ERK inhibitors for use in treating spinal muscular atrophy

Abstract

The present invention relates to a method for treating spinal muscular atrophy and other related neuromuscular disorders in a subject in need thereof, said method comprising administering a therapeutically effective amount of an ERK inhibitor, such as Selumetinib to said subject.

Claims

1. A method of treating spinal muscular atrophy in a subject in need thereof comprising: administering to the subject an effective amount of an ERK inhibitor; wherein said ERK inhibitor is a selective MEK inhibitor; and wherein said spinal muscular atrophy is associated with deficiency in a Survival-Motor-Neuron gene, said deficiency resulting in loss of motor function.

2. The method according to claim 1, wherein said deficiency resulting in loss of motor function is a genetic mutation in Survival-Motor-Neuron 1 gene.

3. The method according to claim 1, wherein said ERK inhibitor is a MEK½ inhibitor.

4. The method according to claim 3, wherein said MEK½ inhibitor is selected from selumetinib, U0126, PD98059, PD0325901, AZD8330, CI-1040 and PD318088.

5. The method according to claim 3, wherein said ERK inhibitor is selected from the group consisting of selumetinib and its derivatives and pharmaceutically acceptable salts thereof.

6. The method according to claim 1, wherein said ERK inhibitor is selected from the group consisting of nucleic acid molecule, siRNA, shRNA and anti-sense oligonucleotide; said nucleic acid molecule reducing the expression of MEK1, MEK2, ERK1 or ERK2.

7. The method according to claim 1, wherein said ERK inhibitor is administered orally to a subject in an amount effective to treat spinal muscular atrophy.

8. A method of treating spinal muscular atrophy in a subject in need thereof comprising: administering to the subject an effective amount of at least one agent selected from selumetinib, U0126, PD98059, PD0325901, AZD8330, CI-1040 and PD318088.

9. The method of claim 3 wherein, the ERK inhibitor has an IC50 of at least 1μM, or less.

10. The method according to claim 3, wherein said ERK inhibitor is selected from the group consisting of U0126 and its derivatives and pharmaceutically acceptable salts thereof.

11. The method according to claim 8, comprising administering to the subject an effective amount of U0126.

12. The method according to claim 8, comprising administering to the subject an effective amount of selumetinib.

Description

FIGURE LEGENDS

(1) FIG. 1:

(2) A. Smn Promoter site 1 sequence (SEQ ID NO: 6)

(3) B. Smn Promoter site 2 sequence (SEQ ID NO: 7)

(4) C and D. Western blot analysis and quantification of Elk-1 protein phosphorylation in the ventral lumbar spinal cord of vehicle- and NMDA-treated control mice at 6 days of age, of vehicle SMA-like mice at 2 days of age and NMDA-treated SMA-like mice at 6 days of age (n=2). Error bars indicate SEM. (*, p<0.05).

(5) E and F. ChIP analysis of Phospho-Elk-1 in the ventral lumbar spinal cord of vehicle- and NMDA-treated control mice at 6 days of age, of vehicle SMA-like mice at 2 days of age and NMDA-treated SMA-like mice at 6 days of age (n=9). Quantitative real time PCR was performed to detect SMN2 promoter site 1 (E) and site 2 (F). Error bars indicate SEM. (*, p<0.05; **, p<0.01; ***, p<0.001).

(6) G and H. ChIP analysis of Phospho-CREB in the ventral lumbar spinal cord of vehicle- and NMDA-treated control mice at 6 days of age, of vehicle SMA-like mice at 2 days of age and NMDA-treated SMA-like mice at 6 days of age (n=9). Quantitative real time PCR was performed to detect SMN2 promoter site 1 (G) and site 2 (H). Error bars indicate SEM. (**, p<0.01; ***, p<0.001).

(7) I and J. ChIP analysis of Histone H3 acetylation in the ventral lumbar spinal cord of vehicle- and NMDA-treated control mice at 6 days of age, of vehicle SMA-like mice at 2 days of age and NMDA-treated SMA-like mice at 6 days of age (n=9). Quantitative real time PCR was performed to detect SMN2 promoter site 1 (1) and site 2 (J). Error bars indicate SEM. (*, p<0.05; **, p<0.01; ***, p<0.001).

