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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
r v
olu
me
(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
r v
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
r v
olu
me
(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
r v
olu
me
(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
r v
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
r v
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 )
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
r v
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
r v
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
r v
olu
me
(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
mo
r v
olu
me
(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
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