Functional Genomics: Implications in Tissue Pathology

René Bernards

Functional genomics:Implications in Tissue Pathology

The Netherlands Cancer InstituteAmsterdam

The Netherlands

Genotype-driven therapy of non-small cell lung cancer

Crizotinib

Kwak et al, N Engl J Med 2010

Erlotinib Gefitinib

Maemondo et al., N Engl J Med 2010

Endless two-way combinations of cancer drugs

1,000 x1,000 2

= 500,000 combinations

Test each combination in 1,000 patients:

500,000,000 patients needed

Three vignettes on the use of functional genetics to find effective combination therapies

• The case of BRAF mutant colon cancer• The case of KRAS mutant lung/colon cancer• The case of RTK mutant lung cancer

N Engl J Med. 2010 363:809-19Kopetz et al., ASCO 2010

Vemurafenib (PLX4032) - A selective BRAFV600E inhibitor

Differential response of BRAF inhibition in BRAF mutant melanoma versus colon cancer

BRAFV600E mutant CRC cell lines are also less responsive to PLX4032 than melanomas having the

same mutation

CRC

Melanoma

Short-term cell viability assay Long-term colony formation assay

Is feedback regulation responsible for the resistanceof CRC cells to BRAF inhibitors?

infect with shRNA kinome library

PCR amplify bar codes

WiDr(BRAFV600E)

Deep sequence Quantify shRNAs

vemurafenibculture to allow selection

control

Synthetic lethal shRNA screen: Inhibition of which kinase synergizes with PLX in BRAF mutant CRC?Synthetic lethal shRNA screen: Inhibition of which kinase synergizes with PLX in BRAF mutant CRC?

Inhibition of EGFR makes BRAF mutant CRC cellsvulnerable to BRAF inhibition

Inhibition of EGFR makes BRAF mutant CRC cellsvulnerable to BRAF inhibition

Grb

BRAF

EGFR

RAS

MEK

ERK

Cetuximab

MutantMutant

Synergistic response of BRAFV600E CRC to EGFR and BRAF inhibition

Synergistic response of BRAFV600E CRC to EGFR and BRAF inhibition

Cetuximab 1.25 g/mlGefitinib 0.125 MCetuximab 1.25 g/mlGefitinib 0.125 M

VACO (BRAFV600E)VACO (BRAFV600E)

Also seen in WiDr andKM20 (BRAFV600E)Also seen in WiDr andKM20 (BRAFV600E)

+Cetuximab

+Gefitinib

BRAFV600E inhibition causes feedback activationof EGFR

BRAFV600E inhibition causes feedback activationof EGFR

VACO (BRAFV600E)VACO (BRAFV600E)

Also seen in WiDr andKM20 (BRAFV600E)Also seen in WiDr andKM20 (BRAFV600E)

EGFR and BRAF inhibition synergize to induce apoptosis and suppress BRAFV600E CRC tumor growth

Also seen in WiDr (BRAFV600E)Also seen in WiDr (BRAFV600E)Prahallad et al, Nature 2012

Feedback regulation of EGFR by BRAF inhibition

Ligand

CDC25CCDC25C

PI3KPI3K

AKTAKT

VemurafenibVemurafenib

Cetuximab

Mechanism-based rational drug combinations: rapid translation to the clinic

8 months frompublication toclinical trial!

EGFR levels determine response to BRAFInhibition in multiple tumor types

EGFR levels determine response to BRAFInhibition in multiple tumor types

Control

Gefitinib

PLX4032

PLX4032+Gefitinib

Trametinib

Trametinib+Gefitinib

EGFR expression in melanoma is sufficient to confer resistance to BRAF and MEK inhibitors

BRAF mutant melanomas upregulate EGFR during the development of drug resistance

EGFR IHC in brown

Patient 7 Pre vemurafenib

Patient 9 Pre vemurafenib

Patient 7 Post vemurafenib

Patient 9 Post vemurafenib

Total:5/12 patients

became EGFR positive

(40%)

Three vignettes on combination therapies to fight resistance

• The case of BRAF mutant colon cancer• The case of KRAS mutant lung/colon cancer• The case of RTK mutant lung cancer

Nat Rev Cancer 3: 459–465. Nat. Rev. Clin. Oncol., 6 (2009) Cancer Research, 69 (10), 4286-4293 Clin Cancer Res 2012;18:2515-2525

Cell survival and proliferation

Inhibitor

Receptors

MEK inhibitorLow efficacy (PDX assay) for

KRAS mutant tumors

Targeting KRAS mutation with MEK inhibitor

Potential for clinic failure:

+Gefitinib (1uM)

Is the combination of MEK and EGFR inhibitors also effective in KRAS mutant cells?

Gefitinib (1uM)

0.25 0.5 1

Selumetinib (uM)

CRC: SW620(KRASG12V)

NSCLC: H358 (KRASG12C)

0.25 0.5 1

Selumetinib (uM)

0 0.125 0.25 0.5 1

Selumetinib(μM)

H747

SW480

SW620

SW837

CRC

H358

H23

H2030

H2122

NSCLC

RNAi screen for enhancers of MEK inhibitors

ERBB3 activation is mostly dependent on heterodimerization

ERBB3 binding partners

ERBB family

shERBB3

shERBB3

H358 (KRASG12C)

Also seen in H2030, H2122,SW837, SW480 and SW620

Targeting EGFR and ERBB2 sensitizes KRAS mutant cells to MEK inhibitor

+Dacometinib (0.5uM)

+Afatinib (0.5uM)

+Gefitinib (0.5uM)

+Pertuzumab (2.5ug/ml)

0.25 0.5 1

Selumetinib (uM)

