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Inside this Issue: September/October 2014

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

October

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.

 

September

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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Inside the March & April Issues

Posted By Connections Editor , Thursday, May 1, 2014
Updated: Wednesday, April 30, 2014
March 
See Syrjänen et al., pages 230–240.

The role of the Trans-membrane (or T cell) immunoglobulin and mucin (TIM) family members in the adult immune system involves the regulation of Th cell responses and removal of apoptotic bodies through phosphatidylserine. However, their expression and role during the development of immune cells is undetermined. To address this, Syrjänen et al. analyzed TIM expression in chicken and mouse hematopoiesis. The most striking finding of the study is the clear restriction of tim-4 expression to fetal liver and especially to fetal liver CD45+F4/80+ cells. Furthermore, two distinct TIM-4+ populations were seen: F4/80hiTIM-4hi and F4/80loTIM-4lo. The authors showthat the F4/80hiTIM-4hi cells had the morphology of mature macrophages and had no in vitro hematopoietic potential, suggesting that they are functional macrophages. On the other hand, F4/80loTIM-4lo cells expressed stem cell markers and had multi-lineage myeloid potential. In addition, the F4/80loTIM-4lo cells included a considerable population
expressing ER-MP12, a known marker for macrophage-colony forming cells and other myeloid progenitors. Therefore, the authors suggest that TIM-4 is a marker for myeloid progenitors. In the future, TIM-4 could be utilized in the isolation or characterization of myeloid progenitor cells. It will also be intriguing to find out whether TIM-4 has a functional role in cell differentiation. 

See Beaudin et al., pages 218–229.

Defining the role of the tyrosine kinase receptor Flk2/Flt3 in hematopoietic differentiation is critical for understanding how aberrant Flk2 activity promotes acute myeloid leukemia. Previous studies focused on the prominent role of Flk2 in lymphoid differentiation suggested that Flk2 regulates cell fate by biasing progenitors toward a lymphoid fate at the expense of megakaryocyte/erythroid potential, but this model fails to explain how activating mutations in Flk2 promote myeloid leukemias. In this issue, Beaudin et al. clarified the role of Flk2 in regulating myeloid development by investigating the effect of Flk2 deficiency on myeloid specification in vivo. Quantification and functional assessment of purified stem and progenitor
populations from Flk2-deficient mice revealed that Flk2 promotes both myeloid and lymphoid development. Furthermore, transplantation of equivalent numbers of WT and Flk2-deficient multipotent progenitors resulted in only a subtle lymphoid defect, indicating that Flk2 promotes the expansion of multipotent progenitor cells. Consistent with this concept, Flk2 deficiency impaired proliferation and cell cycle status of stem and progenitor cells, resulting in reduced sensitivity of Flk2-deficient mice to treatment with the myeloablative agent 5-fluorouracil.
Collectively, the findings by Beaudin et al. support a model in which Flk2 promotes the
expansion of multipotent progenitors that give rise to all myeloid and lymphoid lineages but does not instruct lineage choice. These data yield a fresh perspective regarding the function of Flk2 in normal hematopoiesis and provide new insights into how activating mutations in Flk2 promote leukemogenesis.

See Feng et al., pages 172–182.

The ubiquitin-proteasome system plays a fundamental role in cellular homeostasis. In general, cancer cells have higher levels of proteasome activity compared with normal cells, making this an ideal target for anti-cancer therapies. At present, the proteasome inhibitor bortezomib is used as a first-line therapy for multiple myeloma (MM). However, development of resistance to bortezomib is a major issue, and thus there is a need for the development of new drugs. The small molecule b-AP15 is a novel inhibitor of proteasome deubiquitination activity. Recent studies have shown that b-AP15 displays anti-tumor activity in several pre-clinical solid tumor models. In this study, Feng et al. found that b-AP15 triggers apoptosis of the human MM
cell lines, RPMI8226 and U266. The induction of apoptosis in MM cells was dependent on caspase activation and partially dependent on cathepsin D, a lysosomal protease. The authors further demonstrated that b-AP15 engages the mitochondrial apoptosis pathway in MM cells, as the drug triggered a significant loss of mitochondrial transmembrane potential as well as activation of the pro-apoptotic protein Bax. Notably, the latter effects were caspase independent. Finally, the authors noted that b-AP15 triggers caspase-independent apoptosis in natural killer (NK) cells. Notwithstanding, the study sheds new light on apoptosis induction by b-AP15 and supports the use of b-AP15 or related compounds for the treatment of MM, including in patients who develop resistance to conventional treatments.

See Iwamoto et al., pages 163–171.

Xenotransplantation models using immunodeficient mice are widely used for human stem cell assays. However, this model shows strain-specific differences in the engraftment, and nonobese diabetic (NOD) background strains are the most efficient. This group has previously shown that
the polymorphism of the signal regulatory protein-alpha (Sirpa), which inhibits phagocytic activity of macrophages through binding to its ligand CD47, determines the engraftment
and tolerance of xenotransplants in NOD-scid mice. The BALB/c strain of immunodeficient mice is another strain that can support human hematopoietic reconstitution in vivo. In the present study, Iwamoto et al. tested whether the efficient xenotransplantation capability of the BALB/c
strain is also mediated by the SIRPA-CD47 self-recognition system. The authors sequenced the IgV domain of mouse Sirpa, which contains the binding site of CD47, and compared the binding affinities of C57BL/6, BALB/c, and NOD SIRPA to human CD47. They found
that a BALB/c-specific polymorphism, L29V, confers moderate affinity for human CD47 to mouse SIRPA on the macrophage membrane, which inhibits macrophage-mediated elimination of the human hematopoietic cells. These findings further indicate that the mouse strain effect on xenotransplant efficiency might be explained by the binding affinity between strain-specific SIRPA and human CD47. This information is useful to establish a novel, more efficient immunodeficient mouse model for human cell transplantation by strengthening the binding between mouse SIRPA and human CD47. In addition, the data show that the SIRPA SNPs can influence the binding to CD47 and phagocytic activity, raising an interesting question of whether there might be SIRPA or CD47 SNPs that disrupt the SIRPA-CD47 self-recognition
system and induce human diseases.
April


Sadovnik et al., pages 282–293.
Chronic eosinophilic leukemia (CEL) is a neoplasm characterized by uncontrolled expansion of eosinophils with consecutive organ damage. In many patients, eosinophils express the FIP1L1-PDGFRA oncoprotein. This oncogenic mutant contributes essentially to factor-independent
growth and accumulation of eosinophils in CEL. The PDGFR kinase blocker imatinib is successfully used to suppress growth of neoplastic eosinophils in these patients. Notably, most CEL patients treated with imatinib enter continuous complete remission. However, not all patients with CEL show a long-lasting response to imatinib, but relapse after a certain time interval. For these patients, alternative drugs have to be considered, especially when secondary mutations are detectable in FIP1L1-PDGFRA. In the present study, Sadovnik and colleagues
have examined the effects of various clinically relevant tyrosine kinase inhibitors on growth and survival of EOL-1 cells, a human CEL-related cell line exhibiting the FIP1L1 PDGFRA mutant. The data obtained show that several of the tyrosine kinase inhibitors tested, including ponatinib, sorafenib, masitinib, nilotinib, and dasatinib suppress growth and survival ofEOL-1 cells at a low nM range. In addition, these drugs were found to inhibit cytokine induced migration of eosinophils. However, only ponatinib was found to block the growth of Ba/F3 cells expressing the imatinib-resistant FIP1L1-PDGFRA mutants T674I and D842V. These observations may
have clinical implications. Indeed, based on the authors’ opinion, clinical trials are now warranted to explore the clinical potency of ponatinib in drug-resistant eosinophilic leukemias or other neoplasms expressing drug-resistant mutant forms of PDGFRs.


