Angptl-4 Is Upregulated Under Inflammatory Conditions In The BM Of Mice, Expands Myeloid Progenitors and Accelerates Reconstitution Of Platelets After Myelosuppressive Therapy

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3677-3677
Author(s):  
Anne Schumacher ◽  
Till Braunschweig ◽  
Bernd Denecke ◽  
Tim H. Brümmendorf ◽  
Patrick Ziegler
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Myeloid Cell ◽  
Relative Increase ◽  
T Test ◽  
Gm Csf ◽  
Emergency Situations ◽  
Inflammatory Conditions

Abstract The concerted action of hematopoiesis supporting cytokines such as G-CSF, GM-CSF or IL-6 regulates hematopoiesis during steady state and emergency situations. Respective knockout mice show defects both in production and function of myelopoietic effector cells. However, alternative pathways are likely to exist as mice with single or combined deficiencies for G-CSF, GM-CSF, and IL-6 or G-CSF and GM-CSF are still able to mount reactive neutrophilia responses during inflammatory conditions. In order to identify pathways for inflammation induced enhancement of hematopoiesis as well as to find new cytokines, which enhance myeloid cell regeneration, we analyzed the bone marrow (BM) of lipopolysaccharide (LPS) and vehicle injected wild type (WT) mice (single IP- injection) by gene expression microarray. Focusing on the identification of genes encoding for secreted or membrane proteins, we found 83 candidates to be up- and 14 to be downregulated after LPS treatment. Among known candiates, we found angiopoietin-like 4 (Angptl-4) as a predominantly upregulated gene in the BM of LPS-treated WT-mice. Upregulation was confirmed by RT-PCR as well as by Elisa in the BM of LPS treated mice and bone marrow stromal cells (BMSC) were identified as candidate producer cells. Functionally, we found recombinant Angptl-4 to stimulate the proliferation of myeloid colony-forming units (CFU) in vitro. In mice, repeated injections of Angptl-4 increased BM progenitor cell frequency and this was paralleled by a relative increase in phenotypically defined granulocyte-macrophage progenitors (GMPs). Furthermore, in vivo treatment with Angptl-4 resulted in elevated platelet counts both in untreated animals and after myelosuppressive therapy. After lethal irradiation and transplantation of syngeneic BM cells repetitive injections of recombinant Angptl-4 for 5 consecutive days resulted in an accelerated reconstitution of platelets starting at day 8 after transplantation. The 50% pre-treatment platelet count was reached on day 14 in Angptl-4-treated animals as compared to day 21 for transplanted controls receiving no Angptl-4 (n=8; p=0.03, student´s T test). In contrast, transplantation of BM cells from Angptl-4 pre-treated donor mice had no effect on the recovery of platelets in this setting. The frequency of CD41lowCD61+ immature megakaryocytes was significantly increased in the BM of Angptl-4 injected as compared to control mice (27% vs 19% of total megakaryocytes; p= 0.008, student´s T test). Furthermore, bone marrow cytology revealed local accumulation of megakaryocytes carrying dysplastic features in Angptl-4 injected mice. In summary, our data suggest that Angptl-4 plays a complementary role on hematopoiesis during emergency situations like sepsis. The use of Angptl-4 in the setting of autologous stem cell transplantation could represent a potential approach to accelerate the reconstitution of megakaryopoiesis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Transcription factor Gfi-1 induced by G-CSF is a negative regulator of CXCR4 in myeloid cells

Blood ◽  
2007 ◽  
Vol 110 (7) ◽  
pp. 2276-2285 ◽  
Author(s):  
Maria De La Luz Sierra ◽  
Paola Gasperini ◽  
Peter J. McCormick ◽  
Jinfang Zhu ◽  
Giovanna Tosato
Keyword(s):  
Bone Marrow ◽  
Dna Sequences ◽  
Peripheral Blood ◽  
Cell Function ◽  
Myeloid Cell ◽  
Cxcr4 Expression ◽  
Negative Regulator ◽  
Hematopoietic Stem

