Keiko Okuda

Signaling Domains of the βc Chain of the GM‐CSF/IL‐3/IL‐5 Receptor

Abstract: The granulocyte/macrophage colony‐stimulating factor (GM‐CSF)/interleukin‐3 (IL‐3)/IL‐5 receptors are a family of heterodimeric transmembrane proteins expressed by myeloid lineage cells. Each receptor has a unique ligand‐binding α chain and they share a common β chain (βc chain). Binding of GM‐CSF activates at least one receptor‐associated tyrosine kinase, JAK2, and rapidly induces tyrosine phosphorylation of the GMR βc chain (GMRβ), but not the GMR αchain (GMRα). Mutation of each of the 8 tyrosine residues in the cytoplasmic domain of the human GMRβ to phenylalanine (GMRβF8) reduced tyrosine phosphorylation of GMRβ, SHP2 and SHC, but not JAK2 or STAT5. Interestingly, GMRβ‐F8 was still capable of inducing at least short‐term proliferation and enhancing viability. The role of each individual tyrosine residue was explored by replacing each mutated phenylalanine with the wild‐type tyrosine residue. Tyrosine 577 was found to be sufficient to regenerate GM‐CSF‐dependent phosphorylation of SHC, and any of Y577, Y612, or Y695 were sufficient to regenerate GM‐CSF‐inducible phosphorylation of SHP2. Next, a series of four internal deletion mutants were generated, which deleted small sections from aa 518 to 626. One of these, deleting residues 566‐589 was profoundly defective in signaling and supporting viability, and may identify an important viability signaling domain for this receptor family. Overall, these results indicate that GMRβ tyrosine residues are not necessary for activation of the JAK/STAT pathway, or for proliferation, viability, or adhesion signaling in Ba/F3 cells, although tyrosine residues significantly affect the magnitude of the response. However, internal deletion mutant studies identify critical domains for viability and proliferation.

Suppression of ARG kinase activity by STI571 induces cell cycle arrest through up-regulation of CDK inhibitor p18/INK4c

ARG is a tyrosine kinase closely related to ABL, which is oncogenic when fused to the transcriptional repressor ETV6 (ETS translocation variant 6). In this study, we investigated the growth-inhibitory effect of STI571 (signal transduction inhibitor number 571) on ETV6/ARG-expressing cells and its molecular mechanisms using HT93A, a cell line derived from a patient with AML-M3 carrying t(1;12). STI571 effectively suppressed overall tyrosyl phosphorylation of intracellular proteins including ETV6/ARG fusion protein, as well as the growth of HT93A cells with an IC50 of 200 nM. The growth inhibition was primarily because of cell cycle arrest at G1 phase when cells were treated with 100 nM STI571 for 48 h, and apoptosis was induced after longer exposure (72 h) or by a higher dose (1000 nM). STI571 increased the amount of p18/INK4c after 2 h of culture, when the cell cycle pattern was not yet affected, but not that of other CDK inhibitors (CKI). p18/INK4c was more abundant in G1-enriched fractions than in S- and G2/M-enriched fractions of STI571-treated HT93A cells, suggesting that the upregulation of p18/INK4c expression correlates with the cell cycle arrest. Treatment of HT93A cells with antisense oligonucleotides against the Ink4c gene abrogated the growth inhibition by STI571. These results suggest that leukemogenesis by an aberrant ARG kinase involves the suppression of p18/INK4c, which is ubiquitously expressed and considered the major CKI in hematopoietic stem cells. STI571 can be an effective drug for the treatment of leukemias with deregulated ARG kinase activity.

Signal through gp130 Activated by Soluble Interleukin (IL)‐6 Receptor (R) and IL‐6 or IL‐6R/IL‐6 Fusion Protein Enhances Ex Vivo Expansion of Human Peripheral Blood‐Derived Hematopoietic Progenitors

