Repotrectinib Increases Effectiveness of MEK Inhibitor Trametinib in KRAS Mutant Cancer Models Via Simultaneous SRC/FAK/JAK2 Inhibition

Figure 1. Repotrectinib Kinase Selectivity at 10 µM ATP

• Kirsten Rat Sarcoma viral oncogene homolog (KRAS) is the most frequently mutated oncogene in a broad spectrum of human cancers1, including 25–30% of non-small cell lung cancer (NSCLC), ~45% of colorectal cancer (CRC), and ~75% of pancreatic cancer2

• The MEK inhibitor trametinib, which inhibits signaling downstream of mutant KRAS, has shown limited single agent activity in patients with KRAS-mutant positive NSCLC3,4

J. Jean Cui, Dayong Zhai, Wei Deng, Laura Rodon, Nathan Lee, Brion W. MurrayTurning Point Therapeutics, Inc., San Diego, CA 92121

INTRODUCTION

KINASE SELECTIVITY OF REPOTRECTINIB

Abstract # 1957

• Repotrectinib is a selective kinase inhibitor currently in a registrational clinical trial for ROS1+ and NTRK+ cancer patients

• Repotrectinib potently inhibits SRC/FAK/JAK2 in biochemical assays9 (Figure 1A) and SRC, FAK and STAT3 (JAK2 substrate) phosphorylation in cells (HCT116, Figure 1B)

Presented at: American Association for Cancer Research (AACR), 22 – 24 Jun 2020. Corresponding Contact — Jim Mazzola: jim.mazzola@tptherapeutics.com

1. Haigis, 2017 Trends Cancer 3(10), 686-6972. NCI-SEER database3. Blumenschein et al., 2015 Ann Oncol 26(5), 894–9014. Gadgeel et al., 2019 J Clin Oncol 37, 90215. Lee et al., 2014 Cancer Cell 26(2), 207-2216. Mohrherr et al., 2019 Int J Cancer 145 (12), 3376-33887. Sen et al., 2009 Cancer Res 69(5), 1958-19658. Zhou et al., 2017 J Pharmacol Exp Ther 363(3), 428-4439. Drilon et al., 2018 Cancer Discov 8(10), 1227 - 123610. Ianevski et al., 2017 Bioinformatics 33(15): 2413–241511. Gao et al., 2019 Acta Pharm Sinica 40, 268-278

CONCLUSIONS

REFERENCES & DISCLOSURES

• Repotrectinib/trametinib combination is synergistic in NSCLC, CRC, and pancreatic cancer tumor cell lines

‐ Synergistically inhibits proliferation of a subset of mutant KRAS tumor cell lines ‐ Increases apoptosis of a subset of mutant KRAS tumor cells ‐ Inhibits pS6 to a greater extent than either single agent

• Simultaneous targeting of SRC/FAK/JAK2 by repotrectinib suppresses trametinib-induced AKT and STAT3 feedback activation

• Repotrectinib/trametinib combination enhances efficacy in mutant KRAS xenograft models in vivo

‐ Phosphorylation of SRC, FAK, STAT3 is suppressed at clinically relevant concentrations of repotrectinib

‐ Repotrectinib enhances efficacy of trametinib in Calu-6 KRASQ61K NSCLC ‐ The combination had significantly enhanced efficacy with a robust tumor growth delay in HCT116 KRASG13D CRC

• Preclinical studies are on-going to further define KRAS mutations/tumor types to target with a repotrectinib/trametinib combination

EVALUATION OF REPOTRECTINIB/TRAMETINIB SYNERGY

• KRAS mutation frequency varies by tumor type (Figure 2A). Trametinib potency in cell viability assays is enhanced for a subset of mutant KRAS cell lines when combined with 1 µM repotrectinib (Figure 2B)

• Repotrectinib demonstrates synergy with trametinib (Figure 3A). BLISS synergy analysis10 reveals greatest synergy at clinically relevant concentrations (Figure 3B). Similar results were observed with Calu-6 KRASQ61K, HCT-116 KRASG13D, and PSN-1 KRASG12R cells

EVALUATION OF REPOTRECTINIB/TRAMETINIB SYNERGY (CONTINUED)

Figure 2: Repotrectinib/Trametinib Combination Demonstrates Enhanced Potency in KRAS Mutant Cells

• Repotrectinib suppresses trametinib-induced AKT activation in Calu-6 cells more than other combinations (Figure 4A)

• Repotrectinib suppresses trametinib-induced STAT3 activation in HCT-116 cells (Figure 4B)

REPOTRECTINIB/TRAMETINIB EFFECT ON DOWNSTREAM SIGNALING

Figure 4: Repotrectinib Suppresses Feedback Signaling From Trametinib Treatment

REPOTRECTINIB/TRAMETINIB ENHANCES INDUCTION OF APOPTOSIS

• Repotrectinib enhances trametinib-induced cleavage of full length PARP in both Calu-6 and HCT-116 cancer cells (Figure 6)

Figure 6: PARP Cleavage in Calu-6 and HCT-116 Cancer Cells

REPOTRECTINIB/TRAMETINIB IN KRAS XENOGRAFT TUMOR MODELS

• Repotrectinib enhanced trametinib efficacy and delayed tumor growth in combination (Figure 8A)

• Repotrectinib suppresses SRC and FAK phosphorylation (Figure 8B)

