EORTC10_220_FGFR
1
22nd EORTC-NCI-AACR Symposium on Molecular Targets and Cancer Therapeutics, November 16-19, 2010, Berlin, Germany Table 2. Inhibition of Cell Viability by EXEL-3261: IC 50 Values for Sensitive Cell Lines Cell Line Viability IC 50 (nM) FGFR genotype Tissue NCI-H716 69 FGFR2 gene amplification Colon KATO III 193 FGFR2 gene amplification Gastric SNU-16 250 FGFR2 gene amplification Gastric KG-1 366 FGFR1 translocation AML MFM-223 649 FGFR2 gene amplification Breast RT-112 1223 FGFR3 amplification Bladder OPM-2 1345 FGFR3 translocation and activating mutation Multiple Myeloma AN3 CA 1512 FGFR2 activating mutation Endometrium HuH-7 1852 FGFR4 overexpression Hepatic DMS114 2859 FGFR1 amplification SCLC Ten out of 11 cell lines with EXEL-3261 viability IC • 50 values less than 3000 nM have genetic alterations in FGFR family members These same cell lines are also sensitive to PD173074 and TKI-258 (not shown) • Table 3. Inhibition of FGFR2 Phosphorylation and Cell Viability by FGFR Inhibitors in NCI-H716 Colon Adenocarcinoma Line Compound Biochemical IC 50 (nM) Cellular IC 50 (nM) NCI-H716 FGFR2 pFGFR2 Viability EXEL-3261 11 17 69 EXEL-6484 29 28 27 PD173074 1.8 76 0.4 AZD2171 11 122 103 AG13736 8.4 105 89 TKI-258 12 158 121 EXEL-3261 and EXEL-6484 inhibit FGFR2 activity in vitro and in cells with IC • 50 values less than 30 nM Figure 3. Inhibition of FGFR2 Signaling and Broader Phosphotyrosine Networks by EXEL-6484 pFGFR Y653/Y654 EXEL-6484 (nM) DMSO PD173074 pAKT S473 Inhibition (%) Erlotinib Lapatinib Inhibition (%) pERK T202/Y204 Inhibition (%) pEGFR Y845 pHER2 Y1221/Y1222 Inhibition (%) Inhibition (%) pHER3 Y1197 β-actin Inhibition (%) FGFR2 HER Family AKT ERK 0 99 98 97 85 56 31 27 0 66 83 82 74 50 33 1 0 92 94 92 80 60 –21 –4 0 87 87 83 62 30 19 –13 0 84 80 80 75 40 9 –10 0 93 93 82 88 65 37 28 3000 1000 333 111 37 12 4 Phosphoprotein levels were measured by Western blot following 3 hr compound treatment of NCI-H716 cells in RPMI 1640 containing 10% FCS. Control compounds were PD173074 (3000 nM), erlotinib (5000 nM), and lapatinib (5000 nM). Treatment of NCI-H716 cells with EXEL-6484 inhibited phosphorylation of FGFR2, • ERK, and AKT with an IC 50 value of approximately 50 nM Both EXEL-6484 and PD173074 also reduced phosphorylation of EGFR, HER2, and • HER3 while HER kinase inhibitors erlotinib and lapatinib did not In NCI-H716 cells, FGFR2 overexpression not only promotes activation of • downstream pathways including ERK and AKT, but also HER family receptor tyrosine kinases Table 5. Inhibitors with Selectivity Against FGFR2 over Other Isozymes EXEL-ID Biochemical IC 50 (nM) Cellular IC 50 (nM) FGFR2 FGFR1 FGFR3 FGFR4 pFGFR2 Viability EXEL-3261 11 21 60 137 17 69 EXEL-6484 29 63 116 234 28 27 EXEL-6007 4 31 55 235 28 113 EXEL-4832 4 39 87 161 31 147 EXEL-2825 5 40 114 357 50 128 EXEL-6009 6 44 134 1610 86 103 EXEL-4804 9 147 342 1855 232 288 EXEL-6676 17 199 377 717 153 40 EXEL-9291 17 163 278 2245 517 174 Key: green: IC 50 less than 2× wild type FGFR2 IC 50 ; yellow: IC 50 between 2× and 10× wild type FGFR2 IC 50 ; orange: IC 50 greater than 10× wild type FGFR2 IC 50 pFGFR2 IC 50 values were determined for FGFR2 constructs over-expressed in HEK 293 cells. Lead compounds EXEL-3261 and EXEL-6484 inhibit isozymes FGFR1-3. The rank • order of potencies is FGFR2 > FGFR1 > FGFR3, and IC 50 values are within five-fold