Microtubule Phenotype Research Articles

Interleukin-6 promotes microtubule stability in axons via Stat3 protein–protein interactions

SummaryThe interleukin-6 (IL-6) family of cytokines and its downstream effector, STAT3, are important mediators of neuronal health, repair, and disease throughout the CNS, including the visual system. Here, we elucidate a transcription-independent mechanism for the neuropoietic activities of IL-6 related to axon development, regeneration, and repair. We examined the outcome of IL-6 deficiency on structure and function of retinal ganglion cell (RGC) axons, which form the optic projection. We found that IL-6 deficiency substantially delays anterograde axon transport in vivo. The reduced rate of axon transport is accompanied by changes in morphology, structure, and post-translational modification of microtubules. In vivo and in vitro studies in mice and swine revealed that IL-6-dependent microtubule phenotypes arise from protein-protein interactions between STAT3 and stathmin. As in tumor cells and T cells, this STAT3-stathmin interaction stabilizes microtubules in RGCs. Thus, this IL-6-STAT3-dependent mechanism for axon architecture is likely a fundamental mechanism for microtubule stability systemically.

Tau does not protect microtubules during Klebsiella pneumoniae infections

Hospitals have raised a growing concern for the rampant spread of the bacterial pathogen Klebsiella pneumoniae, which can cause a wide array of symptoms such as pneumonia, urinary tract infections, septicemia, and pyogenic liver abscesses. Moreover, the misuse of antibiotics has contributed to the surge of multidrug‐resistant hypervirulent strains. As a result, therapeutics for K. pneumoniae infections are becoming ineffective leading to lower survival rates and higher mortality rates in highly infected individuals. In our laboratory, we have shown that K. pneumoniae activates katanin microtubule severing enzymes to induce microtubule disassembly in lung epithelial cells. In contrast, microtubule associated proteins (MAPs) in host cells regulate the stability of microtubules and specific MAPs such as Tau (MAPTau) can protect microtubules from katanin‐induced severing. Since Tau plays a role in the stability of microtubules, we hypothesized that Tau protein levels may prevent microtubule disassembly during K. pneumoniae infections. To test this hypothesis, we initially determined if K. pneumoniae targets Tau in infected A549 lung epithelial cells. We lysed uninfected and infected cells and we found that a higher molecular weight Tau species was present in the infected cell lysates. Hyperphosphorylation of Tau typically regulates its binding to microtubules, and thus, we then determined if overexpressing Tau constructs that have different phosphorylation abilities could protect microtubules from K. pneumoniae‐induced katanin‐mediated severing. To do this, we obtained three green fluorescent protein (EGFP)‐tagged tau constructs: wildtype Tau (wt Tau), hyperphosphorylated Tau (E14 Tau), and Tau that cannot be phosphorylated (AP Tau). We then expressed these plasmids in A549 cells and infected the cells with K. pneumoniae. If Tau protects microtubules from katanin‐severing, E14 Tau should have no effect on K. pneumoniae‐induced severing while wild‐type Tau and AP Tau should prevent microtubule severing. During these infections no rescue of the microtubule phenotype was observed thus, neither wild‐type Tau nor AP Tau protected microtubules from K. pneumoniae‐triggered microtubule disassembly. Our findings show that Tau overexpression plays no discernible role in protecting microtubules during Klebsiella pneumoniae infections.Support or Funding InformationThis study was funded by SFU institutional funds.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Dachsous1b cadherin regulates actin and microtubule cytoskeleton during early zebrafish embryogenesis

Dachsous (Dchs), an atypical cadherin, is an evolutionarily conserved regulator of planar cell polarity, tissue size and cell adhesion. In humans, DCHS1 mutations cause pleiotropic Van Maldergem syndrome. Here, we report that mutations in zebrafish dchs1b and dchs2 disrupt several aspects of embryogenesis, including gastrulation. Unexpectedly, maternal zygotic (MZ) dchs1b mutants show defects in the earliest developmental stage, egg activation, including abnormal cortical granule exocytosis (CGE), cytoplasmic segregation, cleavages and maternal mRNA translocation, in transcriptionally quiescent embryos. Later, MZdchs1b mutants exhibit altered dorsal organizer and mesendodermal gene expression, due to impaired dorsal determinant transport and Nodal signaling. Mechanistically, MZdchs1b phenotypes can be explained in part by defective actin or microtubule networks, which appear bundled in mutants. Accordingly, disruption of actin cytoskeleton in wild-type embryos phenocopied MZdchs1b mutant defects in cytoplasmic segregation and CGE, whereas interfering with microtubules in wild-type embryos impaired dorsal organizer and mesodermal gene expression without perceptible earlier phenotypes. Moreover, the bundled microtubule phenotype was partially rescued by expressing either full-length Dchs1b or its intracellular domain, suggesting that Dchs1b affects microtubules and some developmental processes independent of its known ligand Fat. Our results indicate novel roles for vertebrate Dchs in actin and microtubule cytoskeleton regulation in the unanticipated context of the single-celled embryo.

