RESEARCH ARTICLE
Targeting AKT1-E17K and the PI3K/AKT Pathway with an Allosteric AKT Inhibitor, ARQ 092 Yi Yu1, Ronald E. Savage2*, Sudharshan Eathiraj1, Justin Meade3, Michael J. Wick3, Terence Hall2, Giovanni Abbadessa4, Brian Schwartz4 1 Biology, ArQule, Inc., Burlington, Massachusetts, United States of America, 2 Preclinical Development and Clinical Pharmacology, ArQule, Inc., Burlington, Massachusetts, United States of America, 3 Preclinical Research, South Texas Accelerated Research Therapeutics, San Antonio, Texas, United States of America, 4 Clinical Development, ArQule, Inc., Burlington, Massachusetts, United States of America * [email protected]
Abstract OPEN ACCESS Citation: Yu Y, Savage RE, Eathiraj S, Meade J, Wick MJ, Hall T, et al. (2015) Targeting AKT1-E17K and the PI3K/AKT Pathway with an Allosteric AKT Inhibitor, ARQ 092. PLoS ONE 10(10): e0140479. doi:10.1371/journal.pone.0140479 Editor: Jin Q. Cheng, H.Lee Moffitt Cancer Center & Research Institute, UNITED STATES Received: July 17, 2015 Accepted: September 22, 2015 Published: October 15, 2015 Copyright: © 2015 Yu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
As a critical component in the PI3K/AKT/mTOR pathway, AKT has become an attractive target for therapeutic intervention. ARQ 092 and a next generation AKT inhibitor, ARQ 751 are selective, allosteric, pan-AKT and AKT1-E17K mutant inhibitors that potently inhibit phosphorylation of AKT. Biochemical and cellular analysis showed that ARQ 092 and ARQ 751 inhibited AKT activation not only by dephosphorylating the membrane-associated active form, but also by preventing the inactive form from localizing into plasma membrane. In endometrial PDX models harboring mutant AKT1-E17K and other tumor models with an activated AKT pathway, both compounds exhibited strong anti-tumor activity. Combination studies conducted in in vivo breast tumor models demonstrated that ARQ 092 enhanced tumor inhibition of a common chemotherapeutic agent (paclitaxel). In a large panel of diverse cancer cell lines, ARQ 092 and ARQ 751 inhibited proliferation across multiple tumor types but were most potent in leukemia, breast, endometrial, and colorectal cancer cell lines. Moreover, inhibition by ARQ 092 and ARQ 751 was more prevalent in cancer cell lines containing PIK3CA/ PIK3R1 mutations compared to those with wt-PIK3CA/PIK3R1 or PTEN mutations. For both ARQ 092 and ARQ 751, PIK3CA/PIK3R1 and AKT1-E17K mutations can potentially be used as predictive biomarkers for patient selection in clinical studies.
Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: Funding for this work was provided by ArQule, Inc. The funder provided support in the form of salaries for all ArQule, Inc. authors. ArQule contracted START authors to perform some of the studies in the manuscript. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Funding for this work was provided by ArQule, Inc. RES, YY, SE, TH, GA, and BS are employees of ArQule, Inc. JM and MJW have
Introduction AKT (Protein kinase B, PKB) is a serine/threonine kinase that belongs to the AGC family of kinases. It was discovered as the mouse leukemia virus AKT 8 oncogene and in humans is encoded by three genes AKT1, 2 and 3 [1]. The AKT family members affect a wide range of biological functions including cell proliferation and growth, metabolism, protein synthesis, migration, angiogenesis, and anti-apoptotic ability [2, 3]. In order for AKT to modulate these functions, activation is essential. Full activation of AKT requires translocation to the plasma membrane followed by phosphorylation of both the threonine 308 and serine 473 residues
PLOS ONE | DOI:10.1371/journal.pone.0140479 October 15, 2015
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Targeting AKT1-E17K and the PI3K/AKT Pathway with ARQ 092
no competing financial interests. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.
