Niclosamide and its derivatives for use in the treatment of solid tumors

Abstract

The present invention relates to novel therapeutic uses of niclosamide for the treatment of cancer. In particular, a combination of niclosamide or one of its derivatives with an alkylating agent is provided for the treatment of solid tumors. Moreover, niclosamide or one of its derivatives can be used for the treatment of solid tumors characterized by underexpression of NFKBIA. Finally, the invention relates to diagnostic methods for determining whether treatment with niclosamide alone or in combination with an alkylating agent is suitable for a cancer patient.

Claims

1. A method of treating a solid tumor, comprising administering a cytostatic compound according to formula I, II or III ##STR00005## wherein A is carbonyl, methylene, hydroxymethinyl, alkoxymethinyl, aminomethinyl, oxime, hydrazone, arylhydrazone, or semicarbazone; B if present is CR.sub.25R.sub.26, O, S or NR.sub.27; R.sub.1, R.sub.3, R.sub.4, R.sub.8, R.sub.10, and R.sub.11 are independently hydrogen, hydroxyl, alkoxy, halogen or C.sub.1 to C.sub.6 alkyl; R.sub.2 and R.sub.7 are independently halogen, hydroxyl or hydrogen, R.sub.5 if present is hydroxyl, phosphate, hydrogen, halogen, alkyl, cycloalykyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio or amino; R.sub.6 if present is hydrogen or C.sub.1 to C.sub.6 alkyl; R.sub.9 is nitro, hydrogen, hydroxyl, amino, halogen, alkyl, alkenyl, alkynyl, or aryl; and R.sub.21, R.sub.22, R.sub.23, and R.sub.24, if present are independently hydrogen, hydroxyl, halogen, alkyl, alkenyl, alkynyl, cycloalkyl or aryl; R.sub.25 and R.sub.26 if present are independently hydrogen, hydroxyl, halogen, alkyl, alkenyl, alkynyl, cycloalkyl or aryl; and R.sub.27 if present is hydrogen or C.sub.1 to C.sub.6 alkyl; or salt thereof; and an alkylating agent to a subject.

2. The method of claim 1, wherein the alkylating agent is an O.sup.6-alkylating agent.

3. The method of claim 1, wherein the alkylating agent has a structure according to formula IV ##STR00006## wherein X and Y are independently carbonyl, methylene, hydroxymethinyl, alkoxymethinyl, aminomethinyl, oxime, hydrazone, arylhydrazone or semicarbazone, R.sub.31 is alkyl, hydrogen, alkoxy, alkenyl, alkynyl, cycloalkyl or aryl; and R.sub.32 is amino, hydrogen, hydroxyl or halogen or salt thereof.

4. The method of claim 1, wherein the cytostatic compound is niclosamide.

5. The method of claim 1, wherein the alkylating agent is temozolomide.

6. The method of claim 4, wherein the molar ratio between niclosamide and temozolomide is in the range of 10% niclosamide/90% temozolomide to 90% niclosamide/10% temozolomide.

7. The method of claim 1, wherein the solid tumor is glioblastoma.

8. The method of claim 7, wherein the glioblastoma is primary glioblastoma, de novo glioblastoma, secondary glioblastoma, recurrent glioblastoma, glioblastoma with increased methylation of the promoter of the gene O6-Methylguanin-Methyltransferase (MGMT), glioblastoma without increased methylation of the promoter of MGMT, glioblastoma with mutated p53, glioblastoma without mutated p53, glioblastoma with alterations of the gene encoding kappa light polypeptide gene enhancer in B-cells inhibitor (NFκBIA), glioblastoma without alterations of the gene encoding NFκBIA, glioblastoma with alterations of the gene encoding epidermal growth factor receptor (EGFR), glioblastoma without alterations of the gene encoding EGFR, glioblastoma with alterations of the gene encoding platelet-derived growth factor receptor (PDGFRA), glioblastoma without alterations of the gene encoding PDGFRA, glioblastoma with alterations of the gene encoding isocitrate dehydrogenase 1 (IDHI), glioblastoma without alterations of the gene encoding IDHI, glioblastoma with alterations of the gene encoding neurofibromatosis type 1 (NF1) or glioblastoma without alterations of the gene encoding NF1.

9. The method of claim 1, wherein the compounds are formulated for simultaneous or subsequent administration.

10. The method of claim 9, wherein the formulation for simultaneous administration is a mixture of the two compounds.

11. The method of claim 1, wherein the cytostatic compound is formulated for resorption into the central nervous system.

12. A pharmaceutical composition comprising a cytostatic compound according to formula I, II or III ##STR00007## wherein A is carbonyl, methylene, hydroxymethinyl, alkoxymethinyl, aminomethinyl, oxime, hydrazone, arylhydrazone, or semicarbazone; B if present is CR.sub.25R.sub.26, O, S or NR.sub.27; R.sub.1, R.sub.3, R.sub.4, R.sub.8, R.sub.10, and R.sub.11 are independently hydrogen, hydroxyl, alkoxy, halogen or C.sub.1 to C.sub.6 alkyl; R.sub.2 and R.sub.7 are independently halogen, hydroxyl or hydrogen, R.sub.5 if present is hydroxyl, phosphate, hydrogen, halogen, alkyl, cycloalykyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio or amino; R.sub.6 if present is hydrogen or C.sub.1 to C.sub.6 alkyl; R.sub.9 is nitro, hydrogen, hydroxyl, amino, halogen, alkyl, alkenyl, alkynyl, or aryl; and R.sub.21, R.sub.22, R.sub.23, and R.sub.24, if present are independently hydrogen, hydroxyl, halogen, alkyl, alkenyl, alkynyl, cycloalkyl or aryl; R.sub.25 and R.sub.26 if present are independently hydrogen, hydroxyl, halogen, alkyl, alkenyl, alkynyl, cycloalkyl or aryl; and R.sub.27 if present is hydrogen or C.sub.1 to C.sub.6 alkyl: or salt thereof; an alkylating agent; and a pharmaceutically acceptable excipient.

13. A method of treating a solid tumor characterized by a decreased expression level of NFκBIA, comprising administering a cytostatic compound according to formula I, II or III ##STR00008## to a subject, wherein A is carbonyl, methylene, hydroxymethinyl, alkoxymethinyl, aminomethinyl, oxime, hydrazone, arylhydrazone, or semicarbazone; B if present is CR.sub.25R.sub.26, O, S or NR.sub.27; R.sub.1, R.sub.3, R.sub.4, R.sub.8, R.sub.10, and R.sub.11 are independently hydrogen, hydroxyl, alkoxy, halogen or C.sub.1 to C.sub.6 alkyl; R.sub.2 and R.sub.7 are independently halogen, hydroxyl or hydrogen, R.sub.5 if present is hydroxyl, phosphate, hydrogen, halogen, alkyl, cycloalykyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio or amino; R.sub.6 if present is hydrogen or C.sub.1 to C.sub.6 alkyl; R.sub.9 is nitro, hydrogen, hydroxyl, amino, halogen, alkyl, alkenyl, alkynyl, or aryl; and R.sub.21, R.sub.22, R.sub.23, and R.sub.24, if present are independently hydrogen, hydroxyl, halogen, alkyl, alkenyl, alkynyl, cycloalkyl or aryl; R.sub.25 and R.sub.26 if present are independently hydrogen, hydroxyl, halogen, alkyl, alkenyl, alkynyl, cycloalkyl or aryl; and R.sub.27 if present is hydrogen or C.sub.1 to C.sub.6 alkyl.