Dachsous1b cadherin regulates actin and microtubule cytoskeleton during early zebrafish embryogenesis.

Dachsous (Dchs), an atypical cadherin, is an evolutionarily conserved regulator of planar cell polarity, tissue size and cell adhesion. In humans, DCHS1 mutations cause pleiotropic Van Maldergem syndrome. Here, we report that mutations in zebrafish dchs1b and dchs2 disrupt several aspects of embryogenesis, including gastrulation. Unexpectedly, maternal zygotic (MZ) dchs1b mutants show defects in the earliest developmental stage, egg activation, including abnormal cortical granule exocytosis (CGE), cytoplasmic segregation, cleavages and maternal mRNA translocation, in transcriptionally quiescent embryos. Later, MZdchs1b mutants exhibit altered dorsal organizer and mesendodermal gene expression, due to impaired dorsal determinant transport and Nodal signaling. Mechanistically, MZdchs1b phenotypes can be explained in part by defective actin or microtubule networks, which appear bundled in mutants. Accordingly, disruption of actin cytoskeleton in wild-type embryos phenocopied MZdchs1b mutant defects in cytoplasmic segregation and CGE, whereas interfering with microtubules in wild-type embryos impaired dorsal organizer and mesodermal gene expression without perceptible earlier phenotypes. Moreover, the bundled microtubule phenotype was partially rescued by expressing either full-length Dchs1b or its intracellular domain, suggesting that Dchs1b affects microtubules and some developmental processes independent of its known ligand Fat. Our results indicate novel roles for vertebrate Dchs in actin and microtubule cytoskeleton regulation in the unanticipated context of the single-celled embryo.

Abstract 831: BAL101553 (prodrug of BAL27862): A unique microtubule destabilizer active against drug refractory breast cancers alone and in combination with trastuzumab

Abstract Background: BAL101553 is a highly soluble prodrug of BAL27862, a novel, small molecule, microtubule (MT)-depolymerizing agent with a broad in vitro anti-proliferative activity against human tumor lines refractory to standard microtubule-targeting agents (MTAs). The BAL27862 MT phenotype is distinct from standard MTAs (eg taxanes; vinca-alkaloids) and the prodrug has antitumor activity in diverse mouse tumor models when given orally or iv. In this study, activity was assessed in experimental breast cancer models alone and in combination with the ErbB2-targeted therapeutic antibody trastuzumab. Methods: Anti-proliferative activity was analyzed using monolayer or soft agar assays, MT phenotypes by immunofluorescence for α-tubulin and efficacy in mouse xenograft models of human breast tumors. Results: Potent anti-proliferative activity was observed in 8 of 9 breast cancer lines (IC50 range of sensitive lines: 8 - 25 nM) associated with the characteristic BAL27862 MT phenotype (tiny MT asters in G2/M arrested cells). Interestingly, 2 breast cancer lines were sensitive to BAL27862 in a clonogenic assay (IC50/IC70 - MAXF 401: 13/18 nM; MX1: 22/46 nM) despite one being relatively resistant to paclitaxel (MX1: 127/>3500 nM). Moreover, BAL27862 retained activity in 2 Pgp drug efflux pump-expressing breast cancer lines, 50 - 100-fold resistant to paclitaxel and vinblastine (MT-3/ADR, MCF7/TxT50), as well as in MCF7/TxTP50 cells ∼8-fold resistant to paclitaxel through non-Pgp-related mechanisms. When administered iv to mice at well tolerated doses, BAL101553 elicited statistically significant antitumor activity (p<0.05 vs. controls) in chemo-sensitive (MT-3) and chemo-refractory (MT-3/ADR) mammary xenografts, resulting in a final %T/C (ratio of mean tumor volume of treated and control group x 100) superior to comparator cytotoxics (MT-3: 35 % BAL101553, 54% paclitaxel, 104% vincristine; MT-3/ADR: 38% BAL101553, 118% paclitaxel, 103% vincristine). Antitumor activity was also observed in trastuzumab-sensitive (BT474) and-refractory (MaCa3366) ErbB2-overexpressing breast cancer xenografts, and in bulky BT474 tumors insensitive to trastuzumab treatment. Moreover, combination of BAL101553 and trastuzumab was well tolerated, with a striking enhancement of antitumor activity in the trastuzumab refractory MaCa336 model (% T/C - 56% BAL101553, 97% trastuzumab, 32% combination; p<0.05 vs. control or trastuzumab alone) which was associated with a significant delay in tumor growth over an extended time period (p=0.002 vs. controls). Conclusions: BAL101553 is currently undergoing Phase 1 clinical evaluation in advanced cancer patients with solid tumors. Potent antitumor activity alone and in combination with trastuzumab in preclinical chemo-refractory models of breast cancer supports clinical evaluation in breast cancer patients. Citation Format: Felix Bachmann, Karin Burger, George E. Duran, Branimir I. Sikic, Heidi A. Lane. BAL101553 (prodrug of BAL27862): A unique microtubule destabilizer active against drug refractory breast cancers alone and in combination with trastuzumab. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 831. doi:10.1158/1538-7445.AM2014-831

Nek7 kinase accelerates microtubule dynamic instability

The NIMA-related kinases (NRK or Nek) are emerging as conserved and crucial regulators of mitosis and cilia formation. The microtubule (MT) network has long been suspected as a major target of the Neks. However, the underlying mechanism remains unclear. Using the PlusTipTracker software, recently developed by the Danuser group, we followed the consequences of alterations in Nek7 levels on MT dynamic instability. siRNA-mediated downregulation of Nek7 in HeLa cells resulted in lower speeds of MT growth and catastrophe, reduction of the relative time spent in catastrophe, and considerably lowered the overall MT dynamicity. Co-expression of Nek7 with the siRNA treatment rescued the MT phenotypes, while ectopic overexpression of Nek7 yielded inverse characteristics compared to Nek7 downregulation. MT dynamics in mouse embryonic fibroblasts derived from targeted null mutants for Nek7 recapitulated the siRNA downregulation phenotypes. Precise MT dynamic instability is critical for accurate shaping of the mitotic spindle and for cilium formation, and higher MT dynamicity is associated with tumorigenicity. Thus, our results can supply a mechanistic explanation for Nek involvement in these processes.

Abstract 3795: BubR1 function is required for the antiproliferative activity of the novel microtubule-targeting drug BAL27862 (active moiety of the prodrug BAL101553)

Abstract Background: BAL27862 is a novel small molecule that induces apoptosis in cancer cells through microtubule destabilization associated with a distinct microtubule phenotype. BAL27862 has broad antitumor activity in preclinical models of human cancer, and has recently entered Phase I clinical trials in advanced cancer patients administered as a highly soluble lysine prodrug (BAL101553). Methods: Combinatorial RNAi screening using HeLa cervical carcinoma cells and a siRNA library targeting the human kinome/phosphatome was followed by image analysis of cell number and fraction with normal morphology after optimal BAL27862 treatment (50nM, 24h). BubR1 siRNA effects were also assessed by immunoblotting (IB) and 48h crystal violet proliferation assays. Drug-resistant tumor lines were selected in vitro by incubation with increasing BAL27862 concentrations. Patient-derived tumors were maintained in mice and analyzed by immunohistochemistry (IHC). Results: Combinatorial RNAi screening identified BubR1 (BUB1B gene) as a protein required for the antiproliferative and phenotypic effect of BAL27862. This finding was confirmed using single independent siRNAs. BubR1, an evolutionary conserved protein kinase controlling cell division, is a core component of the spindle assembly checkpoint. Consistent with this, a more detailed siRNA analysis across a panel of lines derived from different histological backgrounds (breast, colon, lung, brain) confirmed the essential role of BubR1 in the in vitro response of diverse tumor lines to BAL27862 treatment. For example, non-targeting control siRNA (NTC)-treated MDA-MB-453 breast carcinoma and H460 NSCLC cells entered a cell death program following BAL27862 treatment (∼60 % & ∼40 % cell death resp. vs. starting cell number); a response clearly attenuated by BubR1 down regulation (∼80% cell proliferation. vs. DMSO controls). Further IB analysis of tumor cell variants selected for stable drug resistance to BAL27862 (∼3-24-fold resistant), indicated that BubR1 protein expression was down regulated in 3 of 5 variants as compared to parental lines (SKOV3 ovarian, A549 & H460 non-small cell lung cancer [NSCLC]). Hence, reduced BubR1 expression may modulate development of resistance to BAL27862 under treatment. Strikingly, IHC analysis of paired patient-derived tumors derived from gastric and NSCLC carcinomas, demonstrated that intrinsic resistance to BAL27862 treatment ex vivo can also be associated with reduced BubR1 tumor expression. Conclusions: Reduced BubR1 expression correlates with resistance to BAL27862 treatment in a number of experimental models. Further work to understand the functional significance of BubR1 to the mechanism of action of BAL27862 is ongoing to explore the potential of using this biomarker to support patient stratification efforts during the clinical development of BAL101553. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3795. doi:1538-7445.AM2012-3795

