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Pharmacological Profile of BI 847325, an Orally Bioavailable, ATP-competitive Inhibitor of MEK and Aurora Kinases

Patrizia Sini*1, Ulrich Gürtler4, Stephan K Zahn2, Christoph Baumann1, Dorothea Rudolph1, Rosa

Baumgartinger1, Eva Strauss1, Christian Haslinger1, Ulrike Tontsch-Grunt1 , Irene C Waizenegger1,

Flavio Solca1, Gerd Bader2, Andreas Zoephel2, Matthias Treu2, Ulrich Reiser2, Pilar Garin-Chesa1,

Guido Boehmelt5, Norbert Kraut1, Jens Quant3 and Günther R Adolf1

Departments of 1Pharmacology and Translational Research, 2Medicinal Chemistry, 3Research ADME, 5Research Networking Boehringer Ingelheim RCV GmbH & Co KG, Vienna, Austria

Department of 4R&D Project Management, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach,

Germany

Running title: An ATP-competitive inhibitor of MEK/Aurora kinases

Keywords: BRAF mutation, KRAS mutation, MEK inhibitor, Aurora kinase inhibitor

Corresponding Author: Patrizia Sini, Ph.D., Boehringer Ingelheim RCV GmbH & Co KG, Dr.

Boehringer-Gasse 5-11, A-1121 Vienna, Austria; phone: +43 (1) 80105-2936, Fax: +43 (1) 8040823,

E-mail: [email protected]

Disclosure of potential conflict of interest: All authors are employees of Boehringer Ingelheim

on December 17, 2020. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 5, 2016; DOI: 10.1158/1535-7163.MCT-16-0066

http://mct.aacrjournals.org/

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Abstract Although the MAPK pathway is frequently deregulated in cancer, inhibitors targeting RAF or MEK

have so far shown clinical activity only in BRAF- and NRAS-mutant melanoma. Improvements in

efficacy may be possible by combining inhibition of mitogenic signal transduction with inhibition of

cell cycle progression. We have studied the preclinical pharmacology of BI 847325, an ATP-

competitive dual inhibitor of MEK and Aurora kinases (AK).

Potent inhibition of MEK1/2 and Aurora A/B kinases by BI 847325 was demonstrated in enzymatic

and cellular assays. Equipotent effects were observed in BRAF-mutant cells, while in KRAS-mutant

cells, MEK inhibition required higher concentrations than AK inhibition. Daily oral administration of

BI 847325 at 10 mg/kg showed efficacy in both BRAF- and KRAS-mutant xenograft models.

Biomarker analysis suggested that this effect was primarily due to inhibition of MEK in BRAF mutant

models, but of AK in KRAS-mutant models. Inhibition of both MEK and AK in KRAS-mutant tumors

was observed when BI 847325 was administered once weekly at 70 mg/kg.

Our studies indicate that BI 847325 is effective in in vitro and in vivo models of cancers with BRAF

and KRAS mutation. These preclinical data are discussed in the light of the results of a recently

completed clinical phase I trial assessing safety, tolerability, pharmacokinetics and efficacy of BI

847325 in cancer patients.




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Introduction

Activation of the mitogen-activated protein kinase (MAPK) signaling pathway plays a key role in the

regulation of diverse cellular functions in normal, healthy tissues, including the induction of cell

proliferation. Aberrant pathway activation is frequently observed in human malignancies as a result

of gain-of-function mutations in receptor tyrosine kinases, RAS or RAF (1). These signal-transducing

elements are therefore potentially important therapeutic targets for pharmacological intervention in

multiple types of cancer. To date, numerous attempts to directly inhibit mutant KRAS, one of the

most frequent oncogenic drivers, have not been able to generate compounds with drug-like

properties required for clinical development. Inhibition of downstream transducers, such as the

MAP-ERK kinase (MEK), appeared technically more feasible. Indeed, multiple compounds inhibiting

MEK with high potency and selectivity were synthesized and many have entered clinical

development; interestingly, in contrast to the vast majority of kinase inhibitors, these compounds

were designed as allosteric inhibitors rather than ATP pocket binders. The first compound

administered to cancer patients, CI-1040, provided preliminary evidence of activity in a phase I

study, but development was halted due to lack of efficacy in a subsequent trial (2,3). In general, with

few exceptions, single-agent activity of MEK inhibitors administered to patients with solid tumors

has been disappointing (4,5). Meaningful clinical activity superior to that of chemotherapy was

observed for the allosteric MEK-1/2 inhibitor, trametinib (GSK 1120212), in patients with BRAF-

mutant melanomas (6,7,8); this compound has consequently been approved by regulatory

authorities. Nevertheless, the low rate of objective responses (22%) and short duration of

progression-free survival (4.8 months) clearly indicated that further improvements were required.

More recently, it was shown that dual blockade of the MAPK pathway by concomitant

administration of a MEK inhibitor and a BRAF inhibitor (trametinib plus dabrafenib, cobimetinib plus

vemurafenib) delays the emergence of resistance to single-agent therapy in patients with BRAF-

mutant melanoma, resulting in a higher response rate, improved progression-free survival and

overall survival rate compared to the respective BRAF inhibitor administered as a single agent; both




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drug combinations have been approved by the FDA and other regulatory authorities. Nevertheless,

in view of progression-free survival of less than 1 year for both drug combinations, additional efforts

to optimize signal transduction inhibitor therapy are clearly warranted (9,10,11,12).

