G-CSF and GM-CSF Have Opposite Effects on the Growth and Survival of Cells Containing Abnormal RUNX1.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2760-2760
Author(s):  
Fariborz Mortazavi ◽  
Arun Sharma ◽  
Marie Baraoidan ◽  
Vinzon Ibanez ◽  
SiJun Yang ◽  
...  
Keyword(s):  
Myelodysplastic Syndrome ◽  
Cell Line ◽  
Myeloid Leukemia ◽  
Hematopoietic Cells ◽  
Leukemia Cells ◽  
Growth And Survival ◽  
Gm Csf ◽  
Molecular Abnormalities ◽  
Empty Vector ◽  
Human Cd34

Abstract Primary leukemia cells can respond very differently to cytokines such as G-CSF and GM-CSF, demonstrating marked increases in colony-forming ability to one but not the other (Park et al, Blood1989; 74: 56–65). To examine if such differences might correlate with underlying molecular abnormalities, we examined the growth and survival response of hematopoietic cells containing abnormal RUNX1 variants (a frequent occurrence in leukemia and myelodysplastic syndrome) to treatment with G-CSF and GM-CSF. In the RUNX1-ETO containing cell-line Kasumi-1, GM-CSF decreased colony forming ability whereas G-CSF preserved or increased it. The 32Dcl3 murine myelomonocytic cell-line terminally differentiates in response to G-CSF or mGM-CSF. We transduced 32Dcl3 with RUNX1-ETO or Empty Vector: G-CSF promoted the growth and survival of 32D RUNX1-ETO but caused 32D Empty Vector to terminally differentiate and die. In contrast, 32D RUNX1-ETO cultured in mGM-CSF demonstrated accelerated terminal differentiation and death compared to 32D Empty Vector. Many of the RUNX1 variants seen in leukemia and myelodysplastic syndrome, including RUNX1-ETO, lack the RUNX1 C-terminal transcription regulating domain (TRD). In human CD34+ hematopoietic cells transduced with RUNX1 with TRD deleted (CD34 RUNX1noTRD), colony forming ability was retained even after >40 days of culture in IMDM 10% FBS, SCF 10ng/ml, IL-3 30ng/ml, G-CSF 100ng/ml (CD34 Empty Vector lost colony forming ability after 14 days). However, culture in media supplemented with 100ng/ml GM-CSF instead of G-CSF significantly impaired colony forming ability of CD34 RUNX1noTRD compared to CD34 Empty Vector. In conclusion, RUNX1 variants seen in acute myeloid leukemia and myelodysplastic syndrome promote growth and survival in G-CSF but accelerate differentiation and death in response to GM-CSF. The molecular basis for this effect appears to relate to the presence or absence of the RUNX1 C-terminal transcription regulating domain. These findings may have clinical relevance.

BCL2A1 Is A Survival and Immortalization Factor for Primitive Myeloid Hematopoietic Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3365-3365
Author(s):  
Jean-Yves Metais ◽  
Ashley E. Dunfee ◽  
Rodrigo T. Calado ◽  
Cynthia E. Dunbar
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Gene Product ◽  
Myeloid Leukemia ◽  
Hematopoietic Cells ◽  
Marker Genes ◽  
Empty Vector ◽  
Myeloid Progenitor ◽  
The Impact

