Preclinical Efficacy of Covalent-Allosteric AKT Inhibitor ... · 3/9/2019 · cancer types, highlighting the relevance of these pathways in cancer physiology and . 73. pathology

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Preclinical Efficacy of Covalent-Allosteric AKT Inhibitor Borussertib 1

in Combination with Trametinib in KRAS-mutant Pancreatic and 2

Colorectal Cancer 3

4

Jörn Weisner1,2,*, Ina Landel1,2,*, Christoph Reintjes3,*, Niklas Uhlenbrock1,2,*, Marija 5

Trajkovic-Arsic4,5, Niklas Dienstbier4,5, Julia Hardick1,2, Swetlana Ladigan3, Marius 6

Lindemann1,2, Steven Smith1,2, Lena Quambusch1,2, Rebekka Scheinpflug1,2, Laura 7

Depta1,2, Rajesh Gontla1,2, Anke Unger6, Heiko Müller6, Matthias Baumann6, Carsten 8

Schultz-Fademrecht6, Georgia Günther7, Abdelouahid Maghnouj3, Matthias P. 9

Müller1, Michael Pohl8, Christian Teschendorf9, Heiner Wolters10, Richard Viebahn11, 10

Andrea Tannapfel12, Waldemar Uhl13, Jan G. Hengstler7, Stephan A. Hahn3,§, Jens T. 11

Siveke4,5,§, and Daniel Rauh1,2,§ 12

* These authors contributed equally to this work. 13

14

1 Faculty of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn-15

Straße 4a, D-44227 Dortmund, Germany 16

2 Drug Discovery Hub Dortmund (DDHD) am Zentrum fur Integrierte 17

Wirkstoffforschung (ZIW), D-44227 Dortmund, Germany 18

3 Department of Molecular Gastrointestinal Oncology, Ruhr-University Bochum, 19

Universitätsstraße 150, D-44780 Bochum, Germany 20

4 German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), 21

Heidelberg, Germany 22

5 Division of Solid Tumor Translational Oncology, German Cancer Consortium 23

(DKTK), partner site Essen, West German Cancer Center, University Hospital Essen, 24

Hufelandstraße 55, D-45147 Essen, Germany 25

6 Lead Discovery Center GmbH, Otto-Hahn-Straße 15, D-44227 Dortmund, Germany 26

7 Leibniz Research Centre for Working Environment and Human Factors (IfADo), TU 27

Dortmund University, Ardeystraße 67, D-44139 Dortmund, Germany 28

8 Department of Internal Medicine, Ruhr University Bochum, 29

Knappschaftskrankenhaus, Bochum, Germany 30

9 Department of Internal Medicine, St. Josefs-Hospital, Dortmund, Germany 31

10 Department of Visceral and General Surgery, St. Josefs-Hospital, Dortmund, 32

Germany 33

11 Department of Surgery, Ruhr University Bochum, Knappschaftskrankenhaus, 34

Bochum, Germany 35

Research. on December 9, 2020. © 2019 American Association for Cancercancerres.aacrjournals.org Downloaded from

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12 Institute of Pathology, Ruhr University of Bochum, Bochum, Germany 36

13 Department of Visceral and General Surgery, St. Josef Hospital, Ruhr-University 37

Bochum, Germany 38

39

Present Addresses 40

Steven Smith: agap2 – Life Sciences, D-60323 Frankfurt am Main, Germany. 41

Heiko Müller: Saltigo GmbH Chempark, Building Q 18–2, D-51369 Leverkusen, 42

Germany. 43

44

Running Title 45

Preclinical Efficacy of AKT Inhibitor Borussertib 46

47

§Corresponding Authors 48

Stephan A. Hahn, Department of Molecular Gastrointestinal Oncology, Ruhr-49

University Bochum, Universitätsstraße 150, D-44780 Bochum, Germany. E-mail: 50

[email protected] 51

Jens T. Siveke, Division of Solid Tumor Translational Oncology, West German 52

Cancer Center, German Cancer Consortium (DKTK), partner site Essen, University 53

Hospital Essen, Hufelandstraße 55, D-45147 Essen, Germany. E-mail: 54

[email protected] 55

Daniel Rauh, Faculty of Chemistry and Chemical Biology, TU Dortmund University, 56

Otto-Hahn-Straße 4a, D-44227 Dortmund, Germany. Phone: +49(0)231-755-7080; 57

Fax: +49(0)231-755-7082; E-mail: [email protected]; Web: 58

http://www.ddhdortmund.de; Twitter: @DDHDortmund 59

60

Disclosure of Potential Conflicts of Interest 61

D. Rauh received consultant and lecture fees from Astra-Zeneca, Merck-Serono, 62

Takeda, Pfizer, Novartis, Boehringer Ingelheim, Sanofi-Aventis and BMS. 63

J. Weisner, R. Gontla, and D. Rauh have ownership interest (patent) in borussertib. 64

65

Wordcount: abstract (128 words), statement of significance (29 words), main text 66

(6385 words) 67

Number of figures: 6 68

Number of tables: 0 69




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ABSTRACT (128/150) 70

Aberrations within the PI3K/AKT signaling axis are frequently observed in numerous 71

cancer types, highlighting the relevance of these pathways in cancer physiology and 72

pathology. However, therapeutic interventions employing AKT inhibitors often suffer 73

from limitations associated with target selectivity, efficacy, or dose-limiting effects. 74

Here we present the first crystal structure of auto-inhibited AKT1 in complex with the 75

covalent-allosteric inhibitor borussertib, providing critical insights into the structural 76

basis of AKT1 inhibition by this unique class of compounds. Comprehensive 77

biological and preclinical evaluation of borussertib in cancer-related model systems 78

demonstrated strong antiproliferative activity in cancer cell lines harboring genetic 79

alterations within the PTEN, PI3K, and RAS signaling pathways. Furthermore, 80

borussertib displayed antitumor activity in combination with the MEK inhibitor 81

trametinib in patient-derived xenograft models of mutant KRAS pancreatic and colon 82

cancer. 83




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STATEMENT OF SIGNIFICANCE (29/32) 84

Borussertib, a first-in-class covalent-allosteric AKT inhibitor, displays antitumor 85

activity in combination with the MEK inhibitor trametinib in patient-derived xenograft 86

models and provides a starting point for further pharmacokinetic/-dynamic 87

optimization. 88




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INTRODUCTION 89

With its key roles in various cellular processes including cell proliferation, 90

metabolism, and cell survival, the PI3K/AKT pathway is overactivated in several 91

human cancers, contributing to tumor development, progression, and metastasis (1-92

3). Aberrations among members of this pathway have been described as oncogenic 93

drivers in diverse cancer types, such as activating point mutations in 94

phosphoinositide 3-kinase (PI3K) and gene deletion or loss-of-function mutations in 95

the tumor suppressor PTEN (4). These genetic lesions result in the augmented 96

generation of the second messenger phosphatidylinositol (3,4,5)-trisphosphate, 97

leading to hyperactivation of PDK1 and its substrate AKT, a serine/threonine-specific 98

kinase, also known as protein kinase B. This step acts as a major signaling node in 99

the PI3K/AKT pathway with hundreds of downstream substrates (5,6). 100

In addition to aberrant AKT activity caused by genetic lesions in upstream-acting 101

proteins, overexpression and activating mutations have been observed for all three 102

AKT isoforms, e.g. in lung, prostate, breast, endometrium, and skin carcinomas (7). 103

