Receptor Tyrosine Kinases
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Receptor Tyrosine Kinases

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  • Despite the multitude of genetic and epigenetic alterations found across cancers, a given tumor is likely to be driven by only a select few changes—those that result in the gain of an oncogene or the loss of a tumor suppressor. The phrase “oncogene addiction” was coined to describe the observation that tumor maintenance often depends upon the continued activity of certain oncogenes ( Weinstein, 2002 ). This phenomenon has been demonstrated in vivo for several oncogenes. For example, mouse models using an inducible MYC oncogene have shown that MYC-driven skin papillomas, lymphomas, and osteosarcomas can all be reversed upon MYC withdrawal ( [ Felsher and Bishop, 1999] , [Jain et al., 2002] and [ Pelengaris et al., 1999] ). Similarly, addictions to the HRAS or BCR-ABL oncogenes have been demonstrated in mouse models of melanoma and leukemia, respectively ( [Chin et al., 1999] and [ Huettner et al., 2000] ). In human colorectal cancer cells bearing a KRAS mutation, somatic knockout of the KRAS oncogene results in reversion of the transformed phenotype and abrogates the ability of these cells to form tumors in nude mice ( Shirasawa et al., 1993 ). The subset of oncogenes whose inhibition can lead to tumor cell death, differentiation, arrest, or senescence is of great clinical interest as targets for cancer therapeutics ( Table 1 ). This strategy has proven successful for the protein kinase oncogenes BCR-ABL (imatinib/Gleevec), EGFR (gefitinib/Iressa, erlotinib/Tarceva), and HER2 (trastuzumab/Herceptin) ( [ Druker , 2002] , [Roberts and Der , 2007] and Sharma et al., 2007 S.V. Sharma, D.W. Bell, J. Settleman and D.A. Haber, Epidermal growth factor receptor mutations in lung cancer, Nat. Rev. Cancer 7 (2007), pp. 169–181. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (94) [Sharma et al., 2007] ), and efforts toward inhibition of BRAF, MDM2, and the lipid kinase PI3K are underway. Targeting non-kinase oncogenes such as RAS and MYC , however, has proven more difficult.
  • EGFR is activated by the binding of a variety of ligands [eg EGF, transforming growth factor- α ( TGF α)] to the extracellular domain. This results in receptor dimerisation, leading to activation of the receptor’s tyrosine kinase and subsequent intracellular signalling. EGFR activation has been implicated in the control of cell proliferation, survival and metastasis. 1 There is increasing evidence that EGFR is expressed in a range of human tumours, including NSCLC, and high-level expression has been correlated in many cases with poor prognosis. 2,3 Inhibitors of the EGFR in clinical development include the small molecule EGFR tyrosine kinase inhibitors ZD1839 and OSI-774, and the monoclonal antibody C225. 4-6 A lack of EGFR positivity has been observed in SCLC. 7 References Woodburn J. Pharmacol Ther 1999; 82: 241-250. Salomon D, et al. Crit Rev Oncol Hematol 1995; 19: 183-232. Wells A. Int J Biochem Cell Biol 1999; 31: 637-643. Baselga J, Averbuch S. Drugs 2000; 60 (Suppl 1): 33-40. Hidalgo M, et al. J Clin Oncol 2001; 19: 3267-3279. Baselga J, et al. J Clin Oncol 2000; 18: 904-914. Cerny T, et al. Br J Cancer 1986; 54: 265-269.
