DMSO SRA141 (µM)

0.1 1

Time (h) 3 24 3 6 24 3 6 24

MCM2

pMCM2 S53

pMCM2 S41

P.O.

Collect tumors 12 h postSingle SRA141 dose

Tumor volume - 500 mm2

SRA141(indicated dose)

Background

• Cell division cycle 7 (Cdc7), a serine-threonine kinase, is a novel therapeutic target with emerging clinical validation in oncology. Cdc7 inhibitors demonstrate evidence of anti-tumor activity in both hematological and solid cancers in preclinical models.

• Cdc7, together with its partner proteins Dbf4 or Drf1, is responsible for activating DNA replication during S phase through phosphorylation and activation of the MCM2-7 helicase. Cdc7 also has functions within the DNA Damage Response (DDR) and mitosis.1,2

• While the precise mechanism of Cdc7 inhibitor mediated anti-tumor activity remains to be determined, preclinical studies investigating the inhibition of Cdc7 using siRNA and small molecules demonstrate differential sensitivity of tumor cells as a consequence of a p53-dependent DNA replication checkpoint operational in non-transformed cells. 3,4

• Despite this intriguing observation, the development of first generation Cdc7 inhibitors was limited by a range of pharmaceutical challenges including off-target toxicity and poor pharmacokinetics.

• Therefore, we investigated the novel, potent, orally bioavailable Cdc7 inhibitor SRA141 in biochemical, cellular and in vivo cancer models to better elucidate its potential as an anti-tumor agent.

The novel oral Cdc7 inhibitor, SRA141, demonstrates robust efficacy in preclinical cancer models Abstract #107Ryan J. Hansen, Snezana Milutinovic, Bryan Strouse, Gregg Smith, Christian Hassig

Sierra Oncology, Inc., Vancouver, BC, Canada.

Results

Known Functions of Cdc7/Dbf4

Functions of Cdc7/Dbf4 in S phase

Figure 1: SRA141 is a selective and potent inhibitor of Cdc7. (A) SRA141 demonstrated potent inhibition of Cdc7 in an in vitro biochemical assay (IC50 = 4 nM). Pre-incubation of SRA141 with Cdc7 enzyme for 1 h prior to assay conduct modestly improved the compound potency (IC50 = 1.4 nM), indicating potent inhibition with potentially advantageous inhibitor binding kinetics. (B) Rapid dilution studies indicated a long residence time for SRA141 binding to Cdc7 (t1/2 = 215 min) and slow dissociation kinetics (Koff = 0.0032 at 5 nM). (C) The selectivity of SRA141 for Cdc7 was compared to TAK-931, a Cdc7 inhibitor currently in Phase 1/2 testing. Using the DiscoverX kinome screening assay (~ 430 native and mutant kinases), SRA141 has less off-target activity, compared to TAK-931, at a compound concentration of 500 nM.

SRA141 (µM), 24 h

DMSO 0.033 0.1 0.33 1.0 3.3

CDC7

MCL-1

GAPDH

Figure 2: SRA141 demonstrates on-target selectivity in cells. Colo-205 cells were treated with SRA141 (at concentrations between 0.033 and 3.3 μM) with subsequent assessment of the phosphorylation status of the downstream targets for Cdc7 (pMCM2, S40 and S53), and several potential off-target kinases. Cdc7 levels were unchanged in these studies, while the phosphorylation of its target MCM2 at S40 and S53, was reduced in a concentration-dependent manner starting at 0.033 μM, demonstrating robust on-target inhibition in cells. There was no significant change in the phosphorylation status (or protein level of MCL-1) for any of the cellular substrates at concentrations of SRA141 up to 1 µM. Minimal inhibition of STAT1, YAP1 and MCL-1 was observed at 3.3 μM, although the co-incident reduction of total protein levels (MCM2, STAT1 and YAP1) suggests that these findings may have been due to SRA141 mediated cell death, which is evident at this concentration. Together, these data confirm the selectivity of SRA141 in cellular settings.