(8) K and L. ChIP analysis of Histone H4 acetylation in the ventral lumbar spinal cord of vehicle- and NMDA-treated control mice at 6 days of age, of vehicle SMA-like mice at 2 days of age and NMDA-treated SMA-like mice at 6 days of age (n=9). Quantitative real time PCR was performed to detect SMN2 promoter site 1 (K) and site 2 (L). Error bars indicate SEM. (*, p<0.05; *** , p<0.001).

(9) FIG. 2:

(10) A and B. Western blot analysis and quantification of Elk-1 protein phosphorylation in the ventral lumbar spinal cord of vehicle- and NMDA-treated control mice at 6 days of age, of vehicle SMA-like mice at 2 days of age and U0126-treated SMA-like mice at 6 days of age (n=3). Error bars indicate SEM. (*, p<0.05).

(11) C and D. Western blot analysis and quantification of SMN protein expression in the ventral lumbar spinal cord of vehicle- and NMDA-treated control mice at 6 days of age, vehicle SMA-like mice at 2 days of age and U0126-treated SMA-like mice at 6 days of age (n=2). Error bars indicate SEM. (*, p<0.05).

(12) E. Quantitative analysis of the number of GEMS per motor neuron in the ventral lumbar spinal cord of vehicle- and NMDA-treated control mice at 6 days of age, of vehicle SMA-like mice at 2 days of age and U0126-treated SMA-like mice at 6 days of age (n=2).

(13) F and G. Western blot analysis and quantification of AKT protein phosphorylation in the ventral lumbar spinal cord of vehicle- and NMDA-treated control mice at 6 days of age, of vehicle SMA-like mice at 2 days of age and U0126-treated SMA-like mice at 6 days of age (n=3). Error bars indicate SEM. (*, p<0.05).

(14) H and I. Western blot analysis and quantification of CREB protein phosphorylation in the ventral lumbar spinal cord of vehicle- and NMDA-treated control mice at 6 days of age, of vehicle SMA-like mice at 2 days of age and U0126-treated SMA-like mice at 6 days of age (n=3). Error bars indicate SEM. (*, p<0.05).

(15) J and K. Western blot analysis and quantification of SMN protein expression in vehicle and U0126-treated human SMA cultured myotubes and myoblasts (n=2). Error bars indicate SEM. (*, p<0.05).

(16) FIG. 3:

(17) A and B. Western blot analysis and quantification of ERK protein phosphorylation in the control and SMA spinal cord explants in presence or not of NMDA and of the CREB inhibitor KG501 (n=2). Error bars indicate SEM. (*, p<0.05).

(18) C and D. Western blot analysis and quantification of Elk-1 protein phosphorylation in the control and SMA spinal cord explants in presence or not of NMDA and of the CREB inhibitor KG501 (n=2). Error bars indicate SEM. (*, p<0.05).

(19) E and F. Western blot analysis and quantification of AKT protein phosphorylation in the control and SMA spinal cord explants in presence or not of NMDA and of the CREB inhibitor KG501 (n=3). Error bars indicate SEM. (*, p<0.05).

(20) G and H. Western blot analysis and quantification of SMN protein expression in the control and SMA spinal cord explants in presence or not of NMDA and of the CREB inhibitor KG501 (n=2). Error bars indicate SEM. (*, p<0.05).

(21) FIG. 4:

(22) A. Life span of U0126-treated (n=15) compared to vehicle-treated SMA-like mice (n=10).

(23) B. Weight curve in U0126-treated (n=15) and vehicle-treated SMA-like mice (n=10) compared to U0126-treated (n=15) and vehicle-treated control (n=15).

(24) C-F. Immunodetection of ChAT-positive motor-neurons in the lumbar spinal cord (L1-L5) of 6 days of age vehicle- (C) and U0126-treated control mice (D), and 2 days of age vehicle- (E) and 6 days of age U0126-treated SMA-like mice (F).

