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Developm ental Biology 224, 440 –452 (2000) doi:10.1006/dbio.2000.9775, available online at http://www.idealibrary.com on

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Cyclopam ine Inhibition of Sonic Hedgehog Signal Transduction Is Not Mediated through Effects on Cholesterol Transport

John P. Incardona,* William Gaffield,† Yvonne Lange,‡ Adele Cooney,§ Peter G. Pentchev,§ Sharon Liu,John A. Watson,Raj P. Kapur,ሻ and Henk Roelink*,1

*Departm ent of Biological Structure and Center for Developm ental Biology and ࿣Departm ent of Pathology, U niversity of Washington, Seattle, Washington 98195; †Western Regional Research Center, ARS, U nited States Departm ent of Agriculture, Albany, California 94710; ‡Departm ent of Pathology, Rush–Presbyterian–St. Luke’s Medical Center, Chicago, Illinois 60612; §Developm ental and Metabolic Neurobiology Branch, NINDS, National Institutes of Health, Bethesda, Maryland 20892; and Departm ent of Biochem istry and Biophysics, U niversity of California at San Francisco, San Francisco, California 94143

Cyclopam ine is a teratogenic steroidal alkaloid that causes cyclopia by blocking Sonic hedgehog (Shh) signal transduction. We have tested whether this activity of cyclopam ine is related to disruption of cellular cholesterol transport and putative secondary effects on the Shh receptor, Patched (Ptc). First, we report that the potent antagonism of Shh signaling by cyclopam ine is not a general property of steroidal alkaloids with sim ilar structure. The structural features of steroidal alkaloids previously associated with the induction of holoprosencephaly in whole anim als are also associated with inhibition of Shh signaling in vitro. Second, by com paring the effects of cyclopam ine on Shh signaling with those of com pounds known to block cholesterol transport, we show that the action of cyclopam ine cannot be explained by inhibition of intracellular cholesterol transport. However, com pounds that block cholesterol transport by affecting the vesicular trafficking of the Niem ann–Pick C1 protein (NPC1), which is structurally sim ilar to Ptc, are weak Shh antagonists. Rather than supporting a direct link between cholesterol hom eostasis and Shh signaling, our findings suggest that the functions of both NPC1 and Ptc involve a com m on vesicular transport pathway. Consistent with this m odel, we find that Ptc and NPC1 colocalize extensively in a vesicular com partm ent in cotransfected cells. © 2000 Academ ic Press Key Words: neural developm ent; holoprosencephaly; teratogen; U18666A; progesterone; m em brane trafficking.

required for the induction of ventral cell types in the forebrain (Tanabe and Jessell, 1996; Dale et al., 1997). Loss

INTRODUCTION

of norm al Shh function leads to a loss of ventral cell types all along the neural tube. This has significant consequences, especially in the forebrain, which fails to divide into hem ispheres (Chiang et al., 1996; Roessler et al., 1996). An undivided forebrain, or holoprosencephaly, in its m ost extrem e form is associated with cyclopia (for review, see Siebert et al., 1990; Roessler and Muenke, 1998).
Hedgehog fam ily m em bers are extracellular signaling m olecules involved in em bryonic patterning in m any anim al phyla. In vertebrates, Sonic hedgehog (Shh) plays a critical role in patterning of the neural tube. N otochordderived Shh m ediates both the induction of ventral cell types in the neural tube and the ventral exclusion of dorsal cell types, and Shh released from the prechordal plate is
Cyclopam ine is one of a sm all group of steroidal alkaloids, produced by Veratrum lily species, that induce holoprosencephaly and cyclopia in m am m alian and avian em - bryos. Cyclopam ine causes holoprosencephaly by blocking the response to Shh of cells norm ally sensitive to Shh. The

1

To whom correspondence should be addressed at the Departm ent of Biological Structure, University of Washington at Seattle, Box 357420, HSB G527, Seattle, WA 98195. Fax: (206) 543-1524. E-m ail: [email protected].