The mechanisms underlying granulocyte-colony stimulating factor (G-CSF)–induced mobilization of granulocytic lineage cells from the bone marrow to the peripheral blood remain elusive. We provide evidence that the transcriptional repressor growth factor independence-1 (Gfi-1) is involved in G-CSF–induced mobilization of granulocytic lineage cells from the bone marrow to the peripheral blood. We show that in vitro and in vivo G-CSF promotes expression of Gfi-1 and down-regulates expression of CXCR4, a chemokine receptor essential for the retention of hematopoietic stem cells and granulocytic cells in the bone marrow. Gfi-1 binds to DNA sequences upstream of the CXCR4 gene and represses CXCR4 expression in myeloid lineage cells. As a consequence, myeloid cell responses to the CXCR4 unique ligand SDF-1 are reduced. Thus, Gfi-1 not only regulates hematopoietic stem cell function and myeloid cell development but also probably promotes the release of granulocytic lineage cells from the bone marrow to the peripheral blood by reducing CXCR4 expression and function.

scholarly journals The immunosuppressant rapamycin blocks in vitro responses to hematopoietic cytokines and inhibits recovering but not steady-state hematopoiesis in vivo

Blood ◽  
1994 ◽  
Vol 84 (5) ◽  
pp. 1543-1552 ◽  
Author(s):  
VF Quesniaux ◽  
S Wehrli ◽  
C Steiner ◽  
J Joergensen ◽  
HJ Schuurman ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Steady State ◽  
T Cell ◽  
T Cell Response ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Hematopoietic Recovery ◽  
Stimulating Factor

Abstract The immunosuppressive drug rapamycin suppresses T-cell activation by impairing the T-cell response to lymphokines such as interleukin-2 (IL- 2) and interleukin-4 (IL-4). In addition, rapamycin blocks the proliferative response of cell lines to a variety of hematopoietic growth factors, including interleukin-3 (IL-3), interleukin-6 (IL-6), granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage- colony stimulating factor (GM-CSF), and kit ligand (KL), suggesting that it should be a strong inhibitor of hematopoiesis. In this report, we studied the effects of rapamycin on different hematopoietic cell populations in vitro and in vivo. In vitro, rapamycin inhibited the proliferation of primary bone marrow cells induced by IL-3, GM-CSF, KL, or a complex mixture of factors present in cell-conditioned media. Rapamycin also inhibited the multiplication of colony-forming cells in suspension cultures containing IL-3 plus interleukin-1 (IL-1) or interleukin-11 (IL-11) plus KL. In vivo, treatment for 10 to 28 days with high doses of rapamycin (50 mg/kg/d, orally) had no effect on myelopoiesis in normal mice, as measured by bone marrow cellularity, proliferative capacity, and number of colony-forming progenitors. In contrast, the same treatment strongly suppressed the hematopoietic recovery normally seen 10 days after an injection of 5-fluorouracil (5- FU; 150 mg/kg, intravenously [i.v.]). Thus, rapamycin may be detrimental in myelocompromised individuals. In addition, the results suggest that the rapamycin-sensitive cytokine-driven pathways are essential for hematopoietic recovery after myelodepression, but not for steady-state hematopoiesis.

scholarly journals Thrombopoietin, but not erythropoietin promotes viability and inhibits apoptosis of multipotent murine hematopoietic progenitor cells in vitro

Blood ◽  
1996 ◽  
Vol 88 (8) ◽  
pp. 2859-2870 ◽  
Author(s):  
OJ Borge ◽  
V Ramsfjell ◽  
OP Veiby ◽  
MJ Jr Murphy ◽  
S Lok ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Interleukin 1 ◽  
Calf Serum ◽  
Myeloid Cell ◽  
Promoting Effect ◽  
Multipotent Progenitors

