may also provide functional complementation in co-expressing cells. In this regard, the authors also discuss concepts of functional redundancy and strategies employed to assay transcription factor families for “intramember” compensation.

Global analysis of transcription factors and cofactors during terminal erythropoiesis

See Chen and Lodish, pages 464476.

The adult human generates roughly 2.4 million red blood cells every second, a process that requires the intricately regulated proliferation and differentiation of hematopoietic stem cells into mature erythrocytes. Much of this regulation occurs during terminal erythroid differentiation, and the mRNA level of erythroid-important genes must be tightly regulated during this stage for proper erythroid maturation. Global studies of the changing transcriptional landscape have yielded insight into gene regulatory networks during terminal erythropoiesis, but a comprehensive view of all transcriptional regulators was lacking. To this end, Chen and Lodish used global gene expression analysis to identify 28 transcription factors and 19 transcription cofactors induced during terminal erythroid differentiation. Utilizing protein--protein interaction databases to identify cofactors for each transcription factor, they determined that several co-induced pairs of factors and cofactors, including many known essential erythroid factors, were induced, validating the use of this global study as a resource for finding potential critical transcriptional regulators. The interacting pair of the E2F2 transcription factor and its cofactor TFDP2 was the top hit in the analysis, and thus the authors investigated the function of TFDP2 in detail. In their primary mouse erythroid cell culture system, loss of TFDP2 resulted in ineffective erythropoiesis, with cells accumulating in S phase, likely due to higher than normal levels of cell cycle-important E2F2 target genes. These findings suggest that E2F2 paired with TFDP2 acts as a transcriptional repressor rather than an activator in terminally dividing erythroblasts, a novel model by which cells can coordinate their cell cycle with differentiation. This work also serves as a roadmap for future studies of transcriptional regulators in erythropoiesis that will enhance our molecular understanding of red blood cell production in both physiologic and pathophysiologic states.

Statins potentiate the antileukemic effects of imatinib in chronic myeloid leukemia

See Glodkowska-Mrowka et al., pages 439447.

Tyrosine kinase inhibitors (TKIs) have profoundly changed the therapy of chronic myeloid leukemia (CML) and transformed this disease into a truly chronic ailment for more than a half of CML patients. Unfortunately, the success of TKIs is shadowed by the development of resistance to therapy in a significant number of patients. A major challenge, which has become apparent in recent years, is the resistance of leukemic stem cells to TKIs and their putative role of ticking bombs responsible for treatment failure. Particularly alarming, according to recently published observations, there are also serious side effects, including cardiovascular toxicities of 2nd and 3rd generation TKIs. Since TKIs are not able to cure CML, there is a growing need to introduce new therapeutic modalities, including combination therapies, which remain standard of care in oncology (CML being an exception). In this work, Glodkowska-Mrowka et al. employed statins (with lovastatin as a model compound) to increase the antileukemic efficacy of imatinib, the first and most commonly used TKI in CML. Statins, 3-hydroxy-3methylglutaryl-CoA inhibitors, are among the most commonly prescribed drugs to treat hypercholesterolemia. Since statins exert several pleiotropic effects on both normal and tumor cells, they have been tested in different experimental approaches in oncology. The authors show that statins increased intracellular concentration of imatinib in primary CML cells and cell lines, as measured by uptake of 14C-labeled imatinib, and enhanced the antileukemic activity of imatinib. Statin-induced inhibition of the membrane efflux transporters, ABCB1 and ABCG2, was responsible for these effects. The synergism between statins and imatinib was observed not only in CML cell lines but also in primary CML CD34 + cells from patients in different phases of the disease, including samples from patients in acute (blastic) phase, which is refractory to targeted treatment and still remains a major therapeutic challenge. Importantly, no cumulative cytotoxic effects of such combination were observed in normal CD34 + cells. This work presents a potential and feasible approach to overcome drug resistance to imatinib in CML patients and provides a rationale for a controlled, prospective clinical trial.

Single cell analysis shows mutational heterogeneity in leukemic cells

See Shouval et al., pages 457463.

Recent advances in genomics promote the identification of recurrent somatic mutations in the majority of cytogenetically normal AMLs. However, several key issues in leukemogenesis remain unsolved, including the order of mutation accumulation and complexity of intratumor heterogeneity. This study by Shouval et al. used single-cell analysis, opposite to the regular bulk DNA analysis, to demonstrate that the recurrent FLT3-ITD mutation is more common than previously estimated (∼80% of samples). Both AML and ALL patients considered negative for ITD were found to harbor minor clones with this mutation. The data suggest that FLT3-ITD is a common late event in acute leukemia, most probably associated with a mutational hot spot in this genomic region. The ITD mutation is likely to confer a positive selective advantage mainly in myeloid cells, leading to higher mutant allele frequencies. These results contribute to the increased evidence of branched parallel evolution of leukemia with multiple late events and polyclonal contribution to relapse. The findings of this study have clinical implications and suggest that targeted therapy aimed at eradicating late events (like FLT3-ITD) as part of a combination therapy might be useful in managing leukemia. Single-cell analysis in leukemia is a powerful tool in the study of heterogeneity of the tumor and identification of subtle subpopulations contributing to disease relapse. Better understanding of the mechanisms of recurrent mutations in cancer and the order they occur could significantly improve our ability to prevent leukemia relapse.

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