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Cyclopam ine, Cholesterol Transport, and Shh Signaling

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loss of Shh responsiveness within the developing neural tube, due to early em bryonic exposure to cyclopam ine, results in a phenocopy of the Shh null m utant (Cooper et al., 1998; Incardona et al., 1998). Our previous studies dem onstrated that a suite of Shh-dependent cell types in the neural tube was affected in em bryos with cyclopam ineinduced holoprosencephaly. Furtherm ore, cyclopam ine inhibits m ultiple Shh-m ediated signaling events in other tissues at very different tim es of developm ent, including som ites (Incardona et al., 1998), heart (J.P.I., unpublished observation), gut (Kim and Melton, 1998), lim b/fin (N eum ann et al., 1999), and skin (Chiang et al., 1999). Two decades of work by Keeler and colleagues dem onstrated that unique structural features of cyclopam ine and the related teratogenic com pound jervine (Fig. 1) are required for the induction of holoprosencephaly in whole anim al studies (reviewed in Gaffield and Keeler, 1996a,b). Subtle changes in the structures of cyclopam ine and jervine dram atically affect their teratogenic potency, suggesting that a precise interaction with a target is required for their inhibitory action. Since cyclopam ine blocks the response to Shh, the cyclopam ine target is likely to be involved in Shh signal transduction. Given the extrem e hydrophobicity of cyclopam ine and related com pounds, we expect that their target is a m em brane-associated protein, possibly an elem ent of the Shh receptor com plex. Elem ents of the Shh receptor are the m ultiple m em brane spanning proteins Patched (Ptc) and Sm oothened (Sm o), which act in concert to m ediate the Shh signal (Marigo et al., 1996; Stone et al., 1996; Chen and Struhl, 1998; for review, see Ingham , 1998; Murone et al., 1999). Shh binds to Ptc, which in turn releases the inhibition of Sm o by Ptc, thus activating the Shh response. The structural sim ilarity between cyclopam ine and cholesterol suggests a tantalizing potential m ode of action of cyclopam ine, especially in light of the association of holoprosencephaly, and thus attenuation of Shh signaling, with genetic or pharm acologic im pairm ent of enzym es involved in the term inal steps of cholesterol biosynthesis. These latter conditions produce both reduced levels of tissue cholesterol and high levels of sterol precursors (Tint et al., 1994; Kolf-Clauw et al., 1996). In particular, m utations in the gene encoding ⌬7-dehydrocholesterol (7-DHC) reductase (Fitzky et al., 1998) or inhibition of 7-DHC reductase by the inhibitor AY-9944 prom ote m ild form s of holoprosencephaly (Roux and Aubry, 1966; Kelley et al., 1996). The sim ple hypothesis that both cyclopam ine and AY-9944 inhibit the response of cells in the developing neural tube to Shh through the inhibition of cholesterol synthesis was refuted, however, since AY-9944 is effective only when responding cells are deprived of exogenous cholesterol and therefore have to rely on endogenously synthesized cholesterol, while the action of cyclopam ine is independent of the activity of the cholesterol synthetic pathway (Incardona et al., 1998). Accum ulation of putatively teratogenic cholesterol precursors, rather than cholesterol depletion, m ay underlie Shh-response inhibition by AY-9944. The

FIG. 1. The structures of steroidal alkaloids and steroids tested for inhibition of Shh signaling. The structure of cholesterol is shown at top left and the generic jervane-type steroidal alkaloid structure is at top right. N atural jervane alkaloids and synthetic derivatives are listed below the generic structure, with differences indicated for the functional groups RЈ and RЉ, and for the type of C–C bond at three variant positions. Cycloposine has a single glucose (glc) residue in a glycosidic linkage to the 3-OH. Veratram ine (m iddle, left) occurs naturally and also is derived from cyclopam ine by electrophilic attack on the furan (E) ring under acidic conditions. The spirosolane-type steroidal alkaloids solasodine and tom atidine are represented by a generic structure (m iddle, right). These two alkaloids differ only in the position of the N in the F ring (RЈ and RЉ) and in the level of saturation at C-5, C-6. N ote the differences in the E–F ring attachm ent to ring D between jervanes and spirosolanes, which results in an orientation of the spirofuranopiperidine m oiety of cyclopam ine perpendicular to the planar steroidal fram ework. The glycoalkaloid saponins tom atine and solasonine possess 4- and 3-residue oligosaccharides attached to the 3-OH group of tom atidine and solasodine, respectively. The steroidal am ine U18666A [3␤-(2-diethylam inoethoxy)androstenone] is represented at bottom left and progesterone at bottom right.

holoprosencephaly-inducing activity of cyclopam ine is independent of cholesterol supply and is m ost likely m ediated by another m echanism .

