Progressione tumorale: nuovi target
                   terapeutici


                    Alessandra Gentile
    Institute ...
Outline
• About Invasive Growth
• About the MET gene and Cancer
• A new gene involved in cancer
About Invasive Growth

• Invasive growth is a physiological process that
  occurs during embryonic development and post-
 ...
About Invasive Growth

• Invasive growth is a physiological process that
  occurs during embryonic development and post-
 ...
About Invasive Growth

• Invasive growth is a physiological process that
  occurs during embryonic development and post-
 ...
The MET oncogene, encodes the HGF receptor



HGF (Scatter Factor)        Sema domain
                             500 AA
...
Invasive growth is controlled by specific factors




                                             EGF




      MLP-29 (L...
Invasive growth is controlled by specific factors




                                             HGF




      MLP-29 (L...
HGF



     Met Receptor
   (tyrosine kinase)


  Signal transduction


     Expression of
a selective set of Genes
  (The...
Plexin B1

                                            MET


                                                             ...
HGF



     Met Receptor
   (tyrosine kinase)


  Signal transduction


     Expression of
a selective set of Genes
  (The...
About MET



• The MET oncogene, controls the selective
  expression of a functional cluster of genes (The
  “Invasive Gro...
MET regulated genes
                          ( The Invasive Growth Signature )
HGF (hours)                 Immediate     ...
5
                       years
                                                                  The “metagene”
          ...
About the MET gene
                  and Cancer

• It is over-expressed in response to unfavourable
  micro-environmental ...
Hypoxia promotes invasive growth
by transcriptional activation of the MET Oncogene

                                      ...
About the MET gene
                  and Cancer

• It is over-expressed in response to unfavourable
  micro-environmental ...
MET oncogene mutations in Human Ca.


                                                  Sema


                           ...
MET oncogene amplification in Human Ca.

                                  C   GTL16




      MET gene amplification

   ...
About the MET gene
                   and Cancer

• The oncogene encodes a tyrosine-kinase receptor
  for HGF (“Scatter fa...
Therapeutic inactivation of the MET oncogene
                  can be achieved by:




1.     Small molecules inhibiting t...
In vitro therapy of Met–addicted human gastric Ca
   to a specific kinase inhibitor (small molecule)




            A. Be...
MET Dependency correlates
               with gene amplification




                      <3%                            ...
Gene Therapy with MET antibody




Gene transfer of Lentiviral vector carrying the cDNA for DN30
into the tumor: Cancer ce...
Gene transfer of DN30 RF anti-Met antibodies
  Inhibits growth of U87-MG Glioblastoma
               xenotransplants


   ...
What next ?

• Oa-5d5 antibody, Genetech, preclinical
• DN30 antibody, Metheresis, preclinical
• PF2341066, small molecule...
Receptor Tyrosine Kinase (RTKs) superfamily




                               from Blume-Jensen and Hunter, 2001
Orphan Receptor tyrosine kinase family
            1            2

                             Immunoglobulin-like motif
...
Orphan Receptor protein expression
                            in cancer cell lines
        Screened cancer cells

       ...
Orphan Receptor knock-down impairs cell
             proliferation
     Orphan Recptor*                                Orp...
Orphan Receptor knock-down impairs
        anchorage independent cellular growth

                   NCI-H1993          HS...
Orphan Receptor knock-down impairs tumor growth



                          120,0


                          100,0
% of ...
IRCC – Candiolo (Italy)

                 Director: Paolo M. Comoglio MD

Met and miRNA        Met & Hypoxia           Met...
“Today Science, Tomorrow Medicine”




IRCC: Institute for Cancer Research
          and Treatment
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Gentile Alessandra Torino 13° Convegno Patologia Immune E Malattie Orfane 21 23 Gennaio 2010 [Modalità C

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Gentile Alessandra Torino 13° Convegno Patologia Immune E Malattie Orfane 21 23 Gennaio 2010 [Modalità C

