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Inside the March & April Issues

Posted By Connections Editor , Thursday, May 1, 2014
Updated: Wednesday, April 30, 2014
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.

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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Inside the March and April 2013 Issues

Posted By Connections Editor, Wednesday, April 24, 2013
Updated: Wednesday, April 24, 2013

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

In this Issue – March 2013

#The mitochondrial redox environment during erythroid development affects mitochondria-less mature red blood cells
Mohanty et al.

Manganese superoxide dismutase (SOD2) maintains a low oxidative environment in mitochondria by converting toxic superoxide free radicals to hydrogen peroxide. Mature red blood cells (RBCs) do not contained SOD2 protein as they do not possess mitochondria, however; its deficiency or malfunction during erythroid development results in lasting changes to the mature RBC. In this manuscript, Mohanty et al. used an established hematopoietic specific SOD2 knockout mice chimera model to study the effect of SOD2 deficiency on two characteristics of RBCs: deformability and oxidative stress levels. They report that SOD2-/- RBCs have reduced deformability, higher amounts of heme degradation products, and a higher rate of hemoglobin oxidation compared to controls. These changes may explain the reduced RBC lifetime in circulation leading to increased RBC loss, which is associated with the anemia phenotype previously reported in these mice. These results support and expand the findings published by Case et al. in December 2012 in Free Radic Biol Med, which show that the disruption of the superoxide balance within the mitochondria leads to improper heme formation, gene regulation, and erythrocyte development. . Oxidative stress has been linked to sideroblastic, hemolytic and sickle cell anemia, and thalassemia. Studies such as these ones that further the understanding of how the mitochondrial redox environment affects RBC physiology may lead to novel therapies for those diseases.

#Acute lymphoblastic leukemia cells come out of the bone marrow protective niche for increased sensitivity to chemotherapy
Welschinger et al.

Acute lymphoblastic leukemia (ALL) is the most common cancer in children, and represents 20% of adult acute leukemias. Despite good initial responses to combination chemotherapy, remission remains a significant problem. This lack of efficacy of therapy can be partly attributed to the protection provided to the leukemia cells by the bone marrow (BM) microenvironment, and thus, there is a lot of interest in finding therapies to get the cells out of this protective niche. However, the BM microenvironment also enhances the survival of normal hematopoietic stem and progenitor cells (HSPCs), making it important to determine whether mobilizing therapy by itself or in combination with chemotherapy increases toxicity towards these cells. The interaction of the cytokine SDF-1 with the CXCR4 receptor on the surface of HPSCs and leukemic cells has been implicated in the retention of normal and leukemic cells in the BM, and in this study by Welschinger et al., the authors show that inhibition of the interaction by the CXCR4 antagonist plerixafor (AMD3100) results in the mobilization of ALL cells from the BM and spleen for a longer period of time than normal HSPCs. Unlike what has been reported with acute myeloid leukemia cells, mobilization of ALL cells is associated with increased cell cycle activity, which may be capitalized upon by combination with chemotherapy, as demonstrated by increased survival of ALL xenografts treated with a combination of vincristine and AMD3100. This clinically important information can be leverage to design clinical trials to test the combined use of chemotherapy with AMD3100 for maximal effect on leukemic as opposed to normal hematopoietic cells. Such clinical trials will bring new hope to patients with relapsed or high-risk ALL.

#Mechanistic insights provide scientific justification for the use of Histone Deacetylase Inhibitors in Myeloproliferative Neoplasms
Gao et al., and Caldaza et al.