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Incardona et al.

  • In addition to cyclopam ine and AY-9944, other com -
  • dently on targets within the Shh signaling pathway and the

N PC1-m ediated cholesterol transport pathway with varying efficiency. Our data suggest that the rem arkable sensitivity of Shh signal transduction to cyclopam ine and the less potent effects of other com pounds we tested reflect a prim ary effect on Ptc function. The structural sim ilarities of Ptc and N PC1 lead us to predict that Ptc m ediates a vesicular trafficking process that is differentially sensitive to m any of the sam e pharm acological m anipulations and is crucial for Shh signal transduction. pounds disturbing cholesterol hom eostasis reportedly inhibit the response to Shh (Cooper et al., 1998). These com pounds include other hydrophobic am ines and steroids that interfere with intracellular cholesterol trafficking, such as im ipram ine, progesterone, and U18666A (Liscum and Munn, 1999). This suggested the possibility that Shh signaling through Ptc is linked to cellular cholesterol hom eostasis, in particular to intracellular cholesterol transport (Cooper et al., 1998). A target for the hydrophobic am ines and steroids that block intracellular cholesterol transport m ay be the N iem ann–Pick C1 (N PC1) protein (N eufeld et al., 1999). Treatm ent of cells with these com - pounds induces a cellular cholesterol phenotype indistinguishable from that of cells from N iem ann–Pick type C (N P-C) patients, which lack the N PC1 protein (Liscum and Faust, 1989; Rodriguez-Lafrasse et al., 1990; Roff et al., 1991; Yoshikawa, 1991; Butler et al., 1992; Lange and Steck, 1994). N P-C disease is a neurodegenerative lysosom al storage disorder, with an underlying and severe defect in intracellular transport of cholesterol and sphingolipids. Yet, loss of N PC1 function by m utation is not associated with affects on Shh signaling: N P-C patients and N PC1-null m ice do not have congenital m alform ations. This suggests that the inhibition of Shh signaling by cyclopam ine, hydrophobic am ines, and steroids is not related to their effects on intracellular cholesterol transport. Com parison of Ptc and N PC1 protein sequences dem onstrates a significant sim ilarity, largely, but not com pletely, confined to the m em brane-spanning regions (Carstea et al., 1997; Loftus et al., 1997). This sim ilarity includes a so-called “sterolsensing dom ain,” a pattern of conserved transm em brane regions present in several other proteins involved in cholesterol hom eostasis (Kum agai et al., 1995; Hua et al., 1996; Carstea et al., 1997; Loftus et al., 1997; Bae et al., 1999). The sim ilarity between Ptc and N PC1 suggests another m echanism by which hydrophobic am ines and steroids interfere with Shh reception: not through their docum ented action on N PC1, but rather by acting on the sam e target as cyclopam ine, a critical elem ent of the Shh signal transduction cascade. It rem ains a good possibility that this target is Ptc, given its hom ology to N PC1. A converse prediction of this m odel is that cyclopam ine and related com pounds have an effect on cholesterol hom eostasis through their interaction with N PC1 com parable to that of progesterone or U18666A.

MATERIALS AND METHODS

Materials. Veratrum steroidal alkaloids and their synthetic derivatives were obtained as described previously (Brown and Keeler, 1978; Gaffield et al., 1986). Progesterone was obtained from Sigm a (St. Louis, MO) and U18666A was obtained from BioMol (Plym outh Meeting, PA). Steroidal alkaloids and progesterone were dissolved in 95% ethanol; U18666A was dissolved in water.