Mutations in AKT1 occur most frequently with a mutation rate of 2-3% in urinary and 104

bladder cancer, in which the somatic activating AKT1E17K mutation within the 105

regulatory PH domain is the most prominent genetic lesion and also described as a 106

driver mutation for the rare Proteus syndrome (8-10). Furthermore, overexpression of 107

AKT is associated with resistance to several chemotherapeutics (11). Together, this 108

evidence underlines the crucial role of the PI3K/AKT pathway in cancer progression 109

and highlights the great potential for precisely targeted therapeutic intervention 110

involving this signaling cascade. 111

Traditional ATP-competitive AKT inhibitors such as capivasertib (AZD5363) 112

(12,13) and ipatasertib (GDC-0068) (14,15) are under clinical investigation in phase I 113

and II studies. In contrast to these, allosteric inhibitors, including MK-2206 (16), 114

miransertib (ARQ092) (17), and BAY1125976 (18), which bind to the inactive kinase 115

conformation of AKT, exhibit exquisite target selectivity solely for AKT1-3 while 116

sparing structurally closely related AGC kinases, e.g., p70S6K and protein kinase A. 117

Recently, we reported the development of a covalent-allosteric AKT inhibitor, 118

borussertib. This inhibitor specifically binds to two non-catalytic cysteines in AKT at 119

positions 296 and 310 by decorating allosteric ligands with electrophilic warheads at 120

suitable positions, thus enabling the irreversible stabilization of the inactive 121

conformation (19). Biochemical analyses have revealed superior inhibitory properties 122




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compared to reversible ATP-competitive and allosteric AKT inhibitors. Despite initial 123

concerns associated with covalent kinase inhibitors, recently developed drugs 124

demonstrated tremendous success in the clinics with major beneficial impact on 125

patients’ survival rate (e.g. osimertinib (non-small cell lung cancer) and ibrutinib 126

(chronic lymphocytic leukemia)) (20,21). 127

In the present work, we report the first crystal structure of AKT1 in complex with 128

a covalent-allosteric inhibitor, borussertib (19), showing the unique covalent bond 129

between inhibitor and Cys296. Furthermore, we demonstrate its antiproliferative 130

activity for a panel of cancer cell lines harboring genetic alterations in PI3K, PTEN, 131

and RAS. To investigate cellular pharmacodynamic changes induced by on-target 132

inhibition of AKT, western blot studies were performed, and target selectivity was 133

further corroborated by PathScan® analyses. Subsequently, pharmacokinetic 134

characterization and patient-derived xenograft (PDX) experiments were conducted, 135

with results demonstrating the potential for optimization and development of orally 136

bioavailable, targeted covalent-allosteric AKT inhibitors and their applicability in the 137

mono- or combination therapy of different cancers. 138

139




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MATERIALS AND METHODS 140

Protein Expression, Purification, and Crystallization 141

A gene encoding for Akt1(2-446, E114/115/116A) including an N-terminal His6-142

Tag followed by a TEV protease recognition site was synthesized by GeneArt AG 143

(Regensburg, Germany) and cloned into the pIEx/Bac3 expression vector (Merck 144

Millipore) using NcoI and BamHI restriction sites. Transfection, virus generation, and 145

amplification as well as protein expression were carried out in Spodoptera frugiperda 146

(Sf9) cells (Thermo) following the BacMagic protocol (Merck Millipore). Infected 147

insect cells were grown in Erlenmeyer flasks for 72 hours at 27 °C with shaking at 148

120 rpm, subsequently harvested by centrifugation at 3,000 x g for 15 min and 149

washed once with PBS before being flash frozen in liquid nitrogen. Afterwards, cells 150

were thawed and resuspended in lysis buffer (50 mM Tris, 500 mM NaCl, 1 mM DTT, 151

10% glycerol, 0.1% Triton X-100, pH 8.0, EDTA-free protease inhibitor cocktail 152

(Sigma-Aldrich)). Cells were lysed using a microfluidizer, the lysate was cleared by 153

centrifugation (40,000 x g, 1 h). The supernatant was loaded onto a Ni-NTA 154

Superflow cartridge (Qiagen). Bound protein was eluted in buffer containing 50 mM 155

Tris, 500 mM NaCl, 500 mM imidazole, 1 mM DTT, 10% glycerol, pH 8.0. For 156

cleavage of the hexahistidine-tag, TEV protease was added to the pooled elution 157

fractions and dialyzed overnight into buffer containing 25 mM Tris, 50 mM NaCl, 158

1 mM DTT, 5% glycerol, pH 8.0 at 4 °C. The cleaved protein was further purified by 159

anion-exchange chromatography using a HiTrap Q HP column (GE Healthcare) 160

followed by size-exclusion chromatography on a HiLoad 16/60 Superdex 75 pg 161

column (GE Healthcare) using buffer containing 50 mM HEPES, 200 mM NaCl, 1 mM 162

DTT, 10% glycerol, pH 7.3. Afterwards, the protein was transferred into the storage 163

buffer (25 mM Tris, 100 mM NaCl, 1 mM DTT, 10% glycerol, pH 7.5) using a 164

Superdex 75 10/300 GL column (GE Healthcare), concentrated and stored at -80 °C. 165

For crystallization, purified protein at a concentration of 3 mg/mL was incubated 166

with 3 eq of borussertib on ice for 60 min. The samples were centrifuged at 167

20,000 x g for 10 min before hanging drops were prepared in 15-well crystallization 168

plates (EasyXtal Tool, Qiagen) by mixing protein-ligand complex with reservoir 169

solution (1:1) containing 1.25 mM sodium acetate pH 5.2, 3.75 mM sodium citrate 170

pH 5.2, 15% PEG MME 2000 at 20 °C. Diffraction-grade crystals grew within 3 days 171

and were cryoprotected using 20% ethylene glycol before they were flash cooled in 172

liquid nitrogen. X-ray diffraction data were collected at the PXII-X10SA beam line of 173




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the Swiss Light Source (PSI, Villingen, Switzerland) with wavelengths close to 1 Å. 174

The diffraction data were integrated with XDS (22) and scaled using the program 175

XSCALE (22). The crystal structure was solved by molecular replacement with 176

PHASER (23) using a co-crystal structure of Akt1 in complex with another covalent-177

allosteric inhibitor as template (24). The manual modification of the molecule of the 178

asymmetric unit was performed using the program COOT (25) and with the help of 179

the Dundee PRODRG server (26) the inhibitor topology files were generated. For 180

multiple cycles of refinement phenix.refine (27) was employed and the final structure 181

was evaluated by Ramachandran plot analysis using the server MolProbity (28). Final 182

validation of the model was performed with the help of the PDB_REDO server and 183

the crystal structure was visualized using PyMOL (29,30). 184

185

Cell culture and inhibitors 186

T-47D and ZR-75-1 cell lines were purchased from Sigma-Aldrich/ECACC. 187

KU-19-19 cells were purchased from DSMZ. AN3-CA and HPAF-II cells were 188

obtained from ATCC. BT-474, Dan-G, and MCF-7 were purchased from CLS Cell 189

Lines Service. Cell lines were cultured in DMEM, MEM or RPMI-1640 medium 190

(Gibco) supplemented with 10% fetal bovine serum (FBS) (PAN-Biotech) and 1% 191

penicillin/streptomycin (Gibco). For AN3-CA and MCF-7, 1 mM sodium pyruvate 192

(Gibco) was added to the growth medium and media for BT-474 and T-47D were 193

supplemented with 10 µg/mL insulin (Sigma-Aldrich). Bo103 cells were cultured in a 194

