Enhanced ceramide generation and induction of apoptosis in human leukemia cells exposed to DT(388)-granulocyte-macrophage colony-stimulating factor (GM-CSF), a truncated diphtheria toxin fused to human GM-CSF

DT(388)-GM-CSF, a targeted fusion toxin constructed by conjugation of human granulocyte-macrophage colony-stimulating factor (GM-CSF) with the catalytic and translocation domains of diphtheria toxin, is presently in phase I trials for patients with resistant acute myeloid leukemia. HL-60/VCR, a multidrug-resistant human myeloid leukemia cell line, and wild-type HL-60 cells were used to study the impact of DT(388)-GM-CSF on metabolism of ceramide, a modulator of apoptosis. After 48 hours with DT(388)-GM-CSF (10 nM), ceramide levels in HL-60/VCR cells rose 6-fold and viability fell to 10%, whereas GM-CSF alone was without influence. Similar results were obtained in HL-60 cells. Examination of the time course revealed that protein synthesis decreased by about 50% and cellular ceramide levels increased by about 80% between 4 and 6 hours after addition of DT(388)-GM-CSF. By 6 hours this was accompanied by activation of caspase-9, followed by activation of caspase-3, cleavage of caspase substrates, and chromatin fragmentation. Hygromycin B and emetine failed to elevate ceramide levels or induce apoptosis at concentrations that inhibited protein synthesis by 50%. Exposure to C(6)-ceramide inhibited protein synthesis (EC(50) approximately 5 microM) and decreased viability (EC(50) approximately 6 microM). Sphingomyelinase treatment depleted sphingomyelin by about 10%, while increasing ceramide levels and inhibiting protein synthesis. Diphtheria toxin increased ceramide and decreased sphingomyelin in U-937 cells, a cell line extremely sensitive to diphtheria toxin; exposure to DT(388)-GM-CSF showed sensitivity at less than 1.0 pM. Diphtheria toxin and conjugate trigger ceramide formation that contributes to apoptosis in human leukemia cells through caspase activation and inhibition of protein synthesis.     

Human granulocyte colony-stimulating factor receptors in acute myelogenous leukemia.

Human granulocyte colony-stimulating factor (G-CSF) receptors on human acute leukemia cells were investigated using human G-CSF iodolabeled by the lactoperoxidase method. Among various human leukemic cell lines, only cells of myelogenous lineage including HL-60, THP-1 and U937 had one type of high-affinity receptor for G-CSF, as shown by Scatchard analysis. Fresh leukemia cells from 19 patients with acute myelogenous leukemia (AML) were then studied. Specific receptors for G-CSF were demonstrated on blast cells in all 19 cases, the mean number of G-CSF receptors per AML cell ranging from 95 to 1436. G-CSF receptors on AML cells appeared to be a single affinity type, although some variations were observed. The mean number of G-CSF receptors on leukemic cells from patients with either FAB M3 or FAB M2 was greater than that of cells from patients with M1 (p less than 0.01, p less than 0.10, respectively). Moreover, the mean number of receptors for G-CSF on CD13- and CD34-positive AML cells was higher than that on CD13-negative and CD34-positive AML cells (p less than 0.01), and the mean number of G-CSF receptors on CD7-positive AML cells was lower than that for CD7-negative AML cells (p less than 0.10). Since the FAB classification and surface phenotypes reflect maturation stages, our findings indicate that the distribution of G-CSF receptors, even on AML cells, may be related to the maturation process.     

A urokinase-activated recombinant diphtheria toxin targeting the granulocyte-macrophage colony-stimulating factor receptor is selectively cytotoxic to human acute myeloid leukemia blasts