Figure 8: Evaluation of Repotrectinib/Trametinib in HCT-116 KRASG13D Tumor Model

REPOTRECTINIB/TRAMETINIB IN KRAS XENOGRAFT TUMOR MODELS

• Repotrectinib inhibits tumor growth as a single agent and enhances trametinib efficacy in combination (Figure 7A)

• Repotrectinib suppresses SRC, FAK, AKT, and trametinib-induced STAT3 phosphorylation (Figure 7B)

Figure 7: Evaluation of Repotrectinib/Trametinib in Calu-6 KRASQ61K Tumor Model

Target IC50 (nM) at 10 µM ATP

ROS1 0.07TRKC 0.21TRKB 0.3TRKA 0.53ALK 1.0JAK2 1.0

SRC & SRC family kinases 1.1–5.3

TXK 3.2ARK5 4.5DDR1 5.7

FAK 7.0

B.A.

*Proxy reagents purchased from commercial sources

Trametinib Repotrectinib BLISS Synergy Score

5 nM 333 nM 14.2

14 nM 333 nM 14

41 nM 333 nM 13.9

A. B.

A. B.

This study was sponsored by Turning Point Therapeutics, Inc.

BWM, DZ, JJC, LR, NL, WD: Turning Point Therapeutics – employment/shareholder.

• Compensatory upregulation of PI3K/AKT survival signaling or MEK inhibitor-induced JAK/STAT3 pathway activation have been identified as resistance mechanisms to evade targeted therapy5-8

• Combination of a MEK inhibitor with agents that simultaneously inhibit SRC, FAK and JAK2 may effectively target KRAS-mutant cancers

Figure 3: Synergy of Repotrectinib/Trametinib Combination

• Repotrectinib/trametinib combination effectively suppresses both ERK and AKT phosphorylation as well as S6, a downstream marker of both ERK and PI3K/AKT signaling11. The combination is more effective than either single-agent treatment (Figure 5)

Figure 5. Downstream Signaling is Suppressed by Repotrectinib/Trametinib Combination

Treatment TGI (%)Trametinib 0.4 mg/kg QD 66%

Repotrectinib 15 mg/kg BID 7%Repotrectinib 15 mg/kg BID and Trametinib 0.4 mg/kg QD 81%

Repotrectinib/Trametinib combination vs Trametinib on Day 18: p=0.0401

Treatment TGI (%)Trametinib 0.2 mg/kg QD 38%

Repotrectinib 15 mg/kg BID 55%

Trametinib 0.2 mg/kg QD and Repotrectinib 15 mg/kg BID 73%

Repotrectinib/trametinib combination vs Trametinib on Day 38: p<0.0001

A. B.

NSCLC

CRC

Pancreatic Cancer

Clinically-relevant concentrations of trametinib boxed

A427 (KRASG12D)

*Proxy chemicals purchased from commercial source

Calu-6 (KRASQ61K) HCT-116 (KRASG13D)

A. B.

A. B.

(2h post last dose)

(2h post last dose)

HCT-116 (KRASG13D)Calu-6 (KRASQ61K) PSN-1 (KRASG12R)

PARP cleavage was more pronounced in the combination

PARP cleavage was more pronounced in the combination

Trametinib: 50 nM

Repotrectinib: 1.0 µM

Dasatinib* (SRCi): 0.1 µM

Defactinib* (FAKi): 1.0 µM

Ruxolitinib* (JAKi): 1.0 µM

pSRC Y416

SRC

pSTAT3 Y705

STAT3

pFAK Y397

FAK

B-actin

Trametinib 50 nM - + - + - +Repotrectinib 333 nM - - + + - -

Repotrectinib 1000 nM - - - - + +

pERK Y202/204

ERKpAKT S473

AKTpS6 S235/236

S6B-actin

Trametinib 50 nM - + - + - +Repotrectinib 333 nM - - + + - -

Repotrectinib 1000 nM - - - - + +pERK Y202/204

ERKpAKT S473

AKTpS6 S235/236

S6

B-actin

Trametinib 50 nM - + - + - +Repotrectinib 333 nM - - + + - -

Repotrectinib 1000 nM - - - - + +pERK Y202/204

ERK

pAKT S473AKT

pS6 S235/236S6

B-actin

Trametinib 50 nM - + - + - +Repotrectinib 333 nM - - + + - -

Repotrectinib 1000 nM - - - - + +

pAKT S473

AKT

B-actin

pSTAT3 Y705STAT3B-actin

4h 24hTrametinib 50 nM - + - + - + - + - + - +

Repotrectinib 333 nM - - + + - - - - + + - -Repotrectinib 1000 nM - - - - + + - - - - + +

PARPCleaved PARP

B-actin

4h 24hTrametinib 50 nM - + - + - + - + - + - +

Repotrectinib 333 nM - - + + - - - - + + - -Repotrectinib 1000 nM - - - - + + - - - - + +

Calu-6 KRASQ61K NSCLC

HCT-116 KRASG13D CRC

PARPCleaved PARP

B-actin

4h 48hTrametinib 50 nM - + - + - + - + - + - +

Repotrectinib 333 nM - - + + - - - - + + - -Repotrectinib 1000 nM - - - - + + - - - - + +

DMSO

Tram

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Dasat

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Tram

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Das

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Defac

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Tram

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Def

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Ruxo

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