July’s SHOW will focus on the genetic pathways that drive the development of cancer. Specifically, we will look at melanoma, an aggressive form of skin cancer, which has been on the rise of late. Though making up only five percent of skin cancers cases, melanoma is responsible for a large number of the deaths associates with skin cancers, having a particularly poor prognosis when diagnosed in its later stages. Our special guest speaker, Dr. Craig J. Ceol has been working to identify the genetic defects responsible for the growth of tumors, specifically malignant melanoma.

1. Beyond treading water:
functional analysis of tumor initiation
Science Shaping Our World
July 26, 2012
Craig Ceol, Ph.D.
Assistant Professor
Program in Molecular Medicine
Department of Cancer Biology

2. Why is melanoma research important and cutting-edge?
• Metastatic melanoma has been intransigent for a long time.
• The past decade has seen major advances in understanding
the genetics of melanoma.
• In the past year two drugs for the treatment of late-stage melanoma
were approved.

3. Agenda
• The basics of melanoma
• Genetics underlying melanoma
• Identifying new melanoma genes through functional analysis
• Melanoma therapy

4. Melanocytes and skin biology
skin pigmentation pigmentation disorders
melanocyte
tanning response melanoma

5. Melanocytes reside in the epidermis
Interfollicular
melanocytes
hematoxylin +
eosin stain
Bulge w/
melanocyte
stem cells
melanocyte-specific Bulb w/
stain differentiated
melanocytes
Lin & Fisher, Nature, 2007

6. Melanin is synthesized inside melanosomes
Melanosome maturation Electron micrographs of
melanosome maturation
Marks & Seabra, NRMCB, 2001

7. Melanoma progression
Normal Nevus RGP VGP Metastatic
Epidermis
Dermis
Initiation Invasion and
metastasis
adapted from
Gaggoli & Sahai,
Pig. Cell Res., 2007

8. Melanoma subtypes – classically defined
Superficial spreading
Nodular
Lentigo maligna
Acral lentiginous
Without a primary
Ocular
Superficial spreading Lentigo maligna
common on intermittently common on chronically
sun-exposed skin sun-exposed skin
Nodular Acral lentiginous
Rapid growth typical; volar surfaces, skin w/o
poor prognosis sun exposure
Whiteman, DC, et al. PCMR 2012

9. Clinical staging of melanomas
Depth of invasion:
Breslow depth 5-year survival rate
< 1 mm 95-100% 1 mm depth
1-2 mm 80-96%
2-4 mm 60-75%
> 4 mm 50%
Ulceration: present or absent
Metastatic spread:
local (Stage III)
distant (Stage IV)

10. Patients with advanced disease face a bleak prognosis
• 76,250 new cases and 9,180 deaths est. in 2012 (US only)
• Short window of time between detection and metastasis
Balch, CM, et al. J Clin Oncol 2001

11. Why identify mutant genes in melanoma?
• Understand how mutations contribute to tumor progression:
initiation, migration, metastasis, drug resistance, etc.
• Identify targets for drug development:
target oncogenes that are overactive in tumors

12. Identifying mutations in melanoma
• PRE-GENOME SEQUENCE:
Mapping and cloning familial melanoma genes.
Sequencing of candidate genes.
e.g. CDKN2A, NRAS
Albino, et al., 1984; Kamb, Hussusian et al., 1994
• POST-GENOME SEQUENCE:
Targeted ‘resequencing’ of groups of genes.
e.g. Kinome resequencing discovers frequent BRAF mutations
Davies et al., 2002

13. Mutations that overactivate BRAF
are common in melanoma
• 50-60% of all melanomas have a mutation affecting the BRAF gene, although this
percentage is skewed toward the superficial spreading subtype.
• Of these 90% are BRAFV600E
• The BRAFV600E protein is overactive and can signal independent of upstream
stimulation
stimulus
BRAF BRAFV600E
cell division cell division
Davies et al., Nature, 2002

14. BRAF-mediated signaling is critical
but not sufficient for melanoma
Nevus (mole) Melanoma
~80% have BRAF ~60% have BRAF
mutations mutations
What cooperates with mutant BRAF to initiate melanoma?