Intermediate neural plate explants. Explants of interm ediate

neural plate regions were isolated from Stage 9 –10 chick em bryos and cultured in collagen gel as previously described (Yam ada et al., 1993; Incardona et al., 1998). Explants were distributed in batches of 10 onto pregelled collagen pillows and cultured at 37°C in N eurobasal m edium supplem ented with N 3 m ixture [N 2 plus 10 nM hydrocortisone (Bottenstein, 1992)], MEM nonessential am ino acids, penicillin/streptom ycin, and 10 m M glucose. Test com - pounds were diluted in N eurobasal m edium . In general com pounds were titrated to determ ine the highest concentration in which explants m aintained viability for 12–14 h. Shh signaling was then assayed at the concentrations just below those causing com plete cell death within 12–14 h, but allowing viability to 30 h. Control explants were cultured with an equivalent concentration of solvent (0.1% or less). For assessm ent of the Shh response, explants were harvested after 30 h of incubation, fixed in 4% paraform aldehyde, and processed for HN F-3␤, Isl1/2, and Pax7 im m unofluorescence as described previously (Incardona et al., 1998). The response to Shh was quantified by tallying positive nuclei with a hand counter for at least 8 explants for each individual treatm ent. For 3-hydroxy-3-m ethylglutaryl coenzym e A reductase (HMGR) assays, 15 explants per well were tested in duplicate or triplicate. So that m ore cellular m aterial was assayed, slightly larger explants which included som e dorsal and ventral regions were excised. After 24 h of incubation with the test com pounds in the sam e m edium as above, explants were washed with PBS, then quickly frozen in a dry ice/ethanol bath and stored at Ϫ80°C until assayed.

Shh-N preparation. Assays utilized recom binant Shh-N , either

obtained as conditioned m edium from hum an 293T cells transiently transfected with pShh-N as described previously (Roelink et al., 1995) or partially purified from High-Five (Invitrogen, Carlsbad, CA) cells infected with a recom binant baculovirus expressing Shh-N (H.R., unpublished). The specific activity of each preparation was determ ined by titration in the explant assay, and the Shh-N protein content was estim ated by com parison to a standard after SDS–PAGE and Coom assie blue staining. Som e preparations of Shh-N were shown to contain a fraction of m aterial acylated at an N -term inal Cys, which has a potency 30-fold higher than unm odified Shh-N (Pepinsky et al., 1998). Our preparations m ost likely consisted of such a m ixture, as suggested by the appearance of a doublet band on Western blots (data not shown). Also consis-

To test these hypotheses we evaluated whether the relative potencies of cyclopam ine, structurally related steroidal alkaloids that do not cause holoprosencephaly, U18666A, and progesterone to inhibit Shh signaling correlate with their effects on cholesterol transport. The results show a correlation between the attenuation of the Shh response and the disruption of N PC1-m ediated vesicular trafficking. However, the relative concentrations of these com pounds sufficient to perturb cholesterol trafficking and concentrations sufficient to block Shh signaling are very different. This suggests that the com pounds act indepen-

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Cyclopam ine, Cholesterol Transport, and Shh Signaling

443

tent with this, the baculovirus preparation used for m ost of the experim ents described here proved to be 10-fold m ore potent than the bacterially expressed, unm odified protein originally used to establish the Shh-N dose–response curve (Roelink et al., 1995; Ericson et al., 1997). However, neither the N -term inal acylation nor the C-term inal cholesterol m odification of wild-type Shh is required to assess the inhibitory activity of the com pounds tested, as sim ilar results were obtained using bacterially expressed Shh-N (Cooper et al., 1998).

HMGR and cholesterol esterification assays. N eural plate ex-

plants in collagen gels were processed and assayed for HMGR activity as described previously (Plem enitas et al., 1990). Separate aliquots of the cleared lysate were used for protein determ ination (Giron et al., 1993). For m easurem ent of cholesterol esterification, rat hepatom a cells at confluency were pulse-labeled with [3H]cholesterol and rinsed (Lange et al., 1993). Fresh m edium containing 10% fetal bovine serum plus the test com pounds or a solvent control was added and the cells were incubated for 3 h at 37°C. The cells were dissociated with trypsin, washed, extracted, and analyzed by TLC for the radioactivity in cholesterol and cholesteryl esters (Lange et al., 1993).

Assay of accumulation of low -density lipoprotein (LDL)- cholesterol in lysosomes. N orm al hum an fibroblasts were seeded

into two well cham ber slides at 20,000 cells/well in McCoy’s m edium supplem ented with 5% lipoprotein-deficient serum and antibiotics (LPDS-McCoy’s). After 4 days the cells were cultured for 24 h in LPDS-McCoy’s containing the steroidal alkaloids or progesterone (all dissolved in ethanol; final ethanol concentration was Ͻ0.1% ) and LDL. The cells were then fixed and processed for filipin/cholesterol histochem istry as previously described (Blanchette-Mackie et al., 1988). LPDS and purified hum an LDL were obtained from Intracel (Rockville, MD).

