Semaxinib (SU5416) as a therapeutic agent targeting oncogenic Kit mutants resistant to imatinib mesylate.

Activating mutations in the Kit receptor are frequently observed in various malignancies, pointing Kit as a molecule of interest for drug inhibition. When mutated on Asp 816 (corresponding to Asp 814 in the mouse), as preferentially found in human mastocytosis and acute myeloid leukemia, Kit became non-sensitive to imatinib mesylate (Gleevec). Erythroleukemic cells isolated from Spi-1/PU.1 transgenic mice express Kit mutated at codon 814 (Kit(D814Y) or Kit(D814V)) or codon 818 (Kit(D818Y)). Using these cells in vitro, we demonstrate that the tyrosine kinase inhibitor SU5416 (Semaxinib) induces growth arrest and apoptosis independent of the mutation type by inhibiting the functions of Kit, including Kit autophosphorylation and activation of Akt, Erk1/Erk2 and Stat3 downstream signaling pathways. These findings indicate that SU5416 may be a promising tool to kill cancer cells driven by Kit oncogenic mutations that are resistant to treatment with imatinib mesylate.

Phosphatidylinositol 4-phosphatase type II is an erythropoietin-responsive gene.

The erythroleukemia developed by spi-1/PU.1 transgenic mice is a multistep process. At disease onset, preleukemic cells are arrested in differentiation at the proerythroblast stage (HS1 stage) and their survival and growth are under the tight control of erythropoietin (Epo). During disease progression, malignant proerythroblasts characterized by Epo autonomous growth and in vivo tumorigenicity can be isolated (HS2 stage). During analysis of transcriptional profiling representive of discrete stages of leukemic progression, we found that the phosphatidylinositol 4-phosphatase type II gene was turned off in malignant cells. PI-4-phosphatase II is an enzyme that hydrolyses the 4-phosphate position of phosphatidylinositol-3-4-bisphosphate (PtdIns(3,4)P(2)) to form PtdIns(3)P. Using malignant cells engineered to stably express PI-4-phosphatase II, we showed that PI-4-phosphatase II reduced Akt activation level. Moreover, stimulation of malignant cells with Epo-induced PI-4-phosphatase II transcription pointing this gene as an Epo-responsive gene. This study provides first insight for a physiological role of PI-4-phosphatase II in the proerythroblast by controlling Epo responsiveness through a negative regulation of the PI3K/Akt pathway.

Involvement of the transcription factor PU.1/Spi-1 in myeloid cell-restricted expression of an interferon-inducible gene encoding the human high-affinity Fc gamma receptor.

Induction by gamma interferon (IFN-gamma) of the gene encoding the human high-affinity Fc gamma receptor (Fc gamma R1) in myeloid cells requires an IFN-gamma response region (GRR) and a myeloid cell-activating transcription element (MATE). GRR and MATE interact with factors to form, respectively, an IFN-gamma-activating complex (GIRE-BP), depending on the phosphorylation of the 91-kDa protein (subunit of ISGF3), and a cell-type-specific complex (MATE-BP). Although GIRE-BP is detected in cells of different origins after IFN-gamma treatment, the presence of MATE-BP was found to be restricted to B- and myeloid cell lines. Sequence analysis of a cDNA encoding a polypeptide recognizing specifically the MATE motif led to the identification of this product as the proto-oncogene PU.1/Spi-1, a transcriptional activator expressed in myeloid and B cells. Expression of this factor in nonhematopoietic cells allowed IFN-gamma-induced expression of a reporter gene under control of the GRR and MATE sequences. The presence of these motifs in other gene promoters indicates that the binding of PU.1/Spi-1 and IFN regulatory proteins to their respective motifs could be part of a general mechanism leading to cell-type-restricted and IFN-induced gene expression.

Characterization of Spi-B, a transcription factor related to the putative oncoprotein Spi-1/PU.1.