The recently cloned c-mpl ligand, thrombopoietin (Tpo), has been extensively characterized with regard to its ability to stimulate the growth, development, and ploidy of megakaryocyte progenitor cells and platelet production in vitro and in vivo. Primitive hematopoietic progenitors have been shown to express c-mpl, the receptor for Tpo. In the present study, we show that Tpo efficiently promotes the viability of a subpopulation of Lin-Sca-1+ bone marrow progenitor cells. The ability of Tpo to maintain viable Lin-Sca-1+ progenitors was comparable to that of granulocyte colony-stimulating factor and interleukin-1, whereas stem cell factor (SCF) promoted the viability of a higher number of Lin-Sca-1+ progenitor cells when incubated for 40 hours. However, after prolonged (> 40 hours) preincubation, the viability-promoting effect of Tpo was similar to that of SCF. An increased number of progenitors surviving in response to Tpo had megakaryocyte potential (37%), although almost all of the progenitors produced other myeloid cell lineages as well, suggesting that Tpo acts to promote the viability of multipotent progenitors. The ability of Tpo to promote viability of Lin-Sca-1+ progenitor cells was observed when cells were plated at a concentration of 1 cell per well in fetal calf serum-supplemented and serum-depleted medium. Finally, the DNA strand breakage elongation assay showed that Tpo inhibits apoptosis of Lin-Sca-1+ bone marrow cells. Thus, Tpo has a potent ability to promote the viability and suppress apoptosis of primitive multipotent progenitor cells.

Leishmania donovani infection of bone marrow stromal macrophages selectively enhances myelopoiesis, by a mechanism involving GM-CSF and TNF-α

Blood ◽  
2000 ◽  
Vol 95 (5) ◽  
pp. 1642-1651 ◽  
Author(s):  
Sara E. J. Cotterell ◽  
Christian R. Engwerda ◽  
Paul M. Kaye
Keyword(s):  
Infectious Disease ◽  
Bone Marrow ◽  
Stromal Cell ◽  
Leishmania Donovani ◽  
Protozoan Parasite ◽  
Gm Csf ◽  
Tnf Α ◽  
Macrophage Colony

Alterations in hematopoiesis are common in experimental infectious disease. However, few studies have addressed the mechanisms underlying changes in hematopoietic function or assessed the direct impact of infectious agents on the cells that regulate these processes. In experimental visceral leishmaniasis, caused by infection with the protozoan parasite Leishmania donovani, parasites persist in the spleen and bone marrow, and their expansion in these sites is associated with increases in local hematopoietic activity. The results of this study show that L donovani targets bone marrow stromal macrophages in vivo and can infect and multiply in stromal cell lines of macrophage, but not other lineages in vitro. Infection of stromal macrophages increases their capacity to support myelopoiesis in vitro, an effect mediated mainly through the induction of granulocyte macrophage-colony stimulating factor and tumor necrosis factor-. These data are the first to directly demonstrate that intracellular parasitism of a stromal cell population may modify its capacity to regulate hematopoiesis during infectious disease.

scholarly journals Involvement of p21cip-1 and p27kip-1 in the molecular mechanisms of steel factor-induced proliferative synergy in vitro and of p21cip-1 in the maintenance of stem/progenitor cells in vivo

Blood ◽  
1996 ◽  
Vol 88 (10) ◽  
pp. 3710-3719 ◽  
Author(s):  
C Mantel ◽  
Z Luo ◽  
J Canfield ◽  
S Braun ◽  
C Deng ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Proliferative Response ◽  
Myeloid Cell ◽  
Cyclin Dependent Kinase ◽  
Hematopoietic Progenitor ◽  
Gm Csf ◽  
Myeloid Progenitor ◽  
Myeloid Progenitor Cells

