Download - Jeffrey Toretsky Department of Oncology and Pediatrics Lombardi Comprehensive Cancer Center
Therapeutic Targeting of EWS-FLI1: Small Molecule Protein-Protein
Interaction Inhibitors Prevent Xenograft Growth
Jeffrey ToretskyDepartment of Oncology and Pediatrics
Lombardi Comprehensive Cancer CenterGeorgetown University
Washington, DC
Ewing’s Sarcoma Family of Tumors
• 1992: Breakpoint of t(11;22) cloned; EWS constitutively expressed– Derived chromosome 22 expressed as EWS-FLI1
11 der11
22 der22
•1983: t(11;22) by standard g-banded karyotype
•Elimination of EWS-FLI1 from ESFT cells is lethal
Transcription Factor Biology
Fusion Gene or Message Fusion Protein
Cell Membrane
Translation
Nucleus
2211
Transformation
MGDVKNFLYAWCGKRKMTPSYEIRAVGNKNRQKFMCEVQVEGYNYTGMGNSTNKKDAQSNAARDFVNYLVRINEIKSEEVPAFGVASPPPLTDTPDTTANAEGDLPTTMGGPLPPHLALKAENNSEVGASGYGVPGPTWDRGANLKDYYSRKEEQEVQATLESEEVDLNAGLHGNWTLENAKARLNQYFQKEKIQGEYKYTQVGPDHNRSFIAEMTIYIKQLGRRIFAREHGSNKKLAAQSCALSLVRQLYHLGVVEAYSGLTKKKEGETVEPYKVNLSQDLEHQLQNIIQELNLEILPPPEDPSVPVALNIGKLAQFEPSQRQNQVGVVPWSPPQSNWNPWTSSNIDEGPLAFATPEQISMDLKNELMYQLEQDHDLQAILQERELLPVKKFESEILEAISQNSVVIIRGATGCGKTTQVPQFILDDFIQNDRAAECNIVVTQPRRISAVSVAERVAFERGEEPGKSCGYSVRFESILPRPHASIMFCTVGVLLRKLEAGIRGISHVIVDEIHERDINTDFLLVVLRDVVQAYPEVRIVLMSATIDTSMFCEYFFNCPIIEVYGRTYPVQEYFLEDCIQMTHFVPPPKDKKKKDKDDDGGEDDDANCNLICGDEYGPETRLSMSQLNEKETPFELIEALLKYIETLNVPGAVLVFLPGWNLIYTMQKHLEMNPHFGSHRYQILPLHSQIPREEQRKVFDPVPVGVTKVILSTNIAETSITINDVVYVIDSCKQKVKLFTAHNNMTNYSTVWASKTNLEQRKGRAGRSTAGFCFHL
PPPL---IEACSRARFERLETHMTPEMFRTPLHEIALSIKLLRLGGIGQFLAKAIEPPPLDAVIEAEH
TLRELDALDANDELTPLGRILAKLPIEPRFGKMMIMGCIFYVGDAICTIAAATCFPEPFINEGKRLGYIHRNFAGNRFSDHVALLSVFQAWDDARMGGEEAEIRFCEHKRLNMATLRMTWEAKVQLKEILINSGFPEDCLLTQVFTNTGPDNNLDVVISLLAFGVYPNVCYHKEKRKILTTEGRNALIHKSSVNCPFSSQDMKYPSPFFVFGEKIRTRAISAKGMTLVPPLQLLLFASKKVQSDGQIVLVDDWIKLQISHEAAACITGLRAAMEALVVEVTKQPAIISQLDPVNERMLNMIRQISRPSAAGINLMIGSTRYGDGPRPPKMARYDNGSGYRRGGSSYSGGGYGGGYSSGGYGSGGYGGSANSFRAGYGAGVGGGYRGVSRGGFRGNSGGDYRGPSGGYRGSGGFQRGGGRGAYGTGYFGQGRGGGGY
E9R =PPPLDAVIEA
Progression from Oncogene to Protein Partner to Poison
EWS-FLI1
RHA
Peptide blocks RHA from binding to EWS-FLI
EWS-FLI1
RNA Helicase A
(RHA)
Uren, Biochemistry 2004Toretsky, Cancer Res 2006
5
Mutant RHA fails to enhance EWS-FLI1 dependent transformation
Col
ony
num
bers
600
400
200
0
EWS-FLI1RHA
RHA-D827A
150
75
FLAG-RHA
EWS-FLI1
Anchorage independent growth
RHA peptide inhibits ESFT growth
EWS/FLI1
E9R-P
EGFP E9R
EGFP E9RNES
pGE9R
pGCE9R
P823
P824
P825
L826
D827A828
V829
I830
E831
A832
Peptide Model: Region of Overlap with Lead
Compound
GFP-E9R10 aa peptide
NSC635437in
DruggableSpace
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Screening Library for Potential Binding
Position Sample MW Cycle No.(071106) Binding level (normal by Rmax)Binding stability (nomalized by Rmax) 3rd ka (1/Ms) kd (1/s) KD (M) Rmax (RU)R1H2 1234 550 12 0.92 0.66 7066 0.002485 3.52E-07 39.1R1D7 2345 769 52 0.94 0.70 2301 9.03E-04 3.92E-07 95.6R1B2 43456 523 6 0.46 0.28 1.98E+04 0.01127 7.52E-07 14.6
R1E9 3564 461 71 0.68 0.38 1577 0.002408 1.53E-06 98R1H3 7654 503 21 0.67 0.38 1053 0.005216 4.95E-06 36
Small molecule synthesis and optimizationNCI Compound NSC635437 synthesized at GUIdentified Lead with Homology to Peptide E9RAnalogs were synthesizedYK-4-279 selected based upon binding, EWS-FLI1:RHA disruption, and cytotoxicity profile
YK-4-279 blocks RHA binding to EWS-FLI1
YK-4-279 displaces E9R from EWS-FLI1
YK-4-279 reduces EWS-FLI1 transcription activity
YK-4-279 toxicity against EF compared to non-EF cell lines
YK-4-279 Inhibits ESFT Xenografts
Summary• Validated EWS-FLI1:RHA as molecular target• SPR screen identified small molecule lead• YK-4-279 demonstrates relative specificity with
– 1 microM IC50 against ESFT – Xenograft studies
• Further chemical optimization underway• To be followed by ADME/tox• Hopeful to advance to clinical trials
CollaboratorsCollaborators
•Milton Brown and Yali Kong, GU, LCCC•David Loeb, JHH•Melinda Merchant, NIH•Angela Koehler, Broad Institute•Anton Wellstein, GU, LCCC•Steve Lessnick and Leah Owen, Utah•Olga Tscherkasskaya, GU•Jeff Parvin, Harvard•Michael Grusby, Harvard•Sean Lee, NIH•Marc Landanyi, MSKCC•Tim Cripe, Cincinnati Children’s
SupportSupport
Children’s Cancer Foundation, Baltimore, MD Dani’s Foundation Go4theGoal Liddy Shriver Sarcoma Initiative Amschwand Sarcoma Foundation CureSearch, NCCF NIH, NCI Burroughs-Wellcome
Translational Scientist Award