Visfatin (VF) is a newly discovered adipocytokine thought to play a role in the pathogenesis
of metabolic syndrome–related cancers. Originally identified as a pre B cell colony enhancing factor, it is found in different tissues and organs and performs many functions, including enhancing cell proliferation. The peptide acts as an enzyme, growth factor, and cytokine in
several processes including cell growth, cell survival, DNA replication, and repair.
Currently there are no data about the role of VF in pediatric leukemia and stem cell
transplantation. In this study, Skoczen et al report decrease in VF concentrations in 22
children with leukaemia referred to haematopoietic stem cell transplantation, and normalization of the peptide level after the procedure. It is suggested in the literature that VF is an important factor in carcinogenesis and a potential prognostic marker in several solid tumours. The presented findings suggest that decreased VF in leukaemic children in complete remission
may be caused by myelo- and immunosuppression after many cycles of chemotherapy. Because VF has antiapoptotic activity, reduced levels may result in increased antitumor activity, which may be beneficial for the patients. The authors speculate that the recovery of the plasma VF concentration after transplantation might be caused by a process of immune reconstitution or complications of the procedure, such as prolonged inflammation (infections or GvHD), injury to organs (lungs, gut, and liver), and endocrinology deficiencies. If these findings can be supported by studies with larger cohorts, VF could be an additional marker of leukaemia status and proper reconstitution of cell lineages after haematopoietic stem cell transplantation.

Ueno et al., pages 307–316.

Acute B-cell lymphoblastic leukemia (B-ALL) is the major type of ALL and it can occur at
any age. Despite a good prognosis in most pediatric B-ALL patients, the treatment for adult and
high risk pediatric B-ALL remains challenging. Therefore, exploring new targets or pathways
are essential for the development of better therapeutic options for these patients. SALL4 is an
embryonic stem cell factor which plays an essential role in the maintenance of embryonic stem cell properties. SALL4 is expressed during development, but downregulated and absent in most adult tissues. However, it is re-expressed in various cancers including B-ALL. In this study, Ueno et al. report that SALL4 was constitutively expressed in B-ALL and was a key survival factor for B-ALL cells. Downregulation of SALL4 in B-ALL cells led to decreased cell proliferation, increased apoptosis in vitro, and impaired leukemic engraftment in a xenotransplant model in vivo. SALL4 affected B-ALL cell survival through multiple caspase members. Furthermore, the authors reported that hypomethylation of the SALL4 CpG islands was correlated with its increased expression level. This is the first report to demonstrate that the aberrant expression of SALL4 in B-ALL is associated with hypomethylation, and that SALL4 plays a key role in B-ALL cell survival. The authors further propose that SALL4 could be a novel therapeutic target for B-ALL, and new treatment options such as targeting SALL4 could be developed in the future to treat B-ALL patients.

Jung et al., pages 274–281.

Cellular immunotherapy using dendritic cells (DCs) is emerging as a strategy to treat multiple
myeloma (MM); however, the clinical results have been unsuccessful. Attempts to improve
DC vaccination have included better cytokine combinations to enhance DC function, effective
tumor antigens to induce specific cytotoxic T lymphocytes, and the modification of signal transcriptions to overcome defective DC function. Immunosuppressive cytokine from MM cells,
including transforming growth factor beta, interleukin-10 (IL-10), IL-6, and vascular endothelial growth factor, can modulate host immune responses, including the abrogation ofDCfunction by the constitutive activation of the signal transducer and activator of transcription-3 (STAT-3). A recent study has reported that treating MM cells with the proteosome inhibitor bortezomib could increase the expression of heat-shock protein (HSP) 90 on the myeloma cell surface and
enhance the antitumor immunity against MM in DC-based immunotherapy. In this study, Jung et al. tested whether the synergistic action of bortezomib and JSI-124 (curcurbitacin-I, p-STAT3 inhibitor) could be used to generate highly potent tumor antigens to induce immunogenic death of tumor cell expressing HSPs. The combination treatment resulted in the highest expression of HSP90 and the lowest expression of p-STAT3 in dying myeloma cells. DCs loaded with treated dying myeloma cells recovered from the abnormal cytokine secretions of IL-10, IL-6, and IL-23 without any effect on production of IL-12p70. DCs loaded with JSI-124 and bortezomib-treated dying myeloma cells generated potent myeloma-specific cytotoxic T lymphocytes. This data suggest that pretreatment of myeloma cells with a combination of JSI- 124 and bortezomib can recover DC dysfunction upon loading with dying myeloma cells through the upregulation
of HSP90 and the downregulation of p-STAT3 and inhibitory cytokines. This strategic approach of combining two agents, which target two key survival pathways in the MM cells and neutralize their secretion of released mediators that render DC functions ineffective, is an important incremental step in the ongoing quest for the development of an immunotherapeutic approach to the treatment of MM.

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Inside the January and February 2014 issues

Posted By Connections Editor , Friday, February 28, 2014
Updated: Thursday, February 20, 2014

January 2014-inside this issue

#The role of stroma cells in MDS xenotransplantation

Li and Deeg, pages 410.

There has been a veritable explosion of information on genetic mutations in hematopoietic cells in patients with myelodysplastic syndromes (MDS). Concurrently, several murine models mimicking human MDS have been developed. However, studying the human disease invivo has remained challenging, mainly due to the difficulty of propagating CD34+ cells derived from patients with MDS in mice. There is considerable evidence that for MDS engraftment to be successful, certain microenvironment signals are required. This review by Li and Deeg summarizes data on murine xenotransplantation models of MDS with emphasis on the role of the microenvironment, specifically, stroma and mesenchymal cells. The authors describe strategies to improve engraftment that include, among others, genetic modification of mice to express human growth factors and administration of mesenchymal cells or stroma cells, either directly into the marrow cavity or intravenously. These studies have gained in interest due to the observations that nonclonal cells in the MDS marrow also show altered gene expression and function, presumably related to signals derived from the clone. An interesting byproduct of these studies is the observation that when coinjected intravenously with hematopoietic cells, stroma cells were able to reach bone marrow and spleen. However, none of the mice in the models described in this review have developed clinical features of human MDS, indicating that additional research is needed to generate fully developed MDS in these models. This comprehensive review draws attention to the interactions between marrow stroma and clonal hematopoietic cells in MDS and presents evidence to support the concept that MDS arises from hematopoietic stem cells.

#Mixing T cell development with seven-up

Ichim etal., pages 4658.

The Drosophila protein seven-up (svp) is a key regulator of cell fate decisions in neural development. Recent studies revealed that the orphan nuclear receptor Ear-2, a mammalian homologue of svp, plays a pivotal role at several stages of normal hematopoietic development, and it is an essential negative regulator of granulocytic differentiation in acute myelogenous leukemia. In this article, Ichim etal. investigated the role of Ear-2 in T cell development. They found that hematopoietic stem cells (HSCs) transduced to overexpress Ear-2 failed to repopulate the thymus when infused into lethally irradiated recipients. By using the OP9-DL1 stromal cell line, an invitro system of T cell differentiation, they documented two distinct defects in T cell development initiated with Ear-2-overexpressing HSCs. The first defect was detected at the DN1-DN2 stage, where they observed a severe reduction in proliferation, with widespread changes in the expression of genes associated with early T cell differentiation. The second defect was detected at the DN4 to DP transition, where they observed a block in differentiation accompanied by a wave of apoptosis–a defect identical to that seen in mice with targeted deletion of RORγt, another orphan nuclear receptor. The findings of Ichim etal. indicate that in addition to its roles in hematopoiesis and leukemia, Ear-2 is a novel negative regulator of T cell development, suggesting that this svp homologue may act as a brake in multiple transcriptional programs of differentiation. This discovery has important implications for future development of pharmacologic ligands aimed at regulating Ear-2 activity, which could offer a strategy for therapeutic modulation of both AML and T cell development.

#Memory T cells provide significant advantages for TCR-RNA based immunotherapy

Thomas etal., pages 2838.

Current strategies in cellular immunotherapy of cancer and viral infections include the adoptive transfer of T-cell receptor (TCR) and chimeric antigen receptor engineered T cells. When using transient TCR-RNA expression systems in clinical studies, multiple infusions with receptor-redirected T cells appear necessary. However, in allogeneic hematopoietic stem-cell transplantation, repeated transfer of donor-derived T cells increases the risk of inducing alloreactive graft-versus-host-disease. Considering this, Thomas etal. analyzed in this article alloreactivity of different human CD8+ T-cell subsets transfected with TCR-RNA. They observed in invitro studies that allo-reactivity developed significantly stronger in purified naive compared with memory or EBV-specific T-cell populations. Similar data were obtained after adoptive transfer of CD8+ T-cell subsets into a newly developed invivo alloreactivity test system, including immunodeficient NSG mice previously engrafted with human hematopoietic stem cells. This observation, along with strong effector function after TCR-RNA transfer, suggest CD8+ memory and EBV-specific T cells as promising tools for treating residual leukemia disease and viral infections after allogeneic transplantation.