Philadelphia-negative myeloproliferative neoplasms (MPNs) are a collection of clonal hematological malignant diseases in which more than 50% of patients carry a single mutation (V617F) in the autoinhibitory region of the JAK2 tyrosine. This has lead to the presumption that aberrant gene expression controlled by the deregulated JAK2/STAT pathway is the main cause underlying the disease pathobiology. However, clinical trials with different JAK2 kinase inhibitors have not yet shown eradication of the malignant clone, warranting the search for alternative targets for therapy. Recently, studies have revealed a number of epigenetic alterations that also likely contribute to the pathogenesis of MPNs and determine clinical outcome. Indeed, abnormal histone deacetylase activity has been noted in MPNs and provides a rationale for treatment with Histone deacetylase inhibitors (HDACi). However, the extent and specific role of histone deacetylase-mediated epigenetic aberrations in MPN are still poorly understood. Two articles published in this issue of ExpHem provide mechanistic rationales for the use of HDACi in the treatment of MPN, either alone or in combination with existing therapies.

The article by Gao et al. sheds light on the mechanism of action of the HDACi sodium butyrate (SB) on the JAK/STAT signaling pathway in MPNs. SB is a short-chain fatty acid that induces the accumulation of acetylated histone leading to cell cycle arrest, differentiation and apoptosis in various cancer cells. In this study, the authors found that SB downregulates the JAK/STAT signaling through upregulation of the suppressors of cytokine signaling (SOCS)1 and 3, both of which are potent feedback inhibitors of JAK2/STAT signaling. Their Chip analysis links the downregulation of SOCS1 and 3 to the accumulation of acetylated histones on the genes’ promoter, which is potentially mediated by HDAC8, as demonstrated in a selective siRNA knockdown experiment. Confirmation of these findings is important; if HDAC8 inhibition alone is enough to cause cell death or colony formation inhibition of MPN cells, this concept can be utilized to address the off-target toxicities associated with the utilization of pan-HDAC inhibitors in MPN patients.

The manuscript by Caldaza et al. provides a scientific rationale for combination therapy of the pan-HDACi Givinostat (ITF2357, GVS) and hydroxyurea (HU) for the treatment of MPN patients. GVS has been shown to specifically inhibit proliferation of cells bearing the JAK2V617F mutation and has demonstrated significant activity with good tolerability in clinical studies in MPN. Moreover, a randomized Phase II clinical trial of GVS and HU has recently been completed and has indicated a favourable clinical activity of the combination of these two compounds in the treatment of Policytemia Vera patients. In this study, the authors used MPN cell lines and patients’ cells to demonstrate that a low dose of the two drugs induces synergistic apoptosis, what they link to the inhibition by HU of GVS-mediated induction of p21 and to increased caspase-3 activity. This timely manuscript clarifies the mechanism of the drugs' synergy at the cell cycle and molecular level and provides a scientific justification to their use as combination therapy for the treatment of MPNs.

In this Issue – April 2013

#Gene Set Control Analysis, a new bioinformatic tool to enhance our understanding of blood cell differentiation
Joshi et al.

Transcription factors (TFs) have the unique property that they recognise specific DNA sequence motifs, and therefore have the ability to decipher gene regulatory information encoded in our genome sequence. Recent technological breakthroughs in sequencing technology have made the generation of genome-wide transcription factor binding maps almost routine. However, it is still largely unclear how transcription factor proteins "read” regulatory information when they interact with our genomes. Progress in this area will be vital to enhance our understanding of blood cell differentiation, and, given the complexity and scale of genome-scale binding maps, is likely to depend on new bioinformatic data integration and analysis tools. The paper by Joshi et al reports the generation of a TF binding map compendium, where 142 publicly available datasets for both normal and leukemic murine blood cell types have been re-analysed, integrated and analysis results made available for use by the wider research community ( In addition, the group also report the development of a new bioinformatic web-tool, called Gene Set Control Analysis (GSCA). Experimental biologists commonly generate gene lists, such as differentially expressed genes, from a treatment/control microarray experiment. Such lists are then scrutinized for functional or pathway enrichment by, for example, gene ontology analysis. The new GSCA tool utilises the integrated 142 transcription factor binding maps to predict likely transcriptional regulators for any gene set of interest. Using GSCA, the authors predicted the reactivation of blood stem cell control mechanisms as a likely contributor to LMO2 driven leukemia, and also clarified the recent debate on the role of Myc in leukemia stem cell transcriptional programs. The GSCA tool is freely available, and a web interface for use by the wider community can be found at GSCA accepts both mouse and human gene lists for the prediction of likely upstream regulators. It is anticipated that the underlying compendium will grow further as the community generates ever-increasing numbers of transcription factor binding maps. Nevertheless, even in its current incarnation it is hoped that GSCA will be recognised by the community as a valuable new tool in the bioinformatic toolbox available to us all; hopefully it will increase our ability of extracting biological knowledge from genome-scale datasets.

