Antitumor activity of the selective RAF inhibitor HM95573 in melanoma InHwan Bae, YoungGil Ahn, Namgoong GwangMo, JiYeon Song, TaeHun Song, JaeHo Lee, KyuHang Lee, SuHyeon Kim, Young-Mi Lee, YoungHoon Kim, KweeHyun Suh

Hanmi Research Center, Hanmi Pharmaceutical Co. Ltd., South Korea

Hanmi Hanmi Pharm. Co., Ltd. (http://www.hanmipharm.com)

HM95573 is an orally active, selective and potent 2nd generation RAF inhibitor.

HM95573 offered the opportunity for single agent efficacy in BRAF mutant and RAS mutant cancers.

Potent inhibition of RAS-RAF-MEK-ERK signaling in BRAF or NRAS mutated melanoma cell lines.

Good in vitro DMPK profile and moderate to good systemic exposure in animals.

Potential for minimized cuSCC side effects and resistance.

Currently in phase I clinical trial in patients with advanced solid tumors including melanoma in South Korea.

1. P. Lito, C. Pratilas et al., Cancer Cell, 2012; 22; 668-682.

2. R. Kudchadkar, K. Smalley et al., Clinics in Dermatology, 2013; 31; 200-208.

3. K. Cichowski, P. Jänne, Nature, 2010; 464; 358-359.

4. AS Little et al., Oncogene, 2012; 31; 1-9.

5. Zelboraf-CHMP assessment report (WC500124400)

Inhibition of MEK and ERK phosphorylation in BRAF mutant cell lines

Abstract

Introduction

in-vitro Activity in-vitro Pharmacology

References

Conclusion

The mitogen-activated protein kinase (MAPK) pathway is particularly important for the survival and proliferation of tumor cells. Activation of the MAPK pathway due to mutations in BRAF and NRAS is considered one of the causes of melanoma.1

HM95573 is a novel, highly potent RAF kinase inhibitor. Biochemically assayed for over 120 kinases, HM95573 showed the high selectivity toward BRAF mutant and CRAF kinases. The half maximal inhibition concentrations (IC50) of HM95573 against BRAFWT, BRAFV600E and CRAF kinases were 41nM, 7nM and 2nM, respectively. The strongly inhibited kinases subsequent to RAF kinases appeared to be CSF1R (44nM), DDR1 (77nM) and DDR2 (182 nM).

HM95573 potently inhibited the growth of mutant BRAF melanoma cell lines such as A375 (IC50: 57nM) and SK-MEL-28 (69nM) and of mutant NRAS melanoma cell lines such as SK-MEL-2 (53nM) and SK-MEL-30 (24nM). In addition, the phosphorylations of MEK and ERK downstream kinases associated with cell proliferation were effectively inhibited with treatment of HM95573 in mutant BRAF and mutant NRAS melanoma cells.HM95573 inhibited the downstream signaling in melanoma cells even in the presence of HGF which is known to mediate innate resistance to RAF inhibitors.

HM95573 showed the excellent antitumor activity in mouse models xenografted with both of BRAF mutation cell lines (e.g. A375 and SK-MEL-28) and NRAS mutation cell lines (such as SK-MEL-2 and SK-MEL-30) compared to two RAF inhibitors approved in melanoma which were effective to only BRAF mutation cell lines under conditions tested. Furthermore, HM95573 did not show a potential to paradoxical activation inducing tumor growth in mouse xenograft study using A431 cuSCC (cutaneous squamous cell carcinoma) cancer cell.2

Now, HM95573 is currently in phase I development in patients with advanced solid tumors including melanoma in Korea.

The RAF kinase is a key component of the RAS/RAF/MEK/ERK MAPK signaling pathway that regulates cell proliferation and survival in various tissues.

The important role of RAF kinase family in cancer pathophysiology is supported by the prevalence of 40-50% of abnormal BRAF and 15-20% of abnormal NRAS cellular signaling in melanoma cells.

HM95573 is developed as a novel therapeutic option for BRAF V600 mutant as well as NRAS mutant melanoma.

