Malignant Melanoma and CDKN2A Kate Coffin http://www.liuding.com/blog/uploaded_images/fig8-794407.jpg

Melanoma Genetics and Environment Skin cancer of melanocytes Pre-existing nevus or new nevus Different areas of skin ABCDE assessment system of skin http://www.aad.org/skin-conditions/dermatology-a-to-z/melanoma/signs-symptoms/melanoma-signs-and-symptoms

Dangers of Melanoma Leading cause of death due to skin disease 2012 estimates in US 76,250 new cases 9,180 deaths National Cancer Institute http://www.cancer.gov/cancertopics/types/melanoma http://www.cancer.gov/aboutnci/servingpeople/cancer-statistics/snapshots

Metastasis Spreading to other areas of skin and other tissues 14% 5 year survival rate Miller, A et al. (2006). Melanoma. The New England Journal of Medicine. 355(1), 51-65. PMID:16822996

CDKN2A Cyclin-dependent kinase inhibitor 2A Inhibits CDK4 Tumor Suppressor >30 germline mutations http://en.wikipedia.org/wiki/File:Protein_CDKN2A_PDB_1a5e.png

CDKN2A Domains – Ankyrin repeats Pfam SMART Found in archaea, bacteria, eukarya, and poxvirues In other proteins associated with human disease http://smart.embl-heidelberg.de/ http://pfam.sanger.ac.uk/

CDKN2A Gene Ontology Biological Processes - cell cycle arrest - cell cycle checkpoint - induction of apoptosis - negative regulation of cell growth Cellular Components - nucleus - cytoplasm Molecular Functions - cyclin-dependent kinase inhibitor - protein kinase binding http://amigo.geneontology.org/cgi-bin/amigo/go.cgi

CDKN2A Protein Phylogeny http://www.ebi.ac.uk/Tools/msa/clustalw2/

Human CDKN2A Interaction Network http://string-db.org/

Human CDKN2A Interaction Network http://string-db.org/

Human CDKN2A Interaction Network http://string-db.org/

Human CDK4 Interaction Network http://string-db.org/

CDK4 Domains – Protein Kinase Pfam Conserved from E. coli to humans Catalytic component of Serine/Threonine kinases http://pfam.sanger.ac.uk/

CDK4 Gene Ontology Biological Processes - regulation of cell cycle - regulation of cell proliferation - signal transduction Cellular Components - cyclin-dependent protein kinase holoenzyme complex - transcription factor complex Molecular Functions - cyclin-dependent protein kinase activity - ATP binding http://amigo.geneontology.org/cgi-bin/amigo/go.cgi

Model of CDKN2A and CDK4 CDK4 CDKN2A

Model of CDKN2A and CDK4 CDK4 CDKN2A Regulated Growth http://chungkitblog.files.wordpress.com/2012/01/ellen-family-visual-medium.jpg

Model of CDKN2A and CDK4 CDK4 CDKN2A

Model of CDKN2A and CDK4 CDK4 CDKN2A Unregulated Growth http://upload.wikimedia.org/wikipedia/commons/thumb/6/6c/Melanoma.jpg/250px-Melanoma.jpg

Model of CDKN2A and CDK4 ? CDK4 CDKN2A

? Model of CDKN2A and CDK4 CDK4 CDKN2A Regulated Growth http://chungkitblog.files.wordpress.com/2012/01/ellen-family-visual-medium.jpg

Can I find a compound that mimics CDKN2A by inhibiting CDK4? Question Can I find a compound that mimics CDKN2A by inhibiting CDK4? Question first and then hypothesis – should be it inhibits it by blah blah active site

Can I find a compound that mimics CDKN2A by inhibiting CDK4? Question Can I find a compound that mimics CDKN2A by inhibiting CDK4? Hypothesis If a compound were known to interact with and inhibit CDK4, it could be used as a drug for melanoma

Experiment – Identify Compound Experiment 1 – identify cmpd that binds CDK4 http://pubchem.ncbi.nlm.nih.gov/

Results – Identify Compound 2-Methyl-5-[(4-methylphenyl)amino]benzothiazole-4,7-dione Cdk4 Inhibitor III http://pubchem.ncbi.nlm.nih.gov/

Results – Identify Compound http://pubchem.ncbi.nlm.nih.gov/

Can I find a compound that mimics CDKN2A by inhibiting CDK4? Question Can I find a compound that mimics CDKN2A by inhibiting CDK4? Hypothesis If a compound were known to interact with and inhibit CDK4, it could be used as a drug for melanoma Hypothesis If Cdk4 were inhibited by Cdk4 Inhibitor III in a Cdkn2a CKO mouse, the tumor cells would not proliferate and metastasize

Why choose a mouse? Skin and vascular systems Previous cancer model Cdkn2a – Cdk4 interaction http://info.criver.com/flex_content_area/images/nude_mouse-2.jpg

Why choose a mouse? Skin and vascular systems Previous cancer model Cdkn2a – Cdk4 interaction https://www.aalaslearninglibrary.org/fileupload_content/Course245/14_Nude_tumor.jpg

Why choose a mouse? Skin and vascular systems Previous cancer model Cdkn2a – Cdk4 interaction http://string-db.org/