1:1 mixture of DMEM/F12 (Gibco) and DMEM (Gibco), supplemented with 5% fetal 195

bovine serum (Gibco), 2% penicillin/streptomycin (Gibco), 1.6 µg/mL Amphotericin B 196

(Gibco), 10 µM ROCK inhibitor Y-27632 (LC Labs), 10 µg/mL ciprofloxacin (Sigma-197

Aldrich), 8.4 ng/mL cholera toxin (Sigma-Aldrich), 10 µg/mL insulin (Sigma-Aldrich), 198

20 nM 1-thioglycerol (Sigma-Aldrich), and 0.5 mM sodium pyruvate (Gibco). Cells 199

were cultured in a humidified incubator at 37 °C, 5% CO2 and cell line authenticity 200

was confirmed by STR analysis at Microsynth AG (Balgach, Switzerland) or by SNP 201

profiling at Multiplexion (Heidelberg, Germany). Mycoplasma testing has not been 202

performed. Cells were used for viability and western blot analyses within eight weeks 203

after thawing. 204

Borussertib was synthesized as described elsewhere (24). Reference inhibitors 205

capivasertib, ipatasertib, MK-2206, and miransertib were purchased from 206




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SelleckChem; Staurosporine and Y-27632 were obtained from LC Labs; Trametinib 207

was obtained from LC Labs and Hycultec. 208

209

Cell viability analysis 210

On day 0, cells were plated into white 384-well cell culture plates (Greiner Bio-211

One) using a Multidrop™ reagent dispenser (Thermo) at cell numbers that ensure 212

linear and optimal luminescent signal intensity (AN3-CA: 800 cells/well; BT-474: 213

400 cells/well; Dan-G: 400 cells/well; HPAF-II: 400 cells/well; KU-19-19: 214

400 cells/well; MCF-7: 200 cells/well; T-47D: 800 cells/well; ZR-75-1: 400 cells/well; 215

Bo103: 800 cells/well). Following incubation for 24 h in a humidified atmosphere at 216

37 °C/5% CO2, cells were treated with inhibitors in serial dilutions ranging from 30 µM 217

down to 0.1 nM using an Echo 520 acoustic liquid handler (Labcyte Inc.). Cell viability 218

was analysed on day 5 using the CellTiter-Glo® assay (Promega) as per 219

manufacturer’s instructions. Luminescence was recorded using an EnVision 220

Multilabel 2104 Plate Reader (PerkinElmer) using 500 ms integration time. The 221

obtained data were normalized to the plate positive control (30 µM staurosporine) 222

and negative control (DMSO) and subsequently analysed and fitted with the Quattro 223

Software Suite (Quattro Research) using a four parameter logistic model. As quality 224

control, the Z′-factor was calculated from 16 positive and negative control values. 225

Only assay results showing a Z′-factor ≥0.5 were used for further analysis. All 226

experimental points were measured in duplicates for each plate and were replicated 227

in at least three plates. 228

Combination studies were conducted using the CompuSyn software (Biosoft) for 229

calculating the combination index (CI) equation to determine synergism of drug 230

combinations using fixed drug ratios as described by Chou-Talalay (31). 231

232

Western blot and PathScan analysis 233

For protein isolation, cells were seeded into six-well tissue culture plates 234

(Sarstedt) yielding 80-90% confluency after overnight incubation. Afterwards, cells 235

were treated with various concentrations of inhibitors or DMSO and incubated for 236

additional 24 h before the medium was removed and cells were washed once with 237

ice-cold PBS. Cell lysis was initiated by addition of 100 µL RIPA buffer (Cell Signaling 238

Technology) per well supplemented with phosphatase and protease inhibitor 239

cocktails (Sigma-Aldrich) followed by incubation on ice for 30 min. Cells were then 240




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harvested by scraping and transferred into pre-cooled microcentrifuge tubes. Whole 241

cell lysates were cleared by centrifugation at 14,000 x g/4 °C for 10 min and 242

transferred into fresh, pre-cooled microcentrifuge tubes. Protein concentrations were 243

determined using the Pierce™ BCA protein assay (Thermo) as per manufacturer’s 244

instructions. Equal amounts of protein were separated by SDS-PAGE and transferred 245

to Immobilon-FL PVDF membranes (Merck Millipore) using Pierce™ 1-step transfer 246

buffer (Thermo) and the Pierce™ Power Blotter (Thermo). Membranes were washed 247

for 5 min with ddH2O, blocked with Odyssey® Blocking Buffer TBS (Li-Cor) for 1 h at 248

room temperature and then incubated with primary antibodies diluted in Odyssey® 249

Blocking Buffer TBS overnight at 4 °C with gentle agitation. On the next day, the 250

membranes were washed three times with TBS-T (50 mM Tris, 150 mM NaCl, 0.05% 251

Tween 20, pH 7.4) for 5 min before being incubated with secondary antibodies 252

diluted in Odyssey® Blocking Buffer TBS for 1 h at room temperature with gentle 253

agitation. Finally, the membranes were washed three times for 5 min with TBS-T and 254

then scanned using an Odyssey® CLx imaging system (Li-Cor). 255

For capillary western blot analysis, lysates of the cell pellets were prepared as 256

described above and protein concentration was estimated. Simple Wes assay was 257

performed and analysed according to the manufacturer’s instructions (Protein 258

Simple). For pAKTS473 detection, 0.55 µg of protein was loaded per capillary with 1:20 259

dilution of anti-pAKTS473 antibody while tAKT was detected with anti-tAKT antibody, 260

dilution 1:100 and 0.05 µg of total protein per capillary. 261

For PathScan® Akt Signaling Array (Cell Signaling Technology), 1X array wash 262

buffer, 1X detection antibody cocktail, 1X HRP-linked streptavidin and the multi-well 263

gasket were prepared as per manufacturer’s instructions and glass slides and 264

blocking buffer were calibrated to room temperature. Subsequently, 100 µL array 265

blocking buffer were added to each well for 15 min at RT covered with sealing tape 266

and placed on an orbital shaker (as well as all following incubation steps). After 267

removal of the blocking buffer, 50 µL diluted lysate (0.5 mg/mL protein concentration) 268

were added to each well and incubated for 2 hours at RT. Wells were washed three 269

times with 100 µL 1X array wash buffer for 5 min at RT, followed by an incubation for 270

one hour at RT in 75 µL 1X detection antibody cocktail. Wash steps were repeated 271

four times before adding 75 µL 1X HRP-linked streptavidin to each well for a 272

30 minute incubation at RT. The multi-well gasket was removed from the slides 273

another four wash steps later and the slides were washed with 10 mL 1X array wash 274




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buffer. Slides were then covered with LumiGlo®/Peroxide reagent (as per 275

manufacturer’s instructions) and images with different exposure times were captured 276

using a digital imaging system. 277

278

Half-time Determination (Western blot) 279

For protein isolation, AN3-CA cells were seeded into 10 cm dishes (Sarstedt). 280

Treatment with the inhibitors (borussertib, MK-2206, miransertib) was initiated at a 281

confluency of 60-70% with the indicated concentrations for 24 h. Then the medium 282

was removed and cells were washed twice with PBS. Medium without borussertib 283

was added to start the wash out experiments which were terminated at the indicated 284

time points. Prior to the cell harvest, cells were either not stimulated at all or 285

stimulated with EGF (100 ng/mL, PeproTech), TGF-α (100 ng/mL, R&D Systems), or 286

insulin (100 nM, Sigma-Aldrich) for 15 min. Then the medium was removed and cells 287

were washed twice with ice-cold PBS. Cell lysis was initiated by addition of 500 µL 288