which are regulated by multiple components of the well-known PI3K/AKT/mTOR pathway. In this pathway, PI3K phosphorylates phosphatidylinositol (3,4) bisphosphate (PIP2) to phosphatidylinositol (3,4,5) triphosphate (PIP3). The pleckstrin homology (PH) domains of both PI3Kdependent kinase 1 (PDK1) and AKT bind to PIP3 (located at the plasma membrane) thus allowing PDK1 to phosphorylate the activation loop of AKT at Thr 308. The activation process is completed with the phosphorylation of AKT at the C-terminal Ser 473 of the hydrophobic motif which occurs via the mTORC2/rictor complex [4, 5]. Once activated, AKT phosphorylates downstream target proteins including PRAS40, GSK3, FOXO, and BAD [2]. Additionally, AKT also suppresses the inhibitory effect via phosphorylation of TSC1 and TSC2, thereby activating mTORC1 and ultimately leading to phosphorylation of 4E-BP1 and S6K [6, 7]. Dysregulation of PI3K/AKT/mTOR signaling has been linked to various diseases including cancer and overgrowth syndromes [8–13]. The tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome ten) negatively regulates PI3K activity by dephosphorylating PIP3 to PIP2, which in turn deactivates AKT [14]. Hence, in cancers where PTEN is inactivated via a mutation or deletion, AKT is activated [15, 16]. These activations occur frequently in prostate and endometrial cancers as well as glioblastoma and melanoma [15]. AKT activation can also occur via constitutive activation of PI3K through receptor tyrosine kinase activation and mutations predominantly in the PIK3CA gene. Both PIK3CA activating and PTEN loss mutations have been found to occur frequently in breast, endometrial, and colon cancers with reported incidence rates ranging from 6–35%, 22–54%, and from 9 to 54%, respectively [13, 15]. Consequently, AKT represents an attractive target for therapeutic intervention and as such there are many AKT inhibitors in pre-clinical and clinical development [11, 17–22]. Activating mutations in the PH domain of AKT1, AKT2 and AKT3 which constitutively activate the kinase (E17K) have been reported in a variety of different oncologic and non-oncologic diseases [23–26]. The AKT1-E17K mutant protein has been shown to have increased cell membrane localization and was capable of transforming Rat1 fibroblasts and ultimately inducing leukemia in mice [24]. This mutation has been reported in numerous cancers including breast, colorectal, ovarian and endometrial cancers with the highest incidence being in breast (6–8%), colorectal cancer (2–6%) and meningioma (6%) [23, 24, 27–36]. Though occurring with low frequency rates in cancer, this mutation has been found to be much more prevalent in patients with Proteus syndrome (PS), a rare condition characterized by the overgrowth of bone, fat, skin, and other connective tissues. Lindhurst et al. recently reported that of 29 patients with PS, 26 (90%) carried the AKT1-E17K mutation [10]. Other non-oncologic diseases in which AKT2-E17K and AKT3-E17K mutations cause hypoglycemia and hemimegalencephaly have also been reported [25, 26]. We report data on ARQ 092 and ARQ 751 (S1 Fig) as highly potent and selective allosteric inhibitors of AKT and demonstrate that they are active both in biochemical assays and in cells which contain wild-type (WT) or mutant AKT. In addition, both compounds were shown to potently suppress tumor growth and the AKT pathway in xenograft models harboring either WT or AKT1-E17K, with ARQ 751 consistently demonstrating superior potency compared to ARQ 092. These data support the clinical advancement of both compounds not only in cancers where AKT mutants including AKT1-E17K are prevalent, but also in rare overgrowth diseases like PS where the AKT1-E17K mutant potentially drives disease progression.
Methods Reagents ARQ 092 was synthesized at ArQule, Inc. (Burlington, MA). ARQ 751 was synthesized at Daiichi Sankyo RD Novare Co. Ltd (Daiichi Sankyo Ltd, Japan). MK-2206 was synthesized by SAI
PLOS ONE | DOI:10.1371/journal.pone.0140479 October 15, 2015
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Targeting AKT1-E17K and the PI3K/AKT Pathway with ARQ 092
Life Sciences (Hyderabad, India) or at Daiichi Sankyo RD Novare Co. Ltd. GDC-0068 was purchased from Selleckchem (Houston, TX). Paclitaxel was purchased from Bristol-Myers Squibb (New York, NY) and trastuzumab was purchased from Chugai Pharmaceutical Co. (Tokyo, Japan). Trametinib was purchased from LC Laboratories (Woburn, MA). AN3CA, BT-474, ZR-75-1, MDA-MB-453, NCI-H1650, NIH 3T3 and 293T cells were purchased from American Tissue and Culture Collection (Manassas, VA). KU-19-19 cells were purchased from Creative Bioarray (Shirley, NY). KPL-4 cells were kindly provided by Dr. J. Kurebayashi (Kawasaki Medical School, Okayama, Japan) [37]. Human PDGF-BB was purchased from PeproTech (Rocky Hill, NJ). Methyl cellulose 400cP solution was purchased from Wako Pure Chemical Industries Ltd (Osaka, Japan) and DMA was purchased from Sigma Aldrich (St Louis, MO). All experimental mice were purchased from Taconic USA (Hudson, NY) or CLEA Japan, Inc. (Tokyo, Japan). All other reagents were of the highest grade and obtained from standard suppliers.
