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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
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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
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
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
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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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Page 14 of 30
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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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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