Translational Science

Preclinical Efficacy of Covalent-Allosteric AKTInhibitor Borussertib in Combination withTrametinib in KRAS-Mutant Pancreatic andColorectal CancerJ€orn Weisner1,2, Ina Landel1,2, Christoph Reintjes3, Niklas Uhlenbrock1,2,Marija Trajkovic-Arsic4,5, Niklas Dienstbier4,5, Julia Hardick1,2, Swetlana Ladigan3,Marius Lindemann1,2, Steven Smith1,2, Lena Quambusch1,2, Rebekka Scheinpflug1,2,Laura Depta1,2, Rajesh Gontla1,2, Anke Unger6, Heiko M€uller6, Matthias Baumann6,Carsten Schultz-Fademrecht6, Georgia G€unther7, Abdelouahid Maghnouj3,Matthias P. M€uller1,2, Michael Pohl8, Christian Teschendorf9, Heiner Wolters10,Richard Viebahn11, Andrea Tannapfel12,Waldemar Uhl13, Jan G. Hengstler7,Stephan A. Hahn3, Jens T. Siveke4,5, and Daniel Rauh1,2

Abstract

Aberrations within the PI3K/AKT signaling axis are fre-quently observed in numerous cancer types, highlightingthe relevance of these pathways in cancer physiology andpathology. However, therapeutic interventions employingAKT inhibitors often suffer from limitations associated withtarget selectivity, efficacy, or dose-limiting effects. Here wepresent the first crystal structure of autoinhibited AKT1 incomplex with the covalent-allosteric inhibitor borussertib,providing critical insights into the structural basis of AKT1inhibition by this unique class of compounds. Comprehen-sive biological and preclinical evaluation of borussertib incancer-related model systems demonstrated a strong anti-

proliferative activity in cancer cell lines harboring geneticalterations within the PTEN, PI3K, and RAS signaling path-ways. Furthermore, borussertib displayed antitumor activityin combination with the MEK inhibitor trametinib inpatient-derived xenograft models of mutant KRAS pancre-atic and colon cancer.

Significance: Borussertib, a first-in-class covalent-allostericAKT inhibitor, displays antitumor activity in combinationwith the MEK inhibitor trametinib in patient-derived xeno-graft models and provides a starting point for further phar-macokinetic/dynamic optimization.

IntroductionWith its key roles in various cellular processes including cell

proliferation, metabolism, and cell survival, the PI3K/AKT path-way is overactivated in several human cancers, contributing to

tumor development, progression, and metastasis (1–3). Aberra-tions among members of this pathway have been described asoncogenic drivers in diverse cancer types, such as activatingpoint mutations in PI3K and gene deletion or loss-of-function

1Faculty of Chemistry and Chemical Biology, TU Dortmund University,Dortmund, Germany. 2Drug Discovery Hub Dortmund (DDHD) am Zentrum fürIntegrierte Wirkstoffforschung (ZIW), Dortmund, Germany. 3Department ofMolecular Gastrointestinal Oncology, Ruhr-University Bochum, Bochum,Germany. 4German Cancer Consortium (DKTK) and German Cancer ResearchCenter (DKFZ), Heidelberg, Germany. 5Division of Solid Tumor TranslationalOncology, GermanCancer Consortium (DKTK), partner site Essen,WestGermanCancer Center, University Hospital Essen, Essen, Germany. 6Lead DiscoveryCenter GmbH, Dortmund, Germany. 7Leibniz Research Centre for WorkingEnvironment and Human Factors (IfADo), TU Dortmund University, Dortmund,Germany. 8Department of Internal Medicine, Ruhr-University Bochum,Knappschaftskrankenhaus, Bochum, Germany. 9Department of InternalMedicine, St. Josefs-Hospital, Dortmund, Germany. 10Department of Visceraland General Surgery, St. Josefs-Hospital, Dortmund, Germany. 11Department ofSurgery, Ruhr-University Bochum, Knappschaftskrankenhaus, Bochum,Germany. 12Institute of Pathology, Ruhr-University of Bochum, Bochum,Germany. 13Department of Visceral and General Surgery, St. Josef Hospital,Ruhr-University Bochum, Germany.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

J. Weisner, I. Landel, C. Reintjes, and N. Uhlenbrock contributed equally to thisarticle.

Current address for S. Smith: agap2 – Life Sciences, D-60323 Frankfurt amMain,Germany and current address for H. M€uller, Saltigo GmbH Chempark, Building Q18–2, D-51369 Leverkusen, Germany.

Corresponding Authors: Daniel Rauh, TU Dortmund University, Otto-Hahn-Straße 4a, Dortmund 44227, Germany. Phone: 49-0-231-755-7080;Fax: 49-0-231-755-7082; E-mail: daniel.rauh@tu-dortmund.de; Stephan A.Hahn, Department of Molecular Gastrointestinal Oncology, Ruhr-UniversityBochum, Universit€atsstraße 150, Bochum D-44780, Germany. E-mail:stephan.hahn@rub.de; and Jens T. Siveke, Division of Solid Tumor TranslationalOncology, West German Cancer Center, German Cancer Consortium (DKTK),partner site Essen, University Hospital Essen, Hufelandstraße 55, Essen D-45147,Germany. E-mail: jens.siveke@uk-essen.de

doi: 10.1158/0008-5472.CAN-18-2861

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mutations in the tumor suppressor PTEN (4). These geneticlesions result in the augmented generation of the second mes-senger phosphatidylinositol (3,4,5)-trisphosphate, leading tohyperactivation of 3-phosphoinositide dependent protein kinase1 (PDK1) and its substrate AKT, a serine/threonine-specific kinase,also known as protein kinase B. This step acts as a major signalingnode in the PI3K/AKT pathway with hundreds of downstreamsubstrates (5, 6).

In addition to aberrant AKT activity caused by genetic lesionsin upstream-acting proteins, overexpression and activatingmutations have been observed for all three AKT isoforms, forexample in lung, prostate, breast, endometrium, and skincarcinomas (7). Mutations in AKT1 occur most frequently witha mutation rate of 2%–3% in urinary and bladder cancer, inwhich the somatic activating AKT1E17K mutation within theregulatory PH domain is the most prominent genetic lesion andalso described as a driver mutation for the rare Proteus syn-drome (8–10). Furthermore, overexpression of AKT is associ-ated with resistance to several chemotherapeutics (11). Togeth-er, this evidence underlines the crucial role of the PI3K/AKTpathway in cancer progression and highlights the great poten-tial for precisely targeted therapeutic intervention involving thissignaling cascade.

Traditional ATP-competitive AKT inhibitors such as capivaser-tib (AZD5363; refs. 12, 13) and ipatasertib (GDC-0068; refs. 14,15) are under clinical investigation in phase I and II studies. Incontrast to these, allosteric inhibitors, including MK-2206 (16),miransertib (ARQ092; ref. 17), and BAY1125976 (18), whichbind to the inactive kinase conformation of AKT, exhibit exquisitetarget selectivity solely for AKT1–3 while sparing structurallyclosely related AGC kinases, for example, p70S6K and proteinkinase A.