  • EGFR is activated by the binding of a variety of ligands [eg EGF, transforming growth factor- α ( TGF α)] to the extracellular domain. This results in receptor dimerisation, leading to activation of the receptor’s tyrosine kinase and subsequent intracellular signalling. EGFR activation has been implicated in the control of cell proliferation, survival and metastasis. 1 There is increasing evidence that EGFR is expressed in a range of human tumours, including NSCLC, and high-level expression has been correlated in many cases with poor prognosis. 2,3 Inhibitors of the EGFR in clinical development include the small molecule EGFR tyrosine kinase inhibitors ZD1839 and OSI-774, and the monoclonal antibody C225. 4-6 A lack of EGFR positivity has been observed in SCLC. 7 References Woodburn J. Pharmacol Ther 1999; 82: 241-250. Salomon D, et al. Crit Rev Oncol Hematol 1995; 19: 183-232. Wells A. Int J Biochem Cell Biol 1999; 31: 637-643. Baselga J, Averbuch S. Drugs 2000; 60 (Suppl 1): 33-40. Hidalgo M, et al. J Clin Oncol 2001; 19: 3267-3279. Baselga J, et al. J Clin Oncol 2000; 18: 904-914. Cerny T, et al. Br J Cancer 1986; 54: 265-269.
  • EGFR is activated by the binding of a variety of ligands [eg EGF, transforming growth factor- α ( TGF α)] to the extracellular domain. This results in receptor dimerisation, leading to activation of the receptor’s tyrosine kinase and subsequent intracellular signalling. EGFR activation has been implicated in the control of cell proliferation, survival and metastasis. 1 There is increasing evidence that EGFR is expressed in a range of human tumours, including NSCLC, and high-level expression has been correlated in many cases with poor prognosis. 2,3 Inhibitors of the EGFR in clinical development include the small molecule EGFR tyrosine kinase inhibitors ZD1839 and OSI-774, and the monoclonal antibody C225. 4-6 A lack of EGFR positivity has been observed in SCLC. 7 References Woodburn J. Pharmacol Ther 1999; 82: 241-250. Salomon D, et al. Crit Rev Oncol Hematol 1995; 19: 183-232. Wells A. Int J Biochem Cell Biol 1999; 31: 637-643. Baselga J, Averbuch S. Drugs 2000; 60 (Suppl 1): 33-40. Hidalgo M, et al. J Clin Oncol 2001; 19: 3267-3279. Baselga J, et al. J Clin Oncol 2000; 18: 904-914. Cerny T, et al. Br J Cancer 1986; 54: 265-269.
  • EGFR is activated by the binding of a variety of ligands [eg EGF, transforming growth factor- α ( TGF α)] to the extracellular domain. This results in receptor dimerisation, leading to activation of the receptor’s tyrosine kinase and subsequent intracellular signalling. EGFR activation has been implicated in the control of cell proliferation, survival and metastasis. 1 There is increasing evidence that EGFR is expressed in a range of human tumours, including NSCLC, and high-level expression has been correlated in many cases with poor prognosis. 2,3 Inhibitors of the EGFR in clinical development include the small molecule EGFR tyrosine kinase inhibitors ZD1839 and OSI-774, and the monoclonal antibody C225. 4-6 A lack of EGFR positivity has been observed in SCLC. 7 References Woodburn J. Pharmacol Ther 1999; 82: 241-250. Salomon D, et al. Crit Rev Oncol Hematol 1995; 19: 183-232. Wells A. Int J Biochem Cell Biol 1999; 31: 637-643. Baselga J, Averbuch S. Drugs 2000; 60 (Suppl 1): 33-40. Hidalgo M, et al. J Clin Oncol 2001; 19: 3267-3279. Baselga J, et al. J Clin Oncol 2000; 18: 904-914. Cerny T, et al. Br J Cancer 1986; 54: 265-269.
  • project
  • Median OS was improved by 42.5% and was significantly longer in patients who received TARCEVA compared with patients who received placebo: 6.7 vs 4.7 months, respectively (HR=0.73, P <0.0001). Additionally, OSI used Medical Dictionary for Regulatory Activities (MedDRA) coding in the safety analysis, while NCIC used the National Cancer Institute (NCI) Common Toxicity Criteria (CTC) coding. Data presented with the TARCEVA PI may be different from data presented elsewhere. TARCEVA ™ [package insert]. Melville, NY, and South San Francisco, Calif: OSI Pharmaceuticals, Inc., and Genentech, Inc.; 2004. Data on file (BR.21 study report), Genentech, Inc.
