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James Brugarolas, M.D., Ph.D.Kidney Cancer Program Leader

Virginia Murchison Linthicum Endowed ScholarAssociate Professor of Internal Medicine

University of Texas Southwestern Medical Center

http://www.utsouthwestern.edu/kidneycancer

RCC: Molecular Pathways and Novel Therapies

Brugarolas Lab

Research Funding: Peloton Therapeutics, Inc.

I will discuss investigational use of a HIF-2 inhibitorSiegel et al., CA Cancer J Clin 2015

Kidney Cancer

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https://www.dshs.state.tx.us/tcr/data.shtm

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Sunitinib SorafenibPazopanibAxitinib

Temsirolimus Everolimus

Biological understanding leads to new therapies

Bevacizumab

Shen, C. and Kaelin, W.G. Sem. Can. Biol. 2013

Adapted from Brugarolas J. N Engl J Med 2007

Nivolumab

How do we advance the field?

New targetsNew pathways

HIF‐2

VEGF Myc/Cyclin D1 Oct4 p53

ANGIOGENESIS CELL PROLIFERATION PLURIPOTENCY APOPTOSIS

BevacizumabSunitinibSorafenibPazopanib

Axitinib

HIF2-I

Scheuermann et al. PNAS 2009

B

A

B

A

HRE (DNA)

HIF-2 HIF-1

HRE (DNA)

HIF-1HIF-2

Scheuermann et al. Nat Chem Biol 2013

High-Throughput Screen

HIF2-I

Development of a HIF-2 inhibitor (HIF2-I)

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Testing HIF2-I in kidney cancer

Human tumors in mice do they reproduce: Histological appearance?

Gene expression?

Mutations?

DNA copy number alterations?

Treatment responsiveness?

Sivanand et al., Sci Transl Med 2012Adapted from Sivanand et al., Sci Transl Med 2012

Unsupervised hierarchical clustering of gene expression shows similarities between tumors and corresponding tumorgrafts

HIF2-I is active against human ccRCC transplants in mice

Days

Tum

or v

olum

e (m

m3 )

Vehicle

Sunitinib

HIF2-I

HIF2-I is active in 50% of ccRCC

Inte

rmed

.Se

nsiti

veR

esis

tant

VehicleSunitinibHIF2-I

267 mice from 22 independently derived TG lines

Chen et al., Submitted – do not reproduce

HIF2-I inhibits proliferation and angiogenesis in sensitive ccRCC

XP164

XP469

XP454

XP373

HIF2-Ivehicle

V4239 P4244

P3297V3294

V5407 P5399

V4921 P4924

HIF2-Iveh

XP373

XP144

CD

31H

&E

XP373

Ki67

veh HIF2-I

XP16

4XP

490

XP16

9XP

373

vehicle HIF2‐I

V4237

V5239 V5229 P5231 P5240

V4236 V4241V4234

V3290 V3294 V3287 V3281

V3212 V3224 V3210 V3214

VehicleHIF2-ISunitinib

Chen et al., Submitted – do not reproduce

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HIF2-I dissociates HIF-2 in sensitive & resistant tumors

RE

SIS

TAN

T

S

EN

SIT

IVE

Chen et al., Submitted – do not reproduce

Reformation of HIF-2 dimers with acquired resistance

Input

IP HIF-1

V3290

V3294

V3298

P3283

P3288

P3297

V3290

V3294

V3298

P3283

P3297

Tubulin

post-resistance

HIF‐2

HIF‐1

P3288

pre-resistance

0500100015002000250030003500400045005000

‐20 0 20 40 60 80 100 120 140 160 180 200

Tum

or V

olum

e (m

m3 )

XP164

3283

3288

3296

3295

3299

3286 Veh

Sunitinib

HIF2-I

Chen et al., Submitted – do not reproduce

Phase I Clinical Trial of HIF2-I

Photo, courtesy of Brian Coats

Conclusions (Part I) RCC tumorgrafts reproduce the biological properties of patient

tumors.