Biochemical IC50 determination and kinase profiling Biochemical IC50 determination for ARQ 092, ARQ 751, MK-2206, and GDC-0068 against full-length AKT1, AKT2, and AKT3 was performed at Reaction Biology (Malvern, PA). Compounds were tested in a 10-dose mode with 3-fold serial dilutions starting at 1 μM in the presence of 10 μM ATP and the IC50 was determined. Curve fits were performed where the enzyme activities at the highest concentration of compounds were less than 65% of the enzyme activity (relative to DMSO controls). Kinase profiling was performed for ARQ 092 against 303 kinases and for ARQ 751 against 245 kinases at 5 μM at Carna Biosciences, Inc. (Kobe, Japan). The % inhibition was determined relative to DMSO control. For ARQ 092, a confirmatory IC50 value was determined for any kinase that had greater than 50% inhibition at 5 μM.
Cell Culture Conditions All cancer cell lines were grown in EMEM, DMEM or RPMI-1640 (Life Technologies Corporation, Carlsbad, CA) supplemented with 10% FBS. 293T and NIH 3T3 cells were cultured in DMEM with or without GlutaMAX, respectively, supplemented with 10% FBS. All cell cultures were maintained at 37°C in a humidified atmosphere containing 5% CO2.
Expression DNA Constructs and Transfection Construction of the expression vector was performed at BPS Biosciences (San Diego, CA). WT and E17K mutant forms of the human AKT1 cDNA were fused with green fluorescent protein (GFP) in the c-terminus and cloned into the pcDNA expression vector, generating pcDNAAKT1-WT-GFP and pcDNAAKT1-E17K-GFP. AKT1-WT and AKT1-E17K were validated by sequencing. Cells were transiently transfected with pcDNAAKT1-WT-GFP or pcDNAAKT1-E17K-GFP using Lipofectamine 2000 (Life Technologies, Inc., Grand Island, NY).
Western Blot Analyses Cells (MDA-MB-453: 1.5x106; NCI-H1650: 1x106; KU-19-19: 0.7x106) were plated into 6 well plates, left overnight, and then treated with full media containing different concentrations (0, 0.012, 0.037, 0.11, 0.33, and 1 μM) of AKT inhibitors (ARQ 092, ARQ 751, MK-2206, GDC0068) for 2 hours. Cells were treated under designated conditions and lysates were extracted. Proteins were resolved from extracts using SDS-PAGE followed by immunoblotting. P-AKT
PLOS ONE | DOI:10.1371/journal.pone.0140479 October 15, 2015
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Targeting AKT1-E17K and the PI3K/AKT Pathway with ARQ 092
(T308), p-AKT(S473), total AKT, p-PRAS40(T246), total PRAS40, pGSK3b(S9), pAS160 (S318), pFOXO1(T24)/3a(T32), pBAD(S136), pS6(S235/S236), p4E-BP1(S65), pERK(T202/ Y204), and total ERK, were assessed using the corresponding primary antibodies (Cell Signaling Technology, Beverly, MA). Anti-actin was purchased from Sigma Aldrich (St. Louis, MO). HRP conjugated secondary antibodies were purchased from Cell Signaling Technology. Immunoblotting detection was performed using ECL reagent (GE Healthcare, Piscataway, NJ). Tumor tissues were ground in mortars with liquid nitrogen and lysed. Proteins extracted from tumor tissues were resolved using E-PAGE (Life Technologies Corporation) followed by immunoblotting. P-AKT(T308), p-AKT(S473), total AKT, p-PRAS40(T246) and β-Actin were assessed with corresponding primary antibodies (Cell Signaling Technology). Infrared dye conjugated secondary antibodies (LI-COR Biosciences, Lincoln, NE) were used as secondary antibodies. Immunoblotting detection was performed with a LI-COR scanner (LI-COR Biosciences). The intensity of the bands was quantitated using the accompanying software. The data were expressed as percent inhibition in comparison to vehicle.
