Therapeutic Targeting of TFE3 in Translocation Renal Cell Carcinoma

Nur Damayanti, PhDRoberto Pili, MD

• 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.

tRCC chromosomal rearrangement

17 TFE3 genes fusions identified in tRCC

NONOSFPQPRCCCLTCASPCR1

TFE3

TFE3TFE3

TFE3

RBM10PARP14LUC7L3KHSRPDVL2

TFE3TFE3TFE3TFE3

TFE3

MED15GRIPAP1MALATINEATKAT6ARBMXMALATI TFEB

TFE3TFE3

TFE3

And still counting….

TFE3TFE3TFE3

Generation of tRCC RP-R07 PDX model-NGS based top down approach

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.

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

-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

(+) BEZ-235 250 nM 96 h

F-ac

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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 ]

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(%

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1 0- 1

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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.

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.

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.

RP-R07 UOK-109 UOK-146

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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)

Differential pathways among different fusions even in the same family

Pili lab, SFPQ-TFE3

Linehan lab, PRCC-TFE3

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

-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

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

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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)

Ongoing Work : Establishing functional mechanism of TFE3

How does TFE3 accomplish its function ?

“TFE3 is functional in dimer/multimer state (Beckmann et al, 1991)”

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

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

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

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

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.

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