RESULTS

The Neural Plate Explant Assay

Chick em bryo neural plate explants were cultured in the presence of Shh-N , which leads to the induction of m arkers for floor plate (HN F3␤) and m otor neuron (Isl1/2) differentiation, as well as repression of a m arker for dorsal precursor cells (Pax7) (Roelink et al., 1995; Ericson et al., 1997; Briscoe and Ericson, 1999). In a typical Shh-N dose response, HN F-3␤ϩ floor plate cells are m axim ally induced by 1–2 nM Shh-N , Isl1/2ϩ m otor neurons are m axim ally induced by 0.3–0.5 nM Shh-N , and Pax7ϩ dorsal precursor cells are repressed by 0.05–0.1 nM Shh-N . Thus, at the highest Shh-N concentrations, HN F-3␤ induction predom inates, although this is at the expense of Isl1/2ϩ cells (typically 60 –100 Isl1/2ϩ cells and 1000 –1200 HN F-3␤ϩ cells). Isl1/2ϩ cells are m axim ally induced (typically 200 – 300 cells in a 1500-cell explant) at concentrations of Shh-N that induce few or no HN F-3␤ϩ cells. Even lower concentrations of Shh-N repress Pax7 in the absence of Isl1/2 or HN F-3␤ induction. Therefore, the num ber of HN F3␤ cells is a straightforward, but som ewhat less sensitive indicator of Shh signaling. The Shh-dependent induction of Isl1/2 is m ore sensitive, but m axim um response occurs in a relatively narrow dose range, with the num ber of Isl1/2ϩ cells per explant decreasing both at higher and at lower Shh concentrations and can thus be used as a reliable quantitative indicator of the Shh response only in conjunction with Pax7 or HN F3␤ induction. At a constant high Shh-N concentration (2.5 nM), the cyclopam ine dose response m im ics a reverse of the Shh-N dose response (Roelink et al., 1995; Ericson et al., 1997). Induction of HN F-3␤ϩ cells is reduced and m ore Isl1/2ϩ cells appear at low concentrations of cyclopam ine (12–24 nM); at interm ediate cyclopam ine concentrations HN F-3␤ induction is com pletely blocked (24 –48 nM), while Isl1/2 induction persists; at higher cyclopam ine concentrations both HN F-3␤ and Isl1/2 induction is blocked, while som e Pax7 repression persists (60 nM); all signaling is blocked by 120 nM cyclopam ine (Fig. 3, data not shown and Incardona et al., 1998). Because each m arker (HN F3␤, Isl1/2, and Pax7) responds m axim ally to a different threshold Shh-N concentration, com parisons of the inhibitory activity of the different com pounds are m ost straightforward using a single m arker. Although in general we tested the effects of m ost of the com pounds on all three m arkers, only data for HN F3␤ are shown.

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  • Vismodegib Resistance in Basal Cell Carcinoma: Not a Smooth Fit

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    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Cancer Cell Previews Vismodegib Resistance in Basal Cell Carcinoma: Not a Smooth Fit Todd W. Ridky1 and George Cotsarelis1,* 1Department of Dermatology, Perelman School of Medicine, University of Pennsylvania, BRB 1053, 421 Curie Boulevard, Philadelphia, PA 19104, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.ccell.2015.02.009 In this issue of Cancer Cell, two complementary papers by Atwood and colleagues and Sharpe and colleagues show that basal cell carcinomas resistant to the Smoothened (SMO) inhibitor vismodegib frequently harbor SMO mutations that limit drug binding, with mutations at some sites also increasing basal SMO activity. Basal cell carcinoma (BCC) of the skin is target, Smoothened (SMO), was subse- to mutations downstream of SMO at the the most common human cancer in lightly quently identified in 2000 (Cooper et al., level of Gli2 or SUFU (a Gli regulator). pigmented individuals. Mutations acti- 1998; Incardona et al., 1998; Taipale By aligning the mutations with a vating the Hedgehog (HH) signaling et al., 2000). That initial work defining recently solved crystal structure of the pathway drive BCC. Secreted HH exerts the SMO-cyclopamine interaction also SMO transmembrane domain, the au- its effects through binding to Patched included the observation that some thors were able to separate the resis- (PTCH), a 12-pass membrane bound re- SMO point mutants driving BCCs not tance-associated mutations into two ceptor, which relieves PTCH inhibitory only increase basal SMO activity, but groups: (1) mutations within or immedi- activity on the downstream heptahelical simultaneously confer cyclopamine resis- ately adjacent to the ligand/drug binding transmembrane protein Smoothened tance.
  • The Effects of Inhibiting Hedgehog Signaling Pathways by Using Specific Antagonist Cyclopamine on the Chondrogenic Differentiation of Mesenchymal Stem Cells