Cytoskeletal role in protection of the failing heart by β-adrenergic blockade

Formation of a dense microtubule network that impedes cardiac contraction and intracellular transport occurs in severe pressure overload hypertrophy. This process is highly dynamic, since microtubule depolymerization causes striking improvement in contractile function. A molecular etiology for this cytoskeletal alteration has been defined in terms of type 1 and type 2A phosphatase-dependent site-specific dephosphorylation of the predominant myocardial microtubule-associated protein (MAP)4, which then decorates and stabilizes microtubules. This persistent phosphatase activation is dependent upon ongoing upstream activity of p21-activated kinase-1, or Pak1. Because cardiac β-adrenergic activity is markedly and continuously increased in decompensated hypertrophy, and because β-adrenergic activation of cardiac Pak1 and phosphatases has been demonstrated, we asked here whether the highly maladaptive cardiac microtubule phenotype seen in pathological hypertrophy is based on β-adrenergic overdrive and thus could be reversed by β-adrenergic blockade. The data in this study, which were designed to answer this question, show that such is the case; that is, β(1)- (but not β(2)-) adrenergic input activates this pathway, which consists of Pak1 activation, increased phosphatase activity, MAP4 dephosphorylation, and thus the stabilization of a dense microtubule network. These data were gathered in a feline model of severe right ventricular (RV) pressure overload hypertrophy in response to tight pulmonary artery banding (PAB) in which a stable, twofold increase in RV mass is reached by 2 wk after pressure overloading. After 2 wk of hypertrophy induction, these PAB cats during the following 2 wk either had no further treatment or had β-adrenergic blockade. The pathological microtubule phenotype and the severe RV cellular contractile dysfunction otherwise seen in this model of RV hypertrophy (PAB No Treatment) was reversed in the treated (PAB β-Blockade) cats. Thus these data provide both a specific etiology and a specific remedy for the abnormal microtubule network found in some forms of pathological cardiac hypertrophy.

Pronounced and extensive microtubule defects in a Saccharomyces cerevisiae DIS3 mutant

Subunits of the RNA processing exosome assemble into structurally distinct protein complexes that function in disparate cellular compartments and RNA metabolic pathways. Here, in a genetic, cell biological and transcriptomic analysis, we examined the role of Dis3, an essential polypeptide with endo- and 3'→5' exo-ribonuclease activity, in cell cycle progression. We present several lines of evidence that perturbation of DIS3 affects microtubule (MT) localization and structure in Saccharomyces cerevisiae. Cells with a DIS3 mutant: (a) accumulate anaphase and pre-anaphase mitotic spindles; (b) exhibit spindles that are misorientated and displaced from the bud neck; (c) harbour elongated spindle-associated astral MTs; (d) have an increased G1 astral MT length and number; and (e) are hypersensitive to MT poisons. Mutations in the core exosome genes RRP4 and MTR3 and the exosome cofactor gene MTR4, but not other exosome subunit gene mutants, also elicit MT phenotypes. RNA deep sequencing analysis (RNA-seq) shows broad changes in the levels of cell cycle- and MT-related transcripts in mutant strains. Collectively, the data presented in this study suggest an evolutionarily conserved role for Dis3 in linking RNA metabolism, MTs and cell cycle progression.

Abstract 743: Development of tumor models resistant to the novel microtubule destabilizer BAL27862 (active moiety of the prodrug BAL101553)

