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Inside Experimental Hematology: May and June 2014

Posted By Connections Editor, Monday, June 30, 2014

Inside this issue May

Immunogenicity of ESC-derived hematopoietic progenitors

See Thompson et al., pages 347359.

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

Parsing out the Integrins' work on erythropoiesis

See Ulyanova et al., pages 404409.

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

Is bio-manufacture of human platelets for transfusion possible?

See Haylock et al., pages 332–346.       

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

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

See Uchida et al., pages 369379.

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


Inside this issue June

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

See Pioli and Weis, pages 425430.

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

Global analysis of transcription factors and cofactors during terminal erythropoiesis

See Chen and Lodish, pages 464476.

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

Statins potentiate the antileukemic effects of imatinib in chronic myeloid leukemia

See Glodkowska-Mrowka et al., pages 439447.

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

Single cell analysis shows mutational heterogeneity in leukemic cells

See Shouval et al., pages 457463.

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

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