WO2015179436A1

WO2015179436A1 - Inflammation therapy using mekk3 inhibitors or blocking peptides

Info

Publication number: WO2015179436A1
Application number: PCT/US2015/031626
Authority: WO
Inventors: Jorge MOSCAT-GUILLEN; Marie Teresa Diaz-Meco Conde
Original assignee: Sanford-Burnham Medical Research Institute
Priority date: 2014-05-19
Filing date: 2015-05-19
Publication date: 2015-11-26

Abstract

Provided herein are new therapeutic regimens for treatment of inflammation and cancer with a MEKK3 inhibitor or a blocking peptide, and methods of use thereof. In some embodiments, described herein are methods for treating obesity-induced inflammation in a subject, the method comprising administering to the subject a MEKK3 inhibitor. In some embodiments, the MEKK3 inhibitor blocks activation of JNK cascade. In some embodiments, the MEKK3 inhibitor further blocks M1 polarization of macrophages. In some embodiments, the administration of MEKK3 inhibitor is effective in preventing the onset of type 2 diabetes.

Description

INFLAMMATION THERAPY USING MEKK3 INHIBITORS OR BLOCKING PEPTIDES

CROSS-REFERENCE

[001] This application claims the benefit of U.S. Provisional Application No. : 61/994,996 filed May 19, 2014, which is incorporated by reference herein in its entirety.

SEQUENCE LISTING

[002] The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 14, 2015, is named 14-032-02PCT-42256_718_601_SequenceListing.txt and is 9,569 bytes in size.

BACKGROUND OF THE INVENTION

[003] There is a need for new therapeutic regimens to treat obesity-induced inflammation and cancer.

SUMMARY OF THE INVENTION

[004] In some embodiments, described herein are methods for treating obesity-induced inflammation in a subject, the method comprising administering to the subject a MEK 3 inhibitor. In some embodiments, the MEK 3 inhibitor is selected from a group consisting of

(AT9283),

Crizotinib (PF-02341066), (Hesperadin), (PD 166285),

(Cdkl/2 Inhibitor III), (PP121), osutinib (SKI-606),

IV (NU 6140),

Dasatinib (BMS-354825),

Dovitinib (1X1-258),

Sunitinib Malate (Sutent), and

[005] In some embodiments, the MEK 3 inhibitor is selected from a group consisting of

-02341066),

[006] In some embodiments, described herein are methods for treating obesity-induced inflammation in a subject, the method comprising administering to the subject a MEK 3 inhibitor, wherein the subject is exposed to a high fat diet. In some embodiments, the subject has an elevated expression level of NBR1 in macrophages. In some embodiments, the elevated expression level of NBR1 in macrophages is in macrophages which infiltrate adipose tissue. In some embodiments, the elevated level of NBR1 leads to activation of INK cascade. In some embodiments, the MEKK3 inhibitor blocks activation of INK cascade. In some embodiments, the MEKK3 inhibitor further blocks Ml polarization of macrophages.

[007] In some embodiments, described herein are methods for treating obesity-induced inflammation in a subject, the method comprising administering to the subject a MEKK3 inhibitor, wherein the subject has impaired glucose tolerance. In some embodiments, the subject has insulin resistance.

[008] In some embodiments, described herein are methods for treating obesity-induced inflammation in a subject, the method comprising administering to the subject a MEKK3 inhibitor, wherein the administration of MEKK3 inhibitor results in improved glucose tolerance. In some embodiments, the administration of MEKK3 inhibitor results in reduced insulin resistance.

[009] In some embodiments, described herein are methods for treating obesity-induced inflammation in a subject, the method comprising administering to the subject a MEKK3 inhibitor, wherein the subject is pre-diabetic. In some embodiments, the administration of MEKK3 inhibitor is effective in preventing the onset of type 2 diabetes.

[010] In some embodiments, described herein are methods for treating cancer in a subject, the method comprising administering to the subject a MEKK3 inhibitor. In some embodiments, the MEKK3 inhibitor is selected from a group consistin of

(PF-03814735), (AT9283),

din),

(PP121),

Bosutinib (SKI-606),

Cdk2 Inhibitor IV (NU 6140),

Dasatinib (BMS-354825),

Dovitinib TKI-258),

tinib Malate (Sutent), and

[Oil] In some embodiments, the MEK 3 inhibitor is selected from a group consisting

Crizotinib (PF-02341066), PP121 , and

[012] In some embodiments, described herein are methods for treating cancer in a subject, the method comprising administering to the subject a MEK 3 inhibitor, wherein the subject has an elevated level of p62. In some embodiments, the elevated level of p62 leads to activation of mTORCl, by phosphorylation of p62 by ρ38δ.

[013] In some embodiments, described herein are methods for treating cancer in a subject, the method comprising administering to the subject a MEK 3 inhibitor, wherein the administration of MEK 3 inhibitor blocks the activation of mTORCl by blocking the phosphorylation of p62 by ρ38δ.

[014] In some embodiments, described herein are methods for treating cancer in a subject, the method comprising administering to the subject a MEK 3 inhibitor, wherein the subject is suffering from prostate cancer. In some embodiments, the administration of MEK 3 leads to reduction in tumor volume.

[015] In some embodiments, described herein are methods for treating obesity-induced inflammation in a subject, the method comprising administering to the subject an agent that is capable of blocking the protein-protein interaction between NBR1 and a MEK kinase. In some embodiments, the subject is exposed to a high fat diet.

[016] In some embodiments, described herein are methods for treating obesity-induced inflammation in a subject, the method comprising administering to the subject an agent that is capable of blocking the protein-protein interaction between NBR1 and a MEK kinase, wherein the subject has an elevated expression level of NBR1 in macrophages. In some embodiments, the elevated expression level of NBRl in macrophages is in macrophages which infiltrate adipose tissue. In some embodiments, the elevated level of NBRl leads to activation of JNK cascade.

[017] In some embodiments, described herein are methods for treating obesity-induced inflammation in a subject, the method comprising administering to the subject an agent that is capable of blocking the protein-protein interaction between NBRl and a MEK kinase, wherein the administration of an agent that is capable of blocking the protein-protein interaction between NBRl and a MEK kinase blocks the activation of JNK cascade. In some embodiments, described herein are methods for treating obesity-induced inflammation in a subject, the method comprising administering to the subject an agent that is capable of blocking the protein-protein interaction between NBRl and a MEK kinase, wherein the subject has impaired glucose tolerance. In some embodiments, the subject has insulin resistance.

[018] In some embodiments, described herein are methods for treating obesity-induced inflammation in a subject, the method comprising administering to the subject an agent that is capable of blocking the protein-protein interaction between NBRl and a MEK kinase, wherein the administration of an agent that is capable of blocking the protein-protein interaction between NBRl and a MEK kinase results in improved glucose tolerance. In some embodiments, the administration of an agent that is capable of blocking the protein-protein interaction between NBRl and a MEK kinase results in reduced insulin resistance.

[019] In some embodiments, described herein are methods for treating obesity-induced inflammation in a subject, the method comprising administering to the subject an agent that is capable of blocking the protein-protein interaction between NBRl and a MEK kinase, wherein the subject is pre-diabetic.

[020] In some embodiments, described herein are methods for treating obesity-induced inflammation in a subject, the method comprising administering to the subject an agent that is capable of blocking the protein-protein interaction between NBRl and a MEK kinase, wherein the administration of an agent that is capable of blocking the protein-protein interaction between NBRl and a MEK kinase is effective in preventing the onset of type 2 diabetes. In some embodiments, the agent is a blocking peptide. In some embodiments, the blocking peptide comprises an amino acid sequence selected from SEQ ID NO. 1, SEQ ID NO. 3-SEQ ID NO. 9, and SEQ ID NO. 16-SEQ ID NO. 22.

[021] In some embodiments, described herein are methods for treating obesity-induced inflammation in a subject, the method comprising administering to the subject an agent that is capable of blocking the protein-protein interaction between NBRl and a MEK kinase, wherein the MEK kinase is MEKK3. [022] In some embodiments, described herein are methods for treating cancer in a subject, the method comprising administering to the subject an agent that is capable of blocking the protein- protein interaction between p62 and a MEK kinase. In some embodiments, the subject has an elevated expression level of p62. In some embodiments, the elevated level of p62 leads to activation of mTORC 1 cascade.

[023] In some embodiments, described herein are methods for treating cancer in a subject, the method comprising administering to the subject an agent that is capable of blocking the protein- protein interaction between p62 and a MEK kinase, wherein administration of an agent that is capable of blocking the protein-protein interaction between p62 and a MEK kinase blocks the activation of mTORC 1 cascade. In some embodiments, the agent is a blocking peptide. In some embodiments, the blocking peptide comprises an amino acid sequence selected from SEQ ID NO. 2, SEQ ID NO. 3, and SEQ ID NO. 10-SEQ ID NO. 22.

[024] In some embodiments, described herein are methods for treating cancer in a subject, the method comprising administering to the subject an agent that is capable of blocking the protein- protein interaction between p62 and a MEK kinase, wherein the MEK kinase is MEKK3.

[025] In some embodiments, described herein are peptides comprising an amino acid sequence selected from SEQ ID NO. 1, SEQ ID NO. 3-SEQ ID NO. 9, and SEQ ID NO. 16-SEQ ID NO. 22, wherein the peptide is capable of blocking the protein-protein interaction between NBR1 and a MEK kinase. In some embodiments, the peptide comprises an amino acid sequence selected from SEQ ID NO. 5 and SEQ ID NO. 20. In some embodiments, the peptide is capable of blocking the protein-protein interaction between NBR1 and a MEK kinase, wherein said MEK kinase is

MEKK3. In some embodiments, the peptide is capable of blocking the protein-protein interaction between PB1 domain of NBR1 and PB1 domain of MEKK3. In some embodiments, the peptide is capable of blocking the protein-protein interaction between an acidic residue on the PB1 domain of NBR1 and a basic residue on the PB1 domain of MEKK3. In some embodiments, the peptide is capable of blocking the protein-protein interaction between an acidic residue on the PB1 domain of NBR1 and a basic residue on the PB1 domain of MEKK3, wherein said basic residue is on the N- terminal region of the PB1 domain of MEKK3. In some embodiments, the peptide is capable of blocking the protein-protein interaction between an asparagine residue or a glutamic acid residue on the PB1 domain of NBR1 and a lysine residue on the PB1 domain of MEKK3.

[026] In some embodiments, described herein are peptides comprising an amino acid sequence selected from SEQ ID NO. 1, SEQ ID NO. 3-SEQ ID NO. 9, and SEQ ID NO. 16-SEQ ID NO. 22, wherein the peptides are capable of blocking the protein-protein interaction between NBR1 and a MEK kinase, and wherein the peptides are cell permeable. [027] In some embodiments, described herein are peptides comprising an amino acid sequence selected from SEQ ID NO. 1, SEQ ID NO. 3-SEQ ID NO. 9, and SEQ ID NO. 16-SEQ ID NO. 22, wherein the peptides are capable of blocking the protein-protein interaction between NBRl and a MEK kinase, wherein the length of the peptides are between 5 and 100 amino acids. In some embodiments, described herein are peptides comprising an amino acid sequence selected from SEQ ID NO. 1, SEQ ID NO. 3-SEQ ID NO. 9, and SEQ ID NO. 16-SEQ ID NO. 22, wherein the peptides are capable of blocking the protein-protein interaction between NBRl and a MEK kinase, wherein the length of the peptides are between 35 and 50 amino acids. In some embodiments, described herein are peptides comprising an amino acid sequence selected from SEQ ID NO. 1, SEQ ID NO. 3-SEQ ID NO. 9, and SEQ ID NO. 16-SEQ ID NO. 22, wherein the peptides are capable of blocking the protein-protein interaction between NBRl and a MEK kinase, wherein the length of the peptides are between 9 and 15 amino acids.

[028] Provided herein in some embodiments, are pharmaceutical compositions for treating obesity-induced inflammation, comprising a peptide that is capable of blocking the protein-protein interaction between NBRl and a MEK kinase and a pharmaceutically acceptable carrier. In some embodiments, described herein, are methods of treating obesity-induced inflammation in a subject, the method comprising administering to the subject a pharmaceutical composition comprising a peptide that is capable of blocking the protein-protein interaction between NBRl and a MEK kinase and a pharmaceutically acceptable carrier.

[029] In some embodiments, described herein, are peptides comprising an amino acid sequence selected from SEQ ID NO. 2, SEQ ID NO. 3, and SEQ ID NO. 10-SEQ ID NO. 22, wherein the peptides are capable of blocking the protein-protein interaction between p62 and a MEK kinase. In some embodiments, the peptide comprising an amino acid sequence selected from SEQ ID NO. 11, and SEQ ID NO. 22. In some embodiments, the peptide is capable of blocking the protein-protein interaction between p62 and a MEK kinase, wherein said MEK kinase is MEKK3. In some embodiments, the peptide is capable of blocking the protein-protein interaction between PB1 domain of p62 and PB1 domain of MEKK3. In some embodiments, the peptide is capable of blocking the protein-protein interaction between an asparagine residue or a glutamic acid residue on the PB1 domain of p62 and a lysine residue on the PB1 domain of MEKK3.

[030] In some embodiments, described herein, are peptides comprising an amino acid sequence selected from SEQ ID NO. 2, SEQ ID NO. 3, and SEQ ID NO. 10-SEQ ID NO. 22, wherein the peptides are capable of blocking the protein-protein interaction between p62 and a MEK kinase, and wherein said peptides are cell permeable. In some embodiments, described herein, are peptides comprising an amino acid sequence selected from SEQ ID NO. 2, SEQ ID NO. 3, and SEQ ID NO. 10-SEQ ID NO. 22, wherein the peptides are capable of blocking the protein-protein interaction between p62 and a MEK kinase, and wherein lengths of the peptides are between 5 and 100 amino acids. In some embodiments, described herein, are peptides comprising an amino acid sequence selected from SEQ ID NO. 2, SEQ ID NO. 3, and SEQ ID NO. 10-SEQ ID NO. 22, wherein the peptides are capable of blocking the protein-protein interaction between p62 and a MEK kinase, and wherein lengths of the peptides are between 30 and 60 amino acids. In some embodiments, described herein, are peptides comprising an amino acid sequence selected from SEQ ID NO. 2, SEQ ID NO. 3, and SEQ ID NO. 10-SEQ ID NO. 22, wherein the peptides are capable of blocking the protein-protein interaction between p62 and a MEK kinase, and wherein lengths of the peptides are between 9 and 12 amino acids.

[031] Provided herein in some embodiments, are pharmaceutical compositions for treating cancer, comprising a peptide that is capable of blocking the protein-protein interaction between p62 and a MEK kinase and a pharmaceutically acceptable carrier.

[032] In some embodiments, described herein, are methods of treating cancer in a subject, the method comprising administering to the subject a pharmaceutical composition comprising a peptide that is capable of blocking the protein-protein interaction between p62 and a MEK kinase and a pharmaceutically acceptable carrier.

INCORPORATION BY REFERENCE

[033] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[034] Figure 1 illustrates the correlation between NBRl transcript levels and adipose inflammation in obese patients. These panels show the correlation between transcript levels of NBRl and PPARyl (Figure 1A), PPARy2 (Figure IB), CD68 (Figure 1C), CD163 (Figure ID), MIP-1 (Figure IE), and MCP-1 (Figure IF) in obese patients.

[035] Figure 2 illustrates expression of NBRl in both mice fed a regular chow diet (RD) compared to a high fat diet (HFD), as well as in wild type (WT) compared to NBRl^MyKO (NBRl myeloid knockout) mice. Figure 2A shows immunofluorescence staining of NBRl, F4/80, DAPI, and co-localization of NBRl and F4/80 in epididymal white adipose tissue of RD-fed mice (top) and HFD-fed mice (bottom) (n=5 mice). Figure 2B shows quantitative analysis of relative expression of NBRl, as measured by RT-PCR using total RNA isolated form ATMs of RD-fed mice and HFD-fed mice (n=6-10 mice). Figure 2C shows NBR1 expression in BMDMs (Mo), liver, epididymal white adipose tissue (WAT) and brown adipose tissue (BAT) of WT and NBRl^My KO mice, as assessed by western blotting. Figure 2D shows quantitative analysis of relative expression of makers of inflammation, as measured by RT-PCR assay using total RNA isolated from WAT of WT and NBRl^My KO mice (n=6-8 mice).

[036] Figure 3 illustrates NBR1 deletion in macrophages impaired macrophage polarization toward the pro -inflammatory Ml phenotype. Total RNA was isolated from bone -marrow derived macrophages (BMDMs) of WT and NBRl^My KO mice and incubated with either 100 ng/ml of IFNy for 8 hr or 10 ng/ml of IL-13 for 72 hr. The relative expression of the indicated markers of Ml (Figure 3 A) or M2 (Figure 3B) polarization was measured by quantitative RT-PCR assay (n=6 mice). Figure 3C shows IL-6 secretion by shNT and shNBRl Raw cells, after LPS stimulation for 8 hr (left panel) and the deletion of NBR1 by shNBRl was confirmed by immunoblotting (right panel).

