Phase I study of the novel Cdc2/CDK1 and AKT inhibitor terameprocol in patients with advanced leukemias.

PURPOSE: Inhibiting survivin and Cdc2 (CDK1) has preclinical anti-leukemic activity. Terameprocol is a small molecule survivin and Cdc2/CDK1 inhibitor that was studied in a Phase I dose-escalation trial. PATIENTS AND METHODS: Sixteen patients with advanced acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) were enrolled and 15 treated with Terameprocol in three dose cohorts intravenously three times per week for 2 weeks every 21 days. RESULTS: Patients had AML (n = 11), chronic myelogeneous leukemia in blast phase (CML-BP, n = 2) and one each T-cell acute lymphoblastic leukemia (T-ALL) and MDS. Four, five and six patients were treated at the 1000, 1500 and 2200 mg Terameprocol dose cohorts respectively. Common related treatment emergent adverse events (TEAE) were grade 1 or 2 headache, transaminitis and pruritus, with one grade 4 serious AE (SAE) of pneumonia. No dose limiting toxicity (DLT) was observed, however, due to other observed grade 3 TEAE the recommended phase 2 dose (RP2D) was determined at 1500 mg 3×/week for 2 weeks of a 21-day cycle. Partial remission and transfusion independence in a CML-BP patient (1500 mg cohort) and hematological improvement in erythroid (HI-E) and platelet lineage (HI-P) in an AML patient were observed. Five AML patients had stable disease greater/equal to 2 months. Pharmacodynamic studies showed a reduction of CDK1 and phospho-AKT protein expression. CONCLUSION: Terameprocol can be safely administered to advanced leukemia patients, sufficient drug exposure was obtained and clinical activity and biomarker modulation were observed.

Megakaryocyte growth and development factor. Analyses of in vitro effects on human megakaryopoiesis and endogenous serum levels during chemotherapy-induced thrombocytopenia.

The present study shows that recombinant human megakaryocyte growth and development factor (r-HuMGDF) behaves both as a megakaryocyte colony stimulating factor and as a differentiation factor in human progenitor cell cultures. Megakaryocyte colony formation induced with r-HuMGDF is synergistically affected by stem cell factor but not by interleukin 3. Megakaryocytes stimulated with r-HuMGDF demonstrate progressive cytoplasmic and nuclear maturation. Measurable levels of megakaryocyte growth and development factor in serum from patients undergoing myeloablative therapy and transplantation are shown to be elaborated in response to thrombocytopenic stress. These data support the concept that megakaryocyte growth and development factor is a physiologically regulated cytokine that is capable of supporting several aspects of megakaryopoiesis.

Coinfection with viruses carrying the v-Ha-ras and v-myc oncogenes leads to growth factor independence by an indirect mechanism.

The concomitant expression of certain oncogenes can transform normal diploid rodent cells into transplantable tumorigenic cells. The mechanism by which these oncogenes collaborate is unclear. Recent findings (M. Oshimura, T. M. Gilmer, and J. C. Barrett, Nature [London] 316:636-639, 1985) raise the possibility that karyotypic changes, including monosomy for chromosome 15, are required to induce tumorigenicity in Syrian hamster embryo cells transfected in vitro with v-Ha-ras and v-myc DNAs. We studied the effect of the oncogenes v-Ha-ras and v-myc, introduced by viral infection, on murine hematopoietic cells. The induction of growth factor independence by the two oncogenes was used as an in vitro correlate of tumorigenicity. After a period of reduced growth rate reminiscent of the growth rate of cells in crisis, the doubly infected cells became growth factor independent. These cells showed a great variability in their karyotypes.

Intracellular protein scaffold-mediated display of random peptide libraries for phenotypic screens in mammalian cells.

