Therapeutic Targeting of EWS-FLI1: Small Molecule Protein-Protein

Interaction Inhibitors Prevent Xenograft Growth

Jeffrey ToretskyDepartment of Oncology and Pediatrics

Lombardi Comprehensive Cancer Center

Georgetown 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-FLI111 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

Co

lon

y n

um

ber

s

60

04

00

20

00

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 39.1

R1D7 2345 769 52 0.94 0.70 2301 9.03E-04 3.92E-07 95.6

R1B2 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 98

R1H3 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