  • Just to briefly review some of the roles of VEGF. As you know, secreted by tumors. Increases production of MMPs, increases EC prolif and cap tubeformation. Also leads to the mobilization and differentiation of BM derived precursors that can contribute to tumor neovascularization.
  • Because of critical role in many steps of angiogenesis,
  • Assays establishing the ability of SU11248 to inhibit the functioning of multiple receptor tyrosine kinases (RTKs) of the split-kinase domain family but not that of unrelated RTKs: Biochemical assay: uses isolated receptors to measure the concentration of SU11248 necessary to inhibit receptor kinase function (Ki). Receptor phosphorylation assay: uses whole cells in vitro to determine the concentration that would inhibit 50% (IC 50 ) of the ligand-induced phosphorylation.
  • Laser scanning quantitative analysis was performed on all of the 20 patients with pre and post-treament biopsies. Shown here are the 20 patients analyzed in this study and the % change in levels of phosphorylated PDGFR-b (shown in blue) & VEGFR-2 (shown in red). These are the patients whose tumors displayed PD, SD , and Partial Response. The patients are graphed according to decreasing levels of p-PDGFR-b in each group. Note that the overall change in levels of pPDGFR-b are high in the PD group whereas significant inhibition is observed in the PR group. Overall, p-VEGFR-2 is decreased in the SD and PR group. [These two patients in the SD group actually had the longest time to progression out of the others in the same group.] We are currently investigating the mutational status of the receptors to determine whether this may contribute to the lack of target inhibition in some SD patients. Furthermore, we are in the process of evaluating PDGFR-b activity within the EC compartment to determine whether specific cell types are being targeted. The summary of these data are shown in this table…
  • We can identify at least markers that predict drugs will not work. What changes occur in cases where a drug does have clinical activity?

Receptor Tyrosine Kinases Receptor Tyrosine Kinases Presentation Transcript

  • Receptor tyrosine kinases as therapeutic targets for cancer John Heymach, M.D., Ph.D. Depts. of Thoracic/Head and Neck Medical Oncology and Cancer Biology University of Texas M.D. Anderson Cancer Center March 13, 2009
  • Key points
    • Even though cancer cells are complex, they may be addicted to one or a few key pathways (“oncogene addiction”), often driven by an RTK.
    • 2. Some RTKs are validated or very promising as therapeutic targets. Particularly for tumors with “oncogene addiction”.
    • 3. We are testing RTK inhibitors in the clinic. Some work well for some diseases.
    • 4. Resistance to RTK inhibitor may arise through several different mechanisms. Understanding these mechanisms of resistance is important for designing better combination therapies.
  • Cancer cells are very complex.
    • Genetically unstable.
    • Large number of mutations
    • Multiple deregulated pathways.
    •  Conventional wisdom was: “Targeting any one pathway is not likely to be successful.”
  • Hahn and Weinberg 2002
  • Efficacy of imatinib, an inhibitor of BCR-ABL, in CML (Druker et al, NEJM, 2001)
    • Out of 54 patients treated with imatinib who had failed interferon alfa, at 300 mg or higher bid
      • 53 complete hematological responses
      • 29 cytological responses
  • Efficacy of imatinib, an inhibitor of BCR-ABL, in CML (Druker et al, NEJM, 2001)
    • Out of 54 patients treated with imatinib who had failed interferon alfa, at 300 mg or higher bid
      • 53 complete hematological responses
      • 29 cytological responses
      •  Conventional wisdom: “Perhaps hematological malignancies can be treated by targeting a single pathway. But solid tumors are different.”