    The Effects of Inhibiting Hedgehog Signaling Pathways by Using Specific Antagonist Cyclopamine on the Chondrogenic Differentiation of Mesenchymal Stem Cells

    Int. J. Mol. Sci. 2013, 14, 5966-5977; doi:10.3390/ijms14035966 OPEN ACCESS International Journal of Molecular Sciences ISSN 1422-0067 www.mdpi.com/journal/ijms Article The Effects of Inhibiting Hedgehog Signaling Pathways by Using Specific Antagonist Cyclopamine on the Chondrogenic Differentiation of Mesenchymal Stem Cells Xing Wu 1,*, Zheng-Dong Cai 1, Lei-Ming Lou 1 and Zheng-Rong Chen 2 1 Department of Orthopaedics, Shanghai tenth People’s Hospital, Tongji University School of Medicine, Shanghai 200072, China; E-Mails: [email protected] (Z.-D.C.); [email protected] (L.-M.L.) 2 Department of Orthopaedics, Shanghai Zhongshan Hospital, Fudan University School of Medicine, Shanghai 200032, China; E-Mail: [email protected] * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +86-21-6630-0588; Fax: +86-21-6630-1051. Received: 18 January 2013; in revised form: 5 March 2013 / Accepted: 7 March 2013 / Published: 14 March 2013 Abstract: This study aimed to investigate the effects of cyclopamine, a specific inhibitor of Hedgehog signaling pathways, on the chondrogenic differentiation of mesenchymal stem cells (MSCs). During culture, the experimental groups were treated with cyclopamine and their cell proliferation status was assessed using the MTT test. The extra-bone cellular matrix (ECM) and Collagen II (Col II) was detected by toluidine blue staining and immunohistochemistry of cells. The concentrations of Col II and aggrecan in the culture solution and cytosol were detected using ELISA on the 7th, 14th, and 21st days of cyclopamine induction. Gene and protein expression of Col II and aggrecan were analyzed on the 14th day of cyclopamine induction using real-time PCR and western blot analyses.
  • VIEW Open Access Targeting Hedgehog Signaling in Myelofibrosis and Other Hematologic Malignancies Raoul Tibes* and Ruben a Mesa

    VIEW Open Access Targeting Hedgehog Signaling in Myelofibrosis and Other Hematologic Malignancies Raoul Tibes* and Ruben a Mesa

    Tibes and Mesa Journal of Hematology & Oncology 2014, 7:18 http://www.jhoonline.org/content/7/1/18 JOURNAL OF HEMATOLOGY & ONCOLOGY REVIEW Open Access Targeting hedgehog signaling in myelofibrosis and other hematologic malignancies Raoul Tibes* and Ruben A Mesa Abstract Treatment of myelofibrosis (MF), a BCR-ABL–negative myeloproliferative neoplasm, is challenging. The only current potentially curative option, allogeneic hematopoietic stem cell transplant, is recommended for few patients. The remaining patients are treated with palliative therapies to manage MF-related anemia and splenomegaly. Identification of a mutation in the Janus kinase 2 (JAK2)gene(JAK2 V617F) in more than half of all patients with MF has prompted the discovery and clinical development of inhibitors that target JAK2. Although treatment with JAK2 inhibitors has been shown to improve symptom response and quality of life in patients with MF, these drugs do not alter the underlying disease; therefore, novel therapies are needed. The hedgehog (Hh) signaling pathway has been shown to play a role in normal hematopoiesis and in the tumorigenesis of hematologic malignancies. Moreover, inhibitors of the Hh pathway have been shown to inhibit growth and self-renewal capacity in preclinical models of MF. In a mouse model of MF, combined inhibition of the Hh and JAK pathways reduced JAK2 mutant allele burden, reduced bone marrow fibrosis, and reduced white blood cell and platelet counts. Preliminary clinical data also suggest that inhibition of the Hh pathway, alone or in combination with JAK2 inhibition, may enable disease modification in patients with MF. Future studies, including one combining the Hh pathway inhibitor sonidegib and the JAK2 inhibitor ruxolitinib, are underway in patients with MF and will inform whether this combination approach can lead to true disease modification.
  • The Evolution of Sensory and Neurosecretory Cell Types in Bilaterian Brains