Abstract Background: The synthetic small molecule BAL27862, active component of the prodrug BAL101553, destabilizes microtubules, potently inducing apoptosis in cancer cells. BAL27862 elicits a broad antitumor activity and BAL101553/BAL27862 can be administered orally and intravenously. A unique microtubule phenotype suggests a novel mechanism of action (MoA), which is associated with activity in tumor models refractory to microtubule stabilizers (e.g. taxanes; epothilones) and destabilizers (e.g. Vinca-alkaloids such as vinblastine [VBL]). To further investigate the MoA and provide a platform for the identification of patient stratification biomarkers, BAL27862-resistant tumor models were developed. Methods: Drug-resistant tumor lines (Hela, H460, A549, SKOV3, Jurkat) were selected in vitro by incubation with increasing BAL27862 concentrations. Proliferation was measured by crystal violet assay, apoptosis by flow cytometry. Tubulin polymerization was assessed by immunoblotting or electron microscopy (EM). Tubulin binding experiments used a spin column and [3H]-VBL and [14C]-BAL27862. Results: Selection for BAL27862-resistant tumor cells in vitro proved slow, requiring 8 – 12 months to culture lines with resistance factors (RF) ranging from 4.5 – 16. As an example, the EC50 for induction of apoptosis following BAL27862 treatment in the parental (‘Par’) Jurkat line was 17 nM, whereas the resistant line (‘Res’) had an EC50 of 127 nM. Consistent with BAL27862 not being a Pgp efflux pump substrate, Pgp up regulation could not account for resistance development in any line. Moreover, tubulin polymerization assays indicated a higher proportion of tubulin in the polymerized fraction in ‘Res’ lines, indicative rather of microtubule stabilization. Furthermore, the microtubule network in ‘Res’ lines was significantly less sensitive to the depolymerizing activity of BAL27862 than in the ‘Par’ lines. The Jurkat ‘Res’ line was as sensitive as the ‘Par’ line to VBL treatment (EC50 for induction of apoptosis: 2 nM in both lines), whereas ‘Res’ H460 were equally resistant to BAL27862 (RF: 5.5) and VBL (RF: 4.2). Both Jurkat and H460 ‘Res’ lines had a slightly increased sensitivity to paclitaxel treatment (2-3 fold). Drug cross-resistance in H460 could not be simply due to overlapping binding sites, as BAL27862 and VBL bound independently to tubulin heterodimers. Furthermore, microtubule disassembly analysis by EM showed that BAL27862 did not induce the small oligomeric structures associated with VBL treatment. Conclusion: Selection for BAL27862 resistance in vitro requires prolonged culturing, is associated with more stable microtubules and may be mediated through diverse mechanisms. Results from work in progress to define the molecular changes associated with BAL27862 resistance could facilitate patient stratification during clinical development of the prodrug BAL101553. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 743. doi:10.1158/1538-7445.AM2011-743

Patronin Regulates the Microtubule Network by Protecting Microtubule Minus Ends

Tubulin assembles into microtubule polymers that have distinct plus and minus ends. Most microtubule plus ends in living cells are dynamic; the transitions between growth and shrinkage are regulated by assembly-promoting and destabilizing proteins. In contrast, minus ends are generally not dynamic, suggesting their stabilization by some unknown protein. Here, we have identified Patronin (also known as ssp4) as a protein that stabilizes microtubule minus ends in Drosophila S2 cells. In the absence of Patronin, minus ends lose subunits through the actions of the Kinesin-13 microtubule depolymerase, leading to a sparse interphase microtubule array and short, disorganized mitotic spindles. In vitro, the selective binding of purified Patronin to microtubule minus ends is sufficient to protect them against Kinesin-13-induced depolymerization. We propose that Patronin caps and stabilizes microtubule minus ends, an activity that serves a critical role in the organization of the microtubule cytoskeleton.

Site-specific Microtubule-associated Protein 4 Dephosphorylation Causes Microtubule Network Densification in Pressure Overload Cardiac Hypertrophy

In severe pressure overload-induced cardiac hypertrophy, a dense, stabilized microtubule network forms that interferes with cardiocyte contraction and microtubule-based transport. This is associated with persistent transcriptional up-regulation of cardiac alpha- and beta-tubulin and microtubule-stabilizing microtubule-associated protein 4 (MAP4). There is also extensive microtubule decoration by MAP4, suggesting greater MAP4 affinity for microtubules. Because the major determinant of this affinity is site-specific MAP4 dephosphorylation, we characterized this in hypertrophied myocardium and then assessed the functional significance of each dephosphorylation site found by mimicking it in normal cardiocytes. We first isolated MAP4 from normal and pressure overload-hypertrophied feline myocardium; volume-overloaded myocardium, which has an equal degree and duration of hypertrophy but normal functional and cytoskeletal properties, served as a control for any nonspecific growth-related effects. After cloning cDNA-encoding feline MAP4 and obtaining its deduced amino acid sequence, we characterized by mass spectrometry any site-specific MAP4 dephosphorylation. Solely in pressure overload-hypertrophied myocardium, we identified striking MAP4 dephosphorylation at Ser-472 in the MAP4 N-terminal projection domain and at Ser-924 and Ser-1056 in the assembly-promoting region of the C-terminal microtubule-binding domain. Site-directed mutagenesis of MAP4 cDNA was then used to switch each serine to non-phosphorylatable alanine. Wild-type and mutated cDNAs were used to construct adenoviruses; microtubule network density, stability, and MAP4 decoration were assessed in normal cardiocytes following an equivalent level of MAP4 expression. The Ser-924 --> Ala MAP4 mutant produced a microtubule phenotype indistinguishable from that seen in pressure overload hypertrophy, such that Ser-924 MAP4 dephosphorylation during pressure overload hypertrophy may be central to this cytoskeletal abnormality.

