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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.

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Malignant Melanoma and CDKN2A

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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/Threoninekinases

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 kinaseholoenzyme 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


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 +/+

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 +/+

Cdkn2a -/- (CKO)

2

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


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