We have cloned a human cDNA from a new gene, spi-B, on the basis of its homology with the DNA-binding domain of the Spi-1/PU.1 putative oncogene product. spi-B codes for a protein of 262 amino acids presenting 43% overall identity with Spi-1. Its highly basic carboxy-terminal region exhibits 34% sequence identity with the DNA-binding domain of the Ets-1 protein. We showed that the Spi-B protein is able to bind the purine-rich sequence (PU box) recognized by Spi-1/PU.1 and to activate transcription of a reporter plasmid containing PU boxes. Chromosome in situ hybridization allowed us to map spi-B to the 19q13.3-19q13.4 region of the human genome. spi-B, like spi-1, was found to be expressed in various murine and human hematopoietic cell lines except T lymphoid cell lines.

Spi-1/PU.1 transgenic mice develop multistep erythroleukemias.

Insertional mutagenesis of the spi-1 gene is associated with the emergence of malignant proerythroblasts during Friend virus-induced acute erythroleukemia. To determine the role of spi-1/PU.1 in the genesis of leukemia, we generated spi-1 transgenic mice. In one founder line the transgene was overexpressed as an unexpected-size transcript in various mouse tissues. Homozygous transgenic animals gave rise to live-born offspring, but 50% of the animals developed a multistep erythroleukemia within 1.5 to 6 months of birth whereas the remainder survived without evidence of disease. At the onset of the disease, mice became severely anemic. Their hematopoietic tissues were massively invaded with nontumorigenic proerythroblasts that express a high level of Spi-1 protein. These transgenic proerythroblasts are partially blocked in differentiation and strictly dependent on erythropoietin for their proliferation both in vivo and in vitro. A complete but transient regression of the disease was observed after erythrocyte transfusion, suggesting that the constitutive expression of spi-1 is related to the block of the differentiation of erythroid precursors. At relapse, erythropoietin-independent malignant proerythroblasts arose. Growth factor autonomy could be partially explained by the autocrine secretion of erythropoietin; however, other genetic events appear to be necessary to confer the full malignant phenotype. These results reveal that overexpression of spi-1 is essential for malignant erythropoiesis and does not alter other hematopoietic lineages.

Detection of tumorigenic cells in Friend virus-infected mice: an in vivo methodological investigation.

Tumor formation by subcutaneous transplants of spleens from erythroleukemic mice infected with Friend virus complex inducing polycythemia (FLV-P) is successful only during the late phase of the disease. To determine whether this observation is due to the absence of tumorigenic cells in the early phase of such leukemia or to the inability of standard procedures to detect these cells, the sensitivities of different routes of inoculation in sublethally irradiated or unirradiated syngeneic recipients were examined. The omentum of the sublethally irradiated mouse was found to be a suitable site for the homing and proliferation of recently isolated tumorigenic cells from FLV-P-infected mice, since it proved 1,000 times more sensitive than the usual subcutaneous sites in unirradiated mice. When this sensitive graft in the omentum was applied to detection of tumorigenic cells in the spleens of FLV-P-infected mice, the mean detection time was 20 days after virus infection, compared to 36 days with the usual subcutaneous graft method.

Protooncogene expression in normal, preleukemic, and leukemic murine erythroid cells and its relationship to differentiation and proliferation.

The expression of 18 protooncogenes was examined by Northern blot analysis in preleukemic and leukemic stages of murine erythroleukemias induced by Friend viruses. As controls, erythropoietically stimulated spleens from phenylhydrazine-treated mice were studied. Expression of 10 protooncogenes (c-erb-A, c-erb-B, c-ets, c-sis, c-mos, c-rel, c-src, c-fes, c-fms, N-myc [corrected] was not detectable in Friend erythroleukemias. One protooncogene (c-src) was found expressed in normal erythroid cells but not in erythroleukemias. Four protooncogenes (c-fos, c-abl, N-ras, and c-raf) were expressed at low levels in both steps of erythroleukemia. c-fos and c-abl RNAs were barely detectable in normal erythroid cells. High levels of four protooncogene transcripts (c-H-ras, c-K-ras, c-myc, and c-myb) were detected in preleukemic and leukemic tissues. While c-H-ras RNA was found at similar levels in normal and leukemic erythroid cells, c-myc, c-myb, and c-K-ras were not expressed in normal erythroid cells. To determine whether the elevated levels of c-myc, c-myb, and c-K-ras RNAs in erythroleukemic cells are related to the proliferative state or the undifferentiated state of the cells, the effect of dimethyl sulfoxide-induced differentiation on oncogene expression in two erythroleukemia cell lines was examined. Terminal differentiation was associated with lack of c-myb expression while c-myc and c-K-ras expression was essentially unaffected. These results suggest that the high levels of c-myb transcripts in erythroleukemias may reflect the undifferentiated state of the leukemic cells. In contrast, the elevated expression of c-myc and c-K-ras at both stages of the Friend diseases is probably not related to the stage of differentiation but rather to the uncontrolled proliferation of the cells. Finally among 18 protooncogenes surveyed, only the accumulation of c-myc and c-K-ras RNAs appears to be associated with the Friend erythroleukemic process before the late leukemic phase develops.