Steel factor (SLF) is a hematopoietic cytokine that synergizes with other growth factors to induce a greatly enhanced proliferative state of hematopoietic progenitor cells and factor-dependent cell lines. Even though the in vivo importance of SLF in the maintenance and responsiveness of stem and progenitor cells is well documented, the molecular mechanism involved in its synergistic effects are mainly unknown. Some factor-dependent myeloid cell lines respond to the synergistic proliferative effects of SLF plus other cytokines in a manner similar to that of normal myeloid progenitor cells from bone marrow and cord blood. We show here that SLF can synergize with granulocyte-macrophage colony-stimulating factor (GM-CSF) to induce an enhanced phosphorylation of the retinoblastoma gene product and a synergistic increase in the total intracellular protein level of the cyclin-dependent kinase inhibitor, p21cip-1, which is correlated with a simultaneous decrease in p27kip-1 in the human factor-dependent myeloid cell line, M07e. Moreover, these cytokines synergize to increase p21cip- 1 binding and decrease p27kip-1 binding to cyclin-dependent kinase-2 (cdk2), an enzyme required for normal cell cycle progression; these inverse events correlated with increased cdk2 kinase activity. It is also shown that exogenous purified p21cip-1 can displace p27kip-1 already bound to cdk2 in vitro. These data implicate increased p21cip-1 and decreased p27kip-1 intracellular concentrations and their stoichiometric interplay in the enhanced proliferative status of cells stimulated by the combination of SLF and GM-CSF. In support of these findings, it is shown that hematopoietic progenitor cells from mice lacking p21cip-1 are defective in SLF synergistic proliferative response in vitro. Moreover, the cycling status of marrow and spleen progenitors and absolute numbers of marrow progenitors were significantly decreased in the p21cip-1 -/-, compared with the +/+ mice. We conclude that the cdk threshold regulators p21cip-1 and p27kip- 1 play a critical role in the normal mitogenic response of M07e cells and murine myeloid progenitor cells to these cytokines and particularly in the SLF synergistic proliferative response that is important to the normal maintenance of the stem/progenitor cell compartment.

Modeling the human 8p11-myeloproliferative syndrome in immunodeficient mice

Blood ◽  
2010 ◽  
Vol 116 (12) ◽  
pp. 2103-2111 ◽  
Author(s):  
Helena Ågerstam ◽  
Marcus Järås ◽  
Anna Andersson ◽  
Petra Johnels ◽  
Nils Hansen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Leukemia ◽  
Tyrosine Kinases ◽  
Cellular Proliferation ◽  
Myeloid Cell ◽  
Myeloproliferative Disorder ◽  
Immunodeficient Mice ◽  
Myeloproliferative Syndrome

Abstract The 8p11 myeloproliferative syndrome (EMS), also referred to as stem cell leukemia/lymphoma, is a chronic myeloproliferative disorder that rapidly progresses into acute leukemia. Molecularly, EMS is characterized by fusion of various partner genes to the FGFR1 gene, resulting in constitutive activation of the tyrosine kinases in FGFR1. To date, no previous study has addressed the functional consequences of ectopic FGFR1 expression in the potentially most relevant cellular context, that of normal primary human hematopoietic cells. Herein, we report that expression of ZMYM2/FGFR1 (previously known as ZNF198/FGFR1) or BCR/FGFR1 in normal human CD34+ cells from umbilical-cord blood leads to increased cellular proliferation and differentiation toward the erythroid lineage in vitro. In immunodeficient mice, expression of ZMYM2/FGFR1 or BCR/FGFR1 in human cells induces several features of human EMS, including expansion of several myeloid cell lineages and accumulation of blasts in bone marrow. Moreover, bone marrow fibrosis together with increased extramedullary hematopoiesis is observed. This study suggests that FGFR1 fusion oncogenes, by themselves, are capable of initiating an EMS-like disorder, and provides the first humanized model of a myeloproliferative disorder transforming into acute leukemia in mice. The established in vivo EMS model should provide a valuable tool for future studies of this disorder.