Intrinsic tryptophan fluorescence quench assay The binding of ARQ 092, ARQ 751, MK-2206, and GDC-0068 to AKT1-WT and AKT1-E17K was followed by monitoring intrinsic tryptophan fluorescence quench. Inhibitors were serially diluted (two-fold) with buffer solution (25 mM Tris, pH 7.5, 0.1 M NaCl and 2% DMSO) in 384 well plates. AKT1 or AKT1-E17K (100 nM) was added to each well and diluted to a final volume of 50 μl with buffer. The resulting mixtures were allowed to equilibrate for 30 minutes at room temperature. The change in intrinsic emission was measured on a Tecan microplate reader (Tecan, Mannedorf, Switzerland) at an excitation wavelength of 280 nm and emission wavelength of 345 nm. The fluorescence at 345 nm was plotted versus the inhibitor concentration, and the data were fit to a quadratic equation using nonlinear regression. The following expression relating observed fluorescence (F) to inhibitor concentration [I0] was used to obtain the binding constant for AKT inhibitors: (KDapp), F = F0 + (ΔF/P0) [(KDapp + [P0] + [I0])—the square root of [(KDapp + [P0] + [I0])2–4[P0][I0]]]/2, where P0 is the initial protein concentration (constant), KD is the dissociation constant, ΔF is the total change in fluorescence, and I0 is the inhibitor concentration. Each titration experiment represents an average obtained from four independent experiments.
AKT1 Plasma Membrane Translocation Cells (15,000 cells per chamber) were plated into a Nunc Lab-Tek Chambered Coverglass System (Thermo Fisher Scientific, Waltham, MA) containing full media without antibiotics. Next, the cells were transfected with AKT-GFP plasmids using 1 μL Lipofectamine 2000 (0.81 μg of AKT1-E17K-GFP and 0.6 μg of AKT1-WT-GFP) in the presence of 30 μL Opti-MEM. After 48 hours, cells were incubated in serum free media for an additional 24 hours and then treated with or without inhibitor (1 μM ARQ 092, ARQ 751, MK-2206, or GDC-0068) for 2 hours. Next, the cells were stimulated with PDGF (PeproTech, Rocky Hill, NJ) at 50 ng/ml for 10 minutes. Cells were washed with PBS and then fixed with 200 μl paraformaldehyde fixation buffer (3% paraformaldehyde, 2% sucrose in PBS, pre-warmed at 60–70°C). After washing with PBS, cells were blocked and permeabolized with 0.1% TX-100 + 1% BSA in PBS for 15 minutes at room temperature. Next, 200 μl DAPI (1 μg/ml in PBS) was used to counterstain the nuclei at room temperature in darkness for 15 minutes. Samples were then rinsed with cold PBS and stored at 4°C until imaging. Images were captured using an Olympus inverted epi-fluorescent microscope (Olympus Corporation of America, Center Valley, PA) with a 60x oil immersion objective.
PLOS ONE | DOI:10.1371/journal.pone.0140479 October 15, 2015
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Targeting AKT1-E17K and the PI3K/AKT Pathway with ARQ 092
OncoPanel Analysis Anti-proliferative activity of ARQ 092 and ARQ 751 was assessed using OncoPanel 240 (Eurofins, St. Charles, MO). Cells were seeded into 384-well plates and incubated in a humidified atmosphere of 5% CO2 at 37°C. Compounds were added 24 hours post cell seeding. At the same time, a time zero untreated cell plate was generated. Compounds were serially diluted 3.16-fold and assayed over a range of 10 concentrations in buffer containing 0.1% DMSO. Automated fluorescence microscopy was carried out using an IN Cell Analyzer 1000 (GE Healthcare, Marlborough, MA), and images were collected with a 4X objective. After a 72 hour incubation period, cells were fixed and stained with fluorescently labeled antibodies and nuclear dye to determine relative cell number. Twelve bit tiff images were acquired using the IN Cell Analyzer 1000 3.2 and analyzed with Developer Toolbox 1.6 software (GE Healthcare). GI50 values were calculated using nonlinear regression to fit data to a sigmoidal 4 point, 4 parameter One-Site dose response model, where: y (fit) = A + [(B–A)/(1 + ((C/x) ^ D))]. Curve-fitting, GI50 calculations and report generation are performed using a custom data reduction engine MathIQ based software (AIM).