• Gefitinib: EGFR inhibitor

• Pertuzumab: ERBB2-targeting monoclonal antibody

• Afatinib: EGFR and ERBB2 inhibitor

• Dacometinib: EGFR, ERBB2 and ERBB4 inhibitor

MEK inhibition induces ERBB2 and ERBB3 transcription

EGFR+ERBB2 inhibitor synergizes with MEK inhibitor to induce apoptosis in KRAS mutant cells

Selumetinib

Afatinib

+-+ +

+-- -

Clev. PARP

p-BAD (S112)

p-BAD(S136)

BAD

p-ERK

ERK1/2

p-p90RSK

p90RSK

p-AKT

AKT

H358

p-BIM(S69)

BIM

Targeting EGFR and ERBB2 sensitizes KRAS mutant cells to MEK inhibitor in a xenograft model

25

0%

34

3%

30

6%

H2122 xenografts

BRAF BRAF

Melanoma

Colon cancer

BRAFi/MEKi

BRAFi/MEKi+

EGFRi

Genotype Tissue type Effective therapy

KRAS KRASColon cancerLung cancer

MEKi+

EGFRi+HER2i

Three vignettes on combination therapies to fight resistance

• The case of BRAF mutant colon cancer• The case of KRAS mutant lung/colon cancer• The case of RTK mutant lung cancer

Resistance to TKIs is a frequent problem in the clinic

Crizotinib

GefitinibErlotinib

Acquired resistance to EGFR inhibitors

Sequist et al., Sci Transl Med 2011 Choi et al., NEJM 2010

* L1196M in EML4-ALK

* Other unknown mechanisms

T790M(49%)

Cell line models matching NSCLC genotypes

PC9 (EGFRDelE746A750)H3255 (EGFRL858R)

Erlotinib Gefitinib

TKI-sensitive NSCLC cell lines

H3122(EML4-ALK)

Finding genes that can cause resistance tocrizotinib in EML4-ALK positive NSCLC

culture to allow selection

crizotinibcontrol

infect with shRNA library

PCR amplify bar codes

H3122(EML4-ALK)

Suppression of MED12 causes crizotinib resistance

shMED12#1shMED12#2

MED12

Transcription Mediator complex

CDK8

L1224F, recurrent in Prostate cancer (5%)

Uterine Leiomyomas (70%)

Exon 2

Mäkinen et al., Science Express 2011; Barbieri et al. Nat Genet. 2012

Exon 26

MED12 mutations in cancers

PC9 (EGFRDelE746A750)

MED12 knockdown causes MEK and ERK activation

ERK

MED12

p-ERK

pLKO

shM

ED12#3

shM

ED12#5

pLKO

shM

ED12#3

shM

ED12i#

5

Gefitinib: - - - + + +

p-MEK

MEK

infect with TRC shRNA kinome library MED12KD

(EML4-ALK)

+ Crizotinibculture to allow selection

control

Resistant

Re-sensitization screen: Which kinase to inhibit to restore the crizotinib sensitivity in MED12KD cells?

infect with TRC shRNA kinome library

PCR amplify bar codes

MED12KD

(EML4-ALK)

Deep sequence Quantify shRNAs

+ Crizotinibculture to allow selection

control

Depleted by Crizotinib

Resistant

Re-sensitization screen: Which kinase to inhibit to restore the crizotinib sensitivity in MED12KD cells?

Suppression of TGFR2 restores the crizotinib sensitivity in MED12KD cells

I

II

TGF activation is sufficient to drive drug resistance

H3122(EML4-ALK)

Downregulation of MED12 leads to elevated TGFR signaling in different tumor types

PC9 (EGFRDelE746A750)

H3122(EML4-ALK)

PC9 A375(BARFV6700E)

Melanoma

PC9 SKCO-1(KRASV12)

ColonNSCLCNSCLC

TGF activates the RAS-RAF-MEK-ERK pathway

Zhang, Y. Cell Research 2009:19 p128

MED12KD also causes resistance to chemotherapies through TGF signaling

Generation of a “MED12KD signature”

H3122; NSCLCPC9; NSCLCSKCO1; colon cancerA375; melanomaHuh7; HCC

Wild type+

MED12KD

Differentially expressed genesin 3 out of 5 cell lines (>2-fold);237 genes

MED12 loss induces an EMT-like phenotype

A MED12KD signature predicts resistance to chemotherapy in stage III colon cancer

Three gene profiles specific for intrinsic subtypes

• A-type profile of 32 genes• B-type profile of 53 genes• C-type profile of 102 genes

Three gene profiles specific for intrinsic subtypes

• A-type profile of 32 genes• B-type profile of 53 genes• C-type profile of 102 genes

Colon cancer also has “intrinsic subtypes”

44

Colon cancer subtypes differ in biology and in response to chemotherapy

TGFR2 inhibitor restores the crizotinib sensitivity in MED12KD cells

And by other means?EMT by MED12 loss

TGFsignaling

response to targeted drugs

TGFR inhibitors

potential treatment strategy:

(also true for EGFR inhibitors)

H3122(EML4-ALK)

Precision medicinePrecision medicine

Alterations in pathwaysAlterations in pathways

Cancer genome analysesCancer genome analyses

Cross talk between pathways Cross talk between pathways

Functional genetic analysesFunctional genetic analyses

Acknowledgements

The people involved:Chong SunSid HuangAnirudh PrahalladWipawadee GrernrumLorenza MitempergherFloris GroenendijkTheo KnijnenburgAndy SchlickerLodewijk WesselsWouter NijkampRoderick Beijersbergen

Collaborators:Federica Di NicolantonioAlberto BardelliPaul RoepmanPeter ten Dijke

Our funding sources:Our funding sources: