Beyond treading water:functional analysis of tumor initiation             Science Shaping Our World                   July...
Why is melanoma research important and cutting-edge? • Metastatic melanoma has been intransigent for a long time. • The pa...
Agenda• The basics of melanoma• Genetics underlying melanoma• Identifying new melanoma genes through functional analysis• ...
Melanocytes and skin biologyskin pigmentation                      pigmentation disorders                        melanocyt...
Melanocytes reside in the epidermis                                                                   Interfollicular     ...
Melanin is synthesized inside melanosomesMelanosome maturation           Electron micrographs of                          ...
Melanoma progression            Normal   Nevus          RGP   VGP    MetastaticEpidermisDermis                       Initi...
Melanoma subtypes – classically defined                            Superficial spreading                            Nodula...
Clinical staging of melanomasDepth of invasion:    Breslow depth          5-year survival rate       < 1 mm               ...
Patients with advanced disease face a bleak prognosis   • 76,250 new cases and 9,180 deaths est. in 2012 (US only)   • Sho...
Why identify mutant genes in melanoma?• Understand how mutations contribute to tumor progression:initiation, migration, me...
Identifying mutations in melanoma• PRE-GENOME SEQUENCE:     Mapping and cloning familial melanoma genes.     Sequencing of...
Mutations that overactivate BRAF                      are common in melanoma• 50-60% of all melanomas have a mutation affe...
BRAF-mediated signaling is critical                but not sufficient for melanomaNevus (mole)                            ...
Identifying mutations in melanoma• PRE-GENOME SEQUENCE:     Mapping and cloning familial melanoma genes.     Sequencing of...
The chaos inside a melanoma cell                      Profile of mutations:                         33,345 DNA base substi...
Melanomas contain a high mutation load              as compared to other cancers• High mutation load in melanomas reflecti...
Large-scale melanoma sequencing                                     121 tumorsHow do different melanoma mutations interact...
What does sequencing not provide?• Functional appreciation of how cancer genes work and how mutationsin a given tumor inte...
Copy number varied intervals contain                 important melanoma genesRecurrent copy number variations in set of >9...
Zebrafish are valuable for studying                                      melanocyte biology and melanoma                  ...
A zebrafish model of melanoma                        100Percent melanoma-free                        80                   ...
Zebrafish with designer melanocytesStart with a melanoma-prone but melanocyte-deficient strainRescue melanocytes andspike ...
SETDB1 is the sole enhancer in the                                                chromosome 1q21 interval                ...
SETDB1 is the sole enhancer in the                                                chromosome 1q21 interval                ...
Genetic studies of melanoma-prone cohorts                        independently focus on SETDB1                         -lo...
Large intervals of copy number variation remainRecurrent copy number variations in set of >90 melanomas                   ...
Syntenic dispersal between zebrafish and human genomes                                              Homo sapiens          ...
Designer melanocyte-based analysis informed               by comparative oncogenomics                             Copy gai...
Syntenic intervals recurrently amplified                        in zebrafish and human melanomasHuman                     ...
Targeted therapies take advantage of oncogene addiction    Once tumor is established:                           Withdraw o...
Chronic Myeloid Leukemia (CML)                  ~95% of CML patients have (9;22)                  chromosomal translocatio...
Second site mutations are a frequent cause of resistance                     to kinase inhibitorsCML/GIST  BCR-ABL (CML); ...
Selective BRAF inhibitorsPLX4032 (Vemurafenib) -APPROVED       XL281/BMS908662- Phase 1                                   ...
PLX4032 is effective, but tumors relapsePhase III clinical trials with BRAFV600E inhibitor PLX4032:                       ...
Modes of PLX4032 resistance• Primary resistance:Patient tumors have BRAFV600E mutation, but no response to PLX4032 observe...
Many routes to PLX4032 resistance   Alternative splicing of BRAF                                  -                       ...
What does the future hold for targeted therapy?• Genome sequencing of tumors. Are there actionable mutations?• Match targe...
Acknowledgments        UNIVERSITY of MASSACHUSETTS        MEDICAL SCHOOLLAB MEMBERS:         CHB:             FUNDING:Eli ...
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Science Shaping Our World-SHOW: Beyond Treading Water: Functional Analysis of Tumor Initiation and Maintenance

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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.

