Abstract 1919
Hematopoietic stem cell (HSC) transplantation is a life saving procedure whose applicability is restricted by the lack of suitable donors, by poor responsiveness to mobilization ...regimens in preparation of autologous transplantations, by insufficient HSC numbers in individual cord blood units, and by the inability to sufficiently amplify HSCs ex vivo. Characterization of Stemregenin (SR1), an aryl hydrocarbon receptor (AHR) antagonist that promotes HSC expansion, provided a proof of principle that low molecular weight (LMW) compounds have the ability to promote HSC expansion. To identify novel putative agonists of HSC self-renewal, we initiated a high throughput screen (HTS) of a library comprising more than 5,000 LMW molecules using the in vitro maintenance of the CD34+CD45RA- phenotype as a model system. Our study was based on the fact that mobilized peripheral blood-derived CD34+CD45RA- cells cultured in media supplemented with: stem cell factor, thrombopoietin, FLT3 ligand and interleukin 6, would promote the expansion of mononuclear cells (MNC) concomitant with a decrease in CD34+CD45RA- population and HSC depletion. LMW compounds preventing this loss could therefore act as agonists of HSC expansion. In a 384-well plate, 2000 CD34+cells were initially cultured/well in 50μl medium comprising 1μM test compounds or 0.1% DMSO (vehicle). The proportions of CD34+CD45RA− cells were determined at the initiation of experiment and after a 7-day incubation. Six of 5,280 LMW compounds (0.11%) promoted CD34+CD45RA− cell expansion, and seventeen (0.32%) enhanced differentiation as determined by the increase in proportions of CD34−CD45RA+ cells compared to control (DMSO). The 6 LMW compounds promoting expansion of the CD34+CD45RA− cell population were re-analyzed in a secondary screen. Four out of these 6 molecules suppressed the transcriptional activity of AHR, suggesting that these compounds share the same molecular pathway as SR1 in stimulating HSC expansion, thus they were not further characterized. The remaining 2 compounds promoted, similar to SR1 or better, a 10-fold and 35-fold expansion of MNC during 7 and 12-day incubations, respectively. The expanded cell populations comprised 65–75% of CD34+ cells compared to 12–30% determined for DMSO controls. During 12-day incubation with these compounds, the numbers of CD34+ cells increased ∼25-fold over their input values, or ∼ 6-fold above the values determined for controls. This expansion of CD34+ cells was associated with a ∼5-fold increase in the numbers of multilineage CFC (granulocyte, erythroid, monocyte, and megakaryocyte, or CFU-GEMM) compared to that found in DMSO control cultures. The ability of the 2 newly identified compounds to expand functional HSCs is currently being evaluated in vivo usingimmunocompromised mice. In conclusion, results of our initial screen suggest that other mechanism, besides inhibition of AhR, are at play for expansion of human HSC.
No relevant conflicts of interest to declare.
Abstract 2474
Aberrant expression of Hox genes and their cofactors Pbx and Meis1 has been detected in approximately 50% of all human leukemias, and proteins interacting with these homeodomain factors ...could play a major role in leukemia development. Studies in drosophila showed that hth/MEIS directly interacts with YKI, a component of the Hippo signaling pathway (Peng HW et al., 2009). The core components of this pathway in the mammalian cells are the kinases MST 1 or 2 and LATS 1 or 2, and the downstream transcription cofactors WWTR1 and YAP (homologues of the drosophila Yki). The Hippo pathway has been proposed to play a tumor suppressive role in carcinoma development (Lu L et al. 2010), but little is known about its function in hematopoiesis and leukemia. To address this issue, we first determined the expression levels of the core Hippo pathway constituents in different subpopulations of primitive hematopoietic cells by quantitative RT-PCR. Hematopoietic stem cells (HSC) isolated from day 14.5 fetal liver (FL-HSC, phenotype: CD150+CD48-Lin-), or bone marrow from 3 and 4 week old mice (BM-HSC, phenotype: cKit+CD150+CD48-Lin-) express comparable levels of Lats 1/2 and Mst 1/2. FL-HSC, however, express approximately 3 fold higher levels of Wwtr1 and Yap than the BM-HSC. Expression of all core components of the Hippo pathway was also detected in the Hoxa9+Meis1-induced leukemia named FLA2 in which approximately 70% of cells represent leukemia stem cells (LSC). The role of this pathway in leukemia was assessed using the shRNA-mediated loss of function approach. For each core component, 5 different shRNAs were designed, and 2 achieving ≥40% decrease in the targeted transcript levels were selected for the in vivo experiments. Freshly isolated FLA2 leukemia cells were infected with recombinant retroviruses carrying the control shLuciferase or the targeting shRNA, and green fluorescent protein (GFP), and were transplanted into sub-lethally irradiated recipient mice. The proportions of shRNA transduced (GFP+) cells were determined at the time of transplantation (day 0), and at the time of sacrifice (day 18 ± 2). During this period, the proportions of shWwtr1(GFP+) cells to the leukemic cell populations decreased to 10–20% of the initial day 0 values. Conversely, the Lats1 knockdown leads to > 50% increase over the initial proportion of the