Receptor Tyrosine Kinases

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.

Receptor Tyrosine Kinases

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