Recently, we reported the development of a covalent-allostericAKT inhibitor, borussertib. This inhibitor specifically binds to twononcatalytic cysteines in AKT at positions 296 and 310 by dec-orating allosteric ligands with electrophilic warheads at suitablepositions, thus enabling the irreversible stabilization of the inac-tive conformation (19). Biochemical analyses have revealedsuperior inhibitory properties compared with reversible ATP-competitive and allosteric AKT inhibitors. Despite initial concernsassociated with covalent kinase inhibitors, recently developeddrugs demonstrated tremendous success in the clinics with majorbeneficial impact on patients' survival rate [e.g., osimertinib(non–small cell lung cancer) and ibrutinib (chronic lymphocyticleukemia); refs. 20, 21].

In this article, we report the first crystal structure of AKT1 incomplex with a covalent-allosteric inhibitor, borussertib (19),showing the unique covalent bond between inhibitor andCys296. Furthermore, we demonstrate its antiproliferativeactivity for a panel of cancer cell lines harboring geneticalterations in PI3K, PTEN, and RAS. To investigate cellularpharmacodynamic changes induced by on-target inhibition ofAKT, western blot studies were performed, and target selectivitywas further corroborated by PathScan analyses. Subsequently,pharmacokinetic characterization and patient-derived xeno-graft (PDX) experiments were conducted, with results demon-strating the potential for optimization and development oforally bioavailable, targeted covalent-allosteric AKT inhibitorsand their applicability in the mono- or combination therapy ofdifferent cancers.

Materials and MethodsProtein expression, purification, and crystallization

A gene encoding for AKT1(2-446, E114/115/116A) includingan N-terminal His6-tag followed by a TEV protease recognitionsitewas synthesized byGeneArt AGand cloned into the pIEx/Bac3expression vector (MerckMillipore) usingNcoI andBamHI restric-tion sites. Transfection, virus generation, and amplification aswellas protein expression were carried out in Spodoptera frugiperda(Sf9) cells (Thermo Fisher Scientific) following the BacMagicprotocol (Merck Millipore). Infected insect cells were grown inErlenmeyer flasks for 72 hours at 27�C with shaking at 120 rpm,subsequently harvested by centrifugation at 3,000 � g for 15minutes and washed once with PBS before being flash frozen inliquid nitrogen. Afterwards, cells were thawed and resuspended inlysis buffer [50 mmol/L Tris, 500 mmol/L NaCl, 1 mmol/L DTT,10% glycerol, 0.1% Triton X-100, pH 8.0, EDTA-free proteaseinhibitor cocktail (Sigma-Aldrich)]. Cells were lysed usinga microfluidizer, the lysate was cleared by centrifugation(40,000� g, 1 hour). The supernatant was loaded onto a Ni-NTASuperflowCartridge (Qiagen). Boundproteinwas eluted in buffercontaining 50 mmol/L Tris, 500 mmol/L NaCl, 500 mmol/Limidazole, 1 mmol/L DTT, 10% glycerol, pH 8.0. For cleavageof the His6-tag, TEV protease was added to the pooled elutionfractions and dialyzed overnight into buffer containing 25mmol/L Tris, 50 mmol/L NaCl, 1 mmol/L DTT, 5% glycerol, pH8.0 at 4�C. The cleaved protein was further purified by anion-exchange chromatography using a HiTrap Q HP Column (GEHealthcare) followed by size-exclusion chromatography on aHiLoad 16/60 Superdex 75 pg Column (GE Healthcare) usingbuffer containing50mmol/LHEPES, 200mmol/LNaCl,1mmol/LDTT, 10% glycerol, pH 7.3. Afterwards, the protein was transferredinto the storage buffer (25 mmol/L Tris, 100 mmol/L NaCl, 1mmol/L DTT, 10% glycerol, pH 7.5) using a Superdex 75 10/300GL Column (GE Healthcare), concentrated and stored at �80�C.

For crystallization, purified protein at a concentration of 3mg/mL was incubated with 3 equivalents of borussertib on icefor 60minutes. The samples were centrifuged at 20,000� g for 10minutes before hanging drops were prepared in 15-well crystal-lization plates (EasyXtal Tool, Qiagen) by mixing protein–ligandcomplex with reservoir solution (1:1) containing 1.25 mmol/Lsodium acetate pH 5.2, 3.75 mmol/L sodium citrate pH 5.2, and15% PEG MME 2000 at 20�C. Diffraction-grade crystals grewwithin 3 days and were cryoprotected using 20% ethylene glycolbefore they were flash cooled in liquid nitrogen. X-ray diffractiondata were collected at the PXII-X10SA beam line of the Swiss LightSource (Paul Scherrer Institute, Villingen, Switzerland)withwave-lengths close to 1 Å. The diffraction data were integrated withXDS (X-ray Detector Software) program package (22) and scaledusing the program XSCALE (22). The crystal structure was solvedby molecular replacement with PHASER (23) using a cocrystalstructure of Akt1 in complex with another covalent-allostericinhibitor as template (24). The manual modification of themolecule of the asymmetric unit was performed using the pro-gram COOT (25) and with the help of the Dundee PRODRGserver (26) the inhibitor topology files were generated. For mul-tiple cycles of refinement, phenix.refine (27) was employed andthe final structure was evaluated by Ramachandran plot analysisusing the server MolProbity (28). Final validation of the modelwas performed with the help of the PDB_REDO server and thecrystal structure was visualized using PyMOL (29, 30).

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Cell culture and inhibitorsT-47D and ZR-75-1 cell lines were purchased from Sigma-

Aldrich/ECACC. KU-19-19 cells were purchased from DSMZ.AN3-CA and HPAF-II cells were obtained from ATCC. BT-474,Dan-G, and MCF-7 were purchased from CLS Cell Lines Service.Cell lines were cultured in DMEM, MEM, or RPMI1640 medi-um (Gibco) supplemented with 10% FBS (PAN-Biotech) and1% penicillin/streptomycin (Gibco). For AN3-CA and MCF-7,1 mmol/L sodium pyruvate (Gibco) was added to the growthmedium and media for BT-474 and T-47D were supplementedwith 10 mg/mL insulin (Sigma-Aldrich). Bo103 cells werecultured in a 1:1 mixture of DMEM/F12 (Gibco) and DMEM(Gibco), supplemented with 5% FBS (Gibco), 2% penicillin/streptomycin (Gibco), 1.6 mg/mL Amphotericin B (Gibco), 10mmol/L ROCK inhibitor Y-27632 (LC Laboratories), 10 mg/mLciprofloxacin (Sigma-Aldrich), 8.4 ng/mL cholera toxin (Sigma-Aldrich), 10 mg/mL insulin (Sigma-Aldrich), 20 nmol/L 1-thio-glycerol (Sigma-Aldrich), and 0.5 mmol/L sodium pyruvate(Gibco). Cells were cultured in a humidified incubator at37�C, 5% CO2 and cell line authenticity was confirmed bySTR analysis at Microsynth AG or by SNP profiling at Multi-plexion.Mycoplasma testing has not been performed. Cells wereused for viability and western blot analyses within 8 weeks afterthawing.

Borussertib was synthesized as described previously (24).Reference inhibitors capivasertib, ipatasertib, MK-2206, andmiransertib were purchased from SelleckChem; staurosporineand Y-27632 were obtained from LC Laboratories; trametinibwas obtained from LC Laboratories and Hycultec.