Abstract We recently reported development of an acute myeloid leukemia in a rhesus macaque transplanted with autologous CD34+ cells transduced with a murine stem cell virus-derived replication defective retrovirus vector expressing only marker genes under control of the strong MCSV LTR. This animal had an unusual clonal reconstitution pattern the first year following transplant, with a single transduced myeloid progenitor cell clone accounting for up to 80% of then normal myelopoiesis (Kelly, 2005). The same vector-containing clone then transformed to AML five years following transplantation, and each tumor cell was shown to contain two vector insertions, one localized 20 kb upstream the CDw92 gene on chromosome 9, and the second localized in the first intron of BCL2A1 on chromosome 15 (Seggewiss, 2006), a gene in the anti-apoptotic BCL2 family not previously linked to myeloid leukemia. BCL2A1 was highly expressed in the tumor cells. This tumor was the first hematopoietic malignancy reported in a recipient of primitive cells transduced with a replication-incompetent vector containing only marker genes, and suggested that BCL2A1 could have potent effects on myeloid cell behavior. To investigate the impact of the BCL2A1 gene product on hematopoietic cells, we cloned the murine and human HA-tagged BCL2A1 cDNAs into lentivirus vectors and transduced the murine BaF3 hematopoietic cell line as a model to study the impact of expression of these proteins on hematopoiesis. We confirmed overexpression of the proteins in the producer cell line as well as in transduced cells by western blot using an anti-HA monoclonal antibody. BaF3 cell proliferation and survival are dependant on IL-3, and under IL-3 replete conditions overexpression of murine or human BCL2A1 did alter proliferation compared with untransduced cells or cells transduced with an empty vector. Removal of IL-3 from the cell culture media leads to rapid apoptosis of BaF3 cells, with cell cycle arrest in the G1 and an apoptotic subpopulation appearing within 24 hours of IL-3 removal. 45% untransduced or empty vector cells were apoptotic, and this fraction decreased to 30% and 15% respectively for BaF3 cells expressing murine or human BCL2A1. These results were confirmed by direct analysis of apoptosis. Only BaF3 cells over-expressing human BCL2A1 were still alive and arrested in G1 after 3 days of culture without IL-3. The murine BCL2A1 had similar but less striking effects. Gene expression analyses on the BaF3 cell populations are ongoing, to identify potential downstream targets of the BCL2A1 protein. The BCL2A1 and empty vectors were also utilized in murine bone marrow cell immortalization assay, previously utilized to identify genes impacting on the survival and expansion of primary myeloid progenitor cells (Du, 2005). In an initial set of experiments, clonal clonal expansion was obtained with marrow cells expressing murine (4 clones) and human (5 clones) BCL2A1 but not for empty vector or untransduced murine marrow. Mice have also been transplanted with primary bone marrow cells transduced with the BCL2A1 and control vectors, and are being followed for in vivo expansion of transduced clones and development of leukemia. In conclusion, we have confirmed the role of BCL2A1 as an anti-apoptotic protein, now in myeloid hematopoietic cells, and will continue to investigate the role of this gene product in hematopoiesis and leukemogenesis.

scholarly journals Effects of CCL2/CCR2 Blockade in Acute Myeloid Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1348-1348 ◽  
Author(s):  
Rodrigo Omar Jacamo ◽  
Hong Mu ◽  
Qi Zhang ◽  
Dhruv Chachad ◽  
Wang Zhiqiang ◽  
...  
Keyword(s):  
Cell Line ◽  
Stromal Cells ◽  
Myeloid Leukemia ◽  
Hematopoietic Cells ◽  
Fold Increase ◽  
In Vivo Studies ◽  
Leukemia Cell Line ◽  
Leukemia Cells ◽  
Tumor Associated Macrophages