BACKGROUND: Mammalian cell screens of peptide libraries for changes in cellular phenotype may identify novel functional peptides and their cognate binding partners, and allow identification of signal transduction network members or proteins important in disease processes. RESULTS: Green fluorescent protein (GFP) peptide libraries with different structural biases were tested by retroviral expression in A549 carcinoma cells, HUVEC and other cell types. Three different loop replacement libraries, containing 12 or 18 random residues, were compatible with enhanced GFP (EGFP) folding, as was a C-terminally fused random 20-mer library. Library concentrations in A549 cells ranged from ca. 1 to 54 microM. Replacement of loop 3 with known nuclear localization sequence (NLS) peptides, but not with inactive mutants, directed EGFP to the nucleus. Microscopy-based screens of three different libraries for non-uniform localization revealed novel NLS peptides, novel variants of a peroxisomal localization motif, a variety of partial NLS peptides, peptides localized to the nucleolus, and nuclear-excluded peptides. CONCLUSIONS: Peptides can be presented by EGFP in conformations that can functionally interact with cellular constituents in mammalian cells. A phenotypic screen resulting in the discovery of novel localization peptides that were not cell type-specific suggests that this methodology may be applied to other screens in cells derived from diseased organisms, and illustrates the use of intracellular combinatorial peptide chemistry in mammalian cells.

BCL-2 family proteins as 5-Azacytidine-sensitizing targets and determinants of response in myeloid malignancies.

Synergistic molecular vulnerabilities enhancing hypomethylating agents in myeloid malignancies have remained elusive. RNA-interference drug modifier screens identified antiapoptotic BCL-2 family members as potent 5-Azacytidine-sensitizing targets. In further dissecting BCL-XL, BCL-2 and MCL-1 contribution to 5-Azacytidine activity, siRNA silencing of BCL-XL and MCL-1, but not BCL-2, exhibited variable synergy with 5-Azacytidine in vitro. The BCL-XL, BCL-2 and BCL-w inhibitor ABT-737 sensitized most cell lines more potently compared with the selective BCL-2 inhibitor ABT-199, which synergized with 5-Azacytidine mostly at higher doses. Ex vivo, ABT-737 enhanced 5-Azacytidine activity across primary AML, MDS and MPN specimens. Protein levels of BCL-XL, BCL-2 and MCL-1 in 577 AML patient samples showed overlapping expression across AML FAB subtypes and heterogeneous expression within subtypes, further supporting a concept of dual/multiple BCL-2 family member targeting consistent with RNAi and pharmacologic results. Consequently, silencing of MCL-1 and BCL-XL increased the activity of ABT-199. Functional interrogation of BCL-2 family proteins by BH3 profiling performed on patient samples significantly discriminated clinical response versus resistance to 5-Azacytidine-based therapies. On the basis of these results, we propose a clinical trial of navitoclax (clinical-grade ABT-737) combined with 5-Azacytidine in myeloid malignancies, as well as to prospectively validate BH3 profiling in predicting 5-Azacytidine response.

cDNA clones from autocrine thymic lymphoma cells encode two mitogenic proteins, a serine protease and a truncated T-cell receptor beta-chain.

Cell lines derived from primary X-ray induced T cell lymphomas (PXTL) of C57BL/6 mice secrete into the medium factor(s) required for their growth. These autocrine factor(s) are distinct from previously described growth factors. cDNA cloning experiments were performed in an attempt to identify these autocrine factor(s). cDNA clones were selected by mRNA size, differential expression, and mitogenic activity of their translation products (Xenopus expression system) on PXTL cells. Two different cDNA clones yielded distinct mitogenic proteins. One clone encodes an altered form of the T cell receptor beta-chain which is truncated at the N-terminus to amino acid 49 of the constant region beta 2. The second clone encodes a serine protease which is identical to factor H or granzyme A from cytotoxic T cells. The 5' portion of the cDNA encoding the serine protease derived from PXTL cells differs from that derived from cytotoxic T cells. This difference results in distinct signal peptides. Unlike cytotoxic T cells, PXTL cells do not store the serine protease intracellularly but secrete it.

Localization in mouse-L-Cell chromosomal sites of transferred immunoglobulin genes.