DNA binding specificities of Spi-1/PU.1 and Spi-B transcription factors and identification of a Spi-1/Spi-B binding site in the c-fes/c-fps promoter.

Spi-1/PU.1 and Spi-B encode hematopoietic-specific transcription factors that are the most distantly related members of the Ets family. The Ets proteins share a conserved 85 amino acids DNA binding domain, the Ets domain and recognize various DNA target sites around a common core 5'-GGAA/T-3'. The DNA binding specificities of Spi-1 and Spi-B were investigated by using the method of polymerase chain reaction (PCR)-mediated random site selection. The deduced Spi-1 and Spi-B consensus binding sites are very similar suggesting that the functional activities of Spi-1 and Spi-B cannot be distinguished on the basis of their DNA binding specificities. We identified a putative Spi-1/Spi-B binding site in the promoter region of the c-fes/c-fps protooncogene which encodes a tyrosine kinase expressed predominantly in myeloid cells. In vitro translated Spi-1 and Spi-B proteins were capable to bind this site similarly and to activate the c-fes promoter in HeLa transfected cells. We showed that Spi-1 binds the Spi-1/Spi-B binding site of c-fes in HL-60 cells suggesting that Spi-1 may be involved in the regulation of c-fes transcription in myeloid cells. Intriguingly, we detected only Spi-1 binding to this site in the Raji cell line which express both Spi-1 and Spi-B proteins. This suggests that Spi-1 and Spi-B exhibit different DNA binding activities in vivo although they share similar DNA binding specificities in vitro.

Inhibition of Friend cells proliferation by spi-1 antisense oligodeoxynucleotides.

The spi-1 proto-oncogene encodes the transcription factor PU.1 which is normally expressed in all hematopoietic cell lineages except in T cell lines. During the murine acute erythroleukemia induced by the Friend retrovirus, SFFV, spi-1 deregulation by insertional mutagenesis results in the overexpression of Spi-1/PU.1 in the malignant proerythroblastic cell. To assess the Spi-1 role in the proliferation and the differentiation arrest of the Friend tumor cells we inhibited spi-1 gene expression in two Friend cell lines by using antisense oligodeoxyribonucleotides. Proliferation and cloning efficiency of both cell lines were significantly inhibited by spi1 antisense. This antiproliferative effect was not related to an apparent maturation of erythroleukemic cells demonstrating that repression of spi-1 expression is not sufficient per se to restore the ability of the proerythroblastic cells to spontaneously differentiate in mature erythroblasts. These data suggest that the spi-1 gene would be involved in the Friend leukemic process by promoting the proerythroblast to proliferate.

Differential phosphorylations of Spi-B and Spi-1 transcription factors.

Spi-1/PU-1 and Spi-B are hematopoietic transcription factors, which, in vitro, display similar affinities for DNA target sequences containing the consensus binding site 5'-GGAA-3'. While the role of Spi-1 in the transcriptional regulation of B cell and myeloid specific genes has been largely demonstrated, the biological function of Spi-B still remains to be elucidated. Since Spi-B and Spi-1 are very divergent in their transactivator domain, these domains might acquire functional specificity in vivo by interacting with different co-factors and/or by undergoing different phosphorylations. First, we observed that casein kinase II phosphorylates Spi-B as well as Spi-1, in vitro. Then, by affinity chromatographies and in vitro kinase assays with fusion proteins between glutathione-S-transferase and the transactivator domain of Spi-B, two kinases were identified on their ability to interact and phosphorylate this domain; the MAP kinase ERK1 and the stress activated protein kinase JNK1. The Threonine 56 was defined as the ERK1 phosphorylation site by using phosphoamino-acid analyses and a Spi-B mutant version with the substitution T56 to A56. Strikingly, ERK1 failed to phosphorylate Spi-1, in vitro, whereas JNK1, like CK II, phosphorylated Spi-B and Spi-1. In addition, other purified Spi-B-kinase activities, unidentified as yet, display similar specificity than ERK1 for Spi-B versus Spi-1. Furthermore, the evident interaction of pRb protein with the transactivator domain of Spi-B in an unphosphorylated state disappeared when this domain was first phosphorylated in vitro either by ERK1 or by the purified Spi-B-kinase activities. Our data revealed multiple phosphorylation sites within Spi-B whose some of them appeared specific for Spi-B versus Spi-1 and which may account for differential regulation of their activities.