GFP+ cells. The combined Lats1+Lats2 knockdown enhanced the competitiveness of the transduced cells compared shLuciferase controls. These significant results (p < 0.05, Mann-Whitney-Test) suggest that LATS kinases act as negative regulators of leukemic cell expansion. To exclude the possibility that this effect is limited to FLA2 leukemia we isolated the CD150+CD48-Lin- stem/progenitor cells from FL, co-infected them first with Hoxa9 and Meis1 cDNA carrying retroviruses, and then knocked down Wwtr1 or Lats1. Similar to observations in FLA2 leukemia model, Lats 1 depletion promoted ∼2-fold increase, and Wwtr 1 reduction >80% decrease in proportions of the transduced (GFP+) cells compared to their initial day 0 levels. Together, our observations suggest that LATS kinases act as negative modulators of Hox/Meis-induced leukemia and indicate a possibility for a specific targeting of the Hox/Meis-activated cellular pathways.
No relevant conflicts of interest to declare.
Cholesterol homeostasis has been proposed as one mechanism contributing to chemoresistance in AML and hence, inclusion of statins in therapeutic regimens as part of clinical trials in AML has shown ...encouraging results. Chemical screening of primary human AML specimens by our group led to the identification of lipophilic statins as potent inhibitors of AMLs from a wide range of cytogenetic groups. Genetic screening to identify modulators of the statin response uncovered the role of protein geranylgeranylation and of RAB proteins, coordinating various aspect of vesicular trafficking, in mediating the effects of statins on AML cell viability. We further show that statins can inhibit vesicle-mediated transport in primary human specimens, and that statins sensitive samples show expression signatures reminiscent of enhanced vesicular trafficking. Overall, this study sheds light into the mechanism of action of statins in AML and identifies a novel vulnerability for cytogenetically diverse AML.
•Inhibition of RAB protein function mediates the anti–acute myeloid leukemia activity of statins.•Statin sensitivity is associated with enhanced vesicle-mediated traffic.
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Overexpression of Hoxb4 in bone marrow cells promotes expansion of hematopoietic stem cell (HSC) populations in vivo and in vitro, indicating that this homeoprotein can activate the genetic program ...that determines self-renewal. However, this function cannot be solely attributed to Hoxb4 because Hoxb4-/- mice are viable and have an apparently normal HSC number. Quantitative polymerase chain reaction analysis showed that Hoxb4-/- c-Kit+ fetal liver cells expressed moderately higher levels of several Hoxb cluster genes than control cells, raising the possibility that normal HSC activity in Hoxb4-/- mice is due to a compensatory up-regulation of other Hoxb genes. In this study, we investigated the competitive repopulation potential of HSCs lacking Hoxb4 alone, or in conjunction with 8 other Hoxb genes. Our results show that Hoxb4-/- and Hoxb1-b9-/- fetal liver cells retain full competitive repopulation potential and the ability to regenerate all myeloid and lymphoid lineages. Quantitative Hox gene expression profiling in purified c-Kit+Hoxb1-b9-/- fetal liver cells revealed an interaction between the Hoxa, b, and c clusters with variation in expression levels of Hoxa4,-a11, and -c4. Together, these studies show a complex network of genetic interactions between several Hox genes in primitive hematopoietic cells and demonstrate that HSCs lacking up to 30% of the active Hox genes remain fully competent. (Blood. 2006;108:116-122)
Photodynamic therapy (PDT) treatment of murine EMT6 mammary sarcoma using Photofrin (10 mg/kg) and light (110 J/cm2) cured all these lesions growing in syngeneic BALB/c mice. In contrast, the same ...treatment produced initial ablation but no long-term cures of EMT6 tumors growing in either scid or nude mice, the immunodeficient strains sharing the same genetic background with BALB/c mice. No difference was detected in either the level of Photofrin accumulated per g of tumor tissue or the extent of tumor cell killing during the first 24 h after PDT of EMT6 tumors growing in BALB/c or scid mice. The assumption that the difference in tumor cures could be ascribed to the absence of functional lymphoid cells in scid and nude mice was supported by the results of experiments involving the adoptive T-cell transfer into scid mice or bone marrow transfer between BALB/c and scid mice. The adoptive transfer of splenic virgin T lymphocytes from BALB/c mice into scid mice performed 9 days before PDT of EMT6 tumors growing in the recipients was successful in delaying the recurrence of treated tumors. Adoptive transfer done immediately after PDT or 7 days after PDT had no obvious benefit. Even better improvement and a high cure rate of PDT-treated tumors was obtained with scid mice reconstituted with BALB/c bone marrow. In contrast, a marked drop in tumor cure rate was observed with BALB/c mice reconstituted with scid bone marrow. These results suggest that the activity of host lymphoid populations was essential for preventing the recurrence of EMT6 tumors following the PDT treatment used in this study. The contribution of PDT-induced immune reaction may, therefore, be of critical importance for the cure with at least some tumors.