Cell viability analysisOn day 0, cells were plated into white 384-well cell culture

plates (Greiner Bio-One) using a Multidrop reagent dispenser(Thermo Fisher Scientific) at cell numbers that ensure linearand optimal luminescent signal intensity (AN3-CA: 800 cells/well; BT-474: 400 cells/well; Dan-G: 400 cells/well; HPAF-II:400 cells/well; KU-19-19: 400 cells/well; MCF-7: 200 cells/well; T-47D: 800 cells/well; ZR-75-1: 400 cells/well; andBo103: 800 cells/well). Following incubation for 24 hours ina humidified atmosphere at 37�C/5% CO2, cells were treatedwith inhibitors in serial dilutions ranging from 30 mmol/Ldown to 0.1 nmol/L using an Echo 520 acoustic liquid handler(Labcyte Inc.). Cell viability was analyzed on day 5 usingthe CellTiter-Glo Assay (Promega) as per the manufacturer'sinstructions. Luminescence was recorded using an EnVisionMultilabel 2104 Plate Reader (PerkinElmer) using 500 msintegration time. The obtained data were normalized tothe plate positive control (30 mmol/L staurosporine) andnegative control (DMSO) and subsequently analyzed andfitted with the Quattro Software Suite (Quattro Research) usinga four parameter logistic model. As quality control, the Z0-factor was calculated from 16 positive and negative controlvalues. Only assay results showing a Z0-factor � 0.5 were usedfor further analysis. All experimental points were measuredin duplicates for each plate and were replicated in at leastthree plates.

Combination studies were conducted using the CompuSynSoftware (Biosoft) for calculating the combination index (CI)equation to determine synergism of drug combinations usingfixed drug ratios as described by Chou-Talalay (31).

Western blot and PathScan analysisForprotein isolation, cellswere seeded into 6-well tissue culture

plates (Sarstedt) yielding 80%-90% confluency after overnightincubation. Afterwards, cells were treated with various concentra-tions of inhibitors or DMSO and incubated for additional24 hours before the medium was removed and cells were washedonce with ice-cold PBS. Cell lysis was initiated by addition of100 mL RIPA buffer (Cell Signaling Technology) per well supple-mented with phosphatase and protease inhibitor cocktails (Sig-ma-Aldrich) followed by incubation on ice for 30 minutes.Cells were then harvested by scraping and transferred into pre-cooled microcentrifuge tubes. Whole-cell lysates were cleared bycentrifugation at 14,000 � g/4�C for 10 minutes and transferredinto fresh, precooled microcentrifuge tubes. Protein concentra-tions were determined using the Pierce BCA Protein Assay(Thermo Fisher Scientific) as per the manufacturer's instructions.Equal amounts of protein were separated by SDS-PAGE andtransferred to Immobilon-FL PVDF Membranes (Merck Milli-pore) using Pierce 1-step transfer buffer (Thermo Fisher Scientific)and the Pierce Power Blotter (Thermo Fisher Scientific). Mem-branes were washed for 5 minutes with ddH2O, blocked withOdyssey Blocking Buffer TBS (Li-COR Biosciences) for 1 hour atroom temperature and then incubated with primary antibodiesdiluted in Odyssey Blocking Buffer TBS overnight at 4�C withgentle agitation. The next day, membranes were washed threetimes with TBS-T (50 mmol/L Tris, 150 mmol/L NaCl, 0.05%Tween 20, pH 7.4) for 5 minutes before being incubated withsecondary antibodies diluted inOdyssey Blocking Buffer TBS for 1hour at room temperature with gentle agitation. Finally, themembranes were washed three times for 5 minutes with TBS-Tand then scanned using anOdyssey CLx Imaging System (LI-CORBiosciences).

For capillary western blot analysis, lysates of the cell pelletswere prepared as described above and protein concentration wasestimated. SimpleWes assay was performed and analyzed accord-ing to the manufacturer's instructions (Protein Simple). ForpAKTS473 detection, 0.55 mg of protein was loaded per capillarywith 1:20 dilution of anti-pAKTS473 antibody while tAKT wasdetected with anti-tAKT antibody, dilution 1:100 and 0.05 mg oftotal protein per capillary.

For PathScan Akt Signaling Array (Cell Signaling Technology),1� array wash buffer, 1� detection antibody cocktail, 1� horse-radish peroxidase (HRP)-linked streptavidin, and the multi-wellgasket were prepared as per the manufacturer's instructions andglass slides and blocking buffer were calibrated to room temper-ature. Subsequently, 100 mL array blocking buffer was added toeach well for 15 minutes at room temperature covered withsealing tape and placed on an orbital shaker (as well as allfollowing incubation steps). After removal of the blocking buffer,50 mL diluted lysate (0.5 mg/mL protein concentration) wereadded to each well and incubated for 2 hours at room temper-ature. Wells were washed three times with 100 mL 1� array washbuffer for 5 min at room temperature, followed by incubation for1 hour at room temperature in 75 mL 1� detection antibodycocktail. Wash steps were repeated four times before adding 75 mL1� HRP-linked streptavidin to each well for a 30 minute incu-bation at room temperature. The multi-well gasket was removedfrom the slides another four wash steps later and the slides werewashedwith10mL1� arraywashbuffer. Slideswere then coveredwith LumiGlo/Peroxide reagent (as per the manufacturer's

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instructions) and images with different exposure times werecaptured using a digital imaging system.

Half-time determination (Western blot analysis)For protein isolation, AN3-CA cells were seeded into 10-cm

dishes (Sarstedt). Treatment with the inhibitors (borussertib,MK-2206, and miransertib) was initiated at a confluency of60%–70% with the indicated concentrations for 24 hours. Thenthe medium was removed and cells were washed twice with PBS.Medium without borussertib was added to start the wash outexperiments, which were terminated at the indicated timepoints. Prior to the cell harvest, cells were either not stimulatedat all or stimulated with EGF (100 ng/mL, PeproTech), TGF-a(100 ng/mL, R&D Systems), or insulin (100 nmol/L, Sigma-Aldrich) for 15 minutes. Then the medium was removed andcells were washed twice with ice-cold PBS. Cell lysis wasinitiated by addition of 500 mL RIPA buffer per dish supple-mented with phosphatase (Sigma-Aldrich) and protease inhib-itor cocktails (Roche) followed by harvesting the cells byscraping and transferred into precooled microcentrifuge tubes.Cell lysates were incubated on ice for 30 minutes. After that,cells were lysed by sonication, cleared by centrifugation at14,000 � g at 4�C for 10 minutes and transferred into fresh,precooled microcentrifuge tubes. Protein concentrations of thelysates were determined by the Bradford protein assay system(Bio-Rad). Equal amounts of protein (36 mg protein each lane)were separated by SDS-PAGE and transferred to polyvinylidenedifluoride (PVDF) membranes (Roth). Immunoblots wereblocked with 5% BSA in 1� TBS and Tween-20 (0.1% v/v) for1 hour at room temperature. The membrane was incubatedovernight at 4�C with primary antibodies. Afterwards, themembrane was incubated with the corresponding secondaryantibody conjugated with HRP (Dianova). Bands were visual-ized with enhanced chemiluminescence Western blot detectionsystem (Thermo Fisher Scientific).

AntibodiesAnti-pAkt(Ser473; Cell Signaling Technology, catalog nos.

