A selective and potent pan-RAF inhibitor, HM95573 exhibits high therapeutic potential as a next-generation RAF inhibitor by direct inhibition of RAF kinase activity in BRAF or RAS mutant cancers

Gwangmo Namgoong, Su-Hyeon Kim, Tae-Hun Song, In-Hwan Bae, Young-Gil Ahn, Jae-Ho Lee, Kyungjin Choi, Kyu-Hang Lee, Young-Hoon Kim, Young-Mi Lee and Kwee-Hyun SuhHanmi Research Center, Hanmi Pharmaceutical Co. Ltd., South Korea

28th EORTC-NCI-AACR Symposium on Molecular Targets and Cancer Therapeutics, Munich, Germany; 29 Nov – 2 Dec, 2016

HM95573

BRAF WT 41

BRAFV600E 7

CRAF 2

The biochemical selectivity of HM95573 was profiled at 1 μM against 187 kinases using the kinase panel assay which was performed by Thermo fisher scientific (USA).

(b) Biochemical potency (IC50, nM)(a) Biochemical selectivity (inhibition % at 1 μM)

● BRAF mutant ■ KRAS mutant▲ NRAS mutant ▼ RAS/RAF wild-type

HM95573 Vemurafenib DabrafenibMutation

WTMutation

WTMutation

WTBRAF KRAS NRAS BRAF KRAS NRAS BRAF KRAS NRAS

Sensitive (GI50 < 1 μM) 11 7 5 0 7 0 0 0 11 3 2 1

Resistant (GI50 > 1 μM) 0 0 0 2 4 7 5 2 0 4 3 1

RAS/RAF/MEK/ERK mitogen-activated protein kinase (MAPK) cascade pathway is a key signaling pathway in cancer.Although the BRAF kinase inhibitors such as vemurafenib and dabrafenib showed remarkable anti-tumor activity inBRAFV600E mutant melanoma patients, its clinical efficacy was limited because of paradoxical activation and lowactivity in RAS mutant or BRAF wild-type cancer and acquired resistance in BRAF mutant melanoma. We havedeveloped a novel selective and potent pan-RAF inhibitor HM95573 that has anti-tumor effects in not only in BRAFV600E mutant but also in RAS mutant cancers with minimal paradoxical activation. Although HM95573 induced theBRAF/CRAF heterodimerization, it inhibited BRAF and CRAF kinase activities, which resulted in anti-proliferative andapoptotic effects in NRAS or KRAS mutant melanoma, colorectal cancer (CRC), and non-small-cell lung carcinoma(NSCLC) cells. Additionally HM95573 showed effective growth inhibition in vemurafenib resistant A375 cells by MAPKinhibition. Furthermore, HM95573 exhibited slow dissociation against BRAFV600E and CRAF which representspersistent occupy both RAF dimers by prolonged binding differentiated from vemurafenib and dabrafenib. In addition,HM95573 showed synergistic anti-tumor effect with a MEK inhibitor, EGFR monoclononal antibody, orchemotherapeutic agents in vitro and in vivo in BRAF or RAS mutant cancers. According to these results, wedemonstrates that HM95573 inhibits BRAF and RAS mutant cancers by suppressing the MAPK signaling throughdirect inhibition of RAF kinase activity and by inducing apoptosis. Taken together, a next generation pan-RAF inhibitorHM95573 has the potential role as a candidate treatment for patients with BRAFV600E or RAS mutation harboringmelanoma, CRC and NSCLC.

CompoundsBRAFV600E CRAF

ka (1/Ms) kd (1/s) KD (M) ka (1/Ms) kd (1/s) KD (M)

HM95573 1.35 x 104 2.15 x 10-4 1.60 x 10-8 2.06 x 104 9.99 x 10-5 4.84 x 10-9

Vemurafenib 1.49 x 104 1.10 x 10-2 7.42 x 10-7 1.42 x 104 3.35 x 10-3 2.36 x 10-7

Dabrafenib 9.67 x 104 1.19 x 10-3 1.23 x 10-8 1.35 x 105 1.66 x 10-3 1.23 x 10-8

● 5 μM ● 1 μM ● 0.2 μM ● 0.04 μM ● 0.008 μM

HM95573

VemurafenibHM95573

Dabrafenib

Dabrafenib

BRAFV600E

CRAF

ka, constant rate for association; kd, constant rate for dissociation; KD, dissociation constant (kd/ka)

CSF-1R

CRAF

DDR1

BRAF

ABSTRACT

RESULTS

-3 -2 -1 0 1

2 0

4 0

6 0

8 0

1 0 0

1 2 0

lo g -c o n c . (µ M )

Cel

l gro

wth

(%

)

- 3 -2 -1 0 1

2 0

4 0

6 0

8 0

1 0 0

1 2 0

lo g -c o n c . (µ M )

Cel

l gro

wth

(%

)

Vemurafenib Dabrafenib HM95573 Vemurafenib Dabrafenib HM95573

pMEKMEKpERKERKβ-actin

Con

0.1

1 10 0.1

1 10 0.1

1 10 Con

0.1

1 10 0.1

1 10 0.1

1 10

Parental A375 Vem-R A375 ● HM95573 1 μM● Vemurafenib 1 μM ● Dabrafenib 1 μM

• HM95573 is an orally active, selective and potent next generation RAF inhibitor which has anti-tumoreffect on BRAF mutant as well as RAS mutant cancer.

• HM95573 has anti-tumor effect on RAS mutant cancer cells through direct inhibition of RAF kinaseactivity which might be caused by slow dissociation from RAF.

• HM95573 offered the opportunity of antitumor effect as a monotherapy / combination therapy inBRAF or RAS mutant cancer as well as potential of overcoming acquired resistance by vemurafenib.