This study was designed to investigate the effects of a combination of soluble interleukin (sIL)‐6 receptor (R) and IL‐6 on the ex vivo expansion of human peripheral blood (PB)‐derived hematopoietic progenitor cells in a short‐term serum‐free liquid suspension culture system, using PB‐derived CD34+IL‐6R+/– cells as a target. In combination with stem cell factor (SCF), IL‐3, and sIL‐6R/IL‐6, the expansion efficiency (EE) for granulocyte/macrophage colony‐forming unit (CFU‐GM) reached a peak level on day 10 of incubation. On the other hand, the EE for erythroid burst (BFU‐E) and mixed colony‐forming unit (CFU‐Mix) reached a peak level on day 7 of incubation. Among the cytokine combinations tested, SCF + IL‐3 + sIL‐6R/IL‐6 + flt3 ligand (FL) most effectively expanded CFU‐GM and CFU‐Mix. The maximum EEs for CFU‐GM and CFU‐Mix were 208‐fold and 42‐fold, respectively. While the EE for BFU‐E was 70‐90‐fold in the presence of SCF + IL‐3 + sIL‐6R/IL‐6, FL significantly augmented the EE for CFU‐GM and CFU‐Mix. In contrast, thrombopoietin (TPO) significantly augmented the EE for CFU‐Mix. Interestingly, in combination with IL‐3 and SCF, newly generated IL‐6R/IL‐6 fusion protein (FP) expanded PB‐derived BFU‐E and CFU‐Mix twice more effectively than a combination of sIL‐6R and IL‐6. These results demonstrated that human PB‐derived committed progenitors were effectively expanded in vitro using sIL‐6R/IL‐6 or FP, in combination with IL‐3, SCF and/or FL or TPO, and that FP may transduce a stronger intracellular signal than a combination of sIL‐6R and IL‐6.

Transformation of Ba/F3 cells and Rat-1 cells by ETV6/ARG

ETV6/ARG, a novel fusion gene composed of the ETV6 HLH oligomerization domain and most of sequences of the ARG protein tyrosine, was recently identified in human leukemia cells. The presence of the ETV6/ARG translocation raises the possibility that the resulting fusion protein functions as an oncogene. However, the transforming activity of the ETV6/ARG protein has not been determined and its contribution to leukemogenesis is therefore unknown. Here we address this question by analysing the oncogenic activity of ETV6/ARG in hematopoietic and fibroblast cells. It is demonstrated that expression of ETV6/ARG confers IL3-independent growth to Ba/F3 cells and anchorage independent growth to Rat-1 fibroblasts. It is also shown that multiple signaling molecules, including PI3K, SHC, ras-GAP and CRK-L, are tyrosine phosphorylated in Ba/F3 cells that express ETV6/ARG. Analysis of four different types of ETV6/ARG transcripts previously identified in the AML-M3 leukemia cell line HT93A suggest that ETV6 HLH domain is required for oncogenic activity. Based upon these results it is concluded that ARG can be activated as an oncogene in human malignancy and that the ETV6/ARG oncoprotein triggers some of the same signaling pathways associated with activated ABL oncogenes.

Interleukin 6 receptor expression by human cord blood- or peripheral blood-derived primitive haematopoietic progenitors implies acquisition of different functional properties

The significance of interleukin 6 receptor (IL‐6R) expression by cord blood (CB)‐ and peripheral blood (PB)‐derived primitive haematopoietic progenitors was investigated. IL‐6R was preferentially expressed by PB‐derived myeloid progenitors. Most PB‐derived erythroid bursts (BFU‐E) and mixed colony‐forming cells (CFU‐Mix) did not express this receptor. However, CB‐derived primitive progenitor cells possessed multipotentiality, irrespective of IL‐6R expression. Interestingly, the long‐term culture‐initiating cell (LTC‐IC) population was enriched in PB‐derived CD34+ IL‐6R+ cells, but the extended LTC‐IC (ELTC‐IC) population, which represents a less mature class of haematopoietic progenitors, seemed to be equally distributed in the IL‐6R+ and IL‐6R− cell populations. In contrast, the number of LTC‐ICs and ELTC‐ICs was similar in CB‐derived CD34+ IL‐6R+ or IL‐6R− cells. It is noteworthy that the number of LTC‐ICs and ELTC‐ICs in CB‐derived CD34+ cells was markedly higher than that in PB‐derived CD34+ cells regardless of IL‐6R expression. Telomerase activity was consistently lower in PB‐derived CD34+ IL‐6R− cells than in CD34+ IL‐6R+ cells. In contrast, telomerase activity was similar in CB‐derived CD34+ IL‐6R+ or IL‐6R− cells. The pattern of telomerase induction upon cytokine stimulation differed between CB‐ and PB‐derived CD34+ IL‐6R+ or IL‐6R− cells. However, overall telomerase activity per dish was well correlated with the proliferative potential of both cell populations, suggesting that induction of telomerase plays an important role in the escape from replicative senescence of primitive haematopoietic progenitors. Collectively, these results suggest that CB‐derived primitive progenitors are less mature than PB‐derived progenitors and that the expression of IL‐6R by primitive haematopoietic progenitors may have different implications for PB‐ and CB‐derived CD34+ cells.