  1. 1. Progressione tumorale: nuovi target terapeutici Alessandra Gentile Institute for Cancer Research and Treatment - IRCC, University of Turin School of Medicine
  2. 2. Outline • About Invasive Growth • About the MET gene and Cancer • A new gene involved in cancer
  3. 3. About Invasive Growth • Invasive growth is a physiological process that occurs during embryonic development and post- natal tissue regeneration. • The genetic program includes control of proliferation, cell-scattering, migration and protection from apoptosis. • The MET oncogene, encoding the HGF receptor, is a key regulator of invasive growth
  4. 4. About Invasive Growth • Invasive growth is a physiological process that occurs during embryonic development and post- natal tissue regeneration. • The genetic program includes control of proliferation, cell-scattering, migration and protection from apoptosis. • The MET oncogene, encoding the HGF receptor, is a key regulator of invasive growth
  5. 5. About Invasive Growth • Invasive growth is a physiological process that occurs during embryonic development and post- natal tissue regeneration. • The genetic program includes control of proliferation, cell-scattering, migration and protection from apoptosis. • The MET oncogene, encoding the HGF receptor, is a key regulator of invasive growth.
  6. 6. The MET oncogene, encodes the HGF receptor HGF (Scatter Factor) Sema domain 500 AA MRS G-P rich (PSI) Ig like domains 400 AA Kringles Protease- like Tyrosine Kinase P P P P Met (HGF Receptor)
  7. 7. Invasive growth is controlled by specific factors EGF MLP-29 (Liver stem/progenitor cells)
  8. 8. Invasive growth is controlled by specific factors HGF MLP-29 (Liver stem/progenitor cells)
  9. 9. HGF Met Receptor (tyrosine kinase) Signal transduction Expression of a selective set of Genes (The Met Signature) Invasion & Growth
  10. 10. Plexin B1 MET Integrin α6β 4 CD44v6 Link to cytoskeleton P P PI3K PI3K p85 P p85 CRKL P GAB1 RAS GRB2 PLCγ GRB2P STAT SHP2 P Transient MAP kinase SRC activation (proliferation) Cell-Polarity and Morphogenesis Sustained MAP and PI3 kinase activation (invasion / apoptosis C.Boccaccio and P.M.Comoglio. Nature Rev. Cancer. protection) 2006, 6: 637-645.
  11. 11. HGF Met Receptor (tyrosine kinase) Signal transduction Expression of a selective set of Genes (The Met Signature) Invasion & Growth
  12. 12. About MET • The MET oncogene, controls the selective expression of a functional cluster of genes (The “Invasive Growth Signature”) • The “Signature” can be exploited for the prognosis of some invasive-metastatic cancers
  13. 13. MET regulated genes ( The Invasive Growth Signature ) HGF (hours) Immediate Delayed Early Early Late 1 6 24 Delayed Early Biphasic Immediate Early 2x induced Tonic Biphasic Tonic Late Total = 250 genes 2x suppressed
  14. 14. 5 years The “metagene” Avg.Cluster A - Avg. Cluster B Cluster A Cluster B Tumor samples, ordered by survival time 2x induced 2x suppressed E.Medico et al.(unpublished)
  15. 15. About the MET gene and Cancer • It is over-expressed in response to unfavourable micro-environmental conditions, such as hypoxia : “Oncogene Expedience”. • It is constitutively activated in some cancers, by amplification, mutations or autocrine loops: “Oncogene Addiction”. • It is good candidate for targeted therapies
  16. 16. Hypoxia promotes invasive growth by transcriptional activation of the MET Oncogene Cellular [O2] Proteasome degradation HIF Proline Hydroxylase pVHL HIF-1α α -OH α (regulated) Cytoplasm Nucleus HIF-1αβ HIF-1β β (constitutive) P (-2619) S (-411) S (-345) A (-253) A (-32) START S (+89) A (+353) AP-1 HRE-1 HRE-2 asHRE-1 asHRE-2/ HRE-4 HRE-5 S (-295) HRE-3 The MET promoter Pennacchietti et al. , Cancer Cell 3: 347 (2003)
  17. 17. About the MET gene and Cancer • It is over-expressed in response to unfavourable micro-environmental conditions, such as hypoxia or ionizing radiations: “Oncogene Expedience”. • It is constitutively activated in some cancers, by amplification, mutations or autocrine loops: “Oncogene Addiction”. • It is good candidate for targeted therapies