Abstract Background: A role for the Chemokine (C-C motif) ligand 2 (CCL2) in attracting tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSC) and infiltrating monocytes has been described for many solid tumors in which they play an essential role in modifying the adaptive immune response, ultimately favoring tumor progression. Unfortunately, little is known about the importance of this mechanism for the progression of AML. We recently identified CCL2 as the most prominent chemokine produced by bone marrow (BM) mesenchymal stromal cells (BM-MSC) in response to the interaction with myeloid leukemia cells (PMID: 24599548). In addition, elevated CCL2 plasma levels have been reported in patients of AML (PMID: 17822317), ALL (PMID: 21298741) and CLL (PMID: 22397722) when compared to normal controls. In this study we assessed the effects of blocking the CCR2-CCL2 axis on the migration and signaling of hematopoietic cells as well as on the infiltration of immune-suppressive cells in leukemia-bearing mice. Results: We first studied the efficacy and potency of agents at inhibiting CCL2-mediated migration, using the human monocytic leukemia cell line THP-1. Migration towards human recombinant CCL2 (5 ng/ml) was significantly inhibited by as little as 1 nM of NOX-E36, a human-specific CCL2 Spiegelmer (NOXXON Pharma, Berlin). Spiegelmers are RNA-like molecules built from L-ribose units that are able to bind molecules such as peptides and proteins with an affinity in the pico-to nanomolar range. Similar results were obtained with a CCR2 antagonist (100 ng/ml; Santa Cruz). In anticipation of in vivo studies in mice, we next confirmed the ability of a mouse-specific CCL2 Spiegelmer (mNOX-E36) to inhibit migration and signaling pathway activation in murine hematopoietic cells. For this purpose, we cloned and overexpressed via lentiviral transduction the murine CCL2 receptor (CCR2) in Ba/F3 cells (a murine pro-B cell line). Stimulation of Ba/F3-CCR2 cells with 5 ng/ml of mouse recombinant CCL2 induced a ~2000 fold increase in migration of Ba/F3-CCR2 cells and was successfully blocked with mNOX-E36 in a concentration-dependent manner. Western blot analysis of protein lysates from mCCL2-stimulated cells (30 minutes treatment) indicated activation of AKT, ERK and p38-MAPK. The CCL2-induced phosphorylation of these molecules was completely abrogated by pre-treatment with mNOX-E36. Subsequently, we determined whether the expression of CCL2 by stromal cells in leukemia-resident organs triggers the infiltration of TAMs and possibly other immune-suppressive cells into those organs. We conducted preliminary in vivo studies in non-irradiated immunocompetent C57BL/6 mice (n=5 per group) injected with syngeneic AML1/ETO9a-expressing primary murine leukemia cells (PMID: 19339691). After confirmation of leukemia engraftment by IVIS imaging, mice were treated with mNOX-E36 (14.4 mg/kg, s.c., three times per week) or vehicle control for 3 weeks. At this point, all animals were sacrificed and their tissues (spleens and BM from femurs) were collected for analysis. Although we did not observe differences in leukemia burden by imaging between vehicle and mNOX-E36 treated groups, flow cytometry analysis revealed an increase in the frequency of CD11b+ Ly6Clow MHC IIlow macrophages (2 to 7 fold increase) in spleens of mice engrafted with leukemia (vehicle-treated group) when compared to spleens collected from healthy mice. These MHC IIlow macrophages were previously identified as immunosuppressive M2-like macrophages as opposed to MHC IIhi macrophages which show a pro-inflammatory M1-like phenotype (PMID: 20570887). Importantly, CCL2 inhibitor mNOX-E36 abrogated this macrophage infiltration within the leukemia microenvironment. Conclusions: Our results indicate that blockade of the CCR2-CCL2 axis not only affects migration and signaling of treated cells in vitro, but also interferes with the infiltration of M2-like macrophages into spleens of leukemia-bearing mice. Current in vivo experiments using a combination of standard chemotherapy with mNOX-E36 in AML immunocompetent models are undergoing. We expect that in vivo modulation of CCL2 will improve response to chemotherapy of AML by reducing the marrow infiltration of infiltrating monocytes and tumor-associated macrophages, which would facilitate translation of this novel concept into clinical trials in AML. Disclosures Zuber: Boehringer Ingelheim: Research Funding; Mirimus Inc.: Consultancy, Other: Stock holder. Eulberg:NOXXON Pharma AG: Employment. Kruschinski:NOXXON Pharma AG: Employment.

scholarly journals Granulocyte-macrophage colony-stimulating factor/interleukin-3 fusion protein (pIXY 321) enhances high-dose Ara-C-induced programmed cell death or apoptosis in human myeloid leukemia cells

Blood ◽  
1992 ◽  
Vol 80 (11) ◽  
pp. 2883-2890 ◽  
Author(s):  
K Bhalla ◽  
C Tang ◽  
AM Ibrado ◽  
S Grant ◽  
E Tourkina ◽  
...  
Keyword(s):  
Cell Death ◽  
Myeloid Leukemia ◽  
Colony Growth ◽  
Leukemia Cells ◽  
High Dose ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract High dose Ara-C (HIDAC) induces programmed cell death (PCD) or apoptosis in vitro in human myeloid leukemia cells, which correlates with the inhibition of their clonogenic survival. Hematopoietic growth factors (HGFs) granulocyte-macrophage colony-stimulating factor (GM- CSF) and interleukin-3 (IL-3) have been demonstrated to enhance the metabolism and cytotoxic effects of HIDAC against leukemic progenitor cells. We examined the effect of pIXY 321 (a GM-CSF/IL-3 fusion protein) on HIDAC-induced PCD and related gene expressions as well as HIDAC-mediated colony growth inhibition of human myeloid leukemia cells. Unlike the previously described effects of HGFs on normal bone marrow progenitor cells, exposure to pIXY 321 alone for up to 24 hours did not suppress PCD in HL-60 or KG-1 cells. However, exposure to pIXY 321 for 20 hours followed by a combined treatment with Ara-C plus pIXY 321 for 4 or 24 hours versus treatment with Ara-C alone significantly enhanced the oligonucleosomal DNA fragmentation characteristic of PCD. This was temporally associated with a marked induction of c-jun expression and a significant decrease in BCL-2. In addition, the treatment with pIXY 321 plus HIDAC versus HIDAC alone produced a significantly greater inhibition of HL-60 colony growth. These findings highlight an additional mechanism of HIDAC-induced leukemic cell death that is augmented by cotreatment with pIXY 321 and may contribute toward an improved antileukemic activity of HIDAC.