In humans, three homologous Aurora kinases (AK-A, -B and -C) have overlapping effects on the cell

cycle, in particular cytokinesis; however, there is also evidence of functions specific for individual

isoenzymes. AK-A has been implicated in mitotic entry, separation of centriole pairs, bipolar spindle

assembly and alignment of metaphase chromosomes. AK-B is involved in chromosomal bi-

orientation, regulating the association between kinetochores and microtubules, and phosphorylates

histone H3, which aids chromatin condensation and separation. Several selective AK inhibitors have

entered clinical development; whereas single-agent efficacy was generally disappointing in solid

tumor indications, encouraging activity has been observed in hematological malignancies (13, 14).

Several preclinical studies indicate that concomitant inhibition of MEK and Aurora kinases may

provide superior outcomes compared to single-agent therapy. For example, expression of AK-B was

shown to be upregulated by MEK signaling in melanoma as well as in carcinoma cell lines (15, 16).

Combination therapy with AK and MEK inhibitors would therefore represent a rational approach to

effective pathway blockade by reducing expression of AK-B and inhibiting catalytic activity of

residual enzyme; indeed, in a mouse xenograft model of KRAS-mutant human non-small cell lung

cancer (Calu-6), combination treatment with an AK-B inhibitor (barasertib) and a MEK inhibitor

(selumetinib) resulted in improved efficacy compared with single-agent therapy (17). Moreover,

combination of allosteric MEK inhibitors with an AK-A inhibitor has shown promising activity in

preclinical models. In conventional cell culture as well as in a human skin reconstruction model of

human melanoma (BRAF-mutant A375 cells), cell proliferation and tumor growth, respectively, were

more effectively inhibited when an AK-A inhibitor (alisertib) was added to either a MEK inhibitor

(trametinib) or a BRAF-inhibitor (dabrafenib); the triple-drug combination showed the highest

efficacy (18). In several human carcinoma xenografts in immunodeficient mice, a combination of a

MEK inhibitor (TAK-733) and an AK-A inhibitor (alisertib), both administered at single-agent MTD,




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was tolerated and resulted in additive to synergistic efficacy (19). Similarly, synergistic activity of an

alisertib – TAK-733 (or trametinib) combination was observed in colorectal carcinoma cell lines with

KRAS/PI3KCA3 double mutations in vitro and in a double mutant xenograft model in vivo (20).

We set out to explore the potential of simultaneous inhibition of MEK and AK in tumors with BRAF-

or RAS-mutation by means of a novel, dual-acting compound, the indolinone derivative BI 847325.




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Materials and Methods

Compounds

Compounds were synthesized and/or formulated according to published procedures: BI 847325

(21); trametinib (GSK 1120212; 22) selumetinib (AZD 6244; 23). The structure of the AK-B inhibitor

BI 811283, a 2,3-diamino-pyrimidine, is reported in Supplementary Fig. 1.

X-ray crystallography

The coordinates and structure factors for the following kinase / inhibitor complexes have been

deposited at the PDB: Aurora B / BI 811283 (PDB ID 5K3Y), Aurora B / BI 847325 (PDB ID 5EYK) and

MEK1 / BI 847325 (PDB ID 5EYM). All crystallographic work for the MEK 1 complex structure was

performed by Proteros Biostructures (Martinsried, Germany).

Enzyme assays

AK-B inhibition was assessed using a wild-type Xenopus laevis Aurora B60-361/INCENP790-847 complex

(24). Assays were run in the presence of 100 µmol/L ATP using 10 µmol/L of substrate (biotin-

LRRWSLGLRRWSLGLRRWSLGLRRWSLG). 30 µL PROTEIN-MIX (166 µmol/L ATP, kinase buffer [50

mmol/L Tris/HCl pH 7.5, 25 mmol/L MgCl2, 25 mmol/L NaCl], 10 ng enzyme complex) was added to

10 µL compound solution (serial dilutions, duplicates) in 25% DMSO and incubated for 15 min at RT.

10 µL PEPTIDE-MIX (2x kinase buffer, 5 mmol/L NaF, 5 mmol/L DTT, 1 µCi 33P-ATP, 50 µmol/L

peptide) was added, the mixture was incubated for 60 min at RT and stopped by adding 180 µL 6.4%

TCA (final concentration: 5%). Incorporated phosphate was measured in a scintillation counter and

IC50 values were calculated using a sigmoidal curve analysis program (GraphPad Prism 3.0) with

variable hill slope. AK-A, AK-C and MEK enzyme assays were performed at Invitrogen (SelectScreen®;

Life Technologies). Assays for additional kinases were also performed at Invitrogen.




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Cell Lines

The human cell lines A375, Calu-6, SK-MEL-28, G-361, Colo205, SK-MEL-2, PANC-1, MIA PaCa-2,

AsPC-1, LoVo, HCT 116, DLD-1, A549 and NCI-H460 were obtained from ATCC and were cultured

according to the provider’s instruction. The human cell lines HuP-T4, MEL-JUSO and IPC 298 were

obtained from German Collection of Microorganisms and Cell Cultures (DMSZ) and were cultured

according to the provider’s instruction. The human cell line KP-2 was obtained from the Japanese

Collection of Research Bioresources (JCRB).

All of the cell lines were authenticated by STR profiling at ATCC and JCRB or by multiplex PCR of

minisatellite markers at DMSZ.

The BRO cell line was a kind gift from Prof. Piet Borst (Amsterdam Free University) in 1995, STR data

are from 09.09.2014, no published reference STR profile is available.

Proliferation assay

Cells were plated in 96-well format in recommended growth medium and BI 847325 was added 24

hours after cell seeding. At the same time, a “time zero” untreated cell plate was fixed. Compound

was serially diluted and assayed over 8 concentrations in triplicates. After 72 h incubation, cells were

fixed and stained with fluorescent nuclear dye (CyQuant Direct Cell Proliferation Assay, Invitrogen

Cat. No. C35012). Concentration–response curves were analyzed using a four-parameter log-logistic

function without upper or lower limitation. Half-maximal growth inhibitory concentrations (GI50)

were calculated as the concentration of test drug that provokes a response halfway between the t =

0 h and t = 72 h values.