of one another. A subset of compounds in the series exhibits preferential activity against FGFR2 • with greater than 10-fold selectivity over the other family members (EXEL-4804 and EXEL-6676) Table 6. Differential Activity of FGFR Inhibitors Against Gatekeeper Mutations EXEL-ID Biochemical FGFR2 IC 50 (nM) Cellular pFGFR2 IC 50 (nM) Wild Type FGFR2 Activating Mutation N569K Gatekeeper Mutation V584M Gatekeeper Mutation V584F Gatekeeper Mutation V584I AZD2171 11 64 43 >10,000 >10,000 >10,000 AG13736 8.4 193 106 4056 2885 2822 TKI-258 12 159 74 412 37 2079 EXEL-3261 11 30 36 499 21 89 EXEL-6484 29 74 102 735 42 248 EXEL-6007 3.7 35 45 181 123 68 EXEL-7245 9.2 31 N/A 89 31 76 EXEL-7524 42 327 344 47 16 423 EXEL-7650 578 723 886 105 21 609 Key: green: IC 50 less than 2× wild type FGFR2 IC 50 ; yellow: IC 50 between 2× and 10× wild type FGFR2 IC 50 ; orange: IC 50 greater than 10× wild type FGFR2 IC 50 pFGFR2 IC 50 values were determined for FGFR2 constructs overexpressed in HEK 293 cells. FGFR inhibitors exhibit varying activity against FGFR2 with mutations at • gatekeeper residue V584 ranging from relatively weak activity (AZD2171 and AG13736) to increased potency compared to wild type FGFR2 (EXEL-7524 and EXEL-7650) All tested compounds are equipotent against the activating mutant N569K • compared to wild type FGFR2 CONCLUSIONS/SUMMARY We have identified a series of potent FGFR2 inhibitors with biochemical and • cellular IC 50 values less than 50 nM and greater than 10-fold selectivity over VEGFR2 and PDGFRβ Identified inhibitors bind to the nucleotide binding site of FGFR2 in the inactive • “Phe-out” conformation Distinct subseries were found that are (1) specific for FGFR2 over FGFR1/3/4 or (2) • active against potential resistance mutations at the gatekeeper residue FGFR inhibitors reduce in vitro cell viability for a subset of human tumor lines with • genetic activation of FGFR family members In pharmacodynamic studies, lead compound EXEL-6484 reduces FGFR2 • phosphorylation and downstream signaling in NCI-H716 xenograft tumors EXEL-6484 treatment inhibits NCI-H716 xenograft tumor growth and induces up to • 70% regression at well-tolerated doses These results further validate the use of FGFR inhibitors for the treatment of tumor • types with FGFR pathway activation identified by tumor genomics Acknowledgements This study and the development of this poster were supported by Exelixis, Inc. ABSTRACT Background: Deregulated FGF signaling promotes oncogenesis in several tumor types including gastric, bladder, endometrial, breast and multiple myeloma. Tumor genomic analyses of these cancers has identified amplifications, translocations, and activating mutations in FGFR1, FGFR2, and FGFR3. Inhibitors selective for the FGFR family provide an opportunity to target diverse cancer subtypes driven by FGFR activation while avoiding potential complications from VEGFR/PDGFR inhibition and anti-angiogenic therapy. Methods: Small molecule inhibitors of FGFR family kinases were identified by high-throughput screening and lead optimization using in vitro enzymatic assays. FGFR2 activation was measured in cultured cells and in xenograft tumors using phosphorylation of FGFR and downstream signaling proteins FRS2, ERK, and AKT. Cell viability was assessed by measuring cellular ATP levels. Xenograft tumors were grown in nude mice and compounds