#Novel strategy to generate engineered T cell precursors for immunotherapy
Liu et al.

Adoptive T-cell immunotherapy appears promising to treat T cell immunodeficiency or to improve immune reconstitution after hematopoietic stem cell transplantation. In addition, adoptive transfer of peripheral T cells engineered to express a tumor antigen-specific T cell receptor (TCR) presents a new approach to selectively target malignant cells. Despite recent advances in both stem cell and lymphocyte manipulations, these experimental approaches still have several limitations, particularly in optimal and safe generation, as well as expansion of specific T cell from TCR-transduced stem cells. In this study, Liu et al present a novel strategy for the manipulation of early stage T lymphopoiesis using a two step co-culture system. They show that TCR-transduced hematopoietic progenitors co-cultured on telomerized stromal cells in the presence of SCF, Flt3L and TPO, followed by co-culture on Delta-1- and -4-expressing stromal cells, produce pre-T cell precursors that have the potential to proliferate and differentiate. This study highlights the potential value of engineered human stromal cells to manipulate early T cell development for clinical application, and provides a culture system that could help accelerate the translation of the next generation of adoptive T-cell therapies to the clinical setting.

#Cord blood-derived MSC: a potential tool to accelerate bone marrow recovery after ionizing radiation
Shim et al.

Mesenchymal stem cells (MSCs) have a great capacity for self-renewal while maintaining their multilineage differentiation potential, making them a promising tool for tissue repair. Compared with bone marrow MSCs, human umbilical cord blood-derived MSCs (hUCB-MSCs) are easy to obtain without harm to the donor and have a faster proliferation rate. In this study by Shim et al, the authors investigate the potential of hUCB-MSCs to improve the recovery of hematopoiesis after total body irradiation in comparison with the well-studied agent granulocyte colony stimulating factor (G-CSF). Using sub-lethally irradiated mice, they show that hUCB-MSCs treatment significantly increased the number of peripheral leukocyte counts following a radiation dose for which G-CSF treatment was unsuccessful. Moreover, at some post-irradiation intervals, hUCB-MSCs treatment was more effective than G-CSF in supporting proliferation of various cells in the BM and, in contrast with G-CSF, modulated the plasma level of hematopoietic cytokines. Together, these observations show that hUCB-MSCs treatment is superior to G-CSF, providing support for their use in the treatment of ionizing radiation-induced bone marrow injury. In addition, this study has important implications in the clinical setting, in which hCB-MCSs could be used to accelerate bone marrow recovery after chemotherapy and especially after hematopoietic stem cell transplantation.

#A modified high dose cyclophosphamide regime that could lower the cost of treatment of patients with several aplastic anemia
Zhang et al.

