Colony-forming Unit Granulocyte-macrophage Colony Formation Research Articles

NS-018, a selective JAK2 inhibitor, preferentially inhibits CFU-GM colony formation by bone marrow mononuclear cells from high-risk myelodysplastic syndrome patients

JAK2/STAT signaling promotes survival and expansion of myelodysplastic syndrome (MDS) clones, but little is known about the potential of JAK2/STAT as a therapeutic target in MDS. We investigated the effect of NS-018, a novel antagonist for JAK2, on the colony-forming ability of bone marrow mononuclear cells (BMMNCs) from high-risk MDS patients. NS-018 decreased colony-forming unit-granulocyte/macrophage (CFU-GM) colony numbers from MDS-derived BMMNCs in a dose-dependent manner, and this effect was significantly more potent than against normal BMMNCs. In addition, NS-018 suppressed the phosphorylation of STAT3 in colony-forming cells from MDS patients. Collectively, NS-018 could be a new therapeutic option for high-risk MDS.

The JAK2-Selective Inhibitor NS-018 Preferentially Suppresses CFU-GM Colony Formation By Bone-Marrow Cells From High-Risk Myelodysplastic Syndrome Patients

BackgroundCell signaling mediated by the JAK2-STAT (Janus kinase 2-signal transducer and activator of transcription) pathway plays a critical role in hematopoiesis, and its aberrant activation is associated with the progression of hematological malignancies. For instance, somatic mutations in the JAK2 gene that lead to constitutive activation of STATs are involved in the pathogenesis of myeloproliferative neoplasms (MPN) and refractory anemia with ringed sideroblasts with thrombocytosis, one of the myelodysplastic/myeloproliferative neoplasms, unclassifiable (MDS/MPN, U). In addition, insufficient inactivation of the JAK-STAT pathway due to repression of SOCS-1 (suppressor of cytokine signaling 1) through hypermethylation of the SOCS-1 gene is involved in the disease progression of high-risk myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) (Brakensiek et al.; Br. J. Haematol. 2005;130:209). These observations prompted us to investigate the effect of the JAK2-selective inhibitor NS-018, which is in an early-phase clinical trial for MPN, on the colony formation by bone marrow hematopoietic progenitor cells from high-risk de novo MDS patients and the phosphorylation status of STAT3 in these cells. MethodsBone marrow mononuclear cells (BMMNCs) from six MDS patients and three healthy volunteers were collected with informed consent in accordance with the Declaration of Helsinki and with the approval of the Institutional Review Board. The MDS subtypes of the six patients included two RCMD, three RAEB-1 and one RAEB-2 according to the WHO classification. All MDS patients had complex cytogenetic abnormalities and their prognostic risks were defined to be high (N=1) or very high (N=5) in the IPSS-R. Commercially available normal human CD34+ BM cells were also examined for comparison. Cells were incubated in methylcellulose medium containing cytokines with or without NS-018. Burst forming unit-erythroid (BFU-E) and colony forming unit-granulocyte/macrophage (CFU-GM) were scored on day 14 of culture. Colony-forming cells were then collected and subjected to western blotting analysis. ResultsWe first examined the colony-formation capacity of MDS-derived BMMNCs. CFU-GM colony formation was observed in four of six MDS samples, although the absolute CFU-GM numbers from MDS BMMNCs were less than those from normal BMMNCs and normal CD34+BM cells. MDS-derived BMMNCs produced larger numbers of CFU-GM colonies (78–99% of total colonies) than BFU-E, similar to the findings usually observed in AML, whereas normal BMMNCs and normal CD34+BM cells formed virtually the same numbers of CFU-GM colonies and BFU-E colonies. These results suggest that BMMNCs from four of the MDS patients enrolled in this study had characteristics similar to AML BMMNCs, although they showed less proliferation than AML BMMNCs. We next examined the effect of NS-018 on CFU-GM colony formation. NS-018 treatment decreased the numbers of CFU-GM colonies from MDS-derived BMMNCs in a dose-dependent manner, and this effect was significantly more potent against MDS-derived than against normal cells (57.2% inhibition in MDS cells vs. 12.8% inhibition in normal cells at 0.5 mM NS-018; see Figure). These results indicate that NS-018 is preferentially efficacious in inhibiting CFU-GM formation from BMMNCs from high risk MDS. In addition, the level of phospho-STAT3 in MDS-derived colony-forming cells was twice as high as in colony-forming cells from normal BMMNCs, and 1.0 mM NS-018 completely suppressed the phosphorylation of STAT3 in CFU-GM colony-forming cells from MDS. ConclusionOur results show for the first time that a JAK2 inhibitor, NS-018, potently suppresses the formation of MDS colonies. NS-018 could be a new therapeutic option for high-risk MDS patients. [Display omitted] Disclosures:Kodama:Nippon Shinyaku: Employment. Naito:Nippon Shinyaku: Employment. Nakaya:Nippon Shinyaku: Employment. Taniwaki:Novartis: Honoraria.