Mutation Analysis Mutation analysis was performed based on information from the COSMIC database (http:// cancer.sanger.ac.uk/cancergenome/projects/cosmic/). Cells with GI501 μM were designated as resistant. Mutation analysis was performed based on ArQule’s internal database derived from the Catalogue of Somatic Mutations in Cancer (COSMIC) v41. The Fisher’s exact test was applied. A p
Targeting AKT1-E17K and the PI3K/AKT Pathway with an Allosteric AKT Inhibitor, ARQ 092 Yi Yu1, Ronald E. Savage2*, Sudharshan Eathiraj1, Justin Meade3, Michael J. Wick3, Terence Hall2, Giovanni Abbadessa4, Brian Schwartz4 1 Biology, ArQule, Inc., Burlington, Massachusetts, United States of America, 2 Preclinical Development and Clinical Pharmacology, ArQule, Inc., Burlington, Massachusetts, United States of America, 3 Preclinical Research, South Texas Accelerated Research Therapeutics, San Antonio, Texas, United States of America, 4 Clinical Development, ArQule, Inc., Burlington, Massachusetts, United States of America * [email protected]
Abstract OPEN ACCESS Citation: Yu Y, Savage RE, Eathiraj S, Meade J, Wick MJ, Hall T, et al. (2015) Targeting AKT1-E17K and the PI3K/AKT Pathway with an Allosteric AKT Inhibitor, ARQ 092. PLoS ONE 10(10): e0140479. doi:10.1371/journal.pone.0140479 Editor: Jin Q. Cheng, H.Lee Moffitt Cancer Center & Research Institute, UNITED STATES Received: July 17, 2015 Accepted: September 22, 2015 Published: October 15, 2015 Copyright: © 2015 Yu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
As a critical component in the PI3K/AKT/mTOR pathway, AKT has become an attractive target for therapeutic intervention. ARQ 092 and a next generation AKT inhibitor, ARQ 751 are selective, allosteric, pan-AKT and AKT1-E17K mutant inhibitors that potently inhibit phosphorylation of AKT. Biochemical and cellular analysis showed that ARQ 092 and ARQ 751 inhibited AKT activation not only by dephosphorylating the membrane-associated active form, but also by preventing the inactive form from localizing into plasma membrane. In endometrial PDX models harboring mutant AKT1-E17K and other tumor models with an activated AKT pathway, both compounds exhibited strong anti-tumor activity. Combination studies conducted in in vivo breast tumor models demonstrated that ARQ 092 enhanced tumor inhibition of a common chemotherapeutic agent (paclitaxel). In a large panel of diverse cancer cell lines, ARQ 092 and ARQ 751 inhibited proliferation across multiple tumor types but were most potent in leukemia, breast, endometrial, and colorectal cancer cell lines. Moreover, inhibition by ARQ 092 and ARQ 751 was more prevalent in cancer cell lines containing PIK3CA/ PIK3R1 mutations compared to those with wt-PIK3CA/PIK3R1 or PTEN mutations. For both ARQ 092 and ARQ 751, PIK3CA/PIK3R1 and AKT1-E17K mutations can potentially be used as predictive biomarkers for patient selection in clinical studies.
Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: Funding for this work was provided by ArQule, Inc. The funder provided support in the form of salaries for all ArQule, Inc. authors. ArQule contracted START authors to perform some of the studies in the manuscript. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Funding for this work was provided by ArQule, Inc. RES, YY, SE, TH, GA, and BS are employees of ArQule, Inc. JM and MJW have
Introduction AKT (Protein kinase B, PKB) is a serine/threonine kinase that belongs to the AGC family of kinases. It was discovered as the mouse leukemia virus AKT 8 oncogene and in humans is encoded by three genes AKT1, 2 and 3 [1]. The AKT family members affect a wide range of biological functions including cell proliferation and growth, metabolism, protein synthesis, migration, angiogenesis, and anti-apoptotic ability [2, 3]. In order for AKT to modulate these functions, activation is essential. Full activation of AKT requires translocation to the plasma membrane followed by phosphorylation of both the threonine 308 and serine 473 residues
PLOS ONE | DOI:10.1371/journal.pone.0140479 October 15, 2015
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Targeting AKT1-E17K and the PI3K/AKT Pathway with ARQ 092
no competing financial interests. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.