(25) G and H. Quantitative analysis of the number (G) and the cell body area (H) of motor neurons per ventral horn in the ventral lumbar spinal cord of vehicle- and U0126-treated control mice at 6 days of age, of vehicle SMA-like mice at 2 days of age and U0126-treated SMA-like mice at 6 days of age (n=3). Error bars indicate SEM. (*, p<0.05).

(26) I and J. Western blot analysis and quantification of SMN protein expression in the ventral lumbar spinal cord of vehicle- and AZD6244-treated control mice at 6 days of age, vehicle SMA-like mice at 2 days of age and AZD6244-treated SMA-like mice at 6 days of age (n=3). Error bars indicate SEM. (*, p<0.05).

(27) K and L. Western blot analysis and quantification of AKT protein phosphorylation in the ventral lumbar spinal cord of vehicle- and AZD6244-treated control mice at 6 days of age, of vehicle SMA-like mice at 2 days of age and AZD6244-treated SMA-like mice at 6 days of age (n=3). Error bars indicate SEM. (*, p<0.05).

(28) M. Life span of AZD6244-treated (n=10) compared to vehicle-treated SMA-like mice (n=10).

(29) N. Weight curve in AZD6244-treated (n=10) and vehicle-treated SMA-like mice (n=10) compared to AZD6244-treated (n=10) and vehicle-treated control (n=10).

EXAMPLES

(30) Materials and Methods

(31) Mice and Treatments

(32) The knockout-transgenic SMA-like mice (Smn.sup.−/−, SMN2.sup.+/+) were purchased from the Jackson Laboratory (Bar Harbor, Me.) and genotyped as previously described.sup.15. Vehicle-treated group, NMDA-treated group, U0126-treated group and AZD6244-treated group of type 1 SMA-like mice were randomly constituted in a blind systematic manner to minimize bias. The control mice were heterozygous knock-out for Smn with the human SMN2 transgene (Smn.sup.−/+, to SMN2.sup.+/+).

(33) In order to evaluate phospho-Elk-1 and phospho-CREB role on Smn2 promoter, P1 neonatal control and SMA-like mice, were either injected intrathecally with 5 pmol of N-methyl-D-aspartic Acid 100 μM (NMDA, Sigma, Saint Quentin Fallavier, France) in 0.5 μl/g of 0.9% NaCl dyed in blue Evans per gram. These mice were compared to control and SMA-like mice injected from P1 with 0.5 μl/g of 0.9% NaCl dyed in blue Evans.

(34) In order to evaluate the benefits of phospho-ERK inhibition, P1 neonatal control and type 1 SMA-like mice were injected either intrathecally with 0.5 pmol of 1,4-Diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)butadiene monoethanolate 10 μM (U0126, Sigma, Saint Quentin Fallavier, France) in 0.5 μl/g of 0.9% NaCl 1% DMSO dyed in blue Evans per gram, or per os with 0.5 pmol of Selumetinib 10 μM (AZD6244, Selleck chemicals, Houston, Tex.) in 2 μl/g of 0.9% NaCl 1% DMSO. These mice were compared to control and SMA-like mice either injected from P1 with 0.5 μl/g of 0.9% NaCl 1% DMSO dyed in blue Evans or orally treated with 2 μl/g of 0.9% NaCl 1% DMSO. Body weight and life span recordings were performed every day until the death of the animal. The animals were considered as dead when mice were no longer able to stand up 20 sec after having been placed on their sides.

(35) The care and treatment of animals followed the national authority (Ministère de la Recherche et de la Technologie, France) guidelines for the detention, use and the ethical treatment of laboratory animals.