Novel agents to treat acute myeloid leukemia (AML) are needed with increased efficacy and specificity. We have synthesized a dual-specificity fusion toxin DTU2GMCSF composed of the catalytic and translocation domains of diphtheria toxin (DT) fused to the granulocyte-macrophage colony-stimulating factor (GM-CSF) in which the DT furin cleavage site 163RVRRSV170 is modified to a urokinase plasminogen activator (uPA) cleavage site 163GSGRSA170, termed U2. DTU2GMCSF was highly toxic to the TF1-vRaf AML cell line (proliferation inhibition assay; IC50 = 3.14 pM), and this toxicity was greatly inhibited following pretreatment with anti-uPA and anti-GM-CSF antibodies. The activity of this toxin was then tested on a larger group of 13 human AML cell lines; 5 of the 13 cell lines were sensitive to DTU2GMCSF. An additional 5 of the 13 cell lines became sensitive when exogenous pro-uPA was added. Sensitivity to DTU2GMCSF strongly correlated with the expression levels of uPA receptors (uPARs) and GM-CSF receptors (GM-CSFRs) as well as with total uPA levels. DTU2GMCSF was less toxic to normal cells expressing uPAR or GMCSFR alone, that is, human umbilical vein endothelial cells and peripheral macrophages, respectively. These results indicate that DTU2GMCSF may be a selective and potent agent for the treatment of patients with AML.     

Malignant progenitors from patients with CD87+ acute myelogenous leukemia are sensitive to a diphtheria toxin-urokinase fusion protein

In previous studies, we demonstrated that the diphtheria toxin-urokinase fusion protein DTAT was selectively toxic to acute myeloid leukemia (AML) cell lines overexpressing the CD87 urokinase receptor. In the present study, we analyzed the sensitivity of patient leukemic progenitors to DTAT and correlated the sensitivity with CD87 expression.We isolated leukemic blasts by density gradient centrifugation and performed immunophenotyping by flow cytometry and blast sensitivity measurements by inhibition of cell proliferation and colony formation in semisolid media.We found CD87 overexpression in 18 (25%) of 71 patient leukemic blast samples, including 18 (28%) of 64 myeloid malignancies and 0 (0%) of 7 lymphoid malignancies. DTAT was toxic to patient leukemic blasts by both proliferation inhibition (IC50 85% inhibition by 10 nM DTAT in 11/41 evaluable samples). Only AML and chronic myeloid leukemia (CML) blast crisis blasts (18/61 [30%]) were sensitive to DTAT by the proliferation inhibition assay. Lymphoid leukemia and chronic phase CML/chronic myelomonocytic leukemia (CMML) progenitors were insensitive to DTAT by the proliferation inhibition assay (n = 7 and n = 3, respectively). Similarly, normal marrow progenitors were insensitive to DTAT by both proliferation inhibition (n = 2) and colony inhibition (n = 5) assays. The DTAT toxicity measured by both proliferation inhibition assay and colony inhibition assay correlated with CD87 density (p < 0.0001 and p = 0.001, respectively). DTAT toxicity results were similar for leukemic blasts measured by either of the two assays (p = 0.0002).This study provides the first evidence that a urokinase receptor targeted diphtheria fusion protein is toxic to patient AML blasts. The work also suggests that blast proliferation assays yield similar responses to leukemia colony-forming cell colony assays.     

