Drug-Anchored in vitro and in vivo CRISPR Screens to Identify … · 2020. 6. 25. · rol) CDX and PDX models were treated with MRTX849 @ 100mg/kg PO, ... TSC1/2, and PTEN. Screened

1

Drug-Anchored in vitro and in vivo CRISPR Screens to Identify

Targetable Vulnerabilities and Modifiers of Response

to MRTX849 in KRASG12C-Mutant Models

Lars Engstrom – Principal Scientist

Mirati Therapeutics – San Diego, CA

2

▪ Lars Engstrom is an employee and stock holder of Mirati Therapeutics

Disclosures

3

MRTX849 is a Clinically Active, Irreversible, KRASG12C Inhibitor

600 mg BID Dose Patients: Best Tumor Response*

AACR-NCI-EORTC 10/28/2019

* Based on local radiographic scans every 6 weeks using RECIST 1.1 criteria

‡ Confirmed response (1st scan: -37%, 2nd scan: -47%)

† Response yet to be confirmed (on study but only 1 scan)

§ Patient had confirmed PR post data cut-off (1st scan: -33%, 2nd scan: -43%)

O Patient on study (off study patient: 1 patient withdrawal of consent)

Data cut-off date: 11-Oct-2019

4

MRTX849 Demonstrates Broad Spectrum Tumor Regression in KRASG12C

Nonclinical Tumor Growth Models

Hallin et al. Cancer Discovery 2020

CDX and PDX models were treated with MRTX849 @ 100mg/kg PO, QD in all models shown. % change from baseline control was calculated on ~ day 22 post initiation of dosing.

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6





7

Drug Anchored CRISPR Screen Reveals Drug MOA, Resistance Biomarkers & Combo Targets

TP53

KEAP1

PTPN12

PTPN14

TSC1/2

LU99 In Vivo

Log FC

Vehicle D14 / plasmid

Lo

g F

C

MR

TX

84

9 D

14

/ p

las

mid

“ENRICHMENT” LOF Promotes

Growth / Resistance

“DROP OUT”Dependency Genes

MRTX849

Combination Targets

Negative Control

Positive Control

Target of Interest

Target Gene

GTP-KRASG12C

RSK

MEK

ERK

cRAF

PTEN

AKT

PI3K

mTOR

S6

S6K1

SOS1

SHP2

EGFR

MYC

CCND1

CDK4/6 RB1 E2F

Grb2

GDP-

KRASG12C

NF1

RAS GAPs

RTKsFGFR1

Combination Targets and

Putative Resistance Biomarkers

Library

(Virus)

KRASG12C

+Cas9 cells

DMSO

MRTX849

Vehicle

MRTX849

x 15

in vivo5 replicates

in vitro3 replicates

~5000 sgRNA Library

20 Negative Controls• 20 intronic targets

10 Positive Control Targets• 10 sgRNAs / gene

938 Target Genes• 5 sgRNAs / gene

Transduction

Puro Selection

8

Pro-Survival

Functional Contribution of Top MRTX849 Regulated Pro-survival and Apoptosis Genes

Elucidated by CRISPR Screen

Model MRTX849 MY

C

KR

AS

BIR

C5

BC

L2

L1

MC

L1

CA

SP

9

BC

L2

L1

1

BA

X

CA

SP

3

AP

AF

1

LU99In Vitro

(-)

+

In Vivo(-)

+

H358 In Vitro(-)

+

KYSE410 In Vitro(-)

+

log2 FC

< -2.5 0 2.5 >

CRISPR

log2 FC

< -2.5 0 2.5 >

Tumor Suppressors

Model MRTX849 CB

L

TS

C2

RB

1

TP

53

TS

C1

NF

1

PT

PN

12

PT

EN

KE

AP

1

LU99

In Vitro(-)

+

In Vivo(-)

+

H358 In Vitro(-)

+

KYSE410 In Vitro(-)

+

▪ MRTX849 treatment regulates expression of selected pro-survival and pro-

apoptotic genes which correlates with tumor growth inhibition in multiple

KRASG12C mutant models.