scholarly journals Treatment of newly diagnosed acute myelogenous leukemia with granulocyte-macrophage colony-stimulating factor (GM-CSF) before and during continuous-infusion high-dose ara-C + daunorubicin: comparison to patients treated without GM-CSF

Blood ◽  
1992 ◽  
Vol 79 (9) ◽  
pp. 2246-2255 ◽  
Author(s):  
E Estey ◽  
PF Thall ◽  
H Kantarjian ◽  
S O'Brien ◽  
CA Koller ◽  
...  
Keyword(s):  
Myeloid Leukemia ◽  
Myelogenous Leukemia ◽  
Leukemia Cells ◽  
Newly Diagnosed ◽  
Gm Csf ◽  
Negative Effect ◽  
Acute Myelogenous ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract We gave 56 patients with newly diagnosed acute myelogenous leukemia (AML) granulocyte-macrophage colony-stimulating factor (GM-CSF) 20 or 125 micrograms/m2 once daily subcutaneously before (for up to 8 days or until GM-CSF-related complications developed) and during, or only during (patients presenting with blast counts greater than 50,000 or other leukemia-related complications) ara-C (1.5 g/m2 daily x 4 by continuous infusion) and daunorubicin (45 mg/m2 daily x 3) chemotherapy. Because results seemed independent of GM-CSF schedule, we compared results in these 56 patients with results in 176 patients with newly diagnosed AML given the same dose and schedule of ara-C without GM-CSF (110 patients ara-C alone, 66 patients ara-C + amsacrine or mitoxantrone). Comparison involved fitting a logistic regression model predicting probability of complete remission (CR) and a Cox regression model to predict survival (most patients in all three studies were dead) with treatment included as a covariate in both analyses. After adjusting for other prognostically significant covariates [presence of an antecedent hematologic disorder, an Inv (16), t(8;21), or abnormalities of chromosomes 5 and/or 7, performance status, age, bilirubin], treatment with ara-C + daunorubicin + GM-CSF was predictive of both a lower CR rate and a lower survival probability. There were no treatment-covariate interactions, suggesting that the negative effect of this GM-CSF treatment regime was not an artifact of some imbalance in patient characteristics. The unadjusted Kaplan-Meier hazard rate of the ara-C + daunorubicin + GM-CSF group was not uniquely high during the initial 4 weeks after start of therapy, but was highest among the three treatment groups throughout weeks 5 to 16, suggesting that the negative effect of this treatment was not caused by acute toxicity. Patients who did not enter CR with this treatment tended to have persistent leukemia rather than prolonged marrow aplasia, suggesting that this treatment and, in particular, GM-CSF may increase resistance of myeloid leukemia cells to chemotherapy. To date, relapse rates are similar in all three groups (P = .43) (as are survival rates once patients are in CR) but much of the remission duration data is heavily censored, unlike the survival data. Our results suggest caution in the use of GM-CSF to sensitize myeloid leukemia cells to daunorubicin + ara- C chemotherapy.

Bcr-Abl-Mediated Suppression of Normal Hematopoiesis in Chronic Myeloid Leukemia: Involvement of a Modified Form of NGAL.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2869-2869
Author(s):  
Hui Lin ◽  
Xiaohong Leng ◽  
Tong Sun ◽  
Giuseppe Monaco ◽  
Clifton Stephens ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Hematopoietic Cells ◽  
K562 Cells ◽  
Soft Agar ◽  
Leukemia Cells ◽  
Active Process ◽  
Culture Studies ◽  
Induced Apoptosis

