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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.
5
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.
6
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.
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
“ENRICHMENT” LOF Promotes
Growth / Resistance
“DROP OUT”Dependency Genes
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
“ENRICHMENT” LOF Promotes
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(-)
+
“DROP OUT”Dependency Genes
MRTX849
Combination Targets
12
MRTX849 Demonstrates Broad Spectrum Tumor Regression in KRASG12C
Nonclinical Tumor Growth Models
13
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
MRTX849 Demonstrates Broad Spectrum Tumor Regression
in KRASG12C Nonclinical Tumor Growth Models
EsophagusLung Lung PDX
Pan-HERi Combination – Afatinib SHP2i Combination – RMC-4550
KYSE-410
H2122
LU6405
H2030
LU11692
HCC44
-100
-50
0
50
100
MRTX849 Demonstrates Broad Spectrum Tumor Regression
in KRASG12C Nonclinical Tumor Growth Models
SW1573
KYSE-410
H2122
H358
HCC44
-100
-50
0
50
100
MRTX849 Demonstrates Broad Spectrum Tumor Regression
in KRASG12C Nonclinical Tumor Growth Models
EsophagusLung Lung PDX
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
MRTX849 in Combination with HERi or SHP2i Further Inhibit KRASG12C Resulting in
Dramatic Regression in Models Partially Resistant to Single Agent MRTX849
15
SW1573
KYSE-410
H2122
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
CDK4/6i Combination – PalbociclibmTORi Combination – Vistusertib
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
SW1573
H2122
LU6405
H2030
LU11692
H358
-100
-50
0
50
100
MRTX849 Demonstrates Broad Spectrum Tumor Regression
in KRASG12C Nonclinical Tumor Growth Models
EsophagusLung Lung PDX
SW1573
H2122
LU6405
H2030
LU11692
H358
-100
-50
0
50
100
MRTX849 Demonstrates Broad Spectrum Tumor Regression
in KRASG12C Nonclinical Tumor Growth Models
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
17
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.
18
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