#Transcriptional regulation of the hematopoietic hierarchy by microRNAs

Raghavachari etal., pages 14–27.

Hematopoiesis is a dynamic process that produces circulating blood cells of different lineages through the proliferation, differentiation, and maturation of self-renewing stem cells. Disruption in this well-orchestrated process leads to significant pathologic conditions including anemia, thrombocytopenia, lymphoma, and leukemia. Past studies have identified molecules such as lineage-specific cytokines and transcription factors as critical regulators of hematopoiesis. Recent technological advances in the analysis of noncoding RNAs have led to the understanding that miRNAs are powerful regulators of diverse cellular processes with critical roles in disease pathobiology. In this article, Raghavachari etal. examined the global expression of miRNA in the hematopoietic hierarchy to determine their regulatory roles in the lineage-specific commitment of stem cells. They detected 49 differentially expressed miRNAs, with functional roles in cellular growth, proliferation, and apoptosis during lineage-specific differentiation of stem cells. Target prediction analyses of these differentially expressed miRNAs identified potential miRNA targets associated with the hematopoietic differentiation process. Their integrated analysis of these potential mRNA targets confirmed most of them and provided a library of miRNA-mRNA networks including 87 highly correlated miRNA-mRNA pairs with functional roles in cellular growth and proliferation, hematopoietic system development, and Wnt/Beta-catenin and Flt 3 signaling pathways. This study provides strong evidence that miRNA molecules regulate the hematopoietic process. Future functional analysis of the differentially expressed miRNAs could result in the discovery of regulatory miRNA hubs, which in turn could lead to development of novel therapeutics for the treatment of hematopoietic disorders.

February 2014- inside this issue

#Lenalidomide, drug effect on angiogenesis in chronic lymphocytic leukemia

Maffei etal., pages 126136.

Lenalidomide is an immunomodulatory derivative (IMID) agent that is clinically active in patients with chronic lymphocytic leukemia (CLL). Increasing evidence suggest that angiogenesis can play a role in CLL pathophysiology, providing a supporting and protective milieu inside tissues. Although lenalidomide has been shown to affect angiogenesis invitro, little is known concerning its antiangiogenic properties in patients with CLL. In this study, Maffei etal. inspected the effect of lenalidomide on angiogenesis-related factors in 27 patients with relapsed or refractory CLL treated within a phase II clinical trial and invitro in a coculture system of endothelial and CLL cells. They demonstrated that lenalidomide exerts an antiangiogenic effect in treated patients by inhibiting the release of proangiogenic factors and by decreasing the number of circulating endothelial cells. Lenalidomide was reported to be effective in heavily pretreated or high-risk cytogenetics CLL patients, with response in 32%–47% of cases. The investigators identified vascular endothelial growth factor, bFGF levels, and the activation status of circulating endothelial cells as biological parameters associated with lenalidomide response. Of interest, Maffei etal. also report that lenalidomide interferes with CLL/endothelial cell crosstalk, reducing the survival advantage acquired by leukemic cells. This study provides a novel insight into the pleiotropic effects of lenalidomide in patients with CLL, including the disruption of the tumor-supporting properties ascribed to angiogenesis-related factors and endothelial cell contact. The identification of patients who respond to lenalidomide remains a challenging goal for clinicians. The work presented here indicates easily detectable parameters that could be useful to predict lenalidomide response.

#Pharmacologic activation of p53 impairs thrombopoiesis and leads to thrombocytopenia

Iancu-Rubin etal., pages 137145.

demonstrated to activate p53 and have promising anticancer effects in preclinical studies. RG7112 is the first clinically evaluated MDM2-p53 inhibitor that showed antitumor activities in several phase I clinical trials in solid tumors and hematologic malignancies. However, preliminary data indicate that one of the major hematologic toxicities associated with RG7112 administration in patients is thrombocytopenia. A number of studies in murine megakaryocytes and in human leukemic lines suggest that p53 has a role in megakaryopoiesis. In this study, Iancu-Rubin etal. used RG7112 as a pharmacological probe to activate p53 in human primary megakaryocytes to explore the role of p53 in normal megakaryopoiesis and to decipher the mechanisms responsible for RG7112-induced thrombocytopenia. They demonstrate that RG7112 treatment induces thrombocytopenia in rodents and primates and is deleterious for primary human megakaryopoiesis exvivo. RG7112 induced apoptosis of hematopoietic progenitors during the initial stages of megakaryocyte development and impaired their maturation during the later stages (i.e., inhibited DNA synthesis during megakaryocyte polyploidization and impaired demarcation membrane system formation and platelet granules biogenesis). The authors propose that these two mechanisms of action render the megakaryocytic lineage more susceptible to the drug and contribute to the development of RG7112-induced thrombocytopenia. Importantly, they show that the negative effects of RG7112 on normal thrombopoiesis are reversible, suggesting that intermittent therapeutic dosing of the drug might be a strategy for minimizing the degree of thrombocytopenia yet, retaining its antitumor effects. Furthermore, these studies provide a rationale for exploiting the inhibitory effects of RG7112 to reduce platelet counts in patients with myeloproliferative neoplasms.

#A classification system for hematopoietic stem cells based on reconstitution times

Ema etal., pages 74–82.

In this review, Ema etal. discuss the latest studies on hematopoietic stem cell (HSC) heterogeneity. The current classification systems of HSCs are based on their myeloid and lymphoid reconstitution ratios; however, Ema etal. propose a novel classification—LT-, IT-, and ST-HSCs—based on reconstitution times. They examine the relationship of the various classification systems and discuss how different HSC classes are related to one another in the hematopoietic hierarchy. The authors also make suggestions for how to design experiments based on their proposed classification system. Written by pioneers and leading figures in the field, the topic of this review is timely. The field has evolved quickly, and there is a need for review articles summarizing these new developments in a comprehensive way.

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Inside the November and December 2013 Issues

Posted By Connections Editor, Tuesday, December 31, 2013
Updated: Monday, December 23, 2013

November 2013

#Heterospecific cell hybrids, a powerful system for the study of globin gene switching
See Chang etal., pages 967–979.

Heterospecific cell hybrids provide a useful cellular system to test the dynamic behavior of chromosomally transferred genes upon exposure to new cellular environments. Although the globin expression phenotype of chromosomally transferred human beta globin locus in heterospecific hybrids has been studied previously, the influence of established chromatin domains within the donor cells on the outcome of globin gene expression following chromosomal transfer is still not clear. The availability of cells with documented divergent chromatin states provided this opportunity, as described in this article by Chang etal. Through selective retention of the human chromosome 11 marked with a neomycin resistance gene, the authors created hybrids between human embryonic stem cells (hESCs) or hESC-derived erythroid cells and murine erythroleukemia (MEL) cells and performed long-term observations of the expression of specific human globin genes in the MEL environment. The authors report several interesting findings. First, the entire globin locus of hESCs was mostly inactive and resistant to DNaseI digestion, but when transferred to the adult environment of MEL cells, only beta globin became activated. Second, the epsilon and gamma globin genes of the hESC-derived erythroid cells were actively transcribed. Upon exposure to an adult environment, which is repressive for fetal or embryonic globins, the chromatin state of the epsilon globin gene changed from opened to closed, whereas that of the gamma globin genes remained open, despite the presence of specific gamma globin repressor proteins in the adult environment. Third, in the presence of fully active gamma globin genes, the beta globin gene was not activated within the adult environment, which is consistent with the competitive nature between the transcription of gamma and beta globin genes. However, after prolonged culture, there was a progressive switch from predominant gamma to predominant beta globin expression accompanied by remodeling of the chromatin landscape. This switching process was accelerated by the overexpression of the gamma globin repressor klf1 and partially reversed by the downregulation of Bcl11a in combination with decitabine treatment. Based on the sustained human gamma globin expression within the adult MEL transcriptional environment, the authors predict that erythoid-committed cells with high gamma expression, such as the hESC- or iPSC-derived erythroid cells, are not going to switch quickly upon transplantation into adult recipients. This interesting and important study provides important insights into the interplay between the transcriptional environment and chromatin architecture in governing globin gene expression, and it provides a powerful model system for further research at the molecular level.