Inhibition of arguably the most important driver of ccRCC, the HIF-2 transcription factor, abrogates tumor growth in 56% of ccRCC tumorgrafts, including tumors resistant to sunitinib.

HIF2-I effectively (and specifically) dissociates HIF-2 from HIF-1in human ccRCC implanted in mice.

HIF-2 inhibition results in the downregulation of HIF-2 target genes and decreased circulating levels of tumor-produced VEGF.

Primary resistance occurs despite dissociation of the HIF-2 complex in tumors.

A Phase I clinical trial at UTSW (and elsewhere) with a first-in-class HIF-2 inhibitor has completed accrual.

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Sunitinib SorafenibPazopanibAxitinib

Temsirolimus Everolimus

Bevacizumab

Biological understanding leads to new therapies

How do we go forward?

New targetsNew pathways

Adapted from Brugarolas J. N Engl J Med 2007

PBRM1 mutations in ~50% of ccRCC.

PBRM1 is a two-hit tumor suppressor gene located on chromosome 3p.

Encodes BAF180, a component of a nucleosome remodeling complex - SWI/SNF family (PBAF complex).

Thought to regulate DNA packing and accessibility.

BAP1 is mutated in ~15% of sporadic ccRCC.

BAP1 is a two-hit tumor suppressor gene located on chromosome 3p and mutations abrogate protein expression.

BAP1 is a nuclear DUB of the UCH family implicated in cell cycle regulation, DNA replication and DNA damage repair.

BAP1-mutant tumors tend to be of high grade and associated with mTOR complex 1 activation.

Tumors with simultaneous BAP1 and PBRM1 mutations are underrepresented

p = 0.00003

Pena-Llopis et al., Nat Genet 2012

What are these data telling us?

, deletionI, insertion*, non-senseS, splice site†, missense*L, stop codon lost

Mutation

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Under-representation of tumors with simultaneous BAP1 & PBRM1 in meta-analysis

Pena-Llopis et al., Can Res 2013

Study n PBRM1 BAP1 BAP1/ PBRM1

Expecteddouble mutants p value

Odds Ratio

(95% CI)

Peña-Llopis et al. 176 89 21 3 13 (9-16) 0.00003 0.10 (0.03 - 0.35)

Guo et al. 98 21 8 0 2 (0-4) 0.2 0.19 (0.01 - 3.43)

Hakimi et al. 185 53 10 1 3 (1-5) 0.18 0.23 (0.03 - 1.83)

TCGA 293 101 22 5 10 (7-13) 0.058 0.37 (0.14 - 1.01)

Total 576 175 40 6 14 (11-18) 0.004 0.29 (0.12 - 0.70)

 

A foundation for a molecular genetic classification of ccRCC

Pena-Llopis et al., Nature Genet 2012

High grade(q = 0.0005)

Low grade(q = 0.025)

Pena-Llopis et al., Can Res 2013

BAP1 and PBRM1 genes are on chromosome 3p and one allele is frequently co-deleted with VHL in ccRCC

Chr 3

VH

L

BA

P1

PB

RM

1

VHL intragenic mutation

VH

L

BA

P1

PB

RM

1

Loss of 3p

VH

L

BA

P1

PB

RM

1Brugarolas J. JCO 2014

VH

L

BA

P1

PB

RM

1 BAP1 mutation

High grade Low grade

VH

L

BA

P1

PB

RM

1PBRM1 mutation

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May explain why VHL+/- humans but not Vhl+/- mice develop renal cancer

Chr 6

Vhl Bap1Pbrm1

Chr 14

In the mice:

To test this hypothesis: We inactivated Vhl and one allele of Bap1 in nephron progenitor cells. (Loss of both copies of Bap1 causes renal failure and perinatal death).

