Camkk-beta as a target for treating cancer

Abstract

Provided herein are compounds, compositions, including pharmaceutical compositions, having anti-cancer activity. Also provided are methods for diagnosing, detecting, and treating cancer in a subject, as well as a method for evaluating cancer stage in a subject, wherein the methods include determining the amount of a Ca 2+ /calmodulin dependent kinase kinase (CaMKK) in a sample. Further provided are methods of screening and identifying a compound that inhibits CaMKK.

1 . A method of diagnosing prostate cancer in a subject comprising:
 a) obtaining a sample from the subject;   b) determining an amount of at least one of CaMKKβ, CaMKKβ splice variant 2, CaMKKβ splice variant 7, phosphorylated AMPK, phosphorylated AMPKα1 subunit, and phosphorylated AMPKα2 subunit in the sample from the subject; and   c) comparing the amount of at least one of CaMKKβ, CaMKKβ splice variant 2, CaMKKβ splice variant 7, phosphorylated AMPK, phosphorylated AMPKα1 subunit, and phosphorylated AMPKα2 subunit from the sample from the subject to an amount of the CaMKKβ, CaMKKβ splice variant 2, CaMKKβ splice variant 7, phosphorylated AMPK, phosphorylated AMPKα1 subunit, and phosphorylated AMPKα2 subunit in a control sample;   wherein the subject is diagnosed as having prostate cancer when the amount of at least one of CaMKKβ, CaMKKβ splice variant 2, CaMKKβ splice variant 7, phosphorylated AMPK, phosphorylated AMPKα1 subunit, and phosphorylated AMPKα2 subunit in the sample from the subject is greater than the amount in the control sample.   
     
     
         2 . The method of  claim 1 , wherein the determining comprises detecting the amount of mRNA expression. 
     
     
         3 . The method of  claim 1 , wherein the determining comprises detecting the amount of at least one protein of CaMKKβ, CaMKKβ splice variant 2, CaMKKβ splice variant 7, phosphorylated AMPK, phosphorylated AMPKα1 subunit, and phosphorylated AMPKα2 subunit in the samples. 
     
     
         4 . The method of  claim 1 , wherein the sample from the subject comprises prostate tissue. 
     
     
         5 . The method of  claim 3 , wherein the detecting comprises an antibody that specifically binds to a C-terminal portion of the amino acid sequence encoded by CaMKKβ, CaMKKβ splice variant 2, or CaMKKβ splice variant 7. 
     
     
         6 - 16 . (canceled) 
     
     
         17 . A method of screening a test compound for its anti-cancer activity comprising:
 i) contacting CaMKKβ and a substrate therefor in the presence of the test compound, under conditions that allow for CaMKKβ-dependent phosphorylation of the substrate, and   ii) determining the level of phosphorylation of the substrate resulting from step (i) and comparing the level with a level of phosphorylation of the substrate obtained in the absence of the test compound, wherein a reduction in the level of phosphorylation of the substrate in the presence of the test compound indicates that the test compound has said anti-cancer activity.   
     
     
         18 . The method according to  claim 17  wherein said substrate is a peptide substrate. 
     
     
         19 . The method according to  claim 17  wherein the CaMKKβ and the substrate are present in a cell free system. 
     
     
         20 . The method according to  claim 17  wherein the CaMKKβ and the substrate are present in a cell. 
     
     
         21 . The method according to  claim 17  wherein, in step (i), the CaMKKβ and the substrate are contacted with the test compound in the presence of calmodulin and calcium under conditions such that CaMKKβ-dependent phosphorylation of the substrate can be effected. 
     
     
         22 - 26 . (canceled) 
     
     
         27 . A method of treating prostate cancer in a subject, comprising administering to the subject an effective amount of a compound that inhibits activity of at least one of CaMKKβ, CaMKKβ splice variant 2, or CaMKKβ splice variant 7. 
     
     
         28 . The method of  claim 27 , wherein the compound is selected from an inhibitory RNA or an antibody that specifically binds to a C-terminal portion of CaMKKβ, CaMKKβ splice variant 2, or CaMKKβ splice variant 7. 
     