#Harnessing redox homeostasis for enhanced hematologic recovery after hematopoietic stem cell transplantation
See An etal., pages 944–956.

Understanding the molecules that regulate hematopoietic reconstitution is essential to develop novel approaches for enhancing donor cell engraftment and reducing transplant-related mortality after hematopoietic stem cell (HSC) transplantation. In a previous report, this group demonstrated that AMD3100/plerixafor (a CXCR4 antagonist) enhanced engraftment and chimerism when administered after transplantation in mice. To determine whether this effect was due to the modulation of the microenvironment, the authors used a proteomic approach to identify proteins that are modulated by AMD3100 in the bone marrow supernatants of transplant recipients. They chose to focus on thioredoxin, a small oxidoreductase protein that was upregulated by AMD3100. The key role of thioredoxin in mammals is to function as an antioxidant or reactive oxygen species scavenger. Persistent oxidative stress is one of the major causes for HSC injury during total body irradiation and for graft-versus-host disease during HSC transplantation. The authors demonstrated that thioredoxin can mitigate radiation injury and enhance long-term repopulating HSCs when added to HSC cultures for only 24 hours or administred after transplant. They also showed that giving a combination of thioredoxin and AMD3100 after HSC transplantation promoted donor HSC reconstitution in the secondary transplant recipient mice. This study has important implications because it suggests that thioredoxin has the potential to overcome one of the major hurdles associated with HSC transplantation: delayed or failed donor cell engraftment. Furthermore, recombinant human thioredoxin is readily available and can be administered intravenously, offering the possibility of promptly translating these findings into clinical applications to improve the care and outcome of transplantation patients.

#Double umbilical cord blood transplantation for hematological malignancies
See Wallet etal., pages 924–933.

There are several features that make umbilical cord blood (UCB) a clinically beneficial source of hematopoietic stem and progenitor cells. UCB is abundantly available, and because it is an otherwise discarded material, its use presents no risk or discomfort for the donor. The risk of transmissible infectious diseases is much lower with UCB than with adult bone marrow donations. In addition, the immunologic immaturity of UCB cells allows transplantation in HLA-incompatible hosts and a decrease in the probability and the severity of acute and chronic graft-versus-host disease. However, the low number of hematopoietic stem and progenitor cells in single UCB collections represents a major drawback for its use in transplantation. To overcome this problem, several recent clinical investigations have focused on the use of double UCB for adult patients when cellularity in a single unit is not sufficient. In this study, Labussière Wallet etal. performed a multicenter study from the Société Française de Greffe de Moelle et de Thérapie Cellulaire (SFGM-TC) registry to evaluate the long-term outcomes after adult dUCB transplantation (dUCBT) for hematologic malignancies and to study the different parameters involved in the dominance of one UCB unit after transplantation. Among their findings, the authors report that after a median follow-up of 49.5 months, the 3-year probabilities of overall and progression-free survival were 41% and 35%, respectively. Importantly, they obtained a long-term plateau among patients with reduced intensity conditioning with a 3-years survival probability of 60%, which makes dUCBT a promising treatment strategy for these patients. Another interesting observation was that among patients transplanted with a male and female unit, there was a significant overall survival advantage when male cord engrafted male recipients, which suggests a dominant advantage in using male donor cells for male recipients. Gender has never been considered in previous analyses of dUCBT and needs to be studied in a larger cohort of patients.

December 2013

#Inhibition of GSK-3β promotes naive T cell expansion in bone marrow reconstituted mice.
See Shen etal., pages 1016–1027.

Hematopoietic stem cell transplantation (HSCT) is associated with a high risk of mortality, partially because of the harmful effects of the preconditioning myeloablative regimens. Pretransplant conditioning impairs thymic function, leading to delayed T cell regeneration. In addition, the thymus-independent expansion of mature T cell promotes their differentiation, limiting the diversity of T cell responses, which increases the risk of opportunistic infections and leukemia relapse. Thus, inhibiting the expansion of mature T cell could represent a promising therapeutic approach to improve immune reconstitution after transplantation. In this regard, the Wingless (Wnt) signaling represents a potential target, as it has been shown to inhibit mature T-cell differentiation in transgenic mouse models. In this manuscript, Shen etal. show that pharmacologic activation of Wnt increased the naive T cell pool in mice transplanted with human haematopoietic stem cells. Using a small molecule inhibitor of GSK-3β, 6-bromoindirubin 3’-oxime (BIO), they demonstrate that invitro BIO-treatment promoted naive T cell expansion following mitogenic stimulation and improved proliferative responses of T cells to allogeneic stimuli. Treatment with BIO expanded the IL7Rα+ subset of naive T cells and downregulated the expression of genes that were activated during effector cell differentiation and preserved naive T cell gene expression. The authors propose that pharmacologic inhibition of GSK-3β may increase the potency of T-cells in recipients of HSCT by expanding naive T cell subsets with diverse TCR repertoire, and therefore, significantly improve the outcome of HSCT in which impaired immunity is a serious cause of morbidity and mortality.

#Immunologic pathomechanism of Hodgkin’s lymphoma
See Jona etal., pages 995–1004.

Hodgkin’s lymphoma is a lymphoid malignancy of the immune system, and its cure-rate is one of the greatest success stories of hematology research. Its current first-line treatment gets 80–85% of patients tocomplete metabolic remission; however, treatment of primary refractory and relapsing patients remains challenging. Hence, a deeper understanding of its pathomechanism is needed. This review by Jona etal. provides a comprehensive cross-section of the current knowledge of the molecular mechanisms involved in Hodgkin’s lymphoma pathogenesis and progression. The authors put special emphasis on the immunologic processes and highlight recent therapeutic regimes aimed at improving treatment outcome and reducing late toxicities. This review targets hematologists and research scientists with interests in immunology and experimental hematology. The comprehensible writing allows readers who are new to the field to grasp the complex mechanisms involved in HD pathogenesis.

#Tenascin C regulates hemopoiesis within alternate niches
See Ellis etal., pages 1050–1061.

Microenvironments where hemopoietic stem and progenitor cells develop and reside require further elucidation. Recently, Nakamura-Ishizu etal. (2013) demonstrated that the extracellular matrix molecule Tenascin-C (TNC) was vital in hematopoietic regeneration within the bone marrow (BM) microenvironment. This article by Ellis etal. furthers our understanding of TNC in hemopoiesis by showing that TNC is not only important to steady-state homeostasis within the BM but also plays a role in regulating hemopoiesis within alternate niches such as the thymus. The authors show that TNC knockout mice had a higher proportion of mature T cells within the BM during both steady-state and post--long-term transplant, suggesting a role for TNC in lineage commitment. A decrease in the incidence and total number of thymic progenitor populations with a redistribution of early thymic progenitors to other organs indicated an additional role for TNC in migration and adhesion. Migration was further assessed through short-term homing assays, and evidence for TNC’s role as a cytoadhesive molecule was provided through mobilization assays. Analysis also suggested the integrin α9β1 was integral in these processes. Collectively, the data demonstrate the importance of TNC in steady-state hemopoiesis and highlights this glycoprotein as an important component of hemopoietic stem and progenitor cell niches. By better understanding stem cell niches, we will not only have a better understanding of normal blood formation and the etiology of blood diseases, but also of how to manipulate such niches for improved therapeutic outcomes.

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Inside the September and October 2013 issues

Posted By Connections Editor, Friday, November 1, 2013
Updated: Sunday, October 27, 2013

Inside this issue-September 2013

#Cohesin: a master regulator of hematopoietic transcriptional programs

See Panigrahi et al., pages 967–973.

