Mitoxantrone, etoposide and cytarabine following epigenetic priming with decitabine in adults with relapsed/refractory acute myeloid leukemia or other high-grade myeloid neoplasms: a phase 1/2 study.

DNA methyltransferase inhibitors sensitize leukemia cells to chemotherapeutics. We therefore conducted a phase 1/2 study of mitoxantrone, etoposide and cytarabine following 'priming' with 5-10 days of decitabine (dec/MEC) in 52 adults (median age 55 (range: 19-72) years) with relapsed/refractory acute myeloid leukemia (AML) or other high-grade myeloid neoplasms. During dose escalation in cohorts of 6-12 patients, all dose levels were well tolerated. As response rates appeared similar with 7 and 10 days of decitabine, a 7-day course was defined as the recommended phase 2 dose (RP2D). Among 46 patients treated at/above the RP2D, 10 (22%) achieved a complete remission (CR), 8 without measurable residual disease; five additional patients achieved CR with incomplete platelet recovery, for an overall response rate of 33%. Seven patients (15%) died within 28 days of treatment initiation. Infection/neutropenic fever, nausea and mucositis were the most common adverse events. While the CR rate compared favorably to a matched historic control population (observed/expected CR ratio=1.77), CR rate and survival were similar to two contemporary salvage regimens used at our institution (G-CLAC (granulocyte colony-stimulating factor (G-CSF); clofarabine; cytarabine) and G-CLAM (G-CSF; cladribine; cytarabine; mitoxantrone)). Thus, while meeting the prespecified efficacy goal, we found no evidence that dec/MEC is substantially better than other cytarabine-based regimens currently used for relapsed/refractory AML.

Myeloid cell proliferation stimulated by Steel factor is pertussis toxin sensitive and enhanced by cholera toxin.

Effects of G-protein toxins on Steel factor (SLF) and granulocyte-macrophage colony stimulating factor (GM-CSF) stimulated proliferation of human factor-dependent cell line, M07e, were evaluated. Pertussis toxin pretreatment suppressed GM-CSF- or Steel factor-induced proliferation by 54 +/- 8%; however, proliferation induced by the combination of GM-CSF plus Steel factor was suppressed to a much lesser extent (14 +/- 8%). Pretreatment of M07e cells with cholera toxin, suppressed GM-CSF- and GM-CSF plus Steel factor-stimulated proliferation by 57 +/- 6% and 79%, respectively, but increased the proliferative response to Steel factor alone by twofold. Similar effects of pertussis toxin and cholera toxin were observed on proliferation of normal myeloid progenitor cells from human umbilical cord blood. Pertussis toxin treatment of M07e cells for 4 h resulted in the ADP-ribosylation of 40-42 kDa protein band but did not significantly increase cyclic AMP levels. Cholera toxin pretreatment was associated with a 10-fold increase in intracellular cyclic AMP levels. These results implicate pertussis toxin sensitive pathways for both GM-CSF and Steel factor, but suggest that these pathways may not be required for synergistic proliferation stimulated by the combination. In addition, proliferation stimulated by GM-CSF, +/- Steel factor, is sensitive to cholera toxin pretreatment; whereas cholera toxin pretreatment enhanced proliferation stimulated by Steel factor, possibly via increased cyclic AMP. This suggests divergent signal transduction pathways for the two cytokines.

Ligand-dependent polyubiquitination of c-kit gene product: a possible mechanism of receptor down modulation in M07e cells.

Quantities of proteins in cells are balanced by protein synthesis and degradation. Protein ubiquitination is an important adenosine-triphosphate dependent proteolytic pathway for "short-lived" proteins. We show that soluble steel-factor (SLF) stimulation at 37 degrees C rapidly induced polyubiquitination of c-kit protein in growth-factor-dependent human-myeloid cell line M07e, resulting in smeared, retarded migration of c-kit protein in sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the molecular weight region of 145 kD. Receptor ubiquitination was almost completely absent when cells were treated with SLF at 4 degrees C or at 37 degrees C in the presence of 0.2% sodium azide, or when the cells were pretreated with anti-c-kit monoclonal antibody or genistein, a tyrosine-kinase inhibitor. This suggested that c-kit ubiquitination was ligand dependent and appeared to require intrinsic tyrosine-kinase activation of the c-kit protein. Flow-cytometric analysis of c-kit expression on the cell surface of M07e cells showed down modulation of c-kit within 5 minutes after soluble-SLF treatment at 37 degrees C. However, rapid receptor down modulation was almost completely suppressed when cells were treated with SLF at 4 degrees C or at 37 degrees C in the presence of 0.2% sodium azide, conditions that concomitantly suppressed polyubiquitination of c-kit protein. In addition, these conditions almost completely suppressed radiolabeled SLF (125I-SLF) internalization after ligand-receptor interaction. Pulse-chase studies of 35S-methionine-labeled c-kit protein showed that SLF stimulation at 37 degrees C strikingly enhanced c-kit degradation (T1/2; approximately 20 minutes) compared with that in cells stimulated with SLF at 4 degrees C or at 37 degrees C with 0.2% sodium azide. However, in the presence of chloroquine, which blocks lysosomal degradation, this ligand-induced c-kit degradation at 37 degrees C was only suppressed in part. These data suggest that SLF-induced polyubiquitination of the c-kit receptor protein may play a role in regulation of c-kit-encoded protein-receptor expression in M07e cells.