Expression of either NF-kappaB p50 or p52 in osteoclast precursors is required for IL-1-induced bone resorption.

Interleukin (IL)-1 is implicated in postmenopausal- and inflammation-mediated bone loss. Its expression is regulated by NF-kappaB and vice versa. To examine the role of NF-kappaB p50 and p52 (they are required for osteoclast formation during embryonic development) in IL-1-induced resorption, we used various NF-kappaB knockout (KO) mice, including p50-/- and p52-/- single KO, p50-/- and p52+/- (3/4KO), and p50-/- and p52-/- double KO (dKO) mice. IL-1 increased blood calcium and bone resorption in wild-type (wt), p50, and p52 single KO mice, but not in 3/4KO or dKO mice. Osteoclast formation was impaired in bone marrow cultures from 3/4KO compared with single KO and wt mice treated with IL-1. IL-1 receptor expression was similar in colony forming unit-granulocyte macrophage (CFU-GM) colony cells from wt and dKO mice. However, IL-1 promoted CFU-GM colony formation and survival as well as the formation, activity, and survival of osteoclasts generated from these colonies from wt mouse splenocytes, but not from dKO splenocytes. No difference in expression of the osteoclast regulatory cytokines, RANKL, and OPG, was observed in osteoblasts from wt and dKO mice. Thus, expression of either NF-kappaB p50 or p52 is required in osteoclasts and their precursors, rather than osteoblasts, for IL-1-mediated bone resorption.

Adenovirus-mediated delivery of p53 results in substantial apoptosis to myeloma cells and is not cytotoxic to flow-sorted CD34(+) hematopoietic progenitor cells and normal lymphocytes.

Multiple myeloma (MM) is an incurable disease; therefore, there is a need for new modalities of treatment for this disease. We designed a study to test the sensitivity of MM cell lines, freshly isolated myeloma cells, and CD34(+) hematopoietic progenitor cells to adenovirus-mediated delivery of wild-type p53 (Ad-p53). Replication-deficient Ad-p53, previously used in phase I-II clinical trial for treatment of patients with solid tumors, was used in this study. Myeloma cells from seven MM cell lines with mutated or w.t. p53 and varying expression of bcl-2 were used. Fresh myeloma cells (CD38(bright)CD45(-)) and fresh CD34(+) hematopoietic stem cells and CD34(-) cells were purified by flow sorting of apheresis collections of MM patients undergoing high-dose chemotherapy and stem cell rescue. The effect of Ad-p53 on colony-forming unit granulocyte-macrophage (CFU-GM) and burst-forming unit erythroid (BFU-E) colony formation in methylcellulose was tested on purified CD34(+) and CD34(-) cells to evaluate bone marrow toxicity. Myeloma cells from cell lines, or freshly isolated myeloma cells, were sensitive to Ad-p53 only if they had mutated p53 and had low expression of bcl-2. CD34(+) cells were resistant to Ad-p53-mediated apoptosis, and CFU-GM and BFU-E colony formation was not affected by treatment with Ad-p53.Ad-p53 is a potent inducer of apoptosis in MM cell lines and in freshly isolated myeloma cells expressing low levels of bcl-2. Ad-p53 is not overtly cytotoxic to normal hematopoietic stem cells or normal lymphocytes; therefore, it could be considered for a phase I clinical trial of MM patients with mutated p53.