  18. 18. MET oncogene mutations in Human Ca. Sema PSI IPT S985 Juxtamembrane P Y1003 domain P ATP-binding region P Y1234 KD Activation 1235 Loop PY C-terminal loop P Y1349 Docking site Y1356 P G.Stella, S.Benvenuti et al, submitted
  19. 19. MET oncogene amplification in Human Ca. C GTL16 MET gene amplification C GTL16 MET Protein overexpression
  20. 20. About the MET gene and Cancer • The oncogene encodes a tyrosine-kinase receptor for HGF (“Scatter factor”) • It is over-expressed in response to unfavourable micro- environmental conditions, such as hypoxia or ionizing radiations: “Oncogene Expedience”. • It is constitutively activated in some cancers, by amplification, mutations or autocrine loops: “Oncogene Addiction”. • It is good candidate for targeted therapies
  21. 21. Therapeutic inactivation of the MET oncogene can be achieved by: 1. Small molecules inhibiting the Tyrosine Kinase 2. Monoclonal Antibodies inducing Met “Shedding”
  22. 22. In vitro therapy of Met–addicted human gastric Ca to a specific kinase inhibitor (small molecule) A. Bertotti, L.Trusolino et al., Science Signaling, 2, issue 100, Dec. 2009
  23. 23. MET Dependency correlates with gene amplification <3% MET Cell Line ADDICTION copy N° EBC-1 5.8 MKN-45 6 GTL-16 6.1 97 % HS746T 6.3 EXPEDIENCE P.M.Comoglio and L.Trusolino, Nature Rev. Cancer, In preparation
  24. 24. Gene Therapy with MET antibody Gene transfer of Lentiviral vector carrying the cDNA for DN30 into the tumor: Cancer cells produce the Monoclonal Antibody
  25. 25. Gene transfer of DN30 RF anti-Met antibodies Inhibits growth of U87-MG Glioblastoma xenotransplants . 100 90 % tumor-free animals 80 70 free 60 50 40 DN30 RF MAb cDNA 30 20 ctrl 10 0 2 6 10 14 18 22 26 30 34 38 42 46 50 54 58 Time (days) E.Vigna et al.,Cancer Res. 2008;68:9176
  26. 26. What next ? • Oa-5d5 antibody, Genetech, preclinical • DN30 antibody, Metheresis, preclinical • PF2341066, small molecule, Pfizer, phase I/II clinical • XL880, small molecule, Exelesis, phase I clinical • ARQ197, small molecule, ArQule, phase I/II clinical • MK2461, small molecule, Merck, phase I/II clinical • SGX523, small molecule, SGX pharma., Phase I clinical • JNJ38877605, small molecule, J&J, preclinical From P. Comoglio et al, Nature Rev. Drug Discovery, 7, 504
  27. 27. Receptor Tyrosine Kinase (RTKs) superfamily from Blume-Jensen and Hunter, 2001
  28. 28. Orphan Receptor tyrosine kinase family 1 2 Immunoglobulin-like motif Cysteine rich domain (Frizzled-like domain) Kringle domain Tyrosine kinase domain Serine/Threonine rich domain 1 Proline rich domain 937 aa 943 aa Serine/Threonine rich domain 2
  29. 29. Orphan Receptor protein expression in cancer cell lines Screened cancer cells 85 98% human cancer cell panel 2 phospho-Ror1 positive cells 2% NCI-H1993 HS746T
  30. 30. Orphan Receptor knock-down impairs cell proliferation Orphan Recptor* Orphan Recptor* Control shRNA shRNA(A)_Orphan Receptor 1 shRNA(B)_Orphan Receptor 1
  31. 31. Orphan Receptor knock-down impairs anchorage independent cellular growth NCI-H1993 HS746T PC3 Orphan Recptor* Orphan Recptor* Orphan Recptor Control shRNA shRNA(A)_Orphan Receptor 1 shRNA(B)_Orphan Receptor 1
  32. 32. Orphan Receptor knock-down impairs tumor growth 120,0 100,0 % of tumor free animals HS746T 80,0 Orphan Recptor* 60,0 40,0 Control shRNA 20,0 shRNA(A)_Orphan Receptor 1 shRNA(B)_Orphan Receptor 1 0,0 0 10 20 30 40 50 60 70 Time (day)
  33. 33. IRCC – Candiolo (Italy) Director: Paolo M. Comoglio MD Met and miRNA Met & Hypoxia Met gene therapy S. Giordano P.Michieli E.Vigna S. Corso S.Pennacchietti G.Pacchiana A. Petrelli M.Galluzzo R.Albano C. Migliore C.Basilico Met- signature Met & Stem cells Met Addiction E.Medico C.Boccaccio L.Trusolino L.Lazzari F.De Bacco A.Bertotti P.Luraghi D.Torti S. Gastaldi F.Galimi Met Mutations G.Reato S. Benvenuti Plexins M.F. Di Renzo L.Tamagnone A.Casazza
  34. 34. “Today Science, Tomorrow Medicine” IRCC: Institute for Cancer Research and Treatment

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