3787 and 4060), anti-pAKT (Thr308;Cell Signaling Technology,catalog no. 2965), anti-tAKT (Cell Signaling Technology, catalognos. 2920 and 4691), anti-pS6 ribosomal protein (Ser235/236;Cell Signaling Technology, catalog nos. 2211 and 4858), anti-pPRAS40(Thr246;Cell Signaling Technology, catalog no. 3997),anti-pERK1/2 (Thr202/Tyr204;Cell Signaling Technology, catalogno. 4370), anti-p4E-BP1(Ser65;Cell Signaling Technology,catalog no. 13443), anti-pGSK-3b (Ser9;Cell Signaling Technol-ogy, catalog no. 9323), anti-GSK-3b (Cell Signaling Technology,catalog no. 9315), anti-PARP (Cell Signaling Technology, catalogno. 9542), anti-HSP90, (Cell Signaling Technology, catalog no.4874), anti-b-Actin (Cell Signaling Technology, catalog no. 4970/Sigma-Aldrich, catalog no. A5441), anti-GAPDH (Cell SignalingTechnology, catalog no. 2118), anti-mouse IgG (HþL; DyLight680 Conjugate; Cell Signaling Technology, catalog no. 5470),anti-rabbit IgG (HþL; DyLight 800 4X PEG Conjugate; CellSignaling Technology, catalog no. 5151).

In vitro pharmacokinetic studiesFor determining kinetic solubility, the compound was diluted

from a 10 mmol/L stock in DMSO to a final concentration of500 mmol/L in 50 mmol/L HEPES, pH 7.4. Following an incu-bation of 90 minutes at room temperature on a shaker, the

aqueous dilution was filtered through a 0.2-mm PVDF filter, andthe optical density between 250 and 500 nm was measured atintervals of 10 nm. The kinetic solubility was calculated from theAUCbetween 250 and 500 nmand normalized to absorption of adilution of the compound in acetonitrile.

Metabolic stability under oxidative conditions was mea-sured in human and murine liver microsomes by LC/MS-basedanalysis of depletion of compound at a concentration of 3mmol/L over time up to 50 minutes at 37�C. On the basis ofcompound half-life, t1/2, the in vitro intrinsic clearance, Clint,was calculated.

Plasma stability was measured by LC/MS-based determinationof percent remaining of selected compound at a concentration of5 mmol/L after incubation in 100% plasma obtained from dif-ferent species for 1 hour at 37�C.

Assessment of plasma protein binding was measured by equi-librium dialysis by incubating the compound of interest at aconcentration of 5 mmol/L for 6 hours at 37�C in 50% plasmain buffer (v/v) followed by LC/MS-based determination of finalcompound concentrations. The resulting fraction unbound at50% plasma (fu50%) was extrapolated to the fraction unboundat 100% plasma (fu100%) using the following equation: fu100% ¼fu50%/(2 � fu50%).

In vivo pharmacokinetic studiesFor in vivo pharmacokinetic analysis, RjOrl:SWISS mice (Jan-

vier Labs), age 8–10 weeks, were treated with borussertib byoral gavage (20 mg/kg), intraperitoneal (20 mg/kg), or intra-venous (2 mg/kg) administration. The compound was formu-lated in PBS/PEG200 (60:40) for oral and intraperitonealadministration, whereas it was dissolved in DMSO for intra-venous injection. Blood was collected 5, 15, 45, and 135minutes after compound administration, immediately centri-fuged at 15,000 � g for 10 minutes at 4�C and plasma sampleswere stored at �80�C for subsequent LC-MS/MS analysis. Threemice were analyzed per experimental condition (for each timepoint and route of administration). Mice were fed ad libitumwith Allein-Futter f€ur Ratten-/M€ausehaltung (Sniff Special DietsGmbH). They had free access to water and were kept in a 12hour day/night rhythm. All experiments were approved by thelocal authorities.

Samples and blanks were prepared by adding 2.5 mL blankDMSO and 80 mL of ice-cold acetonitrile containing the internalstandard (griseofulvin, 1 mmol/L) to 20 mL plasma followed bycentrifugation at 13,000 rpm (4�C) for 10 minutes. A total of65 mL of the supernatant were diluted with 65 mL of LC/MSgrade water. Samples were filtered (MSRLN0450, Millipore) andsubjected to LC/MS measurement. Analyte stock solution (10mmol/L in DMSO) was diluted in DMSO to yield DMSO stocksolutions with the following concentrations: 10, 5, 2.5, 1, 0.5,0.25, 0.1, 0.05, 0.025, 0.01, 0.005, and 0.0025 mmol/L. A total of2.5 mL of the corresponding DMSO stock solution were added to20 mL of blank plasma followed by the addition of 80 mL of ice-cold acetonitrile containing the internal standard. The sampleswere centrifuged for 10minutes at 4�C and 13,000 rpm. A total of65 mL of the supernatant were diluted with 65 mL of LC/MS gradewater and subjected to LC/MS analysis. A set of three differentquality controls (n ¼ 3) was prepared by adding 2.5 mL DMSOstock solution (5, 0.5, and 0.05 mmol/L) to 20 mL of plasma.Samples were subsequently handled as described above. Allsamples were analyzed using a Shimadzu LC20ADXR Solvent

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Delivery Unit, a Shimadzu SIL30ACMP autosampler, and anABSciex Qtrap5500 LC-MS/MS system. Therefore, 2 mL of samplewere injected and separated using an Agilent Poroshell C18, 2.7mmcolumn (2.1mm� 50mm) at 60�C starting at 5%of solvent Bfor 0.3 minutes followed by a gradient up to 100% of solvent Bover 0.6 minutes (flow rate 1 mL/min) with 0.1% formic acid inwater as solvent A and 0.1% formic acid in acetonitrile as solventB. Data evaluation was performed using Analyst 1.6.2 Software(Sciex).

Animal models and treatmentsTissue samples to establish the PDX models were collected

from patients following surgical intervention for colon cancer orpancreatic adenocarcinoma at the Ruhr-University Comprehen-sive Cancer Center (Bochum, Germany). From all patientsinformed and written consent were obtained. The studies wereapproved by the Ethics Committees of the Ruhr-UniversityBochum (registry nos. 3534-09, 3841-10, and 16-5792, Bochum,Germany). Animal experiments and care were in accordance withthe guidelines of institutional authorities and approved by localauthorities (nos.: 8.87-50.10.32.09.018, 84-02.04.2012.A328,84-02.04.2012.A360, 84-02.04.2015.A135, and 81-02.04.2017.A423). Nondiagnostic tissue samples were selected by the pathol-ogist within 2–6 hours postsurgery. Selected tumor pieces (1–2mm) were soaked in undiluted Matrigel (Becton Dickinson) for15–30 minutes and subsequently implanted subcutaneouslyonto 5- to 10-week-old female mice (NMRI-Foxn1nu/Foxn1nu,Janvier Labs) at two sites (scapular region, one mouse per tumor)using as many as 4 pieces per site. To establish treatment cohorts,

early passage (�F5 generation) PDX tumor pieceswere implantedas described above into nude mice and were allowed to grow to asize off approximately 100–200 mm3, at which time mice wererandomized in the treatment and control groupswith 3–4mice ineach group. Tumor volumes were estimated from two-dimen-sional tumor measurements by bi-weekly caliper measurementsusing the following formula: tumor volume (mm3) ¼ [length(mm) � width (mm)2]/2. Response was defined in analogy toRECIST 1.1 criteria with at least 30% reduction in mean tumorvolume compared with the mean tumor volume at start oftreatment being a partial response (PR) and an undetectabletumor being a complete response. Disease progression wasdefined as more than 20% increase in mean tumor volume tothe tumor volume at the beginning of treatment. All othermeasurements were defined as stable disease. Mice were treatedwith borussertib by daily intraperitoneal injection dosed at20 mg/kg and trametinib (Hycultec) by oral gavage at 0.5 mg/kgper day with a weekly treatment cycle comprising of 5 consecutivedays of treatment followed by 2 days treatment pause.