Mouse thymidine kinase negative (tk-) L-cells were cotransformed with two different kappa light chain genes (cloned from mouse myeloma) and the tk gene from Herpes simplex virus I. (Transformation is defined as change in the genotype by introduction of foreign DNA.) About 80% of the tk+ -transformants contained varying amounts of transferred kappa light chain sequences, one transformant about 150 copies per genome. The transferred immunoglobulin genes appear to be organized in a nucleosomal substructure, as deduced from digestion experiments with micrococcal nuclease. In situ hybridization experiments revealed, that the transferred genes are not distributed randomly across the chromosomes of the recipient cell. Instead they are clustered at one or a few chromosomal locations.

Characterization of X-chromosome specific satellite DNA of Muntiacus muntjak vaginalis.

A highly repeated DNA (designated satellite IA) was isolated from cultured cells of Muntiacus muntjak vaginalis and its organization analyzed by the use of restriction nucleases and hybridization experiments with cloned DNA-fragments. Several restriction nucleases cleave the satellite IA DNA into a series of fragments, which are multiples of a basic repeat unit of 800 bp. Sequences homologous to the satellite IA DNA were also found in a second highly repetitive DNA component of Muntiacus muntjak vaginalis (satellite IB). Its organization is more complex than the one of satellite IA and does not conform to a simple periodicity of a basic repeat unit.--Hybridization in situ revealed, that both satellites are confined in their entirety to the X-chromosome, where they are located at both arms close to the centromere. No satellite DNA was found at the Y1-chromosome, which is considered to be homologous to the long arm of the X-chromosome. These results have interesting implications for the evolution of the X-chromosome.

Glucocorticoids regulate expression of the v-mycOK10 oncogene in a murine retroviral vector with chimeric MoMuLV-MMTV LTRs.

A murine retrovirus which expresses the v-mycOK10 oncogene under the control of the dexamethasone-regulatable mouse mammary tumor virus (MMTV) promoter has been constructed. In this vector, denoted pMImyc, the Moloney Murine leukemia virus (MoMuLV) sequences required for virus replication, integration and packaging were kept, while all the elements for transcription regulation were derived from the MMTV long terminal repeat (LTR). After transfection of NIH 3T3 fibroblasts with this construct, a cell line was isolated in which the level of v-myc RNAs were increased 60 fold by dexamethasone. Kinetic studies showed that this induction can be maintained for up to 12 hours of hormone treatment. After infection with MoMuLV as a helper virus, and in the presence of dexamethasone, the production of pMImyc RNA, estimated by slot blot analysis, was equivalent to about 10(3) viral particles/ml.

The muntjak satellite IA sequence is composed of 31-base-pair internal repeats that are highly homologous to the 31-base-pair subrepeats of the bovine satellite 1.715.

The nucleotide sequence of a cloned Muntjak satellite IA repeat unit (Muntiacus muntjak vaginalis) was determined. The repeat is 807 base pairs (bp) long. By introducing minor deletions and insertions, the whole sequence of the satellite can be arranged in 27 subrepeats of 31 bp length. Although diverged relative to each other, all subrepeats show a homology of more than 53% with the common consensus sequence. In 29 out of the 31 bp the consensus sequence of the Muntjak satellite subrepeat is identical to the 31-bp subrepeat of the bovine satellite 1.715. This suggests that both satellites are derived from a common ancestral sequence. The results have interesting implications for the evolution of the two satellites.

A highly repetitive DNA component common to all Cervidae: its organization and chromosomal distribution during evolution.