Altered bone marrow cell sensitivity in the lupus-prone NZB/W mouse: regulation of CFU-GM colony formation by estrogen, tamoxifen and thrombopoietin.

Estrogen is thought to contribute to the onset of systemic lupus erythematosus (SLE) in women through mechanisms that are not completely understood. Although estrogen serves as a negative regulator in normal hematopoietic development, little research has been conducted examining alteration in hematopoietic development triggered by estrogen in lupus-susceptible individuals. We examined whether estrogen and other factors could influence colony formation of bone marrow cells obtained from normal and lupus-susceptible mice. Bone marrow cells isolated from New Zealand Black (NZB) and lupus-prone New Zealand Black and New Zealand White cross (NZB/W) mice were cultured in the presence of granulocyte-macrophage colony stimulating factor (GM-CSF) alone or in combination with estrogen, thrombopoietin (TPO), tamoxifen, estrogen and TPO. or estrogen and tamoxifen, and plated in methylcellulose culture medium. Plates were scored for the number of CFU-GM (colony forming unit granulocyte-macrophage) colonies after 6d in culture. For females of both mouse strains, estrogen significantly decreased (P < 0.05) the number of GM colonies. Co-treatment of NZB/W cells, but not NZB cells, with TPO or tamoxifen reversed the suppressive action of estrogen (P < 0.05). In contrast, while estrogen did suppress colony formation from cells of NZB/W males (P < 0.05), neither TPO nor tamoxifen reversed this effect. Our results indicate that the sensitivity of bone marrow cells isolated from both female and male NZB/W lupus-prone mice to hormones/growth factors is qualitatively different from cells of NZB mice, and suggest that hematopoietic alterations at the level of the bone marrow may be related to the pathogenesis of SLE.

A Functional Analysis of Protooncogene Vav's Role in Adult Human Hematopoiesis

The Vav protooncogene is expressed almost exclusively in hematopoietic cells, but its role in regulating adult human hematopoietic cell development remains uncertain. To analyze Vav function in adult blood cell formation, we used antisense (AS) oligodeoxynucleotides (ODN) to disrupt its expression in normal and malignant human hematopoietic cells. Bone marrow or peripheral blood mononuclear cells (MNC) were obtained from consenting normal donors and patients with acute or chronic myelogenous leukemia (AML and CML, respectively) and polycythemia vera (PV). Adherent and T-cell-depleted (A-T-) MNC or CD34+ MNC were exposed to unmodified sense, antisense, or scrambled sequence ODN corresponding to codons 2-7 of Vav's mRNA sequence. Cells were then assayed for Vav mRNA expression by reverse transcription-polymerase chain reaction and Vav protein expression by Western binding. After showing that Vav-targeted AS ODN could specifically diminish Vav mRNA and protein expression, we assessed the ability of Vav-deficient cells to form myeloid and erythroid colonies in methyl-cellulose cultures. When normal CD34+ MNC were exposed to Vav AS ODN, no effect on colony-forming unit-granulocyte-macrophage (CFU-GM) or CFU-megakaryocyte colony formation was observed. In contrast erythroid colony growth was inhibited by a mean +/- SD of 62% +/- 16%. In patients with hematopoietic malignancies. Vav-targeted AS ODN inhibited CFU-GM colony formation in a sequence-specific and dose-dependent manner in 1 of 3 AML, 13 of 17 CML, and 2 of 2 PV patients. At the highest concentration used, the Vav AS ODN inhibited CFU-GM colony formation from 66% to 81% when compared with control cell colony growth. Burst-forming unit-erythroid (BFU-E) colony-formation was also assessed in 7 PV patients. The Vav-targeted AS ODN inhibited BFU-E colony formation in all by a mean +/- SD of 81% +/- 4%. These findings suggest that Vav function may not be easily complemented in a significant subset of normal adult erythroid progenitor cells and may also be necessary for myeloid progenitor cell growth in a variety of hematopoietic malignancies.