[037] Figure 4 illustrates that NBR1 deletion in macrophages down-regulated the MEKK2/3- MKK4-JNK pathway. Western blot analysis with the indicated antibodies in BMDMs from WT and NBRl^My KO mice (Figures 4A and 4C) or in shNT and shNBRltreated Raw cells (Figure 4B) stimulated with LPS (100 ng/ml) for the indicated time. Quantification of pJNK (Figures 4A-B) and pMKK4 (Figure 4C) fold change activation is shown in the right panels. Figure 4D shows a Western blot analysis with the indicated antibodies in BMDMs from WT and NBRl^My KO mice fed a HFD, and stimulated with palmitate (0.8 mM) for the indicated times. Quantification of pJNK fold change activation is shown in the right panel of Figure 4D. Figure 4E shows quantitative analysis of relative expression of markers of inflammation, as measured by RT-PCR using total RNA isolated from BMDMs of WT and NBRl^My KO mice. Data are representative of three experiments. *p < 0.05, **p < 0.01, ***p < 0.001. Results are presented as mean ± SEM.

[038] Figure 5 illustrates that NBR1 is a scaffold for MEKK3 and MKK4. An alignment of amino acid sequence of PB1 domains of mouse NBR1, MEKK2 and MEKK3 is displayed in Figure 5A. The secondary structural elements are shown above the sequences. A conserved basic residue and acidic residues are indicated by boxes respectively. The potential interaction between acidic PB1 (PBQ-A) of NBR1 and basic PB1 (PB1-B) of MEKK3, and the consequence of the D50R substitution in the PB1 domain of NBR1, are shown in the scheme below the sequences. Figure 5B shows quantitative analysis of relative expression of MEKK3, as measured by RT using total RNA isolated from ATMs of RD-fed mice and HFD-fed mice (n = 6-10 mice). Figures 5C and 5D show interaction of NBR1 and MEKK3 through the PB1 domain. This was accomplished by transfecting HEK293 cells with the indicated plasmids, cell lysates were prepared, and lysates and HA immunoprecipitates were analyzed by western blotting for Flag and HA. Endogenous interaction of NBR1 and MEKK3 was determined in immunoprecipitates from Raw cells by anti-MEKK3 antibody or control IgG and cell lysates analyzed by immunoblot (Figure 5E). NBR1 is a scaffold for MEKK3 and MKK4. HEK293T cells were transfected with the indicated plasmids, cell lysates were prepared, and lysates and GST immunoprecipitates were analyzed by western blotting (Figure 5F). Figure 5G shows endogenous interaction of the NBR1-MEKK3-MKK4 complex in LPS- treated cells. HEK-293/hTLR4/MD2/CD14 cells treated with LPS (100 ng/ml) for 30 min were immunoprecipitated with anti-NBRl antibody or control IgG, and cell lysates and immunoprecipitates were analyzed by immunoblotting. Figure 5H shows endogenous interaction of NBR1 and MEKK3 in palmitate-treated BMDMs. BMDMs were serum starved for 1 hr and stimulated with 0.8 mM palmitate for 16 hr. Overexpression of NBR1 but not the PBl-mutant NBR1-D50R mediates TNK activation in cotransfection MEKK3 (Figure 51) or after palmitate treatment (Figure 5 J) in 293T cells. Re-expression of NBR1 WT but not of NBR1-D50R resconstituted palmitate-induced JNK activation in NBR1 -deficient BMDMs (Figure 5K).

[039] Figure 6 illustrates myeloid-specific deletion of NBR1 improved glucose clearance in HFD-fed mice. Glucose tolerance test (GTT) (Figure 6A) and insulin tolerance test (ITT) (Figure 6B) in 8 week old WT and NBRl^MyKO mice fed a HFD for 4 weeks (n=6-8 mice). WT and NBRl^MyKO HFD-fed mice were fasted overnight and then treated by i.p. injection with 1 U/kg insulin (15 min). Representative tissue samples were examined by immunoblot analysis by probing with antibodies to phospho-AKT, AKT, and actin (Figure 6C). Glucose tolerance test (Figure 6D) and insulin tolerance test (Figure 6E) in 12 week old RD-fed WT and NBRl^MyKO mice (mean ± SEM; n = 4 mice). Blood concentration of glucose (Figure 6F) and insulin (Figure 6G) in overnight fasted mice HFD-fed WT and NBRl^MyKO mice (n = 6-8 mice) were measured. Figure 6H shows a quantitative analysis of relative expression of PEPCK and G6Pase, as measures by RT-PCR using total RNA isolated form livers of HFD-fed WT and NBRl^MyKO mice (n=6-8 mice). *p< 0.05, **p < 0.01, ***p < 0.001. Results are presented as mean ± SEM.

[040] Figure 7 illustrates how p62 phosphorylation and MEKK3 are required for mTORCl activation in response to amino acids. HEK293T cells were deprived of amino acids and serum for 50 min and stimulated with amino acids for the indicated times, and cell lysates were analysed by western blot (Figure 7A). HEK293T cells stably expressing Flag- p62^WT or Flag-p62^T269/S272AA were treated as in Figure 7 A, and immunob lotted for the specified proteins (Figures 7B-C). HEK293T cells stably expressing Flag- p62^WT or Flag-p62^T269/S272AA were deprived of serum for 24 hr and stimulated with insulin for the indicated times, and cell lysates were analyzed by western blot (Figure 7D). HEK293T cells stably expressing Flag- p62^WT or Flag-p62^T269/S272AA were treated as in Figure 7 A, and cell lysates and Flag-tagged immunopecipitates were immunoblotted for the indicated proteins (Figure 7E). shNT or shMEK 3 HEK293T cells were treated as in Figure 7A and cell lysates were immunoblotted for the indicated proteins (Figure 7F). HEK293T cells transfected with the indicated plasmids were treated as in Figure 7A, and cell lysates were immunoblotted for the indicated proteins (Figure 7G). shNT or shMEKK3 HEK293T cells were treated as in Figure 7D, and cell lysates were analysed by western blot (Figure 7H). mTOR immunoprecipitates and cell lysates from HEK293T cells, treated as in Figure 7A, were immunoblotted for the indicated proteins (Figure 71). Representative data from two independent experiments are shown.

[041] Figure 8 illustrates that MEK 3 is involved in p62 phosphorylation in response to amino acids. HEK293T cells were transfected with the indicated plasmids, and cell lysates were analysed by western blot (Figure 8A). HEK293T cells transfected with the indicated plasmids, were deprived for amino acids and serum for 50 min and stimulated with amino acids for 20 min. Cells were analysed by western blot (Figure 8B). shNT or shMEKK3 HEK293T cells were treated as in Figure 8B and cell lysates were immunoblotted for the specified proteins (Figure 8C). Scheme of interaction between acidic PB1 of p62 and basic PB1 of MEK 3 (Figure 8D). HEK293T cells were transfected with the indicated plasmids, and cell lysates were immunoblotted for the specified proteins (Figure 8E). HEK293T cells transfected with the indicated plasmids were treated as in Figure 8B, and Myc-tagged immunoprecipitates were analysed by western blot (Figure 8F). Representative data from two independent experiments are shown.

[042] Figure 9 illustrates that ΜΕΚ 3/ΜΚ 3/6-ρ38δ induces p62 phosphorylation in response to amino acids. Figure 9A shows that ρ38δ is required for mTORCl activation in response to amino acids. shNT or shp385 HEK293T cells were deprived of amino acids and serum for 50 min and stimulated with amino acids for the indicated times, and cell lysates were immunoblotted for the indicated proteins (Figure 9B). HEK293T cells were transfected with the indicated plasmids and immunoblotted for the specified proteins (Figures 9C-D). In vitro phosphorylation of p62 by ρ38δ (Figure 9E). Flag-tagged immunoprecipitates from HEK293T cells were phosphorylated in vitro by a recombinant ρ38δ with ATPyS followed of PNBM alkylation and immunoblotted for the indicated proteins (Figure 9F). HEK293T cells and shNT or shMEK 3 HEK293T cells, transfected with the indicated plasmids, were treated as in Figure 9B. In vitro phosphorylation was carried out with the Flag-tagged immunoprecipitates and a p62 recombinant protein (Figures 9G-H). HEK293T cells transfected with scramble siRNA or MEK3 and MEK6 siRNA were treated as in Figure 9B, and cell lysates were immunoblotted for the indicated proteins (Figure 91). Representative data from two independent experiments are shown. [043] Figure 10 illustrates that the ΜΕΚ 3/ρ38δ cascade contributes to cell proliferation and autophagy through mTORCl activation. shNT, shMEK 3 or shp385 PC3 cells were cultured under normal growing conditions, and cell viability was determined by trypan blue exclusion assay. Results are shown as means ± SEM (n=3) (Figures 10A-B). PC3 cells stably expressing Flag- RagB^GTP were infected with shNT, shMEK 3 or shp385 lentiviral vectors. Cell lysates were analysed by western blot, and cell viability was determined as in Figure 10A. Results are shown as means ± SEM (n=3) (Figures 10C-D). For Figures 10E-10H, shNT, shMEK 3 or shp385 PC3 cells were deprived of amino acids and serum for 4 hr in the absence or presence of bafilomycin Al . Cell lysates were immunob lotted for the indicated proteins. Graphs represents LC3-II/actin ratio by densitometry. Figure 101 show images of shNT, shMEK 3 or shp385 A549 cells stably expressing GFP-mCherry-LC3 and treated as in Figure 10E. Figure 10J shows quantification of the number of autophagosomes and autolysosomes per cell is shown. Results are shown as means ± SEM (n=20) **p< 0.01, ***p<0.001. Panels A, B, C, D, E and F show the results of one representative experiment out of two performed independently.

[044] Figure 11 illustrates the critical role of the MEK 3/p385/p62/mTOR cascade in prostate cancer. Figure 11A shows representative images of shNT, shp62, shMEK 3 and shp385 prostate organoids from PTEN-deficient mice after 7 days in culture. Figure 11B shows quantification of number of organoids and size of experiment shown in Figure 11A. Scale bars, 100 μιη. Results are presented as mean ± SEM.(*p < 0.05, **p < 0.01, ** *p < 0.001). Cell lysates from shNT, shp62, shMEKK3 and shp385 prostate organoids were immunob lotted for the indicated proteins (Figure 11C). Consecutive prostate cancer tissue sections were stained for MEK 3, ρ38δ, p62 or phospho- S6. Figure 11D shows representative cases with GS=6 and GS=10. Scale bar 200 μΜ. In Figure HE are box plot graphs showing a statistical analysis of MEKK3 (p=0.006), ρ38δ (p=0.004), p62 (p=0.003) or phospho-S6 (p=0.049) expression in prostate tumour with GS>6 compared to prostate tissue with GS<6. Figure 1 IF shows correlation plots between MEK 3/p62, MEK 3/phosho-S6, ρ38δ/ρ62 and p385/phospho-S6 (arbitrary units). The coefficient of correlation (r) and the p value (p) are indicated.

DETAILED DESCRIPTION OF THE INVENTION

[045] Provided herein, in certain embodiments, are methods for treating obesity-induced inflammation in a subject with a MEK 3 inhibitor. In certain embodiments, the methods described herein, are for treatment of obesity-induced inflammation with a MEK 3 inhibitor, in subjects exposed to high fat diet, wherein the administration of the MEK 3 inhibitor leads to the inhibition of NBR 1 -MEKK3 -JNK signalling pathway. [046] Provided herein, in certain embodiments, are methods for treating cancer in a subject with a MEK 3 inhibitor. In certain embodiments, the methods described herein, are for treatment of cancer with a MEK 3 inhibitor, in subjects exposed to nutrient abundance, wherein the administration of the MEK 3 inhibitor leads to the inhibition of p62-MEK 3-p385-mTORCl signalling pathway.

[047] Also provided herein, are compounds, pharmaceutical compositions and medicaments comprising MEK 3 inhibitor for therapy of obesity-induced inflammation or cancer.

[048] Provided herein, in certain embodiments, are methods for treating obesity-induced inflammation in a subject with a blocking peptide. In certain embodiments, the methods described herein, are for treatment of obesity-induced inflammation with a blocking peptide, in subjects exposed to high fat diet, wherein the administration of blocking peptide blocks the protein-protein interaction between NBR1 and MEK 3.

[049] Provided herein, in certain embodiments, are methods for treating cancer in a subject with a blocking peptide. In certain embodiments, the methods described herein, are for treatment of cancer with a blocking peptide, in subjects exposed nutrient abundance, wherein the administration of blocking peptide blocks the protein-protein interaction between p62 and MEK 3.

[050] Also provided herein, are compounds, pharmaceutical compositions and medicaments comprising a blocking peptide, as described herein, for therapy of obesity-induced inflammation or cancer.

Obesity-Induced Inflammation and Diabetes

[051] The epidemic of overweight and obesity is a major problem because of the plethora of health and economic issues that it induces. Key among these is the sharply increasing prevalence of type 2 diabetes (T2D) and cardiovascular disease. The development of T2D is characterized by two processes: 1) insulin resistance, resulting from impaired insulin signalling and leading to an increased demand for insulin, which must be met by increased insulin production by pancreatic β- cells (compensatory β-cell function); and 2) β-cell dysfunction, with T2D developing when the amount of insulin that is produced is insufficient to meet the demand. Overweight and obesity, especially in case of abdominal fat accumulation, are associated with systemic low-grade inflammation. This low-grade inflammation is characterized by, among other things, higher levels of circulating pro-inflammatory cytokines and fatty acids. These can interfere with normal insulin function and thereby induce insulin resistance, and have also been implicated in β-cell dysfunction.

[052] Impaired insulin signalling , also known as insulin resistance , leads to an increased demand for insulin and this increased demand must be met by an increased insulin production by the pancreatic β-cells, a process known as compensatory β-cell function. Thus, obesity-induced insulin resistance will initially lead to higher circulating insulin concentrations but in case of prolonged and/or worsening insulin resistance, β-cells may no longer be able to meet the high demand. This leads to insufficient hepatic and peripheral glucose disposal, subsequently to higher circulating levels of glucose and eventually to the development of T2D.

[053] Adipocyte hypertrophy, hypoxia and stress may all be involved in adipose tissue inflammation via induction of pro -inflammatory cytokines, as well as of chemokines that attract macrophages. Adipose tissue macrophages may have a pro -inflammatory (Ml), an antiinflammatory (M2), or an intermediate phenotype, depending on the activating cytokines that are present. In obesity, macrophages in adipose tissue were shown to be mainly Ml .

[054] In obesity and type 2 diabetes mellitus (T2DM) insulin signalling via IRS and PI3 kinase is primarily affected. Inflammatory cytokines, e.g. TNF-a and IL-6, as well as saturated fatty acids can all hamper insulin signalling via the IRS and PI3 kinase pathway via activation of intracellular serine/threonine mediators in metabolic insulin resistance. ΙΚΚβ and JNK are important intracellular mediators in metabolic insulin resistance.

NBR1-MEKK3-JNK Signalling in Obesity Induced Inflammation

[055] Obesity is an international healthcare priority due to its increasing prevalence and its association with glucose intolerance. The lack of a complete understanding of the precise regulatory networks that control adipogenesis, energy expenditure, and inflammation is a fundamental problem in metabolic research. It is clear also that obesity-induced inflammation underlies critical aspects of glucose metabolism deregulation and insulin resistance. Many studies have demonstrated that obesity-induced inflammation plays a critical role in the generation of the metabolic syndrome, including a dysfunctional glucose metabolism and insulin resistance.

However, the precise signalling cascades accounting for these effects still need to be identified, which is of great importance for the design of more efficacious treatments of type-2 diabetes. In this regard, TNK was recently recognized as an important factor in obesity-induced inflammation, a condition that contributes significantly to aberrant glucose metabolism in obesity. The initial observations were that TNK activity was abnormally elevated in obese mice, and that whole-body ablation of JNKl resulted in decreases in body weight and adiposity in the context of a high fat diet (HFD) and simultaneous genetic inactivation of both JNKl and JNK2 in the myeloid compartment improved glucose tolerance in HFD mice, although it did not affect the body weight or adiposity (Han et al, 2013). This data demonstrated that JNK activation in macrophages is a critical step in obesity-induced inflammation and glucose intolerance.

[056] Another study has demonstrated that the loss of NBR1, specifically in macrophages, largely prevents obesity-induced inflammation in vivo. Ex vivo and in vitro results link the PB1 domain- mediated interaction between NBRl and MEKK3 to JNK activation and the Ml polarization of macrophages. These observations have to be put in the context of the other PB1 domain proteins, p62 and ΡΚ£ζ, which have been also implicated in the regulation of obesity and the metabolic alterations associated to this physiopathological condition. In this regard, it is quite striking that whereas p62 deficiency results in obesity and that the inflammation associated to the lack of p62 is secondary to increased adiposity, the ΡΚ£ζ loss selectively promotes inflammation in obese mice without effects on adiposity. Interestingly, PKC anti -inflammatory actions are mostly confined to the adipocyte, with no effects on the hematopoietic system. In contrast, NBRl is a proinflammatory signalling adapter in macrophages by nucleating a MEKK3 -driven JNK cascade, not only in response to inflammatory signals such as lipopolysaccharide (LPS), but more importantly in response to saturated fatty acids like palmitate. Overall, these findings reveal a pharmacologically targetable mechanism underlying JNK activation, and suggest that blocking NBRl binding to MEKK3 may represent an opportunity to prevent type 2 diabetes in humans, caused by obesity- induced inflammation.