The human homologue of the putative proto-oncogene Spi-1: characterization and expression in tumors.

Spi-1 is a putative proto-oncogene involved in murine virus-induced acute erythroleukemias. We report here the identification of the human homologue of Spi-1 and its expression in normal and tumorigenic human tissues. Characterization of cDNA clones revealed that the human Spi-1 gene encodes a 216 amino acids protein showing 85% identity with the murine counterpart. By sequencing genomic clones, five exons were identified. To investigate the possible role of Spi-1 gene in human cancers, we studied its expression in a panel of human tumors by Northern blot analysis. Spi-1 expression was detected in all the tumors examined. There was no noticeable evidence of messenger RNA alteration as compared to normal tissues.

The putative oncogene Spi-1: murine chromosomal localization and transcriptional activation in murine acute erythroleukemias.

We have shown previously that spleen focus forming virus integration near the Spi-1 putative oncogene is observed in 95% of erythroid Friend tumors (Moreau-Gachelin et al., 1988). Here we describe how the proviral insertion in the Spi-1 domain is associated with the enhanced transcription of a 1.4 kb mRNA normally expressed at a low level in normal cells. The gene is localized on murine chromosome 2, band E3. The structure of the Spi-1 gene was determined by sequencing genomic and cDNA clones. The gene has an open reading frame encoding a protein of 218 amino acids, extending over five exons. Proviruses always integrate outside, upstream and in the opposite orientation of the protein-encoding domain. This suggests that SFFV integration activates the expression of Spi-2 gene that may contribute to the erythroleukemic process.

Germ-line deletion of p53 reveals a multistage tumor progression in spi-1/PU.1 transgenic proerythroblasts.

Activation of the spi-1/PU.1 proto-oncogene and loss of p53 function are genetic alterations associated with the emergence of Friend malignant erythroleukemic cells. To address the role of p53 during erythroleukemogenesis, spi-1 transgenic mice (spi-1-Tg) which develop erythroleukemia were bred with p53-deficient mice. Three classes of spi-1 transgenic mice differing in their p53 functional status (p53(+/+), p53(+/-) and p53(-/-)) were generated. These mice developed a unique pattern of erythroleukemia. In wild-type p53 spi-1-Tg mice, none of the primary erythroleukemic spleen cells displayed autonomous growth in vitro and in vivo. In contrast, in p53(+/-) spi-1-Tg mice, erythroleukemic cells gave rise to growth factor-independent cell lines and generated tumors in vivo. Malignancy was associated with loss of the wild-type p53 allele. The p53(-/-) spi-1-Tg mice developed erythroleukemia with a total incidence and a reduced latency compared to the two other genotypes. Unexpectedly, 50% of p53(-/-) spi-1-Tg erythroleukemic spleens generated cell lines that were strictly dependent upon erythropoietin (Epo) for proliferation, whereas the remainder proliferated independently of cytokines. Moreover, only 70% of these spleen cells were tumorigenic. These findings indicate that p53 germ-line deletion did not confer malignancy to spi-1-transgenic proerythroblasts. Moreover Epo independence and tumorigenicity appear as separable phenotypic characteristics revealing that the spi-1-Tg proerythroblasts progress towards malignancy through multiple oncogenic events.

Isolation and characterization of a chicken homologue of the Spi-1/PU.1 transcription factor.