(36) Mouse Cell Cultures and Treatments

(37) Co-cultures of spinal cord explants (around 1 mm.sup.3) and muscle cells were performed as described by Kobayashi et al..sup.25 with the following modifications. Spinal cord explants were obtained from control and severe SMA embryonic mice. Explants from the whole transverse slices of 10.5 days-old mice embryo spinal cords including dorsal root ganglia (DRG) were placed on the muscle monolayer. DRG are essential to ensure a good innervation ratio.sup.25. The muscle culture was established through the differentiation of the wild-type muscle cell line C2C12. Myoblast cells were cultured on 35 mm petri dish at 37° C. in 5% CO.sub.2 in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 2 mM glutamine, 20% fetal bovin serum, 2% penicillin/streptomycin (5000 U). All the culture medium reagents were purchased from Invitrogen Life Technologies (Cergy-Pontoise, France). Confluent myoblasts were differentiated into myotubes in DMEM supplemented with 2 mM glutamine, 5% horse serum, 2% penicillin/streptomycin (5000 U) (Differentiation medium, DM). After 5-7 days in DM, spinal cord explants were added on the cultured contracting muscle cells. After co-culture with spinal cord, the culture was kept in DM. All co-cultures were fed three times a week and examined daily by phase-contrast inverted microscopy to check the appearance of the innervation.

(38) Stimulation of the NMDARs was achieved by exposing cells to 100 μM NMDA, as previously described.sup.14. To evaluate the CREB dependency, KG-501 (10 μM, Sigma) was added to the culture. After 5 days of treatment, explants were mechanically removed from the muscle layer, and proteins were purified and analyzed by western blot as described below.

(39) Human Primary Culture of Myogenic Precursor Cells from SMA Patients Biopsies

(40) Muscle biopsies were obtained from the BTR (Bank of Tissues for Research, a partner in the EU network EuroBioBank) in accordance with European recommendations and French legislation. Satellite cells were isolated from biopsies and cultivated as described previously.sup.26 in growth medium consisting of 1 vol 199 Medium/4 vol DMEM (Invitrogen Life Technologies) supplemented with 20% fetal bovin serum (Invitrogen Life Technologies), 2.5 ng/ml hepatocyte growth factor (HGF) (Invitrogen Life Technologies), and 50 μg/ml Gentamycin (Invitrogen Life Technologies). Further expansion was made in growth medium without HGF. The myogenic purity of the populations was monitored by immunocytochemistry using desmin as a marker. Differentiation was induced at confluence by replacing the growth medium with DMEM supplemented with 4% horse serum and 50 μg/ml of gentamycin (Sigma). Specific blockade of MEK phosphorylation was achieved during 5 days using 10 μM of U0126 (Sigma).

(41) Histological and Immunohistochemical Analysis

(42) Spinal cords were dissected and incubated overnight in 4% PFA PBS solution, and washed twice for 2 h with PBS. The lumbar spinal cords (L1 to L5) were embedded in 4% Agarose solution in sterilized water for 30 min at 4° C. 50 μm sections were then performed using a vibratome on the whole length of the sample. One out of every five sections was processed for immunohistochemical analysis. Tissue sections were incubated for 1 h at room temperature in a blocking solution (10% normal donkey serum with 0.5% Triton X-100 and 1% Tween in Tris Buffer Solution (TBS)). Motor neuron and Gemini of coiled bodies immunodetection were performed using a polyclonal goat anti-choline acetyltransferase (ChAT) primary antibody (1:400; Chemicon, Inc., Temecula, Calif.) and a monoclonal mouse anti-SMN primary antibody (1:200; BD Transduction Laboratories, Lexington, Ky.) for 4 days at 4° C. in 3.5% donkey serum with 0.1% Tween TBS. Sections were washed between each subsequent step with 0.1% Tween in TBS. Sections were subsequently incubated with polyclonal C.sub.y™3 conjugated Donkey anti-Goat antibodies (1:400; Jackson ImmunoResearch, West Grove, Pa.) and polyclonal Cy™2-conjugated Donkey anti-Mouse antibodies (1:400; Jackson ImmunoResearch) for 1 h at room temperature in 3.5% donkey serum with 0.1% Tween TBS. The sections were washed three times for 10 min in 0.1% Tween TBS and mounted in Fluoromount G™ (SouthernBiotech, Birmingham, Ala.) mounting medium. The staining specificity was checked in control incubations performed in the absence of the primary antibody.

(43) All counts were performed using ImageJ software v1.37 (National Institutes of Health, Bethesda, Md.). Color images were tinted using Image Pro-Plus software, where identical brightness, contrast, and color balance adjustments were applied to all groups.