14. A method for determining if therapy with a cytostatic compound according to formula I, II or III ##STR00009## wherein A is carbonyl, methylene, hydroxymethinyl, alkoxymethinyl, aminomethinyl, oxime, hydrazone, arylhydrazone, or semicarbazone; B if present is CR.sub.25R.sub.26, O, S or NR.sub.27; R.sub.1, R.sub.3, R.sub.4, R.sub.8, R.sub.10, and R.sub.11 are independently hydrogen, hydroxyl, alkoxy, halogen or C.sub.1 to C.sub.6 alkyl; R.sub.2 and R.sub.7 are independently halogen, hydroxyl or hydrogen, R.sub.5 if present is hydroxyl, phosphate, hydrogen, halogen, alkyl, cycloalykyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio or amino; R.sub.6 if present is hydrogen or C.sub.1 to C.sub.6 alkyl; R.sub.9 is nitro, hydrogen, hydroxyl, amino, halogen, alkyl, alkenyl, alkynyl, or aryl; and R.sub.21, R.sub.22, R.sub.23, and R.sub.24, if present are independently hydrogen, hydroxyl, halogen, alkyl, alkenyl, alkynyl, cycloalkyl or aryl; R.sub.25 and R.sub.26 if present are independently hydrogen, hydroxyl, halogen, alkyl, alkenyl, alkynyl, cycloalkyl or aryl; and R.sub.27 if present is hydrogen or C.sub.1 to C.sub.6 alkyl, is suitable for treating a patient with a solid tumor comprising the steps of a) determining the expression level of NFκBIA in a sample of tumor cells or tumor tissue of the patient; b) comparing the determined expression level with a reference value; c) determining if the therapy with niclosamide is suitable for the patient based on the result of the comparison of step b), wherein underexpression or a deletion of NFκBIA indicates that the combination therapy is suitable for the patient.

15. A method for determining the molar ratio of niclosamide to temozolomide to be administered to a patient with a solid tumor comprising the steps of a) determining the expression level of NFκBIA in a sample of tumor cells or tumor tissue of the patient; b) comparing the determined expression level with a reference value; c) determining the molar ratio of niclosamide to temozolomide based on the result of the comparison of step b), wherein (i) an expression above the reference value indicates that the molar ratio shall be below 40% niclosamide; and (ii) an expression level below the reference value indicates that the molar ratio shall be equal to or larger than 40% niclosamide.

16. The method of claim 13, wherein the solid tumor is glioblastoma.

17. The method of claim 16, wherein the glioblastoma is primary glioblastoma, de novo glioblastoma, secondary glioblastoma, recurrent glioblastoma, glioblastoma with increased methylation of the promoter of the gene O6-Methylguanin-Methyltransferase (MGMT), glioblastoma without increased methylation of the promoter of MGMT, glioblastoma with mutated p53, glioblastoma without mutated p53, glioblastoma with alterations of the gene encoding kappa light polypeptide gene enhancer in B-cells inhibitor (NFκBIA), glioblastoma without alterations of the gene encoding NFκBIA, glioblastoma with alterations of the gene encoding epidermal growth factor receptor (EGFR), glioblastoma without alterations of the gene encoding EGFR, glioblastoma with alterations of the gene encoding platelet-derived growth factor receptor (PDGFRA), glioblastoma without alterations of the gene encoding PDGFRA, glioblastoma with alterations of the gene encoding isocitrate dehydrogenase 1 (IDHI), glioblastoma without alterations of the gene encoding IDHI, glioblastoma with alterations of the gene encoding neurofibromatosis type 1 (NF1) or glioblastoma without alterations of the gene encoding NF1.

Description

FIGURE LEGENDS

(1) FIG. 1: Niclosamide effectively inhibits pGBMs cellular viability. (A) Pharmacodynamic analysis of 21 pGBMs (#'s indicated) at day 5 following niclosamide exposure (concentration indicated). Data as mean±SD of triplicates. (B) Spectrum of IC50 values representing the concentrations that decrease the metabolic activity to 50% of control levels. Data collected from three human non-tumor neural cell populations (hnNCs, see Methods), five commercially available glioma/GBM cell lines (see Methods), and the 21 pGBMs (see (A); Table 3). The inset depicts IC50 data from additional pair-wise comparative experiments (symbol coded) on pGBMs derived from tumor center vs. periphery, from primary vs. recurrent disease, from MGMT promoter hypermethylated vs. unmethylated specimens, and from samples with NFKBIA.sup.+/+ vs. heterozygous NFKBIA deleted genotypes (NFKBIA.sup.+/−). P-values (***p<0.001) were calculated from comparing hnNCs and GBM cell line data with pGBMs, respectively, using the 1-way ANOVA and Tukey's post hoc tests.

(2) FIG. 2: Niclosamide has cytostatic and cytotoxic effects. (A) Cell cycle analysis at 24 hours post application exposing a strongly increasing G1 peak in the niclosamide (niclo)-treated sample. (B) CYCLIN D1 (CCND1)-western blot of cell extracts derived at this time point. (C) Cellular growth kinetics after a single exposure to niclosamide (1.5 μM; squares) or DMSO (0.01%; rhombi) (pGBM #'s 046, 078, 106; mean data±SD). (D) Graph depicting frequency of avital, i.e. Annexin V.sup.+ and/or Hoechst 33258.sup.+ cells at 5 days after application of niclosamide (black bars) or 0.01% DMSO (white bars). Inset: mean data (n=5 pGBMs; **p<0.01). Note the lack of pro-apoptotic effects in the non-malignant human cell sample #155. Right inset: representative scatter plots (#046). (E) The scratch assay (n=2 pGBMs) was performed at day two following exposure of 125 nM niclosamide (dotted line) or of 0.0025% DMSO (solid line). The graph exemplifies the time course of scratch closure for case #046 (mean±SD; triplicate analysis).

(3) FIG. 3: Niclosamide decreases the tumor-initiating potential of pGBMs. (A) Neurosphere assay (n=4 pGBMs; mean±SD). The graph depicts the relative frequency of primary (1°), secondary (2°) and tertiary (3°) neurospheres from niclosamide pre-treated pGBMs (single exposure). Data show a persistent decrease of sphere-forming cells (SFCs). (B) Long-term cell growth data after single application of 1.5 μM niclosamide (niclo; dotted lines) vs. 0.01% DMSO (solid lines) (mean±SD of triplicate analysis). (C) Kaplan-Meier survival curves of xenografts. For experimentation, pGBM #046 cells were pre-treated with niclosamide (dotted) or DMSO (solid). 10.sup.6 vital cells were collected at day 5 and stereotactically injected into the striatum of immunocompromised mice. Distressed animals were euthanized. With one exception (niclosamide; red dot), animals showed intracerebral tumor manifestation.

(4) FIG. 4: Niclosamide affects several cancer regulating signaling pathways simultaneously. Western blot analysis of cleaved-NOTCH1 (A) and phospho-S6-protein (B) was performed at 5 days after single dose application. (C) Data quantification reveals a significant decrease of nuclear phospho-CTNNB1 (Ser552).sup.+ cells (***p<0.001, triplicates, mean±SD). (D) qRT-PCR analysis of WNT/CTNNB1 target genes (***p<0.001, triplicates, mean±SD). Expression levels relative to the DMSO control. P-values calculated using 1-way ANOVA analysis with Bonferroni post-test. Note, all experiments conducted with pGBMs #'s 046, 078, 106, and 118.

(5) FIG. 5: Deletion and expression level of NFKBIA predicts synergistic activity of niclosamide and TMZ. (A) Western blot analysis of pGBMs with NFKBIA.sup.+/− enotype (purple) vs. NFKBIA.sup.+/− samples (green). Levels of phospho-RELA (p65-NF-κB) indicating pathway activity were determined 3 days after niclosamide exposure. (B) Quantification of mRNA levels in NFKBIA.sup.+/+ (046, 078, 138) vs. NFKBIA.sup.+/− (081, 106, 066) pGBMs in response to niclosamide (light) or DMSO (dark) exposure. Data presented as relative to DMSO control. Inset depicting base-line mRNA expression levels of NFKBIA. (C) Combinatorial pharmocodynamics of TMZ and niclosamide in NFKBIA.sup.+/+ (n=4) vs. NFKBIA.sup.+/− (n=3) pGBMs. Increasing concentrations of niclosamide were supplied either in combination with 50 μM TMZ or with 0.05% DMSO as control. Data exemplified by #'s 046 (NFKBIA.sup.+/−, square and triangle) and 106, (NFKBIA.sup.+/+, inverted triangle and diamond) and presented as mean±SD of triplicates. Arrows highlight synergistic activity for the NFKBIA.sup.+/− sample. Note, data for the other investigated pGBMs are listed in Table 2.

(6) FIG. 6: Niclosamide dose-response curves. The metabolic activity as a measure for cellular viability was determined at 5 days after compound exposure (mean data±SD, triplicate analysis). Data relative to control (DMSO) levels. The IC50 was defined as concentration of niclosamide that reduced the metabolic activity to 50% control levels. (A) Comparison of data for commercially available human glioma/GBM cell lines LN229, T89G, U87, U138, and U272 (grey) vs. 21 pGBMs (black; mean±SD, compare with FIG. 2A). (B) Comparison of data for human non-malignant neural (control) cell samples PKI-3, #155, and H9.2 (grey) vs. 21 pGBMs (black; mean±SD, compare with FIG. 2A). (C) Evaluation of paired samples derived from tumor core (squares) vs. tumor periphery (triangles) for pGBM #'s 046, 066, and 078. (D) Evaluation of paired samples derived from primary disease (squares) vs. recurrent disease (triangles) for pGBM #'s 091, 118, and 132. (E) Comparison of data for MGMT promoter hypermethylated pGBM samples #023 and #025 (triangles) vs. MGMT promoter unmethylated pGBM samples #'s 046, 106, and 138 (squares). (F) Comparison of data for NFKBIA.sup.+/+ pGBM samples #'s 066, 081, and 106 (squares) vs. NFKBIA.sup.+/− pGBM samples #'s 046, 078, and 118 (triangles).

(7) FIG. 7: Niclosamide induces a transient G1 phase arrest in pGBMs. Cell cycle analysis revealed similar results for cases #046 and #106 (PI, propidium-iodide). Shown is #046 exposed to 1.5 μM niclosamide vs. 0.01% DMSO at (A) 6 h, (B) 12 h, (C) 24 h, (D) 48 h, (E) 72 h, and (F) 5 d.

(8) FIG. 8: Niclosamide depletes the tumor-initiating potential of pGBM #GNV019. (A) Long-term cell growth data after single dose niclosamide application (1 μM, dotted) vs. DMSO (0.01%, solid). (B) Xenograft experiments were conducted similar to methods described for FIG. 4C, with the exception that neonatal mice were used as recipients. Kaplan-Meier survival curves depict the course of DMSO pre-treated (solid) vs. niclosamide (dotted) pre-treated cell grafts. Intracerebral tumor manifestation was noted in 8/9 animals from the DMSO control group. In contrast, no animal that received niclosamide pre-treated grafts showed evidence for tumor formation (censored events). Calculation of the p-value based on the log-rank test.

(9) FIG. 9: The graph depicts IC50 data from pharmacodynamic analysis of pGBMs vs. ‘standard’ glioblastoma (GBM) cell lines. Niclosamide-treated ‘standard’ cell lines maintained in defined media (dm; n=5; mean±SD) show pGBM-like degrees of sensitivity to niclosamide. P-values were calculated from comparing ‘standard’ glioblastoma cell line data with pGBMs using one-way ANOVA and Tukey post hoc test. n.s., not significant. (sm; standard media conditions)

(10) FIG. 10: Niclosamide dose-response curves (A-E), comparison of results obtained from ‘standard’ GBM cell lines maintained in standard media conditions (sm; solid lines) vs. defined media conditions (dm; dotted lines). It is evident that niclosamide effects are more pronounced (pGBM-like) under dm conditions. (F) Table summarizing the respective IC50 values.

(11) FIG. 11: Coculture experiments combining hnNC case #155 with various pGBMs. (A) CellaVista-based analysis of cocultures. Data were obtained at indicated time points after application of niclosamide (1 μM). Triplicate analysis (***, p<0.001; **, p<0.01). (B) FACS data obtained 5 days after the application of niclosamide (1 μM) to respective cocultures. The inset depicts a representative set of scatter plots. Note that pGBMs cases #046 and #078 are NFKBIA+/−; cases #035 and #106 are NFKBIA+/+ genotypes. LT, lentivirally transduced; CT, CellTracker-labeled populations.

(12) FIG. 12: Niclosamide inhibits NOTCH and mTOR signaling independent of the cellular NFKBIA status (#046=NFKBIA+/−; #106=NFKBIA+/+) (shown here by decreasing levels of phosphorylated S6 protein 5 days after single dose application of ND; n=4 pGBMs analyzed).

(13) FIG. 13: Niclosamide inhibits the malignant potential of pGBMs (NFKBIA+/− genotypes). (A) Neurosphere assay (n=2 pGBMs in triplicates; mean±SD). The graph depicts the relative frequency of primary (1°), secondary (2°), and tertiary (3°) neurospheres from niclosamide preexposed pGBMs (single exposure). Note the persistent decrease of sphere-forming cells (SFC), **p<0.01. (B) Kaplan-Meier survival curves (similar to Additional evidence, FIG. 4). Intracerebral tumors manifested in all but one animal (niclosamide; red dot).

(14) FIG. 14: Deletion and expression level of NFKBIA predicts synergistic activity of niclosamide (Niclo) and temozolomide (TMZ) in pGBMs. (A) Changing mRNA levels in NFKBIA+/+ (gray) versus NFKBIA+/− (black/white) pGBMs in response to niclosamide (relative to DMSO control). Inset, baseline mRNA expression levels of NFKBIA. (B) Combinatorial index (CI) evaluation for application of niclosamide+temozolomide in pGBMs. CIs were expressed as ratio of observed versus expected cell viability. Expected results were calculated according to ref (Chou T C, 2010) as proportion of viable cells after treatment with (only) 1 μM niclosamide multiplied by the proportion of cells following treatment with (only) temozolomide. (CI<1:synergy, CI=1:additive; CI>1:antagonism). (C) Representative combinatorial pharmocodynamics of temozolomide and niclosamide in NFKBIA+/− (left) versus NFKBIA+/+ (right) pGBMs. Increasing concentrations of niclosamide were supplied either in combination with 50 μM temozolomide or with 0.05% DMSO as control. Data presented as mean±SD of triplicates.

(15) FIG. 15: Deletion and expression level of NFKBIA predicts synergistic activity of niclosamide (Niclo) and temozolomide (TMZ) in standard glioblastoma (GBM) cell lines maintained in defined media conditions (dm). (A) changing mRNA levels in NFKBIA+/+ (gray) versus NFKBIA+/− (black/white) in response to niclo (relative to DMSO control). Inset, baseline mRNA expression levels of NFKBIA. (B) CI evaluation for application of niclo+TMZ. CIs were expressed as ratio of observed versus expected cell viability. Expected results were calculated as proportion of viable cells after treatment with (only) 1 μM niclo multiplied by the proportion of cells following treatment with (only) TMZ. (CI<1:synergy, CI=1:additive; CI>1:antogonism). (C) representative combinatorial pharmocodynamics of TMZ and niclo in NFKBIA+/− (left) versus NFKBIA+/+ (right) standard GBM cell lines. Increasing concentrations of niclo were supplied either in combination with 50 μM TMZ or with 0.05% DMSO as control. Data presented as mean±SD of triplicates. It is evident that the niclo-effects are pGBM-like under dm conditions (compare to FIG. 15).

