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
- induction of apoptosis
- cell cycle checkpoint
- 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
- signal transduction
- regulation of cell proliferation
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
CDKN2A
CDK4
Model of CDKN2A and CDK4
CDKN2A
CDK4
Regulated Growth
http://chungkitblog.files.wordpress.com/2012/01/ellen-family-visual-medium.jpg
Model of CDKN2A and CDK4
CDKN2A
CDK4
Model of CDKN2A and CDK4
CDKN2A
CDK4
Unregulated
Growth
http://upload.wikimedia.org/wikipedia/commons/thumb/6/6c/Melanoma.jpg/250px-Melanoma.jpg
Model of CDKN2A and CDK4
?
CDKN2A
CDK4
Model of CDKN2A and CDK4
?
CDKN2A
CDK4
Regulated Growth
http://chungkitblog.files.wordpress.com/2012/01/ellen-family-visual-medium.jpg
Question
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
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/
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 +/+
http://info.criver.com/flex_content_area/images/nude_mouse-2.jpg
http://www.miketiffee.com/uploaded_images/chemoIV-739375.jpg
2
Cdkn2a -/- (CKO)
Experiment – Treatment Groups
1
Cdkn2a +/+
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
2
Cdkn2a -/- (CKO)
Experiment – Treatment Groups
1
Cdkn2a +/+
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
2
Cdkn2a -/- (CKO)
4
Cdkn2a -/- (CKO)
& Cdk4 Inhibitor III
Experiment – Graft Human Melanoma
Cdkn2a +/+
1
Cdkn2a -/- (CKO)
2
Cdkn2a +/+
& Cdk4 Inhibitor III
3
Cdkn2a -/- (CKO)
& Cdk4 Inhibitor III
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
LEAST METASTASIS
4) Cdkn2a -/- (CKO)
& Cdk4 Inhibitor III
Expected Metastasis Results
1) Cdkn2a +/+
2) Cdkn2a -/- (CKO)
MODERATE
METASTASIS
MOST METASTASIS
3) Cdkn2a +/+
& Cdk4 Inhibitor III
4) Cdkn2a -/- (CKO)
& Cdk4 Inhibitor III
LEAST METASTASIS
MODERATE
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