RIPA buffer per dish supplemented with phosphatase (Sigma-Aldrich) and protease 289

inhibitor cocktails (Roche) followed by harvesting the cells by scraping and 290

transferred into pre-cooled microcentrifuge tubes. Cell lysates were incubated on ice 291

for 30 min. After that, cells were lysed by sonication, cleared by centrifugation at 292

14,000 x g at 4 °C for 10 min and transferred into fresh, pre-cooled microcentrifuge 293

tubes. Protein concentrations of the lysates were determined by the Bradford protein 294

assay system (BioRad). Equal amounts of protein (36 μg protein each lane) were 295

separated by SDS-PAGE and transferred to PVDF membranes (Roth). Immunoblots 296

were blocked with 5% BSA in 1X TBS and Tween-20 (0.1% v/v) for 1 hour at room 297

temperature. The membrane was incubated overnight at 4 °C with primary 298

antibodies. Afterwards, the membrane was incubated with the corresponding 299

secondary antibody conjugated with horseradish peroxidase (Dianova). Bands were 300

visualized with enhanced chemiluminescence western blot detection system 301

(Thermo). 302

303

Antibodies 304

anti-pAkt(Ser473) (CST, cat. no. 3787, 4060), anti-pAkt(Thr308) (CST, cat. no. 305

2965), anti-tAkt (CST, cat. no. 2920, 4691), anti-pS6 ribosomal protein (Ser235/236) 306

(CST, cat. no. 2211, 4858), anti-pPRAS40(Thr246) (CST, cat. no. 3997), anti-307

pErk1/2(Thr202/Tyr204) (CST, cat. no. 4370), anti-p4E-BP1(Ser65) (CST, cat. no. 308




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13443), anti-pGSK-3β (Ser9) (CST, cat. no. 9323), anti-GSK-3β (CST, cat. no. 9315), 309

anti-PARP (CST, cat. no. 9542), anti-HSP90, (CST, cat. no. 4874), anti-β-Actin (CST, 310

cat. no. 4970/Sigma-Aldrich, cat. no. A5441), anti-GAPDH (CST, cat. no. 2118), anti-311

mouse IgG (H+L) (DyLight™ 680 Conjugate) (CST, cat. no. 5470), anti-rabbit IgG 312

(H+L) (DyLight™ 800 4X PEG Conjugate) (CST, cat. no. 5151). 313

314

In vitro pharmacokinetic studies 315

For determining kinetic solubility, the compound was diluted from a 10 mM 316

stock in DMSO to a final concentration of 500 μM in 50 mM HEPES, pH 7.4. 317

Following an incubation of 90 min at room temperature on a shaker, the aqueous 318

dilution was filtered through a 0.2 μm PVDF filter, and the optical density between 319

250 and 500 nm was measured at intervals of 10 nm. The kinetic solubility was 320

calculated from the area under the curve (AUC) between 250 and 500 nm and 321

normalized to absorption of a dilution of the compound in acetonitrile. 322

Metabolic stability under oxidative conditions was measured in human and murine 323

liver microsomes by LC-MS-based analysis of depletion of compound at a 324

concentration of 3 μM over time up to 50 min at 37 °C. On the basis of compound 325

half-life t1/2, the in vitro intrinsic clearance Clint was calculated. 326

Plasma stability was measured by LC-MS-based determination of % remaining of 327

selected compound at a concentration of 5 μM after incubation in 100% plasma 328

obtained from different species for 1 h at 37 °C. 329

Assessment of plasma protein binding was measured by equilibrium dialysis by 330

incubating the compound of interest at a concentration of 5 μM for 6 h at 37 °C in 331

50% plasma in buffer (v/v) followed by LC-MS-based determination of final 332

compound concentrations. The resulting fraction unbound at 50% plasma (fu50%) was 333

extrapolated to the fraction unbound at 100% plasma (fu100%) using the following 334

equation: fu100% = fu50%/(2 −fu50%). 335

336

In vivo pharmacokinetic studies 337

For in vivo pharmacokinetic analysis, RjOrl:SWISS mice (Janvier, France), age 338

8-10 weeks, were treated with borussertib by oral gavage (20 mg/kg), intraperitoneal 339

(20 mg/kg), or intravenous (2 mg/kg) administration. The compound was formulated 340

in PBS/PEG200 (60:40) for oral and intraperitoneal administration, whereas it was 341

dissolved in DMSO for intravenous injection. Blood was collected 5, 15, 45 and 342




Page 13 of 30

135 minutes after compound administration, immediately centrifuged at 15,000 x g for 343

10 min at 4 °C and plasma samples were stored at -80 °C for subsequent LC-MS/MS 344

analysis. Three mice were analysed per experimental condition (for each time point 345

and route of administration). Mice were fed ad libitum with Allein-Futter für Ratten-346

/Mäusehaltung (Sniff Special Diets GmbH, Germany). They had free access to water 347

and were kept in a 12 h day/night rhythm. All experiments were approved by the local 348

authorities. 349

Samples and blanks were prepared by adding 2.5 µL blank DMSO and 80 µL of 350

ice-cold acetonitrile containing the internal standard (Griseofulvin, 1 µM) to 20 µL 351

plasma followed by centrifugation at 13.000 rpm (4 °C) for 10 min. 65 µL of the 352

supernatant were diluted with 65 µL of LC-MS grade water. Samples were filtered 353

(MSRLN0450, Millipore) and subjected to LC-MS measurement. Analyte stock 354

solution (10 mM in DMSO) was diluted in DMSO to yield DMSO stock solutions with 355

the following concentrations: 10, 5, 2.5, 1, 0.5, 0.25, 0.1, 0.05, 0.025, 0.01, 0.005, 356

0.0025 µM. 2.5 µL of the corresponding DMSO stock solution were added to 20 µL of 357

blank plasma followed by the addition of 80 µL of ice-cold acetonitrile containing the 358

internal-standard. The samples were centrifuged for 10 min at 4 °C and 13.000 rpm. 359

65 µL of the supernatant were diluted with 65 µL of LC-MS grade water and 360

subjected to LC-MS analysis. A set of 3 different QCs (n = 3) was prepared by adding 361

of 2.5 µL DMSO stock solution (5, 0.5 and 0.05 µM) to 20 µL of plasma. Samples 362

were subsequently handled as described above. All samples were analyzed using a 363

Shimadzu LC20ADXR Solvent Delivery Unit, a Shimadzu SIL30ACMP autosampler 364

and a ABSciex Qtrap5500 LC-MS/MS system. Therefore, 2 μL of sample were 365

injected and separated using an Agilent Poroshell C18, 2.7 µm column (2.1 mm x 366

50 mm) at 60 °C starting at 5% of solvent B for 0.3 min followed by a gradient up to 367

100% of solvent B over 0.6 min (flow rate 1 mL/min) with 0.1% formic acid in water as 368

solvent A and 0.1% formic acid in acetonitrile as solvent B. Data evaluation was 369

performed using Analyst 1.6.2 Software (Sciex). 370

371

Animal models and treatments 372

Tissue samples to establish the PDX models were collected from patients 373

following surgical intervention for colon cancer or pancreatic adenocarcinoma at the 374

Ruhr-University Comprehensive Cancer Center. From all patients informed and 375

written consent was obtained. The studies were approved by the Ethics Committees 376