(16) FIG. 16: TNF-a antagonizes synergistic activity in NFKBIA+/− pGBMs. Graphs present data from combinatorial treatment paradigms in NFKBIA+/− (black) versus NFKBIA+/+ (gray) pGBMs at 3 days after compound application (niclo, 1 μM; temozolomide, 50 μM; TNF-a, 50 ng/ml). Insets, Western blot analyses of pREL A, indicating NF-kB pathway activity 24 hours after exposure to TNF-a, (Ta; 50 ng/ml) or 0.002% bovine serum albumin (C; control). ***p<0.001; **p<0.01; *p<0.05 (triplicates; mean±SD; one-way ANOVA and Tukey post hoc tests).

(17) FIG. 17: List of pGBMs and their respective passage numbers used for the various experimental paradigms in this study. (Pharma, pharmacodynamic analysis; CT/LT, co-culture experiments; WB, Western blots).

EXAMPLES

(18) Reagents

(19) The reagents the experiments set out below can be freely purchased, specifically; alamarBlue®, prodidiumiodide, and Hoechst33258 were purchased from Life Technologies; niclosamide and temozolomide were purchased from Sigma-Aldrich; FITC Apoptosis Detection Kit I were purchased from BD Bioscience.

(20) Mice

(21) The Ethical Committee of the University of Bonn, Medical Centre approved all studies involving animals. Rag2.sup.−/−Il2rg.sup.−/− mice were acquired from Taconic Farm Inc., contractor of the National Institute of Allergy and Infectious Diseases' investigators (42). SCID/Beige mice were purchased from Jackson Laboratory.

(22) Tissue Samples

(23) Tumor tissue derived from GBM surgery and hippocampus tissue (case #155) derived from epilepsy surgery at the Department of Neurosurgery, University of Bonn Medical Centre. Patient characteristics are detailed in Table 3. pGBM case GNV019 derived from surgery of a 9-year-old boy at the University of Florida Department of Neurosurgery. The local Ethics committees at both sites approved the studies; all patients—or their guardians, provided informed consent. Tissue diagnosis and grading based on the current classification of the World Health Organization (43) and confirmed by two independent neuropathologists at the Department of Neuropathology, University of Bonn Medical Centre (the National Brain Cancer Reference Center).

(24) TABLE-US-00001 TABLE 3 Patient data. List of patients and tissue specimens investigated in this study RPA Primary Patient Diagnosis Sex Age Histology Class therapy PFS OS MGMTstatus 021 new m 78 GBM V R, RT/TMZ,  4 12 unmet 2xTMZ (5/28) 023 new f 79 GBM V RA NA  9 meth 025 new m 70 GBM V R, RTA NA NA meth 035 new f 75 GBM IV RB  1  1 unmet 046D new m 76 GBM IV RB  1  1 unmet 066D new f 69 GBM IV R, RT/TMZC  2  2 unmet 078D new m 52 GBM IV R, RT/TMZ,  5 10+ unmet 2xTMZ (5/28) 081 new w 86 GBM IV RA NA 17 unmet 091E new m 52 GBM IV R,  7 10 unmet RT/TMZ, 4xTMZ (5/28) 106 new f 68 GBM IV R, RT/TMZ,  5  5+ unmet 1xTMZ (5/28) 116 new f 67 GBM IV R, RT/TMZ  3  7 unmet 118E new m 63 GBM IV R, RT/TMZ,  7  9 unmet 4xTMZ (5/28) 132E new m 75 GBM IV R, RT/TMZ,  7 n.d. 4xTMZ (5/28) 135 new m 41 GBM IV R, RT/TMZC  8  9 n.d. 138 new w 54 GBM IV R, RT/TMZ, 10 14+ unmet 5xTMZ (5/28) A: Patient denied further treatment; B: Postoperative complications; C: Discontinuation of therapy due to clinical deterioration; D: Two pGBM samples were derived from this patient, one from the tumor core (center) and the second from the tumor periphery (see (10)); E: Two pGBM samples were derived from this patient, one at the time of primary disease and the second at the time of disease recurrence; R: Tumor resection; RT: Standard radiotherapy; RT/TMZ: RT plus continuous daily temozolomide (concomitant); TMZ: Temozolomide (5/28: days 1 to 5 out of a 28-day-cycle) PFS: Progression-free survival; OS: Overall survival; meth: methylated MGMT promoter; unmet: unmethylated MGMT promoter
Tissue Handling and Culture of Primary Cells

(25) Handling of fresh biopsy samples and derivation of pGBMs (10) and hippocampus tissue-derived AHNPs (#155) (11) were performed as described recently. Media conditions for #GNV019 cells are detailed in (44). Media conditions for all other pGBM and AHNP samples are described in (8). Data were generated from culture passages 7 to 13.

(26) Culture of Established Glioma/GBM Cell Lines

(27) LN229, T98G, U87(MG), U138, and U373(MG) cells were maintained and analyzed in DMEM/F12-based 10% fetal calf serum (Hyclone)-supplemented adherent conditions. These conditions are also referred to as “sm conditions”.

(28) Culture of Neural Stem Cells from Human ES and iPS Cells

(29) Together with primary AHNPs (see above), two human long-term self-renewing neural stem cell cultures (lt-NES) were used in this study as non-malignant neural control cells. The lt-NESs were originally derived from the human embryonic stem cell line H9.2 (45) and from the human induced pluripotent stem cell line PKa (46). Conditions for the maintenance of lt-NESs were recently described (45, 47).

(30) For some studies, defined media (dm) were applied to GBM model cell lines for ten days before initiation of experiments. ‘dm’ resemble media compositions used for the culture of pGBMs and hnNCs/AHNPs, i.e. adapted from (Lee J, Kotliarova S, Kotliarov Y, Li A, Su Q, Donin N M, et al. Tumor stem cells derived from glioblastomas cultured in bFGF and EGF more closely mirror the phenotype and genotype of primary tumors than do serum-cultured cell lines. Cancer Cell 2006; 9(5): 391-403): N2/B27-supplemented Neurobasal™ with addition of growth factors every other day (EGF, bFGF; 10 ng/ml each). Handling of tissue and derivation of pGBMs4, hippocampus-derived AHNPs (#155) (Walton N M, Sutter B M, Chen H X, Chang U, Roper S N, Scheffler B, et al. Derivation and large-scale expansion of multipotent astroglial neural progenitors from adult human brain. Development 2006; 133(18): 3671-81.), and #GNV019 cells (Scheffler B, Walton N M, Lin D D, Goetz A K, Enikolopov G, Roper S N, et al. Phenotypic and functional characterization of adult brain neuropoiesis. Proc Natl Acad Sci USA 2005; 102(26): 9353-8.) were recently described. Data presented here were obtained from short-term expanded pGBMs and AHNPs (passage 5-12; FIG. 18). With exception of the neurosphere assay, all cells were cultured adherently on laminin/poly-L-ornithine coated plastic. In addition to AHNPs, two long-term self-renewing neural stem cell cultures (lt-NES) were used as non-malignant neural control. lt-NESs derived from the human embryonic stem cell line H9.218 and from the human induced pluripotent stem cell line PKa19

(31) Co-Culture Experiments

(32) Lentiviral transduction and selection of pGBMs was conducted using the pLenti6.2/V5-DEST Getaway Vector harboring the coding sequence of GFP as suggested by the manufacturer (Life Technologies). Fluorocytometry confirmed stable cellular expression. Alternatively, pGBMs and hnNCs were labeled with CellTracker™ (-CFSE green fluorescent dye or -Red CMTPX; Life Technologies) according to the manufacturer's instructions. For initiation of respective cocultures, cells were mixed at 1:1 ratios and maintained for 24 hours before conducting experimental paradigms. For distinctive monitoring of cell growth, a fluorescence-enabled CellaVista® System Analyzer (Roche Diagnostics) was used. FACS data for end point analysis were obtained using a FACS calibur flow cytometer (BD Bioscience).