▪ Functional role for many genes confirmed in CRISPR data

– Pro-survival genes BIRC5/Survivin, BCL2L1, MCL1: Decreased by MRTX849 & Exhibit

Dropout

– Pro-apoptotic genes BCL2L11: Increased by MRTX849 & Exhibit Enrichment

– Apoptotic regulators APAF1, CASP3/9 enriched in drug-treated CRISPR data, in

particular

▪ Several tumor suppressors enriched suggesting potential for intrinsic resistance

Pro-Apoptosis

Model MRTX849 BIR

C5

BC

L2

L1

MC

L1

BA

D

BM

F

CA

SP

7

CA

SP

9

BB

C3

BC

L2

L1

1

AP

AF

1

MiaPaca2 QDx7 6hr

H1373 QDx7 6hr

H358 QDx7 6hr

H2122 QDx7 6hr

H2030 QDx7 6hr

RNAseq

9

Increased KRASG12C Modification Via Upstream Combinations Improves Response

0 1 0 2 0 3 0 4 0 5 0 6 0 7 00

2 5 0

5 0 0

7 5 0

1 0 0 0

V e h ic le

M R T X 8 4 9 1 0 0 m g /k g

R M C -4 5 5 0 3 0 m g /k g

M R T X 8 4 9 + R M C -4 5 5 0

D o s in g S to p p e d

Model MRTX849 PT

PN

11

FG

FR

1E

RB

B2

EG

FR

FG

FR

3S

OS

1N

F1

LU99In Vitro

(-)

+

In Vivo(-)

+

H358 In Vitro(-)

+

KYSE410 In Vitro(-)

+

log2 FC

< -2.5 0 2.5 >

HERi -- H2122

SOS1i -- MiaPaca-2

SHP2i -- KYSE-410

GTP-KRASG12C

cRAF

SOS1

SHP2

EGFR

Grb2

GDP-

KRASG12C

NF1

RAS GAPs

RTKsFGFR1

0 . 0

0 . 5

1 . 0

1 . 5

A c t i v e R A S E L I S A

K Y S E - 4 1 0 X e n o g r a f t s

No

rm

al

iz

ed

E

xp

re

ss

io

n

MRTX849 (100mg/kg)

RMC-4550 (30mg/kg)+ +

+ +

QD x 1

+ +

+ +

QD x 7

No

rmalized

Acti

ve

RA

S

Active RAS

20 30 40 50

0

200

400

600

800

Miapaca-2

Study Days

Tu

mo

r V

olu

me (

mm

3)

Vehicle

BI-I-13 50mgkg BIDMRTX849 10mgkg QD

Combo

20 30 40 50

0

200

400

600

800

Miapaca-2

Study Days

Tu

mo

r V

olu

me (

mm

3)

Vehicle

BI-I-13 50mgkg BIDMRTX849 10mgkg QD

Combo

10

Model MRTX849 RP

S6

EIF

4E

RA

C1

RP

TO

R

PT

PN

11

GR

B2

MT

OR

ST

K1

1

FG

FR

1

PIK

3C

A

TS

C2

TS

C1

PT

EN

LU99In Vitro

(-)

+

In Vivo(-)

+

H358 In Vitro(-)

+

KYSE410 In Vitro(-)

+

log2 FC

< -2.5 0 2.5 >

Alternative Pathway Activation Through mTOR May Contribute to Adaptive Resistance

GTP-KRASG12C

RSK

MEK

ERK

cRAF

PTEN

AKT

PI3K

mTOR

S6

S6K1

SOS1

SHP2

EGFR

Grb2

GDP-

KRASG12C

NF1

RAS GAPs

RTKsFGFR1

▪ mTOR pathway genes drop out +/- MRTX849 treatment

▪ Loss of TSGs PTEN and TSC1/2 provide growth advantage

▪ mTOR combinations further reduce pS6 activation and leads to increased in vivo

efficacy


Growth / Resistance


MRTX849

Combination Targets

11

log2 FC

< -2.5 0 2.5 >

Cell Cycle Strongly Implicated in CRISPR Screens and CDK4/6 Inhibitors Augment

MRTX849 In Vivo Efficacy

GTP-KRASG12C

RSK

MEK

ERK

cRAF

PTEN

AKT

PI3K

mTOR

S6

S6K1

SOS1

SHP2

EGFR

MYC

CCND1

CDK4/6 RB1 E2F

Grb2

GDP-

KRASG12C

NF1

RAS GAPs

RTKsFGFR1


Growth / Resistance

Model MRTX849 CD

K1

BIR

C5

MY

C

CD

K2

CD

K7

CD

K4

CC

NB

1

AU

RK

B

CD

C25

B

CC

ND

1

CD

K6

E2

F1

CC

NB

2

RB

1

TP

53

LU99In Vitro

(-)

+

In Vivo(-)