ResultsBorussertib binds covalently between the kinase and PHdomain of AKT

Recently, we described a novel class of AKT inhibitors that bindinto the allosteric pocket of AKT and harbor a Michael acceptorto covalently bind to noncatalytic cysteines. These features com-bine the advantage of outstanding selectivity of PH domain–dependent allosteric inhibition with the therapeutic benefit

Figure 1.

Cocrystal structure of Akt1 (2-446) in complex with covalent-allosteric inhibitor borussertib. A, Borussertib binds in the allosteric pocket between the catalytickinase domain (green) and the regulatory PH domain (blue), forming a covalent bond with Cys296 via Michael addition. B, p–p stacking between the 1,6-naphthyridinone scaffold and Trp80 and hydrophobic interactions with Leu210, Leu264, and Ile290. Water-mediated hydrogen bonds between thebenzo[d]imidazolonemoiety and Glu17, Tyr236, and Arg273. The 2FO-FC electron density is depicted as a mesh at 1s (PDB ID 6HHF). The QR codes can bevisualized by the app Augment (35).

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of irreversible modification, leading to increased drug targetresidence time and gain of potency. In this class of inhibitors,we identified a potent lead compound, borussertib, exhibiting anexquisite kinase selectivity profile (19). To investigate the bindingmodeof this novel AKT inhibitor at the atomic level, we solved thecocrystal structure of AKT1 in complex with borussertib to aresolution of 2.9 Å.

The crystal structure discloses the inactive, autoinhibited con-formation with the PH domain folded onto the kinase domain(PH-in conformation) between the N- and the C-lobe, therebydisplacing the regulatory helix aC and simultaneously shapingan allosteric binding pocket at the interface between thesetwo domains (Fig. 1A; Supplementary Fig. S1; SupplementaryTable S1). Borussertib binds to this allosteric pocket and forms akey aromatic p–p stacking interaction between the 1,6-naphthyr-idinone scaffold and the indole side chain of Trp80 in the PHdomain. Additional hydrophobic contacts can be observedbetween the phenyl ring in the 3-position and Leu210, Leu264,and Ile290. Water-mediated hydrogen bonds between Glu17,Arg273, Tyr326, and the benzo[d]imidazolone moiety of borus-sertib foster thehigh-affinity reversible bindingof the ligand to the

kinase (Fig. 1B). Furthermore, the acrylamide moiety is pre-oriented by a hydrogen bond formed between the amide oxygenof the warhead and the backbone NH of Glu85, facilitatingcovalent bond formation between the electrophilic b-carbon andthe thiol side chain of Cys296.

Borussertib potently inhibits proliferation of PI3K/PTEN-mutated cell lines

To investigate the in vitro antiproliferative activity of borusser-tib, we employed breast, bladder, pancreas, and endometriumcancer cell lines harboring genetic alterations in the PI3K/AKT andRAS/MAPK pathways, that is, AN3-CA (endometrium), BT-474(breast), Dan-G (pancreas), HPAF-II (pancreas), KU-19-19 (blad-der), MCF-7 (breast), T-47D (breast), and ZR-75-1 (breast).Genetic alterations include in-frame deletions in PIK3R1, frameshifts and point mutations in PTEN, and (activating) pointmutations in PIK3CA, NRAS, and KRAS (Supplementary TableS2). Each cell line was treated for 96 hours with borussertib orreference inhibitors in a concentration range from 30 mmol/L to0.1 nmol/L. In these cell lines, weobserved outstanding sensitivityto borussertib with EC50 values in the submicromolar range,

Figure 2.

Antiproliferative activities of referenceinhibitors capivasertib (ATP-competitive),ipatasertib (ATP-competitive), MK-2206(allosteric), and miransertib (allosteric)compared with borussertib (quotient ofEC50 values of borussertib and referencecompounds) in bladder, breast,endometrium, and pancreatic cancer celllines (for original data, see SupplementaryTable S1).

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indicating a 1.5- to 43-fold greater potency thanmiransertib, MK-2206, ipatasertib, and capivasertib (Fig. 2). Only in the bladdercancer cell line KU-19-19, which harbors additional activatingmutations in AKT1 (E17K/E49K) and NRAS (Q61R), micromolarantiproliferative activities could be observed (EC50 ¼ 3.1–5.0mmol/L) for the tested compounds being in good correlationwithpreviously published data for the allosteric AKT inhibitor BAY1125976 (18). Furthermore, for pancreatic cancer cell linesDan-Gand HPAF-II, generally lower sensitivities to AKT inhibition wereobserved with only minor differences between the individualinhibitors (Supplementary Table S2). The lack of mutationswithin PI3K and/or PTEN in combination with codon 12 muta-tions in KRAS substantiates the relatively high EC50 valuesobserved for these two cell lines.

Of note, the breast cancer cell line ZR-75-1 exhibited a pro-nounced sensitivity to borussertib, with an EC50 of 5� 1 nmol/Land thus an approximately 7- to 12-fold higher potency comparedwith the reversible allosteric inhibitors miransertib (EC50 ¼ 35�18 nmol/L) and MK-2206 (EC50 ¼ 63 � 21 nmol/L); ATP-competitive inhibitors capivasertib (EC50 ¼ 191 � 68 nmol/L)and ipatasertib (EC50 ¼ 219 � 83 nmol/L) showed a 38- to43-fold lower activity, respectively. The significant differences inantiproliferative activity observed for some of the tested celllines can only to some extent be explained by the biochemicalinhibitory potencies toward AKT1; capivasertib (IC50 ¼ 0.9� 0.1nmol/L) exhibits a higher potency with respect to inhibition ofAKT1 in vitro comparedwith ipatasertib (IC50¼ 3.5�0.6 nmol/L)and MK-2206 (IC50 ¼ 10.0 � 2.1 nmol/L; ref. 24). However,MK-2206 showed a 5- to 9-fold higher activity in MCF-7 cells,whereas capivasertib and ipatasertib inhibited growth of AN3-CA,BT-474, and T-47D cells with similar activities. Differences in

cellular pharmacokinetic properties as well as AKT1 expressionand activity levels might have contributed to these observations.In addition, kinase-independent functions related to specificconformations stabilized by either ATP-competitive or allostericAKT inhibitors could affect in vitro as well as in vivo potency (32,33). Moreover, the molecular impact of AKT isoforms 1–3 on cellsurvival and proliferation is not fully understood, and a potentialinfluence of the compounds' selectivity profiles toward AKT1,AKT2, and AKT3 on their antiproliferative efficacy cannot beexcluded (6). In summary, the experimental inhibitor borussertibexhibited superior antiproliferative properties in our experimen-tal setup compared with the clinical candidates of ATP-compet-itive and allosteric reference compounds, indicating a potentiallybeneficial impact of our approach to irreversibly target AKT.