Molecular basis for stem-cell self-renewal Krosl, Jana K; Faubert, Amelie; Sauvageau, Guy
The hematology journal : the official journal of the European Haematology Association,
2004, Letnik:
5 Suppl 3
Journal Article
We previously showed that HOXB4 is a potent stimulator of hematopoietic stem cell (HSC) proliferation in vivo and ex vivo. As a result, HOXB4 overexpressing HSCs are 20- to 50-times more competitive ...than untransduced cells when transplanted into mice. By knocking down the expression of PBX1 (PBX1
K.D.) in HOXB4 overexpressing cells, we now present the possibility of generating HSCs that are >20-times more competitive than those that overexpress HOXB4. The differentiation activity of these cells appears intact, since they competitively contributed to the reconstitution of normal myeloid and lymphoid compartments in vivo. We also show that the in vivo expansion of HOXB4-PBX1
K.D.-expressing HSCs regenerated normal stem cell pools and did not lead to HSC levels above those detected in unmanipulated mice. The vigorous competitive nature of these cells in vivo compared to HOXB4-transduced HSCs suggests the existence of a distinct, non-cell autonomous mechanism that limits the expansion of HOXB4-transduced hemopoietic stem cells in mice.
The Hoxa9 and Meis1 genes represent important oncogenic collaborators activated in a significant proportion of human leukemias with genetic alterations in the MLL gene. In this study, we show that ...the transforming property of Meis1 is modulated by 3 conserved domains, namely the Pbx interaction motif (PIM), the homeodomain, and the C-terminal region recently described to possess transactivating properties. Meis1 and Pbx1 interaction domain-swapping mutants are dysfunctional separately, but restore the full oncogenic activity of Meis1 when cotransduced in primary cells engineered to overexpress Hoxa9, thus implying a modular nature for PIM in Meis1-accelerated transformation. Moreover, we show that the transactivating domain of VP16 can restore, and even enhance, the oncogenic potential of the Meis1 mutant lacking the C-terminal 49 amino acids. In contrast to Meis1, the fusion VP16-Meis1 is spontaneously oncogenic, and all leukemias harbor genetic activation of endogenous Hoxa9 and/or Hoxa7, suggesting that Hoxa gene activation represents a key event required for the oncogenic activity of VP16-Meis1.
While previous studies with truncated erythropoietin receptors (EpRs) have suggested that the tyrosine phosphorylation of the EpR does not play a role in Ep‐induced proliferation, we have found, ...using a more subtle, full length EpR mutant, designated Null, in which all eight of the intracellular tyrosines have been substituted with phenylalanine residues, that Null cells require substantially more Ep than wild‐type cells in order to proliferate as efficiently. A comparison of Ep‐induced proliferation with Ep‐induced tyrosine phosphorylation patterns, using wild‐type and Null EpR‐expressing cells, revealed that Stat5 tyrosine phosphorylation and activation correlated directly with proliferation. Moreover, studies with a Y343F EpR point mutant and various EpR deletion mutants revealed that both Ep‐induced proliferation and Stat5 activation were mediated primarily through Y343, but that other tyrosines within the EpR could activate Stat5 in its absence.
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