Modulation of myeloid proliferation and differentiation by monoclonal antibodies directed against a protein that interacts with the interleukin-3 receptor

Hematopoietic cells can be transformed through the acquisition of autocrine growth factor production. Because of their ability to inhibit autocrine growth, antibodies directed against the growth factor or its receptor may have therapeutic potential. However, these agents may also inhibit normal cell development. We have developed two monoclonal antibodies, 4G8 and 2F2, directed against a protein of 110 to 150 Kd that interacts with the interleukin-3 (IL-3) receptor (R) complex. These antibodies inhibit IL-3-induced proliferation of nonleukemic and leukemic IL-3-dependent cell lines, as well as the autonomous growth of WEHI-3B in vitro and in vivo. These results suggest the possibility that anti-IL-3R antibodies may be useful in the treatment of some leukemias. However, the effect of anti-IL-3R antibodies on normal myeloid development in vitro has not been examined. We examined the effect of 4G8 and 2F2 on the growth in vitro of colony-forming unit granulocyte-macrophage (CFU-GM) colonies induced by IL-3, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), and macrophage-CSF (M-CSF). Our results show that while 4G8 and 2F2 inhibited CFU-GM colony formation induced by IL-3, they augmented colony formation induced by the other hematopoietins. 4G8 and 2F2 also enhanced G-CSF-induced proliferation of 32Dc13 and GM-CSF-induced proliferation of PT18, confirming that the effect on CFU-GM was a direct effect. Finally, 4G8 and 2F2 inhibited G-CSF-induced differentiation of 32Dc13, similar to low levels of IL-3; yet, neither 4G8 nor 2F2 blocked binding of G-CSF to its receptor. These results indicate that, in the absence of IL-3 and in the presence of other hematopoietins, 4G8 and 2F2 can function as weak IL-3 agonists. These studies suggest that antibodies such as 4G8 and 2F2, directed against components of the IL-3R, could potentially augment myeloid growth in vivo, rather than inhibit myeloid growth.

Modulation of Myeloid Proliferation and Differentiation by Monoclonal Antibodies Directed Against a Protein That Interacts With the Interleukin-3 Receptor

Abstract Hematopoietic cells can be transformed through the acquisition of autocrine growth factor production. Because of their ability to inhibit autocrine growth, antibodies directed against the growth factor or its receptor may have therapeutic potential. However, these agents may also inhibit normal cell development. We have developed two monoclonal antibodies, 4G8 and 2F2, directed against a protein of 110 to 150 Kd that interacts with the interleukin-3 (IL-3) receptor (R) complex. These antibodies inhibit IL-3-induced proliferation of nonleukemic and leukemic IL-3-dependent cell lines, as well as the autonomous growth of WEHI-3B in vitro and in vivo. These results suggest the possibility that anti-IL-3R antibodies may be useful in the treatment of some leukemias. However, the effect of anti-IL-3R antibodies on normal myeloid development in vitro has not been examined. We examined the effect of 4G8 and 2F2 on the growth in vitro of colony-forming unit granulocyte-macrophage (CFU-GM) colonies induced by IL-3, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), and macrophage-CSF (M-CSF). Our results show that while 4G8 and 2F2 inhibited CFU-GM colony formation induced by IL-3, they augmented colony formation induced by the other hematopoietins. 4G8 and 2F2 also enhanced G-CSF-induced proliferation of 32Dc13 and GM-CSF-induced proliferation of PT18, confirming that the effect on CFU-GM was a direct effect. Finally, 4G8 and 2F2 inhibited G-CSF-induced differentiation of 32Dc13, similar to low levels of IL-3; yet, neither 4G8 nor 2F2 blocked binding of G-CSF to its receptor. These results indicate that, in the absence of IL-3 and in the presence of other hematopoietins, 4G8 and 2F2 can function as weak IL-3 agonists. These studies suggest that antibodies such as 4G8 and 2F2, directed against components of the IL-3R, could potentially augment myeloid growth in vivo, rather than inhibit myeloid growth.