Wang et al., PNAS 2014

Six2-Cre induces the loss of Bap1 in renal tubular cells normally expressing Bap1

R26RtdT/-gal/DAPI-galR26RtdT

LTL -gal merge

Bap1expressionloxPFRT

55

413

loxPFRT5 -gal5 6neo 4 4

12 13Gene-trap line

-gal

Reporter line Lineagetracing

+ Six2-Cre

Stopcassette tdTomatotdTomato tdTomato

Knockout line+ Six2-Cre Bap1

knockout

loxPFRT4 5

3 4 5 6

loxP

3 6

Wang et al., PNAS 2014

Targeting Vhl and Bap1 in the mouse kidney causes ccRCC

Ki6

7

CA

IX

Wang et al., PNAS 2014

Chr 3

VH

L

BA

P1

PB

RM

1

VHL intragenic mutation

VH

L

BA

P1

PB

RM

1

Loss of 3p

VH

L

BA

P1

PB

RM

1Brugarolas J. JCO 2014

VH

L

BA

P1

PB

RM

1 BAP1 mutation

High grade Low grade

VH

L

BA

P1

PB

RM

1PBRM1 mutation

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BAP1- and PBRM1-mutant tumors are associated with different outcomes

Time from surgery (years)0 2 4 6 8 10 12

Ove

rall

Surv

ival

0.0

0.2

0.4

0.6

0.8

1.0

PBRM1BAP1

HR, 2.7 (95% CI 0.99-7.6)Log-rank p = 0.044

UTSW cohort

Kapur et al., Lancet Oncology 2013

Time from surgery (years)0 2 4 6 8

Ove

rall

Surv

ival

0.0

0.2

0.4

0.6

0.8

1.0

PBRM1BAP1

HR, 2.8 (95% CI 1.4-5.9)Log-rank p = 0.004

TCGA cohort

Development of a BAP1 IHC test for broader analyses

Pena-Llopis et al., Nat Genet 2012

ID M IHC ID M IHC ID M IHC ID M IHC ID M IHC ID M IHC40 X - 9 + 322 + 9478 + T79 + T157 +63 - 14 + 324 + 9563 + T80 + T158 +78 - 19 + 325 + 9812 + T83 + T160 +

162 - 23 + 425 + 9964 + T84 + T161 +209 - 26 + 572 + 10038 + T91 + T162 +

3397 S - 31 + 619 + 10162 + T92 + T164 +3575 I - 32 + 974 + 10305 + T94 + T165 +9145 XL - 37 + 981 + 13425 + T97 + T170 +9575 X - 39 + 1014 + T4 + T98 + T171 +T16 ¶ - 42 + 1393 + T5 + T106 + T173 +T25 - 44 + 1524 + T6 + T107 + T175 +T26 - 45 + 1637 + T7 + T110 + T183 +T55 - 52 + 1677 + T9 + T116 + T191 +T69 ¶ - 74 + 1791 + T11 + T118 + T192 +T70 X - 75 + 1793 + T15 + T125 + T193 +

T114 - 76 + 2038 + T18 + T126 + T194 +T115 ¶ - 83 + 2077 + T20 + T127 + T197 +T149 - 111 + 2154 + T21 + T128 + T199 +T163 - 113 + 2827 + T22 + T130 + T202 +T166 ¶ - 115 + 3246 + T24 + T131 + T204 +T184 X - 131 + 3483 + T28 + T133 + T205 +T211 X - 139 + 3570 + T37 + T136 + T209 +T145 ¶ + 222 + 3604 + T39 + T142 + T210 +T212 ¶ + 233 + 3750 + T41 + T143 + T213 +

312 - 239 + 3801 + T42 + T144 + T214 +1732 - 240 + 3907 + T52 + T146 + T216 +T195 - 260 + 4077 + T65 + T150 +2368 ? 262 + 4301 + T73 + T151 +

1 + 265 + 4505 + T75 + T153 +4 + 275 + 8885 + T76 + T155 +

BAP1 deficient BAP1 wild-type

Evaluation of BAP1 in 1,400 patients with resectable ccRCC from Mayo Registry

BAP1 loss is associated with reduced RCC-specific survival in the Mayo Registry

RC

C-s

peci

fic s

urvi

val (

%)

Time from surgery (yrs)

BAP1 pos (n=1,196)