Immunoblotting of cell and tumor lysates

Lysates were prepared from cells treated with compounds for 24 h or snap-frozen tumor tissues.

Immunoblots were performed using the following primary antibodies: anti-ERK1/2, anti-phospho-

ERK1/2, anti-MEK 1/2, anti-phospho-MEK-1/2 (Ser217/221), anti-histone H3, anti-Bim (all from Cell




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Signaling Technologies), anti-MEK1 (BD Transduction Lab), anti-phospho histone H3 (Ser10;

Millipore), anti-Cyclin D1 (Biocare Medical), anti-p27 (Leica Mikrosysteme), and anti-ß-actin (Abcam).

Secondary antibodies included HRPO-conjugated goat anti-mouse and anti-rabbit IgG (Dako

Cytomation).

Cell cycle / DNA content analysis

Cells were treated with compounds alone or in combination for 48 h. After fixing, permeabilization

and washing using standard protocols, cells were incubated with 5 µg/mL Hoechst 33342 dye for 1 h

at RT in the dark. Cell cycle analysis was carried out in a Cellomics® ArrayScan® VTI HCS Reader

(Thermo Scientific) applying the Cell Cycle BioApplication program that classifies cells into their cell

cycle phase based on the total nuclear intensity of a DNA binding dye.

Measurement of phospho-ERK and phospho-MEK levels in cell and tumor lysates

Cells were treated with BI 847325 or GSK 1120212 for 24 h. Cell lysates were prepared according to

the MesoScale Discovery protocol and clarified at 15,000 rpm for 30 min at 4°C. Snap-frozen tumor

tissues were homogenized and lysed on ice with buffer containing 20 mmol/L Tris pH 7.5, 150

mmol/L NaCl, 1 mM EDTA, 1 mmol/L EGTA, 10 mmol/L NaF, 1% Triton X-100, supplemented with

protease (Roche) and phosphatase inhibitor cocktails (SIGMA). Homogenates were centrifuged at

15,000 rpm for 30 min at 4°C. Clarified lysates were analyzed with duplex ELISA (MesoScale

Discovery) measuring total and phospho-MEK 1/2 and total and phospho-ERK 1/2 as per

manufacturer’s instructions. In the graphical representations, data was plotted as average with

standard error of the mean (SEM).

Efficacy studies in mouse xenograft models

8-10 week old female BomTac:NMRI-Foxn1nu mice (TaconicArtemis) were grafted subcutaneously

with 5x106 A375 human melanoma cells (ATCC CRL-1619) or 1x106 Calu-6 NSCLC cells (ATCC HTB-56).




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When tumors reached a volume of 100 and 400 mm3, respectively, animals were randomized into

treatment groups of 7 and control groups of 10 mice each. BI 847325 was newly formulated every

third day. The compound was dissolved in 0.5% Natrosol 250 HX (hydroxyethyl-cellulose 250 HX)

with 3% Tween 80 and sonicated until a homogenous suspension was obtained, then 1 mol/L HCl

was added and the suspension was vortexed and sonicated again. MEK inhibitors GSK 1120212 and

AZD 6244 were suspended in 1% or 0.5% Natrosol, respectively. An administration volume of 10

ml/kg body weight was used and compounds were administered orally with a gavage needle at the

indicated dose and schedule. BI 811283 was formulated in 25% HP-β-CD, the pH was adjusted to 7

with 1 mol/L NaOH and the compound was administered weekly using a subcutaneously implanted

Alzet osmotic infusion pump. Tumor volumes were measured three times a week and results were

converted to tumor volumes (mm3) according to the formula“length x width2 x π/6”. Mice were

inspected daily for clinical signs and body weight was determined daily. Data was plotted as average

with standard error of the mean (SEM). Statistical significance (p values) was determined using the

Sidak-Bonferroni method, with alpha=0.05.

Pharmacokinetic analysis

PK studies were carried out on the last day of treatment using tumor-bearing female BomTac:NMRI-

Foxn1nu mice (Taconic) from the xenograft studies. EDTA plasma was prepared by centrifugation of

the collected blood at 10,000 rpm at 4°C. Plasma samples were added to an internal standard and

0.1 mol/L NaOH. BI 847325 was extracted by liquid–liquid extraction using ethyl acetate. The

resulting solution was dried in a warm nitrogen stream and dissolved in 25% methanol/0.1% formic

acid. BI 847325 was quantified by high-performance liquid chromatography/tandem mass

spectrometry (Agilent 1100, Applied Biosystems API3000™, ESI+) with a solvent gradient of 95% A to

10% A in 1.5 min (A: 5 mmol/L ammonium acetate [pH 4.0]; B: acetonitrile with 0.1% formic acid

[Luna C18 3µ 2 x 30]).




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Immunohistochemical staining

Analysis was carried out in formalin-fixed, paraffin-embedded xenograft tumor samples using the

EnVision system (Dako) to measure phospho-ERK1/2 and phospho-histone H3. Diaminobenzidine

was used as a substrate for the immunoreaction and the sections were counterstained with

hematoxylin. Rabbit monoclonal antibodies to phospho-ERK1/2 and phospho-histone H3 (Ser10)

were obtained from Cell Signaling Technologies.