+

H358 In Vitro(-)

+

KYSE410 In Vitro(-)

+


MRTX849

Combination Targets

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Single Agent Activity of MRTX849 in Selected Nonclinical KRASG12C Tumor

Models that Exhibit Intrinsic or Adaptive Resistance

SW1573

KYSE-410

H2122

LU6405

H2030

LU2512

LU11692

H358

HCC44

-100

-50

0

50

100

MRTX849 Demonstrates Broad Spectrum Tumor Regression

in KRASG12C Nonclinical Tumor Growth Models

EsophagusLung Lung PDX

Ch

an

ge f

rom

Bas

eli

ne

(%

of

co

ntr

ol)

CDX and PDX models were treated with MRTX849 @ 100mg/kg PO, QD in all models shown.

% change from baseline control was calculated on ~ day 22 post initiation of dosing.

14

KYSE-410

H2122

LU6405

H2030

LU11692

HCC44

-100

-50

0

50

100




Pan-HERi Combination – Afatinib SHP2i Combination – RMC-4550

KYSE-410

H2122

LU6405

H2030

LU11692

HCC44

-100

-50

0

50

100



SW1573

KYSE-410

H2122

H358

HCC44

-100

-50

0

50

100




SW1573

KYSE-410

H2122

LU6405

H2030

LU2512

LU11692

H358

HCC44

-100

-50

0

50

100



MRTX849 in Combination with HERi or SHP2i Further Inhibit KRASG12C Resulting in

Dramatic Regression in Models Partially Resistant to Single Agent MRTX849

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SW1573

KYSE-410

H2122

H2030

LU2512

LU11692

H358

HCC44

-100

-50

0

50

100




CDK4/6i Combination – PalbociclibmTORi Combination – Vistusertib

SW1573

KYSE-410

H2122

LU6405

H2030

LU2512

LU11692

H358

HCC44

-100

-50

0

50

100



SW1573

H2122

LU6405

H2030

LU11692

H358

-100

-50

0

50

100




SW1573

H2122

LU6405

H2030

LU11692

H358

-100

-50

0

50

100



MRTX849 in Combination with Vistusertib or Palbociclib, Block Downstream

Pathway Activation and Induce Dramatic Regression in Models Partially Resistant

to Single Agent MRTX849

16

0 10 20 300

500

1000

1500

2000

2500

Study Day

Tu

mo

r vo

lum

e (

mm

3)

LU99 sgKEAP1 3-1 Vehicle

LU99 sgKEAP1 3-1 MRTX849

LU99 sgNT Vehicle

LU99 sgNT MRTX849

LU99 sgKEAP1 3-5 Vehicle

LU99 sgKEAP1 3-5 MRTX849

LU99 sgNRF2 Vehicle

LU99 sgNRF2 MRTX849

KEAP1 KO Modifies Response to KRASG12C Inhibition and May Contribute to

Adaptive ResistanceLU99 CRISPR Clones

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Conclusions

▪ MRTX849 is broadly active as a single agent across a panel of KRASG12C-mutant xenograft models.

▪ Executed MRTX849-anchored CRISPR screens targeting ~1,000 genes in 3 KRASG12C cell lines, in

vitro & in vivo.

▪ Tumor suppressor genes that promoted tumor growth also conferred partial drug resistance

including KEAP1, NF1, Rb1, TSC1/2, and PTEN.

▪ Screened ~70 rational compounds in combination with MRTX849 across 8 lung cell lines in vitro that

were partially MRTX849-resistant in vivo.

▪ Top combination targets validated with in vivo combinations are EGFR family, SHP2, SOS1,

mTOR, and CDK4/6.

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Acknowledgments

Mirati Therapeutics

Jamie Christensen

Pete Olson

Laura Waters

Ruth Aranda

Jill Hallin

David Briere

Andrew Calinisan

Niranjan Sudhakar

Lauren Hargis

Vickie Bowcut

Service Providers

Cellecta

Crown Biosciences

Collaborators

Channing Der (UNC)

Adrienne Cox (UNC)

Piro Lito (MSK)

Pasi Janne (DFCI)

XenoSTART

Monoceros Biosystems

Adam Pavlicek

Sole Gatto

Julio Fernandez-Banet

Documents

Drug-Anchored in vitro and in vivo CRISPR Screens to Identify … · 2020. 6. 25. · rol) CDX and PDX models were treated with MRTX849 @ 100mg/kg PO, ... TSC1/2, and PTEN. Screened