The TEL/ARG leukemia oncogene promotes viability and hyperresponsiveness to hematopoietic growth factors.

The TEL/ARG oncogene associated with acute myeloid leukemia is formed by the t(l;12)(q25;pl3) reciprocal transloca-tion, which fuses part of the TEL gene to the tyrosinc kinasc, c-ARG. In an effort to determine the biological effects and investigate signaling of the TEL/ARG fusion protein, multiple sublines of Ba/F3 cells were generated in which a TEL/ARG complementary DNA was expressed under the control of a tetracycline-inducible promoter. Treatment of these cells with doxycycline, a tetracycline analogue, rapidly induced expression of the TEL/ARG protein. TEL/ARG was heavily phospho-rylated on tyrosine residues and was also found to rapidly induce tyrosine phosphorylation of multiple cellular proteins, including rasGAP, CBL, STATS, PI3K, SHP2, Dok, and SHC. The Ba/F3-tet-TEL/ARG cells remained interleukin (IL)-3 dependent without doxycycline but with doxycycline displayed a marked reduction in cell death in the absence of IL-3. TEL/ ARG cells also displayed an enhanced proliferative response to IL-3 and to insulin-like growth factor 1. At least in Ba/F3 cells, although the growth rate was much lower compared to that with IL-3, TEL/ARG appeared to induce some cell proliferation as an immediate consequence. Nonetheless, the hyperresponsiveness to growth factors reported here is more likely to contribute to the pathogenesis of leukemia.

A chimeric receptor/oncogene that can be regulated by a ligand in vitro and in vivo.

The BCR/ABL oncogene encodes an activated tyrosine kinase that causes human chronic myelogenous leukemia. The mechanism of transformation, however, is complex and not well understood. One of the important contributions of BCR to transformation is believed to be dimerization or oligomerization of ABL, thereby activating ABL tyrosine kinase activity. We reasoned that if ABL was dimerized through other mechanisms, activation of the tyrosine kinase activity should also result, and the activated kinase may also be transforming. Erythropoietin is known to activate its receptor by causing dimerization, and therefore a synthetic oncogene was created by linking the extracytoplasmic and transmembrane domains of the EPO receptor with c-ABL. This chimeric receptor was stably expressed in Ba/F3 cells and, in the absence of EPO, had no detectable biological effect on the cells. EPO, however, induced a rapid, dose-dependent activation of ABL tyrosine kinase activity and phosphorylation of several cellular proteins. The major target proteins have been identified, and are very similar to the known substrates of BCR/ABL, including Shc, CBL, CRKL, and several proteins in the cytoskeleton. EPO treatment also resulted in biological effects that were remarkably similar to those of BCR/ABL, including improved viability, altered integrin function, and a weak mitogenic signal. The biological effects were in part dose-dependent, in that low EPO concentrations enhanced viability but did not cause proliferation. At high EPO doses, kinase activation was maximal, and a mitogenic effect was also revealed. In nude mice, Ba/F3 cells expressing this chimeric receptor did not cause detectable disease without administration of pharmacologic doses of EPO. If EPO was given intraperitoneally 5 days a week, however, a dose-dependent lethal leukemia resulted. This ligand-regulatable oncogene mimics some of the biological effects of BCR/ABL, and analysis of ABL mutants in this system will be useful to dissect the signaling pathways that cause CML.

Constitutively active ABL family kinases, TEL/ABL and TEL/ARG, harbor distinct leukemogenic activities in vivo