dosed by oral gavage. Results: FGFR inhibitors were identified with potent biochemical activity against FGFR1, FGFR2, and FGFR3 (IC 50 10-100 nM). X-ray crystallographic studies with FGFR2 demonstrated that the compounds bind in the ATP binding pocket. Cell viability assays were used to identify FGFR-dependent tumor cell lines. Most of these lines have genetic alterations in FGFR family members such as the colon adenocarcinoma line NCI-H716 which contains an amplification of FGFR2. Treatment of NCI-H716 cells with FGFR inhibitors blocks phosphorylation of FGFR2 and the downstream proteins FRS2, ERK, and AKT (IC 50 10-100 nM) as well as a broader phosphotyrosine signaling network that includes the HER family kinases. In vivo pharmacodynamic studies with orally bioavailable compounds demonstrated target inhibition in NCI-H716 xenograft tumors as assessed by a reduction in pFGFR2. Efficacy studies in the NCI-H716 xenograft model showed up to 70% tumor regression at well tolerated doses. Distinct chemical subseries were identified that selectively inhibit FGFR2 vs. other family members or that inhibit FGFR2 with mutations at the gatekeeper residue V584, a common source of resistance to kinase inhibitor therapy. Conclusions: In FGFR-driven tumor models, FGFR selective inhibitors block receptor activation and downstream signaling, reduce cell viability in vitro, and inhibit the growth of xenograft tumors. These results support advancement of FGFR selective inhibitors for the treatment of select cancer subtypes identified by tumor genomics. INTRODUCTION FGF signaling via the FGFR family of receptor tyrosine kinases regulates diverse physiological processes including pattern formation and organogenesis during development as well as homeostatic functions in adults. FGF-promoted receptor transphosphorylation activates downstream signaling pathways including ERK, AKT, and PLCγ via adaptor proteins including FRS2 (FGFR substrate 2) resulting in regulation of cell proliferation, survival, and differentiation. Consistent with the role of FGF ligands in normal development, dysregulation of FGF signaling can cause developmental disorders including craniosynostosis and chondrodysplasia syndromes due to germline activating mutations in FGFR1/2/3. More recently, tumor genomics has provided strong target validation for the FGFR family in multiple cancer types. Common Genomic Alterations of FGFRs in Human Tumors FGFR1 Amplification/overexpression in luminal B breast cancer (23%) • Translocation in EMS (8p11 myeloproliferative syndrome)/AML • FGFR2 Amplification in gastric cancer (10%) • Activating mutations in endometrial (10-16%) and prostate (8%) cancer • Genetic variants associated with breast cancer susceptibility • FGFR3 Translocation t(4;14) in multiple myeloma (15-20%) • Activating mutations in bladder cancer (40-60%) and squamous cell • carcinoma (60%) FGFR4 Activating mutations in rhabdomyosarcoma (7%) • Rationale for FGFR Selective Inhibitors Target diverse tumor types driven by FGFR1/2/3 • Distinguish from numerous VEGFR/FGFR inhibitors in clinical development • Enhanced specificity vs. other tyrosine kinases – Activity against resistance mutations including gatekeeper mutation – Avoid toxicities associated with anti-angiogenic therapy • Cardiovascular complications (hypertension, cardiac damage) – Metabolic dysfunction (hypothyroidism) – Tumor