therapeutic targeting of tfe3 in translocation renal cell ......2019/12/22 · conclusions •...
TRANSCRIPT
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Therapeutic Targeting of TFE3 in Translocation Renal Cell Carcinoma
Nur Damayanti, PhDRoberto Pili, MD
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• tRCC is an aggressive disease
Argani P et al, 2001; Malouf G at al, 2013; Malouf G at al, 2014; Kauffman E et al, 2014; Inamura et al, 2017, Gandhi J et al 2019
A newly defined subtype of RCC characterized by early onset, chromosomal rearrangement, and nuclear expression of TFE3/B (WHO 2016).
~ 20–40% of childhood RCC and 1–4% of adult RCC.
Refractory/resistant to standard treatments for RCC.
• Translocation Renal Carcinoma (tRCC)
• tRCC incidence
• Very low mutational burden
Translocation renal cell carcinoma (tRCC) quick facts
• There is no routine testing for tRCCTrue incidence may be underestimated .
• tRCC molecular mechanisms are not clearly understood.
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tRCC chromosomal rearrangement
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17 TFE3 genes fusions identified in tRCC
NONOSFPQPRCCCLTCASPCR1
TFE3
TFE3TFE3
TFE3
RBM10PARP14LUC7L3KHSRPDVL2
TFE3TFE3TFE3TFE3
TFE3
MED15GRIPAP1MALATINEATKAT6ARBMXMALATI TFEB
TFE3TFE3
TFE3
And still counting….
TFE3TFE3TFE3
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Generation of tRCC RP-R07 PDX model-NGS based top down approach
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Biopsy
sample
RP-R07
PDX
TFE3H&E
RP-R07 PDX
TFE3 ACTIN HOECSHT
RP-R07 CELLS
tRCC RP-R07 fidelity
Damayanti et al 2018, Clin. Cancer Res.
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RP-R07 gene fusion identification by next generation sequencing RNA-seq
RT-PCRRNA-seq
Damayanti et al 2018, Clin Cancer Res
(t(X;1) (p11.2; p34))
Collaboration with Hollenhorst lab, Dr. Justin Budka Indiana University
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-7000 -3500 5’ End 3’ End 3500 7000 -7000 -3500 5’ End 3’ End 3500 7000
-1.1 -0.6 0 2.0 3.9 -1.4 -0.7 0 1.6 3.2
Colorkey Colorkey
TFE3 RP-R07 Cell-line TFE3 RP-R07 PDX
Representative TFE3 target genes out of 3032 total targeted genes
14-3-3;IRS1;PTEN;TSC1;VEGFR-2;ITGB3;TNC LAMC2;PRL;PIK3CB;IGF1R;TCL1B;CCND1;JAK MYC;CREB3L2;AKT3;KDR;TNR;ITGB6;IL6R;JAK;HSP90;IL4R;ITGA3;RPS6;F2R;ITGA1;TSC1;NGF;YWHAZ;COL6A2;COL4A3;DDIT4;SGK1;MET;PHLPP1;PKN3;LAMA1;LAMA3;PIK3R1;EFNA5;THBS2;EGFR;THBS4;PIK3R5;NRAS;BCL2L11;RELN;GNG7;EIF4B;NGFR;FN1;OSM;VEGFC;LAMB1;VEGFA;COL1A1;G6PC3;CDK6;CDK4;PPP2R2B;FGF18;TEK;GNB5;BCL2L1,CHRM2; ITGB1;CRTC2;CSF3R
TFE3 DNA binding targets by NSG ChIP-seq and PI3K/AKT/mTOR
TFE3 Binding site
RP-R07 RP-R07 tTFE3 Binding Motif –E-Box
Collaboration with Hollenhorst lab, Dr. Justin Budka Indiana University
Damayanti et al 2018, Clin. Cancer Res
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(+) BEZ-235 250 nM 96 h
F-ac
tin
Ho
ech
stp
-4e
bp
-1 s
er-
65
faosv
ca
lln
F-ac
tin
P-S
6 r
ibo
som
al
S-2
44
F-ac
tin
P-A
KT
LDH
Ho
ech
stTherapeutic intervention with dual PI3K/mTOR inhibitor
Patient derived cell line
MTT assay In-Vivo Assay
B E Z 2 3 5 L o g [ n M ]
Re
la
tiv
e c
ell g
ro
wth
(%
)
1 0- 1
1 00
1 01
1 02
1 03
1 04
1 05
- 1 5 0
- 1 0 0
- 5 0
0
5 0
1 0 0
1 5 0
2 0 0
Patient derived xenograft
Damayanti et al 2018, Clin. Cancer Res.
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Phospho 4EBP-1 ACTIN HOECHST
siTFE3scramble
Phospho S6 ACTIN HOECHST
N-TFE3 ACTIN HOECHST
Molecular mechanism validation -TFE3 targets PI3K/mTOR
Damayanti et al 2018, Clin. Cancer Res.