In recent work we have isolated and characterized a highly repetitive DNA (MMV satellite IA) from Muntiacus muntjak vaginalis, the species with the most reduced karyotype in the Cervidae family. We have now analysed the genomes of nine related species for the presence of MMV satellite IA components, and have determined their organization and chromosomal distribution. Repetitive satellite IA type DNA is present in all species of the Cervidae, and also in the bovine, but not in a species of the Tragulidae suggesting that these sequences were generated after the phylogenetic separation of Bovidae and Tragulidae. Studies on the organization of the satellite IA DNA in the various species revealed three main repeat lengths: 1400, 1000 and 807 bp. The relative proportion of satellite IA sequences present in any one of the three registers is strikingly different within the various species and can be correlated with the phylogeny of the Cervidae. The chromosomal locations of the satellite IA sequences were determined in seven species by in situ hybridization. It turned out that the chromosomal rearrangements leading to the reduction in the number of chromosomes during karyotype evolution have led to the elimination of satellite I DNA at most locations. In all tandem fusions, the satellite IA sequences located at the centromeres of the ancestral acrocentric chromosomes are lost. In contrast, during the centric fusion that generates the M. m. vaginalis X chromosome satellite IA sequences are amplified. Sequence motifs, which are known to be involved in recombinational events are present in the satellite IA and might have contributed to the unique karyotype variation in the Cervidae.

Differentiation of myeloid dendritic cells into CD8alpha-positive dendritic cells in vivo.

Bone marrow-derived dendritic cells (DC) represent a family of antigen-presenting cells (APC) with varying phenotypes. For example, in mice, CD8alpha(+) and CD8alpha(-) DC are thought to represent cells of lymphoid and myeloid origin, respectively. Langerhans cells (LC) of the epidermis are typical myeloid DC; they do not express CD8alpha, but they do express high levels of myeloid antigens such as CD11b and FcgammaR. By contrast, thymic DC, which derive from a lymphoid-related progenitor, express CD8alpha but only low levels of myeloid antigens. CD8alpha(+) DC are also found in the spleen and lymph nodes (LN), but the origin of these cells has not been determined. By activating and labeling CD8alpha(-) epidermal LC in vivo, it was found that these cells expressed CD8alpha on migration to the draining LN. Similarly, CD8alpha(-) LC generated in vitro from a CD8 wild-type mouse and injected into the skin of a CD8alphaKO mouse expressed CD8alpha when they reached the draining LN. The results also show that CD8alpha(+) LC are potent APC. After migration from skin, they localized in the T-cell areas of LN, secreted high levels of interleukin-12, interferon-gamma, and chemokine-attracting T cells, and they induced antigen-specific T-cell activation. These results demonstrate that myeloid DC in the periphery can express CD8alpha when they migrate to the draining LN. CD8alpha expression on these DC appears to reflect a state of activation, mobilization, or both, rather than lineage. (Blood. 2000;96:1865-1872)

Organization and chromosomal distribution of a novel repetitive DNA component from Muntiacus muntjak vaginalis with a repeat length of more than 40 kb.

The organization and chromosomal distribution of the repetitive DNA component IB from Muntiacus muntjak vaginalis (MMV) was investigated. DNA fragments of component IB were cloned in cosmids and their structure analysed using restriction nucleases and blot-hybridization experiments. Two cosmids were found to be practically identical by restriction enzyme mapping. The repeat unit of component IB DNA is more than 40 kb and contains the 11 and 18 kb Bam HI fragments, which have previously been shown to cross-hybridize with MMV satellite IA. In addition, the repeat unit contains long stretches of DNA sequences which are unique to component IB. In situ hybridization experiments showed that component IB has the properties characteristic of long interspersed repetitive DNA rather than tandemly repeated satellite DNA. Consistent with this conclusion, only a minor fraction of component IB is located on the X chromosome as demonstrated by the analysis of somatic cell hybrids. This is in marked contrast to satellite IA that is specific for the X chromosome. These results have interesting implications for the evolution of the component I DNA family of the MMV genome.

The induction of growth factor-independence in murine myelocytes by oncogenes results in monoclonal cell lines and is correlated with cell crisis and karyotypic instability.