[057] The domain organization of NBRl is remarkably similar to that of p62, featuring PB1, zinc- finger, and UBA domains. The outcomes of overexpression and transfection studies have suggested that NBRl is involved in growth-factor trafficking and/or p62-mediated processes. However, its precise in vivo contribution to the control of metabolic homeostasis and/or the ensuing

inflammation in the context of obesity has not been investigated. It is possible that p62, PKCζ and NBRl play different roles in the control of metabolic homeostasis depending on cell type. The effect of myeloid-specific NBRl ablation has been characterized and it has been established that it plays a critical role in the regulation of macrophage polarization toward the Ml phenotype during obesity-induced inflammation. Our studies in human patients with metabolic syndrome revealed a significantly positive correlation between NBRl transcript levels and markers of metabolic alterations and inflammation. Collectively, these results established that NBRl plays a critical role in obesity-induced glucose intolerance, and that this is due to its impact on macrophage function. They also highlight the relevance of the PB1 network comprising p62, PKC and NBRl at the cellular interface between metabolism and inflammation.

[058] In light of the critical role played by NBRl , via its interaction with the MEKK3 and activation of JNK, in obesity-induced inflammation, it is contemplated that blocking the interaction of MEKK3 and NBRl can lead to reduction of obesity-induced inflammation, improved glucose tolerance and insulin resistance. In some embodiments, blocking the interaction of MEKK3 and NBRl can lead to reduction of obesity-induced inflammation and thereby prevent the onset of type 2 diabetes. [059] In some embodiments, the interaction between MEK 3 and NBR1 is blocked using an agent that mimics the PB1 domain of MEK 3 and blocks the binding of endogenous MEK 3 and NBR1. In some embodiments, the interaction between MEKK3 and NBR1 is blocked using an agent that mimics the PB1 domain of NBR1 and blocks the binding of endogenous NBR1 and MEK 3. In some embodiments the agent is a blocking peptide, or a mimicking peptide. In some embodiments, the blocking peptide, for treatment of obesity-induced inflammation, has an amino acid sequence selected from SEQ ID NO. 1, SEQ ID NO. 3 - SEQ ID NO. 9, and SEQ ID NO. 16 - SEQ ID NO. 22

[060] Further, in light of the critical role played by NBR1 , via its interaction with the MEK 3 and activation of JNK, in obesity-induced inflammation, it is contemplated that inhibiting MEK 3 can also lead to reduction of obesity-induced inflammation, improved glucose tolerance and insulin resistance. In some embodiments, inhibiting MEKK3 can lead to reduction of obesity-induced inflammation and thereby prevent the onset of type 2 diabetes.

[061] In some embodiments, MEK 3 is inhibited using a compound selected from a group consisting of PF-03814735, AT9283, Crizotinib (PF-02341066), Hesperadin, AZD7762, PD 166285, Cdkl/2 Inhibitor III, PP121, BIBF 1120, Bosutinib (SKI-606), Cdk2 Inhibitor IV (NU 6140), BGJ398 (NVP-BGJ398), Dasatinib (BMS-354825), Dovitinib (TKI-258), Sunitinib Malate (Sutent), and WZ3146. The structures for the listed compounds are shown in Table 1.

Cancer and Nutrient Sensing

[062] Cancer also known as a malignant tumor or malignant neoplasm, is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. Not all tumors are cancerous; benign tumors do not spread to other parts of the body. Possible signs and symptoms include: a new lump, abnormal bleeding, a prolonged cough, unexplained weight loss, and a change in bowel movements, among others. While these symptoms may indicate cancer, they may also occur due to other issues. There are over 100 different known cancers that affect humans.

[063] The ability to sense and respond to fluctuations in environmental nutrient levels is a requisite for life. Nutrient scarcity is a selective pressure that has shaped the evolution of most cellular processes. Different pathways that detect intracellular and extracellular levels of sugars, amino acids, lipids and surrogate metabolites are integrated and coordinated at the organismal level through hormonal signals. During food abundance, nutrient-sensing pathways engage anabolism and storage, whereas scarcity triggers homeostatic mechanisms, such as the mobilization of internal stores through autophagy. Nutrient-sensing pathways are commonly deregulated in human metabolic diseases. [064] Cells can sense and respond to fluctuations in nutrient availability. Mutations that disrupt such lines of nutrient communication with the cellular growth machinery seem to contribute to the uncontrolled growth of cancer cells. Nutrient abundance is known to affect the development of cancer.

p62-MEKK3-p38a Signalling in Cancer

[065] Cell metabolism is responsive to the availability of environmental and intracellular nutrients. The mTORC 1 kinase complex is an essential mediator of this response via its actions as a regulator of anabolism and a key nutrient sensor. In addition, it interacts with AMPK in the control of autophagy. The aberrant activation of mTORC 1 has important repercussions in several diseases, including cancer.

[066] The signalling adapter p62, also known as SQSTM1, is central to cell survival and proliferation and has recently been shown to be required for the translocation of mTORC 1 to the lysosomal surface. This is achieved through its interaction with raptor, a distinctive component of the mTORC 1 complex, and, in part, by facilitating the interaction of mTOR with the Rag proteins while also modulating Rag heterodimer formation. The interaction of TRAF6 with p62 facilitates the lysosomal recruitment of mTORC 1 and catalyses its K63-polyubiquitination, which is required for its optimal activation by amino acids. Therefore, the p62/TRAF6 complex must be considered an important modulator of nutrient sensing through mTORC 1. Consistent with this notion, the loss of TRAF6, like that of p62, impaired proliferation and the transforming properties of cancer cells, and led to enhanced autophagy, which could be rescued by the expression of a permanently active RagB mutant. Phosphorylation of p62 at two specific residues, T269/S272, governs the recruitment of TRAF6. Moreover, p62 T269/S272 phosphorylation was shown to be essential for the translocation of mTORC 1 to the lysosome and the subsequent poly-ubiquitination and activation of the mTOR catalytic subunit of the complex.

[067] Based on the observation described above, a new model emerges whereby p62 senses amino acids by undergoing phosphorylation through a newly identified ΜΕΚ 3/ΜΕΚ3/6/ρ38δ cascade. The specificity of this process is provided by the selective interaction of MEK 3 with p62 through their respective PB1 domains.

[068] Collectively, the results described above identify a new sensing pathway that is selectively activated in response to amino acids and that is operative also in cancer cells. This kinase cascade is highly upregulated during cancer progression and is essential for tumour development. Since kinases are eminently druggable, this could open new avenues for designing novel therapeutic interventions in cancer. [069] In light of the critical role played by p62, via its interaction with the MEKK3 and ρ38δ, and activation of mTORCl, in cancer, it is contemplated that blocking the interaction of MEKK3 and p62 can be a treatment for cancer. In some embodiments, blocking the interaction of MEKK3 and p62 can be a treatment for prostate cancer. In some embodiments, blocking the interaction of MEKK3 and p62 can lead to reduction to tumor volumes in prostate cancer.

[070] In some embodiments, the interaction between MEKK3 and p62 is blocked using an agent that mimics the PB1 domain of MEKK3 and blocks the binding of endogenous MEKK3 and p62. In some embodiments, the interaction between MEKK3 and p62 is blocked using an agent that mimics the PB1 domain of p62 and blocks the binding of endogenous p62 and MEKK3. In some embodiments the agent is a blocking peptide, or a mimicking peptide. In some embodiments, the blocking peptide, for treatment of cancer, has an amino acid sequence selected from SEQ ID NO. 2, SEQ ID NO. 3, and SEQ ID NO. 10-—SEQ ID NO. 22

[071] Further, in light of the critical role played by p62, via its interaction with the MEKK3 and ρ38δ, activation of mTORCl, in cancer, it is contemplated inhibiting MEKK3 can be a treatment for cancer. In some embodiments, inhibiting MEKK3 can be a treatment for prostate cancer. In some embodiments, inhibiting MEKK3 can lead to reduction to tumor volumes in prostate cancer.

[072] In some embodiments, MEKK3 is inhibited using a compound selected from a group consisting of PF-03814735, AT9283, Crizotinib (PF-02341066), Hesperadin, AZD7762, PD 166285, Cdkl/2 Inhibitor III, PP121, BIBF 1120, Bosutinib (SKI-606), Cdk2 Inhibitor IV (NU 6140), BGJ398 (NVP-BGJ398), Dasatinib (BMS-354825), Dovitinib (TKI-258), Sunitinib Malate (Sutent), and WZ3146. The structures for the listed compounds are shown in Table 1.

MEKK3

[073] MEKK3 (Mitogen-activated protein kinase kinase kinase 3), also referred to as MAP3K3, is an enzyme that in humans is encoded by the MAP3K3 gene. This gene product is a 626-amino acid polypeptide that is 96.5% identical to mouse MEKK3. Its catalytic domain is closely related to those of several other kinases, including mouse MEKK2, tobacco NPK, and yeast Stel 1. Northern blot analysis revealed a 4.6-kb transcript that appears to be ubiquitously expressed. MEKK3s are involved in regulating cell fate in response to external stimuli. MEKK3 directly regulates the stress- activated protein kinase (SAPK) and extracellular signal-regulated protein kinase (ERK) pathways by activating SEK and MEK1/2 respectively. In cotransfection assays, it enhanced transcription from a nuclear factor kappa-B (NFKB)-dependent reporter gene, consistent with a role in the SAPK pathway. Alternatively spliced transcript variants encoding distinct isoforms have been observed. MEKK3 regulates the p38, JNK and ERKl/2 pathways. MAP3K3 has been shown to interact with [SQSTMl/p62], MAP2K5, YWHAE, GAB1, BRCA1, and Akt. Many studies have described the association of MAP3K3 (or MEK 3) in cancer. Two SNPs in the MAP3K3 gene were found as candidates for association with colon and rectal cancers. MEK 3 is highly expressed in 4 ovarian cancer cell lines (OVCA429, Hey, DOV13, and SKOv3). This expression level is significantly higher in those cancer cells when compared to normal cells. MEK 3 expression levels are comparable to IKK kinase activities, which also relate to activation of NFKB. High expression of MEKK3 in most of these ovarian cancer cells supposedly activate IKK kinase activity, which lead to increased levels of active NFKB. Also, MEKK3 interacts with Akt to activate NFKB. Genes related to cell survival and anti-apoptosis have increased expression in most cancer cells with high levels of MEKK3. This is probably due to constitutive activation of NFKB, which will regulate those genes. In this sense, knockdown of MEKK3 caused ovarian cancer cells to be more sensitive to drugs.

[074] MEKK3 also interacts with BRCA1. Knocking down BRCA1 resulted in inhibited MEKK3 kinase activity. The drug paclitaxel induces MEKK3 activity and it requires functional BRCA1 to do it. It was observed that in a breast cancer cell line BRCA1 -deficient (HCC1937), paclitaxel was unable to activate MEKK3. Paclitaxel may be inducing stress-response through the

ΜΕΚΚ3/ΓΝΚ/ρ38/ΜΑΡΚ pathway, but not in mutated BRCA1 cells.

[075] Normal endothelial cells, but deficient in MEKK3, have reduced cell proliferation and increased apoptosis. MEKK3 -deficient tumors, on the other hand, can grow in the same rate as regular tumors, also producing comparable levels of VEGF and inducing angiogenesis comparably to wild-type tumors. While these results show that MEKK3 is important for normal endothelial cells, MEKK3 may not be necessary for tumor growth and angiogenesis.

[076] MEKK3 expression level is also significantly higher in cervical cancer in comparison with chronic cervicitis and CIN (cervical intraepithelial neoplasia). This high expression correlates with the also high levels of surviving (apoptosis inhibitor), and they both may associate with cervical cancer development and prognosis. Targeted therapy of MEKK3 together with a therapy that promotes apoptosis has been suggested by recent studies as a possible new strategy for treatment of chemotherap eutic-resistant tumors .

[077] Similarly, significantly higher levels of MEKK3 was found as well in esophageal dysplasia and esophageal squamous cell carcinoma (ESCC) when compared to normal esophageal tissue. MEKK3 seems to accumulate even more in ESCC than esophageal dysplasia, which also correlates with poor prognosis of ESCC. Therefore, MEKK3 can be studied for its possible role as an early biomarker of esophageal tumorigenesis.

[078] From all these studies, MEKK3 has become a valuable target for the development of new therapies and diagnosis/prognosis tools. Pharmaceutical Compositions for Treatment of Obesity-Induced Inflammation or Cancer using MEKK3 Inhibitor

[079] Disclosed herein, in certain embodiments, are pharmaceutical compositions and

formulations comprising: (a) a MEK 3 inhibitor; and (b) a pharmaceutically-acceptable excipient. In some embodiments the MEK 3 inhibitor is selected from a group consisting of PF-03814735, AT9283, Crizotinib (PF-02341066), Hesperadin, AZD7762, PD 166285, Cdkl/2 Inhibitor III, PP121, BIBF 1120, Bosutinib (SKI-606), Cdk2 Inhibitor IV (NU 6140), BGJ398 (NVP-BGJ398), Dasatinib (BMS-354825), Dovitinib (TKI-258), Sunitinib Malate (Sutent), and WZ3146. In some embodiments, the MEK 3 inhibitor is PF-03814735. The structures for the listed compounds are shown in Table 1.

[080] In some embodiments, the MEK 3 inhibitor is AT9283. In some embodiments, the

MEK 3 inhibitor is Crizotinib (PF-02341066). In some embodiments, the MEK 3 inhibitor is Hesperadin. In some embodiments, the MEK 3 inhibitor is AT9283. In some embodiments, the MEK 3 inhibitor is AZD7762. In some embodiments, the MEK 3 inhibitor is PD 166285. In some embodiments, the MEK 3 inhibitor is AT9283. In some embodiments, the MEK 3 inhibitor is Cdkl/2 Inhibitor III. In some embodiments, the MEKK3 inhibitor is PP121. In some

embodiments, the MEK 3 inhibitor is BIBF 1120. In some embodiments, the MEK 3 inhibitor is Bosutinib (SKI-606). In some embodiments, the MEKK3 inhibitor is Cdk2 Inhibitor IV (NU 6140). In some embodiments, the MEKK3 inhibitor is BGJ398 (NVP-BGJ398). In some embodiments, the MEKK3 inhibitor is Dasatinib (BMS-354825). In some embodiments, the MEKK3 inhibitor is Dovitinib (TKI-258). In some embodiments, the MEKK3 inhibitor is Sunitinib Malate (Sutent). In some embodiments, the MEKK3 inhibitor is WZ3146. The structures for the listed compounds are shown in Table 1.

Methods of Administering the MEKK3 Inhibitor

[081] In some embodiments of the methods provided herein, a MEKK3 inhibitor is administered to treat obesity-induced inflammation. In some embodiments of the methods provided herein, a MEKK3 inhibitor is administered to treat cancer.

[082] In some embodiments of the methods provided herein, administration of a MEKK3 inhibitor is by injection, transdermal, nasal, pulmonary, vaginal, rectal, buccal, ocular, otic, local, topical, or oral delivery. In certain instances, injection is intramuscular, intravenous, subcutaneous, intranodal, intratumoral, intracisternal, intraperitoneal, or intradermal. In some embodiments, the MEKK3 inhibitor is selected from a group consisting of PF-03814735, AT9283, Crizotinib (PF- 02341066), Hesperadin, AZD7762, PD 166285, Cdkl/2 Inhibitor III, PP121, BIBF 1120, Bosutinib (SKI-606), Cdk2 Inhibitor IV (NU 6140), BGJ398 (NVP-BGJ398), Dasatinib (BMS-354825), Dovitinib (TKI-258), Sunitinib Malate (Sutent), and WZ3146. The structures for the listed compounds are shown in Table 1.

[083] In some embodiments of the methods provided herein, a MEK 3 inhibitor is administered via different routes of administration. In certain embodiments, a MEK 3 inhibitor is administered orally. In some embodiments, a MEK 3 inhibitor is administered in a capsule form. In some embodiments, a MEK 3 inhibitor is administered in about 0.1 to about 12 mg. In some instances, a MEKK3 inhibitor is administered daily. In some embodiments, the MEK 3 inhibitor is selected from a group consisting of PF-03814735, AT9283, Crizotinib (PF-02341066), Hesperadin,

AZD7762, PD 166285, Cdkl/2 Inhibitor III, PP121 , BIBF 1 120, Bosutinib (SKI-606), Cdk2 Inhibitor IV (NU 6140), BGJ398 (NVP-BGJ398), Dasatinib (BMS-354825), Dovitinib (TKI-258), Sunitinib Malate (Sutent), and WZ3146. The structures for the listed compounds are shown in Table 1.