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Results

Chemical structure and target binding mode

BI 847325, a 6-alkylindolinone derivative (21), was discovered in a lead optimization program initially

aiming at the discovery of highly potent AK-B inhibitors and selected for further characterization

based on its distinctive kinase inhibition profile (see below). The target binding mode was

investigated by X-ray crystallography with suitable kinase domain constructs for MEK1 and a

Xenopus laevis Aurora B/INCENP complex. The sequence of X. laevis Aurora B is closely related to

that of its human ortholog (80% identity, 92% homology within the kinase domain with no critical

differences in the ATP-binding site), and therefore was considered to represent a valid surrogate for

structure-based drug design. The co-crystal structures show that BI 847325 is located in the ATP

binding pocket in the hinge region of both kinases and thus acts as an ATP-mimetic. The indolinone

scaffold itself serves as the hinge binding motif; the linear and rigid alkinyl moiety points towards the

inside of the kinases in the direction of the gate-keeper residue (Fig. 1A and B; Supplementary Tables

S1 and S2). The selective AK-B inhibitor BI 811283, a 2,3-diamino-pyrimidine, binds in a similar way

to the hinge region and also acts as a competitive ATP-mimetic (Fig. 1C, Supplementary Table S3).

Potency, selectivity and pathway inhibition in cancer cells BI 847325 inhibited the activity of X. laevis AK-B with an IC50 of 3 nmol/L; the IC50 values for human

AK-A and AK-C were 25 and 15 nmol/L, respectively. A good correlation was observed between the

activity of several inhibitors on X. laevis and human AK-B (data not shown), permitting use of this

assay to determine BI 847325 activity. BI 847325 also inhibited human MEK1 and MEK2 with

respective IC50 values of 25 and 4 nmol/L. In a panel of 29 additional kinases selected to represent

the diversity of the kinome tree, BI 847325 at 1,000 nmol/L inhibited 6 enzymes by more than 50%

(LCK, MAP3K8, FGFR1, AMPK, CAMK1D and TBK1). Subsequent analyses revealed that the IC50 values

were below 100 nmol/L only for LCK (5 nM) and MAP3K8 (93 nmol/L).




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The effects of BI 847325 on target activity in a cellular environment were investigated in a BRAFV600E

mutant melanoma cell line (A375) and a KRASQ61K mutant NSCLC cell line (Calu-6). Proliferation was

inhibited in both cell lines with GI50 values of 7.5 nmol/L and 60 nmol/L, respectively. Western blot

analysis indicated that in A375 cells, BI 847325 potently reduced the concentration of phospho-ERK,

an indicator of MAPK pathway activity (partial to complete inhibition at 10-30 nmol/L), whereas

about 10-fold higher concentrations were required in Calu-6 cells (Fig. 2A). Histone H3

phosphorylation was inhibited at 10 nmol/L in A375 cells and at about 3-fold higher concentration in

Calu-6 cells. MEK phosphorylation in A375 cells was potently but not fully inhibited as well, and to a

lesser extent and at higher concentrations in Calu-6 cells. Interestingly, MEK protein expression was

partially down-regulated in both cell lines at concentrations of 100 nmol/L and higher, whereas ERK

protein expression was not affected.

Independent assays performed using quantitative electrochemoluminescence technology confirmed

these results (Fig. 2B). In these experiments, the allosteric MEK inhibitor GSK 1120212 (trametinib)

was included for comparison. This compound potently inhibited ERK phosphorylation in both cell

lines; MEK phosphorylation was likewise decreased in A375 cells but increased 2-3 fold in Calu-6

cells. In both cell lines, MEK protein levels were markedly reduced by BI 847325 (~90%), but only

moderately down-regulated by GSK 1120212 (~40%).

The effects of BI 847325 on MEK and ERK were then analyzed by Western blot assay across a wider

panel of cell lines with mutations in BRAF, NRAS or KRAS (Supplementary Figs. S2-S4). Modulation of

ERK1/2 phosphorylation was observed in all cell lines tested; higher concentrations (>=100 nmol/L)

were required to strongly down-regulate phospho-ERK1/2 in the majority of KRAS mutant cell lines

compared to BRAF or NRAS mutant cell lines (<100 nmol/L). In 3/3 BRAFV600E-mutant cell lines

(melanoma, colon carcinoma), inhibition of MEK phosphorylation was observed at concentrations

well below 100 nmol/L and a decrease in total MEK1 levels was seen at and above 100 nmol/L.

Inhibition of MEK phosphorylation was also observed in 4/4 NRAS mutant melanoma cell lines but in

only 2/10 KRAS-mutant colon, lung and pancreas carcinoma lines (KP-2, HCT-116). Interestingly,




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increased phospho-MEK (but not phospho-ERK) was observed at high concentrations (1000 nmol/L)

in one NRAS- and several KRAS-mutant cell lines, most clearly visible in MIA PaCa-2 and A-549 cells.

Effect of BI 847325 on cell cycle progression

Changes in the cell cycle profile induced by treatment with BI 847325 for 48 hours were analyzed by

quantitative imaging of cellular DNA content, in comparison with effects induced by GSK 1120212

and BI 811283, a selective AK inhibitor (25). BRAF-mutant A375 cells treated with GSK 1120212 were

apparently arrested in G1 phase, whereas multinucleated and/or polyploid cells accumulated in

cultures treated with BI 811283 (Fig. 3). In contrast, cells treated with BI 847325 at low

concentration (50 nmol/L) showed a distinct profile, with a higher percentage of cells in G2/M and

polyploid/multinucleated states and lower proportion in G1 phase compared with GSK 1120212

alone; at high concentrations (500 nmol/L), the proportion of cells in G1 decreased further whereas

that in G2/M and polyploid/multinucleated states increased. Interestingly, this profile could be

mimicked by treatment with a combination of GSK 1120212 and BI 811283. In KRAS-mutant Calu-6

cells, BI 847325 at 50 nmol/L resulted in accumulation of cells with DNA content >4N, very similar to

the profile of cells treated with BI 811283, whereas at 500 nmol/L the characteristic pattern of dual

inhibition was seen, although the proportion of cells with sub-G1 DNA content, presumably

reflecting cells undergoing apoptosis, increased. Again, a combination of GSK 1120212 and BI

811283 mimicked the effects of BI 847325. These data confirm that BI 847325 acts as a dual MEK

and AK inhibitor in these BRAF- and KRAS-mutant cell lines, although higher concentrations are

required for dual target inhibition in KRAS-mutant cells.

Efficacy in mouse xenograft models

Preliminary studies in female NMRI nude mice had shown that the maximally tolerated dose of BI

847325 upon daily oral administration for 2 weeks is 12 mg/kg; higher doses resulted in




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unacceptable weight loss (data not shown). Daily oral doses of 10 mg/kg were well tolerated for at

least 6 weeks and were therefore used for further studies.

Pharmacokinetic and pharmacodynamic parameters were initially investigated in the A375

melanoma (BRAFV600E) model (Fig. 4). After 10 days of daily treatment at 10 mg/kg,

electrochemoluminescence assays indicated marked inhibition of MEK and ERK phosphorylation in

tumors 2 hours after the last dosing; this was sustained for at least 24 hours post-dosing, when BI

847325 plasma levels were already below the limit of detection (Fig. 4A and 4B).

Immunohistochemistry confirmed reduction of phospho-ERK and phospho-HH3 levels (Fig. 4C) and

rapid induction of the pro-apoptotic protein Bim following compound administration was observed

by Western blot analysis of tumor lysates (Fig. 4D).

In a second, independent experiment, BI 847325 induced gradual regression of A375 tumors that

was sustained for the entire 4-week treatment period (Fig. 5A). In contrast, treatment with an

allosteric MEK inhibitor (AZD 6244, selumetinib) or a selective Aurora kinase inhibitor (BI 811283),

both administered at the respective maximally tolerated dose, resulted in initial tumor stasis but

tumors resumed growth after about two weeks in spite of continuous treatment (adjusted p values:

BI 847325 vs AZD 6244, p<0.0001; BI 847325 vs BI 811283, p<0.0001).

In the Calu-6 model, equivalent, sustained tumor stasis was observed upon treatment with either BI

847325 or the MEK inhibitor, GSK 1120212 (Fig. 5B) (adjusted p value: BI 847325 vs GSK 1120212

p=0.189). In both experiments, tumors were excised following the final dose to assess the effects of

the compounds on pathway modulation. Treatment with BI 847325 resulted in a profound decrease

of phospho-HH3 positive cells in both models, whereas reduction in phospho-ERK was observed only

in BRAF-mutant A375 xenografts. Large, multinucleated cells were observed in Calu-6 sections (Fig.

5C).

We subsequently compared the efficacy of BI 847325 and selective MEK inhibitors in a model of

KRASG12C-mutant pancreatic adenocarcinoma (MIA PaCa-2; Supplementary Fig. S5). Treatment with

BI 847325 at 10 mg/kg daily p.o. resulted in gradual tumor regression that was sustained for at least




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6 weeks. In contrast, initial regression induced by AZD 6244 or GSK 1120212 was followed by tumor

regrowth (adjusted p values: BI 847325 vs AZD 6244, p<0.0001; BI 847325 vs GSK 1120212,

p<0.0001). PD biomarker analysis following treatment suggested that inhibition of tumor growth by

BI 847325 was primarily associated with inhibition of AK rather than MEK (data not shown).

Cmax values of ~500 nmol/L BI 847325 were observed at doses that showed efficacy in both BRAF-

and KRAS-mutant xenograft models (10 mg/kg daily). As our in vitro results indicate that

concentrations of 10-30 nmol/L are sufficient to inhibit ERK phosphorylation in BRAF-mutant A375

cells, but at least 100 nmol/L are required in KRAS-mutant Calu-6 cells (Fig. 2), the failure of BI

847325 to inhibit ERK phosphorylation in KRAS-mutant tumor xenografts may reflect insufficient

drug exposure. Alternative dosing schedules were therefore evaluated (Fig. 6). When BI 847325 was

administered at 70 mg/kg once weekly, the peak plasma concentration was 2,700 nmol/L compared

to 380 nmol/L after a dose of 10 mg/kg daily; 24 h after dosing, plasma concentrations were 460

nmol/L compared to 13 nmol/L. Dosing at 70 mg/kg once weekly resulted in regression of Calu-6

tumors in 6/7 animals (Fig. 6A and 6B), whereas the daily treatment schedule (10 mg/kg), in spite of

delivering the same weekly dose, was less efficacious (1/7 tumors regressing). The more pronounced

efficacy of the high-dose regimen was associated with stronger MEK pathway suppression (Fig. 6C,

6D and 6E); reduction of phospho-MEK and phospho-ERK levels in tumors was evident for at least 48

h (Fig. 6C). Induction of Bim expression was observed at the high dose level (Fig. 6E). An additional

dosing schedule of BI 847325 was tested (15 mg/kg 3 days on, 4 days off); the data on tumor volume

and biomarker modulation demonstrate an intermediate effect between the daily and the weekly

schedule (Fig. 6). GSK 1120212 administered daily initially induced tumor regression, whereas

prolonged treatment was associated with tumor re-growth (Fig. 6A,B) (adjusted p values: BI 847325

at 10 mg/kg vs GSK 1120212, p=0.189; BI 847325 at 15 mg/kg vs GSK 1120212, p= 0.016; BI 847325

at 70 mg/kg vs GSK 1120212, p=0.002) and MEK or ERK phosphorylation in these progressing

tumors, measured 24 hours and 48 hours after the last dose, was not different from that in controls

(data not shown).