GABP Transcription Factor Is Required for Myeloid Cell Development In Vivo.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 374-374 ◽  
Author(s):  
Zhong-fa Yang ◽  
Karen Drumea ◽  
Alan G. Rosmarin
Keyword(s):  
Bone Marrow ◽  
Transcription Factor ◽  
T Cell ◽  
Myeloid Cells ◽  
Myeloid Cell ◽  
Cell Development ◽  
Wild Type ◽  
Ets Domain

Abstract GABP is an ets transcription factor that regulates genes that are required for innate immunity, including CD18 (β2 leukocyte integrin), lysozyme, and neutrophil elastase. GABP consists of two distinct and unrelated proteins. GABPα binds to DNA through its ets domain and recruits GABPβ, which contains the transactivation domain; together, they form a functional tetrameric transcription factor complex. We recently showed that GABP is required for entry into S phase of the cell cycle through its regulation of genes that are required for DNA synthesis and cyclin dependent kinase inhibitors (Yang, et al. Nature Cell Biol9:339, 2007). Furthermore, GABP is an essential component of a retinoic acid responsive myeloid enhanceosome (Resendes and Rosmarin Mol Cell Biol26:3060, 2006). We cloned Gabpa (the gene that encodes mouse Gabpα) from a mouse genomic BAC library and prepared a targeting vector in which the ets domain is flanked by loxP recombination sites (floxed allele). Deletion of both floxed Gabpa alleles causes an early embryonic lethal defect. In order to define the role of Gabpα in myelopoiesis, we bred floxed Gabpa mice to mice that bear the Mx1-Cre transgene, which drives expression of Cre recombinase in response to injection of the synthetic polynucleotide, poly I-C. Deletion of Gabpa dramatically reduced granulocytes and monocytes in the peripheral blood, spleen, and bone marrow, but myeloid cells recovered within weeks. In vitro colony forming assays indicated that myeloid cells in these mice were derived only from Gabpa replete myeloid precursors (that failed to delete both Gabpa alleles), suggesting strong pressure to retain Gabpα in vivo. We used a novel competitive bone marrow transplantation approach to determine if Gabp is required for myeloid cell development in vivo. Sub-lethally irradiated wild-type recipient mice bearing leukocyte marker CD45.1 received equal proportions of bone marrow from wild type CD45.1 donor mice and floxed-Mx1-Cre donor mice that bear CD45.2. Both the CD45.2 (floxed-Mx1-Cre) and CD45.1 (wild type) bone marrow engrafted well. Mice were then injected with pI-pC to induce Cre-mediated deletion of floxed Gabpa. The mature myeloid and T cell compartments were derived almost entirely from wild type CD45.1 cells. This indicates that the proliferation and/or differentiation of myeloid and T cell lineages requires Gabp. In contrast, B cell development was not impaired. We conclude that Gabpa disruption causes a striking loss of myeloid cells in vivo and corroborates prior in vitro data that GABP plays a crucial role in proliferation of myeloid progenitor cells.

Thrombopoietin Stimulation Leads to Enhanced Polyploidization in p53-/- Bone Marrow Megakaryocytes: In Vivo and in Vitro Studies.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2531-2531
Author(s):  
Pani A. Apostolidis ◽  
Stephan Lindsey ◽  
William M. Miller ◽  
Eleftherios T. Papoutsakis
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Platelet Surface ◽  
Platelet Counts ◽  
Reticulated Platelets ◽  
High Ploidy ◽  
And Control ◽  
Platelet Formation

