We have identified a gene, fos B, encoding a nuclear protein of 338 amino acids presenting a 70% homology with c‐fos, whose expression is activated during G0/G1 transition. Growth factor stimulation of quiescent cells leads to a rapid and transient accumulation of fos B mRNA, with kinetics similar to those of c‐fos. The induction of fos B mRNA levels is in part due to a dramatic increase in the transcription of the gene. The half‐life of fos B mRNA is in the order of 10‐15 min. Both transcriptional activation and mRNA stability are substantially increased in the presence of protein synthesis inhibitors. Immunoprecipitation studies showed that fos B as c‐fos protein, forms a complex in vitro with c‐jun and jun B proteins in the absence of a target binding sequence. Gel retardation assays demonstrated that fos B protein positively influences the binding of c‐jun and jun B proteins to an AP‐1 binding consensus sequence, suggesting that fos B protein plays a role in control of gene expression.

Mutations in codon 132 of isocitrate dehydrogenase (IDH) 1 are frequent in diffuse glioma, acute myeloid leukemia, chondrosarcoma and intrahepatic cholangiocarcinoma. These mutations result in a neomorphic enzyme specificity which leads to a dramatic increase of intracellular D-2-hydroxyglutarate (2-HG) in tumor cells. Therefore, mutant IDH1 protein is a highly attractive target for inhibitory drugs. Here, we describe the development and properties of BAY 1436032, a pan-inhibitor of IDH1 protein with different codon 132 mutations. BAY 1436032 strongly reduces 2-HG levels in cells carrying IDH1-R132H, -R132C, -R132G, -R132S and -R132L mutations. Cells not carrying IDH mutations were unaffected. BAY 1436032 did not exhibit toxicity in vitro or in vivo. The pharmacokinetic properties of BAY 1436032 allow for oral administration. In two independent experiments, BAY 1436032 has been shown to significantly prolong survival of mice intracerebrally transplanted with human astrocytoma carrying the IDH1R132H mutation. In conclusion, we developed a pan-inhibitor targeting tumors with different IDH1R132 mutations.

Abstract. UV irradiation of quiescent human fibroblasts immediately triggers the appearance of the nuclear protein cyclin/proliferating cell nuclear antigen (PCNA) as detected by indirect immunofluorescent staining after methanol fixation. This was found to be independent of new synthesis of cyclin/PCNA by twodimensional gel analysis and cycloheximide treatment. The intensity of the immunofluorescent staining of cyclin/PCNA observed in UV-irradiated cells corresponded with the UV dose used and with the DNA repair synthesis detected by autoradiography. The nuclear staining remains as long as DNA repair activity is detected in the cells. By extracting the UV-irradiated quiescent cells with Triton X-100 and fixing with formaldehyde, it was possible to demonstrate by indirect immunofluorescence rapid changes in the cyclin/PCNA population after irradiation, a small proportion (5-10%) of which is tightly associated to the nucleus as determined by high salt extraction. By incubating at low temperature and depleting the ATP pools of the cells before UV irradiation, we have demonstrated that the changes in cyclin/PCNA distribution observed involve at least two different nuclear associations.T hE proliferating cell nuclear antigen (PCNA; 18, 24, 25, 27) 1 and cyclin (reviewed in references 1, 10) have been shown to be identical (17). This protein is present in variable amounts in normal proliferating and transformed cells from a variety of vertebrates (1, 10). The synthesis of cyclin/PCNA is regulated during the cell cycle, increasing during the DNA replication period (2). In quiescent cells, where cyclin/PCNA expression is very low, mitogens induce a coordinate synthesis of this protein and DNA (3).Immunofluorescence studies of cyclin/PCNA have shown that its nuclear distribution changes dramatically through the S-phase and that it is tightly associated with DNA replication sites (4,5,10,18).The observations that cyclin/PCNA is required for SV-40 DNA replication in vitro (20,21) and that it is identical to the auxiliary protein of DNA polymerase/5 (6, 22), a protein that enhances the ability of the polymerase to use templates which contain long single-stranded regions (26), are the most direct proofs that this protein plays a role in DNA synthesis.DNA polymerase/5 is the most recently discovered of the four DNA polymerases known at present (for review see reference 13). It possesses a 3' to 5' exonuclease activity (7) which may contribute to the fidelity of DNA replication (11,15,16). This suggests that both polymerases, ct and/5, could function jointly during DNA replication. Moreover, recent evidence has shown that DNA polymerase/5 plays an impor-1. Abbreviation used in this paper: PCNA, proliferating cell nuclear antigen. rant role during DNA replication and DNA repair synthesis (11,12,15,19). Being cyclin/PCNA, a putative regulatory subunit ofDNA polymerase/5, we found it important to study the effect of UV irradiation in the synthesis and distribution of this protein in quiescent human fibroblasts. Here we present e...

