1 Confidential

MRTX1719: A First-in-class MTA-cooperative PRMT5 Inhibitor that Selectively Elicits Antitumor Activity in MTAP/CDKN2A Deleted Cancer Models

Christopher R. Smith, Lars D. Engstrom, Svitlana Kulyk, Ruth Aranda, Laura Waters, Krystal Moya, Victoria Bowcut, Allan Hebbert, David Trinh, David M. Briere, J. David Lawson, Jeff Clarine, Lisa Rahbaek, James G. Christensen, Matthew A. Marx, Peter Olson*

AACR-NCI-EORTC 2021*Corresponding Author

22

PRMT5

Targeting the PRMT5•MTA Complex: a Synthetic Lethal, Precision Medicine Approach for the Treatment of MTAP Deleted Cancers

PRMT5 was a top hit in large scale functional genomics screens that demonstrated shRNA-mediated PRMT5 inhibition selectively inhibited MTAP del cancer cell line viability1

MTAP is proximal to and co-deleted with CDKN2A, the most commonly deleted gene in human cancer; MTAP deletion increases cellular concentrations of its substrate, MTA

MTA binds to and partially inhibits PRMT5, creating a novel, MTAP del cancer cell-specific target, the PRMT5MTA complex

Current clinical PRMT5 inhibitors do not bind PRMT5MTA and do not exhibit selectivity for MTAP delcancers. Since PRMT5 is critical for the viability of cancer and normal cells, this may result in a narrow therapeutic index2

MRTX1719 selectively binds the PRMT5MTA complex which is elevated in MTAP del cancer cells and is therefore positioned to have an improved therapeutic index based on the concept of synthetic lethality

MRTX1719 was identified as a development candidate which selectively binds the PRMT5MTA complex

1Mavrakis, K, et al, Science, 2016; Marjon, K, et al, Cell Reports, 2016; Kryukov, G, et al, Science, 20162EPZ666 (GSK) – References in 1; JNJ – Brehmer D (Janssen), ASCO, 2017.

33

PRMT5

Confidential

44

PRMT5

MRTX1719 is a potent, selective inhibitor of the PRMT5MTA complex with favorable drug-like properties that selectively targets MTAP del cancers

MRTX1719 is differentiated from previously described PRMT5 inhibitors that inhibit PRMT5 in MTAP WT normal tissue, impact hematopoietic cell viability and may exhibit a narrow TI

MRTX1719 inhibition leads to dysregulated RNA splicing, decreased pRb, decreased proliferation and increased apoptosis

MRTX1719 elicits strong antitumor activity across a panel of MTAP del CDX and PDX models and induces tumor regression in a subset of models as a single agent, including lung and mesothelioma models

Additional mechanistic and combination studies are underway to inform patient populations for clinical development

MRTX1719: A PRMT5 Inhibitor Selectively Targeting MTAP Deleted Cancers

55

PRMT5

Assay MRTX1719 IC50 (nM)

GSK-595 IC50 (nM)

BiochemicalPRMT5MTA 3.6

PRMT5 20

Cellular Activity HCT116

Isogenic Cell Lines

SDMA In-Cell

Western

MTAP del 8 11

MTAP WT 653 12

10-Day Viability

MTAP del 12 189

MTAP WT 890 198

MRTX1719 Selectively Inhibits the PRMT5•MTA Complex, MTAP Deleted Cell Lines and MTAP Deleted Xenograft Tumor Growth

A. A co-crystal structure of the fragment hit (shown in black) with PRMT5 confirmed MTA binding and suggested vectors for further elaboration. Addition of methylpyrazole increased binding to the PRMT•MTA complex by making a key hydrogen bond to Leu312 backbone N-H. Addition of cyanofluorophenyl increased potency through an additional interaction with Phe580 backbone N-H and demonstrated anti-proliferation in MTAP del cells. Further optimization resulted in improvement in antiproliferative activity and high bioavailability in rodent and non-rodent species. B. Compounds were tested in a PRMT5/MEP50 enzymatic assay in which PRMT5 uses SAM to add symmetric dimethyl groups to arginine residues (SDMA) within an H4 peptide (1-15) with and without MTA (Reaction Biology Corp). PRMT5 cellular activity was measured using a 4-day In-Cell Western assay to measure SDMA marks using the SYM11 antibody and normalized using DRAQ5 in HCT116 MTAPdeleted and WT cell lines. Cell viability was measured using a 10-day Cell Titer Glo assay (Promega) in HCT116 MTAP del and MTAP WT cell lines. C. Graph depicting the dose-concentration curves for MRTX1719 and GSK-595 in the 10-day viability assay in HCT116 MTAP del and MTAP WT cell lines. D. MRTX1719, GSK-595 and JNJ-9178 were administered via daily oral gavage to immunocompromised mice bearing established HCT116 MTAP del or MTAP WT xenograft tumors at the indicated doses QD. Data are shown as mean tumor volume (n=10/group) +/- standard error of the mean (SEM). E. Tumors from the tumor growth inhibition study (D) were collected at the end of the study four hours after the last dose and analyzed by western blot and densitometry for SDMA levels. Representative bands were analyzed from 3 tumor lysates per treatment group.