  • 18 FDG-PET of patient with GIST treated initially with SU5416 and later with imatinib mesylate . Heymach et al, CCR, 2004 Pre- and post- treatment with SU5416 Pre- and post- treatment with imatinib
  • Efficacy of imatinib, an inhibitor of c-KIT, in gastrointestinal stromal tumors (Demetri et al, NEJM, 2002)
    • Chemotherapy ineffective
    • Families with inherited GIST found to have activating KIT mutations
    • Imatinib tested in 147 patients
      • 81% with partial response or prolonged stable disease
      •  Selected solid tumors can be treated by targeting a single pathway
  • The Normal Cell Growth factors DNA Apoptosis Senescence Anti-Growth signals Limited nutrients/ microenvironment
  • The Cancer Cell Growth factors DNA Apoptosis Senescence Anti-Growth signals Contact inhibition/ microenvironment
  • The Hallmarks of Cancer
    • Self-sufficiency in growth signals
    • Insensitivity to anti-growth signals
    • Evading apoptosis
    • Limitless replicative potential
    • Sustained angiogenesis
    • Tissue invasion and metastasis
    • (Genetic instability)
    •  RTKs may be involved in all, or almost all, of these traits.
    Hanahan and Weinberg, Cell 2000
  • Hallmarks of cancer Luo et al, Cell 2009
  • “ Oncogene addiction”
    • Despite the multitude of genetic and epigenetic alterations in a cancer, a given tumor is likely to be driven by only a few select changes- i.e. gain of an oncogene or loss of a tumor suppressor.
    • Maintenance often depends on the continued activity of certain oncogenes.
      • Myc
      • K-RAS, HRAS
      • BCR-ABL
      • EGFR
      • Her2
      • Others being studied: BRAF, MDM2, PI3K pathway
    Luo et al, Cell 2009
  • RTK inhibitors
    • Small molecule inhibitors: bind to ATP pocket and prevent receptor phosphorylation (ATP donates phosphate)
      • Examples:
        • EGFR inhibitors: gefitinib and erlotinib
        • Kit and BCR inhibitor: imatinib
        • VEGFR inhibitors: SU5416, SU11248
      • ***Because of structural similarity of different tyrosine kinase domains, small molecule inhibitors typically inhibit several different RTKs
    • Antibodies: typically bind to extracellular domain of receptor or ligand and prevent receptor activation
      • Example:
        • EGFR inhibitor: cetuximab (Erbitux)
        • VEGF: bevacizumab (Avastin)
  • EGFR complexed with Erlotinib Stamos and Eigenbrot, 2002 http://www.ncbi.nlm.nih.gov
  • Structure of wild-type and mutated EGFR tyrosine kinase domain
    • Key structures
      • C and N lobes (ATP cleft in between)
      • P lobe: phosphate binding
      • A loop: activation
    Gazdar et al, Trends Mol Med. 2004 Oct;10(10):481-6.
  • Epidermal Growth Factor Receptors R K R K Signal transduction cascade Ligand Ligand binding Dimerization Phosphorylation
  • EGFR Inhibitors Plasma membrane R K R K Signal transduction cascade Ligand Antibodies: Erbitux (cetuximab), ABX-EGF, ICR62, Omnitarg(pertuzumab) TK inhibitors: Iressa (gefitinib), Tarceva (erlotinib), EKB-569, CI-1033, PKI166
  • EGFR Inhibitor Specificity Plasma membrane R K R K Gefitinib (Iressa) Erlotinib (Tarceva) R K R K R K R K erbB1 erbB2 erbB3 erbB4 R K R K EKB-569 CI-4033
  • EGFR and Tumor Specificity Plasma membrane R K R K R K R K R K R K erbB1 erbB2 erbB3 erbB4 R K R K Gastric, Ovarian H&N NSCLC Prostate Breast: B1,B2,B4
  • Kinase dendrogram for selected RTKIs Adapted from Fabian, M, Nature Biotechnology VOL 23, March 2005
  • Mechanisms of resistance to RTK inhibitors
    • 1. Target not critical for a given tumor (primary resistance)
    • 2. Incomplete receptor inhibition