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Discussion

BI 847325 differs in two major aspects from multiple MEK inhibitors that were extensively profiled in

preclinical studies and have progressed to clinical development. First, allosteric inhibitors such as

trametinib, cobimetinib and selumetinib block MEK activity by binding to sites distant from the

catalytic center, whereas BI 847325 competes with ATP for binding to the catalytic site; these

differences are likely to impact on protein complex formation (e.g., MEK homodimers, MEK-RAF

heterodimers) and may also affect the stability of the target. A recent report has pointed to an

additional distinction, as ATP-competitive inhibitors may not be subject to one of the drug resistance

mechanisms observed in melanoma patients: a secondary mutation in MEK1 renders cells resistant

to both BRAF and allosteric MEK inhibitors (26). Second, BI 847325 also inhibits the activity of Aurora

kinases and thereby directly interferes with cell cycle progression. BI 847325 thus potentially

operates through two parallel mechanisms of action, and the relative impact of these in the cell type

under investigation will depend on its genetic background (e.g., BRAF, NRAS or KRAS mutation).

To explore the preclinical pharmacology of BI 847325 we initially focused on the A375 model of

BRAFV600E-mutant melanoma characterized by strong MAPK pathway activation (high levels of

phospho-MEK and phospho-ERK). BI 847325 fully inhibited ERK phosphorylation at low

concentrations (< 100 nmol/L), and likewise inhibited phosphorylation of histone H3, a direct

substrate of Aurora B, in the same concentration range. Interestingly, BI 847325 also potently

inhibited the phosphorylation of MEK and moreover partially reduced the concentration of MEK

protein; this effect may be a consequence of conformational changes induced by compound binding

and subsequent degradation. Treatment of A375 cells with BI 847325 resulted in a distinctive cell

cycle profile, i.e. a DNA content distribution with features of both MEK and Aurora B inhibition,

which could be mimicked by a combination of MEK- and Aurora-selective kinase inhibitors. In nude

mice bearing subcutaneous A375 tumor xenografts, treatment with BI 847325 induced gradual

tumor regression continuing over a period of at least 4 weeks, whereas the reference MEK and

Aurora inhibitors resulted in initial tumor stasis but eventual regrowth in spite of continuous




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treatment, reminiscent of the clinical situation in melanoma patients treated with BRAF and/or MEK

inhibitors.

Additional BRAFV600-mutant cell lines were studied by Western blot analysis only; two further

melanoma cell lines as well as one colon carcinoma line were as sensitive to BI 847325 as A375 in

terms of suppression of the phospho-ERK/MEK signals as well as partial reduction in total MEK

protein levels. We next investigated the effects of BI 847325 on MAPK pathway activity in four

NRAS-mutant melanoma cell lines and observed inhibition of ERK phosphorylation at concentrations

< 100 nmol/L in all of these. BRAF- as well as NRAS-mutant cancers may therefore be of primary

interest for clinical development of BI 847325. A recent, independent study of BI 847325 in several

well-characterized BRAF-mutant melanoma models has confirmed that this compound shows

efficacy in vitro as well as in vivo; importantly, BRAF inhibitor-naïve as well as resistant models were

sensitive (21). With respect to the mechanism of action, this study has confirmed that BI 847325

down-regulates MEK protein expression and provides evidence that suppression of Mcl-1 and

upregulation of Bim are associated with efficacy.

Across all cancer types, mutations in KRAS are much more frequent drivers of malignant growth than

BRAF or NRAS mutations; however, allosteric MEK inhibitors have so far failed to show efficacy in

late-stage clinical trials. We have initially characterized the activity of BI 847325 using the KRASQ61K

mutant NSCLC cell line Calu-6 as a model. Although ERK phosphorylation in these cells was fully

inhibited by BI 847325, higher concentrations (≥ 100 nmol/L) were clearly required than in

BRAF/NRAS mutant cells. In contrast, histone H3 phosphorylation was inhibited at concentrations

<100 nmol/L, as in A375 cells. The cell cycle profile of cells treated with BI 847325 at low

concentrations (50 nmol/L) indicated accumulation of cells in G2/M and polyploid/multinucleated

states, similar as in cells treated with a selective Aurora B inhibitor; at high concentrations (500

nmol/L), the distribution shifted towards a higher proportion of cells with sub-G1 DNA content,

indicative of induction of apoptosis. In a nude mouse xenograft study, BI 847325 inhibited Calu-6

tumor growth, but did not induce tumor shrinkage; similar tumor growth kinetics were observed in




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mice treated with an allosteric MEK inhibitor. In BI 847325-treated tumors, phospho-ERK staining

was hardly affected; histological examination revealed an accumulation of large, multinucleated

cells, characteristic for cells treated with an Aurora B kinase inhibitor. Thus, in this in vivo model, BI

847325 acted like an Aurora B kinase inhibitor, without evidence for a contribution of MEK inhibition

to the overall outcome, in agreement with the lower sensitivity of Calu-6 cells to MAPK pathway

inhibition in vitro. As dose escalation in the q.d. schedule to increase plasma and tumor

concentrations was not an option due to tolerability limitations we explored alternative schedules

and found that a dose of 70 mg/kg given once weekly, delivering the same total weekly dose as 10

mg/kg q.d., was tolerated by the animals for several weeks. In the Calu-6 model, this dose/schedule

resulted in a stronger suppression of phospho-ERK/MEK levels, a clear induction of Bim, and

importantly, in superior efficacy in terms of tumor shrinkage compared with daily dosing at 10

mg/kg, and was also superior to daily dosing with GSK 1120212. As kinase inhibitors in general are

dosed daily or twice daily to achieve continuous suppression of target activity, it may be surprising to

observe that intermittent dosing can achieve superior outcomes. However, these findings are

consistent with earlier studies using imatinib or dasatinib in BCR-ABL driven CML cell lines as well as

erlotinib in mutant EGFR-driven lung cancer cell lines (27): short-term treatment (as short as 20

minutes) at high inhibitor concentrations conferred similar cytotoxicity as long-term treatment at

low concentrations. Importantly, short-term treatment at high concentrations resulted in

irreversible induction of Bim protein, whereas Bim induction following treatment at low

concentrations for at least several hours was reversible.