A C

D E

0 5 10 15 20 250

500

1000

1500HCT116 MTAP Del

Study Day

Tum

or V

olum

e (m

m3 )

Vehicle

GSK-595 200 mg/kgJNJ-9178 10 mg/kg

MRTX1719 100 mg/kg

0 5 10 15 20 250

500

1000

1500HCT116 MTAP WT

Study Day

Tum

or V

olum

e (m

m3 )

B

NHN

NH2

O

NN

NCF

OCl

MRTX1719 1 10 100 1000 10000 1000000

50

100

150

10-Day Viability Assay

nM

% o

f D

MSO

Con

trol MRTX1719

MRTX1719

GSK-595

GSK-595

HCT116 MTAP Del

HCT116 MTAP WT

66

PRMT5

MRTX1719 Inhibits Growth & SDMA Modification in MTAP Deleted Xenograft Tumors with Minimal SDMA Modulation/Viability Effects in Bone Marrow

A. MRTX1719 (right) and JNJ-9178 (left) were administered via daily oral gavage to immunocompromised mice bearing established MTAP del LU99 xenograft tumors at the indicated doses QD. Data are shown as mean tumor volume (n=5/group) +/- standard error of the mean (SEM). B. Tumors and bone marrow from the tumor growth inhibition study (A) were collected at the end of the study four hours after the last dose and analyzed by western blot and densitometry for SDMA levels. Representative bands were analyzed from 3 or 5 tumor lysates per treatment group. C. MRTX1719 and GSK-595 were administered via daily oral gavage to immunocompromised mice bearing established LU99 xenograft tumors at the indicated doses QD for the indicated number of weeks. Tumor (left) and bone marrow (right) were collected at the end of the study four hours after the last dose and analyzed by western blot and densitometry for SDMA levels. Representative bands were analyzed from 3 tumor lysates per treatment group. D. MRTX1719, JNJ-9178 and GSK-595 were run in human erythroid and myeloid 7-day HemaTox assays (STEMCELL Technologies) and were compared to IC50s from 10-day MTAP del and MTAP WT cell line viability assays.

Human Cancer Cell LinesHCT116 MTAP Isogenic Pair

Human Bone MarrowMTAP WT

10-Day Viability IC50 (nM) 7-Day HemaTox IC50 (nM)

MTAP del MTAP WT Erythroid Myeloid

MRTX1719 12 890 545 559

JNJ-9178 5 5 3 2

GSK-595 189 198 62 58Veh 12.5 25 50 100 Veh 50 100

Tumor SDMA Bone MarrowVeh 10 30 Veh 10 30

Tumor SDMA Bone Marrow

A C

D

LU99 In Vivo PD Timecourse

MRTX1719 100mgkg QDGSK595 200mgkg QD

Vehicle week 1MRTX1719 25mgkg QD

MRTX1719

0 5 10 15 200

500

1000

1500

2000

Study Day

Tum

or V

olum

e (m

m3 )

Vehicle12.5 mg/kg QD25 mg/kg QD50 mg/kg QD100 mg/kg QD

LU99 In Vivo TGI

0 5 10 15 200

500

1000

1500

2000

Study DayTu

mor

Vol

ume

(mm

3 )

Vehicle10 mg/kg QD30 mg/kg QD

JNJ-9178

0

1

2

3

Norm

aliz

ed S

DMA

Sign

al

86% 88% 95% 98%

-64%

-174%

MRTX1719B

0

1

2

3

Norm

aliz

ed S

DMA

Sign

al

99% 99%89% 89%

JNJ-9178

0.0

0.5

1.0

1.5

2.0

WeekSign

al n

orm

aliz

ed to

wee

k 1

veh

5%

52%

99%

41 2 6

Bone Marrow SDMA

0.0

0.5

1.0

1.5

2.0

Week

Sign

al n

orm

aliz

ed to

wee

k 1

veh

96%96%

98%

4 621

Nor

mal

ized

SD

MA

Tumor SDMA

77

PRMT5

MRTX1719 Treatment Decreases Proliferation, Increases Apoptosis and Leads to Dysregulated RNA Splicing

A. LU99 cells were treated with 250 nM MRTX1719 (665B), 250 nM palbociclib or the combination for 7 days and cell cycle distribution analysis was performed using 7-AAD staining on a Guava flow cytometer B. LU99 cells were treated with MRTX1719 at the concentrations indicated for 3 days and apoptotic cells were analyzed for surface Annexin V positive, 7-AAD negative staining. C. RNAseq data was generated from LU99 cells treated with MRTX1719 or DMSO for 3 days and transcripts were analyzed for altered RNA splicing using rMATS. Transcripts with detained introns were increased in MRTX1719-treated vs DMSO-treated cells. ME – Mutually exclusive exon usage, DI – Detained introns, CE – Skipped exons, A3SS – Alternative 3’ splice site, A5SS – Alternative 5’ splice site. D. Gene signature enrichment analysis (GSEA) was performed on RNAseq data from 3 or 5-day MRTX1719-treated LU99 cells compared to DMSO treated. “***” indicates FDR < 0.25. E. Fold changes (1st column) in post translational modifications that were down regulated in selected trypsin-digested peptides from MRX1719 vs DMSO-treated LU99 cell lysates as measured by LC-MS/MS following antibody enrichment using an SDMA motif antibody (top) or the multi-pathway kit that includes phosphorylation (Cell Signaling Technology). Site denotes amino acid. “§” indicates known site of modification.