Abstract Abstract 2531 Poster Board II-508 BACKGROUND AND HYPOTHESIS. We have previously shown that tumor suppressor p53 is activated in differentiating megakaryocytic (Mk) cells and its knock-down (KD) leads to increased polyploidization and delayed apoptosis in CHRF, a human Mk cell line. Furthermore, bone marrow (BM)-derived Mks from p53−/− mice reach higher ploidy classes in culture. Accordingly, we hypothesized that the role of p53 during megakaryopoiesis is to delimit polyploidization and control the transition from endomitosis by inhibiting DNA synthesis and promoting apoptosis. Here, we test this hypothesis by examining the differential effect of mouse thrombopoietin (rmTpo) on the ploidy of p53−/− and p53+/+ mouse Mk cells. METHODS. 8–10 week-old, male p53−/− mice and p53+/+ littermates were injected once with 1.2 μg rmTpo or saline. On days 2 and 5 after Tpo/saline treatment, tail-bleeding assays were performed to measure bleeding times/volumes, mice were bled for platelet counts and sacrificed to harvest BM. We employed flow cytometry to examine baseline ploidy in BM-resident Mks in p53−/− and p53+/+ mice as well as Mk cells generated from BM progenitors after 4 and 6 days of culture with rmTpo. RESULTS. At steady state, ploidy in BM-resident CD41+ Mk cells was similar in p53−/− and p53+/+ mice: 11.8±2.3% and 10.7±1.3% of p53−/− and p53+/+ Mks, respectively, reaching a ploidy of ≥32N (n=3-4). Platelet counts were 1.3×106±1×105/μl (12.5±1.0% reticulated) and 1.1×106±5×104/μl (12.4±1.3% reticulated) in p53−/− and p53+/+ mice, respectively (n=8). Two days following Tpo treatment of the mice, we did not observe significantly increased platelet levels, while ploidy was marginally affected. However, 5 days following Tpo treatment, we found greater ploidy in the BM in the absence of p53: 22±1.6% 16N and 10.1±0.8% ≥32N Mks in the p53−/− versus 18.6±3.3% 16N and 7.1±1.4% ≥32N Mks in the p53+/+ (n=2). This was accompanied by increased platelet formation: 23.6±8.3% reticulated platelets in the p53−/− versus 17.8±2.6% in the p53+/+ (n=2). Culture of BM cells from non-Tpo treated mice with 50ng/ml rmTpo resulted in a 50% increase in total Mks and increased polyploidy by day 6 of culture: 38.6±4.6% of p53−/− versus 19.2±2.3% of p53+/+ Mks reached ploidy classes of ≥32N (n=3-4, p < 0.01). Lack of p53 led to hyperploid Mk cells; by day 6 of culture 10.3±2.2% of p53−/− Mks were in ploidy classes of 128N and higher, while only 0.6±0.1% p53+/+ Mks achieved such high ploidy (n=3-4). In addition, a 6 day culture with Tpo of BM cells derived from p53−/− and p53+/+ mice pre-treated with Tpo 5 days prior to sacrifice led to more profound polyploidization compared to Mks generated from the non-Tpo treated mice but only in the p53−/− Mks: 48.8±1.1% of p53−/− versus only 17.6±0.2% of p53+/+ Mks reached ploidy ≥32N (n=2). Microarray analysis comparing p53KD to control CHRF cells undergoing Mk differentiation revealed down-regulation of genes coding for platelet surface complex CD41/CD61 and CD62P in the p53KD cells. To examine the possibility of altered functionality of platelets in p53−/− mice, we performed tail-bleeding assays on the mice that did not receive Tpo. Bleeding times and volumes were generally prolonged in the absence of p53 (all p53−/− mice exceeded the 10 min duration of the assay; mean p53−/− and p53+/+ blood loss was 17μl and 10μl, respectively, n=3-4). CONCLUSIONS. Our data indicate that in vivo polyploidization and platelet formation from Mks is increased in the p53−/− relative to p53+/+ mice after Tpo administration. These data are in line with our hypothesis that p53 activation decreases the ability of Mks to respond to Tpo and undergo polyploidization. Additionally, our preliminary data on platelet functionality suggest that p53 may have a role in hemostasis. Disclosures: No relevant conflicts of interest to declare.