Aplastic anemia (AA) is a potentially fatal disease characterized by depletion of hematopoietic precursors in bone marrow (BM). The standard therapy for patients with this disease who are not candidates for an allogeneic BM transplantation is immunosuppression with a combination of horse-antithymocyte globulin (ATG) and cyclosporine A (CsA). This treatment results in good overall survival rates. However, relapses and event-free survival are still not satisfying. Furthermore, horse-ATG is not routinely available in developing countries and the costs for ATG-preparations in general are substantial. Therefore, alternative treatments are urgently needed. In this context, this manuscript by Zhang et al reports a retrospective study of patients with severe or very severe AA that received either a modified high dose cyclophosphamide (HD-CTX) regime with CsA or a therapy with rabbit-ATG in combination with CsA. Both treatment groups showed comparable response rates, survival rates and toxicities. In previous clinical trials, HD-CTX showed promising remission rates but unacceptable toxic effects. In this regard, this is a notable study that shows that a lower dose of CTX might overcome the immense toxic effects of the conventional high dose regime. Importantly, the modified HD-CTX plus CsA therapy is significantly cheaper than the ATG plus CsA therapy, thus providing an affordable treatment alternative for severe AA patients that cannot afford ATG therapy. This preliminary study merits further investigation in well-designed, prospective, and randomized clinical trials.

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January and February 2013

Posted By Connections Editor, Wednesday, March 6, 2013
Updated: Wednesday, February 27, 2013

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

In this Issue – January 2013

Role of Wnt antagonists in the regulation of hematopoietic stem cells fate decisions
Cain and Manilay

The Wingless (Wnt) are secreted lipid-modified signaling proteins of 350–400 amino acids in length. The Frizzled (FZD) proteins are seven-pass transmembrane receptors to which Wnts bind. The Wnt signaling is involved in cell fate decisions during hematopoietic differentiation in the bone marrow, in the maintenance of bone homeostasis, and in hematopoietic differentiation in a non-cell autonomous manner. There are 19 different Wnt ligands and 10 FZD receptors identified in humans and mice. In addition, Wnts utilize distinct co-receptor proteins that initiate "canonical” and "noncanonical” Wnt signaling pathways. Additional levels of complexity are added by secreted Wnt antagonists expressed in bone and bone marrow stromal cells that either bind to Wnt ligands directly or block Wnt receptors and co-receptors to halt Wnt-mediated signal transduction in both osteolineage and hematopoietic cell types. In this timely review, Cain et al. compared and contrasted the roles of these Wnt antagonists and their effects on hematopoietic development in mice and also discussed the clinical significance of targeting Wnt antagonists within the context of hematopoietic disease. This manuscript provides an interesting and comprehensive reading for anybody looking to update their knowledge of the field.

An improved method to track kinetics of hematopoietic stem cell reconstitution
Cornils et al.

Integrating viral vectors provide a unique and heritable genomic mark for the identification of single stem cell–derived clones. Linear amplification–mediated (LAM) PCR is an integration-site directed PCR method to sequence the unknown DNA flanking sequences down to the single cell. It has enabled qualitative and quantitative measurements of the clonal kinetics of hematopoietic regeneration in gene transfer studies and uncovered the clonal derivation of non-leukemogenic and leukemogenic insertional side effects in preclinical and clinical gene therapy studies. In this report, Cornils et al. presented a time-course clonal analysis of hematopoietic reconstitution following transplantation of retrovirus and lentivirus transduced cells using a multiarm, 2 restriction enzymes LAM-PCR analysis coupled to next generation sequencing (NGS). They found that the number of clones contributing to hematopoiesis over time were relatively stable for both vectors; however, the change of size of individual clones marked with the lentiviral vector followed wave-like kinetics, whereas retrovirally tag clones showed a tendency for continuous expansion. They also detected that long-term reconstituting clones marked with the retroviral vector had a tendency toward overrepresentation of insertions in the vicinity of proto-oncogenes, which supports the notion that clonal dominance due to insertional mutagenesis is more likely to develop after transduction with retroviral than with lentiviral vector. Finally they performed ligation-mediated (LM)-PCR, a method of sequencing directly from genomic DNA using a specific primer for the region of interest, and found that quantification of LAM-products did not always correlate with results from qPCR. As shown in this and other studies, even the use of two different restriction enzymes for LAM-PCR may not strictly ensure a complete figure of gene-marked clones. Thus, improved technologies are needed to benefit many areas of hematopoietic stem cell research, including in vitro cell expansion, gene therapy, and cancer progression and treatment.