      • Inhibitor not sufficiently potent
      • Secondary mutations that prevent binding of inhibitor (i.e. T790M mutation in lung cancer prevents gefitnib or erlotinib from inhibiting EGFR)
    • 3. Target bypass: the tumor circumvents the inhibited pathway by activation of other pathways (i.e. MET)
  • Potential mechanisms of resistance to targeted agents R R Ligand R R Ligand Pathway X Effect (I.e. tumor cell survival) Pathway X Effect (I.e. tumor cell survival) 1. Incomplete target inhibition Effective target inhibition X (partial) inhibitor
  • Potential mechanisms of resistance to targeted agents R R Ligand R R Ligand Pathway X Effect (I.e. tumor cell survival) R R Ligand Pathway X Effect (I.e. tumor cell survival) Pathway X Effect (I.e. tumor cell survival) R2 R2 Ligand 2 1. Incomplete target inhibition 2. “Target bypass” Effective target inhibition X X Pathway Y A B
  • Approaches to combating resistance R R Ligand R R Ligand Pathway X Effect (I.e. tumor cell survival) Pathway X Effect (I.e. tumor cell survival) R2 R2 Ligand 2 1. Incomplete target inhibition 2. “Target bypass” X Pathway Y A B Add additional inhibitor or try more potent inhibitor for same target
  • Approaches to combating resistance R R Ligand R R Ligand Pathway X Effect (I.e. tumor cell survival) Pathway X Effect (I.e. tumor cell survival) R2 R2 Ligand 2 1. Incomplete target inhibition 2. “Target bypass” X Pathway Y A B Inhibit downstream or bypass pathways
    • EGFR Tyrosine Kinase Inhibitor Therapy in NSCLC
  • Leading causes of cancer death in the U.S. Jemal et al Ca Can J Clin 2003, 53: 5-26 29,000 Pancreatic 31,000 Prostate 42,000 Breast 57,000 Colorectal 155,000 Lung deaths Tumor type
  • Median survival in advanced NSCLC (SEER data) 1970s-1990s Breathnach et al (2001) JCO 19(6): 1734 6.9 months 7.3 months
    • Probably yes.
    • All recent randomized studies have similar results
    • No clear efficacy benefit for nonplatinum combinations or triplet combinations
    Are we approaching the ceiling for improved benefit from combination chemotherapy regimens? Schiller JH et al. N Engl J Med . 2002;346:92-98. Cisplatin/Paclitaxel Cisplatin/Gemcitabine Cisplatin/Docetaxel Carboplatin/Paclitaxel 1.0 0.8 0.6 0.4 0.2 0.0 0 5 10 15 20 25 30 Months Stage IIIB/IV Patient Survival, % ECOG 1594
  • NCI Canada BR.21 Advanced NSCLC Tarceva vs Placebo Phase III Trial Previously Treated Advanced NSCLC N = 638 Stratified by : Center PS, 0/1 vs 2/3 Response to prior Rx (CR/PR:SD:PD) Prior regimens, (1 vs 2) Prior platinum, (Yes vs no) Shepherd, et al. PASCO 2004 Erlotinib 150 mg PO QD R A N D O M I Z E D Placebo PO QD 2:1 randomization
  • Overall Survival *Adjusted for stratification factors at randomization, and HER1/EGFR status. HR = hazard ratio. TARCEVA ™ (erlotinib) PI. TARCEVA (n=488) Placebo (n=243) Median survival (mo) 1 - year survival (%) Months Survival distribution function 1.00 0 5 10 15 20 25 30 0.75 0.50 0.25 0 TARCEVA Placebo 6.7 4.7 31.2 P <0.001 * HR=0.73 (95% CI, 0.61-0.86) 21.5
  • Analysis of tumor from patients with major response to EGFR inhibitors
    • Approximately 10-20% of patients had dramatic tumor shrinkage after treatment (major response)
    • In majority of tumors from patients with major response, mutations in EGFR tyrosine kinase domain observed (exon 19 deletion, point mutation)
    • Mutation led to constitutive activation of EGFR and increased sensitivity to EGFR inhibition.
    • Tumors with amplification of EGFR may also have increased sensitivity
    • Patients who never smoked, or were of Asian origin, had a high frequency of EGFR mutations.