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of the Ruhr-University Bochum (Registry no. 3534-09, 3841-10 & 16-5792). Animal 377

experiments and care were in accordance with the guidelines of institutional 378

authorities and approved by local authorities (numbers: 8.87-50.10.32.09.018, 84-379

02.04.2012.A328, 84-02.04.2012.A360, 84-02.04.2015.A135 & 81-02.04.2017.A423). 380

Non-diagnostic tissue samples were selected by the pathologist within 2 to 6 hours 381

post-surgery. Selected tumor pieces (1-2 mm) were soaked in undiluted Matrigel 382

(Becton Dickinson) for 15 to 30 min and subsequently implanted subcutaneously 383

onto 5- to 10-week-old female mice (NMRI-Foxn1nu/Foxn1nu, Janvier, France) at 384

two sites (scapular region, one mouse per tumor) using as many as 4 pieces per site. 385

To establish treatment cohorts, early passage (≤ F5 generation) PDX tumor pieces 386

were implanted as described above into nude mice and were allowed to grow to a 387

size off approx. 100-200 mm3, at which time mice were randomized in the treatment 388

and control groups with three to four mice in each group. Tumor volumes were 389

estimated from 2-dimensional tumor measurements by bi-weekly caliper 390

measurements using the following formula: Tumor volume (mm3) = [length (mm) x 391

width (mm)2]/2. Response was defined in analogy to RECIST 1.1 criteria with at least 392

30% reduction in mean tumor volume compared to the mean tumor volume at start of 393

treatment being a partial response (PR) and an undetectable tumor being a complete 394

response (CR). Disease progression was defined as more than 20% increase in 395

mean tumor volume to the tumor volume at the beginning of treatment. All other 396

measurements were defined as stable disease. Mice were treated with borussertib by 397

daily intra peritoneal (i.p.) injection dosed at 20 mg/kg and trametinib (Hycultec) by 398

oral gavage at 0.5 mg/kg per day with a weekly treatment cycle comprising of five 399

consecutive days of treatment followed by two days treatment pause. 400

401




Page 15 of 30

RESULTS 402

Borussertib Binds Covalently Between the Kinase and PH Domain of AKT 403

Recently, we described a novel class of AKT inhibitors that bind into the allosteric 404

pocket of AKT and harbor a Michael acceptor to covalently bind to non-catalytic 405

cysteines. These features combine the advantage of outstanding selectivity of PH 406

domain–dependent allosteric inhibition with the therapeutic benefit of irreversible 407

modification, leading to increased drug target residence time and gain of potency. In 408

this class of inhibitors, we identified a potent lead compound, borussertib, exhibiting 409

an exquisite kinase selectivity profile (19). To investigate the binding mode of this 410

novel AKT inhibitor at the atomic level, we solved the co-crystal structure of AKT1 in 411

complex with borussertib to a resolution of 2.9 Å. 412

The crystal structure discloses the inactive, autoinhibited conformation with the 413

PH domain folded onto the kinase domain (PH-in conformation) between the N- and 414

the C-lobe, thereby displacing the regulatory helix αC and simultaneously shaping an 415

allosteric binding pocket at the interface between these two domains (Fig. 1A, 416

Supplementary Fig. S1, Supplementary Table S1). Borussertib binds to this allosteric 417

pocket and forms a key aromatic π-π stacking interaction between the 418

1,6-naphthyridinone scaffold and the indole side chain of Trp80 in the PH domain. 419

Additional hydrophobic contacts can be observed between the phenyl ring in the 420

3-position and Leu210, Leu264, and Ile290. Water-mediated hydrogen bonds 421

between Glu17, Arg273, Tyr326, and the benzo[d]imidazolone moiety of borussertib 422

foster the high-affinity reversible binding of the ligand to the kinase (Fig. 1B). 423

Furthermore, the acrylamide moiety is pre-oriented by a hydrogen bond formed 424

between the amide oxygen of the warhead and the backbone NH of Glu85, 425

facilitating covalent bond formation between the electrophilic β-carbon and the thiol 426

side chain of Cys296. 427

428

Borussertib Potently Inhibits Proliferation of PI3K/PTEN-Mutated Cell Lines 429

To investigate the in vitro antiproliferative activity of borussertib, we employed breast, 430

bladder, pancreas, and endometrium cancer cell lines harboring genetic alterations in 431

the PI3K/AKT and RAS/MAPK pathways, i.e., AN3-CA (endometrium), BT-474 432

(breast), Dan-G (pancreas), HPAF-II (pancreas), KU-19-19 (bladder), MCF-7 433

(breast), T-47D (breast), and ZR-75-1 (breast). Genetic alterations include in-frame 434

deletions in PIK3R1, frame shifts and point mutations in PTEN, and (activating) point 435




Page 16 of 30

mutations in PIK3CA, NRAS, and KRAS (Supplementary Table S2). Each cell line 436

was treated for 96 h with borussertib or reference inhibitors in a concentration range 437

from 30 µM to 0.1 nM. In these cell lines, we observed outstanding sensitivity to 438

borussertib with half-maximal effective concentration (EC50) values in the 439

submicromolar range, indicating a 1.5- to 43-fold greater potency than miransertib, 440

MK-2206, ipatasertib, and capivasertib (Fig. 2). Only in the bladder cancer cell line 441

KU-19-19, which harbors additional activating mutations in AKT1 (E17K/E49K) and 442

NRAS (Q61R), micromolar antiproliferative activities could be observed (EC50 = 3.1-443

5.0 µM) for the tested compounds being in good correlation with previously published 444

data for the allosteric AKT inhibitor BAY 1125976 (18). Furthermore, for pancreatic 445

cancer cell lines Dan-G and HPAF-II, generally lower sensitivities to AKT inhibition 446

were observed with only minor differences between the individual inhibitors 447

(Supplementary Table S2). The lack of mutations within PI3K and/or PTEN in 448

combination with codon 12 mutations in KRAS substantiates the relatively high EC50 449

values observed for these two cell lines. 450

Of note, the breast cancer cell line ZR-75-1 exhibited a pronounced sensitivity to 451

borussertib, with an EC50 of 5 ± 1 nM and thus an approximately 7- to 12-fold higher 452

potency compared to the reversible allosteric inhibitors miransertib 453

(EC50 = 35 ± 18 nM) and MK-2206 (EC50 = 63 ± 21 nM); ATP-competitive inhibitors 454

capivasertib (EC50 = 191 ± 68 nM) and ipatasertib (EC50 = 219 ± 83 nM) showed a 455

38- to 43-fold lower activity, respectively. The significant differences in 456

antiproliferative activity observed for some of the tested cell lines can only to some 457

extent be explained by the biochemical inhibitory potencies towards AKT1; 458

capivasertib (IC50 = 0.9 ± 0.1 nM) exhibits a higher potency with respect to inhibition 459

of AKT1 in vitro compared to ipatasertib (IC50 = 3.5 ± 0.6 nM) and MK-2206 460

(IC50 = 10.0 ± 2.1 nM) (24). However, MK-2206 showed a 5- to 9-fold higher activity 461

in MCF-7 cells, whereas capivasertib and ipatasertib inhibited growth of AN3-CA, BT-462

474, and T-47D cells with similar activities. Differences in cellular pharmacokinetic 463

properties as well as AKT1 expression and activity levels might have contributed to 464

these observations. In addition, kinase-independent functions related to specific 465

conformations stabilized by either ATP-competitive or allosteric AKT inhibitors could 466

affect in vitro as well as in vivo potency (32,33). Moreover, the molecular impact of 467

AKT isoforms 1–3 on cell survival and proliferation is not fully understood, and a 468

potential influence of the compounds’ selectivity profiles towards AKT1, AKT2, and 469




Page 17 of 30

AKT3 on their antiproliferative efficacy cannot be excluded (6). In summary, the 470

experimental inhibitor borussertib exhibited superior antiproliferative properties in our 471

experimental setup compared to the clinical candidates of ATP-competitive and 472

allosteric reference compounds, indicating a potentially beneficial impact of our 473

approach to irreversibly target AKT. 474

475

Borussertib Downregulates AKT-Mediated Signaling 476

To gain further insights into the molecular mode of action and how it affects AKT 477

signaling, we performed PathScan® AKT signaling assays in combination with 478

western blot studies. Furthermore, washout experiments were performed to 479

reconstruct the average half-life of irreversibly inhibited AKT and thus anticipate the 480

mean projected duration of compound treatment. For PathScan® analysis, MCF-7 481

and Dan-G cells were treated with dimethyl sulfoxide (vehicle) or 1 µM borussertib for 482

24 h prior to lysis and array-based readout (Supplementary Fig. S2). For MCF-7 483

cells, the results indicate low basal levels of activated AKT (pAKTS473), whereas 484

PRAS40 as well as GSK-3α/β exhibited pronounced phosphorylation in vehicle-485

treated cells. Upon treatment with 1 µM borussertib, pAKT levels were reduced and 486

phosphorylation of PRAS40 and GSK-3α/β significantly decreased, hinting at potent 487

inhibition of AKT signaling. Moreover, phospho-S6 and phospho-p70 S6 kinase 488

signals were diminished upon inhibitor treatment. Notably, borussertib exhibited no 489

off-target inhibition towards activating kinase PDK1 and RAS/MAPK signaling. 490

Comparable results were observed for Dan-G cells including the downregulation of 491

pAKTS473, pS6S235/236, pPRAS40T246, and pGSK-3αS21. 492

Western blot analyses for ZR-75-1, AN3-CA, Dan-G, T-47D, and MCF-7 cell 493

lines resolved the dose-dependent downregulation of pAKTT308 and pAKTS473 as well 494

as downstream targets pPRAS40T246, pS6S235/236, and p4E-BP1S65 (Fig. 3A, 495

Supplementary Fig. S3). For all cell lines, inhibition of AKT phosphorylation was 496

observed upon drug treatment demonstrating the highest sensitivity for ZR-75-1 and 497

AN3-CA cells, correlating well with the pronounced inactivation of AKT-mediated 498

downstream signaling, as can be seen by the substantial dephosphorylation of 499

PRAS40, S6, and 4E-BP1 (Fig. 3A). Additionally, to investigate the underlying 500

mechanism of borussertib’s antiproliferative activity, cell lysates were probed for 501

cleavage of poly (ADP-ribose) polymerase (PARP) revealing a distinct induction of 502

apoptosis in AN3-CA and ZR-75-1 cells after compound treatment at nanomolar 503




Page 18 of 30

concentrations (Fig. 3A). In contrast, no increase in cleaved PARP (cPARP) level 504

could be observed for KRAS-mutant Dan-G cells at concentrations as high as 10 µM 505

indicating a lower dependence on AKT-mediated signaling. 506

To determine the cellular half-life of covalently inhibited AKT, AN3-CA cells 507

were treated with 0, 100, and 200 nM borussertib, respectively, for 24 h, followed by 508

medium renewal and serum starvation for 0-48 h. Prior to cell lysis, cells were 509

stimulated with epidermal growth factor (EGF) for 15 min. pAKTS473 levels remained 510

significantly downregulated up to 24 h after medium renewal and drug withdrawal 511

(Fig. 3B). Similar results were obtained for unstimulated, EGF-related transforming 512

growth factor-alpha (TGF-α) treated, and insulin treated cells (Supplementary Fig. 513

S4A-D). In contrast to borussertib, higher concentrations were required for MK-2206 514

and miransertib to completely inhibit AKT-mediated signaling, as shown for pGSK-515

3βS9 and pS6S235/236 (Supplementary Fig. S4E), despite efficient downregulation of 516

pAKTS473 at 200 nM. With the slow recovery of pAKTS473 levels upon irreversible 517

inhibition with borussertib, we propose that with respect to in vivo studies, single 518

compound administrations might be efficacious for a relatively long period of time 519

independent of the in vivo pharmacokinetics, provided that a sufficient amount of 520

inhibitor reaches its target before being cleared. However, also reversible inhibition 521

using MK-2206 and miransertib resulted in prolonged downregulation of pAKTS473 522

after compound washout (Supplementary Fig. S5). 523

524

AKTi Borussertib and MEKi Trametinib Act Synergistically in vitro 525

In addition to the potent antiproliferative efficacy of borussertib towards PI3K/PTEN-526

mutated cell lines, we were interested in the identification of potential additive or 527

synergistic effects of AKT inhibition in combination with targeted MEK inhibition or 528

chemotherapy. Therefore, Dan-G cells were treated with borussertib and MEKi 529

trametinib or gemcitabine, respectively, at concentrations close to the respective 530

EC50 (Fig. 4A-B). Employing the Chou-Talalay method (31), combination indices 531

(CIs) were calculated for the tested drug combinations to determine potential additive 532

(CI = 1), synergistic (CI < 1), or antagonistic (CI >1) effects. For the combination of 533

borussertib and trametinib, strong synergy was observed over the entire range of 534

concentrations tested, whereas no significant beneficial effect could be observed for 535

combination treatment with borussertib and gemcitabine even at high doses (Fig. 536

4C). Inhibition of MAPK pathway activity via trametinib has failed so far in clinical 537




Page 19 of 30

trials in KRAS-driven tumors, which may be due to concomitant activity of PI3K/AKT 538

pathway activity or induction of resistance via crosstalk among other reasons. Recent 539

data showed PI3K activation upon complete ablation of KRAS in PDAC cells (30), 540

further supporting strategies to block AKT activation in combination with RAS/MAPK-541

acting drugs. With regard to gemcitabine, efficacy of this chemotherapeutic agent is 542

regulated on multiple levels including expression of transporter genes (e.g. hENT1), 543

intracellular drug metabolism, and cell-cycle state among others. Since the 544

combination of borussertib with gemcitabine showed no clear synergistic signal in 545

PDAC cells, we did not follow this path in more detail but focused on rational drug 546

combinations with favorable combinatory index. 547

To further investigate the potential of AKTi/MEKi combination therapy including 548

our covalent-allosteric inhibitor borussertib, we utilized early passage pancreatic 549

cancer cells (Bo103) harboring a KRAS mutation in codon 12 as the model of interest 550

for viability studies in combination with Western blot analyses. Neither borussertib nor 551

trametinib mono-therapy resulted in complete inhibition of cell viability at 552

concentrations up to 30 µM; remaining cell viabilities at the highest concentrations of 553

borussertib and trametinib were determined to be 51.5 9.5% and 29.3 5.9%, 554

respectively (Fig. 5A). For the combination treatment, both compounds were added 555

to the cells in a 1:1 stoichiometry yielding a highest total concentration of 30 µM 556

(15 µM borussertib/15 µM trametinib); a remaining cell viability of 6.9 2.9% (mean 557

SD) was determined from three independent experiments, indicating a substantial 558

benefit of combination treatment as compared to single agent therapy. The resulting 559