BAL27862: A unique microtubule-targeted agent that severs microtubules and overcomes multifactorial drug resistance.

e13589 Background: Microtubule-targeted agents (MTAs) are widely used to treat human malignancies. A major limitation of their efficacy is the development of resistance modulated by P-gp overexpression or tubulin and/or apoptotic pathway protein modifications. BAL27862 is a novel MTA that triggers apoptosis in cancer cells. In vitro, BAL27862 destabilizes microtubules to produce a microtubule phenotype distinct from that observed with other MTAs, and retains its antiproliferative activity against P-gp overexpressing tumor cells. BAL27862, when administered intravenously or orally, induces significant antitumor responses in a range of animal models of human cancer, including tumors refractory to conventional treatments. Methods: First, we compare the in vitro activity of BAL27862 with conventional MTAs paclitaxel (PTX) and vinblastine (VLB). The antiproliferative potency of BAL27862 was determined in a PTX- and VLB-resistant ovarian cancer cell line. Resistance was mediated by P-gp over-expression, Bcl-2 down-regulation and tubulin modifications. We then analyzed the effects of BAL27862 on microtubule dynamics (MDs) in SKBr3 breast cancer cells compared with PTX. Results: BAL27862 remained fully active against PTX- and VLB-resistant cells when compared with the chemosensitive parental line. The resistance factors observed (defined as EC50 ratio of resistant vs. parental cells) were 1.14 (NS), 147 (p<0.01) and 57 (p<0.01) for BAL27862, PTX and VLB respectively. These results confirm that BAL27862 overcomes P-gp overexpression-mediated resistance, and demonstrate that Bcl-2 status and tubulin modifications do not necessarily affect its antitumor activity. Analysis of MDs shows that BAL27862 elicited effects consistent with its destabilizing activity; suppression of MDs was 2-fold lower than observed with PTX. Strikingly, BAL27862 displayed a unique microtubule severing activity. Conclusions: Besides its microtubule-targeting activity, BAL27862 displays unique features in its mechanism of action that make it a promising compound for clinical investigation. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Basilea Pharmaceutica Basilea Pharmaceutica

BAL27862: A Unique Microtubule Destabilizer Active Against Chemorefractory Breast Cancers.

Abstract Background: BAL27862, a novel, synthetic, small molecule, is a potent inhibitor of tubulin polymerization that induces cancer cell death. BAL27862 elicits a unique microtubule (MT) phenotype, distinct from paclitaxel, vinblastine and colchicine, has broad in vitro anti-proliferative activity against a diverse range of human tumor lines (low nM IC50s) and induces significant antitumor responses in a range of animal models of human cancer when administered orally (p.o.) or intravenously (i.v.). In this study, BAL27862 activity in a panel of experimental breast cancer models was assessed.Materials and Methods: Anti-proliferative activity was analyzed using a monolayer (crystal violet) or soft agar (clonogenic) assay. Effects on MT phenotypes were assessed by immunofluorescence for α-tubulin. Efficacy was assessed in mouse xenograft models bearing chemosensitive and multidrug resistant human breast tumors.Results: The unique BAL27862-associated MT phenotype in interphase cells consisted of a partially collapsed MT network without peripheral MTs. In dividing cells, tiny MT asters were found scattered within the nuclear region. Potent anti-proliferative activity was demonstrated against 8 breast cancer cell lines (crystal violet assay IC50 range: 6.5 – 22 nM for the SKBR3, MCF7, BT474, T47D, BT549, MDA-MB231, MDA-MB453 and MDA-MB468 lines), although one line (HCC1937) appeared relatively insensitive (IC50: &amp;gt;1000 nM). Interestingly, two breast cancer lines were sensitive to BAL27862 treatment in a clonogenic assay (IC50/IC70: MAXF 401 = 13/18 nM; MAXF MX1 = 22/46 nM), despite one being relatively resistant to paclitaxel treatment (MAXF 401 = 11/48 nM; MAXF MX1 = 127/&amp;gt;3500 nM). Moreover, using monolayer assays, BAL27862 activity was retained against five tumor lines overexpressing the Pgp efflux pump (including MT-3/ADR mammary adenocarcinoma cells), which were up to several thousand-fold resistant to paclitaxel and vinblastine. BAL27862 showed little activity against human stem cells or peripheral blood mononucleocytes.When administered p.o or i.v. to mice at well tolerated doses, BAL27862 treatment elicited statistically significant antitumor activity (p≤0.05) in three chemosensitive human breast tumor xenograft models (including MAXF 401, MaCa 4049 and MT-3); resulting in a final %T/C (ratio of mean tumor volume of treated and control group x 100) equivalent to that observed with comparator cytotoxics using MTD schedules (e.g. final %T/C in MT-3 model: 36% BAL27862, 35% paclitaxel, 50% doxorubicin). Strikingly, significant antitumor activity was maintained in the Pgp-overexpressing MT-3/ADR xenograft model, where paclitaxel and doxorubicin were ineffective (final %T/C: 37% BAL27862, 112% paclitaxel, 108% doxorubicin).Conclusions: BAL27862 is a new tubulin-interacting agent with an apparently novel mechanism of action. A potent antitumor activity in experimental models of breast cancer, including chemorefractory models, strongly support further development of BAL27862 as a novel breast cancer treatment modality with a possibility for both i.v. and p.o. administration. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2093.