    The Evolution of Sensory and Neurosecretory Cell Types in Bilaterian Brains

    The evolution of sensory and neurosecretory cell types in bilaterian brains DISSERTATION zur Erlangung des Doktorgrades der Naturwissenschaften Doctor rerum naturalium (Dr. rer. nat.) dem Fachbereich Biologie der Philipps-Universität Marburg vorgelegt von Dipl.-Biol. K. G. Kristin Teßmar–Raible aus Görlitz Marburg/ Lahn 2004 Vom Fachbereich Biologie der Philipps-Universität Marburg als Dissertation am angenommen. Erstgutachterin: Prof. Dr. Monika Hassel Zweitgutachterin : Prof. Dr. Renate Renkawitz-Pohl Tag der mündlichen Prüfung: The evolution of sensory and neurosecretory cell types in bilaterian brains To my parents and my husband for their encouragement and patience Acknowledgements I am very thankful to the following persons in Marburg and Heidelberg that made this study possible: Dr. Monika Hassel at Marburg University for the supervision of my PhD thesis and correction of the text; Dr. Detlev Arendt at the EMBL in Heidelberg for giving me the opportunity to spend time in his lab as a guest and for scientific advice; Dr. Monika Hassel, Dr. Renate Renkawitz-Pohl and the other members of my defense committee for reviewing this thesis. I want to acknowledge all members and guests of the Arendt lab, in particular Sebastian Klaus, Heidi Snyman, and Patrick Steinmetz, for for their help, their useful critical comments, and the interesting scientific discussions; in addition, I want to thank Heidi Snyman for very reliable practical help, especially with degenerated PCRs, minipreps, and restriction digests; and Dr. Detlev Arendt for minipreps of the Pdu-barH1 cloning. Moreover, I thank Dr. Florian Raible and Dr. Detlev Arendt for extensive critical feedback on previous versions of this text.
  • The Hedgehog Signaling Pathway in Cancer

    The Hedgehog Signaling Pathway in Cancer

    Published OnlineFirst June 19, 2012; DOI: 10.1158/1078-0432.CCR-11-2509 Clinical Cancer Molecular Pathways Research Molecular Pathways: The Hedgehog Signaling Pathway in Cancer Ross McMillan and William Matsui Abstract The Hedgehog (Hh) signaling pathway regulates embryonic development and may be aberrantly activated in a wide variety of human cancers. Efforts to target pathogenic Hh signaling have steadily progressed from the laboratory to the clinic, and the recent approval of the Hh pathway inhibitor vismodegib for patients with advanced basal cell carcinoma represents an important milestone. On the other hand, Hh pathway antagonists have failed to show significant clinical activity in other solid tumors. The reasons for these negative results are not precisely understood, but it is possible that the impact of Hh pathway inhibition has not been adequately measured by the clinical endpoints used thus far or that aberrancies in Hh signal transduction limits the activity of currently available pathway antagonists. Further basic and correlative studies to better understand Hh signaling in human tumors and validate putative antitumor mechanisms in the clinical setting may ultimately improve the success of Hh pathway inhibition to other tumor types. Clin Cancer Res; 18(18); 4883–8. Ó2012 AACR. Background and GLI3 represses Hh target genes that include GLI1, PTCH1 Cyclin D1 c-Myc Bcl-2 The Hedgehog (Hh) signaling pathway is highly con- , , , and , whereas GLI2 can act served from flies to humans and is essential for develop- in either a positive or negative manner depending on ment of the normal embryo (1, 2). In mammals, Hh posttranscriptional and posttranslational processing events signaling regulates both patterning and polarity events (4).
  • (IPI-926) Increases Lifespan in a Mouse Medulloblastoma Model