Abstract The BCR-ABL oncogene plays an essential role in chronic myeloid leukemia (CML). In NOD/scid mice injected with soft agar clones of a human CML cell line (K562), we observed a leukemia syndrome involving not only leukemia but also a severe reduction of normal mouse hematopoiesis (Lin et al., Oncogene, 2001). Some of these mice died of a wasting syndrome that involved suppression of hematopoiesis without extensive tumor cell invasion of the spleen and marrow. In CML patients, since normal hematopoietic cells in marrow and spleen are replaced with proliferating leukemic blasts, we postulate that this is an active process mediated by the leukemia cells. The lipocalin 24p3 is secreted by mouse hematopoietic cells deprived of IL-3, resulting in apoptosis induction in a variety of hematopoietic cells including bone marrow cells (Devireddy et al., Science, 2001). We found that BCR-ABL+ mouse hematopoietic cells induce a persistent secretion of a modified form of 24p3 (21 kDa). Co-culture studies show that BCR-ABL+ cells induced apoptosis in BCR-ABL negative cells. Importantly, BCR-ABL+ hematopoietic cells are resistant to apoptosis under the same conditions. Conditioned medium (CM) from BCR-ABL+ cells expressing anti-sense/siRNA 24p3 or CM mixed with 24p3 antibody have reduced apoptotic activity for target cells. We also found that the expression of the Bcr-Abl oncoprotein and its tyrosine kinase are required for induction of 24p3 expression. Leukemic mice induced by BCR-ABL+ cells expressing anti-sense/siRNA 24p3 have increased levels of normal hematopoiesis (marrow and spleen erythropoiesis and blood platelet levels) and reduced invasion of leukemia cells in marrow and spleen tissues, but the leukemia cells readily invade liver and the abdomen as ascites (Lin et al, Oncogene, 2005). These findings indicate that suppression of normal hematopoiesis in BCR-ABL induced leukemia is an active process involving the apoptotic factor 24p3, raising the possibility that similar factors are involved in BCR-ABL+ CML patients. We have found that the K562 clones (Lin et al. 2001) have enhanced expression of NGAL (neutrophil gelatinase-associated lipocalin, human homologue of 24p3) transcripts compared to uncloned K562 cells. We generated additional soft agar K562 clones, each with different expression levels of NGAL transcripts. NOD/scid mice injected with the clone (C5) of K562 cell line expressing a high level of NGAL had severe depression of hematopoiesis and significantly shorter survival time as compared with mice injected with parental K562 cells and a clone (C6) expressing a low level of NGAL. Co-culture studies showed that the C5 K562 clone also induced apoptosis in BCR-ABL negative cells. We detected two glycosylated forms of NGAL/24p3 migrating at 24 kDa and 21 kDa on SDS-PAGE. The 21 kDa form is the major form in CM from mouse BCR-ABL+ cells and K562 clones. Our preliminary data with CML patient samples showed that levels of 21 kDa NGAL protein in bone marrow fluid correlated with BCR-ABL/ABL ratio. Further studies with more patient samples are ongoing to confirm the role of NGAL in suppressing normal hematopoiesis in CML patients and to determine the structural change(s) that leads to the modified form of 24p3/NGAL secreted by CML cells.

Human Plasmacytoid Dendritic Cell like Leukemia Cell Line (PMDC05) Established from a Patient with CD4+CD56+ Acute Leukemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4456-4456
Author(s):  
Miwako Narita ◽  
Nozomi Tochiki ◽  
Norihiro Watanabe ◽  
Anri Saitoh ◽  
Shigeo Hashimoto ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cell ◽  
Acute Leukemia ◽  
Cell Line ◽  
Leukemia Cell ◽  
Leukemia Cell Line ◽  
Leukemia Cells ◽  
Type I ◽  
Gm Csf ◽  
Antigen Presenting