The role of cohesin in mediating chromosomal cohesion between sister chromatids is well established. Recent evidence supports its role as a transcription regulator, as it is able to communicate with tissue-specific transcription factors and basal transcription machinery, mediate architectural chromatin organization, and communicate with chromatin remodeling

and modification molecules. In this review, Panigrahi et al suggested that cohesin may have a central role in the orchestration of hematopoiesis as a ‘‘master regulator’’ of transcriptional programs because of its ability to regulate transcription by various pathways. It will be very interesting to further understand how cohesin accomplishes such a complex process of orchestration of hematopoiesis.

#Tissue-specific T-regs: potential prognostic markers for acute graft-versus-host disease

See Engelhardt et al., pages 974–982.

Acute graft-versus-host disease (aGVHD) is a frequent life-threatening complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Reliable diagnostic and prognostic markers that can predict incidence and survival are urgently needed. Regulatory T cells (T-regs) are a subset of CD4+ T cells capable of suppressing alloimmune responses. In this study, Engelhardt et al reported that after allo-HSCT, patients with high frequencies

of circulating skin (CLA+) or gut tissue-specific (a4b7+) T-regs at engraftment, had significantly lower incidence of aGVHD of skin and gut at day 100. They also had better overall survival and nonrelapse mortality at 2 years. Flow cytometric quantitation of tissue-specific T-regs is a relatively simple test, yet has the potential to be a powerful tool for the clinicians. It will guide them to implement early and aggressive tissue-specific pre-emptive therapies resulting in a decrease in the incidence of clinical aGVHD and improve outcomes in these patients.

#The unexpected role of Pbx3 in leukemic transformation

See Novak et al., pages 1016–1027.

Transgenic mice expressing NUP98-HOXD13 (NHD13) and CALM-AF10 (CA10) oncogenic fusion genes develop AML with a long latency and incomplete penetrance, and therefore, they are useful models to search for collaborating genetic events required for leukemic transformation. In this study, Novak et al showed that both CA10- and NHD13-induced AML acquired a Ras pathway mutation, whereas Flt3 mutations were documented only in CA10-induced AML. Moreover, some Hox genes (Hoxa5, Hoxa9, and Hoxa10 in particular) and mir196b (embedded within the Hoxa locus) were markedly upregulated in both NHD13 and CA10 mice. Interestingly, the Hox cofactors were differentially expressed, Meis1 was increased in CA10- induced AML, whereas Pbx3 was increased in NHD13-induced AML. Furthermore, the investigators demonstrated that loss of Pbx3 is important to the maintenance and survival of cells overexpressing an NHD13 fusion, an unexpected functional role for Pbx3 expression in NHD13-induced leukemic transformation. Such studies searching for collaborating events in leukemic transformation are valuable as they help identify novel therapeutic targets in AML.

Inside this issue-October 2013

#Burst-forming unit–erythroid assays in bone marrow failure

See DeZern et al., pages 808–816.

Patients with cytopenias and a cellular bone marrow (BM) can be a diagnostic and a therapeutic challenge, especially when there is no gross tumor, non-diagnostic morphology, and a normal karyotype. Previous reports have suggested a role for progenitor assays as potentially useful tests for diagnosis and predicting response to therapy. The utility of these assays lies in their ability to provide an assessment of BM growth and differentiation, which other BM tests cannot reliably offer. In this study by DeZern et al, 48 consultative cases of single or multi-lineage cytopenias with cellular marrows had aspirates sent for Burst-forming unit–erythroid (BFU-E) to aid in diagnosis. The data suggest that low BFU-E growth (less than 10 BFU-E /105 cells) is more consistent with a diagnosis of myelodysplastic syndrome (MDS) and helps to exclude the diagnosis of red cell aplasia, large granular lymphocytosis, or cytopenias from systemic autoimmune diseases. More robust BFU-E growth (20 BFU-E per 105 cells) is usually associated with external suppression of hematopoiesis, which occurs in non-large granular lymphocytosis, red cell aplasia, or autoimmunity. In this cohort, non-malignant diseases were well-distinguished from MDS by BFU-E growth. These findings suggest that the BFU-E assay could be a vital clinical adjunct for the patient and the clinician. In clinical settings with cytopenias of unclear significance, this assay may prove particularly useful for further differentiation of the etiology of the BM failure. Future prospective trials may be warranted to incorporate the BFU-E assays into the diagnostic algorithm for BM failure.

#Ciclopirox inhibition of mTOR provides a potential new approach to targeting AML

See Sen et al., pages 799–807.

Acute myelogenous leukemia is initiated and maintained by a chemoresistant population of cells known as leukemia stem cells (LSCs). Parthenolide (PTL) has been described as a selective agent that targets LSCs via inhibition of NF-kappaB and induction of reactive oxygen species (ROS). However, PTL treatment has also been shown to activate the mTOR pathway. The current study by Sen et al. demonstrates that the antifungal drug ciclopirox synergizes with PTL, resulting in potent antileukemia activity against blast, progenitor and LSC populations. This activity was mediated by the ability of ciclopirox to inhibit mTOR, an activity not previously described for this agent in AML. The study also demonstrates the importance of combinatorial strategies to overcome cytoprotective mechanisms. These findings provide a rationale for the use of ciclopirox in clinical trials and in combination therapies that may be improved by inhibition of mTOR.

#A chimeric lentiviral vector for both human and non-human primates hematopoietic repopulating cells

See Uchida et al., pages 779–788.

Innate immune factors, such as TRIM5a and cyclophilin A (CypA), act as major elements restricting retroviral infection among species and potentially limit transduction with retroviral vectors. When HIV1 infects human cells, HIV1 capsid binds to human CypA to escape from human TRIM5 a restriction. However, in rhesus cells, the mismatch between HIV1 capsid and rhesus CypA is recognized by rhesus TRIM5 a and prevents infection, and thus, transduction by HIV1-based lentiviral vectors. To circumvent the mismatch, Uchida and colleagues previously developed a chimeric HIV1 vector (cHIV vector) in which the HIV1 capsid is substituted with the simian immunodeficiency virus (SIV) capsid. The cHIV vector system successfully allowed for the evaluation of HIV1-based vector constructs in non-human primates, an important model in the development of clinical gene therapy trials. Their next question addressed in this study was whether the cHIV vector efficiently transduces human cells, because theoretically, the mismatch between human CypA and SIV capsid in the cHIV vector could be recognized by human TRIM5 a. Expectedly, the cHIV vector produced lower transduction efficiency in a human lymphoblast cell line compared to an HIV1 vector. However, both cHIV and HIV1 vectors showed similar transduction efficiency in human CD34+ cells in vitro and in humanized xenograft mice. In addition, they demonstrated that the cHIV vector is independent of human CypA in escaping from TRIM5 a restriction. These data indicate that the preclinical evaluation of lentiviral vectors targeting human hematopoietic stem cells can be performed using cHIV vectors in the non-human primate.

#The role of the microenvironment during fetal hematopoieisis

See Cao et al., pages 761–768.

Hemopoietic stem cells (HSCs) are sustained in a specific microenvironment known as the stem cell niche. Recent studies in adult bone marrow(BM) have made significant advances in identifying important niche supportive cell types and extracellular components that are critical for HSC regulation. However, the understanding of the role of the microenvironment in the establishment of definitive HSCs and their expansion and maintenance during embryonic development is extremely limited. This review by Cao et al. examines the niche from a developmental perspective and highlights several important issues that merit further study. The

authors focus on the current knowledge of the components of each HSC microenvironment at various developmental stages and their known functional roles. They describe the cells, extracellular matrix, cytokines and signaling pathways that have been identified in different developing hemopoietic organs as well as in the adult BM. The review conveys the message that the microenvironment plays an important part in educating HSCs and modifying hematopoiesis throughout development. Indeed, fetal niches have unique properties, allowing the quick expansion of HSC during early gestation, and assisting in fetal hemopoietic migration during late gestation. Contrary to adult BM, many current studies assessing fetal niches are still reliant on in vitro stromal cell cultures, with the identification of specific cell types and molecular mechanisms regulating fetal HSC needing further investigation. Since reprogramming cells mimic fetal HSC behavior rather than adult HSC, understanding the mechanisms involved in fetal HSC regulation should lead to a better understanding of the mechanisms regulating pluripotency and thus facilitate the engineering of hematopoietic specification.