Abstract C233: BAL27862: A unique microtubule-targeted agent with a potential for the treatment of human brain tumors

Abstract Background: BAL27862 is a synthetic small molecule, potently inducing apoptosis in cancer cells through tubulin depolymerization characterized by a unique microtubule phenotype. BAL27862 has demonstrated a broad in vitro anti-proliferative activity against a range of human tumor histotypes (low nM IC50s). Significant antitumor responses occur in animal models of human cancer after oral or intravenous (i.v.) administration, including tumors refractory to conventional treatments. The purpose of this study was to investigate the tissue distribution of BAL27862 in tumor-bearing mice. Tumor and brain penetration, together with anti-proliferative activity in glioblastoma (GBM) cell lines, is described. Materials and Methods: Anti-proliferative activity and induction of tumor cell death in vitro were analyzed using the YO-PRO® assay (48h incubation). CD1 nu/nu female mice were implanted with human colon carcinoma SW480 cells. For pharmacokinetic studies, tumor-bearing (approx. 150 mm3) mice were administered i.v. with either 2 mg/kg 14C-BAL27862 (n=6, single dose) or 8 mg/kg cold BAL27862 (n=33, once-weekly during 4 weeks). Animals were culled at serial time points. Radioactivity was measured in slices, with Bio-Imaging Analyzer read-outs of the exposed phosphor imaging plates quantified with Aida software. Cold BAL27862 was determined in plasma, brain and tumors using a specific LC-MS/MS method after sample homogenization and protein precipitation. Results: BAL27862 elicited a potent anti-proliferative activity in 6 GBM cell lines (IC50 range: 10–20nM), which was independent of PTEN status. Strikingly, at optimal concentrations, a dramatic loss of cell viability was observed (% cell death at 50nM BAL27862: &amp;gt;15% in 5 lines, &amp;gt;30% in 2 lines), demonstrating that BAL27862 potently drives GBM cells into a cell death program..In mice, after i.v. administration of 14C-BAL27862, radioactivity was distributed to all organs: notably brain and tumor. A delayed peak was observed in slowly perfused organs such as skin, tumor and fat-tissue. There was no tissue-specific retention of radioactivity, as halflives were comparable between tissues and blood. 48h after administration, radioactivity was almost undetectable in most tissues. Following administration of 8 mg/kg i.v. cold BAL27862 to mice, the excellent brain and tumor penetration was confirmed. Specifically, similar levels of BAL27862 were found in brain and plasma (Cmax of 6.86 µg/g and 7.34 µg/mL, respectively). The ratio tumor/plasma was also around 1. There was no brain or tumor accumulation over time, as concentrations paralleled those in plasma. Conclusions: BAL27862 is efficiently distributed to tissues and tumor in a mouse model of human cancer. Significant brain penetration, coupled with cytotoxic activity in GBM tumor cell lines, would support further evaluation of BAL27862 for the treatment of human brain cancers. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C233.

Overexpressing Temperature-Sensitive Dynamin Decelerates Phototransduction and Bundles Microtubules in Drosophila Photoreceptors

shibire(ts1), a temperature-sensitive mutation of the Drosophila gene encoding a Dynamin orthologue, blocks vesicle endocytosis and thus synaptic transmission, at elevated, or restrictive temperatures. By targeted Gal4 expression, UAS-shibire(ts1) has been used to dissect neuronal circuits. We investigated the effects of UAS-shibire(ts1) overexpression in Drosophila photoreceptors at permissive (19 degrees C) and restrictive (31 degrees C) temperatures. At 19 degrees C, overexpression of UAS-shi(ts1) causes decelerated phototransduction and reduced neurotransmitter release. This phenotype is exacerbated with dark adaptation, age and in white mutants. Photoreceptors overexpressing UAS-shibire(ts1) contain terminals with widespread vacuolated mitochondria, reduced numbers of vesicles and bundled microtubules. Immuno-electron microscopy reveals that the latter are dynamin coated. Further, the microtubule phenotype is not restricted to photoreceptors, as UAS-shibire(ts1) overexpression in lamina cells also bundles microtubules. We conclude that dynamin has multiple functions that are interrupted by UAS-shibire(ts1) overexpression in Drosophila photoreceptors, destabilizing their neural communication irreversibly at previously reported permissive temperatures.