[084] In some embodiments of the methods provided herein, a MEKK3 inhibitor is administered orally. In some embodiments, a MEKK3 inhibitor is administered periodically every three weeks. In some embodiments, a MEKK3 inhibitor is administered weekly. In some embodiments, a MEKK3 inhibitor is administered daily. In some embodiments, the MEKK3 inhibitor is selected from a group consisting of PF-03814735, AT9283, Crizotinib (PF-02341066), Hesperadin,

[085] In some embodiments, the administration is over a period of time selected from the group consisting of at least about 3 weeks, at least about 6 weeks, at least about 9 weeks, at least about 12 weeks, at least about 15 weeks, at least about 18 weeks, at least about 21 weeks, at least about 24 weeks, at least about 27 weeks, at least about 30 weeks, at least about 33 weeks, at least about 36 weeks, at least about 39 weeks, at least about 42 weeks, at least about 45 weeks, at least about 48 weeks, at least about 51 weeks, at least about 54 weeks, at least about 57 weeks, at least about 60 weeks, at least about 75 weeks, at least about 90 weeks, and at least about 120 weeks.

[086] In some embodiments of the methods provided herein, a MEKK3 inhibitor provided in a kit. In some embodiments, a kit may comprise a MEKK3 inhibitor and a container (e.g., a vial, ampule, bottle, syringe, and/or dispenser package, or other suitable container). In some embodiments, provided kits may optionally further include a second container comprising a pharmaceutical excipient for dilution or suspension of a provided pharmaceutical composition or compound, such as a MEKK3 inhibitor. In some embodiments, a provided pharmaceutical composition or compound, such as a MEK 3 inhibitor, is provided in the container and the second container are combined to form one unit dosage form. In some embodiments, a provided kit further includes instructions for use. In some embodiments, the inhibitor targeting the MEKK3 inhibitor is selected from a group consisting of PF-03814735, AT9283, Crizotinib (PF-02341066), Hesperadin,

AZD7762, PD 166285, Cdkl/2 Inhibitor III, PP121, BIBF 1120, Bosutinib (SKI-606), Cdk2 Inhibitor IV (NU 6140), BGJ398 (NVP-BGJ398), Dasatinib (BMS-354825), Dovitinib (TKI-258), Sunitinib Malate (Sutent), and WZ3146. The structures for the listed compounds are shown in Table 1.

[087] For oral administration, a MEK 3 inhibitor can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers or excipients well known in the art. Such carriers enable the compounds described herein to be formulated as tablets, powders, pills, dragees, capsules, liquids, gels, syrups, elixirs, slurries, suspensions and the like, for oral ingestion by a patient to be treated.

[088] Pharmaceutical preparations for oral use can be obtained by mixing one or more solid excipient with one or more of the compounds described herein, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as: for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methylcellulose, microcrystalline cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose; or others such as: polyvinylpyrrolidone (PVP or povidone) or calcium phosphate. If desired, disintegrating agents may be added, such as the cross linked croscarmellose sodium, polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[089] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

[090] Pharmaceutical preparations which can be used orally include push fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration. In some embodiments, a MEK 3 inhibitor is in powder form and is directly filled into hard gelatin capsules.

[091] For buccal or sublingual administration, the compositions may take the form of tablets, lozenges, or gels formulated in conventional manner.

[092] In some embodiments, a MEK 3 inhibitor is an injectable composition. Injectable compositions may involve for bolus injection or continuous infusion. In some embodiments, the MEK 3 inhibitor may be in a form suitable for parenteral or any other type of injection as a sterile suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. The composition may be formulated for intramuscular, intravenous, subcutaneous, intranodal, intratumoral, intracisternal, intraperitoneal, and/or intradermal injection. Pharmaceutical formulations for injection administration include aqueous solutions of the active compounds in water soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[093] In various embodiments, a MEK 3 inhibitor composition is in liquid form for ocular or otic delivery. Liquid forms include, by way of non-limiting example, neat liquids, solutions, suspensions, dispersions, colloids, foams and the like and can be formulated by known methods.

[094] A MEK 3 inhibitor can be administered topically and can be formulated into a variety of topically administrable compositions, such as solutions, suspensions, lotions, gels, pastes, medicated sticks, balms, creams or ointments. Such pharmaceutical compounds can contain solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives.

[095] Formulations suitable for transdermal administration of a MEK 3 inhibitor may employ transdermal delivery devices and transdermal delivery patches and can be lipophilic emulsions or buffered, aqueous solutions, dissolved and/or dispersed in a polymer or an adhesive. Such patches may be constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents. Still further, transdermal delivery of the MEK 3 inhibitor can be accomplished by means of iontophoretic patches and the like. Additionally, transdermal patches can provide controlled delivery of the MEKK3 inhibitor. The rate of absorption can be slowed by using rate-controlling membranes or by trapping the compound within a polymer matrix or gel. Conversely, absorption enhancers can be used to increase absorption. An absorption enhancer or carrier can include absorbable pharmaceutically acceptable solvents to assist passage through the skin. For example, transdermal devices are in the form of a bandage comprising a backing member, a reservoir containing the compound optionally with carriers, optionally a rate controlling barrier to deliver the compound to the skin of the host at a controlled and predetermined rate over a prolonged period of time, and means to secure the device to the skin.

[096] For administration by inhalation for pulmonary or nasal delivery, a MEK 3 inhibitor may be in the form of an aerosol, a mist or a powder. Pharmaceutical compositions of a MEK 3 inhibitor is conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, such as, by way of example only, gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[097] A MEK 3 inhibitor may also be formulated in rectal or vaginal compositions such as enemas, douches, gels, foams, aerosols, suppositories, jelly suppositories, or retention enemas, containing conventional suppository bases such as cocoa butter or other glycerides, as well as synthetic polymers such as polyvinylpyrrolidone, PEG, and the like. In suppository forms of the compositions, a low-melting wax such as, but not limited to, a mixture of fatty acid glycerides, optionally in combination with cocoa butter is first melted.

[098] One may administer a MEK 3 inhibitor in a local rather than systemic manner, for example, via injection of the compound directly into an organ, often in a depot or sustained release formulation. Furthermore, one may administer pharmaceutical composition containing a MEK 3 inhibitor in a targeted drug delivery system, for example, in a liposome coated with organ-specific antibody. The liposomes will be targeted to and taken up selectively by the organ. Pharmaceutical compositions of a MEKK3 inhibitor may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. Any of the well-known techniques, carriers, and excipients may be used as suitable and as understood in the art. Pharmaceutical compositions comprising a MEK 3 inhibitor may be manufactured in a conventional manner, such as, by way of example only, by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or compression processes.

[099] The pharmaceutical compositions will include at least one pharmaceutically acceptable carrier, diluent or excipient and a MEK 3 inhibitor described herein as an active ingredient in free- acid or free-base form, or in a pharmaceutically acceptable salt form. In addition, the methods and pharmaceutical compositions described herein include the use of N-oxides, crystalline forms (also known as polymorphs), as well as active metabolites of these compounds having the same type of activity. In some situations, a MEK 3 may exist as a tautomer. All tautomers are included within the scope of the compounds presented herein. Additionally, a MEK 3 inhibitor described herein can exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like. The solvated forms of a MEK 3 inhibitor presented herein are also considered to be disclosed herein. In addition, the pharmaceutical compositions may include other medicinal or pharmaceutical agents, carriers, adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure, and/or buffers. In addition, the pharmaceutical compositions can also contain other therapeutically valuable substances.

[0100] Methods for the preparation of compositions comprising a MEK 3 inhibitor described herein include formulating the MEK 3 inhibitor with one or more inert, pharmaceutically acceptable excipients or carriers to form a solid, semi-solid or liquid. Solid compositions include, but are not limited to, powders, tablets, dispersible granules, capsules, cachets, and suppositories. Liquid compositions include solutions in which a compound is dissolved, emulsions comprising a compound, or a solution containing liposomes, micelles, or nanoparticles comprising a compound as disclosed herein. Semi-solid compositions include, but are not limited to, gels, suspensions and creams. The compositions may be in liquid solutions or suspensions, solid forms suitable for solution or suspension in a liquid prior to use, or as emulsions. These compositions may also contain minor amounts of nontoxic, auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and so forth.

Further forms of pharmaceutical compositions of a MEK 3 inhibitor can be integrated with other active agents in a unitary dosage form for combination therapies. The unitary dosage forms can be formulated to release where both agents are released simultaneously or where there is sequential release of each agent via known modified release mechanisms including but not limited to timed release, delayed release, pH release, pulsatile release and the like. [0101] In some embodiments, a MEK 3 inhibitor is administered in a dose of about 1 mg/kg to about 5 mg/kg, about 10 mg/kg to about 20 mg/kg, about 30 mg/kg to about 50 mg/kg, about 60 mg/kg to about 75 mg/kg, about 100 mg/kg to about 125 mg/kg to about 150 mg/kg, about 200 mg/kg to about 250 mg/kg, about 300 mg/kg to about 350 mg/kg, about 400 mg/kg to about 500 mg/kg, about 600 mg/kg to about 700 mg/kg, about 800 mg/kg to about 900 mg/kg, or about 950 mg/kg to about 1 gm/kg. One skilled in the art would appreciate that a MEK 3 inhibitor can be administered in a dose that falls within the ranges described above.

[0102] In some embodiments, the dose of a MEK 3 inhibitor is based on determination of maximum tolerated dose and dose limiting toxicity, in a patient cohort.

Selection and Evaluation of Subjects for Administering Pharmaceutical Compositions for Treatment of Obesity-Induced Inflammation or Cancer using MEKK3 inhibitor

[0103] In certain embodiments of the method provided herein, the subject is preselected for administration of MEK 3 inhibitor for treatment of obesity-induced inflammation. In some embodiments, preselection is by assessment of genetic mutations in NBR1-MEKK3-JNK pathway genes. In certain instances, preselection is by assessment of amino acid compositions of the PB1 domains for MEK 3 and NBR1. In other embodiments of the methods provided herein, the methods further comprise evaluating the treated subject, wherein the evaluation comprises determining at least one of: (a) glucose tolerance levels, (b) insulin sensitivity, (c) blood glycated haemoglobin (HbAlC) levels, or (d) blood glucose levels.

[0104] In certain embodiments of the method provided herein, the subject is preselected for administration of MEK 3 inhibitor for treatment of cancer. In further embodiments of the methods provided herein, the subject is preselected for having completed first-line anti-cancer therapy. In other embodiments, subject is preselected for sensitivity to administration of the compound. In certain instances, preselection is by assessment of genetic mutations in MEK 3-p62 pathway genes. In certain instances, preselection is by assessment of genetic mutations in MEK 3 or p62 genes. In certain instances, preselection is by assessment of amino acid compositions of the PB1 domains of MEK 3 and p62.In other embodiments of the methods provided herein, the methods further comprise evaluating the treated subject, wherein the evaluation comprises determining at least one of: (a) tumor size, (b) tumor location, (c) nodal stage, (d) growth rate of the cancer, (e) survival rate of the subject, (f) changes in the subject's cancer symptoms, (g) changes in the subject's S-100B concentration, (h) changes in the subject's S-100B concentration doubling rate, (i) changes in the subject's biomarkers, or (j) changes in the subject's quality of life. Blocking Peptides

[0105] Blocking peptides, also known as mimicking peptides, can mimic the structures of the binding domains within proteins, and competitively inhibit protein-protein interactions. Several methods can be adopted to generate the blocking peptides.

[0106] In some embodiments, the blocking peptides are derived from the interacting domains of NBRl, p62, or MEKK3 proteins. In some embodiments, the blocking peptides are derived from the PB1 domains of NBR2, p62, or MEKK3 proteins.

[0107] In some embodiments, the blocking peptide has an amino acid sequence comprising SEQ ID NO. 1. In some embodiments, the blocking peptide has an amino acid sequence comprising SEQ ID NO. 2. In some embodiments, the blocking peptide has an amino acid sequence comprising SEQ ID NO. 3.

[0108] In some embodiments, derived from NBRl, the blocking peptide has an amino acid sequence comprising SEQ ID NO. 4. In some embodiments, the blocking peptide, derived from NBRl, has an amino acid sequence comprising SEQ ID NO. 5. In some embodiments, derived from NBRl, the blocking peptide has an amino acid sequence comprising SEQ ID NO. 6. In some embodiments, the blocking peptide, derived from NBRl, has an amino acid sequence comprising SEQ ID NO. 7. In some embodiments, the blocking peptide, derived from NBRl, has an amino acid sequence comprising SEQ ID NO. 8. In some embodiments, the blocking peptide, derived from NBRl, has an amino acid sequence comprising SEQ ID NO. 9.

[0109] In some embodiments, the blocking peptide, derived from p62, has an amino acid sequence comprising SEQ ID NO. 10. In some embodiments, the blocking peptide, derived from p62, has an amino acid sequence comprising SEQ ID NO. 11. In some embodiments, the blocking peptide, derived from p62, has an amino acid sequence comprising SEQ ID NO. 12. In some embodiments, the blocking peptide, derived from p62, has an amino acid sequence comprising SEQ ID NO. 13. In some embodiments, the blocking peptide, derived from p62, has an amino acid sequence comprising SEQ ID NO. 14. In some embodiments, the blocking peptide, derived from p62, has an amino acid sequence comprising SEQ ID NO. 15. In some embodiments, the blocking peptide, derived from MEK 3, has an amino acid sequence comprising SEQ ID NO. 16. In some embodiments, the blocking peptide, derived from MEK 3, has an amino acid sequence comprising SEQ ID NO. 17. In some embodiments, the blocking peptide, derived from MEK 3, has an amino acid sequence comprising SEQ ID NO. 18. In some embodiments, the blocking peptide, derived from MEKK3, has an amino acid sequence comprising SEQ ID NO. 19. In some embodiments, the blocking peptide, derived from MEK 3, has an amino acid sequence comprising SEQ ID NO. 20. In some embodiments, the blocking peptide, derived from MEKK3, has an amino acid sequence comprising SEQ ID NO. 21. In some embodiments, the blocking peptide, derived from MEK 3, has an amino acid sequence comprising SEQ ID NO. 22.

[0110] In some embodiments, the blocking peptide can have substitutions in one or more amino acid residues in the peptide sequences comprising SEQ ID NO. 1 - SEQ ID NO. 22. In some embodiments, amino acid substitutions include any naturally occurring or man-made amino acid modifications known or later discovered in the field. In some embodiments, amino acid substitutions include, e.g., substitution, deletion, addition, insertion, etc. of one or more amino acids. In some embodiments, amino acid substitutions include replacing an existing amino acid with another amino acid. In some embodiments, amino acid substitutions include replacing one or more existing amino acids with non-natural amino acids, or inserting one or more non-natural amino acids. In some embodiments, amino acid substitutions that substitute a given amino acid with another amino acid are of similar characteristics. In some embodiments, the substitutions are conservative substitutions. Conservative substitutions include, among the aliphatic amino acids, interchange of alanine, valine, leucine, and isoleucine; interchange of the hydroxyl residues serine and threonine, exchange of the acidic residues aspartate and glutamate, substitution between the amide residues asparagine and glutamine, exchange of the basic residues lysine and arginine, and replacements among the aromatic residues phenylalanine and tyrosine. In some embodiments, amino acid substitutions may be made in 1 or more (e.g, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 20 or more, 40 or more, 50 or more, 70 or more, 80 or more, 90 or more) amino acid residues in the peptide sequences comprising SEQ ID NO. 1 - SEQ ID NO. 22. The one or more amino acid changes can confer various properties to the blocking peptides, e.g., affecting the stability, binding activity and/or specificity, etc.

Blocking Peptide Sequences

[0111] SEQ ID NO. 1:

QVTLNVTFKNEIQSFLVSDPENTTWADIEAMVKVSFDLNTIQIKYLDEENEEVSINSQGEYE

EALKMAVKQGNQLQMQVHEG

SEQ ID NO. 2:

SLTVKAYLLGKEDAAREIRRFSFCCSPEPEAEAEAAAGPGPCERLLSRVAALFPALRPGGFQ

AHYRDEDGDLVAFSSDEELTMAMSYVKDDIFRIYIKEK

SEQ ID NO. 3:

DVRIKFEHNGERRIIAFSRPVKYEDVEHKVTTVFGQPLDLHYMNNELSILLKNQDDLDKAI DILDRS S SMKSLRILLLSQ SEQ ID NO. 4: NTIQIKYLDEENEEVSINSQGEYEEALKMAVKQGNQLQMQVHEG

SEQ ID NO. 5: NTIQIKYLDEENEEVSINSQGEYEEALKMAVKQGN

SEQ ID NO. 6: QVTLNVTFK EIQSFLVSDPENTTWADIEAMVKVSFDL

SEQ ID NO. 7: QVTLNVTFK

SEQ ID NO. 8: NEIQSFLVSDPENT

SEQ ID NO. 9: TWADIEAMVKVSFDL

SEQ ID NO. 10: PGGFQAHYRDEDGDLVAFSSDEELTMAMSYVKDDIFRIYIKEK

SEQ ID NO. 11: PGGFQAHYRDEDGDLVAFSSDEELTMAMSYVKDD

SEQ ID NO. 12: IFRIYIKEK

SEQ ID NO. 13:

SLTVKAYLLGKEDAAREIRRFSFCCSPEPEAEAEAAAGPGPCERLLSRVAALFPALPv

SEQ ID NO. 14: SLTVKAYLLGKE

SEQ ID NO. 15: DAAREIRRFSFCCSPEPEAEAEAAAGPGPCERLLSRVAALFPALR

SEQ ID NO. 16: QPLDLHYMNNELSILLK QDDLDKAIDILDRSSSMKSLRILLLSQ

SEQ ID NO. 17: QPLDLH YMNNELSILLK QDDLDKAIDILDRS S SMK

SEQ ID NO. 18: SLRILLLSQ

SEQ ID NO. 19: DVRIKFEHNGERRIIAFSRPVKYEDVEHKVTTVFG

SEQ ID NO. 20: DVRIKFEHNG

SEQ ID NO. 21: ERRIIAFSRPV

SEQ ID NO. 22: KYEDVEHKVTTVFG

[0112] In some embodiments the blocking peptides are generated using combinatorial screening methods. In some embodiments, the combinatorial screening is carried out by screening of a phage display library. In some embodiments, the combinatorial screening is carried out by yeast two hybrid screening between NBR1, p62, or MEK 3 protein and random peptide or peptide aptamer libraries. In some embodiments, the blocking peptides are generated by screening libraries of naturally occurring peptides.