Abstract Human dendritic cell precursors are commonly divided into two distinct subsets: myeloid DC and Plasmacytoid DC (pDC). The pDC, which show plasma cell like morphology, have been defined as the population that produce a large amount of type I interferon in response to viruses. The surface phenotypes of human pDCs are defined as CD4+, DC11c−, CD45RA+, IL3Rα (CD123)+, CD1c (BDCA-1)−, CD303 ((BDCA-2)+ and lineage negative. On the other hand, leukemia/lymphoma cells in CD4+CD56+ leukemia/lymphoma have been proposed to be of pDC lineage. CD4+CD56+ pDC leukemia/lymphoma are a rare hematological malignancy, totally only about 100 cases in the world by the literatures. We established a pDC like leukemia cell line (PMDC05) from leukemia cells of a patient with CD4+CD56+ acute leukemia. PMDC05 showed a complex hypoploid chromosomal abnormalities (44, XY) including add(5)(q22), add(15)(q26) and del(15)(q11q15), which is identical to original leukemia cells. Abnormalities including 5q and 15q are reported as the frequent aberrations in CD4+CD56+ pDC leukemia/lymphoma. PMDC05, which morphology was similar to plasma cells, was positive for CD4, CD56, CD123, CD33, CD86, HLA-ABC, HLA-DR, CD1a, CD40, and CD83 but negative for linage markers. Cytokine receptors for GM-CSF, IL3Rα and IL-6Rα were positive on PMDC05. The expression of Trail and Flt-3L was positive. By the culture with IL-3, CPG-A/B, GM-CSF, molecules associated with antigen presentation such as CD1a and CD40 were up-regulated. Besides, the addition of LPS increased the expression of CD40, CD80 and CD83 on PMDC05. PMDC05 by itself possessed a potent antigen presenting ability to naïve T cells and the treatment of PMDC05 with IL-3, CPG-A/B, or GM-CSF enhanced the antigen presenting ability to naïve T cells. TLR7, TLR 8 and TLR 9 as well as TLR1, TLR2, TLR4 were demonstrated to be expressed on PMDC05 by RT-PCR and RQ-PCR showed that the expression of TLR7 and TLR9 was most characteristic. λ-like 14.1 and preTα was also demonstrated to be expressed on PMDC05 by RT/RQ-PCR. PMDC05 possessed an ability to uptake the antigens like FITC-dextran and lucifer yellow. Although IFN-α was not identified to be secreted from PMDC05 by the stimulation of influenza virus, IFN-γ and TNF-α was demonstrated to be secreted to the similar level in pDC, which was examined simultaneously with PMDC05 by CBA assay. These data demonstrated that newly established leukemia cell line PMDC05 is involved in pDC lineage and PMDC05 provides invaluable tools not only for the elucidation of pathophysiology of CD4+CD56+ leukemia/lymphoma but also for the investigation of differntiation and regulation of pDC. In addition, PMDC05 could be applied for generating tumor-specific CTL clone, which may be used for anti-tumor cellular immunotherapy.

Negative Effects of GM-CSF Signaling In t(8;21)-Induced Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3163-3163
Author(s):  
Shinobu Matsuura ◽  
Ming Yan ◽  
Eun-Young Ahn ◽  
Miao-Chia Lo ◽  
David Dangoor ◽  
...  
Keyword(s):  
Myeloid Leukemia ◽  
Sex Chromosome ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Hematopoietic Cells ◽  
Receptor Activation ◽  
Pseudoautosomal Region ◽  
Wild Type ◽  
Gm Csf