Macrophage inflammatory protein (MIP)-1 beta abrogates the capacity of MIP-1 alpha to suppress myeloid progenitor cell growth.

The effects of recombinant murine macrophage inflammatory protein (MIP)-1 beta and MIP-2 on the suppressive activity of MIP-1 alpha were tested using colony formation by human and murine bone marrow burst-forming unit-erythroid (BFU-E), colony-forming unit-granulocyte erythroid macrophage, megakaryocyte (CFU-GEMM), and colony-forming unit-granulocyte macrophage (CFU-GM) progenitor cells. MIP-1 beta, but not MIP-2, when added with MIP-1 alpha to cells, blocked the suppressive effects of MIP-1 alpha on both human and murine BFU-E, CFU-GEMM, and CFU-GM colony formation. Similar results were observed regardless of the early acting cytokines used: human rGM-CSF plus human rIL-3, and two recently described potent cytokines, a genetically engineered human rGM-CSF/IL-3 fusion protein and MGF, a c-kit ligand. The more potent the stimuli, the greater the suppressive activity noted. Pulse treatment of hu bone marrow cells with MIP-1 alpha at 4 degrees C for 1 h was as effective in inhibiting colony formation as continuous exposure of cells to MIP-1 alpha, and the pulsing effect with MIP-1 alpha could not be overcome by subsequent exposure of cells to MIP-1 beta. Also, pulse exposure of cells to MIP-1 beta blocked the activity of subsequently added MIP-1 alpha. For specificity, the action of a nonrelated myelosuppressive factor H-ferritin, was compared. MIP-1 alpha and H-ferritin were shown to act on similar target populations of early BFU-E, CFU-GEMM, and CFU-GM. MIP-1 beta did not block the suppressive activity of H-ferritin. Also, hemin and an inactive recombinant human H-ferritin mutein counteracted the suppressive effects of the wildtype H-ferritin molecule, but did not block the suppressive effects of MIP-1 alpha. These results show that MIP-1 beta's ability to block the action of MIP-1 alpha is specific. In addition, the results suggest that MIP-1 alpha and MIP-beta can, through rapid action, modulate early myeloid progenitor cell proliferation.

Transforming growth factor beta inhibits formation of osteoclast-like cells in long-term human marrow cultures.

Transforming growth factor beta (TGF-beta), a polypeptide present in abundant amounts in bone matrix, was examined for its effects on osteoclast formation by using a human bone marrow culture system in which multinucleated cells (MNCs) with osteoclast characteristics form. TGF-beta strongly inhibited MNC formation at concentrations as low as 1 ng/ml. TGF-beta also completely suppressed the effects of osteotropic factors known to stimulate MNC formation. The inhibitory effect of TGF-beta on osteoclast-like cell formation was more pronounced during the first week of culture, which corresponds to the period of proliferation of mononuclear osteoclast precursors. To examine whether the inhibitory effects of TGF-beta on MNC formation could be due to inhibition of the granulocyte/macrophage progenitor cell [colony-forming unit granulocyte/macrophage (CFU-GM)], the probable precursor for MNC, we tested the effects of TGF-beta on CFU-GM formation in presence of a source of colony-stimulating factor. Unexpectedly, TGF-beta at concentrations (0.1-1 ng/ml) that were inhibitory for MNC formation enhanced day 7 CFU-GM colony formation. This increase in CFU-GM colony formation seen in cultures containing TGF-beta resulted from significantly more granulocytic colonies being formed in the cultures, suggesting that TGF-beta may induce CFU-GM to differentiate preferentially to cells of the granulocytic lineage. Differentiation of CFU-GM to granulocytes rather than osteoclast precursors in response to TGF-beta would result in inhibition of MNC formation by depleting the precursor pool for MNC. These data suggest that inhibition of osteoclast-like cell formation by TGF-beta may be an important mechanism of control of local bone resorption.