* in-vitro growth inhibition (GI50, mean ± SD) of melanoma cell lines (n=3)

In vitro enzyme activity

In vitro cellular activity

RAF immunoprecipitation (IP) kinase assay in SK-MEL-2 (NRAS Q61R) cell line 3, 4

Inhibition of cell proliferation

Study of A431 (BRAF WT, cuSCC) cell line

HM95573 BRAF WT 41

BRAF V600E 7 CRAF* 2

- FRET assay * RAF-1 Y340D Y341D

Biochemical potency (IC50, nM) Fold selectivity for BRAF V600E HM95573

BRAF WT 5.8 CRAF 0.28

CSF1R(FMS) 6.2 DDR1 11 DDR2 26 MEK1 >100 ERK1 >100

- The biochemical selectivity of HM95573 was profiled at 1.0 μM against 123 kinases using the kinase panel assay which was performed by Invitrogen (USA).

0 4 8 1 2 1 6 2 0 2 4 2 80

8 0 0

1 6 0 0

2 4 0 0

3 2 0 0

4 0 0 0

4 8 0 0C o n tro lD a b ra fe n ib , 1 0 0 m g /k g , Q D x 2 9H M 9 5 5 7 3 , 3 m g /k g , Q D x 2 9H M 9 5 5 7 3 , 1 0 m g /k g , Q D x 2 9H M 9 5 5 7 3 , 3 0 m g /k g , Q D x 2 9

D a y s

Tu

mo

r v

olu

me

(m

m3)

Cell line Mutation IC50, nM *

HM95573 Vemurafenib Dabrafenib

A375 BRAF V600E 57 ± 7 75 ± 12 <0.1

SK-MEL-28 BRAF V600E 69 ± 6 77 ± 17 <0.1

SK-MEL-2 NRAS Q61R 53 ± 23 >10,000 >10,000

SK-MEL-30 NRAS Q61K 24 ± 6 >10,000 >10,000 in-vivo Efficacy

A375

p-MEK

MEK

p-ERK

-ERK

Actin

DM

SO

HM95573

0.001

0.01

0.1

1 10

0.001

0.01

0.1

1 10

Vemurafenib

p-MEK

MEK

p-ERK

ERK

Actin

DM

SO

HM95573

0.001

0.01

0.1

1 10

0.001

0.01

0.1

1 10

Vemurafenib

SK-MEL-28

Inhibition of MEK and ERK phosphorylation in NRAS mutant cell lines SK-MEL-2 SK-MEL-30

p-MEK

MEK

p-ERK

ERK

Actin

DM

SO

HM95573

0.001

0.01

0.1

1 10

0.001

0.01

0.1

1 10

Vemurafenib

p-MEK

MEK

p-ERK

ERK

Actin

DM

SO

HM95573

0.001

0.01

0.1

1 10

0.001

0.01

0.1

1 10

Vemurafenib

Xenograft efficacy study in BRAF mutant cell lines

Reduced potential for acquired resistance by HGF exposure

ERK activation in A431 cell line

0 2 4 6 8 1 0 1 2 1 40

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

1 2 0 0

1 4 0 0

1 6 0 0 C o n tro lH M 9 5 5 7 3 , 3 0 m g /k g , Q D x 1 4V e m u ra fe n ib , 1 0 0 m g /k g , B ID x 1 4

D a y s

Tu

mo

r vo

lum

e (

mm

3)

A431 (cuSCC) xenograft model 5

Xenograft efficacy study in NRAS mutant cell lines

0 3 6 9 1 2 1 5

6 0 0

9 0 0

1 2 0 0

1 5 0 0

1 8 0 0

2 1 0 0C o n tro lV e m u ra fe n ib , 1 0 0 m g /k g , B ID x 1 5D a b ra fe n ib , 1 0 0 m g /k g , Q D x 1 5H M 9 5 5 7 3 , 3 0 m g /k g , Q D x 1 5

D a y s

Tu

mo

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olu

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(m

m3)

0 3 6 9 1 2 1 5

4 0 0

6 0 0

8 0 0

C o n tro lV e m u ra fe n ib , 1 0 0 m g /k g , B ID x 1 5H M 9 5 5 7 3 , 3 m g /k g , Q D x 1 5

D a y s

Tu

mo

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olu

me

(m

m3)

0 3 6 9 1 2 1 50

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0C o n tro lD a b ra fe n ib , 1 0 0 m g /k g , Q D x 1 5H M 9 5 5 7 3 , 1 0 m g /k g , Q D x 1 5H M 9 5 5 7 3 , 3 0 m g /k g , Q D x 1 5

D a y s

Tu

mo

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olu

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(m

m3)

W ith o u t H G F in S K -M E L -5(B R A F V 6 0 0 E )