Pharmaceutical compositions for Treatment of Obesity-Induced Inflammation or Cancer using a Blocking Peptide

[0113] Disclosed herein, in certain embodiments, are pharmaceutical compositions and

formulations comprising: (a) a blocking peptide; and (b) a pharmaceutically-acceptable excipient.

[0114] Disclosed herein, in certain embodiments, are pharmaceutical compositions and

formulations comprising: (a) a blocking peptide that mimics the PB1 domain of NBR1 and blocks the interaction between endogenous NBR1 and MEK 3; and (b) a pharmaceutically-acceptable excipient. [0115] Disclosed herein, in certain embodiments, are pharmaceutical compositions and formulations comprising: (a) a blocking peptide that mimics the PBl domain of MEK 3 and blocks the interaction between endogenous NBRl and MEK 3; and (b) a pharmaceutically- acceptable excipient.

[0116] Disclosed herein, in certain embodiments, are pharmaceutical compositions and

formulations comprising: (a) a blocking peptide that mimics the PBl domain of p62 and blocks the interaction between endogenous p62 and MEK 3; and (b) a pharmaceutically-acceptable excipient.

[0117] Disclosed herein, in certain embodiments, are pharmaceutical compositions and

formulations comprising: (a) a blocking peptide that mimics the PBl domain of MEK 3 and blocks the interaction between endogenous p62 and MEK 3; and (b) a pharmaceutically- acceptable excipient.

[0118] Disclosed herein, in certain embodiments, are pharmaceutical compositions and

formulations comprising: (a) a blocking peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO. 1- SEQ ID NO. 22; and (b) a pharmaceutically-acceptable excipient.

[0119] In some embodiments, the pharmaceutical composition comprising a blocking peptide is contained in nanoparticles. In some embodiments, the nanoparticles containing the pharmaceutical compositions comprising a blocking peptide are fullerenes, liquid crystals, liposome, quantum dots, superparamagnetic nanoparticles, dendrimers, or nanorods. In some embodiments the

pharmaceutical composition comprising a blocking peptide is in liposomes. In some embodiments, the blocking peptides are conjugated to the surface of liposomes. In some embodiments, the blocking peptides are encapsulated within the shell of a liposome. In some embodiments, the liposome is a cationic liposome.

[0120] In some embodiments, the pharmaceutical composition comprising a blocking peptide, selected from a group consisting of peptides that mimic the PBl domains of NBRl, MEK 3, or p62, is contained in nanoparticles. In some embodiments, the nanoparticles containing the pharmaceutical compositions comprising a blocking peptide, selected from a group consisting of peptides that mimic the PBl domains of NBRl, MEK 3, or p62, are fullerenes, liquid crystals, liposome, quantum dots, superparamagnetic nanoparticles, dendrimers, or nanorods. In some embodiments the pharmaceutical composition comprising a blocking peptide, selected from a group consisting of peptides that mimic the PBl domains of NBRl, MEKK3, or p62, is contained in liposomes. In some embodiments, the blocking peptides, selected from a group consisting of peptides that mimic the PBl domains of NBRl, MEK 3, or p62, are conjugated to the surface of liposomes. In some embodiments, the blocking peptides, selected from a group consisting of peptides that mimic the PB1 domains of NBR1, MEK 3, or p62, are encapsulated within the shell of a liposome. In some embodiments, the liposome is a cationic liposome.

[0121] In some embodiments, the pharmaceutical composition comprising a blocking peptide, comprising an amino acid sequence selected from SEQ ID NO. 1- SEQ ID NO. 22, is contained in nanoparticles. In some embodiments, the nanoparticles containing the pharmaceutical compositions comprising a blocking peptide, comprising an amino acid sequence selected from SEQ ID NO. 1- SEQ ID NO. 22, are fullerenes, liquid crystals, liposome, quantum dots, superparamagnetic nanoparticles, dendrimers, or nanorods. In some embodiments the pharmaceutical composition comprising a blocking peptide, comprising an amino acid sequence selected from SEQ ID NO. 1- SEQ ID NO. 22, is contained in liposomes. In some embodiments, the blocking peptide, comprising an amino acid sequence selected from SEQ ID NO. 1- SEQ ID NO. 22, are conjugated to the surface of liposomes. In some embodiments, the blocking peptide, comprising an amino acid sequence selected from SEQ ID NO. 1- SEQ ID NO. 22, are encapsulated within the shell of a liposome. In some embodiments, the liposome is a cationic liposome.

Methods of Administering the Blocking Peptides

[0122] In some embodiments of the methods provided herein, a blocking peptide is administered by an intramuscular, intravenous, subcutaneous, intranodal, intratumoral, intraperitoneal or intradermal injection. In some embodiments, the pharmaceutical composition comprising a blocking peptide is administered by an intramuscular, intravenous, subcutaneous, intranodal, intratumoral, intraperitoneal or intradermal injection.

[0123] In some embodiments of the methods provided herein, a blocking peptide, selected from a group consisting of peptides that mimic the PB1 domains of NBR1, MEK 3, or p62, is administered by an intramuscular, intravenous, subcutaneous, intranodal, intratumoral, intraperitoneal or intradermal injection. In some embodiments, the pharmaceutical composition comprising a blocking peptide is administered by an intramuscular, intravenous, subcutaneous, intranodal, intratumoral, intraperitoneal or intradermal injection.

Methods of treating Cancer using the MEKK3 inhibitors or Blocking Peptides

[0124] Cancers treatable by methods described herein include, but are not limited to, administration of MEKK3 inhibitor or blocking peptide to treat breast cancer, lung cancer, head and neck cancer, brain cancer, abdominal cancer, colon cancer, colorectal cancer, esophageal cancer, parapharyngeal cancer, gastrointestinal cancer, glioma, liver cancer, tongue cancer, neuroblastoma, osteosarcoma, ovarian cancer, renal cancer, pancreatic cancer, retinoblastoma, cervical cancer, uterine cancer, Wilm's tumor, multiple myeloma, skin cancer, lymphoma, leukemia, blood cancer, anaplastic thyroid tumor, sarcoma of the skin, melanoma, adenocystic tumor, hepatoid tumor, non-small cell lung cancer, chondrosarcoma, pancreatic islet cell tumor, prostate cancer including castration resistant forms, ovarian cancer, and/or carcinomas including but not limited to squamous cell carcinoma of the head and neck, colorectal carcinoma, glioblastoma, cervical carcinoma, endometrial carcinoma, gastric carcinoma, pancreatic carcinoma, leiomyosarcoma and breast carcinoma. In some embodiments, the therapies described herein treat prostate cancer.

[0125] The methods described herein treat various stages of cancer including stages which are locally advanced, metastatic and/or recurrent. In cancer staging, locally advanced is generally defined as cancer that has spread from a localized area to nearby tissues and/or lymph nodes. In the Roman numeral staging system, locally advanced usually is classified in Stage II or III. Cancer which is metastatic is a stage where the cancer spreads throughout the body to distant tissues and organs (stage IV). Cancer designated as recurrent generally is defined as the cancer has recurred, usually after a period of time, after being in remission or after a tumor has visibly been eliminated. Recurrence can either be local, i.e., appearing in the same location as the original, or distant, i.e., appearing in a different part of the body. In certain instances, a cancer treatable by methods described herein is unresectable, or unable to be removed by surgery. In further instances, a cancer treatable by the therapies described herein is incurable, i.e., not treatable by current treatment methods.

[0126] In some embodiments, the methods described herein are administered as a first-line or primary therapy, i.e. subjects are treatment naive. Other subjects suitable for treatment by the therapies described herein include those that have completed first-line anti-cancer therapy. First- line anti-cancer therapies include chemotherapy, radiotherapy, immunotherapy, gene therapy, hormone therapy, surgery or other therapies that are capable of negatively affecting cancer in a patient, such as for example, by killing cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or cancer cells, promoting an immune response against cancer cells or a tumor, preventing or inhibiting the progression of cancer, or increasing the lifespan of a subject with cancer.

[0127] In additional embodiments, subjects suitable for treatment by the therapies described herein include those that are administered a MEK 3 inhibitor or a blocking peptide in combination with one or more than one additional therapy selected from chemotherapy, radiotherapy, immunotherapy, gene therapy, hormone therapy, surgery and/or other therapies that are capable of negatively affecting cancer in a patient, such as for example, by killing cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or cancer cells, promoting an immune response against cancer cells or a tumor, preventing or inhibiting the progression of cancer, or increasing the lifespan of a subject with cancer.

[0128] Chemotherapies for first-line and subsequent therapy include, but are not limited to, hormone modulators, androgen receptor binding agents (e.g., anti-androgens, bicalutamide, flutamide, nilutamide, MDV3100), gonadotropin-releasing hormone agonists and antagonists (e.g., leuprolide, buserelin, histrelin, goserelin, deslorelin, nafarelin, abarelix, cetrorelix, ganirelix degarelix), androgen synthesis inhibitors (abiraterone, TOK-001), temozolomide, mitozolomide, dacarbazine, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, anthracyclines (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin), bleomycin, plicomycin, mitomycin, etoposide (VP 16), tamoxifen, raloxifene, estrogen receptor binding agents, cabazitaxel, paclitaxel, gemcitabine, navelbine, farnesyl-protein transferase inhibitors, transplatinum, 5- fluorouracil, capecitabine, vincristin, vinblastin and methotrexate, topoisomerase inhibitors (e.g., irinotecan, topotecan, camptothecin, etoposide) or any derivative related agent of the foregoing. Many of the above agents are also referred to as hormone therapy agents such as, for example, androgen receptor binding agents, gonadotropin-releasing hormone agonists and antagonists, androgen synthesis inhibitors, estrogen receptor binding agents as well as aromatase inhibitors.

[0129] Radiotherapies for first-line and subsequent therapy include factors that cause DNA damage and include what are commonly known as γ-rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors include microwaves and UV- irradiation. It is likely that all of these factors affect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes. Dosage ranges for X-rays may range from daily doses of 50 to 200 roentgens for prolonged periods of time (e.g., 3 to 4 weeks), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.

[0130] Immunotherapies generally rely on the use of immune effector cells and molecules to target and destroy cancer cells. The immune effector may be, for example, a tumor antigen or an antibody specific for some marker on the surface of a tumor cell. The tumor antigen or antibody alone may serve as an effector of therapy or it may recruit other cells to actually affect cell killing. An antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells. Alternatively, a tumor antigen may stimulate a subject's immune system to target the specific tumor cells using cytotoxic T cells and NK cells. Immunotherapies include Sipuleucel-T (Provenge®), bevacizumab and the like.

[0131] A gene therapy includes a therapeutic polynucleotide is administered before, after, or at the same time as a combination therapy. Therapeutic genes may include an antisense version of an inducer of cellular proliferation (oncogene), an inhibitor of cellular proliferation (tumor suppressor), or an inducer of programmed cell death (pro-apoptotic gene).

[0132] Surgery of some type is performed for resectable cancers. Surgery types include preventative, diagnostic or staging, curative and palliative surgery and can be performed as a first- line and subsequent therapy.

[0133] In some embodiments, the MEKK3 inhibitor or blocking peptide administration described herein are administered as a second-line therapy after a first-line therapy becomes ineffective or the cancer is recurrent. In other embodiments, the MEKK3 inhibitor or blocking peptide described herein administered as a third-line therapy after the first- and second-line therapy fails. In further embodiments, individuals are preselected for having completed a first- or second-line therapy.

[0134] In some embodiments, the methods described herein comprise the administration of

MEKK3 inhibitor, selected from the group consisting of PF-03814735, AT9283, Crizotinib (PF- 02341066), Hesperadin, AZD7762, PD 166285, Cdkl/2 Inhibitor III, PP121, BIBF 1120, Bosutinib (SKI-606), Cdk2 Inhibitor IV (NU 6140), BGJ398 (NVP-BGJ398), Dasatinib (BMS-354825), Dovitinib (TKI-258), Sunitinib Malate (Sutent), and WZ3146, for treatment of cancer. The structures for the listed compounds are shown in Table 1.

[0135] In some embodiments, the methods described herein comprise the administration of a blocking peptide comprising an amino acid sequence selected from SEQ ID NO. 2, SEQ ID NO. 3, and SEQ ID NO. 10 - SEQ ID NO. 22, for treatment of cancer.

Certain Definitions

[0136] The terms "blocking peptides" and "mimicking peptides" as used herein refer to peptides that mimic certain domains of proteins or their binding partners and are able to inhibit protein- protein interactions.

[0137] The terms "insulin resistance" and "insulin sensitivity" as used herein, refer to a

physiological condition in which cells fail to respond to the normal actions of the hormone insulin.

[0138] The term "impaired glucose tolerance" as used herein refers to a pre-diabetic state of hyperglycemia that is associated with insulin resistance and increased risk of cardiovascular pathology. EXAMPLES

Example 1: Down regulation of NBR1-MEKK3-JNK signalling cascade results in improved glucose tolerance and insulin resistance, caused by obesity-induced inflammation, in subjects exposed to a high fat diet

Materials and Methods:

[0139] Mice

[0140] NBR1" mice were described previously (Yang et al, 2010). NBR1' mice were bred to LysM-cre mice to generate myeloid-specific NBR1-KO (NBRlmYKO). All genotyping was done by PCR. Animals were maintained under controlled temperature (22.5 °C) and illumination (12 hr dark/light cycle). Mice had free access to water and were fed either standard chow or high fat diets (45%-HFD; D12451 or 60%-HFD, D12492; Research Diets Inc.) ad libitum. To assess glucose tolerance and insulin sensitivity 8 -week-old mice were fed a standard chow or a 60% high fat diet for 4 weeks. Mice were injected intraperitoneally with 2 g glucose/kg body weight after overnight fasting (25% 0-glucose [Fisher Scientific] in 0.9%> saline) for GTT assay. For ITT, mice were injected with 0.5-0.75 U insulin/kg body weight (100 U/ml Novolin R [Novo Nordisk]) after 6 hr fasting. Tail-blood glucose levels were measured by using an ACCU-CHEK Aviva (Roche) glucometer. Insulin concentration was measured in plasma with a kit purchased from Crystal Chem. Measurements of energy expenditure were performed using the indirect calorimetry

Oxymax system of the Comprehensive Lab Animal Monitoring System (CLAMS; Columbus Instruments) at UCSD Animal Care Program in mice fed a 45% HFD for 12 weeks. After adaptation for 24 hr, recordings were collected for 72 hr. The Institutional Animal Care and Utilization Committee approved all procedures, in accordance with the NIH guide for the care and use of laboratory animals.

[0141] Human studies

[0142] The clinical studies were reviewed and approved by the Pennington Biomedical Research Center Institutional Review Board and all subjects provided written confirmation of informed consent.

[0143] Stromal vascular cell isolation

[0144] Epididymal adipose tissue was excised and minced in 10 ml of HBSS solution containing 0.5%

[0145] BSA-Fatty acid free (BSA-FAF). Collagenase II (Sigma C6885, 0.5 mg/ml) was added and the tissue was incubated at 37 °C with shaking (30 min). 10 mM EDTA was added 5 min before the end of the incubation. Larger particles were removed using a 250 prn nylon sieves and the filtrates were centrifuged at 500 g for 5 min to separate floating adipocytes. The pelleted SVCs were suspended in erythrocyte lysis buffer (155 mM NH4CI, 10 niM KHC03, 0.1 niM EDTA) and incubated at room temperature for 5 min. The erythrocyte-depleted SVCs were centrifuged at 500 g for 5 min, and the pellet was suspended in FACS buffer (PBS containing 25 mM HEPES, 2 mM EDTA and 0.5% FBS). Enrichment of F4/80+ cells was performed by magnetic immunoaffinity with F4/80-APC antibody (BM8, eBioscience) and APC positive selection kit (StemCell

Technologies).