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NEW! Experimental Hematology now accepts brief communications

Posted By Connections Editor, Tuesday, October 29, 2013
Experimental Hematology welcomes articles that can be reported in a shorter format. Brief communications are intended to allow publication of highly significant findings reported concisely, and not of incomplete or preliminary data. As with regular submissions, brief communications are expected to provide mechanistic insights, elucidate novel disease biology or approaches to therapy. Click here to learn more.

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Inside the July and August 2013 issues

Posted By Connections Editor, Monday, September 9, 2013
Updated: Thursday, August 22, 2013

Inside this issue July 2013

#Single-cell Raman spectroscopy demonstrates mechanochemical differences between normal, sickle, and cord red blood cells

See Liu et al., pages 656–661.

Sickle cell disease, a blood disorder characterized by abnormal rigid, sickle-shaped red blood cells (RBCs), can lead to various complications that can degrade a patient’s quality of life or be life-threatening. To better understand both the mechanical and biochemical properties of these cells, in this study Liu etal. applied laser tweezers Raman spectroscopy (LTRS), a novel label-free single-cell analytical tool, to measure the oxygenation response of RBCs to an applied mechanical force. LTRS was used to acquire the oxygenation-specific Raman spectra of individual optically trapped normal adult, sickle, and cord blood RBCs subjected to different forces by varying the laser power of the optical trap. For all cell types, an increase in laser power induced a greater deoxygenation of the cell. However, sickle RBCs deoxygenated more readily than normal RBCs when subjected to the same optical forces. Conversely, cord blood RBCs were able to maintain their oxygenation better than normal RBCs. These results suggest that differences in the chemical or mechanical properties of fetal, normal, and sickle cells affect the degree to which applied mechanical forces can deoxygenate the cell. Populations of normal, sickle, and cord RBCs were identified and discriminated based on this mechanochemical phenomenon. With its ability to characterize the functional properties (e.g., mechanical deformability, oxygen binding) of normal and diseased RBCs at the single-cell level, LTRS has the potential to be used for many applications, such as determining the efficacy of patient treatments in-vitro, assessing the effectiveness of sickle cell gene therapy approaches to restore normal RBC function, and in fundamental RBCresearch.

#Involvement of the aldehyde dehydrogenase 1 family member A2 in AraC resistance

See Kawasoe et al., pages 597–603.

1-β-D-arabinofuranosylcytosine (AraC) is one of the most effective drugs in the treatment of acute myeloid leukemia. It is also active against other hematologic malignancies, such as acute lymphoblastic leukemia and non-Hodgkin's lymphoma. However, AraC resistance remains a critical problem, underscoring the urgent need to clarify the underlying mechanisms. In this manuscript, Kawasoe etal. set out to identify candidate proteins associated with drug resistance. Using an unbiased proteomics-based approach to compare the protein expression profiles of K562 cells sensitive and resistant to AraC, the authors demonstrate that expression of aldehyde dehydrogenase 1 family member A2 (ALDH1A2), otherwise known as retinaldehyde dehydrogenase 2, is increased in resistant cells. They also show that the siRNA knockdown of ALDH1A2 in K562-resistant cells promoted the recovery of sensitivity to AraC, whereas ALDH1A2 overexpression in K562-sensitive cells induced AraC resistance. The role of ALDH1A2 in AraC resistance is still unclear; however, the enzyme catalyzes the synthesis of retinoic acid, which has been shown to have anti-apoptotic properties, and thus, may protect cells from the apoptotic-signaling trigger by AraC. This study provides useful insights into the mechanisms of AraC resistance and, if confirmed, points at ALDH1A2 as a potential therapeutic target in combination with AraC for the treatment of AraC-resistant neoplasms.

#New insights into the biology of endothelial selectins in hematopoietic stem/progenitor cell migration

See Nabors et al., pages 588–596.

It has been known for some time that endothelial (E- and P-) selectins are constitutively expressed in bone marrow (BM) sinusoidal endothelial cells, and that at least one of these molecules is critically involved in the rapid homing of hematopoietic stem/progenitor cells (HSPC) to BM in the context of BM transplantation. The distinct patterns of expression of E- versus P-selectin suggest the possibility that they mediate distinct functions. Moreover, it has never been established whether homing to BM requires both or only one of the two selectins. In this study, Nabors etal. examined the role of the individual endothelial selectins and found that either one was sufficient to rescue lethally irradiated mice that were transplanted with a limited number of cells. These data demonstrate, at least for homing and rescue, an "either/or” requirement for the endothelial selectins to support recruitment to BM. In addition, the authors found heterogeneity of expression of selectin ligands on HSPC, with higher levels of selectin ligands on progenitors (lineage-negative/sca-1+/c-kit-lo; LSK) than on stem cells (LSK CD48+CD150-negative). Acquisition of E- and P-selectin ligands did not appear to involve upregulation of glycoproteins known or proposed to function as selectin ligands, but rather, involved a gradual increase in expression of one or more glycosyltransferases responsible for biosynthesis of the glycans that function as selectin ligands. These results clarify the role of E- and P-selectin in HSPC migration to BM and document heterogeneity of selectin ligand expression on HSPC.

#Telomere shortening in Philadelphia-negative chronic myeloproliferative neoplasms

See Ruella et al., pages 627–634.

Among noncoding DNA structures, telomeres and the telomere length (TL) have been investigated in cancer cells with increasing interest. TL typically shortens with age and is therefore a marker of cellular aging. It also shortens as consequence of both DNA damages and oncogene activation. Moreover, telomere sequences are variably lost following exposure to chemotherapy. The observation that TL is shortened in most malignancies has pointed out the many biological links between telomere and neoplastic cell growth. In this manuscript, Ruella etal. investigated this connection further in Philadelphia-negative (Ph-neg) chronic myeloproliferative neoplasms (CMNs). Their study shows that among Ph-neg-CMNs, polycythemia vera (PV) and myelofibrosis (MF) presented a pronounced TL shortening, while essential thrombocytosis (ET) and secondary erythrocytosis had TL similar to the aged-matched normal population. On multivariate analysis, shortened TLs correlated with JAK2-V617F allele burden >50%, while no correlation was found with disease duration nor with Hydroxycarbamide therapy, which further supports the preferential use of this safe and cheap drug in patients requiring cytoreduction. Of note, in this study patients were not exposed to the new generation of JAK1-2 inhibitors, so these data may provide a useful historical control to understand the impact that these innovative drugs will have on the biology of telomeres. Altogether, these results support further studies to validate the use of TL in Ph-neg-CMNs to distinctly characterize PV and MF patients, to offer prognostic insights during the disease course, and to monitor DNA damage induced by potential therapeutic drugs under investigation. The study also shows the marked difference in TL between two apparently similar neoplasms such as PV and TE, suggesting the need for further biological studies to fully understand the pathogenetic steps leading to the various CMN subtypes.

Inside this issue August 2013

#Engraftable CD34+ cells from human embryonic stem cells

See Kim et al., pages 749–758.

Until recently, protocols for generating CD34+ cells from human embryonic stem cells (hESCs) have mostly relied on either embryoid body formation or coculture on mouse feeder cell lines, both of which pose significant challenges for their clinical utility. Moreover, the level of engraftment achieved after transplantation of hESC-derived CD34+ cells has remained low, raising concerns regarding their invivo potential. Based on a previous study from this group, Kim etal. hypothesized that a combination of mesenchymal stromal cells and macrophages could recapitulate a bone marrow microenvironment invitro, inductive to the generation of CD34+ cells from hESCs. In this study, these two types of human cells were used as feeder cells to direct the differentiation of hESCs into CD34+ cells. The hESC-derived CD34+ cells generated in this manner express cell surface markers and genes associated with hematopoietic stem cells. Most importantly, they demonstrate high levels of engraftment and development of multilineage blood cells following transplantation into the pre-immune fetal sheep model. To humanize the differentiation protocol further, the authors cultured undifferentiated hESCs in a human serum–based matrix to replace Matrigel, a mouse sarcoma tumor-derivative matrix. This study provides a clinically applicable methodology to generate engraftable CD34+ HSCs from hESCs and offers a framework for the design of more effective invitro platforms for efficient differentiation of hESCs.