Gpr171: a possibly new regulator of HSC differentiation
Rossi et al.

The G protein-coupled receptor (GPR) 171 is structurally and evolutionally related to a subfamily of the GPR family of purinergic receptors (P2YRs) for extracellular nucleotides, a group of mediators that regulate hematopoietic progenitor cells. GPR171 is an orphan receptor, and its signaling pathway and biological role remain unknown. In this study, Rossi et al. investigated the role of Gpr171 in the murine hematopoietic system. Their phylogenetic analysis confirmed that Gpr171 is evolutionally related to members of a P2Y gene-cluster that is localized on mouse chromosome 3. Interestingly, examination of its expression profile showed that, as opposed to other P2YRs, Gpr171 is downregulated in monocytes and granulocytes. Moreover, its enforced over-expression resulted in decreased expression of myeloid markers and increased the generation of colonies in vitro. Conversely, Gpr171 silencing diminished the expression of myeloid markers and the clonogenic potential of cells. In addition, mice transplanted with HPCs over-expressing Gpr171 displayed a significant reduction in the percentage of myeloid cells. Together, these elegant experiments demonstrated that Gpr171 plays a role in myeloid-monocytic lineage development and suggested that Gpr171 may have followed a separate evolutionary pathway as compared to other P2YRs belonging to the same gene-cluster. This work provides the framework for the identification of Gpr171 agonists and antagonists, which will not only help achieve a deeper understanding of the role of this orphan receptor, but also of the molecular mechanisms underlying the multi-step process that leads from committed progenitors to terminally differentiated cells.

Sca-1 as a possible early-response marker to identify hematopoietic stem/progenitor cells with increased self-renewal capacity in response to HDIs in vitro
Walasek et al.

Histone deacetylase inhibitors (HDIs) are epigenetic modifiers that control gene expression by altering the acetylation status of chromatin and other non-histone proteins. These small molecules can enhance the ex vivo self-renewal and propagation of hematopoietic stem/progenitor cells (HSPCs) as well as modify the process of differentiation. In a paper published in Blood in March 2012, this group reported the molecular and functional changes induced on HSPCs by a 7-day treatment with valproic acid (VPA), a widely used HDI. In the current study, Walasek et al. performed microarrays after short-term, 24h VPA stimulation, to identify early response VPA-targets in HSPCs. Their genome-wide gene expression studies identified the well-known and widely used HSPC marker Sca-1 as an early VPA-responsive gene. Early up-regulation of Sca-1 was also seen with two additional HDIs, Apicidin and MS-275, which differ from VPA in their chemical structure and their specificity. Strikingly, VPA not only strongly preserved Sca-1 expression and the LSK (Lin-Sca1+cKit+) phenotype of isolated HSPCs after 24h culture, but it also re-induced Sca-1 on committed common myeloid and granulocyte macrophage progenitor cells that had lost its expression in the process of differentiation, reverting those cells to a more immature LSK phenotype. Moreover, Sca-1 re-induction coincided with induced self-renewal capacity as measured by in vitro re-plating assays, while Sca-1 itself was dispensable for the functional effects of VPA. Together, the authors provided the first analysis of direct VPA targets in primitive hematopoietic cells and showed that Sca-1 is a faithful marker to identify cells with increased self-renewal capacity in response to HDIs in vitro. Studies like this one, aimed at understanding the spectrum of both biological and molecular effects of VPA, are crucial to determine the potential of this HDI to improve hematopoietic reconstitution and engraftment of ex vivo expanded HSPCs, a vital goal for transplantation, gene, and cellular therapies

In this Issue – February 2013

Improved therapeutic outcome of patients with Multiple Myeloma requires novel treatments and a better understanding of the mechanistic underlying drug resistance
See Salem et al. and Wolschke et al.