Proliferating cardiac microtubules

identifying the mechanisms underlying the transition from adaptive cardiac hypertrophy to maladaptive cardiac failure has long been a central goal of basic and clinical cardiovascular science. Such studies at the level of the cardiocyte have focused largely on the cardiac motor, i.e., the

Rapid microtubule bundling and stabilization by the Streptococcus pneumoniae neurotoxin pneumolysin in a cholesterol‐dependent, non‐lytic and Src‐kinase dependent manner inhibits intracellular trafficking

Streptococcus pneumoniae is the most frequent cause of bacterial meningitis, leading to permanent neurological damage in 30% and lethal outcome in 25% of patients. The cholesterol-dependent cytolysin pneumolysin is a major virulence factor of S. pneumoniae. It produces rapid cell lysis at higher concentrations or apoptosis at lower concentrations. Here, we show that sublytic amounts of pneumolysin produce rapid bundling and increased acetylation of microtubules (signs of excessive microtubule stabilization) in various types of cells--neuroblastoma cells, fibroblasts and primary astrocytes. The bundling started perinuclearly and extended peripherally towards the membrane. The effect was not connected to pneumolysin's capacity to mediate calcium influx, macropore formation, apoptosis, or RhoA and Rac1 activation. Cellular cholesterol depletion and neutralization of the toxin by pre-incubation with cholesterol completely inhibited the microtubule phenotype. Pharmacological inhibition of Src-family kinases diminished microtubule bundling, suggesting their involvement in the process. The relevance of microtubule stabilization to meningitis was confirmed in an experimental pneumococcal meningitis animal model, where increased acetylation was observed. Live imaging experiments demonstrated a decrease in organelle motility after toxin challenge in a manner comparable to the microtubule-stabilizing agent taxol, thus proposing a possible pathogenic mechanism that might contribute to the CNS damage in pneumococcal meningitis.

Spinophilin Facilitates Dephosphorylation of Doublecortin by PP1 to Mediate Microtubule Bundling at the Axonal Wrist

The axonal shafts of neurons contain bundled microtubules, whereas extending growth cones contain unbundled microtubule filaments, suggesting that localized activation of microtubule-associated proteins (MAP) at the transition zone may bundle these filaments during axonal growth. Dephosphorylation is thought to lead to MAP activation, but specific molecular pathways have remained elusive. We find that Spinophilin, a Protein-phosphatase 1 (PP1) targeting protein, is responsible for the dephosphorylation of the MAP Doublecortin (Dcx) Ser 297 selectively at the "wrist" of growing axons, leading to activation. Loss of activity at the "wrist" is evident as an impaired microtubule cytoskeleton along the shaft. These findings suggest that spatially restricted adaptor-specific MAP reactivation through dephosphorylation is important in organization of the neuronal cytoskeleton.

LET-711, theCaenorhabditis elegansNOT1 Ortholog, Is Required for Spindle Positioning and Regulation of Microtubule Length in Embryos

Spindle positioning is essential for the segregation of cell fate determinants during asymmetric division, as well as for proper cellular arrangements during development. In Caenorhabditis elegans embryos, spindle positioning depends on interactions between the astral microtubules and the cell cortex. Here we show that let-711 is required for spindle positioning in the early embryo. Strong loss of let-711 function leads to sterility, whereas partial loss of function results in embryos with defects in the centration and rotation movements that position the first mitotic spindle. let-711 mutant embryos have longer microtubules that are more cold-stable than in wild type, a phenotype opposite to the short microtubule phenotype caused by mutations in the C. elegans XMAP215 homolog ZYG-9. Simultaneous reduction of both ZYG-9 and LET-711 can rescue the centration and rotation defects of both single mutants. let-711 mutant embryos also have larger than wild-type centrosomes at which higher levels of ZYG-9 accumulate compared with wild type. Molecular identification of LET-711 shows it to be an ortholog of NOT1, the core component of the CCR4/NOT complex, which plays roles in the negative regulation of gene expression at transcriptional and post-transcriptional levels in yeast, flies, and mammals. We therefore propose that LET-711 inhibits the expression of ZYG-9 and potentially other centrosome-associated proteins, in order to maintain normal centrosome size and microtubule dynamics during early embryonic divisions.