(33) Primary Drug Screening and Pharmacodynamic Analysis

(34) The tested compounds were supplied to cells proliferating in a linear-exponential phase. For all used cell samples, respective titration experiments were conducted before analysis. 24 hours after seeding 2-3×10.sup.3 cells/well into laminin/poly-L-ornithine coated 96-well plates, cells were treated with 1 μM of each compound (stock solution 10 mM in DMSO). Control cells were treated with 0.01-0.1% DMSO. Five days after application, metabolic activity as a measure of cell viability was determined using the alamarBlue® assay according to the manufacturers recommendations (Life Technologies). Fluorescence was measured using an Infinite200 microplate reader (Tecan) at λ.sub.ex=540 nm and λ.sub.em=590 nm. Experiments were performed in triplicates for each sample.

(35) For pharmacodynamic analysis, 5×10.sup.4 cells were plated in 12-well-plates at 24 hours before application of compound-series and compound combinations, respectively. alamarBlue®-based analysis was conducted at 3-5 days post treatment. Experiments were performed in triplicates. IMC50 was defined as the compound concentration that reduced the metabolic activity by 50% compared to control conditions and determined via data analysis in GraphPad Prism 4.0.

(36) Proliferation Kinetics

(37) Five days post treatment, 4.7×10.sup.4 vital cells were plated into 3.5 cm laminin/poly-L-ornithine coated plastic dishes, and four to six days later trypsinized, harvested, counted, and re-plated at a density of 4.7×10.sup.4. The procedure was repeated 4-5 times. For long-term monitoring of niclosamide-induced alterations to cellular growth, cell confluence was determined using the CellaVista® system Analyzer (Roche Diagnostics) according to the manufacturer's instructions

(38) Cell Migration Analysis

(39) 5×10.sup.4 cells were plated into 12-well-plates coated with laminin/poly-L-ornithine. Cells were treated with 125 nM niclosamide every 24 hours for 4 days. Three days after plating (at a cell density of 70%), a scratch/wound was inflicted with a sterile pipette tip. Thereafter, culture media was exchanged to remove non-adherent cells. The Plaque Assay application of the CellaVista® system (Roche Diagnostics) was used according to the manufacturer's instructions to monitor the scratch/wound size over time. Triplicate analysis data±SEM.

(40) Cell Cycle Analysis

(41) Cells (5×10.sup.4 per well) were grown in 12-well plates, and collected after treatment at times indicated. Cells were re-suspended in phosphate-buffered saline (PBS), fixed with ice-cold methanol and incubated for a minimum of 24 hours at 4° C. Cell pellets were collected by centrifugation and re-suspended in PBS solution, containing 50 μg/ml propidium iodide and 50 μg/ml RNase. Following incubation for 30 min at 37° C., cells were analyzed for DNA content using a FACS calibur flow cytometer (BD Bioscience).

(42) Annexin V-Based FACS Analysis

(43) 1×10.sup.5 cells were collected at 5 days following compound application, settled by centrifugation, re-suspended in 100 μl AnnexinV buffer and incubated with 5 μl Annexin V-FITC for 1 hour at room temperature. To distinguish between living and dead cells, labeling with 1.2 μg/ml Hoechst 33258 was used Annexin V presence was determined using standard conditions in a LSRII equipped with FACSDiva Software (BD Bioscience). 2×10.sup.4 cells were counted per measurement. The term ‘avital cells’ was used for Annexin V.sup.+−, Annexin V.sup.+/H33258.sup.+, and H33258.sup.+ cells.

(44) Neurosphere Assay

(45) The neurosphere assay was performed to estimate the frequency of self-renewing clonogenic cells according to established protocols (10, 44). Neurospheres were quantified at 21 days in culture, triturated to a single cell suspension, and re-plated for analysis of the secondary and tertiary neurospheres. Multipotency was determined by plating a representative fraction of 3° neurospheres onto laminin/poly-L-ornithine coated glass coverslips allowing differentiation for 2-3 weeks before fixation in 4% paraformaldehyde (PFA).

(46) Fluorescence Analysis

(47) Immunofluorescence analysis was performed on PFA-fixed samples according to standard protocols (44, 48) using antibodies against βIII tubulin (Promega; monoclonal mouse, 1:1000), GFAP (DAKO, polyclonal rabbit, 1:600), β-catenin, and phospho-β-catenin (Ser552) antibody (both Cell signaling, 1:400). Cell nuclei were visualized with DAPI (Sigma).

(48) Western Blot Analysis

(49) Cell extracts were prepared at 24 to 144 hours following compound application and processed as described (49). Blot membranes were incubated overnight at 4° C. with antibodies against Cyclin-D1 (1:1000; BD Pharmingen), cleaved-Notch1 (1:1000), or phospho-S6 protein (1:1000; all Cell signaling) respectively. After washing, peroxidase-coupled secondary antibodies (Santa Cruz) were added for 1 hour. After washing, blots were developed using the ECL system (Millipore). To confirm equal loading, blots were re-probed with an β-actin antibody (Sigma; 1:5000).

(50) Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR)

(51) Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. Quantification of RNA concentration was performed with Nanodrop (Peqlab) and 400 ng total RNA was reversely transcribed with oligo-dT primers in a reaction mix (1×RT-Puffer, 10 mM DTT, 500 μM pooled dNTPs, 1 U/μl RNase inhibitor, 2.5 U/μl Expand Reverse Transcriptase; all from Roche Diagnostics). Reaction occurred at 42° C. for 1 h. The cDNA product was amplified in a total volume of 10 μl in 96 well plates using the realplex 4 Mastercylcer Epp Gradient S (Eppendorf) and the following PCR conditions: 95° C. for 2 minutes, followed by 40 cycles of 95° C. for 15 seconds, 60° C. for 20 seconds and 72° C. for 30 seconds. For quantification, the following primers were used:

(52) TABLE-US-00002 S100A4 forward: (SEQ ID NO: 1) 5′-CTCAGCGCTTCTTCTTTC-3′; S100A4 reverse: (SEQ ID NO: 2) 5′-GGGTCAGCAGCTCCTTTA-3′; c-Myc forward: (SEQ ID NO: 3) 5′-TTCGGGTAGTGGAA-AACCAG-3; c-Myc reverse: (SEQ ID NO: 4) 5′-CAGCAGCTCGAATTTCTTCC-3′; Cyclin D1 forward: (SEQ ID NO: 5) 5′-CCGTCCATGCGGAAGATC-3′; Cyclin D1 reverse: (SEQ ID NO: 6) 5′-ATGGCCAGCGGGAAGAC-3′; NFKBIA forward: (SEQ ID NO: 7) 5′-ACACCAGGTCAGGATTTTGC-3′; NFKBIA reverse: (SEQ ID NO: 8) 5′-GCTGATGTCAATGCTCAGGA-3.

(53) For cDNA quantification of the house keeping gene glycerinaldehyd-3-phosphat-dehydrogenase (GAPDH) the following primers were used: forward: 5′-TGCACCACCAACTGCTTAGC-3 (SEQ ID NO: 9); reverse: 5′-GGCATGGACTGTGGTCATGAG-3′ (SEQ ID NO: 10). Data analysis was performed with the Mastercycler Epp Realplex Software (Eppendorf). Mean values were calculated from triplicate qRT-PCR reactions. Each mean value of the expressed gene was normalized to the respective mean amount of the GAPDH cDNA.