Borussertib downregulates AKT-mediated signalingTo gain further insights into the molecular mode of action and

how it affects AKT signaling, we performed PathScan AKT signal-ing assays in combination with Western blot studies. Further-more, washout experiments were performed to reconstructthe average half-life of irreversibly inhibited AKT and thus antic-ipate the mean projected duration of compound treatment. ForPathScan analysis, MCF-7 and Dan-G cells were treated withdimethyl sulfoxide (vehicle) or 1 mmol/L borussertib for 24 hoursprior to lysis and array-based readout (Supplementary Fig. S2).For MCF-7 cells, the results indicate low basal levels of activatedAKT (pAKTS473), whereas PRAS40 as well as GSK-3a/b exhibitedpronounced phosphorylation in vehicle-treated cells. Upon treat-ment with 1 mmol/L borussertib, pAKT levels were reduced andphosphorylation of PRAS40 and GSK-3a/b significantlydecreased, hinting at potent inhibition of AKT signaling.

Figure 3.

In vitro cellular pharmacodynamicstudies. A,Western blot analyses forcancer cell lines ZR-75-1 (breast),AN3-CA (endometrium), and Dan-G(pancreas) treated with indicated dosesof borussertib for 24 hoursdemonstrating dose-dependentdownregulation of pAKTT308, pAKTS473,and phosphorylation of downstreamtargets 4E-BP1, S6 ribosomal protein,and PRAS40. Induction of apoptosis isindicated by cleavage of PARP (cPARP)for ZR-75-1 and AN3-CA cells. B, Half-life determination of AKT in AN3-CAcells treated with indicated doses ofborussertib for 24 hours prior towashout and medium renewal.Subsequently, cells were grown forindicated time periods, followed bystimulation with EGF for 15 minutesprior to cell lysis. Efficientdownregulation of pAKTS473 can beobserved up to 24 hours after mediumrenewal.

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Moreover, phospho-S6 and phospho-p70 S6 kinase signals werediminished upon inhibitor treatment. Notably, borussertibexhibited no off-target inhibition toward activating kinase PDK1and RAS/MAPK signaling. Comparable results were observed forDan-Gcells including thedownregulationofpAKTS473, pS6S235/236,pPRAS40T246, and pGSK-3aS21.

Western blot analyses for ZR-75-1, AN3-CA, Dan-G, T-47D,and MCF-7 cell lines resolved the dose-dependent downregula-tion of pAKTT308 and pAKTS473 as well as downstream targetspPRAS40T246, pS6S235/236, and p4E-BP1S65 (Fig. 3A; Supple-mentary Fig. S3). For all cell lines, inhibition of AKT phos-phorylation was observed upon drug treatment demonstratingthe highest sensitivity for ZR-75-1 and AN3-CA cells, correlat-ing well with the pronounced inactivation of AKT-mediateddownstream signaling, as can be seen by the substantialdephosphorylation of PRAS40, S6, and 4E-BP1 (Fig. 3A). Inaddition, to investigate the underlying mechanism of borus-sertib's antiproliferative activity, cell lysates were probed forcleavage of PARP revealing a distinct induction of apoptosis inAN3-CA and ZR-75-1 cells after compound treatment at nano-molar concentrations (Fig. 3A). In contrast, no increase incleaved PARP (cPARP) level could be observed for KRAS-mutant Dan-G cells at concentrations as high as 10 mmol/L,indicating a lower dependence on AKT-mediated signaling.

To determine the cellular half-life of covalently inhibited AKT,AN3-CA cells were treated with 0, 100, and 200 nmol/L borus-

sertib, respectively, for 24 hours, followed by medium renewaland serum starvation for 0–48 hours. Prior to cell lysis, cellswere stimulated with EGF for 15 minutes. pAKTS473 levelsremained significantly downregulated up to 24 hours after medi-um renewal and drug withdrawal (Fig. 3B). Similar results wereobtained for unstimulated, EGF-related TGF-a–treated, and insu-lin-treated cells (Supplementary Fig. S4A-S4D). In contrast toborussertib, higher concentrations were required for MK-2206andmiransertib to completely inhibit AKT-mediated signaling, asshown for pGSK-3bS9 and pS6S235/236 (Supplementary Fig. S4E),despite efficient downregulation of pAKTS473 at 200 nmol/L.Withthe slow recovery of pAKTS473 levels upon irreversible inhibitionwith borussertib, we propose that with respect to in vivo studies,single-compound administrations might be efficacious for arelatively long period of time, independent of the in vivo phar-macokinetics, provided that a sufficient amount of inhibitorreaches its target before being cleared. However, also reversibleinhibition using MK-2206 andmiransertib resulted in prolongeddownregulation of pAKTS473 after compound washout (Supple-mentary Fig. S5).

AKTiborussertib andMEKi trametinib act synergistically in vitroIn addition to the potent antiproliferative efficacy of borusser-

tib toward PI3K/PTEN-mutated cell lines, we were interested inthe identification of potential additive or synergistic effects of AKTinhibition in combination with targeted MEK inhibition or

Figure 4.

Synergistic inhibitory effect studies of borussertib in combination with MEK inhibitor trametinib and chemotherapeutic agent gemcitabine in Dan-G cells. Cellviability was measured after 72 hours treatment with either single agent or drug combination borussertib and trametinib (A); borussertib and gemcitabine (B) atindicated doses (EC50,borussertib¼ 2.07 mmol/L; EC50,trametininb¼ 0.008 mmol/L; EC50,gemcitabine¼ 0.023 mmol/L). C, CI calculation was performed with CompuSynSoftware; strong synergism of borussertib and trametinib in pancreatic cancer cell line Dan-G was observed while no synergistic effects were observed forborussertib in combination with gemcitabine.

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chemotherapy. Therefore, Dan-G cells were treated with borus-sertib and MEKi trametinib or gemcitabine, respectively, at con-centrations close to the respective EC50 (Fig. 4A and B). Employ-ing the Chou-Talalay method (31), combination indices (CI)were calculated for the tested drug combinations to determinepotential additive (CI¼1), synergistic (CI<1), or antagonistic (CI> 1) effects. For the combination of borussertib and trametinib,strong synergy was observed over the entire range of concentra-tions tested, whereas no significant beneficial effect could beobserved for combination treatment with borussertib and gem-citabine even at highdoses (Fig. 4C). InhibitionofMAPKpathwayactivity via trametinib has failed so far in clinical trials in KRAS-driven tumors, which may be due to concomitant activity ofPI3K/AKT pathway activity or induction of resistance via cross-talk among other reasons. Recent data showed PI3K activationupon complete ablation of KRAS in PDAC cells (30), furthersupporting strategies to block AKT activation in combinationwith RAS/MAPK-acting drugs. With regard to gemcitabine,efficacy of this chemotherapeutic agent is regulated on multiplelevels including expression of transporter genes (e.g., hENT1),intracellular drug metabolism, and cell-cycle state amongothers. Because the combination of borussertib with gemcita-bine showed no clear synergistic signal in PDAC cells, we didnot follow this path in more detail but focused on rational drugcombinations with favorable combinatory index.