[0146] Analysis of tissue sections

[0147] Histology was performed on WAT following fixation in 10% formalin for 24 hr, dehydration, and embedding in paraffin. Sections (5 pm) were cut and stained using hematoxylin and eosin (H&E). For immunohistochemical detection of F4/80, sections were deparaffmized, rehydrated and treated for antigen retrieval. The Vector Mouse on Mouse (M.O.M.)

immunodetection kit was then used according to the manufacturer's protocol (Vector Laboratories). After blocking of endogenous peroxidase activity, the sections were incubated in avidin/biotin blocking solution and M.O.M. mouse Ig-blocking reagent, and then with mouse monoclonal F4180 antibody (#17— '4801, eBioscience) for 30 min. The binding of primary antibody was detected using M.O.M. biotinylated anti-mouse IgG, and visualized using diaminobenzidine as the chromogen. For immunofluorescence, sections were deparafmized as described above and incubated with anti- NBR1 antibody (Thermo Scientific) 1 : 100 and anti-F4/80 (eBioscience) 1 :50, overnight at 4°C. Secondary antibodies were then applied (Alexa fluor 568, Al 1011; Alexa fluor 488, Al 1006) 1 :500 (Invitrogen). Stained sections were examined under an inverted laser scan microscope (LSM 710 NLO, Zeiss, Germany)

[0148] Cell Culture

[0149] Raw and HEK-293T cells were from ATCC. HEK-293 hTLR4A/MD2/CD14 cells were from InvivoGen (San Diego, CA). Cells were cultured in DMEM supplemented with 10%> FCS, 2 mM L-glutamine, 100 U/ml penicillin and 100 pg/ml streptomycin. In the case of the

hTLR4 AIMD2/CD 14 HEK-293 cells, 10 pg/ml blasticidin and 50 pg/ml hygromycin b were added to growth medium as recommended by the manufacturer.

[0150] Preparation of Lentiviruses for the target genes

[0151] shNBRl- and shMEKK3 -encoding plasmids were cotransfected with psPAX2 (Addgene; plasmid 12260) and pMD.2G (Addgene; plasmid 12259) packaging plasmids, into actively growing HEK-293T cells, using FUGENE 6 transfection reagent. Retroviruses for the target genes were made by transfecting pWZL-Flag-NBRl or pWZL-Flag-NBRl(D50R) mutant into actively growing Phoenix cells, using Lipofectamine 2000 transfection reagent. Virus-containing supernatants were collected 24, 48, and 72 hr after transfection, filtered and used to infect target cells in the presence of 10 μ§/ηιΙ, polybrene. Cells were selected after infection, using puromycin or hygromycin. For co-transfection experiments, 0.9 x 10⁶ HEK293T cells were plated into 6cm tissue-culture dishes. After 24 hr, the cells were transfected with 2.5 μg of different expression plasmids in the presence of 15mM calcium chloride and BES-buffered saline (Sigma- Aldrich). DNA in the transfection mixes were brought to the same total amount by adding empty vector. HEK293 hTLR4 A/MD2/ CD 14 cells were transfected with the Flag-NBRl construct at the time of seeding, in 6-cm tissue-culture dishes, with 3 μg of either expression vector or empty vector and the X-tremeGENE HD reagent (ratio 1 : 1). After 48 hr, the cells were washed with PBS and lysed in ice-cold lysis buffer (20 mM Tris-HCL [pH 7.4], 150 mM NaCl, 3 mM EDTA, 3 mM EGTA, 1% Nonident P-40, 10 mM glycerophosphate, 0.3 mM sodium orthovanadate, 2 μΜ PMSF, 1 μg/ml leupeptin, 10 mM sodium fluoride, 10 μg/ml aprotinin and 1 mM DTT. The soluble fraction of each cell lysates were obtained by centrifugation at 13,000 rpm for 15 min. For pull-down assays HA- NBR1 or HA-NBR1(D50R) mutation were over-expressed in HEK293T cells as described above and immunopurified using EZview red anti-HA affinity gel. After 3 washes with lysis buffer, proteins were eluted with 50 μΐ of 0.1M Glycine, pH 2.5 and neutralized with 3 μΐ of 1M Tris-HCl, pH 9.2 to be used in in vitro pull-down assay.

[0152] Bone marrow macrophages

[0153] Bone marrow-derived macrophages (BMDMs) were prepared by crushing mouse leg bones in DMEM supplemented with 20% FBS, 100 U/ml penicillin, 100 pg/ml streptomycin, and 2 mM L-glutamine. After being filtered through a 70 pm nylon mesh, cells were plated in 10 cm dishes and incubated for 24 hr. Supernatants containing bone marrow precursors were collected, centrifuged at 1,500 rpm and cultured in differentiation media (DMEM supplemented with 30% L929 supernatant, 15% fetal bovine serum, 5% horse serum, 100 U/ml penicillin, 100 pg/ml streptomycin, 2 mM L-glutamine, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate and 0.5 mM 13-mercaptoethanol). Polarization studies were performed using BMDMs (8 days in culture) incubated with 100 ng/ml LPS, 0.8 mM Palmitate, 100 ng/ml IFNy (Ml) or 10 ng/ml IL-13 (M2) for the indicated times.

[0154] Retroviral transduction of murine bone marrow

[0155] After 2 days of differentiation adherent and non-adherent BM cells were suspended in virus supernatants containing 5 pg/ml polybrene, and seeded in six -well plates (2 x 106 cells per well). Plates were centrifuged 1 hr at 1000 g and incubated at 37 °C and 5% C02. After 24 hr the transduction was repeated as before, suspending non-adherent cells in new virus supernatants that were combined with the other adherent cells. After 24 hr virus supernatants were replaced with differentiation media and selection antibiotics. Cells were replated after 4 days for the final experiment

[0156] Cytokine secretion

[0157] BMDMs or shNBRl Raw cells were seeded into 6 well plates at 3 x 106 cells/well. The next day, the cells were incubated for 1 hr with serum- free DMEM, followed by 3 hr (BMDMs) or 8 hr (shNBRl Raw cells) with or without 100 ngfml LPS. Supernatants were collected, filtered through a 0.45 pm mesh and subjected to IL-6 measurement using BD OptEIATM Mouse IL-6 [LISA Kit (BD Biosciences). Plasma levels of cytokines (MCP-1, TNFa, IL-6) were subjected to measurement using BD CptEIATM Mouse IL-6 ELISA Kit and Mouse TNFa ELISA kit (BD Biosciences), and eBioscience Mouse MCP-1 ELISA Ready-SET-Go! TM.

[0158] RNA analysis

[0159] Total RNA from mouse tissues and cultured cells was isolated using the TRI reagent (Molecular Research Center) and the RNeasy Mini Kit (Qiagen), followed by DNAse treatment. After quantification using a Nanodrop 1000 spectrophotometer (Thermo Scientific), 1 tag of RNA was reverse-transcribed using random primers and MultiScribe Reverse Transcriptase (Applied Biosystems). Gene expression was analyzed by amplifying 50 ng of the complementary DNA, using the CFX96 Real Time PCR Detection System with SYBR Green Master Mix (BioRad) and primers described in Table 52. The amplification parameters were set at 95 °C for 30 s, 58 °C for 30 s and 72 °C for 30 s (40 cycles total). Gene expression values for each sample were normalized to the 185 RNA.

[0160] Immunoblot analysis

[0161] Protein extracts and immunoprecipitates were separated by SDS-PAGE and transferred to Immobilon-P PVDF membranes (Millipore). After blocking with 5% nonfat dry milk in Tris- buffered saline and 0.1% Tween (TBS-T), the membranes were incubated with the indicated antibodies overnight at 4°C. After 2 hr incubation with the appropriate horseradish peroxidase- conjugated antibodies, the immune complexes were detected by chemiluminescence (Thermo Scientific).

[0162] Immunoprecipitation

[0163] For the immunoprecipitation experiments, 1 to 3 mg of cell lysates were pre-cleared with 30 μΐ of a 50% slurry of protein G or protein A-sepharose (GE healthcare) for 30 min. Then 1 μg of primary antibody or control immunoglobulin (Santa Cruz Biotech) was added to the lysates and incubated with rotation overnight at 4°C. The next day, 30 μΐ of a 50% slurry of protein G- sepharose or protein A-sepharose was added and the incubation was continued for 1 hr. In the case of the GST-pulldown experiments, lysates were pre-cleared with protein A-sepharose, as described above, prior to overnight incubation at 4°C with glutathione sepharose beads. After a 15 minute centrifugation at 2,500 rpm at 4°C, the immunoprecipitates were washed several times with lysis buffer and high salt buffer (HEPES 40 mM, 500 mM NaCl, and 0.5% Triton X-100).

Immunoprecipitated proteins were denatured by adding 10 μΐ of sample buffer followed by boiling for 5 min, and subjected to immunoblotting.

[0164] Pull-down assay

[0165] Recombinant His-MK 4 (5 μg) was immobilized on HisPur Cobalt Resin in 500 μΐ of pulldown buffer (TBS, pH 7.4, 10 mM imidazole). After 3 hr of incubation at 4°C, the resin was washed three times with 1 ml of pull-down buffer, and incubated with purified HA-NBR1, HA- NBR1(D50R) or recombinant GST-MEK 3 for an additional 2hr at 4°C. Samples were washed three times with 500 μΐ of pull-down buffer, once with 20 mM Tris-HCl [pH 7.4], 150 mM NaCl, 3 mM EDTA, 3 mM EGTA, 1% Nonidet P-40, 10 mM imidazole, and once with pull-down buffer. Proteins were eluted with 25 μΐ of TBS, pH 7.4, 200

[0166] MLK3 kinase assay

[0167] BMDM incubated with 0.8 mM of PA for the indicated times were lysed with protein extraction buffer and endogenous MLK3 was immunoprecipitated by using an antibody raised against the C-terminal peptide of MLK3. An in vitro kinase assay was then performed and the incorporation of 32 P into SEK1 (K-R) was detected by autoradiography and normalized with the expression of MLK3 levels.

[0168] Statistical analyses

[0169] Data are presented as the mean ± SEM. Differences between groups were examined for statistical significance using non-parametric Mann- Whitney test. The level of statistical

significance was set at p < 0.05. All error bars represent SEM.

Results:

[0170] High NBR1 levels correlate with obesity in human patients

[0171] A positive and significant statistical correlation (r=0.36, p= 0.003) was found between transcript levels of NBR1 and PPARy from adipose tissue, in a first study, by analyzing the transcript levels of NBR1 and PPARy in a cohort of 63 young and healthy men and women with a wide range of body mass index (BMI) and body fat values. The positive correlation suggested that NBR1 and PPARy could act in the same pathway to control metabolic homeostasis in adipose tissue of obese but generally healthy patients. A second study was conducted using a different cohort of 44 middle-aged men with NCEP defined "metabolic syndrome", characterized by more adipose tissue inflammation than the healthy participants in the cohort described above. Here again, a significant positive correlation was found between transcript levels of NBR1, PPARyl, PPARy2, markers of monocyte and macrophage lineage, such as CD68 and CD 163, transcripts encoding chemokines and pro -inflammatory proteins like MCP-1 ad MIP-1, as shown in Figures 1 A-F. Collectively these results suggested a potential role of NBRl in obesity-induced inflammation.

[0172] NBRl in the myeloid compartment plays a critical role in obesity-induced inflammation

[0173] Based on the potential macrophage based link between NBRl, adipose-tissue inflammation and metabolic syndrome, the next step was to investigate the expression of NBRl in the adipose tissue of obese wild type mice fed a high fat diet (HFD) as compared to lean mice fed a regular chow diet (RD). The NBRl levels were evaluated using double immunofluorescence with the macrophage marker F4/80. Results indicated NBRl co-localization with F4/80 exclusively in crown-like structures (Figure 2A), mostly in the adipose sections from the HFD mice, suggesting that NBRl is specifically expressed in macrophages that infiltrate obese adipose tissues. Consistent with the observation, NBRl mRNA levels were also found to be higher in adipose tissue macrophages (ATM) isolated from obese mice than in lean mice (Figure 2B).

[0174] In order to understand the role of NBRl in adipose-tissue inflammation in vivo, mice were generated in which NBRl was genetically and selectively inactivated in the myeloid cell compartment, following the steps described earlier in the Materials and Methods section. The deletion of NBRl from macrophages was confirmed by western blotting, as shown in Figure 2C.

[0175] The NBRl knockout mice exhibited impaired macrophage recruitment to the adipose tissue as well as reduced levels of inflammatory markers in white adipose tissue (WAT) and adipose tissue macrophages (ATMs), as shown in Figure 2D.

[0176] NBRl is critical for Ml differentiation and macrophage activation ex vivo

[0177] In order to gain a better understanding of the mechanisms whereby NBRl regulates macrophage function in obesity, an ex vivo system was used, wherein bone marrow derived macrophages (BMDMs) were differentiated along the Ml or M2 lineage. BMDMs from NBRl^MyKO and wild type mice were incubated with cytokines that are known to induce differentiation of macrophages along the Ml (induced by IFNy) or M2 lineage (induced by IL-13, IL-14, type 2 cytokines etc.), and afterwards the expression of specific markers of each lineage were determined by qRT-PCR. The results indicated that BMDMs from NBRl^MyKO mice had dramatically reduced ability to respond to IFNy as evidenced by the low levels of induced transcription of Ml lineage specific markers IL-6, TNFa and NOS. The BMDMs from NBRl^MyKO however were sensitive to IL-13, as demonstrated by high levels of induced transcription of M2 lineage specific markers, Argl and Mgl2. The results are shown in Figures 3A and 3B. [0178] The results discussed above are consistent with NBRl being required for the differentiation of macrophages along the Ml, but not the M2 lineage, and therefore being an important transducer of pro-inflammatory signals.

[0179] The reduced ability of BMDMs to induce the production of Ml lineage specific marker IL-6 was confirmed by depleting NBRl from macrophage derived raw cells, using a NBRl specific shRNA (shNBRl). As a control, the raw cells were also exposed to a control shRNA. It was found that depletion of NBRl in the raw cells resulted in impaired production of IL-6 in response to lipopolysachharide (LPS) stimulation, shown in Figure 3C, reinforcing the hypothesis that NBRl plays a critical role in the inflammatory activation of macrophages.

[0180] NBRl regulates the MEKK2/3-MKK4-JNK pathway in macrophages

[0181] To address the signalling pathway whereby NBRl governs macrophage functions, BMDMs were stimulated with LPS for various periods ranging up to 1 hr, and a number of signalling parameters were evaluated. JNK activation was found to be reproducibly inhibited in the NBRl^MyKO BMDMs, however neither the activation of ERK (pER ) nor the phosphorylation and degradation of ΙκΒ were affected, as shown in Figure 4A. NBRl knock-down in the macrophage- derived raw cell lines also led to a suppression of the JNK in response to LPS stimulation, as shown in Figure 4B. Based on these data, it was concluded that NBRl deficiency resulted in impaired JNK activation in macrophages.

[0182] It is known that JNK is the main MAPK activated upon inflammatory stress, is associated with glucose-intolerance in mice fed with HFD, required for the polarization of BMDMs to the proinflammatory MI phenotype, and it's deletion in macrophages reduces ATM infiltration, inflammation and enhances the efficiency of glucose metabolism in the context of obesity.

[0183] Therefore, the results described above, demonstrating that NBRl deficiency resulted in impaired JNK activation in macrophages, and improved glucose tolerance in NBRl^MyKO, established NBRl as a key regulator of JNK function in the context of obesity-induced inflammation.

[0184] The molecular mechanism by which NBRl regulated the activation of JNK was next investigated. The activation of JNK upstream kinase MKK4 was found to be severely suppressed in in NBRl^MyKO BMDMs, but the myeloid specific knockout of NBRl did not have any effect on the activation of MKK3/6, as shown in Figure 4C. This was further corroborated by another experiment, wherein the macrophages were stimulated by palmitate, a saturated fatty acid abundant in obesity, and a robust activation of JNK was seen in wild type cultures but not in the NBRl^MyKO macrophages (Figure 4D). Notably, the synthesis of IL-6 and TNFa was likewise induced by palmitate treatment in wild type but not in the NBRl^MyKO macrophages (Figure 4E). These results strongly suggested that NBRl positively regulated JNK through a direct or indirect interaction with MKK4 and/or any of its upstream activators (i.e., MAP Kinase Kinase Kinases, MAPKKKs).

[0185] PBl mediated scaffold role of NBRl in macrophages

[0186] As NBRl contains a PBl domain, it was reasoned that its most likely immediate target would be a PBl -containing MAPKKK, MEKK2 or MEKK3. It was further hypothesized that such an interaction might involve direct contact between an acidic residue in the NBRl P131 domain and a basic residue in the N-terminal region of the MEKK2 or MEKK3 PBl domain (Figure 5 A). Interestingly, MEKK3 (Figure 5B), similar to NBRl (Figure 2B), was upregulated in ATMs isolated from HFD-fed mice as compared to those from mice fed a regular chow diet.