- 0 .3 3 0 .3 30

5 0

1 0 0

1 5 0

2 0 0

2 5 0

3 0 0

p-E

RK

/ T

ota

l E

RK

(%

)

HM95573 Vemurafenib

W ith H G F in S K -M E L -5(B R A F V 6 0 0 E )

- 0 .3 3 0 .3 30

5 0

1 0 0

1 5 0

2 0 0

2 5 0

3 0 0

p-E

RK

/ T

ota

l E

RK

(%

)

HM95573 Vemurafenib

Acquired resistance & cuSCC Side effect

A375 SK-MEL-28

SK-MEL-30 SK-MEL-2

Abstract No. : 2606

1 2 3 4 5 6 7 80

1 0 0

2 0 0

3 0 0

4 0 0

p-E

RK

/ER

K (

%)

Control

0.001

0.01

0.1

1 10

1 10

HM95573 Vemurafenib

A-RAF B-RAF

C-RAF Lysates

NRAS

MEK

ERK

RTK

Transcription Factor

Proliferation / Survival / Transformation

Cytoplasm

Membrane

Nucleus

B-RAF C-RAF B-RAF

SOS

Mutation

1st generation RAFi －Vemurafenib －Dabrafenib

HM95573 B-RAF

C-RAF

Antitumor activity of the selective RAF Inhibitor HM95573 in solid tumors and hematologic malignancies Young-Mi Lee, InHwan Bae, Namgoong GwangMo, JaeHo Lee, KyuHang Lee, SuHyeon Kim, JiYeon Song, TaeHun Song, YoungGil Ahn, YoungHoon Kim, KweeHyun Suh

Hanmi Research Center, Hanmi Pharmaceutical Co. Ltd., South Korea

Hanmi Hanmi Pharm. Co., Ltd. (http://www.hanmipharm.com)

HM95573 is an orally active, selective and potent 2nd generation RAF inhibitor.

HM95573 offered the opportunity for single agent efficacy in BRAF mutant and RAS mutant cancers.

Potent inhibition of RAS-RAF-MEK-ERK signaling in BRAF or RAS mutated advanced solid tumors cell lines.

Good in vitro DMPK profile and moderate to good systemic exposure in animals.

Currently in phase I clinical trial in patients with advanced solid tumors including KRAS mutation NSCLC and mCRC in South Korea.

1. J. Xing et al., Biochemical and Biophysical Research Communications, 2011; 404; 958–962.

2. A. Cox and C. Der, Cancer cell, 2012; 21; 147-149 .

3. H. Yang, B. Higgins, et al., Cancer Res, 2012; 72; 779-789.

Inhibition of MEK and ERK phosphorylation in KRAS mutant cell lines

Abstract

Introduction

in-vitro Activity

in-vitro Pharmacology

References

Conclusion

Abstract No. : 2607

The mitogen-activated protein kinase (MAPK) pathway is particularly important for the survival and proliferation of tumor cells. Activation of the MAPK pathway due to mutations in BRAF, NRAS and KRAS is considered one of the causes of solid tumors (NSCLC, CRC,HCC, and thyroid cancers) and hematologic malignancies.

HM95573 is a novel, highly potent RAF kinase inhibitor. Biochemically assayed for over 120 kinases, HM95573 showed the high selectivity toward BRAF mutant and CRAF kinases. The half maximal inhibition concentrations (IC50) of HM95573 against BRAF WT, BRAF V600E and CRAF kinases were 41nM, 7nM and 2nM, respectively. The strongly inhibited kinases subsequent to RAF kinases appeared to be CSF1R (44nM), DDR1 (77nM) and DDR2 (182 nM).

HM95573 potently inhibited the growth of BRAF mutation CRC cell lines (e.g. IC50: 118nM for Colo-205) and thyroid cancer cell lines (43nM for B-CPAP) 1; KRAS mutation NSCLC cell lines (297nM for Calu-6),CRC cell lines (65nM for HCT-116) and thyroid cancer cell lines (479nM for CAL-62); and NRAS mutation HCC cell lines (28nM for HepG2) and leukemia cell lines (39nM for HL-60). HM95573 effectively inhibited the phosphorylations of MEK and ERK, downstream kinases associated with cell proliferation in tumor cell lines mutated in BRAF, KRAS and NRAS. In addition, the phosphorylation of downstream kinases of RAF such as MEK and ERK was effectively inhibited with treatment of HM95573 in mutant KRAS NSCLC and CRC cells. 2

HM95573 showed the excellent antitumor activity in mouse models xenografted with BRAF mutation cell line (Colo-205), KRAS mutation cell lines (Calu-6 and HCT-116) and NRAS mutation cell line (HepG2 ). The in vivo antitumor activity of HM95573 was potentiated with MEK inhibitors. Furthermore, HM95573 did not show a potential to paradoxical activation inducing tumor growth in mouse xenograft study using A431 cuSCC cancer cell.