Experimental Design: Derivatives of L-glutamate were investigated in cell competition assays to characterize the responsible transporter. An automated radiosynthesis was established for the most promising candidate. The resulting 18 F-labeled PET tracer was characterized in a panel of in vitro and in vivo tumor models. Tumor specificity was investigated in the turpentine oil-induced inflammation model in rats.Results: A fluoropropyl substituted glutamate derivative showed strong inhibition in cell uptake assays. The radiosynthesis was established for (4S)-4-(3-[ 18 F]fluoropropyl)-L-glutamate (BAY 94-9392).Tracer uptake studies and analysis of knockdown cells showed specific transport of BAY 94-9392 via the cystine/glutamate exchanger designated as system x C À . No metabolites were observed in mouse blood and tumor cells. PET imaging with excellent tumor visualization and high tumor to background ratios was achieved in preclinical tumor models. In addition, BAY 94-9392 did not accumulate in inflammatory lesions in contrast to FDG.Conclusions: BAY 94-9392 is a new tumor-specific PET tracer which could be useful to examine system x C À activity in vivo as a possible hallmark of tumor oxidative stress. Both preclinical and clinical studies are in progress for further characterization.

No abstract

The distinguishing characteristic of vampire bat (Desmodus rotundus) salivary plasminogen activators (DSPAs) is their strict requirement for fibrin as a cofactor. DSPAs consist of structural modules known from urokinase (u-PA) and tissue-type plasminogen activator (t-PA) such as finger (F), epidermal growth factor (E), kringle (K), and protease (P), combining to four genetically and biochemically distinct isoenzymes, exhibiting the formulas FEKP (DSPA␣1 and ␣2) and EKP and KP (DSPA␤ and DSPA␥). Only DSPA␣1 and ␣2 bind to fibrin. All DSPAs are single-chain molecules, displaying substantial amidolytic activity. In a plasminogen activation assay, all four DSPAs are almost inactive in the absence of fibrin but strongly stimulated by fibrin addition. The catalytic efficiency (k cat /K m ) of DSPA␣1 increases 10 5 -fold, whereas the corresponding value of t-PA is only 550. The ratio of the bimolecular rate constants of plasminogen activation in the presence of fibrin versus fibrinogen (fibrin selectivity) of DSPA␣1, ␣2, ␤, ␥, and t-PA was found to be 13,000, 6500, 250, 90, and 72, respectively. Whereas all DSPAs are therefore more fibrin dependent and fibrin selective than t-PA, the extent depends on the respective presence of the various domains. The introduction of a plasmin-sensitive cleavage site in a position akin to the one in t-PA partially obliterates fibrin cofactor requirement. Fibrin dependence and fibrin selectivity of DSPAs are accordingly mediated by fibrin binding, which involves the F domain, as yet undefined determinants within the K and P domains, and by the absence of a plasmin-sensitive activation site. These findings transcend the current understanding of fibrin-mediated stimulation of plasminogen activation: in addition to fibrin binding, specific protein-protein interactions come into play, which stabilize the enzyme in its active conformation.

Neomorphic mutations in isocitrate dehydrogenase 1 (IDH1) are frequently found in several human cancer types including acute myeloid leukemia (AML) and lead to the production of high levels of the oncometabolite (R)-2-hydroxyglutarate (R-2HG). Here we report the characterization of BAY1436032, a novel pan-mutant IDH1 inhibitor, both in vitro and in vivo. BAY1436032 specifically inhibits R-2HG production and colony growth, and induces myeloid differentiation of AML cells carrying IDH1R132H, IDH1R132C, IDH1R132G, IDH1R132L and IDH1R132S mutations. In addition, the compound impacts on DNA methylation and attenuates histone hypermethylation. Oral administration of BAY1436032 led to leukemic blast clearance, myeloid differentiation, depletion of leukemic stem cells and prolonged survival in two independent patient-derived xenograft IDH1 mutant AML mouse models. Together, BAY1436032 is highly effective against all major types of IDH1 mutant AML.

The lactate transporter /monocarboxylate transporter 1 (MCT1) plays a central role in tumor cell energy homeostasis. In a cell-based screen, we identified a novel class of MCT1 inhibitors, including BAY-8002, which potently suppress bidirectional lactate transport. We investigated the antiproliferative activity of BAY-8002 in a panel of 246 cancer cell lines and show that hematopoietic tumor cells, in particular diffuse large B-cell lymphoma cell lines, and subsets of solid tumor models are particularly sensitive to MCT1 inhibition. Associated markers of sensitivity were, among others, lack of MCT4 expression, low pleckstrin homology like domain family A member 2, and high pellino E3 ubiquitin protein ligase 1 expression. The antitumor effect of MCT1 inhibition was less pronounced on tumor xenografts, with tumor stasis being the maximal response. BAY-8002 significantly increased intratumor lactate levels and transiently modulated pyruvate levels. In order to address potential acquired resistance mechanisms to MCT1 inhibition, we generated MCT1 inhibitor-resistant cell lines and show that resistance can occur by upregulation of MCT4 even in the presence of sufficient oxygen, as well as by shifting energy generation toward oxidative phosphorylation. These findings provide insight into novel aspects of tumor response to MCT1 modulation and offer further rationale for patient selection in the clinical development of MCT1 inhibitors..