Preferential eradication of acute myelogenous leukemia stem cells by fenretinide

Leukemia stem cells (LSCs) play important roles in leukemia initiation, progression, and relapse, and thus represent a critical target for therapeutic intervention. However, relatively few agents have been shown to target LSCs, slowing progress in the treatment of acute myelogenous leukemia (AML). Based on in vitro and in vivo evidence, we report here that fenretinide, a well-tolerated vitamin A derivative, is capable of eradicating LSCs but not normal hematopoietic progenitor/stem cells at physiologically achievable concentrations. Fenretinide exerted a selective cytotoxic effect on primary AML CD34⁺ cells, especially the LSC-enriched CD34⁺CD38⁻ subpopulation, whereas no significant effect was observed on normal counterparts. Methylcellulose colony formation assays further showed that fenretinide significantly suppressed the formation of colonies derived from AML CD34⁺ cells but not those from normal CD34⁺ cells. Moreover, fenretinide significantly reduced the in vivo engraftment of AML stem cells but not normal hematopoietic stem cells in a nonobese diabetic/SCID mouse xenotransplantation model. Mechanistic studies revealed that fenretinide-induced cell death was linked to a series of characteristic events, including the rapid generation of reactive oxygen species, induction of genes associated with stress responses and apoptosis, and repression of genes involved in NF-κB and Wnt signaling. Further bioinformatic analysis revealed that the fenretinide–down-regulated genes were significantly correlated with the existing poor-prognosis signatures in AML patients. Based on these findings, we propose that fenretinide is a potent agent that selectively targets LSCs, and may be of value in the treatment of AML.Acute myelogenous leukemia (AML) represents a group of clonal hematopoietic stem cell disorders, in which a small subpopulation of leukemia stem cells (LSCs) are responsible for the accumulation of large numbers of immature myeloblasts in the bone marrow of AML patients. In addition to their crucial roles in leukemia initiation and progression, LSCs are also responsible for the high frequency of relapse that is characteristic of current AML therapies. Of patients receiving treatment with curative intent, less than one-half will achieve long-term survival (1). Similar to normal hematopoietic stem cells (HSCs), LSCs exhibit stem cell-like characteristics such as the capacity for self-renewal, differentiation potential, and relative quiescence (2, 3). The quiescent feature renders LSCs resistant to conventional chemotherapeutic agents that predominantly target proliferating rather than quiescent cells (1). For this reason, it is not surprising that relapse occurs in the majority of cases; this is further supported by recent studies showing that AML patients with LSCs enrichment have worse clinical outcomes (4–7). It is therefore crucial that therapies be developed targeting the quiescent and drug-resistant LSCs.Despite the similarities shared by LSCs and HSCs, LSCs often possess several unique features as well, which may provide important hints for designing LSC-targeted therapy. For instance, LSCs are usually associated with the abnormal expression of CD markers (e.g., CD44, CD47, CD96, and CD123), constitutive activation of nuclear factor κB (NF-κB), active Wnt/β-catenin signaling, and elevated levels of interferon regulatory factor-1 (IRF-1) and death-associated protein kinase (DAPK) (8–12). Most recently, emerging evidence points to oxidative signaling as being a two-edged sword in AML: moderate levels of reactive oxygen species (ROS) are important for driving disease, whereas higher levels result in cell death (13–15). The dual roles of oxidative signaling suggest that LSCs, in comparison with normal HSCs, are more vulnerable to ROS-generating agents. Accordingly, pharmacological agents favoring the generation of ROS are worth exploring in LSC-targeted therapy. Indeed, ROS induction has been shown as a critical mechanism for the selective eradication of LSCs by several compounds, such as parthenolide (PTL), dimethyl-aminoparthenolide (DMAPT), 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8), and 4-hydroxynonenal (HNE) (16–19).Another promising agent that could be used in this regard is fenretinide, a synthetic retinoid that lacks a carboxyl functional group likely necessary for retinoid receptor activity (20). We and others have previously demonstrated that fenretinide, unlike classical retinoids that often induce differentiation, triggers apoptotic effects; it is largely achieved through the generation of ROS (21–24), enhanced cellular ceramide, and/or ganglioside D3 (25). Moreover, several key stem cell survival-associated signaling pathways, such as NF-κB, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK), have been reported to be inactivated in the fenretinide-induced apoptosis in different cancer cell types (25, 26); this further suggests the therapeutic value of fenretinide in targeting cancer stem cells.Fenretinide has been used clinically for some time as an effective chemopreventive agent for various cancers (27). It can significantly reduce the risk of breast cancer and small cell lung cancer (28, 29), suggesting an ability to prevent the development of cancer and/or eliminate early-stage malignant cells (likely cancer-initiating cells). Furthermore, long-term clinical trials have demonstrated only minimal side effects in patients receiving fenretinide (28, 30–32). In particular, no significant hematopoietic toxicity has been observed in patients treated with fenretinide (28). To illustrate the potential value of fenretinide in AML therapies, in this study, we defined the fenretinide effects on primary AML CD34⁺ cells and LSC-enriched AML CD34⁺CD38⁻ cells.     

Dysregulated gene expression networks in human acute myelogenous leukemia stem cells

We performed the first genome-wide expression analysis directly comparing the expression profile of highly enriched normal human hematopoietic stem cells (HSC) and leukemic stem cells (LSC) from patients with acute myeloid leukemia (AML). Comparing the expression signature of normal HSC to that of LSC, we identified 3,005 differentially expressed genes. Using 2 independent analyses, we identified multiple pathways that are aberrantly regulated in leukemic stem cells compared with normal HSC. Several pathways, including Wnt signaling, MAP Kinase signaling, and Adherens Junction, are well known for their role in cancer development and stem cell biology. Other pathways have not been previously implicated in the regulation of cancer stem cell functions, including Ribosome and T Cell Receptor Signaling pathway. This study demonstrates that combining global gene expression analysis with detailed annotated pathway resources applied to highly enriched normal and malignant stem cell populations, can yield an understanding of the critical pathways regulating cancer stem cells.     