    (IPI-926) Increases Lifespan in a Mouse Medulloblastoma Model

    Hedgehog pathway inhibitor saridegib (IPI-926) increases lifespan in a mouse medulloblastoma model Michelle J. Leea,b,1, Beryl A. Hattona,1, Elisabeth H. Villavicencioa,c, Paritosh C. Khannad, Seth D. Friedmand, Sally Ditzlera, Barbara Pullara, Keith Robisone, Kerry F. Whitee, Chris Tunkeye, Michael LeBlancf, Julie Randolph-Habeckerg, Sue E. Knoblaughg, Stacey Hansena, Andrew Richardsa, Brandon J. Wainwrighth, Karen McGoverne, and James M. Olsona,b,c,2 aClinical Research Division, fPublic Health Sciences Division, gComparative Medicine, Fred Hutchinson Cancer Research Center, Seattle, WA 98109; bNeurobiology and Behavior Program, Departments of dRadiology and cPediatrics, University of Washington and Seattle Children’s Hospital, Seattle, WA 98105; eInfinity Pharmaceuticals, Cambridge, MA 02139; and hDivision of Molecular Genetics and Development, Institute for Molecular Biosciences, University of Queensland, Brisbane QLD 4072, Australia Edited by Dennis A. Carson, University of California at San Diego, La Jolla, CA, and approved April 4, 2012 (received for review September 13, 2011) The Sonic Hedgehog (Shh) pathway drives a subset of medullo- drug resistance (4–6). It remains to be determined whether these blastomas, a malignant neuroectodermal brain cancer, and other drugs confer a survival benefit to medulloblastoma patients. The cancers. Small-molecule Shh pathway inhibitors have induced tu- identification of Smo inhibitors that remain active against cells mor regression in mice and patients with medulloblastoma; how- that develop resistance to other agents in this class could benefit ever, drug resistance rapidly emerges, in some cases via de novo patients, particularly if acquired resistance is limited or slower mutation of the drug target. Here we assess the response and to develop.
  • Efficacy and Safety of Sonic Hedgehog Pathway Inhibitors in Cancer

    Efficacy and Safety of Sonic Hedgehog Pathway Inhibitors in Cancer

    HHS Public Access Author manuscript Author ManuscriptAuthor Manuscript Author Drug Saf Manuscript Author . Author manuscript; Manuscript Author available in PMC 2020 February 01. Published in final edited form as: Drug Saf. 2019 February ; 42(2): 263–279. doi:10.1007/s40264-018-0777-5. Efficacy and safety of sonic hedgehog pathway inhibitors in cancer Richard L Carpenter1,2,3,* and Haimanti Ray2 1Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, 1001 E. 3rd St, Bloomington, IN 47405 2Medical Sciences, Indiana University School of Medicine, 1001 E. 3rd St, Bloomington, IN 47405 3Simon Cancer Center, Indiana University School of Medicine, 1001 E. 3rd St, Bloomington, IN 47405 Abstract The hedgehog pathway, for which sonic hedgehog (Shh) is the most prominent ligand, is highly conserved and is tightly associated with embryonic development in a number of species. This pathway is also tightly associated with development of several types of cancer, including basal cell carcinoma and acute promyelocytic leukemia (APL) among many others. Inactivating mutations in Patched 1 (PTCH1), leading to ligand-independent pathway activation, are frequent in several cancer types but most prominent in basal cell carcinoma. This has led to the development of several compounds targeting this pathway as a cancer therapeutic. These compounds target the inducers of this pathway in Smoothened (SMO) and the GLI transcription factors, although targeting SMO has had the most success. Despite the many attempts at targeting this pathway, there are only three FDA-approved drugs for cancers that affect the Shh pathway. Two of these compounds, vismodegib and sonidegib, target SMO to suppress signaling from either PTCH1 or SMO mutations that lead to upregulation of the pathway.