Recombinant human interleukin-9 induces protein tyrosine phosphorylation and synergizes with steel factor to stimulate proliferation of the human factor-dependent cell line, M07e.

Human interleukin-9 (IL-9) was originally identified and cloned based on its stimulatory effect on proliferation of human myeloid cell line, M07e. IL-9 synergized with Steel factor, the ligand for the c-kit product, to stimulate M07e cell proliferation. To investigate potential mechanisms for this, IL-9 was assessed for effects on protein tyrosine kinase activities in M07e cells by immunoblotting with anti-phosphotyrosine monoclonal antibody; results were compared with those of Steel factor alone and in combination with IL-9, and those of 12-0-tetradecanoyl phorbol-13-acetate (TPA). Recombinant human IL-9 (10 ng/mL) rapidly and transiently induced or enhanced at least four tyrosine phosphorylated protein bands with molecular weights of 105, 97, 85, and 81 Kd. This tyrosine phosphorylation pattern was different from that generated by recombinant murine Steel factor or TPA stimulation and the combination of IL-9 and Steel factor did not change the IL-9-induced pattern. IL-9-induced tyrosine phosphorylated bands were completely blocked by treatment of IL-9 with anti-IL-9 antibody under conditions that also neutralized the synergistic effect of IL-9 with Steel factor on M07e cell proliferation. Genistein, a tyrosine kinase inhibitor, blocked phosphorylation of IL-9 and Steel factor-induced bands. Unlike Steel factor or TPA, IL-9 did not appear to stimulate phosphorylation of 42-Kd mitogen-activated protein (MAP) kinase or Raf-1, or enhance MAP kinase activity. MAP kinase and Raf-1 are serine/threonine kinases that are phosphorylated and activated by many growth factors and by agonists for protein kinase C. While the combination of IL-9 plus SLF did not appear to induce phosphorylation of new bands not already seen with either IL-9 or SLF alone, or enhance the phosphorylation of those bands seen with either cytokine alone, the results suggest that IL-9 activates specific and unique signal transduction pathways.

The kit receptor and its ligand, steel factor, as regulators of hemopoiesis.

Mouse strains carrying mutations at the Dominant White Spotting (W) locus or the Steel (Sl) locus are anemic and display defects in pigmentation and gametogenesis. In W mutants the anemia is due to a deficiency of hemopoietic stem cells and, in Sl mutants, to a deficiency of supporting stromal cells in the bone marrow. The W locus encodes the c-kit proto-oncogene product, a cell surface receptor with protein-tyrosine kinase activity, and the Sl locus encodes its ligand, a hemopoietic cytokine known variously as Steel factor (SLF), mast cell growth factor, stem cell factor, and Kit ligand. SLF can synergize with a number of other cytokines to stimulate growth of hemopoietic progenitors in vitro and stimulates blood cell production in vivo in animals. Here we review the biological activities of SLF, with particular emphasis on its effects on hemopoietic stem and progenitor cells. We also discuss present knowledge of the molecules involved in SLF-triggered signal transduction, and speculate on potential therapeutic applications for SLF in human disease.

Comparative analysis of signaling pathways between mast cell growth factor (c-kit ligand) and granulocyte-macrophage colony-stimulating factor in a human factor-dependent myeloid cell line involves phosphorylation of Raf-1, GTPase-activating protein and mitogen-activated protein kinase.

Mast cell growth factor (MGF, the ligand for c-kit receptor) can stimulate proliferation of factor dependent myeloid cell line, M07e, and MGF synergizes with granulocyte-macrophage colony-stimulating factor (GM-CSF) or IL-3 in this effect. The effect of MGF on protein tyrosine kinase activity in M07e cells was investigated by immunoblotting with anti-phosphotyrosine mAb and this was compared with effects of GM-CSF. MGF stimulation rapidly induced or enhanced at least 12 tyrosine phosphorylated bands. Major bands had molecular weights of 145, 120, 110, 98, 62, 55 and 42 kD. P145, the most prominent phosphorylated protein, was identified as c-kit product using anti-c-kit-mAb (YB5.B8), suggesting ligand-dependent receptor autophosphorylation. Five of six tyrosine phosphorylated bands induced or enhanced by GM-CSF stimulation comigrated with those tyrosine phosphorylated by MGF (138, 120, 76, 55 and 42 kD). P42 was identified, at least in part, as mitogen-activated protein (MAP) kinase. MGF induced tyrosine phosphorylation of a complex of GTPase-activating protein (GAP, 120 kD) and GAP associated proteins (p62/p190) as detected by anti-GAP Ab immunoprecipitation followed by immunoblotting with anti-phosphotyrosine mAb. GM-CSF also stimulated slightly but consistently tyrosine phosphorylation of GAP and p190 but not p62. Both MGF and GM-CSF enhanced Raf-1 phosphorylation and increased Raf-1 associated kinase activity in vitro. Phosphoamino acid analysis revealed Raf-1 phosphorylation by these two growth factors occurred almost exclusively on serine residues. No tyrosine phosphorylation of Raf-1 protein was detected. These data suggest shared and unshared components of signaling pathways of both factors, which may be involved in cell proliferation.