The effect of granulocyte-macrophage colony-stimulating factor on undifferentiated and mature acute myelogenous leukemia blast progenitors.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been used recently to recruit undifferentiated acute myelogenous leukemia (AML) blasts into the S-phase of the cell cycle and increase the fraction of cells killed by cell cycle-specific drugs. Using three AML blast colony assays combined with a suspension culture (delta assay), we determined the in vitro effect of GM-CSF on mature and undifferentiated AML blast progenitors obtained from bone marrow aspirates of six AML patients. GM-CSF stimulated AML blast colony proliferation at a concentration of 5 ng/ml in the methylcellulose and the agar clonogenic assays in six of six AML marrow samples. However, in the delta assay, which selects for immature AML progenitors, GM-CSF did not affect AML blast colony-forming cells in five of six AML marrow samples at concentrations ranging from 5 to 300 ng/ml. Our data imply that GM-CSF stimulates mature but not undifferentiated AML blast progenitors. It is therefore possible that GM-CSF may not be beneficial as a recruiting agent in most AML patients.     

Blasts from patients with acute myelogenous leukemia express functional receptors for stem cell factor.

Stem cell factor (SCF) acts in concert with lineage-specific growth factors to stimulate the growth of hematopoietic colonies. To determine if neoplastic human hematopoietic cells would also respond to SCF, we cultured marrow mononuclear cells from 20 patients with newly diagnosed acute myelogenous leukemia (AML) and two normal donors with SCF, interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or combinations of growth factors in semisolid medium, and assessed colony growth. SCF receptors (c-kit receptors) were quantitated by equilibrium binding studies with 125I-SCF, and binding parameters were estimated using the ligand program. The cellular distribution of c-kit receptors was determined by autoradiography. Our results show that SCF alone or in combination with IL-3 or GM-CSF increased both the number and size of colonies in 10 of the patients. Receptors for SCF were identified on the blasts from all 20 AML patients. The number of receptors ranged from 600 to 29,000 per cell. In the majority of patients, both high- and low-affinity binding sites were identified. Neither the number of receptors per cell nor the finding of one or two classes of receptors correlated with growth response to SCF. Autoradiographic analysis of 125I-SCF binding to normal marrow mononuclear cells revealed grains associated with blasts and megakaryocytes. Grain counts on blasts from 10 AML patients and on normal marrow blasts suggested that high-affinity c-kit receptor expression on AML blasts is lower than or similar to that of normal blasts. These results identify c-kit receptors on human AML blasts, and indicate that SCF acts synergistically with IL-3 or GM-CSF to stimulate colony growth from the marrow cells of a portion of patients with AML.     

TIM-3 as a novel therapeutic target for eradicating acute myelogenous leukemia stem cells

Acute myelogenous leukemia (AML) originates from self-renewing leukemic stem cells (LSCs), which represent the ultimate therapeutic target for AML. Recent studies have identified several AML LSC-specific surface antigens as candidate targets of therapeutic molecules. T cell immunoglobulin mucin-3 (TIM-3) is expressed on LSCs in most types of AML, with the exception of acute promyelocytic leukemia, but not on normal hematopoietic stem cells (HSCs). In xenograft models reconstituted with human AML LSCs or HSCs, an anti-human TIM-3 mouse IgG2a antibody with cytotoxic activities eradicates AML LSCs in vivo, but does not affect normal human hematopoiesis. Thus, TIM-3 is a promising therapeutic target for the eradication of AML LSCs.     