EC50 of 82.7 26.0 nM additionally highlights the supreme efficacy of the 560

combination therapy tested herein. EC50 values for either monotherapy were not 561

calculated due to incomplete inhibition of cell viability (Fig. 5A). 562

For correlation of antiproliferative activity with downregulation of pS6S235/236 and 563

p4E-BP1S65 induced by either AKT or MEK inhibition, western blots were prepared for 564

pancreatic cancer Bo103 cells treated with single agent or drug combination (Fig. 565

5B). Pronounced downregulation of pAKTS473 or pERK1/2T202/Y204 was detected upon 566

treatment with borussertib and trametinib, respectively, correlating with decreasing 567

amounts of detectable pS6S235/S236. However, p4E-BP1S65 was not affected by 568

treatment with either of the two compounds. Similar effects were observed for cells 569

treated with drug combination, yet showing a more distinct decrease of pS6S235/S236. 570

These observations might explain the superior antiproliferative efficacy of 571




Page 20 of 30

combination compared to single agent treatment and thus indicate the enormous 572

potential of covalently targeting AKT. 573

574

Borussertib Exerts Antitumor Activity in KRAS-mutant Patient-Derived 575

Xenografts in Combination with Trametinib 576

To investigate the potential of borussertib as a drug candidate in preclinical studies, 577

we performed in vitro and in vivo pharmacokinetic (PK) analyses for borussertib 578

(Supplementary Fig. S6). Besides an unfavorable low solubility in aqueous media 579

(13 µM), in vitro analyses in both human and murine samples revealed promising 580

features with generally low intrinsic clearance (Clint,human = 7 µL/min/µg, 581

Clint,murine = 31 µL/min/µg), high plasma stability (human: 99% remaining, murine: 582

100% remaining) and high plasma protein binding (PPB) (human: 100%, murine: 583

99%) (Supplementary Fig. S6A). Subsequent PK studies were carried out in mice (2 584

mg/kg intravenous; 20 mg/kg oral gavage; 20 mg/kg, intraperitoneal) 585

(Supplementary Fig. S6B). Despite a rather low oral bioavailability (<5%), reaching 586

only a maximum plasma concentration of 78 ng/mL (0.13 µM), we found a 587

significantly higher bioavailability upon intraperitoneal administration (39.6%), with 588

maximum plasma levels of 683 ng/mL (1.14 µM), indicating sufficient absorption of 589

the compound to potentially exert antitumor activity in xenografts. 590

Given the promising pharmacokinetic and pharmacodynamic properties, we next 591

examined the antitumor activity of borussertib in mouse xenograft studies, using 592

implanted xenograft models derived from KRAS-mutant primary pancreas and colon 593

cancers. These tumor entities are characterized by RAS/MAPK activity but 594

considerable resistance to single-agent MAPK pathway inhibition clinically. 595

Encouraged by our in vitro analyses indicating synergistic effects, we therefore 596

focused on combined activity of MEK and AKT inhibition with trametinib and 597

borussertib, respectively. For PDX studies, borussertib was administered at 20 mg/kg 598

per intraperitoneal injection once daily, either as monotherapy or in combination with 599

the targeted MEK inhibitor trametinib at 0.5 mg/kg administered perorally for 5 days 600

per week. 601

First, PDAC PDX models were used to evaluate borussertib for its antitumor 602

activity (Fig. 6A, Supplementary Fig. S7A, B). Borussertib monotherapy resulted in 603

insignificant tumor growth delays in Bo103 PDX (Fig. 6A), Bo73 (Supplementary Fig. 604

S7A), and Bo85 (Supplementary Fig. S7B) compared to untreated control mice. In 605




Page 21 of 30

contrast, trametinib monotherapy induced a decelerated tumor growth in all tested 606

PDX models. However, only the combination of borussertib with trametinib resulted in 607

a durable partial response in Bo103, whereas progressive diseases were observed 608

for Bo73 and Bo85. 609

Additionally, we employed KRAS-mutant colorectal carcinoma PDX models to 610

further evaluate the antitumor activity of borussertib (Fig. 6B-D, Supplementary Fig. 611

S7C, D). In four of five established model systems, borussertib monotherapy did not 612

affect tumor growth as compared to untreated control mice (Fig. 6B, D, 613

Supplementary Fig. S7C, D). Nevertheless, the PDX cohort engrafted with BoC105 614

exhibited significantly delayed tumor growth upon borussertib treatment (Fig. 6C). 615

This effect was additionally augmented in mice treated with borussertib in 616

combination with trametinib, resulting in a durable partial response. In total, the 617

combination of the AKT inhibitor borussertib with the MEK inhibitor trametinib yielded 618

three stable diseases (Fig. 6B, D, Supplementary Fig. S7C) and two partial 619

responses (Fig. 6C, Supplementary Fig. S7D), highlighting the potential benefit of this 620

combination compared to MEK inhibitor monotherapy for treating colorectal cancer. 621

Taken together, these results underscore the general applicability of covalent-622

allosteric AKT inhibitors in vivo. Although the KRAS-mutant PDX models employed in 623

this study did not show any response to AKTi monotherapy, alternative in vivo model 624

systems harboring, e.g., genetic lesions in PI3K or PTEN, could be more suitable for 625

evaluation of borussertib monotherapy. 626

627




Page 22 of 30

DISCUSSION 628

Numerous signaling cascades rely on AKT as a central integrator of diverse stimuli 629

relevant for physiological and pathobiological processes. To date, few small molecule 630

AKT modulators have entered preclinical and clinical trials, largely because of 631

selectivity issues caused by structurally similar kinases, lack of efficacy, and 632

mechanism-based adverse effects. Moreover, aberrant AKT signaling resulting from 633

activating mutations in PI3K or functional loss of PTEN might not give rise to 634

oncogene addiction per se (14). However, (co-)targeting AKT in a clinical setting may 635

result in beneficial therapy outcomes, as demonstrated for ipatasertib, capivasertib, 636

MK-2206, and miransertib, respectively. Of note, low prevalent hyperactive AKT1E17K 637

has recently been described to act as a classical driver oncogene in patients 638

suffering from gynecologic and estrogen receptor–positive breast cancers, thus 639

eliciting pronounced therapeutic responses upon administration of ATP-competitive 640

AKT inhibitor capivasertib (34). The efficacy of borussertib for such indications 641

remains to be determined. 642

Borussertib proved to be a highly selective and irreversible allosteric inhibitor of 643

AKT with potent in vitro antiproliferative activity and the ability to synergize with other 644

targeted therapies such as MEKi in KRAS-mutant colon and pancreatic cancer PDX 645

models thereby overcoming potential limitations regarding therapeutic efficacy 646

observed for MEKi monotherapy in the types of cancer mentioned above. The X-ray 647

crystallographic complex structure presented here supports the anticipated binding 648

mode and will foster the rational derivatization and optimization of our lead molecule 649

borussertib concerning binding affinity and inhibitory potency. We provide evidence 650

for the potent inhibition of cancer cell proliferation, especially for cell lines featuring 651

genetic alterations in the PI3K/AKT signaling cascade, resulting from the targeted 652

downregulation of pAKT and downstream effectors, including pS6, p4E-BP1, and 653

pPRAS40, as deduced from immunoblot analyses. Future efforts will be directed 654

towards the profiling of cancer cell lines from additional primary sites and the 655

evaluation of potential drug combination strategies in combination with expanded 656

comprehensive pharmacodynamic analyses. In addition, we provide proof-of principle 657

data for the in vivo efficacy of what is to our knowledge the first-in-class covalent-658

allosteric AKT inhibitor, as shown for KRAS-mutant pancreatic ductal 659

adenocarcinoma and colorectal carcinoma PDX models. Additional efforts will be 660

directed towards the optimization of aqueous solubility in order to generate an oral 661