Now, HM95573 is currently in phase I development in patients with advanced solid tumors including KRAS mutation NSCLC and mCRC in Korea.

The RAF kinase is a key component of the RAS/RAF/MEK/ERK MAPK signaling pathway that regulates cell proliferation and survival in various tissues.

The important role of RAF kinase family in cancer pathophysiology is supported by the abnormal BRAF and RAS cellular signaling in various solid tumors.

HM95573 is developed as a novel therapeutic option for RAS mutant as well as BRAF mutant advanced solid tumors.

In vitro enzyme activity

In vitro cellular activity Inhibition of cell proliferation

- FRET assay * RAF-1 Y340D Y341D

Selectivity for other kinases Biochemical potency (IC50, nM)

** The biochemical selectivity of HM95573 was profiled at 1.0 μM against 123 kinases using the kinase panel assay which was performed by Invitrogen (California, USA).

in-vivo Efficacy

* Abbreviation : Vem (Vemurafenib), Dab(Dabrafenib), HM573 (HM95573)

Xenograft efficacy study in Calu-6 (K-RAS,Q61K) NSCLC cell line

Xenograft efficacy study in HCT116 (KRAS, G13D) mCRC cell line

Single-Therapy Combination with MEKi

Combination with Capecitabine 3 Combination with MEKi

H T -29

C o lo-2

0 5

B -CP A P

F R OA R O

8 5 0 5C

H e p G2

H L 6 0

H C T 1 1 6L o v o

S W6 2 0

L S 1 7 4 T

L S 5 1 3

C a lu-1

C a lu-6

C A L -62

MD A -2

3 1

C a p a n -1

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

3 0 0 0

Ce

llu

alr

IC

50 (

nM

)V e m u ra fe n ib

H M 9 5 5 7 3

B R A F m u t N R A S m u t K R A S m u t

0 3 6 9 1 2 1 50

3 0 0

6 0 0

9 0 0

1 2 0 0

1 5 0 0

C o n tro lH M 9 5 5 7 3 , 3 0 m g /k g , Q D x 1 4

M E K i, 1 0 m g /k g , B ID x 1 4

H M 9 5 5 7 3 (3 0 m g /k g ) + M E K i (1 0 m g /k g )

D a y s

Tu

mo

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olu

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(m

m3)

0 3 6 9 1 2 1 50

3 0 0

6 0 0

9 0 0

1 2 0 0

1 5 0 0C o n tro lH M 9 5 5 7 3 , 3 m g /k g , Q D x 1 5H M 9 5 5 7 3 , 1 0 m g /k g , Q D x 1 5H M 9 5 5 7 3 , 3 0 m g /k g , Q D x 1 5

D a y s

Tu

mo

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olu

me

(m

m3)

Calu-6 (K-RASQ61K) HCT116 (K-RASG13D) Lovo (K-RASG13D)

(μM) - 0.1 1 10 - - - - - - - - - - 0.1 1 10 - - - - - - - - - - 0.1 1 10

2 hr

- 0.1 1 10 - - - - - - - - - - 0.1 1 10 - - - - - - - - - - 0.1 1 10

24 hr

- 0.1 1 10 - - - - - - - - - - 0.1 1 10 - - - - - - - - - - 0.1 1 10

2 hr

- 0.1 1 10 - - - - - - - - - - 0.1 1 10 - - - - - - - - - - 0.1 1 10

24 hr

- Vem Dab HM573 - Vem Dab HM573 - Vem Dab HM573 - Vem Dab HM573 pMEK

- Vem Dab HM573 - Vem Dab HM573

MEK

pERK

ERK

Actin - 0.1 1 10 - - - - - - - - - - 0.1 1 10 - - - - - - - - - - 0.1 1 10

2 hr

- 0.1 1 10 - - - - - - - - - - 0.1 1 10 - - - - - - - - - - 0.1 1 10

24 hr

0 3 6 9 1 2 1 50

3 0 0

6 0 0

9 0 0

1 2 0 0

1 5 0 0C o n tro lD a b ra fe n ib , 1 0 0 m g /k g , Q D x 1 4

H M 9 5 5 7 3 , 1 0 m g /k g , Q D x 1 4H M 9 5 5 7 3 , 3 0 m g /k g , Q D x 1 4

M E K i, 2 5 m g /k g , B ID x 1 4

D a y s

Tu

mo

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(m

m3)