    • (Paez et al, Science, 2003; Lynch et al, NEJM, 2003)
  • Never smokers: overall survival- erlotinib vs Placebo 1.0 0.8 0.6 0.4 0.2 0 0 5 10 15 20 25 Survival rate Erlotinib Placebo Months on study Herbst et al., ASCO 2004
  • Secondary mutations in EGFR (T790M) lead to acquired resistance to EGFR TKIs Kobayashi et al, NEJM 2005
  • Secondary mutations in EGFR (T790M) lead to acquired resistance to EGFR TKIs Kobayashi et al, NEJM 2005 T790M mutation
  • Amplifications of MET receptor lead to resistance in previously EGFR-addicted NSCLC cells. Engelman et al, Science, 2007
  • Pivotal role of VEGF in tumor angiogenesis 2. VEGF increases endothelial protease expression 1. Tumor secretes VEGF 4.VEGF induces mobilization and differentiation of BM- derived CEPs 3. Endothelial cell migration proliferation, and capillary tube formation promoted by VEGF Bone marrow
  • Different approaches to inhibition of VEGF signaling Ligand sequestration: MAbs, soluble receptors (i.e. bevacizumab) GRB2 SOS ras PLC  p85 Inhibition of tyrosine phosphorylation and downstream signaling inhibition Transcription factor inhibition Tyrosine kinase inhibition: TKIs TKI  tyrosine kinase inhibitor Receptor blocking: MAbs
  • SU11248: Multitargeted Receptor Tyrosine Kinase Inhibitor VEGFR-1 VEGFR-2 VEGFR-3 Fms PDGFR  PDGFR  CSF1R KIT FLT3 Split Kinase Domain RTKs Mendel DB, et al. Clin Cancer Res 2003;9:327–37 *Receptor phosphorylation FLT3 (WT) 0.25 KIT 0.01 VEGFR-2 0.009 *Cellular IC 50 (µM) PDGFR  0.008 EGFR 8.9 MET 12.0 VEGFR-2 0.009 PDGFR  0.008 FGFR1 0.83 EGFR >10 Enzymatic K i (µM) VEGFR-3 0.017 N H O N H F N H O N OH COOH HOOC H
  • LSC analysis of PDGFR-beta and VEGFR-2 phosphorylation after treatment of GIST patients with SU11248 for 10-14 days Patients (n=20) PD SD PR PD = progressive disease; SD = stable disease; PR = partial response % change in phosphorylation post-treatment Davis et al, ECCO 2005 - 
  • Lessons from tumor-based biomarkers of activity
    • For SU5416/SU6668, lack of clinical activity likely due, at least in part, to incomplete target inhibition.
    • It is critical to not only identify important targets, but also to determine the duration and degree of target inhibition needed for anti-tumor activity.
    • 2. SU11248-treated GIST patients with clinical benefit had a:
      • greater post-treatment induction in TEC apoptosis (9.55 vs 1.78) and TC apoptosis than patients with progressive disease
      • greater degree (but not complete) inhibition of PDGFR- β and VEGFR phosphorylation.
    • There is likely to be room for improvement with more potent inhibitors or combinations.
  • Key points (again)
    • Even though cancer cells are complex, they may be addicted to one or a few key pathways (“oncogene addiction”), often driven by an RTK.
        • CML: addicted to BCR-ABL
        • Gastointestinal stromal cancer: Addicted to C-KIT
        • Some NSCLC: EGFR mutations.
    • 2. Some RTKs are validated or very promising as therapeutic targets. Particularly for tumors with “oncogene addiction”.
      • BCR-ABL (CML)
      • KIT (GIST)
      • PDGFR (GIST)
      • EGFR (lung cancer)
      • VEGFR (multiple diseases)
      • RET (medullary thyroid)
    • 3. We are testing RTK inhibitors in the clinic. Some work well for some diseases.
        • Imatinib: GIST, CML
        • Erlotinib: NSCLC
        • Sunitinib: GIST, RCC
    • 4. Resistance to RTK inhibitor may arise through several different mechanisms. Understanding these mechanisms of resistance is important for designing better combination therapies.
        • Secondary mutations or other changes that prevent the inhibitor from binding.
        • Activation of a “bypass” pathway.