To broaden the database for KRAS-mutant tumors, we tested a panel of 10 additional KRAS-mutant

pancreatic and colorectal carcinoma cell lines for sensitivity to MAPK pathway inhibition by BI

847325 and observed a broad spectrum of outcomes. Whereas a few cell lines were as sensitive as

BRAF/NRAS mutant cells lines in terms of ERK inhibition, the majority, such as Calu-6, were less

sensitive. Heterogeneous sensitivity to allosteric MEK inhibitors of KRAS mutant cell lines has been




of 28

reported previously and it has been demonstrated that gene expression signatures are more reliable

predictors of sensitivity than KRAS mutation status (28-30).

The results of a clinical phase I trial to determine safety, maximally tolerated dose (MTD) and

pharmacokinetics of BI 847325 in patients with refractory solid tumors have recently been published

(31). Two dosing schedules were studied: A, daily oral administration for 2 weeks, followed by a one-

week break; B, daily treatment for five days, followed by 2 days off drug, for 3 weeks. The MTDs

were defined as 120 mg/day for schedule A and 150 mg/day for schedule B; higher doses (150

mg/day for schedule A and 180 mg/day for schedule B) were tested but not tolerated. The safety

profile was considered to be acceptable, with reversible hematological (mainly neutropenia) and

gastrointestinal adverse events as the most common dose-limiting toxicities, consistent with earlier

studies of Aurora kinase inhibitors; MEK-inhibitor class effects such as visual disturbances or skin

rash were observed in only a small number of patients. Although patients were not selected for

tumors with activating mutations in BRAF, NRAS or KRAS, signs of efficacy were observed as follows:

(1) one patient with esophageal cancer (negative for RAS- and BRAF mutations) experienced a

confirmed partial response and a progression-free survival of 128 days; (2) 30% of patients achieved

stable disease, among them a patient with KRAS-mutant thymic carcinoma with PFS of 251 days and

four patients with PFS > 120 days.

Pharmacokinetic studies showed highly variable results even in the narrow dose range between 120

mg and 180 mg, with group mean cmax values from 12 nmol/L to 147 nmol/L and AUC values from 80

to 1200 nmol*h/L (Supplementary Table S4). For comparison, pharmacokinetic analysis of samples

from xenograft experiments in mice indicated a mean cmax of 377 nmol/L and an AUC of 3700

nmol*h/L (Supplementary Table S4) is fully effective. Considering differences in plasma protein

binding (free fraction in mouse plasma 1.2%, in human plasma 2.4%; data not shown), it seems that

humans are somewhat more sensitive to BI 847325 than mice. Nevertheless, the toxicity profile

characteristic for Aurora inhibitors and evidence of efficacy in several patients indicate that

engagement of at least the Aurora target has been achieved.




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Attempts to analyze pharmacodynamic effects of BI 847325 in skin (but not in tumor) biopsies did

not provide evidence for target inhibition. These results should be interpreted with caution: the

phospho-histone H3 marker used to evaluate Aurora kinase inhibition was not affected in a dose-

dependent manner, although the toxicity profile defining the MTD was clearly compatible with BI

847325 – mediated Aurora kinase inhibition in the bone marrow and gastrointestinal tract. The

negative results of phospho-ERK analysis in skin biopsies thus also appear to be of limited value.

Although the safety profile of BI 847325 was acceptable, the efficacy readout was not deemed

satisfactory and the low frequency of adverse events relating to MEK inhibition (skin rash, visual

disturbances) was taken as evidence that drug exposure was insufficient to achieve dual MEK-Aurora

pathway inhibition; further development of the compound was halted. However, the observation

that in normal tissues, BI 847325 acts primarily as an Aurora kinase inhibitor is not surprising in view

of our preclinical results: potent suppression of MAPK pathway activity (phospho-ERK biomarker)

was observed in all BRAF- and NRAS-mutant cancer cell lines (8/8), but in only 2 out of 11 cell lines

with wild-type BRAF and NRAS; efficacy in mouse xenograft models at 10 mg/kg q.d. was associated

with strong MAPK pathway inhibition in a BRAF-mutant model (A375), but was due mainly to Aurora

kinase inhibition in two KRAS-mutant models (Calu-6, MIA PaCa-2). If future trials of BI 847325 are

considered, these should therefore focus on patients with activating BRAF or NRAS mutations as

these display the strongest dependence on MEK signaling and thus the highest potential for

equipotent MEK and Aurora pathway inhibition in the tumors. Additional options for treating

patients with KRAS-mutant cancers will depend on the identification of robust, clinically feasible

biomarkers to select those most sensitive to MEK inhibition. Finally, clinical exploration of

alternative schedules allowing for higher doses administered for shorter periods of time (e.g. once

weekly) may offer further opportunities for improving treatment outcomes.