Multiple myeloma (MM) is a rare but devastating plasma cell malignancy that affects mainly older adults. In the last ten years, induction regimens that combine the glucocorticoid-analog dexametasone (Dex) with novel chemotherapeutic agents, such as the proteasome-inhibitor bortezomib (BTZ) or the antiangiogenetic and immunomodulator drugs thalidomide and its derivative lenalidomide, have significantly increased the complete response (CR) rate before and after Allogenic Stem Cell Transplantation (ASCT) with a positive impact on progression-free survival. Maintenance therapy with thalidomide and lenalidomide may further prolong remission duration. However, in spite of these advances, MM remains incurable, as the disease ultimately acquires resistance to therapies. Thus, novel treatment approaches and a better understanding of the mechanism(s) underlying resistance are essential to improve the therapeutic outcome of patients with relapsed MM.

The manuscript by Salem et al. provides a mechanistic rationale for combining Dex and BTZ, which is one of the most effective and widely used treatments for MM. Both drugs are inhibitors of the nuclear factor (NF)-κB pathway, which is chronically active in myeloma cells and can induce stromal cells to produce myeloma growth factors such as interleukin (IL)-6. Concomitantly, aberrant NF-kB activation has been associated with the emergence of resistance to anti-cancer drugs and radiation in MM, and elevated IL-6 serum levels correlate with poor prognosis. Dex and BTZ inhibit NF-κB by different pathways, and the authors found that the combination potentiated direct cell death of both the drug-sensitive, and, importantly, drug-resistant MM cells. Furthermore, combination therapy down-regulated the NF-κB targeted gene expression of IL-6 and manganese superoxide dismutase, which can induce chemo- and radio-resistance in MM, supporting potential combinations of these drugs with radiotherapy and additional chemotherapeutic drugs for the clinical benefit of MM patients.

The manuscript by Wolschke et al. reports a prospective phase I/II study to define the dose-limiting toxicity and the immunological effects of lenalidomide given to MM patients early post-ASCT. Lenalidomide is a more potent analog of thalidomide, developed to enhance immunomodulatory properties with improved safety. Its antitumor activity seems to be mediated through the modulation of cytokines and activation of T- and natural killer (NK) cell-mediated antitumor response. Increasing evidence of NK cell allo-reactivity in MM patients after ASCT provides a rationale for an early application of lenalidomide after transplantation. In this context, two recent clinical trials published by McCarthy et al. and Attal et al. in May 2012 in the New England Journal of Medicine showed that lenalidomide maintenance therapy significantly lengthens disease-free progression and improves overall survival among patients with MM. However, as described in the first study, this was associated with more toxicity and second cancers. Undesired side effects were also found in the HOVON 76 Trial published in Blood in September 2011, which excluded the feasibility of using lenalidomide as maintenance after ASCT due to Graft versus Host Disease (GvHD). Although encouraging, these clinical trials underscore the need to determine lenalidomides maximum tolerated dose as well as the mechanism underlying its unwanted side effects. In this study, the authors found that 5 mg of lenalidomide given daily for 21 days was the maximal tolerable dosage early after ASCT, with the major limiting factor being the occurrence of acute liver and gut toxicity due to GvHD, supporting the results of the HOVON trial. In agreement with the two New England Journal of Medicine papers, they also reported that lenalidomide treatment strongly increased complete remission rate, which they linked to its immunomodulatory effect.

The current five-year survival rate for patients with MM is 40% and thus, studies such as these that search for mechanistic insights of current therapies, drug resistance and side effects, offer great hope for MM patients who are in desperate need of treatments with a better response and survival rate. Such studies might also provide better target therapies and new immunomodulator regimes to supply physicians with a wider variety of treatment options to tailor the most appropriate and efficacious treatment to their patients' disease.