(44) Microscopy

(45) All immunofluorescence images were collected with a CCD camera (QImaging Retiga 2000R Fast, Cooled Mono 12 bit) mounted on Olympus microscope (BX51) using the Image Pro-Plus v6.0 software (MediaCybernetics Inc., Bethesda, Md.) with ×40 (4× Olympus objective UPlan FL N 0.13), 100 (10× Olympus objective UPlan FL N 0.3), 200 (20× Olympus objective FL N 0.5), 400 (40× Olympus objective UPlan FL N 0.75), 600 (60× Olympus objective UPlanS Apo 1.35 oil) and 1000 (100× Olympus objective UPlanS Apo 1.4 oil) magnifications.

(46) Protein and Western Blot Analysis

(47) Ventral lumbar spinal cord samples (2 to 5 mg) were homogenized in 100 μl/5 mg tissues of ice-cold RIPA buffer (50 mM Tris HCl pH=8.0, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP40, 5 mM EDTA pH 8.0, 2 mM PMSF (phenyl-methylsulfonyl fluoride, Sigma-Aldrich), 50 μg/ml leupeptin, 50 μg/ml pepstatin A and 50 μg/ml aprotinin). Protein concentration of the clarified homogenates (4° C., 15 min, 13,500 rev.Math.min-1) was determined on all samples using the Bradford protein assay (Biorad Laboratories, CA). 10 μg protein samples for SMN analysis and 30 μg samples for other analysis of each homogenate were submitted to 12.5% SDS-PAGE electrophoresis (1.5 M Tris pH 8.3, 12.5% Acrylamide, 0.07% Bis, 0.1% SDS, 0.05% APS, 0.06% TEMED). The separated proteins were transferred on PVDF membranes (Biorad) according to Towbin et al..sup.27. Equal loading of samples was checked by Ponceau dye staining of the transferred gels. Western blot analysis was performed on membranes overnight at 4° C. in 4% BSA, 0.05% TWEEN 20, TBS pH 7.4. Each of the following primary antibodies, including monoclonal mouse anti-SMN (1:5,000; Santa Cruz Biotechnology, Inc.), polyclonal rabbit anti-Ser473 phospho-AKT (1:1000; Cell signaling Technology, Inc, Boston, Mass.), polyclonal rabbit anti phospho-ERK1/2 (1:500; Cell Signaling, Inc.), polyclonal rabbit anti-Ser133 phospho-CREB (1:1,000; Millipore), monoclonal mouse anti-Ser183 phospho-Elk-1 (1:1,000; Santa Cruz Biotechnology, Inc) was incubated overnight at 4° C. in the above blocking medium. Membranes were rinsed in 0.1% TWEEN 20 in TBS for 3×10 min at room temperature and then incubated in horseradish peroxydase-conjugated Goat secondary antibody directed against Mouse Immunoglobulins (1:5,000; Biorad Laboratories, CA) and in horseradish peroxydase-conjugated Goat secondary antibody directed against Rabbit Immunoglobulins (1:10,000; Jackson ImmunoResearch) in 0.1% TWEEN 20 in TBS for 1 h at room temperature. Bound antibody complexes were developed using the ECL system (Amersham Biotech., Saclay, France) and exposed to hyperfilm ECL-plus X ray film (Amersham Biotech.).

(48) In some instances, membranes were stripped after immunoblotting with phospho-AKT, phospho-ERK1/2, phospho-CREB and phospho-Elk-1 by incubation in stripping buffer (100 mM β-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl, pH 6.7) for 30 min at 55° C. with agitation, and membranes were then blocked and reprobed with polyclonal rabbit anti-AKT (1:1,000; Cell Signaling, Inc.), polyclonal rabbit anti-ERK1/2 (1:500; Cell Signaling, Inc.), polyclonal rabbit anti-CREB (1:1,000; Millipore), monoclonal mouse anti-Elk-1 (1:1,000; Santa Cruz Biotechnology, Inc.) and monoclonal mouse anti-glyceraldehyde-3-phosphate dehydrogenase antibody (GAPDH) (1:5,000; Chemicon). Films were quantified with ImageJ v1.37 (National Institutes of Health, Bethesda, Md.) and the results reported as means±SEM.