which are regulated by multiple components of the well-known PI3K/AKT/mTOR pathway. In this pathway, PI3K phosphorylates phosphatidylinositol (3,4) bisphosphate (PIP2) to phosphatidylinositol (3,4,5) triphosphate (PIP3). The pleckstrin homology (PH) domains of both PI3Kdependent kinase 1 (PDK1) and AKT bind to PIP3 (located at the plasma membrane) thus allowing PDK1 to phosphorylate the activation loop of AKT at Thr 308. The activation process is completed with the phosphorylation of AKT at the C-terminal Ser 473 of the hydrophobic motif which occurs via the mTORC2/rictor complex [4, 5]. Once activated, AKT phosphorylates downstream target proteins including PRAS40, GSK3, FOXO, and BAD [2]. Additionally, AKT also suppresses the inhibitory effect via phosphorylation of TSC1 and TSC2, thereby activating mTORC1 and ultimately leading to phosphorylation of 4E-BP1 and S6K [6, 7]. Dysregulation of PI3K/AKT/mTOR signaling has been linked to various diseases including cancer and overgrowth syndromes [8–13]. The tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome ten) negatively regulates PI3K activity by dephosphorylating PIP3 to PIP2, which in turn deactivates AKT [14]. Hence, in cancers where PTEN is inactivated via a mutation or deletion, AKT is activated [15, 16]. These activations occur frequently in prostate and endometrial cancers as well as glioblastoma and melanoma [15]. AKT activation can also occur via constitutive activation of PI3K through receptor tyrosine kinase activation and mutations predominantly in the PIK3CA gene. Both PIK3CA activating and PTEN loss mutations have been found to occur frequently in breast, endometrial, and colon cancers with reported incidence rates ranging from 6–35%, 22–54%, and from 9 to 54%, respectively [13, 15]. Consequently, AKT represents an attractive target for therapeutic intervention and as such there are many AKT inhibitors in pre-clinical and clinical development [11, 17–22]. Activating mutations in the PH domain of AKT1, AKT2 and AKT3 which constitutively activate the kinase (E17K) have been reported in a variety of different oncologic and non-oncologic diseases [23–26]. The AKT1-E17K mutant protein has been shown to have increased cell membrane localization and was capable of transforming Rat1 fibroblasts and ultimately inducing leukemia in mice [24]. This mutation has been reported in numerous cancers including breast, colorectal, ovarian and endometrial cancers with the highest incidence being in breast (6–8%), colorectal cancer (2–6%) and meningioma (6%) [23, 24, 27–36]. Though occurring with low frequency rates in cancer, this mutation has been found to be much more prevalent in patients with Proteus syndrome (PS), a rare condition characterized by the overgrowth of bone, fat, skin, and other connective tissues. Lindhurst et al. recently reported that of 29 patients with PS, 26 (90%) carried the AKT1-E17K mutation [10]. Other non-oncologic diseases in which AKT2-E17K and AKT3-E17K mutations cause hypoglycemia and hemimegalencephaly have also been reported [25, 26]. We report data on ARQ 092 and ARQ 751 (S1 Fig) as highly potent and selective allosteric inhibitors of AKT and demonstrate that they are active both in biochemical assays and in cells which contain wild-type (WT) or mutant AKT. In addition, both compounds were shown to potently suppress tumor growth and the AKT pathway in xenograft models harboring either WT or AKT1-E17K, with ARQ 751 consistently demonstrating superior potency compared to ARQ 092. These data support the clinical advancement of both compounds not only in cancers where AKT mutants including AKT1-E17K are prevalent, but also in rare overgrowth diseases like PS where the AKT1-E17K mutant potentially drives disease progression.
Methods Reagents ARQ 092 was synthesized at ArQule, Inc. (Burlington, MA). ARQ 751 was synthesized at Daiichi Sankyo RD Novare Co. Ltd (Daiichi Sankyo Ltd, Japan). MK-2206 was synthesized by SAI
PLOS ONE | DOI:10.1371/journal.pone.0140479 October 15, 2015
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Targeting AKT1-E17K and the PI3K/AKT Pathway with ARQ 092
Life Sciences (Hyderabad, India) or at Daiichi Sankyo RD Novare Co. Ltd. GDC-0068 was purchased from Selleckchem (Houston, TX). Paclitaxel was purchased from Bristol-Myers Squibb (New York, NY) and trastuzumab was purchased from Chugai Pharmaceutical Co. (Tokyo, Japan). Trametinib was purchased from LC Laboratories (Woburn, MA). AN3CA, BT-474, ZR-75-1, MDA-MB-453, NCI-H1650, NIH 3T3 and 293T cells were purchased from American Tissue and Culture Collection (Manassas, VA). KU-19-19 cells were purchased from Creative Bioarray (Shirley, NY). KPL-4 cells were kindly provided by Dr. J. Kurebayashi (Kawasaki Medical School, Okayama, Japan) [37]. Human PDGF-BB was purchased from PeproTech (Rocky Hill, NJ). Methyl cellulose 400cP solution was purchased from Wako Pure Chemical Industries Ltd (Osaka, Japan) and DMA was purchased from Sigma Aldrich (St Louis, MO). All experimental mice were purchased from Taconic USA (Hudson, NY) or CLEA Japan, Inc. (Tokyo, Japan). All other reagents were of the highest grade and obtained from standard suppliers.
