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Inside Experimental Hematology: November/December 2014

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


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

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

Mixed chimerism in C3a-deficient mice

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

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

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

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

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



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

See Heath et al., pages 1022-1030.

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


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

See Li et al., pages 995-1012.

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

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

See Zhou et al., pages 1041-1052.

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


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