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Science Shaping Our World-SHOW: Beyond Treading Water: Functional Analysis of Tumor Initiation and Maintenance

  1. 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. 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. 3. Agenda• The basics of melanoma• Genetics underlying melanoma• Identifying new melanoma genes through functional analysis• Melanoma therapy
  4. 4. Melanocytes and skin biologyskin pigmentation pigmentation disorders melanocytetanning response melanoma
  5. 5. Melanocytes reside in the epidermis Interfollicular melanocyteshematoxylin +eosin stain Bulge w/ melanocyte stem cellsmelanocyte-specific Bulb w/stain differentiated melanocytes Lin & Fisher, Nature, 2007
  6. 6. Melanin is synthesized inside melanosomesMelanosome maturation Electron micrographs of melanosome maturation Marks & Seabra, NRMCB, 2001
  7. 7. Melanoma progression Normal Nevus RGP VGP MetastaticEpidermisDermis Initiation Invasion and metastasis adapted from Gaggoli & Sahai, Pig. Cell Res., 2007
  8. 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. 9. Clinical staging of melanomasDepth 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. 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. 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. 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. 13. Mutations that overactivate BRAF are common in melanoma• 50-60% of all melanomas have a mutation affecting the BRAF gene, although thispercentage is skewed toward the superficial spreading subtype.• Of these 90% are BRAFV600E• The BRAFV600E protein is overactive and can signal independent of upstreamstimulation stimulus BRAF BRAFV600E cell division cell division Davies et al., Nature, 2002
  14. 14. BRAF-mediated signaling is critical but not sufficient for melanomaNevus (mole) Melanoma ~80% have BRAF ~60% have BRAF mutations mutationsWhat cooperates with mutant BRAF to initiate melanoma?
  15. 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. 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. 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. 18. Large-scale melanoma sequencing 121 tumorsHow do different melanoma mutations interact?Will there be additional value to further sequencing? Hodis, Chin et al., Cell, 2012
  19. 19. What does sequencing not provide?• Functional appreciation of how cancer genes work and how mutationsin a given tumor interact with each other.• Identification of genes that promote cancer, but which are not mutated.
  20. 20. Copy number varied intervals contain important melanoma genesRecurrent copy number variations in set of >90 melanomas Lin et al., 2008
  21. 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 TerminallyNeural 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. 22. A zebrafish model of melanoma 100Percent 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. 23. Zebrafish with designer melanocytesStart with a melanoma-prone but melanocyte-deficient strainRescue melanocytes andspike in gene of interestRescued melanocytes withgene of interest expressedMelanomas develop withgene of interest expressed;all phases of melanomaprogression captured
  24. 24. SETDB1 is the sole enhancer in the chromosome 1q21 interval 100 ARNT CDC42SE1 ECM1 ENSAPercent 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. 25. SETDB1 is the sole enhancer in the chromosome 1q21 interval 100 SETDB1 average vs. EGFP average p=3.1x10-6Percent 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. 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. 27. Large intervals of copy number variation remainRecurrent copy number variations in set of >90 melanomas Lin et al., 2008
  28. 28. Syntenic dispersal between zebrafish and human genomes Homo sapiens Mus musculus Danio reriozebrafish map to human mouse map to human 50 mya
  29. 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 spreadTumor 60 and otheronset 40 histological miniCoopR-EGFP 20 miniCoopR-oncogene 0 features 5 8 11 14 17 20
  30. 30. Syntenic intervals recurrently amplified in zebrafish and human melanomasHuman Human 0 MB 64 MB 42 MB 78 MB Chr 20 Chr 17 Mapping onto Mapping onto fish genome fish genomeFish Fish Chr 13 Chr 10 Chr 17 Chr 15 Chr 23 Chr 11 Chr11 Chr 5 AMPLIFIED NOT AMPLIFIED
  31. 31. Targeted therapies take advantage of oncogene addiction Once tumor is established: Withdraw oncogene tumor cell Tumor initiation: Oncogene mutation normal cellOncogene addiction: the phenomenon by which some cancers that containmultiple genetic and epigenetic abnormalities remain dependent on(addicted to) one or a few genes for both maintenance of the malignantphenotype and cell survival.
  32. 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. 33. Second site mutations are a frequent cause of resistance to kinase inhibitorsCML/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.jpgNSCLC EGFR Gefitinib/Erlotinib (T790M) EML4-ALK Crizotinib (L1196M) Soda M., et al. Nature, 2007.
  34. 34. Selective BRAF inhibitorsPLX4032 (Vemurafenib) -APPROVED XL281/BMS908662- Phase 1 RAF265- PHASE 1GSK2118436/SB590885- PHASE 3 GDC-0879-Preclinical
  35. 35. PLX4032 is effective, but tumors relapsePhase 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. 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 areresistant; treatment selects for these minor subpopulations and relapse is drivenby them. Prior to Response After treatment phase resistance Wagle N et al. JCO 2011;29:3085-3096
  37. 37. Many routes to PLX4032 resistance Alternative splicing of BRAF - Corcoran RB et al. Oncotarget, 2011
  38. 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 tocombat drug resistance.• Couple with non-specific therapies where advantageous.
  39. 39. Acknowledgments UNIVERSITY of MASSACHUSETTS MEDICAL SCHOOLLAB MEMBERS: CHB: FUNDING:Eli Freiman Yariv HouvrasJustin Hess Len Zon Worcester Foundation forSharanya Iyengar Biomedical ResearchMelissa KashetaJames NeiswenderAna NetoArvind Venkatesan

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