Biochemical IC50 determination and kinase profiling Biochemical IC50 determination for ARQ 092, ARQ 751, MK-2206, and GDC-0068 against full-length AKT1, AKT2, and AKT3 was performed at Reaction Biology (Malvern, PA). Compounds were tested in a 10-dose mode with 3-fold serial dilutions starting at 1 μM in the presence of 10 μM ATP and the IC50 was determined. Curve fits were performed where the enzyme activities at the highest concentration of compounds were less than 65% of the enzyme activity (relative to DMSO controls). Kinase profiling was performed for ARQ 092 against 303 kinases and for ARQ 751 against 245 kinases at 5 μM at Carna Biosciences, Inc. (Kobe, Japan). The % inhibition was determined relative to DMSO control. For ARQ 092, a confirmatory IC50 value was determined for any kinase that had greater than 50% inhibition at 5 μM.
Cell Culture Conditions All cancer cell lines were grown in EMEM, DMEM or RPMI-1640 (Life Technologies Corporation, Carlsbad, CA) supplemented with 10% FBS. 293T and NIH 3T3 cells were cultured in DMEM with or without GlutaMAX, respectively, supplemented with 10% FBS. All cell cultures were maintained at 37°C in a humidified atmosphere containing 5% CO2.
Expression DNA Constructs and Transfection Construction of the expression vector was performed at BPS Biosciences (San Diego, CA). WT and E17K mutant forms of the human AKT1 cDNA were fused with green fluorescent protein (GFP) in the c-terminus and cloned into the pcDNA expression vector, generating pcDNAAKT1-WT-GFP and pcDNAAKT1-E17K-GFP. AKT1-WT and AKT1-E17K were validated by sequencing. Cells were transiently transfected with pcDNAAKT1-WT-GFP or pcDNAAKT1-E17K-GFP using Lipofectamine 2000 (Life Technologies, Inc., Grand Island, NY).
Western Blot Analyses Cells (MDA-MB-453: 1.5x106; NCI-H1650: 1x106; KU-19-19: 0.7x106) were plated into 6 well plates, left overnight, and then treated with full media containing different concentrations (0, 0.012, 0.037, 0.11, 0.33, and 1 μM) of AKT inhibitors (ARQ 092, ARQ 751, MK-2206, GDC0068) for 2 hours. Cells were treated under designated conditions and lysates were extracted. Proteins were resolved from extracts using SDS-PAGE followed by immunoblotting. P-AKT
PLOS ONE | DOI:10.1371/journal.pone.0140479 October 15, 2015
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Targeting AKT1-E17K and the PI3K/AKT Pathway with ARQ 092
(T308), p-AKT(S473), total AKT, p-PRAS40(T246), total PRAS40, pGSK3b(S9), pAS160 (S318), pFOXO1(T24)/3a(T32), pBAD(S136), pS6(S235/S236), p4E-BP1(S65), pERK(T202/ Y204), and total ERK, were assessed using the corresponding primary antibodies (Cell Signaling Technology, Beverly, MA). Anti-actin was purchased from Sigma Aldrich (St. Louis, MO). HRP conjugated secondary antibodies were purchased from Cell Signaling Technology. Immunoblotting detection was performed using ECL reagent (GE Healthcare, Piscataway, NJ). Tumor tissues were ground in mortars with liquid nitrogen and lysed. Proteins extracted from tumor tissues were resolved using E-PAGE (Life Technologies Corporation) followed by immunoblotting. P-AKT(T308), p-AKT(S473), total AKT, p-PRAS40(T246) and β-Actin were assessed with corresponding primary antibodies (Cell Signaling Technology). Infrared dye conjugated secondary antibodies (LI-COR Biosciences, Lincoln, NE) were used as secondary antibodies. Immunoblotting detection was performed with a LI-COR scanner (LI-COR Biosciences). The intensity of the bands was quantitated using the accompanying software. The data were expressed as percent inhibition in comparison to vehicle.