Experiment – Treatment Groups 1 Cdkn2a +/+ http://info.criver.com/flex_content_area/images/nude_mouse-2.jpg http://www.miketiffee.com/uploaded_images/chemoIV-739375.jpg

Experiment – Treatment Groups 1 Cdkn2a +/+ 2 Cdkn2a -/- (CKO) http://info.criver.com/flex_content_area/images/nude_mouse-2.jpg http://www.miketiffee.com/uploaded_images/chemoIV-739375.jpg

Experiment – Treatment Groups 1 Cdkn2a +/+ 2 Cdkn2a -/- (CKO) 3 Cdkn2a +/+ & Cdk4 Inhibitor III http://info.criver.com/flex_content_area/images/nude_mouse-2.jpg http://www.miketiffee.com/uploaded_images/chemoIV-739375.jpg

Experiment – Treatment Groups 1 Cdkn2a +/+ 2 Cdkn2a -/- (CKO) 3 Cdkn2a +/+ & Cdk4 Inhibitor III 4 Cdkn2a -/- (CKO) & Cdk4 Inhibitor III http://info.criver.com/flex_content_area/images/nude_mouse-2.jpg http://www.miketiffee.com/uploaded_images/chemoIV-739375.jpg

Experiment – Graft Human Melanoma Cdkn2a +/+ Cdkn2a -/- (CKO) 1 2 Cdkn2a +/+ & Cdk4 Inhibitor III Cdkn2a -/- (CKO) & Cdk4 Inhibitor III 3 4 https://www.aalaslearninglibrary.org/fileupload_content/Course245/14_Nude_tumor.jpg http://www.miketiffee.com/uploaded_images/chemoIV-739375.jpg

Data Collection Microarray analysis of Cdkn2a and Cdk4 expression in cells of mice https://commons.wikimedia.org/wiki/File:DNA_microarray.svg

Expected Microarray Results 1) Cdkn2a +/+ 2) Cdkn2a -/- (CKO) 3) Cdkn2a +/+ & Cdk4 Inhibitor III 4) Cdkn2a -/- (CKO) & Cdk4 Inhibitor III Left Well: Cdkn2a Right Well: Cdk4

Expected Microarray Results 1) Cdkn2a +/+ 2) Cdkn2a -/- (CKO) 3) Cdkn2a +/+ & Cdk4 Inhibitor III 4) Cdkn2a -/- (CKO) & Cdk4 Inhibitor III Left Well: Cdkn2a Right Well: Cdk4

Expected Microarray Results 1) Cdkn2a +/+ 2) Cdkn2a -/- (CKO) 3) Cdkn2a +/+ & Cdk4 Inhibitor III 4) Cdkn2a -/- (CKO) & Cdk4 Inhibitor III Left Well: Cdkn2a Right Well: Cdk4

Expected Microarray Results 1) Cdkn2a +/+ 2) Cdkn2a -/- (CKO) 3) Cdkn2a +/+ & Cdk4 Inhibitor III 4) Cdkn2a -/- (CKO) & Cdk4 Inhibitor III Left Well: Cdkn2a Right Well: Cdk4

Expected Microarray Results 1) Cdkn2a +/+ 2) Cdkn2a -/- (CKO) 3) Cdkn2a +/+ & Cdk4 Inhibitor III 4) Cdkn2a -/- (CKO) & Cdk4 Inhibitor III Left Well: Cdkn2a Right Well: Cdk4

Expected Metastasis Results 1) Cdkn2a +/+ 2) Cdkn2a -/- (CKO) 3) Cdkn2a +/+ & Cdk4 Inhibitor III 4) Cdkn2a -/- (CKO) & Cdk4 Inhibitor III

Expected Metastasis Results 1) Cdkn2a +/+ 2) Cdkn2a -/- (CKO) 3) Cdkn2a +/+ & Cdk4 Inhibitor III 4) Cdkn2a -/- (CKO) & Cdk4 Inhibitor III LEAST METASTASIS

Expected Metastasis Results 1) Cdkn2a +/+ 2) Cdkn2a -/- (CKO) MOST METASTASIS 3) Cdkn2a +/+ & Cdk4 Inhibitor III 4) Cdkn2a -/- (CKO) & Cdk4 Inhibitor III LEAST METASTASIS

Expected Metastasis Results 1) Cdkn2a +/+ 2) Cdkn2a -/- (CKO) MODERATE METASTASIS MOST METASTASIS 3) Cdkn2a +/+ & Cdk4 Inhibitor III 4) Cdkn2a -/- (CKO) & Cdk4 Inhibitor III MODERATE METASTASIS LEAST METASTASIS

Conclusion CDK4 Inhibitor III could be used as an antimelanoma drug by mimicking the tumor suppressor function of CDKN2A in patients with CDKN2A loss of function mutations

Future Directions Test other modes of drug administration Topical cream vs Intravenous solution Different dosages/times of administration Study other proteins in interaction network Look for other tumor suppressors Study effects of UV exposure on CDKN2A How does SPF affect this interaction? Look for compounds in other mutated melanoma pathways

Questions? http://www.liuding.com/blog/uploaded_images/fig8-794407.jpg