Granulocyte-macrophage colony-stimulating factor induces expression of heparin-binding epidermal growth factor-like growth factor/diphtheria toxin receptor and sensitivity to diphtheria toxin in human neutrophils.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a widely expressed EGF superfamily member that induces mitogenic and/or chemotactic activities toward different cell types through binding to EGF receptors 1 or 4. Membrane-bound HB-EGF exerts growth activity and adhesion capabilities and possesses the unique property of being the receptor for diphtheria toxin (DT). Using molecular and functional techniques, we show that human polymorphonuclear granulocytes (PMN), which did not express HB-EGF in resting conditions, expressed it at mRNA and protein level, following incubation with granulocyte-macrophage colony-stimulating factor (GM-CSF). Other classic agonists for PMN (including lipopolysaccharide, phagocytable particles, tumor necrosis factor-alpha, or G-CSF) failed to induce HB-EGF. The effects of GM-CSF on HB-EGF mRNA levels were concentration-dependent, reached a plateau after 1 to 2 hours of stimulation, and did not require protein synthesis. After GM-CSF treatment, membrane-bound HB-EGF was detected by flow cytometry. At the same time, PMN acquired sensitivity to the apoptosis-promoting effect of DT, which, moreover, specifically suppressed the GM-CSF-induced priming of formyl-methionyl-leucyl-phenylalanine-stimulated superoxide anion release. Finally, soluble HB-EGF was detected in the PMN culture medium by a specific enzyme-linked immunosorbent assay. Thus, we provide evidence that HB-EGF is specifically inducible by GM-CSF in PMN and represents a novel peptide to be included in the repertoire of PMN-derived cytokines.     

Acute myeloid leukemia cells in G0 phase of the cell cycle that are unresponsive to conventional chemotherapy are sensitive to treatment with granulocyte-macrophage colony-stimulating factor/diphtheria toxin fusion proteins

OBJECTIVE: Unresponsiveness to chemotherapy is a major problem in the treatment of leukemia, which can be caused by unresponsiveness of noncycling cells to cell cycle-dependent cytotoxic agents. Targeted toxins consisting of a targeting and activating cytokine (granulocyte-macrophage colony-stimulating factor [GM-CSF]) and diphtheria toxin (DT) can be used to overcome this kind of resistance of leukemic cells. In this study we manipulated the cell cycle and proliferative status of leukemic cells, explored the effect on sensitivity to DT, and determined the ability of DT388GMCSF fusion proteins to activate and subsequently kill leukemic cells. MATERIALS AND METHODS: We used the GM-CSF-dependent myeloid leukemic cell line AML-193 as a model. GM-CSF or granulocyte colony-stimulating factor (G-CSF) was used to manipulate the cell cycle and proliferative state of AML-193 cells. Cell death was quantified by 51Cr release assays. The results obtained in the AML-193 cell line model were confirmed using primary leukemic blasts. RESULTS: Similar to treatment with chemotherapy and immunotherapy, leukemic cells in resting G0 phase were relatively resistant to DT-induced cell death. Synchronized recruitment of leukemic cells into activated phases of the cell cycle by low concentrations of GM-CSF or G-CSF resulted in significant increased DT sensitivity. DT388GMCSF fusion proteins specifically targeted GM-CSF receptor-expressing cells, resulting in recruitment of leukemic cells from G0 phase of the cell cycle and subsequent kill of these cells. CONCLUSION: Leukemic cells in G0 phase, which are resistant to conventional chemotherapy, Fas-induced immunotherapy, and DT alone, can be synchronically activated and subsequently killed by DT388GMCSF fusion proteins.     

Autologous peripheral blood stem cell transplantation with granulocyte colony-stimulating factor combined conditioning regimen as a postremission therapy for acute myelogenous leukemia in first complete remission

We retrospectively analyzed the outcomes of 81 patients with non-M3 acute myelogenous leukemia (AML) in first complete remission (CR1) who were treated with high-dose chemotherapy (HDCT) and autologous peripheral blood stem cell transplantation (Auto-PBSCT) by the Fukuoka Blood and Marrow Transplantation Group between 1989 and 2005. Cytogenetically, 16 patients were defined as good risk, 56 as intermediate risk, and nine as poor risk, following the Southwest Oncology Group criteria. The pre-transplant conditioning regimen consisted of high-dose busulfan, etoposide, and cytarabine (BEA regimen), combined with priming by granulocyte colony-stimulating factor (G-CSF). Disease-free survival (DFS) and overall survival at 5 years were 64.0 % (95 % CI 52.5–73.4) and 66.4 % (95 % CI 54.9–75.6) after Auto-PBSCT at a median follow-up time of 103 months (range 3–240 months), respectively. Two patients died of transplant-related pulmonary complications 6 months after Auto-PBSCT without relapse. The 5-year DFS rates of patients in the genetically good-, intermediate-, and poor-risk groups were 80.8, 64.3, and 33.3 %, respectively, but there was no significant difference statistically among the risk groups (log-rank p = 0.0579). These observations suggest that HDCT supported by Auto-PBSCT with the BEA regimen combined with G-CSF priming is a therapeutic option for postremission therapy of AML in CR1.     