Abstract Abstract 3163 The t(8;21)(q22;q22) translocation is one of the most common chromosomal translocations in de novo acute myeloid leukemia (AML). The 8;21 translocation is often associated with additional cytogenetic abnormalities. The loss of the sex chromosome (LOS) is by far the most frequent abnormality found in association with the t(8;21) leukemia, accounting for 32–59% of patients, in contrast to other types of AML in which the LOS occurs in less than 5% of patients. To evaluate the role of sex chromosome deletion in t(8;21)-related leukemogenesis, hematopoietic cells from a mouse line with only one sex chromosome were used in retrovirus-mediated t(8;21) (AML1-ETO) expression and transplantation assays. The absence of leukemia in those animals suggested that a gene present in the pseudoautosomal region of sex chromosomes in humans but not in mice may be the target gene in LOS. The granulocyte-macrophage colony-stimulating factor receptor α (GM-CSFRα) gene is one such gene and is also known to be involved in myeloid cell survival, proliferation and differentiation. The GM-CSFRα gene is specifically down-regulated in AML patients with t(8;21), but not in other common translocations (Valk PJM et al, NEJM, 2004). The GM-CSFR complex is composed of α and βc subunits that assemble into a complex for receptor activation and signaling. To investigate the role of GM-CSFR signaling in t(8;21)-mediated leukemogenesis, GM-CSFR common β subunit knockout (GM-CSFRβc-/-) mice were used in our studies as a model for deficient GM-CSFR signaling. Transduction of AML1-ETO in hematopoietic cells from GM-CSFRβc-/- resulted in myeloid leukemia of a median survival time of 225 days, high percentage of blasts in peripheral blood and bone marrow, anemia, thrombocytopenia, hepatomegaly and splenomegaly. Comparison of wild-type and GM-CSFRβc-/- cells in the same transplantation resulted in development of AML1-ETO-induced leukemia at higher penetrance in GM-CSFRβc-/- cells (28.5% vs 100%). Moreover, the latency of leukemia was shorter in GM-CSFRβc-/- cells than in wild-type cells after transduction of AML1-ETO9a. Analysis of the hematopoietic compartment of healthy GM-CSFRβc-/- mice detected no significant abnormalities in the immature hematopoietic compartment (LSK, CMP, GMP, MEP), suggesting that AML1-ETO expression is required for leukemia to occur. In vitro, expression of AML1-ETO alone is sufficient for the immortalization of normal hematopoietic cells, as demonstrated by serial replating capacity of cells in methylcellulose colony assay. Addition of mGM-CSF to the basic cytokine cocktail (mIL-3, hIL-6, mSCF, hEPO) did not significantly affect number, type, size, and cell composition of colony cells. In contrast, the addition of mGM-CSF eliminated the replating capacity of AML1-ETO expressing cells, although they survived longer than control vector-infected cells. The results suggest that activation of GM-CSF signaling can specifically abrogate the self-renewal ability of potential leukemic stem cells in the early immortalization phase. These results support a possible tumor suppressor role of GM-CSF in leukemogeneis by AML1-ETO and may provide clues to understand how AML1-ETO corrupts normal GM-CSF signals to its own advantage for leukemogenic transformation. Disclosures: No relevant conflicts of interest to declare.

Targeting the Mammalian Mitochondrial Clpp (mClpP) As a Novel Therapeutic Strategy for Acute Myeloid Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3603-3603
Author(s):  
Alicia Cole ◽  
Zezhou Wang ◽  
Rachel Mattson ◽  
Rose Hurren ◽  
Fengshu Lin ◽  
...  
Keyword(s):  
Therapeutic Target ◽  
Myeloid Leukemia ◽  
Mitochondrial Protein ◽  
Hematopoietic Cells ◽  
Leukemia Cells ◽  
Enzyme Complex ◽  
Wild Type ◽  
Rt Pcr ◽  
Chaperone Protein ◽  
Acute Myeloid