Treatment with a MEK Inhibitor Improves Myeloproliferation, Anemia and Survival in a Mouse Model of CMML and JMML.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 966-966
Author(s):  
Natalya Lyubynska ◽  
Jennifer Lauchle ◽  
Kevin Shannon ◽  
Benjamin S. Braun
Keyword(s):  
Bone Marrow ◽  
Mouse Model ◽  
Mek Inhibitor ◽  
Colony Growth ◽  
Dramatic Improvement ◽  
Low Concentration ◽  
Gm Csf ◽  
Myeloid Progenitors

Abstract Abstract 966 Mutations that deregulate cellular signaling are a hallmark of myeloproliferative neoplasms (MPNs), and pharmacologic inhibitors of MPN-associated proteins have redefined therapy for some MPNs. However, this strategy cannot yet be applied to juvenile- and chronic myelomonocytic leukemias (JMML and CMML). These diseases are characterized by aberrant N-Ras, K-Ras, Cbl, and SHP-2 proteins that are not easily targeted by drugs. An attractive alternative approach is to inhibit downstream effector pathways, which include the Raf/MEK/ERK, phosphoinositide-3-OH kinase (PI3K)/Akt, and Ral-GDS/Ral-A cascades. However, it is not known which of these pathways are crucial for the aberrant growth and survival of JMML and CMML cells and might therefore provide the best targets for therapy. To address these questions, we developed an accurate mouse model of JMML and CMML by expressing a conditional “knock-in” KrasLSL-G12D oncogene in bone marrow. We administered PD0325901, a potent and selective MEK inhibitor, to Mx1-Cre, KrasG12D mutant mice to test the hypothesis that the Raf→MEK→ERK cascade is necessary for MPN initiated by KrasG12D expression. Oral administration of PD0325901 5 mg/kg caused deep and durable MEK inhibition in primary bone marrow progenitors. Mx1-Cre, KrasG12D mice with established MPN and wild-type (WT) littermates were randomly assigned to receive PD0325901 5 mg/kg/day or a control vehicle. Treated Mx1-Cre, KrasG12D mice demonstrated rapid correction of leukocytosis and anemia, and reduction in splenomegaly. Treatment was also associated with dramatic improvement in the survival of Mx1-Cre, KrasG12D mice (8.1 vs. 2.0 weeks after entry, p=0.003). Two of three Mx1-Cre, KrasG12D mice that were treated for 12 weeks ultimately died with KrasG12D T-lineage leukemia/lymphoma, but none succumbed with progressive MPN. Flow cytometry of bone marrow and peripheral populations showed that PD0325901 reversed the granulocyte/monocyte progenitor bias and ineffective erythropoiesis in KrasG12D mice. However, PD0325901 did not eliminate the rearranged mutant Kras allele in myeloid progenitors, and these cells remained hypersensitive to GM-CSF in methylcellulose cultures. Therefore, PD0325901 did not eliminate Kras mutant cells, but rather modified their behavior in vivo so as to restore a normal output of the hematopoietic system. To further address the biologic effects of PD0325901 on growth of primary progenitor cells in vitro, we examined colony growth over a range of GM-CSF concentrations. Importantly, whereas in vitro exposure to PD0325901 did not selectively abrogate colony growth from bone marrow of naïve Mx1-Cre, KrasG12D mice in the presence of saturating doses of GM-CSF, a low concentration of PD0325901 eliminated the growth of cytokine-independent progenitor colonies. Even more strikingly, this also restored a normal GM-CSF dose response curve in clonogenic progenitors, eliminating the hypersensitive growth pattern that is a hallmark of MPN. Finally, even at saturating doses of GM-CSF, a low concentration of PD0325901 was sufficient to normalize the numbers and types of cells within the colonies. Together, these data show that a low concentration of PD0325901 is sufficient to impart a normal program of proliferation and differentiation in KrasG12D myeloid progenitors. These findings are highly consistent with the in vivo data. Collectively, our data suggest that aberrant MEK activation mediates most aspects of the MPN phenotype in the progenitor compartment and support the development of clinical trials to evaluate MEK inhibitors in patients with JMML and CMML. Disclosures: No relevant conflicts of interest to declare.