ABL (ABL1) and ARG (ABL2) are highly homologous to each other in overall domain structure and amino-acid sequence, with the exception of their C termini. As with ABL, translocations that fuse ARG to ETV6/TEL have been identified in patients with leukemia. To assess the in vivo leukemogenic activity of constitutively active ABL and ARG, we generated a bone marrow (BM) transplantation model using the chimeric forms TEL/ABL and TEL/ARG, which have comparable kinase activities. TEL/ABL rapidly induced fatal myeloid leukemia in recipient mice, whereas recipients of TEL/ARG-transduced cells did not develop myeloid leukemia, instead, they succumbed to a long-latency infiltrative mastocytosis that could be adoptively transferred to secondary recipients. Swapping of the C termini of ABL and ARG altered disease latency and phenotypes. In a detailed in vitro study, TEL/ARG strongly promoted mast cell differentiation in response to stem cell factor or interleukin-3, whereas TEL/ABL preferentially induced myeloid differentiation of hematopoietic stem/progenitor cells. These results indicate that ABL and ARG kinase activate distinct differentiation pathways to induce specific diseases in vivo, that is, myeloid leukemia and mastocytosis, respectively. Further elucidation of the differences in their properties should provide important insight into the pathogenic mechanisms of oncogenes of the ABL kinase family.

A new model to evaluate Raf signaling in hematopoietic cells

The Raf/MEK/ERK pathway is thought to be critical in mediating cell survival and proliferation by cytokine receptors. However, the exact contribution of Raf is complex and not well understood. A better understanding of Raf signaling is important because of the recent observation that B-Raf is frequently mutated in various human cancers. We have generated a new model system that activates Raf directly by linking the extracytoplasmic and transmembrane domains of the erythropoietin receptor (EPOR) with the catalytic domain of Raf (CR3). This synthetic oncogene in which dimerization can be controlled by an exogenous ligand, is fixed at the cellular membrane, while the endogenous Raf is normally activated by binding with Ras. The chimeric receptor EPOR/CR3 was stably expressed in Ba/F3 cells which lack EPO receptors. Although the lines remained dependent on IL-3 for proliferation, EPO treatment reduced the rate of cell death in the absence of IL-3. Also, EPO was synergistic with sub-optimal concentrations of IL-3 in inducing long-term cell proliferation, but did not augment proliferation of cells cultured with full concentrations of IL-3. EPO induced a rapid activation of ERK and also phosphorylation of endogenous Raf. It also induced tyrosine phosphorylation of several cellular proteins. The MEK1 inhibitor PD98059 reduced EPO-induced tyrosine phosphorylation, suggesting these substrates are downstream of MEK kinase. Interestingly, PD98059 also reduced the phosphorylation of endogenous Raf, indicating there is a positive feedback mechanism in Raf activation. We conclude that Raf can be activated by a mechanism that induces clustering at the cell membrane, and that this leads directly to activation of MEK and ERK. This EPOR/CR3 system may serve as a useful model to evaluate the unknown Raf kinase pathway and the effects of signal transduction inhibitors for Raf as a target.

BCR/ABL and Signal Transduction Pathways

BCR/ABL transforms hematopoietic cells in vitro and in vivo and exerts a wide variety of biological effects, including induction of factor independence, reduction of apoptosis, and altering adhesion of CML cells to marrow stroma. However, at a biochemical level, the mechanisms by which BCR/ABL transforms myeloid cells are poorly understood. p210BCR/ABL has elevated ABL tyrosine kinase activity, relocates to the cytoskeleton, and phosphorylates several cellular proteins, including c-BCR, pl20rasGAP, c-CBL, p52SHC, p93FES, p95VAV, pl25FAK, p68paxillin, and p72SH PTP2. In addition, BCR/ABL has been shown to bind directly to GRB2 at Y177 of BCR, therefore potentially activating p21ras. However, it has been difficult to determine the significance of any of these potential BCR/ABL substrates, in part due to the complexity of studying a large protein with many potential signaling motifs, and in part due to the fact that there are so many BCR/ABL substrates in these cell lines. One approach to simplifying BCR/ABL biology has been to examine primary human CML cells, rather than cell lines made to overexpress BCR/ABL. Interestingly, in primary leukemic cells, there are only a few proteins which either intereact with BCR/ABL or are phosphorylated by BCR/ABL. This suggests that studies in primary CML cells, rather than tissue culture cell lines, may be more reliable in terms of identifying important signaling pathways. One of the major tyrosine phosphoproteins complexed with BCR/ABL in CML neutrophils has recently been identified as CRKL, an SH2/SH3 “adapter” protein. CRKL binds to BCR/ABL at least partly through its SH3 domain, and in cell lines may link BCR/ABL to some cytoskeletal proteins. The hypothesis hypothesis is set forth that BCR/ABL functions in part to disrupt signals going to, or coming from integrins in hematopoietic cells, and that this event is important in the pathogenesis of stable phase CML.