#G6PD deficiency in a Zebrafish model

See Patrinostro et al., pages 697–710.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy and genetic mutation worldwide. The zebrafish has been used in the modeling and discovery of new mechanisms involved with the development of the hematopoietic system, of which erythropoiesis is a foremost process. The zebrafish allows for unparalleled insight and visualization into the early developmental events governing the blood system because of its relatively rapid development that is external to any maternal environment. In this article by Patrinostro etal., the authors use morpholino-based downregulation of g6pd expression to reduce G6pd protein and activity levels significantly in the developing zebrafish. G6pd-deficient animals developed erythrocytic hemolysis when exposed to several prooxidant compounds, including naphthol, menthol, and primaquine, which typically lead to hemolytic crises in persons with G6PD deficiency. Animals showed high levels of reactive oxygen species (ROS) and subsequently developed significant red cell apoptosis, resulting in anemia and substantial cardiac edema, similar to the severe clinical presentation of G6PD-deficient individuals during a hemolytic crisis. This model system will be useful in trying to elucidate the nature of ROS and its effects on the developing organism, as well as the biological response to increased ROS. It can also be used to study the effects of ROS-induced hemolysis on the hematopoietic system. Finally, researchers can use this system to search for new and novel compounds or drugs to reduce ROS and its deleterious effects.

#Myelodysplastic syndrome and the genomic instability hypothesis

See Zhou et al., pages 665–674.

Myelodysplastic syndrome (MDS) has received increased attention from both the scientific community and the public, in part because the number of people who are most susceptible to developing this hematologic malignancy, in particular the elderly and cancer survivors, is expanding dramatically. Therefore, there is a pressing need to understand more about the molecular pathogenesis and underlying biology of this disease, so that unique or improved therapeutic strategies can be developed. In this review, Zhou etal. highlight recent advances in our understanding of MDS, including the identification and functional characterization of many novel gene perturbations that might be acquired by patients with the disease. As a result, the authors discuss the emerging notion that MDS is a genomic instability syndrome, possibly reflecting an underlying problem in DNA repair. They also summarize several seminal studies on the maintenance of genomic integrity within hematopoietic stem cells, believed to be the cell of origin in MDS, and their differentiating progeny. In addition to raising intriguing new questions, these studies provide rare insights into the response of hematopoietic stem cells to DNA damage. The review provides a balance between clinical findings and the appropriate mouse phenotypes, as well as from hematopoietic stem cell research with and without links to aging. The article is authored by specialists in all crucial areas under discussion and should be interesting reading for researchers in the field.

#Efficient platelet production from human pluripotent stem cell–derived megakaryocytes using a two-directional flow bioreactor

See Nakagawa et al., pages 742–748.

The underlying mechanisms of platelet biogenesis from megakaryocytes remain elusive. Under conditions recapitulating platelet release based on invivo behavior of thrombopoiesis within mouse bone marrow, shear stress induced by blood flow in capillary vessels of bone marrow appears to stimulate the adjacent megakaryocytes to promote platelet yield. Recently, a series of articles have reported the application of shear stress to invitro thrombopoiesis using one directional flow. In this study, Nakagawa etal. propose a novel bioreactor system to recapitulate the invivo situation of platelet production. They derived megakaryocytes successfully from human pluripotent stem cells by applying in a three-dimensional bioreactor a one-directional flow and a second flow-mediated fixation of megakaryocytes in appropriate positions. The pressure flow held the megakaryocytes in the chambers of the bioreactor, and the main flow produced shear stress on the megakaryocytes' surface. The authors concluded that the angle between the main flow direction and the megakaryocyte surface was one of the critical factors for efficient platelet production. In a 90-degree situation, platelet production seemed to be slightly better than in static culture; however, in a 60-degree situation, platelet production efficiency increased 3.6-fold compared with static cultures. This novel finding will definitively influence the future planning of exvivo platelet production systems from human pluripotent stem cell–derived megakaryocytes for transfusion medicine.

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Inside the May and June 2013 Issues

Posted By Connections Editor, Sunday, June 30, 2013
Updated: Friday, June 28, 2013

Take a quick tour of the May and June issues and get a feel for the publication's cutting-edge scientific content. Read the full issues online.

In this Issue – May 2013

#Migration of hematopoietic precursors during development and its clinical implications
Ciriza et al.

Fetal hematopoietic stem cells (HSCs) need to migrate through different niches located in diverse anatomic microenvironments to gain the necessary molecule repertoire to engraft and home into the bone marrow. This review by Ciriza etal. comprehensively summarizes early and recent work related to multipotential cells in the yolk sac, placenta, intraembryonic sites including the AGM and the fetal liver, as well as factors that affect interaction of these cells with the local microenvironment. These factors include a host of adhesion molecules, chemokines, cytokines, and ECM molecules, which are discussed in much detail and without significant bias in a presentation of opposing models. The authors also devote a significant amount of discussion to the important issue of embryonic stem cell differentiation into transplantable, long-term self-renewing HSCs, and they propose intriguing ideas for why this process might not be efficient. This timely review is highly relevant to readers looking to further their understanding of fetal HSC migration and of the potential application of that knowledge to stem cell and transplantation biology.

#A step further toward the generation of a viable in-vitro stem cell niche
Deneault et al.

The importance of extrinsic regulation of hematopoietic stem cell (HSC) activity is increasingly acknowledged. Invivo, the stem cell niche integrates a complex mixture of different cells and signaling molecules. This microenvironment is not easily imitable invitro, making it difficult to maintain HSC activity in culture for extended periods of time. In this study, Deneault etal. engineered niche cells to overexpress several nuclear factors agonists of HSC activity, which they identified in a previous report. They find that most of the tested intrinsic factors have actually a non–cell autonomous effect on HSC activity. In addition, they explore the transcriptional regulatory networks that have been rewired within engineered niche cells. Interestingly, the identification of the involvement of FOS, SPI1, KLF10, TFEC, and PRDM16, which consistently interact epistatically, reveal a novel non–cell autonomous network in engineered niche cells that support HSC activity. Moreover, these five factors are normally involved in osteoclastogenesis invivo, supporting the hypothesis that osteoclasts might also be part of the functional HSC niche. Finally, the authors uncover an important contrast between mouse and human HSC biology, because the engineered niches failed to enhance activity of human HSCs, as opposed to mouse HSCs. This finding suggests that HSC self-renewal determinants are poorly conserved between the two species. These findings contribute to characterize the functional niche that supports HSC activity and pave the way for the identification of new secreted growth factors that expand HSC numbers in culture to improve transplantation-based therapies.

#The hematopoietic role of the sodium-dependent phosphate import protein Sodium-Dependent Phosphate Import Protein, PiT1, is not associated with phosphate transport
Liu et al.

Emerging data suggest that the sodium-dependent phosphate import protein, PiT1 (SLC20A1), modulates cellular proliferation. In this article, Liu L. etal. characterize the hematopoietic phenotype of mice with conditional deletion of Pit1 and show that the animals develop a severe macrocytic anemia with a block in terminal erythroid differentiation around the proerythroblast stage. The mice also have a profound B cell deficiency, which primarily reflects a block at the pro-B stage in development, mild neutropenia, and thrombocytopenia. Importantly, the mice have a general hematopoietic cell defect in cell cycle progression in the absence of a change in cellular phosphate uptake, consistent with a phosphate import–independent role for PiT1 in governing cellular proliferation. This work contributes to the currently limited published data on the role of metabolite transporters in hematopoiesis and raises the intriguing possibility that PiT1 could have separable phosphate transport and cellular proliferation signaling functions. Moreover, as the hematopoietic phenotype of Pit1-deleted mice shares several features with low-grade myelodysplastic syndrome (MDS), and MDS is associated with Gibbon Ape leukemia virus and feline leukemia virus B infections (both utilize PiT1 for cell entry) in nonhuman primates and cats, respectively, future studies of these Pit1-deleted mice and PiT1-associated pathways could also provide insight into the pathobiology of these disorders.

#KI haplotype A: A novel predictive factor of complete molecular response in chronic myeloid leukemia patients treated with tyrosine kinase inhibitors
La Nasa et al.