(49) Chromatin Immunoprecipitation

(50) Ventral lumbar Spinal Cord samples were chopped into small pieces with a scalpel and were fixed for 15 min with 1% formaldehyde. Tissues were washed 3 times in cold PBS containing protease inhibitors (2 mM PMSF, 50 μg/ml leupeptin, 50 μg/ml pepstatin A and 50 μg/ml aprotinin) and collected by centrifugation. Cell pellet were resuspended and incubated on ice for 10 min in 300 μl of lysis buffer (5 mM piperazine-N,N′-bis(2-ethanosulfonic acid) (PIPES) pH 8.0, 85 mM KCL, 0.5% NP-40) and protease inhibitors. Cells were pelleted by centrifugation and resuspended in 300 μl of 1% SDS, 10 mM EDTA and 50 mM Tris-HCL (pH 8.0) containing protease inhibitors. After incubation on ice for 10 min, cells were sonicated 6 times for 30 sec using Bioruptor (Diagenode, Philadelphia, Pa.). Lysates were cleared by centrifugation and DNA concentration was determined using a nanodrop spectrophotometer. ChIP-Adembeads (Ademtech SA, Pessac, France) were incubated for 15 min at room temperature with blocking buffer on a rotating wheel. Beads were resuspended in 125 μl of ChIP Dilution buffer (0.01% SDS, 1% Triton X100, 1.2 mM EDTA, 16.7 mM Tris-HCl (pH 8.1)), and after a 1 h incubation, equal amounts of DNA diluted 10 times in dilution buffer were added. DNA was incubated overnight at 4° C. on a rotating wheel with 1 μg of the following antibodies: polyclonal rabbit anti-Ser133 phospho-CREB (Millipore), monoclonal mouse anti-Ser183 phospho-Elk-1 (Santa Cruz Biotechnology, Inc), polyclonal rabbit anti-acetyl-Histone H3 Lys9 (Millipore) and polyclonal rabbit-acetyl-Histone H4 Lys 8 (Upstate Biotechnology, Inc., Lake Placid, N.Y.). Beads were washed sequentially in TSE (0.1% SDS, 1% Triton X100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1) with 150 mM NaCl, TSE with 500 mM NaCl, buffer A (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-Hcl (pH 8.1), and 2 times with tris-EDTA and then eluted with 200 μl 1% SDS and 0.1 M NaHCO3. Cross-links were reversed by heating at 65° C. for 4 h after adding NaCl to 200 mM final concentration. After treatment with proteinase K (50 μg/ml) for 1 h at 37° C., DNA was purified using Geneclean Turbo Kit (Q-Biogene, MP Biomedicals, Illkirch, France). Real time PCR analysis of inputs or immunoprecipitated DNAs was performed.

(51) Quantitative Real Time PCR Analysis

(52) Quantitative real time PCR was performed with standard protocols using SYBR®Green ROX Mix (ABgene, Courtaboeuf, France) as a fluorescent detection dye in ABI PRISM® 7000 in a final volume of 10 μl which also contains 300 nM of primers (Operon, Cologne, Germany)

(53) The relative amounts of DNA in samples were determined on the basis of the threshold cycle for each PCR product (Ct).

(54) Statistical Analysis

(55) All values are displayed as means and standard error of the mean (SEM) within each group (Systat v 8.0, SPSS Inc., Chicago, Ill.). Statistical analysis was performed and comparison between groups were done using ANOVA and post-hoc test LSD. Survival analysis was performed using Kaplan-Meier analysis.

(56) Results

(57) The mode of action of therapeutic molecules for the treatment of SMA may include the increase of SMN expression particularly in motor neurons through activating the SMN2 promoter, increasing exon-7 inclusion in SMN transcripts, or extending the half-life of SMN mRNA or protein. It may also include the promotion of motor neuron survival through the activation of anti-apoptotic pathways. Over the years, a number of groups have identified SMN2 gene-inducing compounds using cultured fibroblasts derived from SMA patients, and which benefits were often further tested in vivo in SMA mouse models.sup.5. Among those, SMN inducer compounds were identified based on their supposed ability to increase general gene expression, such as histone deacetylase inhibitors.sup.6, 7, 8, or by high throughput screenings, such as quinazoline derivatives.sup.9, 10. Unfortunately, to date, many of these compounds were disappointing in clinical trials with no substantial clinical benefit demonstrated.sup.11, 12, 13. Ultimately, none of these compounds provide efficient anti-apoptotic potential for motor neurons.