(54) Single Nucleotide Polymorphism Array Analysis (SNP)

(55) For evaluation of the NFKBIA locus whole-genome genotyping analysis was performed. Genotyping of 299,140 SNPs was conducted using the Illumina HUMANCytoSNP-12 v2.1 according to the manufacturer's Infinium HD assay (Illumina, San Diego, USA). Data was analyzed with Illumina GenomeStudio (2011.1) software including the Genotyping and GenomeViewer modules. Chromosomal aberrations were identified by examination of Log R ratios and B-allele frequencies.

(56) MGMT Promoter Methylation Status

(57) The methylation status of the MGMT gene was determined by pyrosequencing as recently described (50). In brief, 0.5 μg genomic DNA was treated with sodium bisulfite using the EpiTect Bisulfite kit (Qiagen, Hilden, Germany) according to the manufacturers recommendations. For pyrosequencing, a 265 bp region was amplified from bisulfite modified genomic DNA using primers MGMT-Py forward, 5′-biotin-GGATATGTTGGGATAGTT-3′ (SEQ ID NO: 11) (GenBank accession number AL355531, nucleotides 46891 to 46908) and MGMT-Bis reverse, 5′-AAACTAAACAACACCTAAA-3′ (SEQ ID NO: 12) (GenBank accession number AL355531, nucleotides 47138 to 47156) with biotin attached to the 5′-end of the forward primer. The primer used for the extension reaction was 5′-CCCAAACACTCACCAAA-3′ (SEQ ID NO: 13) which allowed sequencing of a 63 bp fragment containing 12 CpG sites. The pyrosequencing assay was designed to target CpG sites with strong methylation in GBM. Pyrosequencing was performed using PyroGold Reagents (Qiagen, Hilden, Germany) on the Pyromark Q24 instrument (Biotage, Uppsala, Sweden), according to the manufacturer's instructions. Pyrogram outputs were analyzed by the PyroMark Q24 software (Biotage, Uppsala, Sweden), using the CpG quantification software to determine the percentage of methylated versus unmethylated alleles according to percentage relative peak height. Tumor samples were scored methylated or unmethylated after measuring CpG methylation at individual positions and comparing with methylation data obtained from age matched normal brain tissues. Human reference DNA in vitro methylated by SssI methylase was used as positive control for methylation.

(58) Tumor Xenograft Experiments

(59) Cells were harvested, counted and re-suspended in 0.1% DNase/PBS. Cell vitality was confirmed via trypan blue exclusion. For case #046, 10.sup.6 DMSO-control (n=5)- or niclosamide (n=5)-pretreated pGBMs were injected stereotactically into the striatum of 12-week old Rag2Il2rg.sup.−/− mice (0.8 mm anterior, 2 mm lateral, 3 mm deep). For case #GNV019, 2.5×10.sup.4 sham control (Killer Plates® compound 2F05; n=9)- or niclosamide (n=6)-pretreated pGBMs were injected intracranially into P2 to P3 old Scid Beige mice. Mice were monitored daily and euthanized upon presentation with signs of distress/neurological symptoms or significant weight loss. The #019GNV experiment was terminated at day 169 with one remaining animal that did not appear distressed. For subsequent histological analysis, brains were removed, cryoprotected, and serially cut on a cryostat (Leica) at 20 μm thickness. Every fifth section underwent routine H&E staining for histological analysis of tumor formation.

(60) Statistical Analysis

(61) GraphPad Prism 4.0 software was used for statistical analysis. Data presented with error bars represent mean±SD from triplicate experiments unless otherwise noted. For pharmacodynamic analysis, p-values were calculated using the 1-way ANOVA and Tukey's post-hoc tests (FIG. 1). For multiple comparisons, p-values were calculated using the 1-way ANOVA with Bonferroni post-hoc test. If applicable, the two-sided Student's t test was used to determine statistical significance. A p-value of <0.05 was considered significant.

(62) Results

(63) Niclosamide is a Previously Unrecognized Candidate for GBM Therapy.

(64) A library comprising of 160 synthetic and natural toxic substances was used for the screening approach. Four pGBMs, previously shown to maintain patient- and GBM-specific signatures and to contain sub-populations of tumor cells with and without stem cell qualities served as a discovery platform (#'s 023, 035, 046, 106; see (10)). Primary screening was conducted based on the alamarBlue® assay determining the metabolic activity as a measure for cellular viability at day 5 following single application of the library's compounds. In the experiments, every compound that reduced the mean metabolic activity of the 4 pGBMs below 50% of control levels was considered as a ‘hit’. 31 compounds fulfilled this criterion, amongst them niclosamide. Moreover, niclosamide indicated a sufficient potential to address inter-patient heterogeneity by impacting effectively on all four of the tested pGBMs (Table 1) and it demonstrated a cancer-specific potential, as it did not appear to similarly affect hnNCs sample #155, a control case of non-malignant primary adult human neural progenitor cells (AHNPs; (11), Table 1). Niclosamide, revealed a selective pGBM-anticancer potential that had not yet been suggested for treating brain tumors. Niclosamide is known for decades and approved by many regulatory agencies as antihelminthic. Recent work in extra-neural, e.g. preclinical colorectal cancer models suggested some activity of this drug (12, 13). However, given the scarcity of effective cytostatic compounds for the treatment of glioblastoma, its efficacy in this tumor entity was surprising.

(65) TABLE-US-00003 TABLE 1 Metabolic activity of different cell lines after application of 1 mM niclosamide for 5 days (results of triplicate analysis), the metabolic inhibition the cells is indicated in % of the activity of a control without niclosamide pGBMs pGBMs.sup.1 average Controls 023 035 046 106 155.sup.2 U87.sup.3 32.3 5.6 34.6 33.0 26.4 75.4 66.5 .sup.1primary glioblastoma cell lines used in this study .sup.2non-malignant primary adult human neural progenitor cells .sup.3a commonly investigated glioma cell line
Niclosamide is Effectively and Selectively Inhibiting pGBMs Cellular Viability.

(66) To validate the results obtained from primary screening of the library, pharmacodynamic analysis was conducted using a formulation of niclosamide obtained from Sigma-Aldrich. A total of 21 pGBMs were investigated, including the four cases already used in the screening experiments. The obtained dose-response curves showed consistent courses for all samples (FIG. 1A). The concentrations at which niclosamide induced a 50% reduction of the relative metabolic activity (IC50) ranged from 300 nM to 1.2 μM. This contrasted to the more resistant performance observed in reference- and control-cell samples. The IC50 values of five commonly investigated glioma/GBM cell lines (LN229, T98G, U87, U138, and U373, see methods), here used as a reference, were calculated at 2.4 to 4.2-fold higher concentrations (FIG. 1B; FIG. 6). The lower sensitivity of glioma/GBM cell lines to niclosamide exposure may be due to their standard conditions of maintenance (i.e. serum-containing, mitogen-free), a major factor that in the past might have interfered with many results of drug screening at early developmental stages (7). Further experiments revealed that this assumption is correct. When ‘standard GBM model’ cell lines (i.e. LN229, T98G, U87(MG), U138, U373(MG)) are maintained under ‘dm conditions’, i.e. the culture conditions used for maintenance of pGBMs, ND effects are highly similar to findings obtained from pGBMs.

(67) Notably, however, analysis of the three non-malignant hnNCs that were maintained in similar defined culture conditions as pGBMs also revealed a significantly lower level of sensitivity (see methods; FIG. 1B; FIG. 6). This suggested a pGBM-specific activity of niclosamide. When pGBMs and non-malignant hnNCs are co-cultured under dm conditions, the lower sensitivity of hnNCs can be confirmed. These data suggest low levels of ND toxicity on non-malignant neural cells and selective activity against pGBMs.