Figure 3: SRA141 demonstrates potent anti-proliferative activity in numerous cells lines comparable or superior to other Cdc7 inhibitors and demonstrates potential therapeutic index. (A) The anti-proliferative activity of SRA141 was characterized in several cell lines (Colo-205, SW620, SNU-398, NCI-H716, MDA-MB-231, NCI-H1573 and SW1116) using four orthogonal assays. The relative sensitivities of cell lines within each assay format were generally concordant. A consistent increase of SRA141 potency was observed with a longer treatment duration of 144 h when compared with 72 h across all assays and cell lines tested, suggesting that extended target coverage, as has been observed with other kinase inhibitors, may facilitate optimal anti-tumor activity. (B) SRA141 demonstrates comparable or superior in vitro activity in numerous cell lines versus other Cdc7 inhibitors, TAK-931 and LY-3177833, across two viability assays. Taken together, SRA141 demonstrated potent anti-proliferative activity in numerous cell lines across several indications. (C) The differential cytotoxic activity of SRA141 in Colo-205 colon cancer cells versus normal human dermal fibroblast cells (NHDF) was assessed. Following treatment, cells labelled with propidium iodide were analyzed by flow cytometry to determine the percentage of dying cells found in sub-G1 fractions (indication of cell death). A marked difference in sensitivity to SRA141 between cancer and normal cells was observed. These findings are consistent with published data demonstrating a DNA replication checkpoint in healthy cells that is absent in cancer cells.5 This supports a potential therapeutic index for SRA141 between tumor and non-transformed tissues.

Figure 4: Anti-proliferative potency of SRA141 increases with longer treatment of cancer cells. SRA141 IC50s were also determined at 144 h following differing durations of exposure to SRA141 (24, 48 or 72 h) followed by replenishment with drug-free media. Consistent with the earlier assessment, results demonstrate SRA141 potency increases with treatment duration beyond 24 h.

Figure 5. SRA141 displays good oral bioavailability and favorable pharmacokinetics in vivo. Non-tumor bearing female nude rats were dosed orally with SRA141 (n = 3 per dose group) at 50 mg/kg (QD x 12 days) or 100 or 150 mg/kg (5 days on/2 days off/5 days on). The vehicle was 0.5% CMC-Na/1% Lutrol in water. SRA141 plasma concentrations were determined by LC-MS/MS following the last dose. Systemic exposure increased in a broadly dose-concordant manner over the investigated dose range.

Figure 7: SRA141 demonstrates potent on-target engagement and anti-tumor activity in MV-4-11 xenografts. (A) Rats bearing subcutaneous biphenotypic B myelomonocytic leukemia MV-4-11 tumors were treated orally with SRA141 or vehicle for four cycles of 5 days on/2 days off. Significant tumor growth inhibition (92% at day 27 and 85% at day 34) was observed following 75 mg/kg BID. A complete tumor regression (1/7) and a partial regression (1/7) were achieved. (B) MV-4-11 tumor bearing rats were treated with SRA141 (75 mg/kg) or vehicle, BID, or SRA141(100 mg/kg), QD, for 5 days and the tumors were collected 12 h post last dose. Formalin fixed paraffin-embedded tumor and/or skin were assessed by immunohistochemistry for on-target, pMCM2 (S40 and S53) and pMCM2 (S41), a target of Cdk, effects at steady-state. Substantial decreases in both pMCM2 (S40) in tumor correlated with changes in surrogate tissue, suggesting skin biopsies could be utilized to demonstrate on-target activity of SRA141. (C) Tumors were analyzed by immunohistochemistry for pHH3, a marker of mitotic cells, or stained with H&E. After 5 days of dosing, SRA141 resulted in fewer mitotic cells and an increased presence of apoptotic cells (white arrowheads), which was associated with anti-tumor activity in MV-4-11 tumors.