progression (local invasion and distant metastasis) – Discovery of Selective Inhibitors of Fibroblast Growth Factor Receptor (FGFR): Antitumor Activity in Cellular and Xenograft Tumor Models with FGFR Activation David Markby, Matt Williams, Jonathan Li, Artur Plonowski, Sean Wu, Julie Lougheed, Mike Ollmann Exelixis, South San Francisco, CA, USA 220 FGFR2 inhibitors were identified by a biochemical high-throughput screen of 4.6 million compounds, which yielded 1100 compounds with IC 50 < 5 µM from multiple chemical classes. EXEL-3261 was identified in the initial screen, and further optimization of the scaffold led to the identification of EXEL-6484 and other lead compounds. Table 1. Biochemical Selectivity of Inhibitors for FGFR1-4, VEGFR2, and PDGFRβ Compound Biochemical IC 50 (nM) FGFR2 FGFR1 FGFR3 FGFR4 VEGFR2 PDGFRβ EXEL-3261 11 21 60 137 317 >3600 EXEL-6484 29 63 116 234 614 3679 PD173074 1.8 3.6 5.4 438 65 >3500 AZD2171 11 37 19 829 <1.6 139 AG13736 8.4 19 27 995 <1.6 <1.6 TKI-258 12 29 21 293 21 13 IC 50 : concentration required for 50% inhibition EXEL-3261 and EXEL-6484 inhibit FGFR1-3 with IC • 50 values between 10 nM and 120 nM. Both compounds are greater than 10-fold selective for FGFR2 vs. VEGFR2 and PDGFRβ. Figure 1. Structure of the FGFR2:EXEL-3261 Complex (A) The structure of the kinase domain of FGFR2 in complex with EXEL-3261 at 2.45Å resolution. (B) The FGFR2 nucleotide complex with bound ATP analog and peptide substrate (PDB ID 2PVF). EXEL-3261 binds to the nucleotide binding site of FGFR2 in the inactive “Phe-out” • conformation EXEL-3261 interacts with the linker region and packs against the Phe of DFG and • the C-helix Figure 2. Identification of FGFR1/2/3 Dependent Tumor Cell Models: Inhibition of Cell Viability by EXEL-3261 0 5000 10000 15000 20000 25000 30000 77 Cell Lines Viabililty IC 50 (nM) Cell viability was measured by CellTiterGlo after 72-hour compound treatment in complete growth media. Figure 4. Inhibition of FGFR2 Phosphorylation and Downstream Signaling in NCI-H716 Xenograft Tumors by EXEL-6484 pFGFR (% Inhibition) 0 82 39 59 94 pFRS2 (% Inhibition) 0 62 23 44 80 pERK (% Inhibition) 0 73 5 40 92 pAkt (% Inhibition) 0 84 46 79 90 [Inhibitor] Plasma (μM) 0.9 0.0 0.1 1.2 [Inhibitor] Tumor (μM) 1.7 0.0 0.1 1.3 – 30 EXEL-3261 EXEL-6484 Vehicle EXEL-6484 EXEL-6484 30 100 10 pFGFR Y653/Y654 FGFR2 FRS2 pERK T202/Y204 ERK pAkt S473 Akt pFRS2 Y196 mg/kg Phosphoprotein levels in tumor xenografts were measured at 4 hours following a single oral dose of compound. EXEL-6484 inhibits FGFR2 phosphorylation and downstream activation of AKT and • ERK in NCI-H716 tumor xenografts. Inhibition is > 90% at 100 mg/kg at 4 hours post-dose. Figure 5. Inhibition of NCI-H716 Xenograft Tumor Growth by EXEL-6484 0 500 1000 1500 0 5 10 15 20 25 30 35 Days Post Implantation Mean Tumor Weight (mg) Vehicle 30 mg/kg bid 100 mg/kg bid 200 mg/kg bid 100 mg/kg qd 300 mg/kg qd Tumor xenograft growth was monitored over a 14-day period with EXEL-6484 dosed orally at the indicated schedules. Table 4. Inhibition of NCI-H716 Xenograft Tumor Growth by EXEL-6484 Group % TGI % regression Vehicle – – 100 mg/kg qd 53 – 300 mg/kg qd 80 – 30 mg/kg bid 23 – 100 mg/kg bid 78 – 200 mg/kg bid >100 70 TGI: tumor growth inhibition EXEL-6484 inhibits NCI-H716 xenograft tumor growth with 70% regression at • 200 mg/kg bid EXEL-6484 is well tolerated at efficacious doses with less than 7% body weight • loss and no dose skipping (not shown) RESULTS