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Acknowledgements

We gratefully acknowledge the contributions of the entire BI 847325 research and development

team. In particular, we thank Matthew Kennedy, Sonja Porits, Astrid Jeschko, Ursula Strobl, Stefan

Fischer, Kai Zuckschwerdt, Irene Kothbauer, Janine Rippka, Susy Straubinger, Ines Kaupe, Martina

Scherer, Karin Bosch, Oliver Bergner, Nicole Budano, Christina Puri, Alexander Wlachos, Christoph

Albrecht, Reiner Meyer, Andreas Schrenk, Monika Leber for their excellent, dedicated contributions

to experiments described in this paper.




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Figure Legends

Figure 1. Crystal structure of BI 847325 and BI 811283 bound to Aurora B and of BI 847325 bound

to MEK1.

A, X-ray structure of Xenopus laevis Aurora B (green) in complex with INCENP (magenta) and BI

847325 bound to the ATP binding site. The structure was solved at a resolution of 1.93 Å.

B, X-ray structure of human MEK 1 (blue) in complex with BI 847325 solved at 2.7 Å resolution. The

orientation of the kinase domain is identical to that in Figure 1A.

C, X-ray structure of Xenopus laevis Aurora B (orange) in complex with INCENP (magenta) and BI

811283 bound to the ATP binding site.

Figure 2. Analysis of MAPK pathway inhibition by BI 847325.

A, Western blot analysis of pathway inhibition in BRAFV600E melanoma cells (A375) and KRASQ61K

NSCLC cells (Calu-6) treated with BI 847325 for 24 h;

B, Electrochemoluminescence assays for expression of MEK, phospho-MEK and phospho-ERK

expression in A375 and Calu-6 cells treated with BI 847325 or GSK 1120212 for 24 h. * two tailed p

value ≤ 0.05 using a student’s t- test.

Figure 3. DNA content analysis of A375 and Calu-6 cells treated with BI 847325, GSK 1120212 and

BI 811283.

Cell were treated with the indicated compounds for 48 h, fixed and stained with Hoechst 33342. The

DNA content analysis was performed using a Cellomics® ArrayScan® VTI HCS Reader and applying

the Cell Cycle BioApplication program.

Figure 4. Pharmacokinetic and pharmacodynamic profile of BI 847325 following oral

administration of 10 mg/kg for 10 days to mice bearing A375 (BRAFV600E) xenografts.




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A and B, MEK 1/2 phosphorylation and ERK 1/2 phosphorylation, respectively, as determined by

electrochemoluminescence assay; * two tailed p value ≤ 0.05 using a Student’s t- test.

C, ERK and histone H3 phosphorylation visualized by immunohistochemistry;

D, Bim expression detected by Western blot.

Figure 5. Efficacy of BI 847325 in nude mouse xenograft models.

BomTac:NMRI-Foxn1nu mice bearing human A375 (BRAFV600E) or Calu-6 (KRASQ61K) tumors were

treated with vehicle, BI 847325 at 10 mg/kg daily p.o., AZD 6244 at 25 mg/kg twice daily p.o., GSK

1120212 at 0.5 mg/kg daily p.o. or BI 811283 once weekly per subcutaneously implanted osmotic

mini-pump delivering 20 mg/kg during a period of 24 h.

A, Tumor growth kinetics of human melanoma A375 (BRAFV600E). Data is plotted as average tumor

volume with standard error of the mean (SEM). Adjusted p values: BI 847325 vs AZD 6244 p<0.0001

(statistically significant difference); BI 847325 vs BI 811283 p<0.0001 (statistically significant

difference).

B, Tumor growth kinetics of human NSCLC Calu-6 (KRASQ61K). Data is plotted as average tumor

volume with standard error of the mean (SEM). Adjusted p values: BI 847325 vs GSK 1120212

p=0.189 (not statistically significant).

C, 24h after the last oral administration of BI 847325, tumors were excised, fixed in formalin,

embedded in paraffin and stained with hematoxylin/eosin or antibodies to phospho-ERK or

phospho-HH3.

Figure 6. Efficacy of BI 847325 in a Calu-6 (KRASQ61K) xenograft model of human NSCLC.

BomTac:NMRI-Foxn1nu mice bearing Calu-6 (KRASQ61K) tumors were treated orally with vehicle, GSK

1120212 at 0.5 mg/kg daily BI 847325 at 10 mg/kg daily, or 15 mg/kg daily for three consecutive

days per week or 70 mg/kg once weekly.

A, Tumor growth kinetics. Data is plotted as average tumor volume with standard error of the mean




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(SEM). Adjusted p values: BI 847325 at 10 mg/kg vs GSK 1120212 p=0.189 (not statistically

significant); BI 847325 at 15 mg/kg vs GSK 1120212 p= 0.016 (not statistically significant); BI 847325

at 70 mg/kg vs GSK 1120212 p=0.002 (statistically significant difference)

B, Individual tumor volumes as percent change from baseline/prior treatment,

C and D, Drug plasma levels and phospho-marker expression determined by electrochemo-

luminescence assay and immunohistochemistry, respectively, 24 and 48 hours after the last dose.

* two tailed p value ≤ 0.05 using a Student’s t- test. E, Bim expression detected by Western blot;

each lane represents one tumor sample.




Sini et al. Figure 1

A B

C





A Calu-6 (KRASQ61K)

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B I 8 4 7 3 2 5 7 0 m g /k g q 7 d .

G S K 1 1 2 0 2 1 2 0 .5 m g /k g q .d .

Calu-6




Published OnlineFirst August 5, 2016.Mol Cancer Ther Patrizia Sini, Ulrich Gürtler, Stephan K Zahn, et al. ATP-competitive Inhibitor of MEK and Aurora KinasesPharmacological Profile of BI 847325, an Orally Bioavailable,

Updated version

10.1158/1535-7163.MCT-16-0066doi:

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