Spi-1/PU.1 is a member of the Ets family of transcription factors important in regulation of hematopoiesis. We have isolated a chicken cDNA homologuous to the mammalian Spi-1/PU.1 gene with an open reading frame of 250 amino acids (aa). The chicken Spi-1/PU.1 protein is 14 aa and 16 aa shorter than its human and mouse counterparts but is extremely well conserved with 78.8% and 75.2% identity respectively. The carboxy terminal DNA binding region, or ETS binding domain, is 100% identical to that of human and mouse. Some differences with the mammalian homologues are seen in the N-terminal part of the protein and in the PEST connecting domain. However, the differences are mainly conservative and all the features underlying functional aspects seem preserved. The major discrepancy lies in a 12 aa deletion in an already poorly conserved part of the PEST sequence. Spi-1/PU.1 transcripts were detected at high levels in spleen and Fabricius bursa of chick embryos by Northern blot and in situ hybridization. Our results show that the chicken Spi-1/PU.1 protein behaves like a bonafide Spi-1/PU.1 transcription factor in its DNA binding and transactivating properties.

Spi-1 transgenic mice develop a clonal erythroleukemia which does not depend on p53 mutation.

Spi-1 transcriptional activation and wild-type p53 extinction are two oncogenic alterations involved in the malignant transformation of erythroblasts during the Friend acute erythroleukemia. To dissect the contribution of these alterations in the deregulation of the differentiation and proliferation of erythroblasts, we generated spi-1 transgenic mice. Analysis of these animals revealed that Spi-1 overexpression was directly involved in the block of proerythroblast differentiation. However, the erythroleukemia that develops in these animals evolved in two steps. During the early step (HS1 step), non tumorigenic proerythroblasts remained strictly dependent upon erythropoietin (Epo) for their survival and proliferation. Later on, Epo-independent and tumorigenic proerythroblasts emerged (HS2 step) suggesting that other oncogenes cooperate with Spi-1 to lead to a fully malignant phenotype. By provirus tagging, we demonstrate that the HS1 step was clonal indicating that a cell selection must occur in vivo. Analysis of the nature of p53 in both the in vivo HS1 and HS2 proerythroblasts and in cultured erythroblastic cell lines showed that--p53 was normal in the HS1 primary tissues but was mutated in the HS1 cultured cell lines--p53 was frequently altered in HS2 primary tissues but was found normal in some mice. These data indicate that (i) the blockage of the erythroblast differentiation by Spi-1 occurs independently of p53 alteration (ii) p53 alteration is not necessary to confer Epo independence and tumorigenicity to spi-1 transgenic proerythroblasts.

PU.1 (Spi-1) autoregulates its expression in myeloid cells.

PU.1 (Spi-1), a member of the Ets transcription factor family, is predominantly expressed in myeloid (granulocytes, monocytes and macrophages) and B cells. PU.1 is upregulated early during commitment of multipotential progenitors to the myeloid lineages and inhibition of PU.1 function in human CD34+ progenitors prior to this upregulation blocks myeloid colony formation. Since PU.1 expression appears to play a role in hematopoietic development, we characterized the PU.1 promoter. Here we report that the murine PU.1 promoter, as well as the human promoter, demonstrate tissue-specific reporter gene expression in myeloid cell lines but not in T cells and HeLa (non-hematopoietic cells) cells. Deletion analysis of the PU.1 promoter indicates that tissue-specific functional elements are encoded in the -61 to -39 bp and -7 to +34 bp regions. The first region contains a functional octamer (Oct) site at -54 bp and an Sp1 site at -39 bp. The second contains a binding site at +20 bp for both PU.1 itself and the related ets family member Spi-B. In vivo footprinting assays demonstrate that a hypersensitive band was detected at the PU.1 site in myeloid cells but not in HeLa. A mutation of the PU.1 site which abolished PU.1 binding caused a significant decrease in promoter activity. Mutation of the Oct and/or Sp1 site results in a lesser decrease of promoter activity in myeloid cells. Co-transfection of PU.1 or Spi-B in cells lacking PU.1 and Spi-B specifically transactivated a minimal promoter containing the PU.1 binding site, indicating that PU.1 can activate its own promoter elements in an autoregulatory loop. Positive autoregulation of the PU.1 promoter may play an important role in the function of PU.1 in myeloid cells.

