Marjo Simonen

Identification and characterization of NVP-BEZ235, a new orally available dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor with potent in vivo antitumor activity

The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin inhibitor (mTOR) pathway is often constitutively activated in human tumor cells, providing unique opportunities for anticancer therapeutic intervention. NVP-BEZ235 is an imidazo[4,5-c]quinoline derivative that inhibits PI3K and mTOR kinase activity by binding to the ATP-binding cleft of these enzymes. In cellular settings using human tumor cell lines, this molecule is able to effectively and specifically block the dysfunctional activation of the PI3K pathway, inducing G1 arrest. The cellular activity of NVP-BEZ235 translates well in in vivo models of human cancer. Thus, the compound was well tolerated, displayed disease stasis when administered orally, and enhanced the efficacy of other anticancer agents when used in in vivo combination studies. Ex vivo pharmacokinetic/pharmacodynamic analyses of tumor tissues showed a time-dependent correlation between compound concentration and PI3K/Akt pathway inhibition. Collectively, the preclinical data show that NVP-BEZ235 is a potent dual PI3K/mTOR modulator with favorable pharmaceutical properties. NVP-BEZ235 is currently in phase I clinical trials. [Mol Cancer Ther 2008;7(7):1–13 [Mol Cancer Ther 2008;7(7):1851–13]

Systemic deletion of the myelin-associated outgrowth inhibitor Nogo-A improves regenerative and plastic responses after spinal cord injury.

To investigate the role of the myelin-associated protein Nogo-A on axon sprouting and regeneration in the adult central nervous system (CNS), we generated Nogo-A-deficient mice. Nogo-A knockout (KO) mice were viable, fertile, and not obviously afflicted by major developmental or neurological disturbances. The shorter splice form Nogo-B was strongly upregulated in the CNS. The inhibitory effect of spinal cord extract for growing neurites was decreased in the KO mice. Two weeks following adult dorsal hemisection of the thoracic spinal cord, Nogo-A KO mice displayed more corticospinal tract (CST) fibers growing toward and into the lesion compared to their wild-type littermates. CST fibers caudal to the lesion-regenerating and/or sprouting from spared intact fibers-were also found to be more frequent in Nogo-A-deficient animals.

Nogo-A-Deficient Mice Reveal Strain-Dependent Differences in Axonal Regeneration

Nogo-A, a membrane protein enriched in myelin of the adult CNS, inhibits neurite growth and regeneration; neutralizing antibodies or receptor blockers enhance regeneration and plasticity in the injured adult CNS and lead to improved functional outcome. Here we show that Nogo-A-specific knock-outs in backcrossed 129X1/SvJ and C57BL/6 mice display enhanced regeneration of the corticospinal tract after injury. Surprisingly, 129X1/SvJ Nogo-A knock-out mice had two to four times more regenerating fibers than C57BL/6 Nogo-A knock-out mice. Wild-type newborn 129X1/SvJ dorsal root ganglia in vitro grew a much higher number of processes in 3 d than C57BL/6 ganglia, confirming the stronger endogenous neurite growth potential of the 129X1/SvJ strain. cDNA microarrays of the intact and lesioned spinal cord of wild-type as well as Nogo-A knock-out animals showed a number of genes to be differentially expressed in the two mouse strains; many of them belong to functional categories associated with neurite growth, synapse formation, and inflammation/immune responses. These results show that neurite regeneration in vivo, under the permissive condition of Nogo-A deletion, and neurite outgrowth in vitro differ significantly in two widely used mouse strains and that Nogo-A is an important endogenous inhibitor of axonal regeneration in the adult spinal cord.

Nogo-A expressed in Schwann cells impairs axonal regeneration after peripheral nerve injury

Înjured axons in mammalian peripheral nerves often regenerate successfully over long distances, in contrast to axons in the brain and spinal cord (CNS). Neurite growth-inhibitory proteins, including the recently cloned membrane protein Nogo-A, are enriched in the CNS, in particular in myelin. Nogo-A is not detectable in peripheral nerve myelin. Using regulated transgenic expression of Nogo-A in peripheral nerve Schwann cells, we show that axonal regeneration and functional recovery are impaired after a sciatic nerve crush. Nogo-A thus overrides the growth-permissive and -promoting effects of the lesioned peripheral nerve, demonstrating its in vivo potency as an inhibitor of axonal regeneration.

A microarray-based, integrated approach to identify novel regulators of cancer drug response and apoptosis.

DNA microarrays are powerful tools for the analysis of gene expression on a genomic scale. The importance of individual regulatory events for the process under study can however not be deduced unequivocally without additional experiments. We devised a strategy to identify central regulators of cancer drug responses by combining the results of microarray experiments with efficient methods for phenotypic testing of candidate genes. We exposed murine FL5.12 pro-B cells to cisplatin, camptothecin, methotrexate or paclitaxel, respectively and analysed the patterns of gene expression with cDNA microarrays. Drug-specific regulatory events as well as intersections between different apoptotic pathways, including previously studied responses to staurosporine and interleukin-3 (IL-3) deprivation, were identified. Genes shared by at least three pathways were chosen for further analysis. Ectopic expression of three such genes, TEAP, GP49B, and Lipin1 was found to have an anti-proliferative effect on pro-B cells. Interestingly, we identified hemoglobin alpha as a strong pro-apoptotic regulator. While hemoglobin-expressing cells were growing normally in the presence of IL-3, they displayed accelerated apoptosis with similar kinetics as Bax overexpressing cells upon IL-3 removal. The pro-apoptotic effect of hemoglobin was suppressed by Bcl-2 and was characterized by enhanced stimulation of caspase activity.