Abstract Abstract 3603 The mammalian mitochondrial caseinolytic protease (mClpP) is a nuclear-encoded enzyme complex responsible for degrading excess proteins in the mitochondria. It is functionally similar to the proteasome complex in the cytoplasm and structurally homologous to the bacterial ClpP enzyme complex. While the proteasome has been well characterized as a therapeutic target for the treatment of hematologic malignancy, little is known regarding mClpP as a potential therapeutic target in malignant cells. Therefore, we evaluated the expression of mClpP in acute myeloid leukemia (AML) and normal hematopoietic cells by immunoblotting. Strikingly, mClpP was robustly expressed in AML cell lines (n=3/4) and primary AML patients (n=4/4), but was undetectable in normal bone marrow samples (n = 3) and G-CSF-mobilized peripheral blood mononuclear cells from consenting volunteers donating stem cells for allotransplant (n = 5). We also demonstrated over-expression of mClpP mRNA in these AML samples compared to normal by Q-RT-PCR. Next, we knocked down mClpP and its regulatory chaperone protein mClpX in OCI-AML2 and TEX human leukemia cells using 3 independent shRNA in lentiviral vectors. Target knockdown was confirmed by Q-RT-PCR and immunoblotting. Compared to cells infected with control sequences, knockdown of mClpP reduced the growth and viability of these leukemia cells by > 90%. Knockdown of the chaperone protein mClpX also reduced the growth and viability of these cells, but with less potency than mClpP knockdown. Rho-zero cells have been depleted of mitochondrial DNA by treatment with ethidium bromide and therefore lack mitochondrial protein synthesis. Through feedback mechanisms, expression of nuclear encoded proteins that contribute to oxidative metabolism are also reduced. Thus, these cells have lower rates of mitochondrial protein accumulation and turnover. We demonstrated that wild type and rho-zero 143B rhabdomyosarcoma cells both express mClpP protein, but levels were slightly lower in the rho-zero cells. We then tested the effects of mClpP knockdown in rho-zero cells and demonstrated that knockdown of mClpP reduced the growth and viability of wild type 143B cells, but had little effect on their rho-zero counterparts. As a chemical approach to evaluate the effects of mClpP inhibition on AML and normal hematopoietic cells, we synthesized a derivative of recently reported beta-lactone bacterial ClpP inhibitor and generated (3RS,4RS)-3-(non-8-en-1-yl)-4-(2-(pyridin-3-yl)ethyl)oxetan-2-one that we termed A2–32–01. We confirmed that A2–32–01 inhibited the enzymatic activity of recombinant bacterial ClpP similar to the activity of the reported beta-lactone inhibitors. A2–32–01 induced cell death in TEX, OCI-AML2, and K562 leukemia cells that express mClpP as measured by trypan blue staining. We also isolated mitochondria from these cells after treatment with A2–32–01 and demonstrated that the compound reduced the enzymatic activity of the mClpP protease as measured by cleavage of the fluorogenic substance N-succinyl-Leu-Tyr-7-amidomethylcoumarin (Suc-LY-AMC). In contrast, A2–32–01 was not cytotoxic to HL60 cells that had undetectable mClpP. Likewise, A2–32–01 induced death in 143B wild type cells, but not the rho-zero counterparts. Finally, we evaluated the effects of mClpP inhibition on primary AML and normal hematopoietic cells. Primary AML and normal hematopoietic cells were treated with increasing concentrations of A2–32–01 and cell viability was measured after 48 hours incubation by Annexin V/PI staining followed by flow cytometry. A2–32–01 did not kill normal hematopoietic cells (n = 3 samples), but was cytotoxic to 5/6 tested primary AML cells. In summary, we have used genetic and chemical approaches to highlight the mitochondrial protease, mClpP, as a novel therapeutic target for AML. Disclosures: No relevant conflicts of interest to declare.

A Novel Non-Genetic Photostable Approach to Labelling Acute Myeloid Leukemia and Bone Marrow Cells with Quantum Dots

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4818-4818
Author(s):  
Yanwen Zheng ◽  
Zhengwei Mao ◽  
Bin Yin
Keyword(s):  
Bone Marrow ◽  
Quantum Dots ◽  
Acute Myeloid Leukemia ◽  
Blood Cells ◽  
Myeloid Leukemia ◽  
Hematopoietic Cells ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Dependent Manner ◽  
Acute Myeloid

Abstract Abstract 4818 Acute myeloid leukemia (AML) is a detrimental disease with difficult diagnosis and treatment. Understanding the biology of AML at the molecular and cellular levels would be essential to successful management of the disease. However, the notoriously known difficulty in manipulation of leukemia cells has long hindered the dissection of AML pathogenesis. The advent of CdSe/ZnS quantum dots (QDs) represents an important advancement in the research field of nanotechnology, which have recently also been applied for imaging of live cells. Here, we have introduced a non-genetic approach of marking blood cells, by taking advantage of QD technology. We compared QDs complexed with different vehicles, including a peptide Tat (QDs-Tat), cationic polymer Turbofect (QDs-Tf) and liposome Lipofectamine 2000 (QDs-Lip), in their abilities to mark cells. QDs-Tat showed the highest efficiency in delivery into hematopoietic cells, among the three vehicles. We then examined QDs-Tat labelling of leukemia cell lines, and found that QDs-Tat could label 293T, bone marrow (BM) cells, THP-1, MEG-01 and HL-60 with a decreasing efficiency. The efficiency of QDs-Tat delivery was dependent on the concentration of QDs-Tat applied, but not the length of incubation time. In addition, more uniform intracellular distributions of QDs in 293T and leukemia cells were obtained with QDs-Tat, compared with the granule-like formation obtained with QDs-Lip. Clearly, QD fluorescence was sharp and tolerant to repetitive photo excitations, and could be detected in 293T for up to one week following labelling. In summary, our results suggest that QDs have provided a photostable, non-genetic and transient approach that labels normal and malignant hematopoietic cells in a cell type-, vehicle-, and QD concentration-dependent manner. We expect for potentially wide applications of QDs as an easy and fast tool assisting investigations of various types of blood cells in the near future. Disclosures: No relevant conflicts of interest to declare.

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