Mast cell growth factor (c-kit ligand) enhances cytokine stimulation of proliferation of the human factor-dependent cell line, M07e.

Murine mast cell growth factor (muMGF), a c-kit ligand, has additive to greater-than-additive effects on in vitro colony formation of murine and human myeloid progenitor cells stimulated with erythropoietin, granulocyte-macrophage colony-stimulating factor (GM-CSF), and/or interleukin (IL)-3. To confirm direct-acting effects on responding cells, MGF was assessed alone and in combination with other cytokines for effects on the proliferation of the human factor-dependent cell line, M07e. Proliferation was assayed in liquid culture by [3H]thymidine uptake and in semisolid medium by colony formation. Purified recombinant (r) muMGF (25-50 ng/ml) by itself had proliferative activity but less than r human (hu) GM-CSF. In combination with rhuGM-CSF (250 U/ml) or IL-3 (500 U/ml), rmuMGF (25 ng/ml) enhanced [3H]thymidine uptake two- to sevenfold greater than the sum of the effects of each factor alone. Similar enhancement was seen in the number and size of colonies formed. When MGF was used in combination with rhuIL-4 (500-1000 U/ml), rhuIL-6 (5 ng/ml), rhuIL-9 (5-10 U/ml), or rhu interferon gamma (IFN-gamma; 250-500 U/ml) (factors that alone stimulate little proliferation), [3H]thymidine uptake and colony formation were respectively increased 2- to 11- and 3- to 55-fold over the sum of each of the effects of the factors alone. Exposure of 5 x 10(5) cells/ml to 50 ng/ml MGF for 24 h, a time during which synergism is noted with MGF plus either GM-CSF or IL-3, did not change GM-CSF or IL-3 receptor binding affinity or the number of binding sites. Exposure of cells to MGF for 48 h did not alter subsequent GM-CSF- or IL-3-stimulated proliferation. The results suggest that M07e cells will be useful as a model for the analysis of intracellular biochemical mechanisms of the direct-acting proliferative and synergistic effects of MGF.

CD45 cell surface antigens are linked to stimulation of early human myeloid progenitor cells by interleukin 3 (IL-3), granulocyte/macrophage colony-stimulating factor (GM-CSF), a GM-CSF/IL-3 fusion protein, and mast cell growth factor (a c-kit ligand).

CD45 antigens are protein tyrosine phosphatases. A possible link was evaluated between expression of CD45 antigens on human myeloid progenitor cells (MPC) (colony-forming unit-granulocyte/macrophage [CFU-GM], burst-forming unit-erythroid [BFU-E], and colony-forming unit-granulocyte/erythroid/macrophage/megakaryocyte [CFU-GEMM]) and regulation of MPC by colony-stimulating factors (CSF) (interleukin 3 [IL-3], GM-CSF, G-CSF, M-CSF, and erythropoietin [Epo]), a GM-CSF/IL-3 fusion protein, and mast cell growth factor (MGF; a c-kit ligand). Treatment of cells with antisense oligodeoxynucleotides (oligos) to exons 1 and 2, but not 4, 5, or 6, of the CD45 gene, or with monoclonal anti-CD45, significantly decreased CFU-GM colony formation stimulated with GM-CSF, IL-3, fusion protein, and GM-CSF + MGF, but not with G-CSF or M-CSF. It also decreased GM-CSF, IL-3, fusion protein, and MGF-enhanced Epo-dependent BFU-E and CFU-GEMM colony formation, but had little or no effect on BFU-E or CFU-GEMM colony formation stimulated by Epo alone. Similar results were obtained with unseparated or purified (greater than or equal to one of two cells being a MPC) bone marrow cells. Sorted populations of CD343+ HLA-DR+ marrow cells composed of 90% MPC were used to demonstrate capping of CD45 after crosslinking protocols. Also, a decreased percent of CD45+ cells and CD45 antigen density was noted after treatment of column-separated CD34+ cells with antisense oligos to exon 1 of the CD45 gene. These results demonstrate that CD45 cell surface antigens are linked to stimulation of early human MPC by IL-3, GM-CSF, a GM-CSF/IL-3 fusion protein, and MGF.