15. Identifying mutations in melanoma
• PRE-GENOME SEQUENCE:
Mapping and cloning familial melanoma genes.
Sequencing of candidate genes.
e.g. CDKN2A, NRAS
Albino, et al., 1984; Kamb, Hussusian et al., 1994
• POST-GENOME SEQUENCE:
Targeted ‘resequencing’ of groups of genes.
e.g. Kinome resequencing discovers frequent BRAF mutations
Davies et al., 2002
• ADVENT OF MASSIVELY PARALLEL (a.k.a. DEEP) SEQUENCING:
Sequence entire genome or all protein-coding DNA (i.e. the exome).
e.g. common PREX2 and GRIN2A, GRM3 glutamate receptor mutations
Berger et al., 2012; Wei et al., 2011

16. The chaos inside a melanoma cell
Profile of mutations:
33,345 DNA base substitutions
(187 predicted to affect proteins)
frequent copy number variation
several rearrangements
Pleasance et al., Nature 2010

17. Melanomas contain a high mutation load
as compared to other cancers
• High mutation load in melanomas reflective of UV-induced mutations.
Samuels, PCMR, 2012

18. Large-scale melanoma sequencing
121 tumors
How do different melanoma mutations interact?
Will there be additional value to further sequencing?
Hodis, Chin et al., Cell, 2012

19. What does sequencing not provide?
• Functional appreciation of how cancer genes work and how mutations
in a given tumor interact with each other.
• Identification of genes that promote cancer, but which are not mutated.

20. Copy number varied intervals contain
important melanoma genes
Recurrent copy number variations in set of >90 melanomas
Lin et al., 2008

21. Zebrafish are valuable for studying
melanocyte biology and melanoma
melanin
retention
single cell
resolution
conserved mutants
development Non-mesenchymal Differentiating Terminally
Neural Crest Neural Crest Melanoblast Melanocyte Differentiated
kit albino
Non-mesenchymal Differentiating Terminally
Neural Crest Neural Crest Melanoblast Melanocyte Differentiated
sox2 sox2sox10 sox10
mitfa sox10
sox10
mitfa
sox10
mitfa
sox10
ednrb sox10 mitfa sox10
dct dct
tyr
mitfa mitfa mitfa
dct dct
tyr

22. A zebrafish model of melanoma
100
Percent melanoma-free
80
Tg(mitfa:BRAFV600E);p53(lf)
Survival
60
Tg(mitfa:BRAFV600E)
40
p53(lf)
20
0
0 10 20 30 40 50 60 70
Age (weeks)

23. Zebrafish with designer melanocytes
Start with a melanoma-
prone but melanocyte-
deficient strain
Rescue melanocytes and
spike in gene of interest
Rescued melanocytes with
gene of interest expressed
Melanomas develop with
gene of interest expressed;
all phases of melanoma
progression captured

24. SETDB1 is the sole enhancer in the
chromosome 1q21 interval
100
ARNT
CDC42SE1
ECM1
ENSA
Percent melanoma-free survival
80
FAM63A
LASS2
MLLT11
MRPL9
60
PIK4CB
PIP5K1A
POGZ combined
PRUNE
40
PSMBeta1
PSMD4
RFX5
TARS2
SETDB1 2008-01-16
20
SETDB1 2008-01-29
GFP 2007-09-13
GFP 2007-11-01
GFP 2007-11-27
0
5
10
15
20
25
Age (weeks)

25. SETDB1 is the sole enhancer in the
chromosome 1q21 interval
100
SETDB1 average vs. EGFP average
p=3.1x10-6
Percent melanoma-free survival
80
60
40
SETDB1 2008-01-16
20
SETDB1 2008-01-29
GFP 2007-09-13
GFP 2007-11-01
GFP 2007-11-27
0
5
10
15
20
25
Age (weeks)

26. Genetic studies of melanoma-prone cohorts
independently focus on SETDB1
-log10(P value)
Amos et al., 2011
-log10(P value)
Macgregor
et al., 2011

27. Large intervals of copy number variation remain
Recurrent copy number variations in set of >90 melanomas
Lin et al., 2008

28. Syntenic dispersal between zebrafish and human genomes
Homo sapiens
Mus musculus
Danio rerio
zebrafish map to human mouse map to human
50 mya