To further investigate the potential of AKTi/MEKi combina-tion therapy including our covalent-allosteric inhibitor borus-sertib, we utilized early passage pancreatic cancer cells (Bo103)harboring a KRAS mutation in codon 12 as the model of

interest for viability studies in combination with Western blotanalyses. Neither borussertib nor trametinib monotherapyresulted in complete inhibition of cell viability at concentra-tions up to 30 mmol/L; remaining cell viabilities at the highestconcentrations of borussertib and trametinib were determinedto be 51.5% � 9.5% and 29.3% � 5.9%, respectively (Fig. 5A).For the combination treatment, both compounds were addedto the cells in a 1:1 stoichiometry yielding a highest totalconcentration of 30 mmol/L (15 mmol/L borussertib/15mmol/L trametinib); a remaining cell viability of 6.9% �2.9% (mean � SD) was determined from three independentexperiments, indicating a substantial benefit of combinationtreatment as compared with single-agent therapy. The resultingEC50 of 82.7 � 26.0 nmol/L additionally highlights thesupreme efficacy of the combination therapy tested herein.EC50 values for either monotherapy were not calculated dueto incomplete inhibition of cell viability (Fig. 5A).

For correlation of antiproliferative activity with downregula-tion of pS6S235/236 and p4E-BP1S65 induced by either AKT orMEK inhibition, Western blots were prepared for pancreaticcancer Bo103 cells treated with single agent or drug combina-tion (Fig. 5B). Pronounced downregulation of pAKTS473 orpERK1/2T202/Y204 was detected upon treatment with borusser-tib and trametinib, respectively, correlating with decreasingamounts of detectable pS6S235/S236. However, p4E-BP1S65 wasnot affected by treatment with either of the two compounds.Similar effects were observed for cells treated with drug com-bination, yet showing a more distinct decrease of pS6S235/S236.These observations might explain the superior antiproliferative

Figure 5.

Combination of borussertib andtrametinib shows synergistic inhibitoryeffects in early passage pancreatic cancercells Bo103 (KRAS-mutant). A, Bo103cells were treated with either single agentor drug combination at the indicatedconcentrations, and cell viability wasmeasured after 96 hours of treatment.The mean cell viabilities and SDs fromthree independent experiments areplotted relative to DMSO-treated controlcells. B, Early passage Bo103 cells weretreated with either single agent or drugcombination at indicated concentrationsfor 24 hours prior to preparation ofwhole-cell lysates and subsequentimmunoblotting to detect pAKTS473,pErk1/2T202/Y204, p4E-BP1S65, pS6S235/236,and b actin (loading control).

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efficacy of combination compared with single-agent treatmentand thus indicate the enormous potential of covalently target-ing AKT.

Borussertib exerts antitumor activity in KRAS-mutant PDXs incombination with trametinib

To investigate the potential of borussertib as adrug candidate inpreclinical studies, we performed in vitro and in vivo pharmaco-kinetic analyses for borussertib (Supplementary Fig. S6). Besidesan unfavorable low solubility in aqueous media (13 mmol/L),in vitro analyses in both human and murine samples revealedpromising features with generally low intrinsic clearance (Clint,human ¼ 7 mL/min/mg, Clint,murine ¼ 31 mL/min/mg), high plasmastability (human: 99% remaining, murine: 100% remaining) andhigh plasma protein binding (human: 100%, murine: 99%;Supplementary Fig. S6A). Subsequent pharmacokinetic studieswere carried out in mice (2 mg/kg i.v.; 20 mg/kg oral gavage; and20mg/kg, i.p.; Supplementary Fig. S6B). Despite a rather low oralbioavailability (<5%), reaching only a maximum plasma con-

centration of 78 ng/mL (0.13 mmol/L), we found a significantlyhigher bioavailability upon intraperitoneal administration(39.6%), with maximum plasma levels of 683 ng/mL (1.14mmol/L), indicating sufficient absorption of the compound topotentially exert antitumor activity in xenografts.

Given the promising pharmacokinetic and pharmacodynam-ic properties, we next examined the antitumor activity ofborussertib in mouse xenograft studies, using implanted xeno-graft models derived from KRAS-mutant primary pancreasand colon cancers. These tumor entities are characterized byRAS/MAPK activity but considerable resistance to single-agentMAPK pathway inhibition clinically. Encouraged by our in vitroanalyses indicating synergistic effects, we therefore focused oncombined activity of MEK and AKT inhibition with trametiniband borussertib, respectively. For PDX studies, borussertib wasadministered at 20 mg/kg per intraperitoneal injection oncedaily, either as monotherapy or in combination with thetargeted MEK inhibitor trametinib at 0.5 mg/kg administeredperorally for 5 days per week.

Figure 6.

In vivo antitumor efficacy of borussertib, trametinib, and their combination in subcutaneous PDXmouse models. Tumor volumewas recorded for KRAS-mutantpancreatic (A) and colorectal (B–D) PDX over the indicated time periods. Data represent the mean� SD (n� 3). Dashed lines indicate partial response (PR;�30% from baseline) and progressive disease (PD;þ20% from baseline) according to RECIST 1.1 criteria. ns, nonsignificant; � , P < 0.05; ��, P < 0.01; ��� , P < 0.001;���� , P < 0.0001; two-tailed unpaired t test. QD, once daily (quaque die); p.o., orally.

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First, PDAC PDX models were used to evaluate borussertib forits antitumor activity (Fig. 6A; Supplementary Fig. S7A and S7B).Borussertib monotherapy resulted in insignificant tumor growthdelays in Bo103 PDX (Fig. 6A), Bo73 (Supplementary Fig. S7A),and Bo85 (Supplementary Fig. S7B) compared with untreatedcontrol mice. In contrast, trametinib monotherapy induced adecelerated tumor growth in all tested PDX models. However,only the combination of borussertib with trametinib resulted in adurable partial response in Bo103, whereas progressive diseaseswere observed for Bo73 and Bo85.

In addition, we employed KRAS-mutant colorectal carcino-ma PDX models to further evaluate the antitumor activity ofborussertib (Fig. 6B–D; Supplementary Fig. S7C and S7D). Infour of five established model systems, borussertib monother-apy did not affect tumor growth as compared with untreatedcontrol mice (Fig. 6B and D; Supplementary Fig. S7C and S7D).Nevertheless, the PDX cohort engrafted with BoC105 exhibitedsignificantly delayed tumor growth upon borussertib treatment(Fig. 6C). This effect was additionally augmented in micetreated with borussertib in combination with trametinib,resulting in a durable partial response. In total, the combina-tion of the AKT inhibitor borussertib with the MEK inhibitortrametinib yielded three stable diseases (Fig. 6B and D; Sup-plementary Fig. S7C) and two PRs (Fig. 6C; Supplementary Fig.S7D), highlighting the potential benefit of this combinationcompared with MEK inhibitor monotherapy for treating colo-rectal cancer.

Taken together, these results underscore the general appli-cability of covalent-allosteric AKT inhibitors in vivo. Althoughthe KRAS-mutant PDX models employed in this study did notshow any response to AKTi monotherapy, alternative in vivomodel systems harboring, for example, genetic lesions in PI3Kor PTEN, could be more suitable for evaluation of borussertibmonotherapy.