Intrinsic tryptophan fluorescence quench assay The binding of ARQ 092, ARQ 751, MK-2206, and GDC-0068 to AKT1-WT and AKT1-E17K was followed by monitoring intrinsic tryptophan fluorescence quench. Inhibitors were serially diluted (two-fold) with buffer solution (25 mM Tris, pH 7.5, 0.1 M NaCl and 2% DMSO) in 384 well plates. AKT1 or AKT1-E17K (100 nM) was added to each well and diluted to a final volume of 50 μl with buffer. The resulting mixtures were allowed to equilibrate for 30 minutes at room temperature. The change in intrinsic emission was measured on a Tecan microplate reader (Tecan, Mannedorf, Switzerland) at an excitation wavelength of 280 nm and emission wavelength of 345 nm. The fluorescence at 345 nm was plotted versus the inhibitor concentration, and the data were fit to a quadratic equation using nonlinear regression. The following expression relating observed fluorescence (F) to inhibitor concentration [I0] was used to obtain the binding constant for AKT inhibitors: (KDapp), F = F0 + (ΔF/P0) [(KDapp + [P0] + [I0])—the square root of [(KDapp + [P0] + [I0])2–4[P0][I0]]]/2, where P0 is the initial protein concentration (constant), KD is the dissociation constant, ΔF is the total change in fluorescence, and I0 is the inhibitor concentration. Each titration experiment represents an average obtained from four independent experiments.
AKT1 Plasma Membrane Translocation Cells (15,000 cells per chamber) were plated into a Nunc Lab-Tek Chambered Coverglass System (Thermo Fisher Scientific, Waltham, MA) containing full media without antibiotics. Next, the cells were transfected with AKT-GFP plasmids using 1 μL Lipofectamine 2000 (0.81 μg of AKT1-E17K-GFP and 0.6 μg of AKT1-WT-GFP) in the presence of 30 μL Opti-MEM. After 48 hours, cells were incubated in serum free media for an additional 24 hours and then treated with or without inhibitor (1 μM ARQ 092, ARQ 751, MK-2206, or GDC-0068) for 2 hours. Next, the cells were stimulated with PDGF (PeproTech, Rocky Hill, NJ) at 50 ng/ml for 10 minutes. Cells were washed with PBS and then fixed with 200 μl paraformaldehyde fixation buffer (3% paraformaldehyde, 2% sucrose in PBS, pre-warmed at 60–70°C). After washing with PBS, cells were blocked and permeabolized with 0.1% TX-100 + 1% BSA in PBS for 15 minutes at room temperature. Next, 200 μl DAPI (1 μg/ml in PBS) was used to counterstain the nuclei at room temperature in darkness for 15 minutes. Samples were then rinsed with cold PBS and stored at 4°C until imaging. Images were captured using an Olympus inverted epi-fluorescent microscope (Olympus Corporation of America, Center Valley, PA) with a 60x oil immersion objective.
PLOS ONE | DOI:10.1371/journal.pone.0140479 October 15, 2015
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Targeting AKT1-E17K and the PI3K/AKT Pathway with ARQ 092
OncoPanel Analysis Anti-proliferative activity of ARQ 092 and ARQ 751 was assessed using OncoPanel 240 (Eurofins, St. Charles, MO). Cells were seeded into 384-well plates and incubated in a humidified atmosphere of 5% CO2 at 37°C. Compounds were added 24 hours post cell seeding. At the same time, a time zero untreated cell plate was generated. Compounds were serially diluted 3.16-fold and assayed over a range of 10 concentrations in buffer containing 0.1% DMSO. Automated fluorescence microscopy was carried out using an IN Cell Analyzer 1000 (GE Healthcare, Marlborough, MA), and images were collected with a 4X objective. After a 72 hour incubation period, cells were fixed and stained with fluorescently labeled antibodies and nuclear dye to determine relative cell number. Twelve bit tiff images were acquired using the IN Cell Analyzer 1000 3.2 and analyzed with Developer Toolbox 1.6 software (GE Healthcare). GI50 values were calculated using nonlinear regression to fit data to a sigmoidal 4 point, 4 parameter One-Site dose response model, where: y (fit) = A + [(B–A)/(1 + ((C/x) ^ D))]. Curve-fitting, GI50 calculations and report generation are performed using a custom data reduction engine MathIQ based software (AIM).
Mutation Analysis Mutation analysis was performed based on information from the COSMIC database (http:// cancer.sanger.ac.uk/cancergenome/projects/cosmic/). Cells with GI501 μM were designated as resistant. Mutation analysis was performed based on ArQule’s internal database derived from the Catalogue of Somatic Mutations in Cancer (COSMIC) v41. The Fisher’s exact test was applied. A p