Infection of established (IL-3)-dependent hematopoietic cell lines with Abelson murine leukemia virus (A-MLV) abrogates their requirement for IL-3 and leads to non-autocrine growth factor-independent cells. We were interested to determine whether A-MLV can induce IL-3 independence also in non-established cells. To obtain long-term cultures of diploid myelocytes, splenic hematopoietic cells were first infected with MMCV, a murine retrovirus carrying the avian v-myc oncogene. These cultures were superinfected with A-MLV. In three independent experiments, the first growth factor-independent cells appeared between 18 and 43 days after superinfection with A-MLV and represented .02-1% of the population. Furthermore, the cultures that became growth factor-independent were monoclonal for integration of the v-abl gene. These results indicate that the acquisition of growth factor-independence after superinfection of v-myc-expressing cells with A-MLV is a rare event. The low frequency of growth factor-independent cells was not due to a low percentage of infected cells, since 15-25% of the cells were infected with A-MLV after 7 days. The first appearance of growth factor-independent cells coincided with crisis in the cultures, as indicated by a high incidence of cell death and a reduced overall growth rate of the cell populations. These growth factor-independent cells exhibited variable karyotypes, including many that were near-triploid to near-tetraploid. In summary, growth factor-independence induced by super-infection with A-MLV, like that induced by double-infection with v-myc- and v-H-ras-containing viruses, is associated with unstable karyotypes. The growth factor-independent cells show variable ploidy characteristic of cells which survived crisis.

TPO and IL-3 induce overlapping but distinct protein tyrosine phosphorylation in a myeloid precursor cell line.

TPO is a recently cloned cytokine which appears to play a central role in megakaryopoiesis and platelet production. It has been shown to be the ligand for c-mpl, a member of the hematopoietic receptor superfamily. We examined intracellular tyrosine phosphorylation events induced by TPO and compared them with events stimulated by IL-3, a pleiotropic cytokine which also has activity on the megakaryocyte lineage. The overall pattern of tyrosine phosphorylation stimulated by TPO and IL-3 in myeloid precursor cells revealed an overlapping but not identical pattern reflecting their distinct but partially redundant biological effects. We identify Shc and Vav as intracellular targets of TPO-induced tyrosine phosphorylation. Moreover, we demonstrate that the tyrosine kinase Jak2 is phosphorylated after TPO stimulation. Whereas phosphorylation of these proteins was induced by both cytokines, phosphorylation of Jak1 was induced only by IL-3 and not by TPO, distinguishing the signal transduction of the two cytokines on a molecular level.

[Significance of thrombopoietin and its receptor c-Mpl in regulation of thrombocytopoiesis in thrombocytopenia]. / Die Bedeutung von Thrombopoietin (TPO) und seines Rezeptors c-Mpl in der Regulation der Thrombozytopoese bei Thrombozytopenien.

Thrombopoietin (TPO) belongs to the family of hematopoietic growth factors. It supports the growth and differentiation of megakaryocytic progenitors and precursors in vitro and in vivo. The predominant site of production of TPO is the liver. However, regulation of TPO serum levels seems to be not due to transcriptional regulation in the liver but due to degradation of circulating TPO by platelets. Thrombopoietin serum levels in children with thrombocytopenia associated with aplastic anemias, Fanconi anemia and TAR syndrome are elevated whereas in children with idiopathic thrombocytopenia the TPO levels are normal. The defective activation of platelets by TPO in a children with TAR syndrome suggests a defective response of megakaryocytopoiesis to TPO.

Neutron diffraction of chromatin in interphase nuclei and metaphase chromosomes.