Page 23 of 30

bioavailable derivative of borussertib with improved biochemical potency, cellular 662

antiproliferative activity and in vivo efficacy. Furthermore, detailed in vivo PD 663

analyses, optimal dosage identification and toxicity profiling are mandatory for the 664

subsequent development of covalent-allosteric AKT inhibitors as drug-like 665

candidates. Eventually, besides combination studies, it will be of interest to employ 666

borussertib or its optimized derivatives in PDX models harboring genetic alterations 667

in the PI3K/AKT signaling axis to enable characterization of its potential as a 668

monotherapeutic agent in immediately relevant disease settings, e.g., breast or 669

endometrium cancer. 670

671




Page 24 of 30

Acknowledgments 672

We thank Axel Choidas and Bert Klebl for helpful discussions and we are thankful to 673

Prof. Dr. Philippe I. H. Bastiaens for granting access to the Odyssey® CLx Imaging 674

System (Li-Cor). This work was supported by the MERCATOR Foundation (Pr-2016-675

0014). D. Rauh is thankful for support from the German Federal Ministry for 676

Education and Research (NGFNPlus and e:Med) (Grant No. BMBF 01GS08104, 677

01ZX1303C), the Deutsche Forschungsgemeinschaft (DFG) and the German federal 678

state North Rhine Westphalia (NRW) and the European Union (European Regional 679

Development Fund: Investing In Your Future) (EFRE-800400). J.T. Siveke is 680

supported by the European Union s Seventh Framework Programme for research, 681

technological development and demonstration (FP7/CAM-PaC) under grant 682

agreement n° 602783, the German Cancer Aid (grant 70112505), the Erich and 683

Gertrud Roggenbuck Foundation and the German Cancer Consortium (DKTK). 684

685




Page 25 of 30

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30. Muzumdar MD, Chen PY, Dorans KJ, Chung KM, Bhutkar A, Hong E, et al. 771

Survival of pancreatic cancer cells lacking KRAS function. Nat Commun 772

2017;8:1090 773

31. Chou TC. Drug combination studies and their synergy quantification using the 774

Chou-Talalay method. Cancer Res 2010;70:440-6 775

32. Rauch J, Volinsky N, Romano D, Kolch W. The secret life of kinases: functions 776

beyond catalysis. Cell Commun Signal 2011;9:23 777

33. Vivanco I, Chen ZC, Tanos B, Oldrini B, Hsieh WY, Yannuzzi N, et al. A 778

kinase-independent function of AKT promotes cancer cell survival. eLife 779

2014;3:e03751 780

34. Hyman DM, Smyth LM, Donoghue MTA, Westin SN, Bedard PL, Dean EJ, et 781

al. AKT Inhibition in Solid Tumors With AKT1 Mutations. J Clin Oncol 782

2017;35:2251-9 783

784




Page 29 of 30

Figure 1. Co-crystal structure of Akt1 (2-446) in complex with covalent-allosteric 785

inhibitor borussertib. A, Borussertib binds in the allosteric pocket between the 786

catalytic kinase (green) and the regulatory PH domain (blue) forming a covalent bond 787

with Cys296 via Michael addition. B, π-π-stacking between the 1,6-naphthyridinone 788

scaffold and Trp80 and hydrohobic interactions with Leu210, Leu264 and Ile290. 789

Water-mediated hydrogen bonds between the benzo[d]imidazolone moiety and 790

Glu17, Tyr236 and Arg273. The 2FO-FC electron density is depicted as a mesh at 1σ 791

(PDB ID 6HHF). The QR codes can be visualized by the app Augment. 792

793

Figure 2. Antiproliferative activities of reference inhibitors capivasertib (ATP-794

competitive), ipatasertib (ATP-competitive), MK-2206 (allosteric), and miransertib 795

(allosteric) compared with borussertib (quotient of EC50 values of borussertib and 796

reference compounds) in bladder, breast, endometrium and pancreatic cancer cell 797

lines (for original data see Supplementary Table 1). 798

799

Figure 3. In vitro cellular pharmacodynamic studies. A, Western blot analyses for 800

cancer cell lines ZR-75-1 (breast), AN3-CA (endometrium) and Dan-G (pancreas) 801

treated with indicated doses of borussertib for 24 h demonstrating dose-dependent 802

downregulation of pAKTT308, pAKTS473 and phosphorylation of downstream targets 803

4E-BP1, S6 ribosomal protein, and PRAS40. Induction of apoptosis is indicated by 804

cleavage of poly (ADP-ribose) polymerase (cPARP) for ZR-75-1 and AN3-CA cells. 805

B, Half-life determination of AKT in AN3-CA cells treated with indicated doses of 806

borussertib for 24 h prior to washout and medium renewal. Subsequently, cells were 807

grown for indicated time periods followed by stimulation with epidermal growth factor 808

(EGF) for 15 min prior to cell lysis. Efficient downregulation of pAKTS473 can be 809

observed up to 24 h after medium renewal. 810

811

Figure 4. Synergistic inhibitory effect studies of borussertib in combination with MEK 812

inhibitor trametinib and chemotherapeutic agent gemcitabine in Dan-G cells. Cell 813

viability was measured after 72 h treatment with either single agent or drug 814

combination (A, borussertib and trametinib; B, borussertib and gemcitabine) at 815

indicated doses (EC50,borussertib = 2.07 µM; EC50,trametininb = 0.008 µM; 816

EC50,gemcitabine = 0.023 µM). C, Combination index (CI) calculation was performed with 817

CompuSyn Software; strong synergism of borussertib and trametinib in pancreatic 818




Page 30 of 30

cancer cell line Dan-G was observed while no synergistic effects were observed for 819

borussertib in combination with gemcitabine. 820

821

Figure 5. Combination of borussertib and trametinib shows synergistic inhibitory 822

effects in early passage pancreatic cancer cells Bo103 (KRAS-mutant). A, Bo103 823

cells were treated with either single agent or drug combination at the indicated 824

concentrations, and cell viability was measured after 96 hours of treatment. The 825

mean cell viabilities and standard deviations from three independent experiments are 826

plotted relative to DMSO-treated control cells. B, Early passage Bo103 cells were 827

treated with either single agent or drug combination at indicated concentrations for 828

24 hours prior to preparation of whole cell lysates and subsequent immunoblotting to 829

detect pAKTS473, pErk1/2T202/Y204, p4E-BP1S65, pS6S235/236 and β-Actin (loading 830

control). 831

832

Figure 6. In vivo antitumor efficacy of borussertib, trametinib, and their combination 833

in subcutaneous PDX mouse models. Tumor volume was recorded for KRAS-mutant 834

pancreatic (A) and colorectal (B-D) PDX over the indicated time periods. Data 835

represent the mean SD (n ≥ 3). Dashed lines indicate partial response (PR, -30% 836

from baseline) and progressive disease (PD, +20% from baseline) according to 837

RECIST 1.1 criteria. ns, non significant. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, 838

p < 0.0001, two-tailed unpaired t-test. QD, once daily (quaque die). 839






















Published OnlineFirst March 11, 2019.Cancer Res Jörn Weisner, Ina Landel, Christoph Reintjes, et al. Pancreatic and Colorectal CancerBorussertib in Combination with Trametinib in KRAS-mutant Preclinical Efficacy of Covalent-Allosteric AKT Inhibitor

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