50 mg/kg 100 mg/kg 150 mg/kg

Mean (SD) Mean (SD) Mean (SD)

Tmax (h) 1 1 1

Cmax (ng/mL) 690 (174) 1074 (289) 1463 (368)

AUClast (ng•h/mL) 5801 11920 16479

Figure 6: SRA141 results in tumor regressions and potent on-target pharmacodynamics in Colo-205 colorectal xenografts. (A) Rats bearing subcutaneous colorectal Colo-205 tumors were treated orally with SRA141 or vehicle for six cycles of 5 days on/2 days off. Significant tumor growth inhibition (93%) was observed following administration of 75 mg/kg BID. Complete tumor regressions were achieved in 4/7 (57%) rats. (B) Colo-205 tumor bearing rats were treated with a single dose of SRA141 or vehicle and the tumors were collected 12 h later. Tumor homogenates prepared from three animals per treatment group were analyzed by LC-MS/MS or Western blot. Tumor SRA141 levels of approximately 1.2 μM resulted in an approximate 50% decrease in pMCM2 (S53) as compared to vehicle treated rats suggesting that selective inhibition of Cdc7 is incompatible with tumor cell survival. These results demonstrate the profound monotherapy activity of SRA141 in the colorectal Colo-205 rat xenograft model at well-tolerated doses.

Kinase Cellular Substrate

CDK1 pMCM2 S41

CDK7,9 pRNAPII S2

CDK8 pSTAT1 S727

LATS2 pYAP1 S127

Cdc7 and Cdk Phosphorylation Sites pMCM2 (S41) – Cdk

pHH3 (S10)

Vehicle

Tum

orTu

mor

Skin

Vehicle Vehicle

Vehicle

Vehicle

SRA141(75 mg/kg)

Day

Day

0.0

0.5

1.0

1.5

2.0

[SRA

141]

tum

or (µ

M)

pMCM2 (S53)

GAPDH

Vehicle

SRA141 Treated

SRA141 Treated SRA141 Treated

SRA141 Treated

SRA141 75mg/kg

SRA141 Treated

H&E

pMCM2 (S53) – Cdc7 pMCM2 (S40) – Cdc7

C

BA

A

C

0%

Percent Control

0.1%

0.1-1%1-5%

5-10%10-35%

>35%

A B

C

B

SRA141 (µM), 8 h

DMSO 0.033 0.1 0.33 1.0 3.3

pMCM2 S40/41

MCM2

pSTAT1 S727

STAT1

pRNAP II S2

RNAP II

pYAP1 S127

YAP1

GAPDH

Summary of Hits% of Control SRA141 TAK-931

Less < 35% 38 52

Less < 20% 26 39

Less < 10% 21 29

Less < 5% 14 20

Less < 1% 1 11

MCM2MCM4MCM6

ClaspinRAD18

Eco1Hp1

Cdc7/Dbf4

DNA Damage ResponseDNA Replication Mitosis

Phosphorylation of MCM

Normal cells

Completion of DNA replicationInduction of DNA replication

through the phosphorylation of MCM2 in the MCM

complex

MCM complex

Cdc7

P

S phase

Conclusions

References:

• SRA141 is a potent, highly-selective, orally-bioavailable, small-molecule inhibitor of cell division cycle 7 kinase (Cdc7).

• SRA141 inhibits Cdc7 in biochemical kinase assays with an IC50 of 1.4-4.0 nM and potently inhibits the phosphorylation of the Cdc7 substrate MCM2 (EC50 ~ 33 nM) in cells.

• SRA141 displays excellent selectivity with less cross-reactivity than TAK-931, another Cdc7i currently in Phase 1/2 clinical trials demonstrating early anti-tumor activity. Cellular selectivity of SRA141 was confirmed against a subset of potential off-target kinases.

• Consistent with its biochemical and cellular inhibition of Cdc7, SRA141 displayed potent in vitro anti-proliferative activity against various tumor cell lines, including several highly sensitive colorectal tumor lines. Activity was comparable or superior to other Cdc7 inhibitors in the majority of lines tested.

• Further mechanistic studies demonstrated increased potency with prolonged compound exposure to elicit maximal anti-proliferative activity as is commonly observed with kinase inhibitor drugs.

• Importantly, while a majority of Colo-205 cancer cells were dying and found in the sub-G1 fraction at SRA141 concentrations between 1 µM and 10 µM; fewer than 10% of the normal non-transformed cells were apoptotic. These findings support a potential therapeutic index for SRA141 between tumor and non-transformed tissues, consistent with prior reports of Cdc7 inhibition.