DMSO 10 50 250MRTX1719 (nM)

MRTX1719:DMSO Protein Name SDMA PTMScan; Site Description-195.4 snRNP D3 §112, §114, §118 Small nuclear ribonucleoprotein Sm D3-68.4 MYO1E;MYO1F 739;737 Unconventional myosin-Ie-52.5 snRNP B1 §181, §209;181, 209;181, 209 Small nuclear ribonucleoprotein-associated proteins B and B'-33.7 LSM4 §115, §117 U6 snRNA-associated Sm-like protein LSm4-23.5 KANSL1 1101;432;369 KAT8 regulatory NSL complex subunit 1

MRTX1719:DMSO Protein Name Phosphorylation PTMScan; Site Description-35.0 CAF-1A §767, §770, §780 Chromatin assembly factor 1 subunit A-30.3 TIMELESS §1149;1148 Protein timeless homolog-23.2 SBNO1 §819, §828;818, 827;§817, 826 Protein strawberry notch homolog 1-21.9 Rb §805, §811 Retinoblastoma-associated protein-20.7 CD2AP §541, §550, §553 CD2-associated protein-16.3 AurB §236;195;237;204;§202;168;183 Aurora kinase B

G0/G1 S G2/M0

20

40

60

80

% C

ells

DMSOMRTX1719 250 nM

50%

65%

18% 20%10%10% 9% 7%

12%

Palbociclib 250 nMMRTX1719 + Palbo

65% 64%

8%

Cell CycleLU99 Day 7

Day 3 Day 5

GSEAA

B

DDetained Introns

increased

RNA Splicing

DI

CE

A5SS

A3SS

ME

0

2

4

6

8

10

% A

nnex

in(+

) / 7

-AAD

(-) C

ells

Early ApoptosisLU99 Day 3

CCell CycleLU99 Day 7

E

88

PRMT5

MRTX1719 Demonstrates Broad Activity in vitro and in vivo and Induces Regression in a Subset of Cell Line- and Patient-derived Xenograft Models

A. MRTX1719 in vitro activity across a panel of MTAP WT and MTAP del cell line models (5-day viability assay, Crown Biosciences). B. Dot plots showing median with 95% confidence intervals of IC50 values for MRTX1719 (left) and GSK-595 (right) in MTAP WT and MTAP del cell line models. A smaller cohort of models was tested with GSK-595. Median values: MRTX1719 - MTAP del - 100 nM; MTAP WT - 2.2 µM; GSK-595 - MTAP del - 284 nM; MTAP WT - 437 nM. Of note, in internal studies, IC50 values were 3-4-fold lower in the preferred 10-day viability assay format (e.g. MRTX1719 LU99 5-day viability IC50 – 72 nM; 10-day viability IC50 – 20 nM (n=13, both formats)) C. MRTX1719 was tested in a panel of cell line-and patient derived-xenograft tumor models at 50 or 100 mg/kg administered by oral gavage QD and a range of activity was observed including minimal response, tumor growth delay, tumor stasis and tumor regression. Individual tumor growth plots from selected models shown in which average tumor volume is plotted +/- SEM (n=3/treatment group).

0 10 20 30 400

1000

2000

3000

PXF 537

Study Day

Tum

or V

olum

e (m

m3 )

Mesothelioma PDXPXF 537

0 10 20 300

200

400

600

800

NCI-H2228

Study Day

Tum

or V

olum

e (m

m3 )

Lung CDXNCI-H2228

0 5 10 15 20 250

200

400

600

800

1000

1200

PA0372

Study Day

Tum

or V

olum

e (m

m3 )

Pancreatic PDXPA0372

VehicleMRTX1719 100 mg/kg QD

A C

B

-2

-1

0

1

MRT

X171

9Re

lativ

e IC

50 (l

ogµM

)

LungPancreasMesotheliomaOther

MTAP WT MTAP Del

MTAP WT MTAP DEL1

10

100

1000

10000

100000

MRTX1719

Rela

tive

IC50

val

ues

(nM

)

MTAP WT MTAP Del MTAP WT MTAP DEL1

10

100

1000

10000

100000

GSK-595

Rela

tive

IC50

val

ues

(nM

)

MTAP WT MTAP Del

0 5 10 15 20 250

200

400

600

800

1000

LU6408

Study Day

Tum

or V

olum

e (m

m3 )

Lung PDXLU6408

99

PRMT5

Reaction Biology Corp

Crown Biosciences

Charles River Laboratories

Monoceros Biosystems

STEMCELL Technologies

Cell Signaling Technology

Acknowledgements