DiscussionNumerous signaling cascades rely on AKT as a central inte-

grator of diverse stimuli relevant for physiologic and patho-biologic processes. To date, few small-molecule AKT modula-tors have entered preclinical and clinical trials, largely becauseof selectivity issues caused by structurally similar kinases, lackof efficacy, and mechanism-based adverse effects. Moreover,aberrant AKT signaling resulting from activating mutations inPI3K or functional loss of PTENmight not give rise to oncogeneaddiction per se (14). However, (co-)targeting AKT in a clinicalsetting may result in beneficial therapy outcomes, as demon-strated for ipatasertib, capivasertib, MK-2206, and miransertib,respectively. Of note, low prevalent hyperactive AKT1E17K hasrecently been described to act as a classical driver oncogene inpatients suffering from gynecologic and estrogen receptor–positive breast cancers, thus eliciting pronounced therapeuticresponses upon administration of ATP-competitive AKT inhib-itor capivasertib (34). The efficacy of borussertib for suchindications remains to be determined.

Borussertib proved to be a highly selective and irreversibleallosteric inhibitor of AKT with potent in vitro antiproliferativeactivity and the ability to synergize with other targeted therapiessuch as MEKi in KRAS-mutant colon and pancreatic cancer PDXmodels thereby overcoming potential limitations regarding ther-apeutic efficacy observed for MEKi monotherapy in the types of

cancer mentioned above. The X-ray crystallographic complexstructure presented here supports the anticipated binding modeandwill foster the rational derivatization and optimization of ourlead molecule borussertib concerning binding affinity and inhib-itory potency. We provide evidence for the potent inhibition ofcancer cell proliferation, especially for cell lines featuring geneticalterations in the PI3K/AKT signaling cascade, resulting from thetargeted downregulation of pAKT and downstream effectors,including pS6, p4E-BP1, and pPRAS40, as deduced from immu-noblot analyses. Future efforts will be directed toward the pro-filing of cancer cell lines from additional primary sites and theevaluation of potential drug combination strategies in combina-tion with expanded comprehensive pharmacodynamic analyses.In addition, we provide proof-of principle data for the in vivoefficacy of what is to our knowledge the first-in-class covalent-allosteric AKT inhibitor, as shown for KRAS-mutant pancreaticductal adenocarcinoma and colorectal carcinoma PDX models.Additional efforts will be directed toward the optimization ofaqueous solubility to generate an oral bioavailable derivative ofborussertib with improved biochemical potency, cellular anti-proliferative activity, and in vivo efficacy. Furthermore, detailed invivo pharmacodynamic analyses, optimal dosage identificationand toxicity profiling are mandatory for the subsequent develop-ment of covalent-allosteric AKT inhibitors as drug-like candidates.Eventually, besides combination studies, it will be of interest toemploy borussertib or its optimized derivatives in PDX modelsharboring genetic alterations in the PI3K/AKT signaling axis toenable characterization of its potential as a monotherapeuticagent in immediately relevant disease settings, for example, breastor endometrium cancer.

Disclosure of Potential Conflicts of InterestJ. Weisner has ownership interest (including stock, patents, etc.) in chemical

patent (borussertib). R. Gontla has ownership interest (including stock, patents,etc.) in chemical patent (borussertib). M. Pohl has received speakers bureauhonoraria from Roche Pharma AG, Amgen AG, Sanofi Aventis, Servier, Dres.SchlegelþSchmidt, and AbbVie and is a consultant/advisory board member forRoche Pharma AG, Amgen AG, Lilly, MSD Sharp & Dome, MCI DeutschlandGmbH,Merck Serono, SanofiAventis, BMS, Baxalta Shire, Chugai, and Celgene.D. Rauh has received speakers bureau honoraria from Astra-Zeneca, Merck-Serono, Takeda, Pfizer, Novartis, Boehringer, Sanofi, and BMS. No potentialconflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: J. Weisner, I. Landel, C. Reintjes, N. Uhlenbrock,C. Schultz-Fademrecht, S.A. Hahn, J.T. Siveke, D. RauhDevelopment of methodology: C. Reintjes, R. Scheinpflug, A. Maghnouj,J.T. Siveke, D. RauhAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J. Weisner, I. Landel, C. Reintjes, N. Uhlenbrock,M. Trajkovic-Arsic, N. Dienstbier, J. Hardick, S. Ladigan, M. Lindemann,S. Smith, L. Quambusch, R. Scheinpflug, L. Depta, R. Gontla, G. G€unther,M. Pohl, C. Teschendorf, H. Wolters, R. Viebahn, A. Tannapfel, W. Uhl,J.G. Hengstler, S.A. Hahn, J.T. SivekeAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J. Weisner, I. Landel, C. Reintjes, N. Dienstbier,J. Hardick, A. Unger, H. M€uller, M. Baumann, C. Schultz-Fademrecht,M.P. M€uller, A. Tannapfel, J.G. Hengstler, S.A. Hahn, J.T. Siveke, D. RauhWriting, review, and/or revision of the manuscript: J. Weisner, I. Landel,C. Reintjes, N. Uhlenbrock, M. Trajkovic-Arsic, N. Dienstbier, J. Hardick,M. Lindemann, S. Smith, L. Quambusch, R. Gontla, G. G€unther,M.P. M€uller, M. Pohl, H. Wolters, R. Viebahn, W. Uhl, J.G. Hengstler,S.A. Hahn, J.T. Siveke, D. Rauh

Preclinical Efficacy of AKT Inhibitor Borussertib

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Administrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): J. Weisner, I. Landel, N. Uhlenbrock,A. Maghnouj, M.P. M€uller, C. TeschendorfStudy supervision: J. Weisner, I. Landel, C. Reintjes, J.T. Siveke, D. Rauh

AcknowledgmentsWe thank Axel Choidas and Bert Klebl for helpful discussions and we are

thankful to Prof. Dr. Philippe I. H. Bastiaens for granting access to theOdyssey CLx Imaging System (Li-Cor). This work was supported by theMERCATOR Foundation (grant no. Pr-2016-0014). D. Rauh is thankful forsupport from the German Federal Ministry for Education and Research(NGFNPlus and e:Med; grant No. BMBF 01GS08104, 01ZX1303C), theDeutsche Forschungsgemeinschaft and the German federal state NorthRhine Westphalia, and the European Union (European Regional Develop-

ment Fund: Investing In Your Future; grant no. EFRE-800400). J.T. Siveke issupported by the European Union's Seventh Framework Programmefor Research, Technological Development and Demonstration (FP7/CAM-PaC) under grant agreement no. 602783, the German Cancer Aid (grant no.70112505), the Deutsche Forschungsgemeinschaft (DFG; KFO337/SI 1549/3-1), and the Erich and Gertrud Roggenbuck Foundation and the GermanCancer Consortium (DKTK).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received September 11, 2018; revised January 18, 2019; accepted March 6,2019; published first March 11, 2019.

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2019;79:2367-2378. Published OnlineFirst March 11, 2019.Cancer Res   Jörn Weisner, Ina Landel, Christoph Reintjes, et al.   Colorectal Cancer

-Mutant Pancreatic andKRASin Combination with Trametinib in Preclinical Efficacy of Covalent-Allosteric AKT Inhibitor Borussertib

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