We have used neutron diffraction to study chromatin structure in interphase nuclei and metaphase chromosomes as a function of decreasing ion concentration. Aliquots of a suspension of rat liver nuclei prepared in a polyamine-free buffer were washed in buffers of 1/3, 1/6 and 1/12 if the original concentration of monovalent and divalent cations (40 mM KCl; 20 mM NaCl; 1.2 mM MgCl2). After the first dilution step (1/1 to 1/3), only small changes occurred in the diffraction pattern. They can be interpreted by a loosening of the original structure, i.e. by the formation of isolated buffer-filled spaces with an overall size of the order of 35-45 nm. Drastic changes in the diffraction pattern were observed, however, when the nuclei were washed in the more diluted buffers (1/6 and 1/12). The profiles of the distances distribution functions indicate the formation of supranucleosomal particles with an overall diameter of 40-50 nm. The compact chromatin structure disassembled directly into these fundamental structural units. Structural transformations in the Chinese hamster ovary metaphase chromosomes were induced by diminishing the Ca2+ ion concentration of the buffer from originally 3.0 mM to 0.3 mM and/or by increasing the pH value of the buffer from originally 7.0 up to 8.0. The neutron diffraction patterns remained essentially unchanged during these treatments, i.e. the decondensation of the chromosomes as observed in the light microscope is not accompanied by disassembly at the ultrastructural level between 2 nm and 150 nm.

Optimization of regulated LTR-mediated expression.

Retroviral vectors are ideally suited to the study of gene function, allowing efficient, stable expression. Many biological systems (e.g., cell cycle, apoptosis) require the use of regulated expression systems. We therefore developed a regulated retroviral vector system, TRA99, based on a tetracycline transactivator-dependent LTR, where the MMLV enhancer was replaced with a tetracycline-response element. Using fluorescence-activated flow cytometric analysis of a destabilized green fluorescent protein to monitor expression levels, we optimized the minimal promoter configuration with respect to both activated and repressed transcription. The TRA99 vectors demonstrate regulated expression with activated levels comparable to those of standard retroviral vectors and repressed levels indistinguishable from background. This was achieved without using an internal promoter cassette, thus retaining the cis-packaging elements requisite for helper-mediated transfer.

Chromosome assignment of two cloned DNA probes hybridizing predominantly to human sex chromosomes.

In situ hybridization experiments were carried out with two clones, YACG 35 and 2.8, which had been selected from two genomic libraries strongly enriched for the human Y chromosome. Besides the human Y chromosome, both sequences strongly hybridized to the human X chromosome, with few minor binding sites on autosomes. In particular, on the X chromosome DNA from clone YACG 35 hybridized to the centromeric region and the distal part of the short arm (Xp2.2). On the Y chromosome, the sequence was assigned to one site situated in the border region between Yq1.1 and Yq1.2. DNA from clone 2.8 also hybridized to the centromeric region of the X and the distal part of the short arm (Xp2.2). On the Y, however, two binding sites were observed (Yp1.1 and Yq1.2). The findings indicate that sex chromosomal sequences may be localized in homologous regions (as suggested from meiotic pairing) but also at ectopic sites.

Thrombopoietin in patients with congenital thrombocytopenia and absent radii: elevated serum levels, normal receptor expression, but defective reactivity to thrombopoietin.

The pathophysiology of thrombocytopenia in the syndrome of thrombocytopenia with absent radii (TAR) is not yet understood. We examined thrombopoietin (TPO) serum levels and the in vitro reactivity of platelets to TPO in five patients affected with TAR syndrome. We found elevated TPO serum levels in all patients tested, excluding a TPO production defect as cause for thrombocytopenia in TAR syndrome. In addition, we found similar expression of the TPO receptor c-Mpl on the surface of platelets from TAR patients (5 of 5) and a similar molecular weight of the receptor as compared with healthy controls (4 of 4). Platelet response to adenosine diphosphate or thrombin receptor agonist peptide SFLLRN (TRAP) was normal in TAR patients. However, in contrast to results with healthy controls we could show absence of in vitro reactivity of platelets from TAR patients to recombinant TPO as measured by testing TPO synergism to adenine diphosphate and TRAP in platelet activation. TPO induced tyrosine phosphorylation of platelet proteins was completely absent (3 of 4) or markedly decreased (1 of 4). Our results indicate that defective megakaryocytopoiesis/thrombocytopoiesis in TAR syndrome is not caused by a defect in TPO production but a lack of response to TPO in the signal transduction pathway of c-Mpl.