High-Content Assay to Study Protein Prenylation

The mevalonate pathway leads to synthesis of cholesterol and isoprenoid lipids. Prenyltransferases attach the isoprenoid lipids to the C-terminus of several small guanosine triphosphate—binding proteins. The prenyl groups are essential for the biological activity of these proteins. The prenyltransferases and other components of the mevalonate pathway are either present or potential drug targets for cancer, osteoporosis, restenosis, or high serum cholesterol level. Until recently, cellular assays to study protein prenylation have been tedious, low-throughput assays. The authors have developed a high-content imaging-based assay to study protein prenylation. The assay is based on a green fluorescent protein (GFP) reporter, which is tagged with the prenylation motif of human H-Ras. The C-terminus of H-Ras targets GFP to the plasma membrane. When protein prenylation is inhibited, the tagged GFP cannot be localized to plasma membrane but is soluble in the cells. The localization of the GFP reporter can be analyzed in the 96- or 384-well format using automated microscopy and automated image analysis. Information about cell number and nuclear intensity can be obtained from the same images. In compound screening, these readouts provide valuable information about the toxicity of the compounds. The authors have validated their assay using several inhibitors of the mevalonate pathway as well as siRNA against farnesyl pyrophosphate synthase, a critical enzyme in the synthesis of the isoprenoid lipids. (Journal of Biomolecular Screening 2008:456-467)

Upregulation of alpha globin promotes apoptotic cell death in the hematopoietic cell line FL5.12

The function of alpha globin in the context of oxygen transport in erythroid cells is well described. Recently the expression of alpha globin was shown to be upregulated upon specific apoptotic stimuli like cytokine deprivation or cisplatin treatment in the hematopoietic pro-B cell line, FL5.12. In contrast to alpha globin, beta globin or globin-like genes were expressed at a very low level or were not expressed at all. Further, we found that alpha globin was not associated with heme. Apoptotic cells neither produced hemoglobin nor displayed a phenotype of cells differentiating down the erythroid lineage. Also other cell lines of variable differentiation status (NIH3T3, HeLa, K562) upregulated alpha globin during treatment with apoptosis-inducing agents. Under IL-3-deprived conditions GFP-alpha globin accelerated the progression of apoptosis comparable to GFP-Bax. GFP-alpha globin was expressed at a low level and enrichment of FL5.12 cells expressing GFP-alpha globin was difficult even in the presence of IL-3. Caspase-8, -9 and -3 as well as the proapoptotic factor Bax and cytochrome c were activated. Antisense alpha globin downregulated the expression of endogenous alpha globin und reduced caspase activity. Taken together these data indicate that alpha globin is a new and crucial factor in apoptosis especially supporting the mitochondrial pathway.

Comparison of Variability and Sensitivity between Nuclear Translocation and Luciferase Reporter Gene Assays

High-content screening (HCS), a technology based on subcellular imaging by automated microscopy and sophisticated image analysis, has emerged as an important platform in small-molecule screening for early drug discovery. To validate a subcellular imaging assay for primary screening campaigns, an HCS assay was compared with a non—image-based readout in terms of variability and sensitivity. A study was performed monitoring the accumulation of the forkhead transcription factor of the O subfamily (FOXO3a) coupled with green fluorescent protein in the nucleus of human osteosarcoma (U-2 OS) cells. In addition, the transcription of a luciferase gene coupled with a FOXO3a-responsive promoter was monitored. This report demonstrates that both assay formats show good reproducibility in primary and concentration response screening despite differences in statistical assay quality. In primary screening, the correlation of compound activity between the 2 assays was low, in contrast to the good correlation of the IC50 values of confirmed compounds. Furthermore, the high-content imaging assay showed a mean shift of 2.63-fold in IC50 values compared with the reporter gene assay. No chemical scaffold was specifically found with 1 of the technologies only, however these results validate the HCS technology against established assays for screening of new molecular entities. (Journal of Biomolecular Screening 2009:59-65)

Hematopoietic transcription factor GATA-2 promotes upregulation of alpha globin and cell death in FL5.12 cells

Recently we showed that alpha globin is a novel pro-apoptotic factor in programmed cell death in the pro-B cell line, FL5.12. Alpha globin was also upregulated in various other cell lines after different apoptotic stimuli. Under withdrawal of IL-3, overexpression of alpha globin accelerated apoptosis in FL5.12. Here, we have studied how transcription of alpha globin is placed in the broader context of apoptosis. We used Affymetrix chip technology and RT QPCR to compare expression patterns of FL5.12 cells growing with or without IL-3 to search for transcription factors which were concomitantly upregulated with alpha globin. The erythroid-specific transcription factor GATA-2 was the earliest and most prominently upregulated candidate. GATA-1 was expressed at low levels and was weakly induced while GATA-3 was completely absent. To evaluate the influence of GATA-2 on alpha globin expression and cell viability we overexpressed GATA-2 in FL5.12 cells. Interestingly, high expression of GATA-2 resulted in cell death and elevated alpha globin levels in FL5.12 cells. Transduction of antisense GATA-2 prevented both increase of GATA-2 and alpha globin under apoptotic conditions and delayed cell death. We suggest a role of GATA-2 in apoptosis besides its function in maintenance and proliferation of immature hematopoietic progenitors.