Stimulation of the biosynthesis of membrane glycoproteins from Zajdela ascites hepatoma cells by Robinia lectin.

Membrane glycoprotein biosynthesis of ascites hepatoma cells is followed by [14C]glucosamine and [3H]leucine incorporation into cells in culture. The rate of incorporation is strongly increased by the addition of Robinia lectin in culture medium. Labeled glycoproteins are released from lectin stimulated and non-stimulated cells by trypsin digestion. Studies of labeled trypsinates on sodium dodecyl sulfate gel electrophoresis and Sephadex G-200 filtration exhibit two fractions both labeled with [14C]glucosamine and [3H]leucine and having different molecular weights, one over 200000 and the other about 2000. Identical results are obtained when external membrane glycoproteins are solubilized by sodium deoxycholate. Comparison of surface glycoproteins isolated by trypsinization from control cells labeled with [3H]-glucosamine and from lectin stimulated cells labeled with [14C]glucosamine displays no significant qualitative differences between glycoprotein fractions released from both cell groups.

Spi-1 oncogene activation in Rauscher and Friend murine virus-induced acute erythroleukemias.

The Friend viruses, like the Rauscher virus, cause murine acute erythroleukemias which evolve in a similar multistep process. In previous studies it has been described that the late malignant proerythroblastic transformation induced by the polycythemia-inducing strain of Friend spleen focus-forming virus (SFFVP) is correlated with Spi-1 oncogene activation by insertional mutagenesis. In this paper we report that Spi-1 genomic rearrangements were also observed in 90% of tumors induced by the anemia-inducing strain of Friend spleen focus-forming virus (SFFVA) and in all Rauscher-induced tumors analyzed. SFFVA and Rauscher proviral insertions occurred in the viral integration cluster previously characterized in SFFVP-induced tumors. The Spi-1 1.4-Kb messenger RNA was found highly expressed in all SFFVA and Rauscher-induced malignant cells as compared to normal tissues. The nucleotide sequence of Spi-1 cDNA isolated from a library constructed from SFFVA-induced tumor cells revealed no difference between the Spi-1 gene transcripts expressed in both SFFVP and SFFVA-induced leukemic cells. These results indicate that Spi-1 gene activation is a general feature in the malignant proerythroblastic transformation which occurs in mice infected with Friend and Rauscher viruses.

Incapacity of hematopoietic stem cell-deprived mice to produce tumor colonies induced by Friend virus-infected cells.

The role of host hematopoietic stem cells in the formation of tumor colonies in the spleen of (C57BL/6 X DBA/2) F1 mice after grafts of spleen cells from Friend virus (FVP)-infected donors has been investigated. Hematopoietic stem cell compartments of recipient mice were destroyed by Myleran treatment or gamma-ray irradiation. A single injection of Myleran reduced the pluripotent hematopoietic stem cells (CFU) and the erythropoietin responsive cells (ERC) in polycythemic mice to around 1% of that of controls. Repeated injections of erythropoietin (EPO) restored the erythropoietic precursor cell (ERC) population. Pretreatment of polycythemic hosts with Myleran totally suppressed the tumor colony forming ability of grafted Friend virus-infected spleen cells, whereas it had no effect on tumor colonies produced by inoculation of true tumoral Friend cells. After EPO injections in such Myleran-treated recipients, with a consequent appreciable ERC repopulation, splenic colonies again occurred. Similar results were obtained in hosts whose ERC populations were damaged by irradiation. These data strongly suggest that splenic colonies result from the proliferation of the host cells transformed by virus released by Friend virus-infected cells and not from the proliferation of donor tumor cells.

Phosphorylation of the Spi-B transcription factor reduces its intrinsic stability.

The Spi-B transcription factor is an Ets protein expressed in B lymphoid cells and closely related to the Spi-1/PU.1 oncoprotein. By mutational analysis, we showed that Spi-B is phosphorylated by casein kinase II in vitro on four serine residues. Mutation of these four serines to alanines prevented the phosphorylation of Spi-B in vivo, increased the ability of Spi-B to transactivate expression of a reporter gene and led to a decrease of Spi-B stability. We propose that the phosphorylation of Spi-B may participate in the modulation of Spi-B functional activity by controlling its intracellular protein level.