29. Designer melanocyte-based analysis informed
by comparative oncogenomics
Copy gain in human melanoma:
Determine CNV in
zebrafish melanomas
Make designer melanocytes
and melanomas that express
candidates
100
80 Metastatic spread
Tumor 60 and other
onset 40
histological
miniCoopR-EGFP
20 miniCoopR-oncogene
0
features
5 8 11 14 17 20

30. Syntenic intervals recurrently amplified
in zebrafish and human melanomas
Human Human
0 MB 64 MB 42 MB 78 MB
Chr 20 Chr 17
Mapping onto Mapping onto
fish genome fish genome
Fish Fish
Chr 13 Chr 10
Chr 17 Chr 15
Chr 23 Chr 11
Chr11 Chr 5
AMPLIFIED NOT AMPLIFIED

31. Targeted therapies take advantage of oncogene addiction
Once tumor is established:
Withdraw oncogene
tumor cell
Tumor initiation:
Oncogene mutation
normal cell
Oncogene addiction: the phenomenon by which some cancers that contain
multiple genetic and epigenetic abnormalities remain dependent on
(addicted to) one or a few genes for both maintenance of the malignant
phenotype and cell survival.

32. Chronic Myeloid Leukemia (CML)
~95% of CML patients have (9;22)
chromosomal translocation, which
generates a new oncoprotein BCR-
ABL.
BCR-
ABL Increased BCR-
ABL expression
~85% of patients are Imatinib
in CP at diagnosis
Melo JV and Barnes DJ, et al, Nat Rev Cancer, 2007

33. Second site mutations are a frequent cause of resistance
to kinase inhibitors
CML/GIST
BCR-ABL (CML); KIT/PDGFRα (GIST);
Imatinib resistance in CML
>90 different resistant variants
85% mutations found in 9 AA
BCR-ABL (T315I)
KIT (T670I)
http://www.cmlalliance.com/
PDGFRα (T674I) images/BCR-ABL-protein-cml.jpg
NSCLC
EGFR
Gefitinib/Erlotinib
(T790M)
EML4-ALK
Crizotinib
(L1196M)
Soda M., et al. Nature, 2007.

34. Selective BRAF inhibitors
PLX4032 (Vemurafenib) -APPROVED XL281/BMS908662- Phase 1
RAF265- PHASE 1
GSK2118436/SB590885- PHASE 3
GDC-0879-Preclinical

35. PLX4032 is effective, but tumors relapse
Phase III clinical trials with BRAFV600E inhibitor PLX4032:
Resistant
tumors
+PLX4032 develop
PLX4032(n=336)
Overall Survival, %
Dacarbazine (n=336)
Months After Beginning Treatment Chapman et al., NEJM 2011

36. Modes of PLX4032 resistance
• Primary resistance:
Patient tumors have BRAFV600E mutation, but no response to PLX4032 observed.
Resistance is present in most cells of the tumor prior to treatment.
• Secondary resistance:
Patient tumors initially regress, but within months progression occurs.
Most of the tumor cells are sensitive, but minor subpopulations of cells are
resistant; treatment selects for these minor subpopulations and relapse is driven
by them.
Prior to Response After
treatment phase resistance
Wagle N et al. JCO 2011;29:3085-3096

37. Many routes to PLX4032 resistance
Alternative splicing of BRAF
-
Corcoran RB et al. Oncotarget, 2011

38. What does the future hold for targeted therapy?
• Genome sequencing of tumors. Are there actionable mutations?
• Match targeted therapy to mutations present in tumor.
PLX4032 for BRAFV600E; Imatinib for BCR-ABL, KIT mutant tumors
Note: different tumor types may be targetable by same drug
because of similar genetics, e.g. BRAFV600E mutations also found in
some colorectal, multiple myeloma, adult lymphoblastic leukemia.
• Try combination therapies or effective second line therapies to
combat drug resistance.
• Couple with non-specific therapies where advantageous.

39. Acknowledgments
UNIVERSITY of MASSACHUSETTS
MEDICAL SCHOOL
LAB MEMBERS: CHB: FUNDING:
Eli Freiman Yariv Houvras
Justin Hess Len Zon Worcester Foundation for
Sharanya Iyengar Biomedical Research
Melissa Kasheta
James Neiswender
Ana Neto
Arvind Venkatesan