Identification of Non-Cardiotoxic hERG1 Blockers to Overcome Chemoresistance in Acute Lymphoblastic Leukemias

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1506-1506
Author(s):  
Marika Masselli ◽  
Serena Pillozzi ◽  
Massimo D'Amico ◽  
Luca Gasparoli ◽  
Olivia Crociani ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mouse Model ◽  
Map Kinases ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Significant Proportion ◽  
Leukemic Cells ◽  
K Channel

Abstract Abstract 1506 Although cure rates for children with acute lymphoblastic leukemia (ALL), the most common pediatric malignancy, have markedly improved over the last two decades, chemotherapy resistance remains a major obstacle to successful treatment in a significant proportion of patients (Pui CH et al. N Engl J Med., 360:2730–2741, 2009). Increasing evidence indicates that bone marrow mesenchymal cells (MSCs) contribute to generate drug resistance in leukemic cells (Konopleva M et al., Leukemia, 16:1713–1724, 2002). We contributed to this topic, describing a novel mechanism through which MSCs protect leukemic cells from chemotherapy (Pillozzi S. et al., Blood, 117:902–914, 2011.). This protection depends on the formation of a macromolecular membrane complex, on the plasma membrane of leukemic cells, the major players being i) the human ether-a-gò-gò-related gene 1 (hERG1) K+ channel, ii) the β1integrin subunit and iii) the SDF-1α receptor CXCR4. In leukemic blasts, the formation of this protein complex activates both the ERK 1/2 MAP kinases and the PI3K/Akt signalling pathways triggering antiapoptotic effects. hERG1 exerts a pivotal role in the complex, as clearly indicated by the effect of hERG1 inhibitors to abrogate MSCs protection against chemotherapeutic drugs. Indeed, E4031, a class III antiarrhythmic that specifically blocks hERG1, enhances the cytotoxicity of drugs commonly used to treat leukemia, both in vitro and in vivo. The latter was tested in a human ALL mouse model, consisting of NOD/SCID mice injected with REH cells, which are relatively resistant to corticosteroids. Mice were treated for 2 weeks with dexamethasone, E4031, or both. Treatment with dexamethasone and E4031 in combination nearly abolished bone marrow engraftment while producing marked apoptosis, and strongly reducing the proportion of leukemic cells in peripheral blood and leukemia infiltration of extramedullary sites. These effects were significantly superior to those obtained by treatment with either dexamethasone alone or E4031 alone. This model corroborated the idea that hERG1 blockers significantly increase the rate of leukemic cell apoptosis in bone marrow and reduced leukemic infiltration of peripheral organs. From a therapeutic viewpoint, to develop a pharmacological strategy based on hERG1 targeting we must consider to circumvent the side effects exerted by hERG1 blockers. Indeed, hERG1 blockers are known to retard the cardiac repolarization, thus lengthening the electrocardiographic QT interval, an effect that in some cases leads to life threatening ventricular arrhythmias (torsades de points). On the whole, it is mandatory to design and test non-cardiotoxic hERG1 blockers as a new strategy to overcome chemoresistance in ALL. On these bases, we tested compounds with potent anti-hERG1 effects, besides E4031, but devoid of cardiotoxicity (e.g. non-torsadogenic hERG1 blockers). Such compounds comprise erythromycin, sertindole and CD160130 (a newly developed drug by BlackSwanPharma GmbH, Leipzig, Germany). We found that such compounds exert a strong anti-leukemic activity both in vitro and in vivo, in the ALL mouse model described above. This is the first study describing the chemotherapeutic effects of non-torsadogenic hERG1 blockers in mouse models of human ALL. This work was supported by grants from the Associazione Genitori contro le Leucemie e Tumori Infantili Noi per Voi, Associazione Italiana per la Ricerca sul Cancro (AIRC) and Istituto Toscano Tumori. Disclosures: No relevant conflicts of interest to declare.

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