Heat shock protein 90 inhibition sensitizes acute myelogenous leukemia cells to cytarabine

Previous studies demonstrated that ataxia telangiectasia mutated- and Rad3-related (ATR) kinase and its downstream target checkpoint kinase 1 (Chk1) facilitate survival of cells treated with nucleoside analogs and other replication inhibitors. Recent results also demonstrated that Chk1 is depleted when cells are treated with heat shock protein 90 (Hsp90) inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG). The present study examined the effects of 17-AAG and its major metabolite, 17-aminogeldanamycin (17-AG), on Chk1 levels and cellular responses to cytarabine in human acute myelogenous leukemia (AML) cell lines and clinical isolates. Cytarabine, at concentrations as low as 30 nM, caused activating phosphorylation of Chk1, loss of the phosphatase Cdc25A, and S-phase slowing. Conversely, treatment with 100 to 300 nM 17-AAG for 24 hours caused Chk1 depletion that was accompanied by diminished cytarabine-induced S-phase accumulation, decreased Cdc25A degradation, and enhanced cytotoxicity as measured by inhibition of colony formation and induction of apoptosis. Additional studies demonstrated that small inhibitory RNA (siRNA) depletion of Chk1 also sensitized cells to cytarabine, whereas disruption of the phosphatidylinositol 3-kinase (PI3k) signaling pathway, which is also blocked by Hsp90 inhibition, did not. Collectively, these results suggest that treatment with 17-AAG might represent a means of reversing checkpoint-mediated cytarabine resistance in AML.     

Granulocyte colony-stimulating factor enhances the in vitro cytotoxicity of gemtuzumab ozogamicin against acute myeloid leukemia cell lines and primary blast cells

OBJECTIVE: To evaluate the effects of granulocyte colony-stimulating factor (G-CSF) on the in vitro sensitivity of acute myeloid leukemia (AML) cell lines and primary AML blast cells to gemtuzumab ozogamicin (GO). MATERIALS AND METHODS: AML cell lines and primary blasts from 10 patients with AML were first incubated for 72 hours in the presence of G-CSF (5 or 100 ng/mL) and then exposed to increasing concentrations of GO (1-1,000 ng/mL) for an additional 72 hours. RESULTS: Pretreatment with G-CSF translated into significant enhancement of GO-induced cytotoxicity in the GO-sensitive HL-60 and NB-4 cells. Conversely, the response of GO-insensitive KG-1a, TF-1, and K562 cells was unaffected by in vitro priming with G-CSF. In vitro exposure to G-CSF augmented GO-induced apoptosis in 7 of 10 primary AML samples and rendered blast cells from three refractory patients sensitive to killing effect of GO. The G-CSF-induced increase of the cytocidal activity of GO was independent of effects on the cell cycle and on the expression levels of CD33 antigen. Of potential interest, G-CSF induced dose-dependent inhibition of P-glycoprotein (P-gp/ABCB1) function in the GO-sensitive HL-60 and NB-4 cells and in blasts from three patients with AML that we tested. CONCLUSION: Collectively, our findings point to G-CSF as a potential sensitizing agent that can be exploited therapeutically to improve the clinical efficacy of GO.     

Growth factor priming in therapy of acute myelogenous leukemia

Treatment of acute myelogenous leukemia (AML) incorporates the use of growth factors towards several objectives, including abbreviating the time of neutropenia, reducing susceptibility to infections, reducing length of hospitalization, and increasing susceptibility of the blasts to chemotherapy drugs, or priming. Priming is defined as the driving of leukemia cells into cell cycle in order to increase response to S-phase-specific drugs such as cytarabine (Ara-C). Growth factors that have been used in priming include both filgrastim (G-CSF) and sagramostim (GM-CSF). Priming has often been applied in the treatment of older patients, who do not respond as well to standard treatment regimens, and whose disease was transformed from a myelodysplastic syndrome (MDS), or preleukemia. Recent, large randomized trials have demonstrated that although subgroups of patients may benefit, there does not appear to be any improvement in the complete remission rate sustained by inclusion of growth factor priming as part of the initial therapy of acute myelogenous leukemia.