1. Malignant Melanoma and CDKN2A
Kate Coffin

2. Melanoma
Genetics and Environment Skin cancer of melanocytes Pre-existing nevus or new nevus Different areas of skin ABCDE assessment system of skin

3. 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

4. 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), PMID:

5. CDKN2A Cyclin-dependent kinase inhibitor 2A
Inhibits CDK4 Tumor Suppressor >30 germline mutations

6. CDKN2A Domains – Ankyrin repeats
Pfam
SMART
Found in archaea, bacteria, eukarya, and poxvirues
In other proteins associated with human disease

7. 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

8. CDKN2A Protein Phylogeny

9. Human CDKN2A Interaction Network

10. Human CDKN2A Interaction Network

11. Human CDKN2A Interaction Network

12. Human CDK4 Interaction Network

13. CDK4 Domains – Protein Kinase
Pfam
Conserved from E. coli to humans
Catalytic component of Serine/Threonine kinases

14. 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

15. Model of CDKN2A and CDK4
CDK4
CDKN2A

16. Model of CDKN2A and CDK4 CDK4 CDKN2A Regulated Growth

17. Model of CDKN2A and CDK4
CDK4
CDKN2A

18. Model of CDKN2A and CDK4 CDK4 CDKN2A Unregulated Growth

19. Model of CDKN2A and CDK4 ?
CDK4
CDKN2A

20. ? Model of CDKN2A and CDK4 CDK4 CDKN2A Regulated Growth

21. 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

22. 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

23. Experiment – Identify Compound
Experiment 1 – identify cmpd that binds CDK4

24. Results – Identify Compound
2-Methyl-5-[(4-methylphenyl)amino]benzothiazole-4,7-dione
Cdk4 Inhibitor III

25. Results – Identify Compound

26. 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

27. Why choose a mouse?
Skin and vascular systems Previous cancer model Cdkn2a – Cdk4 interaction

28. Why choose a mouse?
Skin and vascular systems Previous cancer model Cdkn2a – Cdk4 interaction

29. Why choose a mouse?
Skin and vascular systems Previous cancer model Cdkn2a – Cdk4 interaction

30. Experiment – Treatment Groups1
Cdkn2a +/+

31. Experiment – Treatment Groups1
Cdkn2a +/+ 2
Cdkn2a -/- (CKO)

32. Experiment – Treatment Groups1
Cdkn2a +/+ 2
Cdkn2a -/- (CKO) 3
Cdkn2a +/+
& Cdk4 Inhibitor III

33. Experiment – Treatment Groups1
Cdkn2a +/+ 2
Cdkn2a -/- (CKO) 3
Cdkn2a +/+
& Cdk4 Inhibitor III 4
Cdkn2a -/- (CKO)
& Cdk4 Inhibitor III

34. Experiment – Graft Human Melanoma
Cdkn2a +/+
Cdkn2a -/- (CKO) 1 2
Cdkn2a +/+
& Cdk4 Inhibitor III
Cdkn2a -/- (CKO)
& Cdk4 Inhibitor III 3 4

35. Data Collection
Microarray analysis of Cdkn2a and Cdk4 expression in cells of mice

36. 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

37. 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

38. 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

39. 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

40. 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

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

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

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

44. 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

45. 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

46. 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

47. Questions?