Chromophobe RCC

Elizabeth (Lisa) Petri Henske, MDDirector, Center for LAM Research and Clinical Care,

Brigham and Women’s Hospital, Boston MA

Professor of Medicine, Harvard Medical School

Medical Oncologist, Dana Farber Cancer Institute

Associate Member, The Broad Institute of MIT and Harvard

Ehenske@BWH.Harvard.edu

Carmen Priolo, MD PhDAssistant Professor of Medicine, BWH, Harvard Medical School

Brigham and Women’s Hospital/Dana-Farber Cancer Institute

Harvard Medical School

Growing chRCC Team!

BWH: Carmen Priolo

David KwiatkowskiDamir Khabibulllin

Long ZhangRachel Yu

DFCI:David BraunToni Choueiri

Sabina SignorettiMatthew Freedman

+ Others!

Wei Shi (MGH)John Asara (Beth Israel, Boston)

Ed Reznik (MSKCC)

National Cancer Institute/NIH (RO1)

US Department of Defense Kidney Cancer Program (Hypothesis Award)

The Tuttle Family

Lymphangioleiomyomatosis (LAM)

Angiomyolipomas

ChRCC can occur in Tuberous Sclerosis Complex and Birt-Hogg-Dube Syndrome

Renal Cell Carcinoma

Tuberous Sclerosis Complex

Birt-Hogg-Dube

5% of all sporadic renal cancers

Occur in BHD and TSC

Filled with abnormal mitochondria

Losses of chromosomes 1, 2, 6, 10, 13 and 17

Few driver mutations (TCGA: ~50%: p53, Tsc1, PIK3CA)

4

Chromophobe RCC

• 367 measured metabolites

• 188 differential metabolites

TumorNormal

*Glucose metabolism

*Glutathione metabolism

Metabolomics: Nine chRCC vs. matched normal kidney

7.00

6.00

5.00

4.00

3.00

2.00

1.00

0.00

-1.00

-lo

g10

(p)

log2(FC)-8.00 -7.00 -6.00 -5.00 -4.00 -3.00 -2.00 -1.00 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00

5-oxoproline

Ɣglutamylleucine

Ɣglutamylglutamate

Top differential metabolites in ChRCC compared to normal kidney

Features selected by volcano plot with fold change threshold 2

(FC; x) and t-tests threshold p <0.01 (y). The red circles

represent features above the threshold. The further the red

circle is away from the (0,0), the more significant the feature is.

Normal kidney

Tumor36 metabolites decreased in tumor vs normal

Decreased expression of gamma-glutamyl

transferase 1 (GGT1) in ChRCC

nsp<0.001

GGT1 gene locus= Chr 22q11.23 RNA seq data from TCGA

n=25 n=66 n=18 n=82

The glutathione salvage pathway (or gamma-glutamyl cycle)

• gamma-glutamyl transferase 1

(GGT1): membrane

transpeptidase

• transfers gamma-glutamyl

moieties from extracellular

GSH, GSSG, or GSH

conjugates to an amino acid

acceptor (gamma-glutamyl

amino acid)

• GGT-deficient mice: lower

GSH levels, increased DNA

damage (Rojas et al, 1999),

and dysfunctional mitochondria

(Will et al, 2000)

GCLC= Glutamate-cysteine ligase catalytic subunit

GSS= glutathione synthetase

SLC7A11= cystine-glutamate antiporter

GCLC

GSS

Enhanced expression of glutathione biosynthetic enzymes in ChRCC

10

GGT1 downregulation increases De Novo Glutathione Synthesis

CB-839

Priolo C, Khabibullin D,… Henske EP. PNAS 2018HEK293,72h

GGT1 down-regulation sensitizes HEK203 to inhibition

of glutathione biosynthesis

Damir Khabibullin

BSO=buthionine sulfoximine

48-hr treatment after 72-hr transfection with siGGT1 or siControl

Working model and Future Directions

Hypothesis: Defects in the Ɣ-glutamyl cycle lead to accumulation of damaged

mitochondria and tumorigenesis

Compensatory metabolic reprogramming Impaired response to oxidative stress

• Role in renal tumorigenesis• Increased sensitivity to agents that inhibit glutathione synthesis

p-AMPK(Thr172)

GGT1

p-ULK1(S555)

GGT1 downregulation in HEK293: Erk activation and suppression of LKB1, AMPK and ULK1

13

siCtrl siGGT1

siCtrl siGGT1

siCtrl siGGT1

p-LKB1(Ser428)

actin

p-Erk1/2(Thr202/Tyr204)

ATG7

ATG5

β-actin

Conan GK. Nature Medicine. 2019

HEK293T, 72hAmbion siGGT1 5725

Rachel Yu, unpublished

Major Roadblock: chRCC Models

• Additional cell lines

• Additional animal models

15

• Chromophobe RCC with sarcomatoid differentiation

• Clonal losses of chromosomes 1, 3, 4, 11, 13, 14, and 19 and gains of X, Y, 6, 8, and 20.

– (vs ChRCC losses of chromosomes 1, 2, 6, 10, 13 and 17)

• TP53 missense mutation

Genes Chromosomes and Cancer, 2017

H E K 2 9 3 U O K 2 7 6

0 .0

0 .5

1 .0

1 .5

Re

lati

ve

GG

T1

mR

NA

Low GGT1 in UOK276 relative to HEK293

GGT1Chromo1039 cellsChromo1039 Tissue

GGT1

ChromoA cells

ChRCC-derived primary cell lines: low GGT1 expression

A

B

(Gerharz et al., Am J Path, 1995)

SNP arrays, 250K

Big Questions

Additional mouse models of chRCC

Which nephron segment?

Ksp (Kidney specific cadherin)

Aqp2 (Aquaporin 2, Principal cells of collecting duct)

ATP6V1B1 (alpha-Intercalated cells of collecting duct )

Which genes?

GGT1 and PTEN (20% ChRCC has PTEN mutation)

GGT1 and TSC2

Take Home Hypothesis:

Defective glutathione salvage pathway (or gamma-glutamyl cycle)

Role in Pathogenesis and Therapy of chRCC