     
         29 . The method of  claim 27 , wherein the compound has Formula III: 
       
         
           
           
               
               
           
         
         wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 7a , R 8 , R 9 , R 10 , and R 11  are each independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, cycloalkylalkenyl, cycloalkylalkynyl, heterocyclo, heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl, aryl, arylalkyl, arylalkenyl, arylalkynyl, heteroaryl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, alkoxy, halo, mercapto, azido, cyano, formyl, carboxylic acid, hydroxyl, nitro, acyl, aryloxy, alkylthio, amino, alkylamino, arylalkylamino, disubstituted amino, acylamino, acyloxy, ester, amide, sulfoxyl, sulfonyl, sulfonate, sulfonic acid, sulfonamide, urea, alkoxylacylamino, and aminoacyloxy; or a pharmaceutically acceptable salt or prodrug thereof. 
       
     
     
         30 . The method of  claim 29 , wherein the compound is STO-609. 
     
     
         31 . The method of  claim 1 , wherein the method further comprises administering an effective amount of a second agent selected from anti-androgens, Selective Androgen Receptor Modulators (SARMs), Selective Androgen Receptor Degraders (SARDs), CYP17 inhibitors, suphatase inhibitors, Src inhibitors, anti-estrogens, estrogens, Selective Estrogen Receptor Modulators (SERMs), Selective Estrogen Receptor Degraders (SERDs), ERb antagonists, aromatase inhibitors, and a vaccine. 
     
     
         32 . The method of  claim 31 , wherein the second agent is selected from MDV3100, ARN-509, bicalutamide, and flutamide. 
     
     
         33 - 41 . (canceled) 
     
     
         42 . A method of inhibiting proliferation of a prostate cancer cell in a subject, the method comprising administering to the subject an effective amount of an inhibitor of AMPK, AMPKα1 subunit, or AMPKα2 subunit, or any combination thereof. 
     
     
         43 . The method of  claim 42  wherein the inhibitor is an inhibitory RNA. 
     
     
         44 . A method of determining the efficacy of therapy in a patient being treated for prostate cancer, the method comprising:
 a) obtaining a series of samples from the subject;   a) determining an amount of at least one of CaMKKβ, CaMKKβ splice variant 2, CaMKKβ splice variant 7, phosphorylated AMPK, phosphorylated AMPKα1 subunit, and phosphorylated AMPKα2 subunit in the series of samples from the subject, where the samples are taken from the subject at different time points during the therapy; and   b) comparing the amount of at least one of CaMKKβ, CaMKKβ splice variant 2, CaMKKβ splice variant 7, phosphorylated AMPK, phosphorylated AMPKα1 subunit, and phosphorylated AMPKα2 from the series of samples from the subject;   wherein when the amount of at least one of CaMKKβ, CaMKKβ splice variant 2, CaMKKβ splice variant 7, phosphorylated AMPK, phosphorylated AMPKα1 subunit, and phosphorylated AMPKα2 in the series of samples is about the same or increases the therapy is determined to be not effective.   
     
     
         45 - 47 . (canceled) 
     
     
         48 . A nucleic acid molecule comprising a sequence that binds under stringent conditions to a region that is about 2.3 kb upstream (5′) relative to a CaMKKβtranscriptional start site. 
     
     
         49 . The nucleic acid molecule of  claim 48  wherein the region is from about −2231 to about −1632 upstream (5′) relative to a CaMKKβ transcriptional start site. 
     
     
         50 . The nucleic acid molecule of  claim 48  wherein the region is from about −2019 to about −1632 upstream (5′) relative to a CaMKKβ transcriptional start site. 
     
     
         51 . The nucleic acid molecule of  claim 50  comprising the sequence 5′-GTA ACA TGA TGT AAA-3′. 
     
     
         52 . The nucleic acid molecule of  claim 48 , wherein the nucleic acid molecule is selected from decoy RNA, dsRNA, a nucleic acid aptamer, an antisense nucleic acid molecule, and an enzymatic nucleic acid molecule. 
     
     
         53 . A nucleic acid molecule comprising a double stranded siRNA that down-regulates expression of a CaMKKβ gene via RNA interference (RNAi), wherein: a) each strand of the siRNA molecule is independently about 18 to about 28 nucleotides in length; and b) one strand of the siRNA molecule comprises a sequence that binds under stringent conditions to a CaMKKβ RNA of the CaMKKβ gene and directs cleavage of the CaMKKβ RNA via RNA interference. 
     
     
         54 . The nucleic acid molecule of  claim 53  comprising the siRNA sequences of SEQ ID NOs. 105-113.