CONCLUSIONS

#387

In vitro Activity of HM95573

HM95573 shows potent and selective inhibitory effects on pan-RAF kinases among 187 kinases1

HM95573 is sensitive in BRAF mutant and RAS mutant cancer cells among 25 cancer cell lines 2

Mode of Action of HM95573

HM95573 shows slow dissociation from BRAFV600E and CRAF3

HM95573 suppresses the MAPK signaling through directinhibition of RAF kinase activity in RAS mutant cancer cells4

Anti-tumor Potential of HM95573

HM95573 has a potential to overcome the resistance in vemurafenib-resistant A375 cells6

HM95573 shows synergistic effect with MEK inhibitor, EGFR mAb andchemotherapeutic agent in BRAF or RAS mutant cancer7

pMEK

MEK

(μM, 2h)

pMEK

MEK

Con

0.00

10.

010.

11 10

HM95573

0.00

10.

010.

11 10

Dabrafenib

IP:BRAF

IP:CRAF

Con

0.00

10.

010.

11 10

HM95573

0.01

0.1

1 10

Dabrafenib

Con

0.00

10.

010.

11 10

HM95573

0.00

10.

010.

11 10

Dabrafenib

0.00

1

CRAF

BRAFDimerization

(IP:BRAF)

SK-MEL-30 (NRASQ61K) HCT116 (KRASG13D) Calu-6 (KRASQ61K)

RAF

kin

ase

activ

ity

pMEK

pERKβ-actin

Cell lysates

-3 .0 -2 .5 -2 .0 -1 .5 -1 .0

2 0

4 0

6 0

8 0

1 0 0

1 2 0

lo g -c o n c . (µ M )

Cel

l g

row

th (

%)

1 4 7 1 1 1 4 1 8 2 10

3 0 0

6 0 0

9 0 0

1 2 0 0

1 5 0 0

1 8 0 0

D a y s

Tu

mo

r v

olu

me

(m

m3

)

EGFR mAb in HT-29(BRAFV600E, CRC)

Cell growth inhibition Cell growth inhibition

Cytotoxic agent in HCT116(KRASG13D, CRC)

MEKi in SK-MEL-30(NRASQ61K, melanoma)

Cell growth inhibition

-3 .0 -2 .5 -2 .0 -1 .5 -1 .0 -0 .50

2 0

4 0

6 0

8 0

1 0 0

1 2 0

lo g -c o n c . (µ M )

Ce

ll g

row

th (

%)

-2 .0 -1 .5 -1 .0 -0 .5 0 .0 0 .50

2 0

4 0

6 0

8 0

1 0 0

1 2 0

lo g -c o n c . (µ M )

Ce

ll g

row

th (

%)

● HM95573 ● 5-FU (2.5 μM)● Combination

● HM95573 ● MEKi (20 nM)● Combination

● HM95573 ● Cetuximab (100 μg/mL)● Combination

1 4 8 1 1 1 5 1 8 2 20

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

D a y s

Tu

mo

r vo

lum

e (m

m3

)

1 4 8 1 1 1 5 1 9 2 30

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

D a y s

Tu

mo

r vo

lum

e (m

m3

)

● Control ● HM95573 (10 mpk)

● Control● HM95573 (30 mpk)

● Control● HM95573 (10 mpk)

● Capecitabine (400 mpk) ● Combination

● MEKi (10 mpk)● Combination

● Cetuximab (40 mpk)● Combination

Xenograft tumor inhibition (n=5) Xenograft tumor inhibition (n=5) Xenograft tumor inhibition (n=5)

11 BRAF mutant cell lines, 7 KRAS mutant cell lines, 5 NRAS mutant cell lines, 2 RAS/RAF wild-type cell lines

187 kinases

1 0 -1

1 0 0

1 0 1

1 0 2

1 0 3

1 0 4

GI 50

(nM

)

S e n s itiv e

R e s is ta n t

H M 9 5 5 7 3 V e m u ra fe n ib D a b ra fe n ib

1 µ M

HM95573 has anti-tumor effect on BRAF or RAS mutant cancercells by inducing apoptosis5

DMSO HM95573 0.1 μM HM95573 1 μM

Annexin-VAnnexin-V Annexin-V

PI PI PI

DMSO HM95573 0.1 μM HM95573 1 μM

11.5% 20.4% 47.6%

A375 (BRAFV600E)

Calu-6 (KRASQ61K)

LoVo (KRASG13D)

DMSO HM95573 0.1 μM HM95573 1 μM

BR

AF

CR

AF

Active RAS

i

RAS Mutant

X Xi

MEK

ERK

Tumor inhibition

HM95573

BR

AF

CR

AF

Active RAS

i

X

MEK

ERK

Proliferation

BRAF inhibitor

X

X

Gro

wth

Inhi

bitio

n

10.5%

PI

Annexin-V

22.7%

PI

Annexin-V

40.4%

Annexin-V

9.2%

PI

43.9%

PI

20.7%

PI

Annexin-V Annexin-V Annexin-V

Cleaved PARP

Cleaved caspase-3

PARP

Caspase-3

Bim0 0.1 1

β-actin

HM95573 (μM)

Cleaved PARP

Cleaved caspase-3

PARP

Caspase-3

Bim0 0.1 1

β-actin

HM95573 (μM)

Cleaved PARP

Cleaved caspase-3

PARP

Caspase-3

Bim0 0.1 1

β-actin

HM95573 (μM)

PI

*Administration of in vivo model : Once daily for HM95573, MEK inhibitor and capecitabine; twice a week for cetuximab

Vemurafenib

Cel

l gro

wth

(%)

Cel

l gro

wth

(%)

HM95573 Vemurafenib Dabrafenib