BAP1 neg (n=148)

HR: 3.06; 95% CI (2.28 - 4.10)P = 6·10-14

Joseph, R.* and Kapur, P.* et al., Cancer 2013

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BAP1 and PBRM1 loss in different tumors regions

BAP1 and PBRM1 loss in same tumor regions

IHC identifies tumors with simultaneous inactivation of BAP1 and PBRM1

BAP1-

BAP1+

PBRM1+

PBRM1-

Joseph, R.* and Kapur, P.* et al., J. Urol. 2015

Four molecular subtypes of ccRCC with different outcomes (Mayo cohort)

PBRM1+ BAP1+

PBRM1- BAP1+

PBRM1+ BAP1-

PBRM1- BAP1-Expected double mutants: 5.3%Observed: 1.8%OR, 0.18; CI 0.11-0.28, p<0.00001

Joseph, R.* and Kapur, P.* et al., J. Urol. 2015

A foundation for the first molecular genetic classification of ccRCC

WT PBRM1 BAP1 BAP1/PBRM1

Deadliness

DR

UG

A

DR

UG

B

DR

UG

C

DR

UG

D

The Future:

Tumorgrafts tissue microarray:• 58 ccRCC

o 22 PBRM1-deficiento 7 BAP1-deficiento 3 BAP1/PBRM1-deficient

Integrated genomics (Exome, RNAseq) ~70 TG lines

TG: a platform for evaluation of BAP1 & PBRM1 pathway-targeting drugs

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Photo, courtesy of Brian Coats

BAP1 and PBRM1 mutations define 4 subtypes of renal cancer with different biology (gene expression) and prognosis.

These discoveries underlie the foundation for the first molecular genetic classification of sporadic ccRCC.

These findings pave the way for the development of subtype-specific treatments of previously unrecognized subtypes.

Co-linear arrangement of multiple ccRCC two-hit tumor suppressor genes on chromosome 3p may explain human predisposition to ccRCC.

More broadly, these findings provide a potential explanation for the differential tumor predisposition across species.

Conclusions (Part II)

http://www3.utsouthwestern.edu/brugarolaslab

http://www.utsouthwestern.edu/kidneycancer

Brugarolas LabYifeng GuHaley HillFarrah Homayoun Meghan KondaEric MaRenee McKay Andrea Pavia-JimenezNick C. WolffHui YeAnum Yousuf

Alana Christie Xian-Jin Xie

Wenfang ChenShannon CohnEboni Holloman Blanka KucejovaSamuel Peña-LlopisSharanya SivanandVanina ToffessiTram Anh T. TranSilvia Vega-Rubin de CelisShanshan WangToshinari Yamasaki

Deni Von Merveldt Vincy AlexDebbie Harvey

FundingVirginia Murchison Linthicum EndowmentCancer Prevention and Research Institute of TexasNIH, National Cancer Institute

Kidney Cancer ProgramUrologyJeff CadedduJeff GahanYair LotanVitaly MargulisGanesh RajArthur I. Sagalowsky

Medical OncologyYull ArriagaKevin CourtneyEugene Frenkel

Radiation OncologyRaquibul HannanNathan KimDavid PistenmaaRobert Timmerman

PathologyPayal KapurDinesh Rakheja

RadiologyIvan PedrosaLori Watumull

Clinical GeneticsMegan Farley

Illumina Inc. Arnold LiaoNan LengChristian HaudenschildMark RossDavid Bentley

Mayo ClinicRichard W. JosephDaniel J. SerieJeanette Eckel-PassowThai HoJohn C. ChevilleAlexander Parker

GenentechAnwesha DeySteffen DurinckEric W. StawiskiZora ModrusanSekar Seshagiri

NCILaura SchmidtMarston Linehan

Other CollaboratorsThomas CarrollRalph DeBerardinisRobert E. HammerTae Hyun Hwang Min KimBruce PosnerDipti RanganathanNoelle WilliamsYang XieJin YeYonghao Yu

GENENTECHPELOTON THERAPEUTICS