[0187] To test this possibility, HA-tagged MEKK2 or MEKK3 with Flag-tagged NBRl were ectopically expressed in human HEK-293T cells, and then interactions between NBRl and these kinases by were assessed by immunoprecipitation followed by immunoblotting. The results in Figure 5C showed that, Flag-tagged NBRl was efficiently immunoprecipitated with HA-tagged MEKK3, but this was not the case for HA-tagged MEKK2, establishing that NBRl interacts specifically with MEKK3. Notably, a D50R mutation in the PBl domain of NBRl completely abolished this interaction (Figure 5D). Figure 5E demonstrated that endogenous NBRl and MEKK3 likewise interact physically.

[0188] In agreement with the in vitro results, pull-down of ectopically expressed GST-MKK4 resulted in co-immunoprecipitation of ectopically expressed NBRl and MEKK3 (Figure 5F). This demonstrated that MEKK3 acts as a bridge to bring MKK4 and NBRl together. To test the existence of this complex in an endogenous setting, HEK-293 cells stably expressing hTLR4, MD2 and CD14 were stimulated with LPS, and then endogenous NBRl was immunoprocepitated.

Western blotting revealed that the interaction between NBRl and MEKK3 was not inducible, but the interaction with MKK4 was (Figure 5G). Notably, treatment of BMDM with palmitate resulted in enhanced expression of NBRl and MEKK3 (Figure 6H), in accordance with the data of Figure 2B and 6B in ATMs from obese mice. More importantly, under these conditions a clear

endogenous NBR1-MEKK3 interaction was observed (Figure 5H). Thus, NBRl appeared to function as an organizer of a MEKK3/MKK4 cassette that is required to activate JNK and an obesity-induced inflammatory response.

[0189] Consistent with a functional role for the PBl domains in the interaction between NBRl and MEKK3, it was found that the overexpression of NBRl but not of a PBl mutant (D50R) activated JNK in cotransfection with MEKK3 (Figure 5L). Interestingly, similar results were obtained in palmitate-activated cells (Figure 5M). Furthermore, the re-expression of NBRl WT but not of NBR1-D50R reconstituted JNK activation in response to palmitate in NBRl -deficient BMDMs (Figure 5N).

[0190] NBRl deficiency in macrophages improves metabolism

[0191] In order to address whether the defects in macrophage polarization of mutant mice affects glucose homeostasis, HFD -fed NBRl ^MyKO mice were compared to identically treated WT controls in terms of glucose tolerance and insulin response. As demonstrated in Figure 6A, NBRl deficiency in macrophages resulted in improved glucose clearance in glucose tolerance tests.

Likewise, NBRl deficiency in macrophages resulted in improved insulin responses in insulin tolerance tests (Figure 6B), and activation of Akt in liver, muscle and WAT (Figure 6C). In contrast, no differences were found in glucose tolerance and insulin response assays between both mouse genotypes when fed regular chow diet (Figures 6D and 6E). These findings suggested that a lack of NBRl in the myeloid compartment improved glucose metabolism in the context of chronic HFD exposure. Consistent with this, fasting basal glucose (Figure 6F) and insulin (Figure 6G) levels were significantly reduced in the mutant mice, as were the transcript levels of liver PEPCK and G6Pase (Figure 6H).

Example 2: Down regulation of p62-MEKK3-p38delta-mTORCl signalling cascade results in reduction in size of prostate organoids

Materials and Methods:

[0192] Isolation and culture of prostate epithelial cells

[0193] Murine prostates were isolated from 8 weeks old PTENfl/fl-PBcre male mice and were placed in 5 mg ml-1 collagenase type II in ADMEM/F12 and digested for 1 to 2 hr at 37°C.

Glandular structures were washed with ADMEM/F12 and centrifuged at 100 G. Subsequently structures were digested in 5 ml TrypLE with the addition Y-27632 10 μΜ for 15 min at 37°C. Trypsinized cells were washed and seeded in growth factor reduced Matrigel. Murine prostate epithelial cells were cultured in ADMEM/F12 supplemented with B27, 10 mM HEPES, Glutamax and Penicillin/Streptomycin and contained following growth factors: EGF 50 ng/ml, R-spondinl conditioned medium or 500 ng/ml recombinant R-spondinl, Noggin conditioned medium or 100 ng/ml recombinant Noggin and the TGF-p/Alk inhibitor A83-01. Murine prostate organoids were passaged either via trituration with a glass Pasteur pipet or trypsinization with TrypLE for 5 min at 37°C. Lentiviral infections were performed as described previously using pLKO. l-puro targeting p62, MEKK3, ρ38δ or control scramble. In short, 100,000 single cells were infected with an MOI 1 * 103. Infection was done during centrifugation for 1 hr at 600 G RT. Cells were subsequently placed at 37°C. 5% C02 for 3 hr to recover. Cells were plated in Matrigel and 24 hr post seeding 1 μg/ml puromycin was applied for 2 days to ensure only infected cells remained. [0194] Mammalian lentiviral shRNAs, siRNAs and retroviral transduction

[0195] TRC lentiviral shRNAs targeting human MEK 3 (TRCNOOOOO 10692, TRCN0000002305), human ρ38δ (TRCN0000055428), mouse MEKK3 (TRCN0000025250), mouse p62

(TRCN0000098616) and mouse ρ38δ (TRCN0000023092) were obtained from Sigma. shRNA- encoding plasmids were co-transfected with psPAX2 (Addgene; plasmid 12260) and pMD2.G (Addgene; plasmid 12259) packaging plasmids into actively growing HEK293T cells by using FuGENE 6 transfection reagent. Virus containing supematants were collected 48 hr after transfection, filtered to eliminate cells, and then used to infect target cells in the presence of 8 μg/ml polybrene. Cells were analyzed on the third day after infection. Small interfering RNAs (siRNA) for MAPKs were obtained from Ambion. siRNAs were co-transfected into actively growing cells by using Lipofectamine transfection reagent. Cells were analyzed on the second day after transfection. Retroviruses were produced in Phoenix cells by transient transfection using Lipofectamine. Culture supematants were collected 24, 48, and 72 hr post-transfection, filtered, and supplemented with 8 μg/ml polybrene. Cells were infected with three rounds of viral supematants and selected with hygromycin (75 μg/ml).

[0196] Cell lysis, immunoprecipitations and immunoblotting

[0197] Cells were rinsed once with ice-cold PBS and lysed in ice-cold lysis buffer (40 mM HEPES [pH 7.4], 120 mM NaCl, 1 mM EDTA, 10 mM pyrophosphate, 10 mM glycerophosphate, and 0.3% CHAPS, and one tablet of EDTA- free protease inhibitors [Roche] per 25 ml). The soluble fractions of cell lysates were isolated by centrifugation at 13,000 rpm for 15 minutes. For immunoprecipitations, primary antibodies were added to the lysates and incubated with rotation overnight at 4°C. 40 μΐ of a 50% slurry of protein G-sepharose or protein A-sepharose was then added and the incubation continued for an additional 1 hr. Immunoprecipitates were washed three times with lysis buffer. Cell extracts or immunoprecipitated proteins were denatured by the addition of 20 μΐ of sample buffer and boiling for 5 minutes, resolved by 8%— 14% SDS-PAGE, and then transferred to nitrocellulose-ECL membranes (GE Healthcare). The immune complex was detected by chemiluminescence (Thermo Scientific).

[0198] In vitro kinase-assay and MS/MS phosphopeptide identification

[0199] For in vitro phosphorylation assays, 1 μg of recombinant MBP-p62 was incubated at 30 °C for 60 minutes in kinase assay buffer containing 25 mM Tris-HCl (pH 7.5), 5 mM MgCl₂, 0.5 mM EGTA, 1 mM DTT and 100 μΜ ATP in the presence of recombinant MEKK3 or ρ38δ. For phosphorylation detection by radioactivity, 50 μθ of [γ- 32 P]-ATP were added to the reaction. For ATP analog-based phosphorylation detection, the protocol described previously⁵⁰ was followed with minor modifications. Briefly, 100 μΜ of ATPyS (Bio log) was added to the reaction, after which PNBM (Abeam) and EDTA were added to a final concentration of 2.5 mM and 20 mM, respectively, and incubated for 1 hour at room temperature. Immunoblotting detection was performed with anti-thiophosphate ester antibody from cell signalling.

[0200] Statistical Analysis

[0201] Data are presented as the mean ± SEM. Significant differences between groups were determined using a Student's t-test (two-tailed) when the data meet the normal distribution tested by D'Agostino test. If the data did not meet this test, Mann- Whitney was used. The significance level for statistical testing was set at p < 0.05. All experiments were performed at least two or three times. No statistical method was used to predetermine sample size for the in vitro experiments. Investigators were not blinded to allocation during experiments and outcome assessment. The experiments were not randomized, except the TMA analysis that was performed by Aperio software algorithm.

Results:

[0202] p62 phosphorylation is required for the activation of mTORCl by amino acids

[0203] Several studies have suggested that phosphorylation maybe a mechanism for the control of p62 function. Therefore, it was hypothesized that nutrient-driven p62 phosphorylation might underlie p62-mediated regulation of mTORCl activation in response to amino acids. To address this possibility, HEK293T cells stably expressing Flag-tagged p62 or Flag control were generated. Cells were stimulated with amino acids, after which anti-Flag immunoprecipitates were subjected to in-gel trypsin digestion followed by phosphopeptide enrichment using titanium dioxide columns and liquid chromatography coupled to mass spectrometry (LC-MS). It was found that p62 exhibited low-abundance, baseline phosphorylation at residues S28, T221, and S224 that was not altered by amino acid stimulation. In contrast, phosphorylation at residues T269 and S272 was markedly induced by amino acids. These sites, and their surrounding sequences, were highly conserved across species. To establish the relevance of these phosphorylation events in mTORCl signalling, a phospho-specific antibody generated against the peptide SRLT(P)PVS(P)PES(C) of human p62 was used, which allowed detection of phospho-T269/S272. Interestingly, immunoblotting analysis revealed a strong phosphorylation of T269/S272 in p62 upon the stimulation of HEK293T cells with amino acids (Figure 7A). Mutation of these sites to alanine (p62T269/S272AA) abolished p62 phosphorylation, demonstrating that these are bona fide nutrient-sensitive p62 phosphorylation residues (Figure 7B). To test the functional relevance of these phosphorylations, Flag-tagged p62WT or Flag-tagged p62T269/S272AA were stably expressed in HEK293T cells, after which cells were treated with amino acids, or vehicle control, at different times, and the activation of downstream targets of mTORCl was determined by immunoblotting. Results shown in Figure 7C demonstrate that mTORC 1 activation by amino acids was markedly reduced in cells that expressed the p62T269/S272AA mutant, as compared to cells expressing p62WT. Interestingly, insulin- induced mTORC 1 activation was independent of p62 phosphorylation (Figure 7D), indicating that this selectively regulates mTORC 1 activity in response to amino acids. Furthermore, expression of the p62T269/S272AA mutant severely abrogated the interaction of p62 with different components of the mTORC 1 complex, including mTOR, raptor, and TRAF6 in response to amino acids (Figure 7E), in keeping with the notion that p62 phosphorylation is a key event to orchestrate the nutrient- induced complex.

[0204] Important role of MEKK3 in mTORCl activation by amino acids

[0205] Based on previous evidence which strongly suggested that MEK 3 selectively binds the PB1 domain of p62, it was hypothesized that MEK 3 is required for the activation of mTORCl, that it acts as a partner of p62 in this pathway, and that it is involved, directly or indirectly, in the phosphorylation of p62 in response to amino acids.

[0206] Lentiviral infection with a specific short hairpin RNA (shRNA) was used to knock down MEK 3 in HEK293T cells, and a non-targeting (shNT) lentivirus was used as control. Cells were starved of serum and amino acids, and then stimulated with amino acids for different durations. Notably, the knockdown of MEK 3 severely impaired S6K and 4EBP1 phosphorylation in response to amino acids (Figure 7F). Subsequently it was determined whether the kinase activity of MEK 3 is required to promote mTORCl activation. HEK293T cells were transfected with either WT HA-tagged MEK 3 or a kinase-dead mutant (HA-MEK 3-KD), and stimulated with amino acids as above. Of note, the expression of the inactive MEK 3 mutant severely impaired the amino acid- stimulated activation of mTORCl (Figure 7G) but not that by insulin (Figure 7H).

Furthermore, endogenous immunoprecipitation experiments showed the recruitment of MEK 3 to the mTOR complex in response to amino acids (Figure 71). These results indicate that MEK 3, like p62 and TRAF6, is also a component of the mTORCl complex and selectively regulates its activation in the amino acid-response pathway.

[0207] MEKK3 is a critical kinase for p62 phosphorylation by amino acids

[0208] Since MEK 3 and the phosphorylation of p62 are two important events for mTORC 1 activation by amino acids, it seemed conceivable that MEK 3 might directly or indirectly regulate p62 T269/S272 phosphorylation. In agreement with this hypothesis, MEKK3 overexpression but not that of a kinase-dead mutant was able to induce the phosphorylation of p62 at T269/S272 but not of the p62 non-phosphorylatable mutant (Figures 8A-B), which correlated with mTORCl activation (Figure 8B). Furthermore, p62 phosphorylation was severely impaired in HEK293T cells with MEK 3 knocked down (Figure 8C). These results demonstrate that p62 is phosphorylated in response to amino acids through a MEK 3 -dependent mechanism, and that this event is critical for mTORCl activation.

[0209] The role of the PB1 domain interaction was next tested in studying the in the ability of MEK 3 to promote p62 phosphorylation. The rationale for this experiment was based on the fact that the acidic region of the p62 PB1 domain interacts with the basic region of the MEK 3 PB1 (Figure 8D). Therefore, D69A/D73A double mutation in the PB1 domain of p62 was generated, which suppresses p62-MEK 3 interaction, and determined the effect of this mutation on p62 phosphorylation induced by MEK 3 overexpression. Interestingly, not only was MEK 3 overexpression unable to promote the phosphorylation of the D69A/D73A p62 mutant (Figure 8E) but, more importantly, this could not be phosphorylated upon amino acid stimulation (Figure 8F). Collectively, these results establish that the physical interaction between p62 and MEKK3 through their respective PB1 domains is required for p62 phosphorylation in response to amino acids. Based on these results, it is possible that p62 could be targeted directly by MEKK3. However, when bacterially expressed recombinant p62 was incubated with active recombinant MEK 3 in an in vitro kinase assay, we found that MEK 3 was not able to directly phosphorylate p62 (data not shown). These results strongly suggest the existence of another/other kinase(s) that act(s) downstream of MEK 3 to phosphorylate p62 in response to amino acids.

[0210] MEK3/6-p38delta channels MEKK3 induced phosphorylation of p62 by amino acids

[0211] To identify the kinase, it was reasoned that since MEK 3 is a MAP3K, it is possible that a MAP2K/MAPK cascade could act downstream of MEK 3 in the direct phosphorylation of p62. To address this possibility, we the five distinct groups of MAPKs characterized in mammals were knocked down. These were the extracellular signal -regulated kinases ER 1 , ER 2, ER 3, ER 4, ER 5 and ER 8; the c-Jun amino-terminal kinases XNKl, XNK2, and XNK3; the p38 isoforms α, β, γ, and δ; and NLK. A mini-screening was used with short interfering RNAs (siRNA) specific for each MAPK and a non-targeting siRNA as control. Cells were stimulated with amino acids, as above, and the activation of mTORCl was determined. Notably, only depletion of ρ38δ (MAPK13) severely impaired amino acid-induced activation of mTORCl, suggesting that ρ38δ is likely the direct p62 kinase in the MEK 3 pathway (Figure 9A). Importantly, knocked down of ρ38δ by using a specific shRNA completely abolished p62 phosphorylation and mTORCl activation in cells stimulated with amino acids (Figure 9B). Interestingly, it was also found that the overexpression of ρ38δ was able to induce the phosphorylation of p62 at T269/S272 in p62WT but not in

p62T269/S272AA (Figure 9C). However, the expression of a kinase-inactive ρ38δ (T180A/Y182F) mutant did not promote p62 phosphorylation (Figure 9D). Taken together, these results demonstrate that ρ38δ is responsible for p62 phosphorylation and mTORCl activation by amino acids. To determine whether p62 is actually a direct substrate of ρ38δ, bacterially expressed recombinant p62 was incubated with active ρ38δ in an in vitro kinase assay, and it was found that ρ38δ directly phosphorylated p62 at T269/S272 (Figure 9E). To determine whether these residues account for p62 phosphorylation by ρ38δ, purified p62WT and p62T269/S272AA were phosphorylated in vitro with ATP- γ -S and recombinant active ρ38δ. Results of Figure 9F demonstrate that p62 phosphorylation by ρ38δ was completely abolished in the p62T269/S272AA mutant as compared to p62WT, indicating that ρ38δ is a bona fide direct p62 T269/S272 kinase.