• In vivo characterization of SRA141 demonstrates favorable PK properties and excellent anti-tumor activity in several tumor models representing solid (colorectal, CRC) and hematologic (biphenotypic B myelomonocytic leukemia, AML) cancers, including complete and partial regressions.

• Pharmacodynamic assessment of SRA141 treated tumors reveals potent on-target inhibition in both tumor and surrogate skin tissues, in addition to fewer mitotic cells and increased presence of apoptotic cells in tumors.

• The demonstrated biological, pharmacological and pharmaceutical properties of SRA141 support its clinical development as an oncology therapeutic agent.

1. Sawa, M. Drug design with Cdc7 kinase : a potential novel cancer therapy target and checkpoint regulation. Drug Des. Devel. Ther. 4, 255 -264 (2008).

2. Larasati & Duncker, B. P. Mechanisms Governing DDK Regulation of the Initiation of DNA Replication. Genes (Basel). 8, 3 (2016).

3. Huggett, M. T. et al. Cdc7 is a potent anti-cancer target in pancreatic cancer due to abrogation of the DNA origin activation checkpoint. Oncotarget 7, 18495 -18507 (2016).

4. Bonte, D. et al. Cdc7-Dbf4 kinase overexpression in multiple cancers and tumor cell lines is correlated with p53 inactivation. Neoplasia 10, 920 -31 (2008).

5. Montagnoli, A. et al. Cdc7 Inhibition Reveals a p53-Dependent Replication Checkpoint That Is Defective in Cancer Cells. Cancer Res. 64, 7110 -7116 (2004).

2001501005000

100

200

300

400

500

600

700

Time (min)

DMSOStaurosporineSRA141

Prod

uct (

nM)

1000100

SRA141 Concentration (nM)

1 h pre-incubation

Cdc7/Dbf4

1010.1

Perc

ent

Disp

lace

men

t

0

20

40

60

80

100

120

+ = IC50 > 33 μM

454035302520

10

20

0

-10

4000

10000

8000

6000

4000

2000

0

3000

2000

1000Complete tumor regressions achieved in 4/7 (57%) rats.

00 5 10 15 20 25 30 35 40 45

-20151050

Body

Wt C

hang

e (%

)fr

om B

asel

ine

Tum

or V

olum

e (m

m3 )

Tum

or V

olum

e (m

m3 )

Study sponsored by Sierra Oncology. For more information, email info@sierraoncology.com or visit www.sierraoncology.com

Contact

SRA141 (75 mg/kg) BID

SRA141 (75 mg/kg) BID, 5 on, 2 off, p.o.

Vehicle

Vehicle

00

100

1000

2000

6 12 18 24Time (h)

100 mg/kg150 mg/kg

50 mg/kg

0 5 10 15 20 25 30 35

144 h

CyQuantCTG

0.1

1

10

100

24 h

48 h

72 h

SRA

141

IC50

(µM

)

Colo-205

Colo-205

SW620

SW620

NCI-H716

NCI-H716

SW1116

SW1116

MDA-MB-231

MDA-MB-213

A

SRA

141

IC50

(µM

)

Colo-205

Colo-205

Colo-205

Colo-205

SW620

SW620

SW620

SW620

NCI-H716

NCI-H716

NCI-H716

NCI-H716

NCI-H1573

NCI-H1573

NCI-H1573

NCI-H1573

SW1116

SW1116

SW1116

SW1116

SNU-398

SNU-398

SNU-398

SNU-398

MDA-MB-2

31

MDA-MB-231

MDA-MB-2

31

MDA-MB-2

31

+ = IC50 > 33 μM

B

SRA

141

IC50

(µM

)

Colo-205

Colo-205

SW620

SW620

NCI-H716

NCI-H716

NCI-H1573

NCI-H1573

SNU-398

SNU-398

MDA-MB-231

MDA-MB-231

0.1

1

10

100

72 hr144 hr

SRA141 @ 500nm TAK-931 @ 500nm [SR

A14

1] pl

asm

a (n

g/m

L)