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EORTC10_220_FGFR
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22nd EORTC-NCI-AACR Symposium on Molecular Targets and Cancer Therapeutics, November 16-19, 2010, Berlin, Germany
Table 2. Inhibition of Cell Viability by EXEL-3261: IC50 Values for Sensitive Cell Lines
Cell LineViability IC50
(nM) FGFR genotype Tissue
NCI-H716 69 FGFR2 gene amplification Colon
KATO III 193 FGFR2 gene amplification Gastric
SNU-16 250 FGFR2 gene amplification Gastric
KG-1 366 FGFR1 translocation AML
MFM-223 649 FGFR2 gene amplification Breast
RT-112 1223 FGFR3 amplification Bladder
OPM-2 1345 FGFR3 translocation and activating mutation Multiple Myeloma
AN3 CA 1512 FGFR2 activating mutation Endometrium
HuH-7 1852 FGFR4 overexpression Hepatic
DMS114 2859 FGFR1 amplification SCLC
Ten out of 11 cell lines with EXEL-3261 viability IC 50 values less than 3000 nM have genetic alterations in FGFR family membersThese same cell lines are also sensitive to PD173074 and TKI-258 (not shown)
Table 3. Inhibition of FGFR2 Phosphorylation and Cell Viability by FGFR Inhibitors in NCI-H716 Colon Adenocarcinoma Line
Compound
Biochemical IC50 (nM)
Cellular IC50 (nM) NCI-H716
FGFR2 pFGFR2 Viability
EXEL-3261 11 17 69EXEL-6484 29 28 27PD173074 1.8 76 0.4AZD2171 11 122 103AG13736 8.4 105 89TKI-258 12 158 121
EXEL-3261 and EXEL-6484 inhibit FGFR2 activity in vitro and in cells with IC 50 values less than 30 nM
Figure 3. Inhibition of FGFR2 Signaling and Broader Phosphotyrosine Networks by EXEL-6484
pFGFRY653/Y654
EXEL-6484 (nM)
DM
SO
PD17
3074
pAKTS473
Inhibition (%)
Erlo
tinib
Lapa
tinib
Inhibition (%)
pERKT202/Y204
Inhibition (%)
pEGFRY845
pHER2Y1221/Y1222
Inhibition (%)
Inhibition (%)
pHER3Y1197
-actin
Inhibition (%)
FGFR2
HERFamily
AKTERK
0 99 98 97 85 56 31 27
0 66 83 82 74 50 33 1
0 92 94 92 80 60 21 4
0 87 87 83 62 30 19 13
0 84 80 80 75 40 9 10
0 93 93 82 88 65 37 28
3000 1000 333 111 37 12 4
Phosphoprotein levels were measured by Western blot following 3 hr compound treatment of NCI-H716 cells in RPMI 1640 containing 10% FCS. Control compounds were PD173074 (3000 nM), erlotinib (5000 nM), and lapatinib (5000 nM).
Treatment of NCI-H716 cells with EXEL-6484 inhibited phosphorylation of FGFR2, ERK, and AKT with an IC50 value of approximately 50 nMBoth EXEL-6484 and PD173074 also reduced phosphorylation of EGFR, HER2, and HER3 while HER kinase inhibitors erlotinib and lapatinib did notIn NCI-H716 cells, FGFR2 overexpression not only promotes activation of downstream pathways including ERK and AKT, but also HER family receptor tyrosine kinases
Table 5. Inhibitors with Selectivity Against FGFR2 over Other Isozymes
EXEL-ID
Biochemical IC50 (nM) Cellular IC50 (nM)
FGFR2 FGFR1 FGFR3 FGFR4 pFGFR2 Viability
EXEL-3261 11 21 60 137 17 69EXEL-6484 29 63 116 234 28 27EXEL-6007 4 31 55 235 28 113EXEL-4832 4 39 87 161 31 147EXEL-2825 5 40 114 357 50 128EXEL-6009 6 44 134 1610 86 103EXEL-4804 9 147 342 1855 232 288EXEL-6676 17 199 377 717 153 40EXEL-9291 17 163 278 2245 517 174Key: green: IC50 less than 2 wild type FGFR2 IC50; yellow: IC50 between 2 and 10 wild type FGFR2 IC50; orange: IC50 greater than 10 wild type FGFR2 IC50pFGFR2 IC50 values were determined for FGFR2 constructs over-expressed in HEK 293 cells.