[0212] To determine whether ρ38δ is be activated by amino acids in a MEK 3 -dependent manner, HEK293T cells were transfected with Flag-tagged ρ38δ, after which cells were treated with amino acids, or vehicle control, at different times as described above. Transfected ρ38δ was

immunoprecipitated with an anti-Flag antibody, and its ability to phosphorylate recombinant p62 was determined in an in vitro kinase assay. Interestingly, ρ38δ from shNT cells that were stimulated with amino acids displayed higher enzymatic activity towards recombinant p62 than ρ38δ from unstimulated shNT cells (Figure 9G). The finding that amino acid stimulation did not increase the activity of ρ38δ in shMEK 3 cells (Figure 9H) clearly established that ρ38δ is a critical downstream target of MEK 3 in the nutrient-sensing cascade that activates mTORCl through p62 phosphorylation. To further determine the upstream kinases for this pathway, MEK3 and MEK6 were simultaneously knocked down in HEK293T, after which cells were stimulated by amino acids. Notably, the simultaneous depletion of MEK3 and MEK6 severely impaired amino acid-induced mTORCl activation and p62 phosphorylation (Figure 91). These data demonstrate that MEKK3 is the apical kinase in an amino acid-sensing cascade that includes MEK3/MEK6 and ρ38δ, and that leads to p62 phosphorylation, which is a critical step for mTORCl activation in response to amino acids.

[0213] The MEKK3/p38delta cascade contributes to cell proliferation and autophagy through mTORCl activation

[0214] Since mTORCl activation promotes cell proliferation and transformation while inhibiting autophagy, it was hypothesized that MEK 3 and ρ38δ are key upstream regulators of mTORCl 's effects on these two key cellular functions. In keeping with this notion, it was found that the knockdown of MEKK3 or ρ38δ in PC3 prostate cancer cells significantly reduced cell proliferation under normal growing conditions (Figures 10A-B), which was rescued by the expression of a constitutively active mutant of RagB, which bypasses the requirement of p62 for the activation of mTORCl (Figures 10C-D). It is known that nutrient starvation induces autophagy through inhibition of mTORC 1 and knockdown of p62 or TRAF6 is also known to promote autophagy in response to nutrient deprivation. Interestingly, the knockdown of MEK 3 or ρ38δ synergistically enhanced LC3 processing, which was even more apparent when cells were incubated with bafilomycin Al, an inhibitor of autophagosomal and lysosomal fusion (Figures 10E-H).

Autophagic flux using the reporter GFP-mCherry-LC3, which allows the identification of autolysosomes (mCherry positive/GFP negative; red dots) and autophagosomes (mCherry- positive/GFP-positive; yellow dots) was also analyzed. The total number of autophagosomes and autolysosomes under basal and amino acid starvation were higher in the MEK 3- and p3 SB- deficient cells (Figures 101- J). Altogether, these results demonstrated that the ΜΕΚ 3/ρ38δ cascade modulates autophagy in response to nutrient starvation, consistent with its role in the regulation of mTORCl activation.

[0215] Critical role of the MEKK3/p38delta/p62/mTOR axis in prostate cancer

[0216] To investigate the relevance of the ρ62/ΜΕΚΚ3/ρ38δ cascade in the activation of mTOR in prostate cancer, prostate 3D organoid cultures were used. Using the system, prostate epithelial cells were isolated from PTEN-deficient mice, and subjected to lentiviral infection to selectively knockdown MEK 3, ρ38δ or p62 and the cells were cultured in the 3D organoid conditions.

Interestingly, it was found that the inactivation of MEK 3, ρ38δ or p62 led to decreases in the number and the size of prostate organoids (Figures 11 A-B), strongly suggesting an important role of the ΜΕΚΚ3/ρ38δ cascade in prostate cancer. Notably, the knockdown of p62, MEK 3 or ρ38δ severely impaired S6K and 4EBP1 phosphorylation in this model (Figure 11C).

[0217] To assess the relevance of these findings to human disease, we analysed the levels of the MEK 3/p386/p62/mTOR cascade in a human prostate tissue microarray (TMA). Stainings for MEK 3, ρ38δ, p62 and phospho-S6 were much stronger in aggressive tumours with high Gleason score (GS >6) than in low Gleason score (GS <6) samples (Figures 11D-E). Importantly, correlation analysis demonstrated that MEKK3 and ρ38δ expression in human prostate tumours correlated strongly with p62 and phospho-S6 (Figure 1 IF).

[0218] To further extend these observations, human prostate cancer datasets were interrogated. Analysis of a dataset from the University of Michigan (GSE35988), including matched benign prostate tissues (n=28) and localized prostate cancer (n=59), demonstrated that ρ38δ, p62, and mTOR were significantly upregulated in tumours compared with normal tissue. Similar results were obtained using a different dataset from MSKCC Prostate Oncogenome Project (GSE21032). In this study, 181 primary tumour samples, 37 metastatic prostate cancer samples, and 12 prostate cancer cell lines and xenografts were analysed. A positive and significant correlation was found between p62 gene-expression levels and the levels of the ΜΕΚ 3/ρ38δ and mTOR in primary tumours, which demonstrated that p62 and the ΜΕΚ 3/ρ38δ cascade act in the same pathway to regulate mTOR signalling. Taken together, these results established that the PB1 -driven MEK 3/p385/p62/mTOR kinase pathway is relevant to prostate cancer.

Example 3: Specificity of Inhibitors towards MEKK3 and determination of IC50 values

[0219] Small molecule inhibitors were screened for activity against target kinase MEK 3 and another kinase TAOK3, using a TR-FRET (time resolved-fluorescence resonance energy transfer) based HTRF® Transcreener® ADP Assay (Cisbio Assays). The IC₅₀ values for the inhibitors, against MEK 3 and TAOK3 (serine/threonine protein kinase TA03) are listed in Table 1.

Table 1: MEKK3 Inhibitors

Example 4: Improved glucose tolerance and insulin resistance by inhibiting the NBR1- MEKK3-JNK signalling cascade using a MEKK3 inhibitor

[0220] A patient cohort, exposed to high-fat diet, is administered a therapeutically effective dose of a pharmaceutical composition comprising a MEK 3 inhibitor and the following parameters are measured post-administration: (a) glucose tolerance and (b) insulin resistance. It is observed that administration of a MEK 3 inhibitor, leading to inhibition of the NBR1-MEKK3-JNK signalling pathway, is effective in improving glucose tolerance and reducing insulin sensitivity. The MEK 3 inhibitor for the study is selected from the compounds listed in Table 1.

Example 5: Reduction in tumor volume by inhibiting the p62-MEKK3-p38 delta-mTORCl signalling cascade using a MEKK3 inhibitor

[0221] A patient cohort, suffering from cancer and exposed to nutrient abundance, is administered a therapeutically effective dose of a pharmaceutical composition comprising a MEK 3 inhibitor and the tumor volumes are measured post-administration. It is observed that administration of a MEK 3 inhibitor, leading to inhibition of the p62-MEK 3-p385-mTORCl signalling pathway, is effective in reducing tumor volumes. The MEK 3 inhibitor for the study is selected from the compounds listed in Table 1. Example 6: Improved glucose tolerance and insulin resistance by inhibiting the NBR1- MEKK3 protein-protein interaction using a blocking peptide

[0222] A patient cohort, exposed to high-fat diet, is administered a therapeutically effective dose of a pharmaceutical composition comprising a blocking peptide and the following parameters are measured post-administration: (a) glucose tolerance and (b) insulin resistance. It is observed that administration of a blocking peptide, leading to inhibition of the protein-protein interaction between NBR1 and MEK 3, is effective in improving glucose tolerance and reducing insulin sensitivity. The blocking peptide for the study has an amino acid sequence selected from SEQ ID NO. 1, SEQ ID NO. 3 - SEQ ID NO. 9, and SEQ ID NO. 16 -SEQ ID NO. 22.

Example 7: Reduction in tumor volume by inhibiting the p62-MEKK3 protein-protein interaction using a blocking peptide

[0223] A patient cohort, suffering from cancer and exposed to nutrient abundance, is administered a therapeutically effective dose of a pharmaceutical composition comprising a blocking peptide and the tumor volumes are measured post-administration. It is observed that administration of a blocking peptide leading to inhibition of the protein-protein interaction between p62 and MEKK3, is effective in reducing tumor volumes. The blocking peptide for the study has an amino acid sequence selected from SEQ ID NO. 2, SEQ ID NO. 3, and SEQ ID NO. 10 -SEQ ID NO. 22.

[0224] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A method for treating obesity-induced inflammation in a subject, the method comprising administering to the subject a MEK 3 inhibitor.

2. The method of claim 1, wherein the MEK 3 inhibitor is selected from a group

762),

(PD 166285), 6 ^: (Cdkl/2 Inhibitor III ,

(PP121), (BIB

Cdk2 Inhibitor IV (NU 6140),

Dovitinib (TKI-258), Malate (Sutent), and

3. The method of claim 1, wherein the MEKK3 inhibitor is selected from a group

consisting

Crizotinib

(PF-02341066), PP121 , and

4. The method of claim 1, wherein the subject is exposed to a high fat diet.

5. The method of claim 4, wherein the subject has an elevated expression level of NBRl in macrophages.

6. The method of claim 5, wherein the elevated expression level of NBRl in macrophages is in macrophages which infiltrate adipose tissue.

7. The method of claim 6, wherein the elevated level of NBRl leads to activation of JNK cascade.

8. The method of claim 7, wherein the MEKK3 inhibitor blocks activation of JNK

cascade.

9. The method of claim 8, wherein the MEKK3 inhibitor further blocks Ml polarization of macrophages.

10. The method of claim 4, wherein the subject has impaired glucose tolerance.

11. The method of claim 4, wherein the subject has insulin resistance.

12. The method of claim 10, wherein the administration of MEK 3 inhibitor results in improved glucose tolerance.

13. The method of claim 11, wherein the administration of MEK 3 inhibitor results in reduced insulin resistance.

14. The method of claim 4, wherein the subject is pre-diabetic.

15. The method of 14, wherein the administration of MEK 3 inhibitor is effective in preventing the onset of type 2 diabetes.

16. A method for treating cancer in a subject, the method comprising administering to the subject a MEKK3 inhibitor.

17. The method of claim 16, wherein the MEK 3 inhibitor is selected from a group

(Cdkl/2 Inhibitor III), (PP121),

sutinib (SKI-606),

hibitor IV (NU 6140),

Dasatinib (BMS

-354825), Dovitinib (TKI-258), itinib Malate (Sutent), and

18. The method of claim 16, wherein the MEK 3 inhibitor is selected from a

PP121 , and

19. The method of claim 16, wherein the subject has an elevated level of p62.

20. The method of claim 19, wherein the elevated level of p62 leads to activation of

mTORCl, by phosphorylation of p62 by ρ38δ.

21. The method of claim 20, wherein the administration of MEK 3 inhibitor blocks the activation of mTORCl by blocking the phosphorylation of p62 by ρ38δ.

22. The method of claim 21, wherein the method comprises administering to the subject a MEKK3 inhibitor, wherein the subject is suffering from prostate cancer.

23. The method of claim 22, wherein the administration of MEKK3 leads to reduction in tumor volume.

24. A method of treating obesity-induced inflammation in a subject, the method comprising administering to the subject an agent that is capable of blocking the protein -protein interaction between NBRl and a MEK kinase.

25. The method of claim 24, wherein the subject is exposed to a high fat diet.

26. The method of claim 25, wherein the subject has an elevated expression level of NBRl in macrophages.

27. The method of claim 26, wherein the elevated expression level of NBR in macrophages is in macrophages which infiltrate adipose tissue.

28. The method of claim 27, wherein the elevated level of NBRl leads to activation of JNK cascade.

29. The method of claim 28, wherein the administration of an agent that is capable of

blocking the protein-protein interaction between NBRl and a MEK kinase blocks the activation of JNK cascade.

30. The method of claim 25 wherein the subject has impaired glucose tolerance.

31. The method of claim 25, wherein the subject has insulin resistance.

32. The method of claim 30, wherein the administration of an agent that is capable of

blocking the protein-protein interaction between NBRl and a MEK kinase results in improved glucose tolerance.

33. The method of claim 31, wherein the administration of an agent that is capable of

blocking the protein-protein interaction between NBRl and a MEK kinase results in reduced insulin resistance.

34. The method of claim 25, wherein the subject is pre-diabetic.

35. The method of 34, wherein the administration of an agent that is capable of blocking the protein-protein interaction between NBRl and a MEK kinase is effective in preventing the onset of type 2 diabetes.

36. The method of claim 24, wherein the agent is a blocking peptide.

37. The method of claim 36, wherein the blocking peptide has an amino acid sequence selected from SEQ ID NO. 1, SEQ ID NO. 3 - SEQ ID NO. 9, and SEQ ID NO. 16 - SEQ ID NO. 22

38. The method of claim 24, wherein the MEK kinase is MEKK3.

39. A method of treating cancer in a subject, the method comprising administering to the subject an agent that is capable of blocking the protein-protein interaction between p62 and a MEK kinase.

40. The method of claim 39, wherein the subject has an elevated expression level of p62.

41. The method of claim 40, wherein the elevated level of p62 leads to activation of

mTORCl cascade.

42. The method of claim 41, wherein administration of an agent that is capable of blocking the protein-protein interaction between p62 and a MEK kinase blocks the activation of mTORCl cascade.

43. The method of claim 39, wherein said agent is a blocking peptide.

44. The method of claim 43, wherein the blocking peptide has an amino acid sequence selected from SEQ ID NO. 2, SEQ ID NO. 3, and SEQ ID NO. 10 -SEQ ID NO. 22

45. The method of claim 39, wherein the MEK kinase is MEKK3.

46. A peptide comprising an amino acid sequence selected from SEQ ID NO. 1 , SEQ ID NO. 3 - SEQ ID NO. 9, and SEQ ID NO. 16 -SEQ ID NO. 22, wherein the peptide is capable of blocking the protein-protein interaction between NBR1 and a MEK kinase.

47. The peptide of claim 46, wherein the peptide has an amino acid sequence of SEQ ID NO. 5 or SEQ ID NO. 20.

48. The peptide of claim 46, wherein the peptide is capable of blocking the protein-protein interaction between NBR1 and a MEK kinase, wherein said MEK kinase is MEKK3.

49. The peptide of claim 48, wherein the peptide is capable of blocking the protein-protein interaction between PB1 domain of NBR1 and PB1 domain of MEKK3.

50. The peptide of claim 49, wherein the peptide is capable of blocking the protein-protein interaction between an acidic residue on the PB1 domain of NBR1 and a basic residue on the PB1 domain of MEKK3.

51. The peptide of claim 50, wherein the peptide is capable of blocking the protein-protein interaction between an acidic residue on the PB1 domain of NBR1 and a basic residue on the PB1 domain of MEKK3, wherein said basic residue is on the N-terminal region of the PB1 domain of MEKK3.

52. The peptide of claim 51, wherein the peptide is capable of blocking the protein-protein interaction between an asparagine residue or a glutamic acid residue on the PB1 domain of NBR1 and a lysine residue on the PB1 domain of MEKK3.

53. The peptide of claim 46, wherein said peptide is cell permeable.

54. The peptide of claim 46, wherein length of the peptide is between 5 and 100 amino acids.

55. The peptide of claim 46, wherein length of the peptide is between 35 to 50 amino acids.

56. The peptide of claim 46, wherein length of the peptide is between 9 to 15 amino acids.

57. A pharmaceutical composition for treating obesity-induced inflammation, comprising a peptide according to claim 46 and a pharmaceutically acceptable carrier.

58. A method of treating obesity-induced inflammation in a subject, the method comprising administering to the subject a pharmaceutical composition according to claim 46.

59. A peptide comprising an amino acid sequence selected from SEQ ID NO. 2, SEQ ID NO. 3, and SEQ ID NO. 10-SEQ ID NO. 22, wherein the peptide is capable of blocking the protein-protein interaction between p62 and a MEK kinase.

60. The peptide of claim 59, wherein the peptide has an amino acid sequence of SEQ ID NO. 11 or SEQ ID NO. 20.

61. The peptide of claim 59, wherein the peptide is capable of blocking the protein-protein interaction between p62 and a MEK kinase, wherein said MEK kinase is MEKK3.

62. The peptide of claim 61, wherein the peptide is capable of blocking the protein-protein interaction between PB1 domain of p62 and PB1 domain of MEKK3.

63. The peptide of claim 61, wherein the peptide is capable of blocking the protein-protein interaction between an asparagine residue or a glutamic acid residue on the PB1 domain of p62 and a lysine residue on the PB1 domain of MEKK3.

64. The peptide of claim 59, wherein said peptide is cell permeable.

65. The peptide of claim 59, wherein length of the peptide is between 5 and 100 amino acids.

66. The peptide of claim 59, wherein length of the peptide is between 30 to 60 amino acids.

67. The peptide of claim 59, wherein length of the peptide is between 9 to 12 amino acids.

68. A pharmaceutical composition for treating cancer, comprising a peptide according to claim 59 and a pharmaceutically acceptable carrier.

69. A method of treating cancer in a subject, the method comprising administering to the subject a pharmaceutical composition according to claim 59.