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D
UOK-146
Nuclear speckle
UMR-C2RP-R07 UOK-109
Internal -TFE3 Hoechst
Differential nuclear TFE3 pattern among cells with fusions in the same family
SFPQ-TFE3 NONO-TFE3 PRCC-TFE3 TFE3
UOK-109 and UOK-146 were gifts from Linehan Lab (NCI/NIH)
Damayanti et al 2018, Clin. Cancer Res.
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RP-R07 UOK-109 UOK-146
Mit
otr
ack
er-
red
FM
F-a
ctin
E-C
ad
he
rin
Cy
tok
era
tin
A
B
Vim
en
tin
α-T
ub
uli
nF
-act
in
C
D
E
RP-R07 UOK-109 UOK-146 UMR-C2
Differential phenotype among fusions in the same family
2D CELL CULTURE 3D CELL CULTURE
UOK-109 and UOK-146 were gifts from Linehan Lab (NCI/NIH)
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Differential pathways among different fusions even in the same family
Pili lab, SFPQ-TFE3
Linehan lab, PRCC-TFE3
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17 TFE3 genes fusions identified in tRCC
NONOSFPQPRCCCLTCASPCR1
TFE3
TFE3TFE3
TFE3
RBM10PARP14LUC7L3KHSRPDVL2
TFE3TFE3TFE3TFE3
TFE3
MED15GRIPAP1MALATINEATKAT6ARBMX
TFE3TFE3
TFE3
And still counting….
TFE3TFE3TFE3
TFE3
TFE3 is the common denominator
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-100 kD-75 kD
-25 kD
Mouse anti-GFP
The Protein The Partner
The “Exotic” Fusion
Tale of three proteins : is TFE3 protein different from its partner and fusion?
mClover TFE3
mClover SFPQTFE3
mClover SFPQ
HEK-293 cells
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0 3 6 912
15
18
21
24
27
30
33
36
39
42
45
48
51
54
57
60
63
66
69
72
0
2 0 0 0
4 0 0 0
6 0 0 0
8 0 0 0
1 0 0 0 0
Co
un
ts (
/we
ll)
S F P Q C L O V E R
S F P Q -T F E 3 C L O V E R
T F E 3 C L O V E R
C L O V E R
TFE3-clover SFPQ-clover
SFPQTFE3-clover
Tale of three proteins : is TFE3 protein different from its partner and fusion?
Proliferative phenotype in HEK-293 cells
The fusion protein is the driver for acquisition of oncogenic phenotype
Time (hours)
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Ongoing Work : Establishing functional mechanism of TFE3
How does TFE3 accomplish its function ?
“TFE3 is functional in dimer/multimer state (Beckmann et al, 1991)”
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partner Donor
TFE3 Acceptor
partner
TFE3
Dimerization –FRET
Intermolecular FRET biosensing platform
Establishing mechanism of action of TFE3 by Fluorescence Resonance Energy Transfer (FRET)
High Energy Donor Lower Energy Acceptor
Donor Acceptor
mClover SFPQ-TFE3 Ruby TFE3 SFPQTFE3/TFE3
Interaction FRET
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Establishing mechanism of action of TFE3 by Fluorescence Resonance Energy Transfer (FRET)
mClover SFPQ-TFE3
Ruby TFE3
SFPQTFE3/TFE3
% FRET MAP
50
TFE3/ SFPQTFE3 FRETTFE3 alone
Fusion protein dimerizes with TFE3 wild type
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Drug discovery using High Throughput Screening combined with real time TFE3 –FRET biosensor
Real-time FRET
TFE3/ SFPQTFE3 FRETTFE3 alone
mClover SFPQ-TFE3
Ruby TFE3
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IU -115T
Establishing another PDX model of tRCC for lead compound testing
IU -115 CELLS
IU -115 PDX
Internal -TFE3 F-Actin
Hoechst
H&E TFE3
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Conclusions
• Translocation RCC remains a clinical challenge.
• We have established two patient derived models of tRCC; RP-R07 with SFPQ-TFE3 fusion and IU-115 with unknown fusion.
• PI3K-mTOR may represent a potential targeted signaling pathway in tRCC patient with SFPQ-TFE3 fusion.
• Different fusion partners of TFE3 may exhibit different biological phenotype.
• We are developing high throughput screening to discover novel drugs to inhibit TFE3.
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Acknowledgements
Pili Lab Dr. Nur DamayantiDr. Justin BudkaDr. Eri BannoDr. Kirk StasckheDr. Chinghai KaoDr. Ben ElzeyDr. May ElbannaDenise BraunLumin ZhangRicardo CordovaSherri Lipps
Hollenhorst Lab
Linehan Lab (NIH)
DOD KC170209