(68) Considering the cellular and genetic diversity that characterizes GBM, we next investigated niclosamide's pharmacological effect in pGBMs representing key clinical constellations (10, 14-16). Comparative experiments were therefore conducted with samples derived from (i) the tumor core (center) vs. periphery region of the same GBM patient, (ii) primary vs. recurrent disease of the same GBM patient, (iii) MGMT-promoter hypermethylated vs. unmethylated tissue as well as from (iv) GBMs with heterozygous deleted NFKBIA vs. undeleted NFKBIA genotypes (see below). The strong inhibitory activity of niclosamide could be demonstrated similarly in all of these pGBM samples (inset FIG. 1B; FIG. 6). Together, these data confirmed and validated our primary screening results, portraying niclosamide as a highly effective and selective inhibitor of pGBMs.

(69) Niclosamide has Cytostatic, Cytotoxic, and Anti-Migratory Effects in pGBMs.

(70) To further classify the inhibitory activity of niclosamide in pGBMs, studies on cell cycle, vitality, and migratory function were conducted subsequent to a single exposure to the compound. Propidium iodide (PI)-based flow cytometry analysis at revealed a transient G1 phase arrest of pGBMs peaking at 24 to 48 hours (FIG. 2A; FIG. 7). This coincided with a strong decrease of Cyclin D1 expression, a regulator of cell cycle transition from G1 to S phase (FIG. 2B). Evidence for an immediate and transient cytostatic activity was similarly revealed upon examination of pGBM's growth kinetics (FIG. 2C). With a resulting growth delay of 5 days, an additional cytotoxic response became apparent. At this time, pro-apoptotic effects of niclosamide caused a strong and pGBM-selective decrease of vital cells—as observed by phase contrast microscopy (not shown) and as quantified by Hoechst/Annexin V flow cytometric analysis (FIG. 2D). Intriguingly, application of niclosamide at sub-toxic concentrations additionally caused anti-migratory effects on pGBMs, similar to recent findings described for colon cancer cells (12) (FIG. 2E). Thus, niclosamide induced combined cytotoxic, cytostatic, and antimigratory effects in pGBMs.

(71) Niclosamide Inhibits pGBMs Tumor-Initiating Activity.

(72) In a next series of experiments it was aimed to determine the influence of niclosamide on the activity of tumor-initiating cells (TICs). TICs embody a severe functional consequence of intra-tumor heterogeneity as, at least in human GBM, it is anticipated that they are represented by a small subpopulation of stem-like, i.e. self-renewing and multipotent cells (e.g. (17-19)). However, their precise phenotypic characteristics remain elusive (20). We thus applied a combination of assays to measure their responses to niclosamide indirectly. First, the neurosphere assay (NSA) was used to estimate potential alterations to the pool of self-renewing and multipotent cells. In previous studies, we established their frequencies in the range from 0.25 to 1% among culture passage 5-10 pGBMs (see (10)). In the present study, three of these heterogeneous pGBM samples (#'s 046, 078, 106) were exposed to niclosamide, and vital cells were collected at day 5 for processing in the NSA (see methods). Quantification of primary, secondary and tertiary spheres from DMSO-vs. niclosamide pre-treated cells indicated that a single application of niclosamide reduced the frequencies of self-renewing, multipotent cells among pGBMs strongly (FIG. 3A). Because niclosamide did not abolish the multipotent potential among the remaining self-renewing pGBMs at the applied concentration of 1.5 μM (FIG. 3A, inset), it was tempting to speculate that this setting could be used to similarly demonstrate a measurable reduction of tumorigenic cell frequencies in vivo. Parallel long-term growth analysis (CellaVista®, cell confluence-based, see methods) of pGBMs indicated that at this concentration, the recovery of vital cells from cytostatic niclosamide effects had to be expected with a delay of 14-23 days (FIG. 3B). Orthotopic xenotransplantation studies demonstrated, however, that animals engrafted with niclosamide pre-treated vital cells survived considerably longer than expected (FIG. 3C; FIG. 8). The statistic significance of these results was paired with a lower extent to which tumor formation was observed in niclosamide pre-treated cell grafts. In one transplantational series (#GNV019), a single exposure to niclosamide completely extinguished the tumor forming capacity of pGBMs (FIG. 8). In a second experimental series (#046), a strong reduction could be observed. Here, DMSO pre-treated #046 pGBMs that were grafted unilaterally into the striatum elicited severe signs of distress in recipient animals after 88±5d (n=5). Subsequent histological analysis revealed in every of these cases massive intracerebral tumor formation and a strong invasive capacity of engrafted cells along white matter tracts into the contra-lateral hemisphere. By contrast, the tumors that developed in 4/5 animals from niclosamide pre-treated #046 pGBMs at 153±23d after engraftment were smaller of size, with cells accumulating in areas adjacent to the striatal transplant site. Proliferative pGBMs were found clustered in the subventricular zone and dispersed throughout the corpus callosum (FIG. 3E, inset), with individual cells reaching the contra-lateral hemisphere. This corresponded to early post-transplantational stages of DMSO pre-treated #046 cells. Apparently, the diffusely invasive nature of pGBMs sufficed during the long-term experiments to induce neurological dysfunction/distress in the animals that required their euthanization even before the manifestation of an expanding tumor mass. The combined data of our experiments, regardless, suggested strongly that already a single exposure of niclosamide did lead to an effective reduction of tumor initiating activity in pGBMs.

(73) Niclosamide Interferes with Cancer-Driving Signaling Cascades.

(74) It is known that a circumscribed number of transcription factors and associated signaling pathways are overactive in human cancer cells (21). Evidence from previous studies had already suggested that niclosamide interfered with several of these in blood, breast, and colon cancer cells, specifically with Notch-, mTOR-, Wnt-/β-catenin-, and NF-κB-signaling (12, 13, 22-24). Hence, the study focused on this array of pathways for mode of action analysis in pGBMs. Cells were investigated at day 5 after a single-dose exposure to niclosamide (n=4 cases: #'s 046, 078, 81, 106). Western blots demonstrated a concentration-dependent inhibition of Notch pathway activity in the pGBMs, as indicated by decreasing levels of the cleaved Notch 1-protein (FIGS. 4A, 12A and 12C). Similarly, levels of the phosphorylated S6-protein as a major indicator of active mTOR signaling (25) could be shown to decrease in all samples (FIGS. 4B, 12B and 12D). This effect is independent of the NFKBIA gene status. The pleiotropic activity also explains the strong antitumor potential of ND in pre-exposed pGBMs upon orthotopic engraftment in animal models of disease (FIG. 13). Exploration of the Wnt-/β-catenin pathway furthermore suggested a specific interference of niclosamide with the non-canonical (alternative) Akt-dependent regulation of β-catenin's transcriptional activity. Characteristic for the active state of this mechanism, known to play an important role for tumor invasion, is an enhanced nuclear accumulation of β-catenin, phosphorylated at Ser.sup.552 (26). The respective immunocytochemical exposure and quantification in pGBMs demonstrated a strong decrease of the nuclear phospho-β-catenin (Ser552) antigen in response to application of niclosamide. Consequently, the expression of characteristic β-catenin target genes appeared significantly decreased in the pGBMs (12, 27, 28). Thus, niclosamide revealed a pleiotropic mode of action in pGBMs, inhibiting major cancer-driving signaling cascades simultaneously.

(75) NFKBIA Predicts Synergistic Effects of Niclosamide and Temozolomide.