H M 9 5 5 7 3 ,(1 0 m g /k g )+ M E K i (2 5 m g /k g )

0 3 6 9 1 2 1 50

3 0 0

6 0 0

9 0 0

1 2 0 0

1 5 0 0C o n tro l

H M 9 5 5 7 3 , 1 0 m g /k g , Q D x 1 5H M 9 5 5 7 3 , 3 0 m g /k g , Q D x 1 5

C a p e c ita b in e , 4 0 0 m g /k g , Q D x 1 5

H M 9 5 5 7 3 (3 0 m g /k g )+ C a p e c ita b in e (4 0 0 m g /k g )

D a y s

Tu

mo

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olu

me

(m

m3)

0 3 6 9 1 2 1 5

1 5 0

3 0 0

4 5 0

6 0 0

7 5 0

9 0 0C o n tro l

H M 9 5 5 7 3 , 1 0 m g /k g , Q D x 1 5H M 9 5 5 7 3 , 3 0 m g /k g , Q D x 1 5

M E K i, 2 5 m g /k g , B ID x 1 5

D a y s

Tu

mo

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olu

me

(m

m3)

H M 9 5 5 7 3 ,(1 0 m g /k g )+ M E K i (2 5 m g /k g )

Xenograft efficacy study in BRAF mutant mCRC cell lines

Colo-205 (BRAF, V600E) HT-29 (BRAF, V600E) HM95573

BRAF WT 41 BRAF V600E 7

CRAF* 2 CSF1R (FMS) 44

DDR1 77 DDR2 182

0 3 6 9 1 2 1 50

2 0 0

4 0 0

6 0 0

8 0 0C o n tro l

H M 9 5 5 7 3 , 3 m g /k g , Q D x 1 5H M 9 5 5 7 3 , 1 0 m g /k g , Q D x 1 5H M 9 5 5 7 3 , 3 0 m g /k g , Q D x 1 5

D a y s

Tu

mo

r v

olu

me

(m

m3)

V e m u ra fe n ib , 7 5 m g /k g , B ID x 1 5

Study of A431 (BRAF WT, cuSCC) cell line

cuSCC Side effect

ERK activation in A431 cell line

0 2 4 6 8 1 0 1 2 1 40

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

1 2 0 0

1 4 0 0

1 6 0 0 C o n tro lH M 9 5 5 7 3 , 3 0 m g /k g , Q D x 1 4V e m u ra fe n ib , 1 0 0 m g /k g , B ID x 1 4

D a y s

Tu

mo

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olu

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(m

m3)

A431 (cuSCC) xenograft model 5

1 2 3 4 5 6 7 80

1 0 0

2 0 0

3 0 0

4 0 0

p-E

RK

/ER

K (

%)

Control

0.001

0.01

0.1

1 10

1 10

HM95573 Vemurafenib

Xenograft efficacy study in HepG2 (NRAS, Q61K) HCC cell lines

0 3 6 9 1 2 1 50

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

1 2 0 0

1 4 0 0C o n tro l

S o ra fe n ib , 1 0 0 m g /k g , Q D x 1 5H M 9 5 5 7 3 , 3 0 m g /k g , Q D x 1 5

D a y s

Tu

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(m

m3)

§ : Colon cancer, + : Thyroid cancer, ^ : HCC, ¤ : Leukemia, Ж : NSCLC, ‡ : Breast cancer, √ : Pancreatic cancer

RAS

MEK

ERK

RTK

Transcription Factor

Proliferation / Survival / Transformation

Cytoplasm

Membrane

Nucleus

B-RAF C-RAF B-RAF

SOS

Mutation

Vemurafenib Dabrafenib

HM95573

• 50-60% melanoma • 40-50% thyroid cancer • 25-30% ovarian cancer • 10-15% colon cancer

• 70-90% pancreatic cancer • 30-40% colon cancer • 25-30% lung cancer • 15-20% melanoma

(NRAS, KRAS)

Melanoma