(58) One promising, as yet unexplored, therapeutic development for SMA could involve the pharmacological correction of molecular mechanisms, specifically altered in SMA neuromuscular system, potentially capable of modulating either SMN expression, or motor-neuron survival or both. In this context, the inventors paid further attention to the activation pattern of the AKT/CREB signalling pathway. Constitutively down-regulated in mouse SMA spinal cord, the AKT/CREB pathway is able to remarkably alleviate SMA symptoms in mice as long as it is reactivated.sup.14. In very severe SMA-like mice.sup.15, the reactivation of AKT/CREB pathway by NMDA resulted in an increased in the total amount of SMN transcripts in the SMA spinal cord without modifying its splicing pattern suggesting a SMN2 gene regulation at the transcriptional level.sup.14. Furthermore, considered as a common and powerful antiapoptotic pathway.sup.16 notably for spinal motor neurons.sup.17, the AKT/CREB pathway activation likely represents an important clue for motor neuron resistance to cell death in SMA spinal cord. Thus, identifying therapeutic agents that could lead to the reactivation of the AKT/CREB pathway in SMA spinal cord is of a paramount importance.

(59) Interestingly, the activation profile of another major intracellular signaling pathway in neurons.sup.18, namely the ERK1/2 signaling pathway, was in opposite contrast to that of AKT in SMA spinal cord. Constitutively over-activated in the spinal cord of two different severe mouse models of SMA, characterized by a weak SMN expression, ERK1/2 was inhibited when AKT is reactivated and this change in ERK/AKT activation balance correlated with an increase in SMN expression.sup.14. These data raise important questions regarding 1) the respective roles of ERK and AKT pathways in modulating SMN2 gene expression and 2) the potential cross relationships in the activation profile of these two signaling pathways in SMA spinal cord.

(60) Interestingly, the sequence analysis of the human SMN promoter (GenBank accession AF187725) revealed that several CREB binding sites are flanked by putative response elements for transcription factors that are direct target of ERK.sup.19, 20, 21, namely the transcription factors of the ETS family Elk-1 (FIG. 1a-b). Yet, the CREB binding site 2 (+244 to +248 bp), considered as a positive regulator of SMN gene expression.sup.22, contains also putative response elements for Elk-1 (+356 to +429 bp), including a binding site for the Serum Response Factor (SRF). We identified an additional putative CRE site, which we named site 1, that includes two putative CREB binding sites (−2572 to −2569 by and −2525 to −2522 bp) also containing putative SRF binding site (−2556 to −2548 bp). ChIP experiments showed that the two transcription factors effectively bound to the two CRE sites but with an efficacy that correlated to their levels of activation. Elk-1, over-activated in the spinal cord of type 1 SMA-like mice (FIG. 1c-d), as expected for a direct target of ERK, displayed an increased binding on the two CRE sites in SMA spinal cord compared to controls (FIG. 1e-f). In contrast, ChIP experiments revealed a dramatic decrease in the binding of CREB to the two CRE sites (FIG. 1g-h). Interestingly, the ratio of CREB and Elk-1 binding on the CRE sites was completely reversed in SMA spinal cords when SMN expression is promoted i.e. after a direct NMDA-receptor activation (FIG. 1e-h). To gain further insight into the potential role of Elk-1 in the control of SMN2 gene expression, we analysed by ChIP the acetylation profiles of histones H3 and H4 in the two CRE sites in the spinal cord of SMA and control mice. Elk-1 recruitment to the two CRE sites correlated to a marked decrease of H3 and H4 acetylation, compared to controls, whereas CREB recruitment induced a marked increase of H3 and H4 acetylation (FIG. 1i-l). Taken together, these results suggested that the ERK/Elk-1 pathway activation resulted in the repression of SMN2 gene expression in SMA spinal cord, contrasting with the results found in a non SMA neuronal context.sup.23.