(76) In contrast to the consistent inhibitory impact on the Notch-, mTOR-, and Wnt-/β-catenin-mediated pathways, niclosamide exhibited a variable effect on NF-κB-signaling in pGBMs. Among the four cases used for mode of action analysis, Western blots revealed for only two (#046 and #078) a pathway inhibition as indicated by decreased levels of the phospho-p65-NFκB protein (FIG. 5A). Subsequent genomic analysis (see methods) demonstrated for these two cases a heterozygous deletion of the NFKBIA locus (NFKBIA.sup.+/−) at 14q13 that encodes for a major repressor of intracellular NF-κB-signaling. As recent work had suggested that deletion and low expression of NFKBIA were associated with unfavorable clinical outcome in GBM patients (16), additional NFKBIA.sup.+/− and NFKBIA.sup.+/+ pGBMs and standard GBM models were identified from the cohort for further investigation. These samples (pGBMs: n=3 for each group; GBMs: n=2″ for each group) revealed baseline expression levels that coincided with the respective genomic status of NFKBIA (inset FIG. 5B; insets of FIGS. 14A and 15A). However, upon exposure to niclosamide, NFKBIA.sup.+/− pGBMs as well as standard GBM models under dm conditions were shown to strongly up-regulate their NFKBIA expression (FIGS. 5B, 14A and 15A). As similar responses were not observed in NFKBIA.sup.+/+ samples, the variable effects of niclosamide on NF-κB-signaling in pGBMs could be explained by a differential stimulation of NFKBIA expression in the NFKBIA.sup.+/− samples. This observation intrigued, as it is known that down-regulation of NFKBIA in GBM cells is associated with a lack of response to alkylating agents, e.g. the standard GBM chemotherapeutic TMZ (29). On the other hand, it is known that the inhibition of NF-κB alone may not severely affect most solid tumors, rather that it may help to prevent resistance of cancer cells to chemotherapy (30, 31). Thus, a potential benefit that a combined application of niclosamide and TMZ might have in this setting was investigated.

(77) TABLE-US-00004 TABLE 2 Combinatorial index evaluation for treatment with niclosamide plus TMZ in pGBMs indicates synergistic activity. Expected Observed NFKBIA survival survival Combinatorial Case # status proportion proportion index 046 +/− 0.562 0.319 0.567 078 +/− 0.375 0.283 0.754 118 +/− 0.806 0.392 0.486 138 +/− 0.417 0.261 0.625 66 +/+ 0.705 0.720 0.979 81 +/+ 0.406 0.363 0.886 106 +/+ 0.819 0.731 0.901

(78) The combinatorial indices (CIs) for niclosamide and TMZ were expressed as ratio of observed vs. expected cell viability. Expected results were calculated as the proportion of viable cells following treatment with (only) 1 μM niclosamide multiplied by the proportion of cells following treatment with (only) TMZ. (CI<1:synergy, CI=1:additive; CI>1:antagonism). NFKBIA status +/− (heterozygous deletion), +/+ (not deleted).

(79) Experiments employed a cohort of 7 pGBMs (n=4, NFKBIA.sup.+/−; n=3 NFKBIA.sup.+/+; Table 2) and four standard GBM cell lines. All of these samples showed an unmethylated MGMT promoter status, a condition that indicates poor clinical responses to standard radio/TMZ-chemotherapy (2, 16). Combinatorial index analysis of niclosamide was conducted in the presence of 50 μM TMZ. Combinatorial indizes of pGBMs and GBMs are given in FIGS. 14B and 15B. The concentration of TMZ was chosen based on the reported plasma peak levels in patients (32), which in many previous studies had shown to impact very little on the viability of glioma cells maintained in vitro (10, 33, 34). Similarly, we here observed that application of 50 μM TMZ to the pGBMs reduced their metabolic activity of pGBMs to only 94±4% of control levels (n=7, triplicate analysis; data not shown) and the activity of GBMs to only 89±8%. In combination with niclosamide, however, TMZ showed a particular effect on NFKBIA.sup.+/− pGBM as well as GBM samples. Their dose-response curves showed a remarkable left-shift indicating stronger inhibitory activity of combined niclosamide/TMZ application compared to the NFKBIA.sup.+/+ samples (FIGS. 5C, 14C and 15C). Calculation of the combinatorial index (CI; (35)) suggested for NFKBIA.sup.+/+ pGBMs approximately additive effects (CI=0.92±0.05), and a clear synergistic activity of niclosamide/TMZ in all NFKBIA.sup.+/− samples (CI=0.61±0.11) (Table 1).

(80) Similar results were obtained for standard cell lines (see FIG. 15).

(81) To directly demonstrate the involvement of NFκB in the observed synergistic activity, we performed control studies using the NFκB activator TNFα (Peprotech). Application of TNFα activated NFκB in pGBMs, and in accordance to our hypothesis, counteracted synergy effects in NFKBIA+/− genotypes (FIG. 16).

(82) These data suggested that niclosamide augments the anticancer effects of TMZ, the current GBM standard chemotherapeutic. Based on determining the genomic status of NFKBIA in GBM cells, a synergistic effect of niclosamide and TMZ may furthermore become predictable.

(83) Discussion

(84) The combined data of this study indicate that the pleiotropic anticancer effects of niclosamide are ideally suited to inhibit pGBMs from a variety of key clinical constellations. Cytostatic, cytotoxic, and anti-migratory effects are elicited, and the stem-like/tumorigenic cell fraction among pGBMs is strongly reduced. Thus, the issues of inter- and intra-patient tumor heterogeneity as well as the invasive nature of glial tumor cells that complicate any therapeutic approach in GBM (36) may become accessible by one drug. Several unique features of this compound nevertheless warrant future investigation for translation to brain tumor therapy. Niclosamide is a common, by many regulatory agencies approved antihelminthic that has not yet been considered for the treatment of brain tumors. It is a salicylanilide, a chemical derivative of salicylic acid that was introduced by Bayer as a molluscide in 1959. For medical use in animals and humans oral application is preferred causing only little toxicity. Studies in animals suggested no mutagenic, oncogenic, or embryotoxic activity and no cumulative effects. Its rate of absorption from the intestinal tract was estimated at 33% (for cumulative review, see (37)). The in vitro data suggest that niclosamide inhibits GBM core and periphery cells from primary disease, from disease recurrence, from MGMT promoter methylated and unmethylated, as well as from NFKBIA.sup.+/+ and NFKBIA.sup.+/− GBM samples in concentration ranges that only marginally affect human non-malignant neural (control) cells.

(85) For mode of action analysis, this study hays mostly relied on evidence from previous work in the fields of hemato-oncology, colon, and breast cancer research (12, 13, 22-24). The findings of the present study have confirmed the results of these studies, exposing pleitropic inhibitory effects of niclosamide on Wnt-/β-catenin-, Notch-, and mTor-signaling, which are known to play a pivotal role for GBM malignancy as well (16, 38-40). Of particular interest for future clinical application is niclosamide's hitherto unrecognized attribute to stimulate NFKBIA expression in NFKBIA.sup.+/− cancer genotypes. While the responsible mechanism remains yet unclear, the resulting inhibition of NF-κB activity could be used to overcome resistance to alkylating agents such as TMZ (29). The here demonstrated synergistic activity of niclosamide with the current standard GBM chemotherapeutic TMZ in NFKBIA.sup.+/− pGBMs provides evidence for this assumption. It is highly probable that other cancer entities presenting with specific single-nucleotide polymorphisms and haplotypes of NFKBIA, e.g. Hodgkin's lymphoma, colorectal cancer, melanoma, hepatocellular carcinoma, breast cancer, and multiple myeloma (for collective references, see (16)) might profit from combining niclosamide with alkylating chemotherapeutic regimens.

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