Lead compounds EXEL-3261 and EXEL-6484 inhibit isozymes FGFR1-3. The rank order of potencies is FGFR2 > FGFR1 > FGFR3, and IC50 values are within five-fold of one another.
A subset of compounds in the series exhibits preferential activity against FGFR2 with greater than 10-fold selectivity over the other family members (EXEL-4804 and EXEL-6676)
Table 6. Differential Activity of FGFR Inhibitors Against Gatekeeper Mutations
EXEL-ID
Biochemical FGFR2
IC50 (nM)
Cellular pFGFR2 IC50 (nM)
Wild Type FGFR2
Activating Mutation
N569K
Gatekeeper Mutation
V584M
Gatekeeper Mutation
V584F
Gatekeeper Mutation
V584IAZD2171 11 64 43 >10,000 >10,000 >10,000AG13736 8.4 193 106 4056 2885 2822TKI-258 12 159 74 412 37 2079EXEL-3261 11 30 36 499 21 89EXEL-6484 29 74 102 735 42 248EXEL-6007 3.7 35 45 181 123 68EXEL-7245 9.2 31 N/A 89 31 76EXEL-7524 42 327 344 47 16 423EXEL-7650 578 723 886 105 21 609Key: green: IC50 less than 2 wild type FGFR2 IC50; yellow: IC50 between 2 and 10 wild type FGFR2 IC50; orange: IC50 greater than 10 wild type FGFR2 IC50pFGFR2 IC50 values were determined for FGFR2 constructs overexpressed in HEK 293 cells.
FGFR inhibitors exhibit varying activity against FGFR2 with mutations at gatekeeper residue V584 ranging from relatively weak activity (AZD2171 and AG13736) to increased potency compared to wild type FGFR2 (EXEL-7524 and EXEL-7650)
All tested compounds are equipotent against the activating mutant N569K compared to wild type FGFR2
CONCLUSIONS/SUMMARY
We have identified a series of potent FGFR2 inhibitors with biochemical and cellular IC50 values less than 50 nM and greater than 10-fold selectivity over VEGFR2 and PDGFRIdentified inhibitors bind to the nucleotide binding site of FGFR2 in the inactive Phe-out conformation Distinct subseries were found that are (1) specific for FGFR2 over FGFR1/3/4 or (2) active against potential resistance mutations at the gatekeeper residueFGFR inhibitors reduce in vitro cell viability for a subset of human tumor lines with genetic activation of FGFR family membersIn pharmacodynamic studies, lead compound EXEL-6484 reduces FGFR2 phosphorylation and downstream signaling in NCI-H716 xenograft tumorsEXEL-6484 treatment inhibits NCI-H716 xenograft tumor growth and induces up to 70% regression at well-tolerated dosesThese results further validate the use of FGFR inhibitors for the treatment of tumor types with FGFR pathway activation identified by tumor genomics
AcknowledgementsThis study and the development of this poster were supported by Exelixis, Inc.
ABSTRACTBackground: Deregulated FGF signaling promotes oncogenesis in several tumor types including gastric, bladder, endometrial, breast and multiple myeloma. Tumor genomic analyses of these cancers has identified amplifications, translocations, and activating mutations in FGFR1, FGFR2, and FGFR3. Inhibitors selective for the FGFR family provide an opportunity to target diverse cancer subtypes driven by FGFR activation while avoiding potential complications from VEGFR/PDGFR inhibition and anti-angiogenic therapy.
Methods: Small molecule inhibitors of FGFR family kinases were identified by high-throughput screening and lead optimization using in vitro enzymatic assays. FGFR2 activation was measured in cultured cells and in xenograft tumors using phosphorylation of FGFR and downstream signaling proteins FRS2, ERK, and AKT. Cell viability was assessed by measuring cellular ATP levels. Xenograft tumors were grown in nude mice and compounds dosed by oral gavage.