Growing evidence suggests that the occurrence of chronic myeloid leukemia (CML), the disease course, and the response of patients to therapy with tyrosine kinase inhibitors can be influenced by inhibitory and activating receptors regulating the function of natural killer (NK) cells. NK cells are important mediators of both innate and adaptive immune responses. NK cell recognition and lysis of tumor cells is regulated by the interaction of killer cell immunoglobulin-like receptors (KIRs) expressed on the NK cell surface and human leukocyte antigen (HLA) class I alleles expressed on target cells. It is speculated that changes in the balance of inhibitory and activating KIR signals could lead to NK cell deficiency, contributing to the occurrence of CML and hampering patients’ response to treatment. In this study by La Nasa etal., the authors present a retrospective study of KIR profiles and response to therapy in an ethnically and genetically homogeneous cohort of CML patients and control subjects. They show that patients homozygous for the KIR haplotype A have a significantly higher probability of achieving early and sustained complete molecular response (CMR) than do patients with AB or BB genotypes. Achievement of CMR is also associated with a reduced frequency of KIR2DL2 and a low number of inhibitory KIR genes. Considering that group A haplotypes have only six inhibitory genes and that KIR2DL2 is exclusively carried by group B haplotypes, it is possible that the KIR haplotype AA could represent a valuable marker of durable CMR. The identification of such markers, combined with the currently known prognostic factors for CMR, should help clinicians make informed decisions regarding the modulation and possible withdrawal of tyrosine kinase inhibitor treatment in patients with CML.

In this Issue – June 2013

#A mouse model of Hoxa9-mediated pre-T Lymphoblastic Leukemia
Beachy et al.

Despite the strong association of Hoxa9 expression with T cell and myeloid leukemia, enforced expression of Hoxa9 in murine bone marrow or thymus has only modest malignant transforming abilities. To gain a better understanding of the relationship between Hoxa9 overexpression and hematopoietic malignancies, Beachy etal. generated transgenic mice that ectopically expressed Hoxa9, by targeting transgene expression to hematopoietic tissues with Vav regulatory elements. When studying four lines of Hoxa9 transgenic mice from different potential founders, two major findings were observed. First, overexpression of the Hoxa9 transgene was associated with embryonic lethality, as two of the four potential founders did not transmit the transgene to surviving offspring, but analysis of embryos from timed pregnancies indicated that the embryos were viable until at least E9.5. Second and somewhat unexpected, a fraction of viable Hoxa9 mice developed pre-T lymphoblastic leukemia (LBL) or lymphoma, all with Notch1 mutations, but none of the mice developed acute myeloid leukemia (AML). Notably, Hoxa9 mice with pre-T LBL lacked global upregulation of other Hoxa cluster genes or mir-196b, which expression has been shown to be increased in other genetically engineered mice that express leukemic fusion genes (e.g., MLL-AF9, CALM-AF10, NUP98-HOXD13). Interestingly, Hoxa9 bone marrow could be immortalized invitro with the addition of Meis1, suggesting that lack of Meis1 overexpression in the Hoxa9 mice might partially explain why AML was not observed in this study. The authors propose that this Hoxa9 transgenic mouse might be a useful tool for studying collaborative events needed for the development of pre-T LBL or AML.

#Increased sensitivity of acute myeloid leukemia CD34+ cells to bortezomib by targeting NF-κB and MCL-1
Bosman et al.

Despite treatment with intensive chemotherapy, many patients with acute myeloid leukemia (AML) still have unfavorable outcomes. Various innovative therapeutic options have been explored to target the small population of leukemic stem cells that is responsible for the development of leukemia and relapse after treatment. Constitutive activation of NF-κB has been observed in AML blasts, and proteasome inhibition, which results in degradation of NF-κB, has been suggested to specifically induce apoptosis in AML CD34+/CD38 cells. Clinical studies evaluating the effect of the proteasome inhibitor bortezomib in patients with AML have been initiated. However, the efficacy of bortezomib in targeting AML stem cells has not been investigated thoroughly. This article by Bosman etal. provides evidence that the leukemic stem cell–enriched CD34+ fraction is resistant to bortezomib, using invitro stem cells assays on CD34+ samples from patients with AML. Interestingly, bortezomib resistance in CD34+ AML cells was largely dependent on inadequate inhibition of NF-κB, which might be related to differences in NF-κB activation in AML CD34+ versus CD34 cells. Intriguingly, proteasome inhibition resulted in the upregulation of the important antiapoptotic protein MCL-1. Inhibition of MCL-1 strongly increased the sensitivity of AML CD34+ cells to bortezomib. Future research should therefore be focused on combining bortezomib with MCL-1 inhibitors for the treatment of patients with AML. Furthermore, it will be important to evaluate whether the second-generation proteasome inhibitors that bind the proteasome in an irreversible manner, such as carfilzomib and oprozomib, can, in contrast to bortezomib, reduce the frequency of AML stem cells as single agents.

#Cytokine-induced killer cells as therapy against multiple myeloma
Liu et al.

Multiple myeloma (MM) remains an incurable plasma cell malignancy, and innovative therapeutic approaches are urgently needed. In addition to small molecules, immunotherapy using adoptively transferred immune cells, cancer vaccines, and virotherapy using tumor-selective oncolytic viruses are being pursued in clinical trials. This study by Liu etal. explores the potential of cytokine-induced killer (CIK) cells as therapy against MM. CIK cells are a heterogeneous subset of ex-vivo expanded T lymphocytes that present phenotypic and functional properties of both natural killer and T cells, and have major histocompatibility complex–unrestricted antitumor activity against both solid tumors and hematologic malignancies. The authors show that allogeneic CIK cells have antitumor activity against subcutaneous and disseminated animal models of human myeloma (KAS-6/1). The antimyeloma activity of CIK cells can be further enhanced by pretreating the myeloma cells with ionizing radiation or by loading CIK cells with a virotherapy agent—an oncolytic measles virus (MV) that is currently in phase I clinical testing in patients with relapsed or refractory myeloma. Irradiation of the myeloma cells enhanced the delivery of CIK to the irradiated tumors and induced mRNA and protein expression of natural killer group 2 D (NKG2D) ligands, which mediate the antitumor activity of CIK cells. CIK cells can also serve as carriers to protect and deliver the oncolytic virus to tumors to initiate viral induced oncolysis, resulting in superior antitumor activity. This study demonstrates the potential of CIK against MM, and that combination of virotherapy with radiation could be used to enhance therapeutic outcome using CIK cells. CIK therapy is already a clinical reality; therefore, the clinical translation of this promising delivery platform is highly feasible and worth pursuing in future studies.

#Narrowing down the origin of endogenic mesoderm
Kataoka et al.

Hematopoietic precursor cells (HPCs) develop from hemogenic endothelial cells (EC), a specialized type of ECs undergoing hematopoietic transition. Despite their importance, the origin of hemogenic ECs has not been defined clearly. Ets variant 2 (Etv2)/ER71 is a master regulator generating ECs/HPCs from mesoderm. Etv2 deficiency causes complete EC or HPC loss in mouse embryos and differentiated ES cells. In this study by Kataoka etal., the authors tested regional deletion of Etv2 to narrow the critical mesoderm subset that gives rise to hemogenic ECs. They report that among region-specific Cre deleters, Etv2 ablations by Hoxb6 Cre and CreER revealed that caudal-lateral mesoderm including allantois is a critical mesoderm source of intraembryonic hematopoiesis. Explant cultures before hematopoietic cells circulate showed that Etv2 deletion in Hoxb6+ E7.5–8.5 mesoderm causes severe intraembryonic hematopoietic defect with intact yolk sac hematopoiesis. Etv2 deletion in that area also resulted in significant reduction in intraaortic c-Kit+ clusters and umbilical cord Runx1+ cells. Labeling of the relevant population in differentiated ES cells (Flk-1+/Hoxb6+ cells) showed that those cells have hemogenic characteristics over Hoxb6 negative population, including T lymphocyte generation. These findings suggest that Hoxb6+ mesoderm will be a promising source for inducing definitive hematopoietic cells in developing embryos and invitro–differentiated embryonic stem cells. Currently, conventional hematopoietic differentiation methods are failing to efficiently generate Hoxb6+/Flk-1+ cells. Future efforts are necessary to optimize the culture condition for efficiently inducing Hoxb6+ mesoderm

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