(61) Therefore, it could be speculated that inhibiting the ERK pathway would abolish the Elk-1-induced inhibition of SMN2 gene transcription and would lead consequently to an increase of SMN expression in SMA spinal cord. To test this hypothesis, a population of type 1 SMA-like mice was treated daily from birth by intrathecal injection of U0126, a specific MAPK Kinase (MEK) inhibitor. The in vivo ERK inhibition, that induced a marked decrease of Elk-1 activation in SMA (FIG. 2a-b), resulted in a remarkable increase in SMN protein concentration in the spinal cord of type 1 SMA-like mice (FIG. 2c-d). These data are further emphasized by the significant increase of gemini of coiled bodies (gems) in the motor-neuron nuclei of U0126-treated SMA-like mice (data not shown and FIG. 2e). Unexpectedly, the ERK inhibition resulted in a significant activation of AKT (FIG. 2f-g) and CREB (FIG. 2h-i) in SMA spinal cord, likely acting synergistically with the Elk-1 inhibition to increase SMN expression. Consistent with our findings in SMA-like mice, the inhibition of the ERK pathway by U0126 in myotube culture of paravertebral muscles from type 2 SMA patient resulted in a significant increase of SMN expression (FIG. 2j-k).

(62) In order to substantiate this crosstalk hypothesis at the level of the kinases ERK and AKT in the signaling cascades, we tested in vitro the effects of CREB inhibition on the ERK/Elk-1 pathway activation profile in a SMA context in which the AKT pathway was significantly activated i.e. following NMDA-receptor activation. We found that CREB inhibition resulted in the activation of ERK1/2 (FIG. 3a-b) and Elk-1 (FIG. 3c-d) as hypothesized. More surprisingly, the CREB inhibition resulted in a significant inhibition of the AKT (FIG. 3e-f), suggesting a negative feedback from the transcription factor to its activating kinase. The concomitant activation of the ERK/Elk-1 pathway and inhibition of the AKT/CREB pathway expectedly resulted in a significant decrease in SMN expression (FIG. 3g-h). Taken together, these data strongly suggest the existence of a dynamic equilibrium between ERK and AKT pathways in SMA spinal cord. This equilibrium could be displaced by reciprocal blockades, opening thus a promising way for reactivating the AKT/CREB pathway in SMA spinal cord.

(63) Finally, in vivo ERK inhibition resulted in a remarkable improvement in the phenotype and survival of severe SMA-like mice compared to vehicle-treated counterparts. The mean survival increased from 1.60±0.48 days for the vehicle-treated SMA-like mice to 4.13±1.07 days for the U0126-treated mice (FIG. 4a), representing a 2.5 fold increase in lifespan (p<0.01), which remains, to date, the best pharmacological treatment ever reported in this SMA mouse model. In addition, the U0126 treatment led to a significant and progressive increase in the body weight of SMA-like mice, until death (FIG. 4b). These benefits were associated with the significant increase in the number and the surface of motor-neuron in lumbar spinal cord of U0126-treated SMA-like mice compared to placebos (FIG. 4c-h).

(64) These results prompted us to test whether a pre-approved MEK inhibitor could provide the same effects of U0126 on SMN expression and severe SMA-like mouse lifespan. We chose to test a new drug, Selumetinib (AZD6244), a well known specific MEK inhibitor, which is currently in phase II clinical trial.sup.4, successfully tested in the Pediatric Preclinical Testing.sup.24. Expectedly, oral Selumetinib treatment reproduced the effects obtained with U0126, including an activation of SMN expression in the spinal cord of SMA mice (FIG. 4i-j), an activation of AKT (FIG. 4k-l) and a remarkable increase in the life span of SMA mice (FIG. 4m) associated with a progressive gain of body weight (FIG. 4n).

(65) Taken together, all these results indicate that the pharmacological inhibition of ERK pathway, notably through the use of Selumetinib, could be considered as an efficient treatment to alleviate SMA symptoms in patients.

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