Results: FGFR inhibitors were identified with potent biochemical activity against FGFR1, FGFR2, and FGFR3 (IC50 10-100 nM). X-ray crystallographic studies with FGFR2 demonstrated that the compounds bind in the ATP binding pocket. Cell viability assays were used to identify FGFR-dependent tumor cell lines. Most of these lines have genetic alterations in FGFR family members such as the colon adenocarcinoma line NCI-H716 which contains an amplification of FGFR2. Treatment of NCI-H716 cells with FGFR inhibitors blocks phosphorylation of FGFR2 and the downstream proteins FRS2, ERK, and AKT (IC50 10-100 nM) as well as a broader phosphotyrosine signaling network that includes the HER family kinases. In vivo pharmacodynamic studies with orally bioavailable compounds demonstrated target inhibition in NCI-H716 xenograft tumors as assessed by a reduction in pFGFR2. Efficacy studies in the NCI-H716 xenograft model showed up to 70% tumor regression at well tolerated doses. Distinct chemical subseries were identified that selectively inhibit FGFR2 vs. other family members or that inhibit FGFR2 with mutations at the gatekeeper residue V584, a common source of resistance to kinase inhibitor therapy.
Conclusions: In FGFR-driven tumor models, FGFR selective inhibitors block receptor activation and downstream signaling, reduce cell viability in vitro, and inhibit the growth of xenograft tumors. These results support advancement of FGFR selective inhibitors for the treatment of select cancer subtypes identified by tumor genomics.
INTRODUCTION
FGF signaling via the FGFR family of receptor tyrosine kinases regulates diverse physiological processes including pattern formation and organogenesis during development as well as homeostatic functions in adults. FGF-promoted receptor transphosphorylation activates downstream signaling pathways including ERK, AKT, and PLC via adaptor proteins including FRS2 (FGFR substrate 2) resulting in regulation of cell proliferation, survival, and differentiation. Consistent with the role of FGF ligands in normal development, dysregulation of FGF signaling can cause developmental disorders including craniosynostosis and chondrodysplasia syndromes due to germline activating mutations in FGFR1/2/3. More recently, tumor genomics has provided strong target validation for the FGFR family in multiple cancer types.
Common Genomic Alterations of FGFRs in Human TumorsFGFR1
Amplification/overexpression in luminal B breast cancer (23%)
Translocation in EMS (8p11 myeloproliferative syndrome)/AML
FGFR2Amplification in gastric cancer (10%)
Activating mutations in endometrial (10-16%) and prostate (8%) cancer
Genetic variants associated with breast cancer susceptibility
FGFR3Translocation t(4;14) in multiple myeloma (15-20%)
Activating mutations in bladder cancer (40-60%) and squamous cell carcinoma (60%)
FGFR4Activating mutations in rhabdomyosarcoma (7%)
Rationale for FGFR Selective InhibitorsTarget diverse tumor types driven by FGFR1/2/3
Distinguish from numerous VEGFR/FGFR inhibitors in clinical development
Enhanced specificity vs. other tyrosine kinases Activity against resistance mutations including gatekeeper mutation
Avoid toxicities associated with anti-angiogenic therapy
Cardiovascular complications (hypertension, cardiac damage) Metabolic dysfunction (hypothyroidism) Tumor progression (local invasion and distant metastasis)
Discovery of Selective Inhibitors of Fibroblast Growth Factor Receptor (FGFR): Antitumor Activity in Cellular and Xenograft Tumor Models with FGFR ActivationDavid Markby, Matt Williams, Jonathan Li, Artur Plonowski, Sean Wu, Julie Lougheed, Mike Ollmann
Exelixis, South San Francisco, CA, USA
220
FGFR2 inhibitors were identified by a biochemical high-throughput screen of 4.6 million compounds, which yielded 1100 compounds with IC50 < 5 M from multiple chemical classes. EXEL-3261 was identified in the initial screen, and further optimization of the scaffold led to the identification of EXEL-6484 and other lead compounds.
Table 1. Biochemical Selectivity of Inhibitors for FGFR1-4, VEGFR2, and PDGFR
Compound
Biochemical IC50 (nM)
FGFR2 FGFR1 FGFR3 FGFR4 VEGFR2 PDGFR
EXEL-3261 11 21 60 137 317 >3600
EXEL-6484 29 63 116 234 614 3679
PD173074 1.8 3.6 5.4 438 65 >3500
AZD2171 11 37 19 829