Azacitidine and decitabine are "coming of age” in treatment, but great challenges in understanding how they work remain
See Tsujioka et al.and Bernal et al.

Low dose regimes of the DNA demethylating agents Azacytidine (AZA) and its derivative decitabine (DAC) were approved in 2004 and 2006, respectively, by the US Food and Drug Administration for the treatment of myelodysplastic syndrome (MDS), a group of hematopoietic disorders characterized by ineffective hematopoiesis and increased risk of leukemic progression. In Europe, AZA is the only drug approved for the treatment of high-risk MDS not eligible for stem cell transplantation. These drugs are proposed to work by inducing functional re-expression of aberrantly hypermethylation-silenced tumor suppressor genes. However, whether epigenetic effects of the drugs account for all, or even some, of their clinical efficacy is still under debate. The mechanisms of resistance to AZA and DAC, which develops during treatment, are also still unclear, with some studies suggesting methylation-independent pathways. The importance of unravelling the mechanisms underlying AZA and DAC clinical efficacy and resistance is underscored by a recent clinical trial sponsored by Lung Cancer SPORE and Stand Up to Cancer. The results of the trial, published by Jurgens et al. in Cancer Discovery in December 2011, demonstrate the benefit of low doses of the drugs in patients with refractory lung cancer, and thus support the potential use of DAC and AZA for a wider management of cancer. Moreover, clinical trials in breast cancer have already begun in patients with advanced disease, and trials in colon cancer are planned. Searching for mechanistic insights, the paper by Tsujioka et al. show a positive relationship between growth suppression and demethylation by DAC or AZA in the MDS-derived cell line developed in their lab. Interestingly, DAC-induced cell death was preceded by DNA damage-induced G2 arrest via a p53-independent pathway, whereas AZA did not influence the pattern of the cell cycle. Their gene expression profiling suggest that at very low concentration, DAC significantly affect various gene biogroups, which include categories such as cellular movement, inflammatory response, hematological system development and function, hematopoiesis, and cell death. This work supports the finding of a recent important study by Tsai et al., published in Cancer Cell in March 2012, showing that transient low doses of AZA and DAC had antitumor effects on leukemic and epithelial tumor cells, which were accompanied by genome-wide changes in promoter DNA methylation and gene expression, which affected multiple key regulatory pathways.

Looking for mechanisms of drug resistance, the paper by Bernal et al. analyzes the effect of AZA on Matrix-Metalloproteinase (MMP)-9 expression. MMP-9 is a member of the MMP family of zinc-dependent proteolytic enzymes, which play a key role in angiogenesis and metastasis. MMP-9 regulates extramedullary disease in acute leukemia, and it is overexpressed in MDS cells, a phenomenon that in other cancers has been linked to its aberrant methylation. This article describes a dose-response increase of MMP-9 mRNA expression in two human cell lines established from patients with acute leukemia evolved from MDS and in six patients with MDS that relapsed after AZA treatment. This increment was associated with decreased DNA methylation levels in the MMP-9 promoter region and correlated with a higher invasiveness of the cells in vitro. Although the number of patient's samples analysed was small, the findings are interesting for their practical and speculative aspects, providing further evidence of the epigenetic regulation of MMP9, and suggesting that its aberrant methylation may play a role in the resistance to azacitidine in MDS patients. If these findings are confirmed in a larger cohort of patients, the role of MMP-9 in the therapeutic management of MDS or as a prognosis tool will warrant further investigation.

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Experimental Hematology offers fast publication time

Posted By Connections Editor, Wednesday, March 6, 2013
Updated: Wednesday, February 27, 2013

Experimental Hematology offers one of the fastest publication times in the field

*Articles published online 5 days after acceptance

*Initial review of manuscripts averages under 4 weeks

*Fast-Track Submission for research articles that have been previously considered by other top ranked journals and for which the previous reviewers' comments and the appropriate responses can be provided. Decisions are made within 7-10 days of submission.


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