RESUMEN
Rare DRAM2 coding variants cause retinal dystrophy with early macular involvement via unknown mechanisms. We found that DRAM2 is ubiquitously expressed in the human eye and expression changes were observed in eyes with more common maculopathy such as Age-related Macular Degeneration (AMD). To gain insights into pathogenicity of DRAM2-related retinopathy, we used a combination of in vitro and in vivo models. We found that DRAM2 loss in human pluripotent stem cell (hPSC)-derived retinal organoids caused the presence of additional mesenchymal cells. Interestingly, Dram2 loss in mice also caused increased proliferation of cells from the choroid in vitro and exacerbated choroidal neovascular lesions in vivo. Furthermore, we observed that DRAM2 loss in human retinal pigment epithelial (RPE) cells resulted in increased susceptibility to stress-induced cell death in vitro and that Dram2 loss in mice caused age-related photoreceptor degeneration. This highlights the complexity of DRAM2 function, as its loss in choroidal cells provided a proliferative advantage, whereas its loss in post-mitotic cells, such as photoreceptor and RPE cells, increased degeneration susceptibility. Different models such as human pluripotent stem cell-derived systems and mice can be leveraged to study and model human retinal dystrophies; however, cell type and species-specific expression must be taken into account when selecting relevant systems.
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Mutations in the GBA1 gene encoding glucocerebrosidase (GCase) are linked to Gaucher (GD) and Parkinson's Disease (PD). Since some GD and PD patients develop ocular phenotypes, we determined whether ocular phenotypes might result from impaired GCase activity and the corresponding accumulation of glucosylceramide (GluCer) and glucosylsphingosine (GluSph) in the Gba1D409V/D409V knock-in (Gba KI/KI; "KI") mouse. Gba KI mice developed age-dependent pupil dilation deficits to an anti-muscarinic agent; histologically, the iris covered the anterior part of the lens with adhesions between the iris and the anterior surface of the lens (posterior synechia). This may prevent pupil dilation in general, beyond an un-responsiveness of the iris to anti-muscarinics. Gba KI mice displayed atrophy and pigment dispersion of the iris, and occlusion of the iridocorneal angle by pigment-laden cells, reminiscent of secondary open angle glaucoma. Gba KI mice showed progressive thinning of the retina consistent with retinal degeneration. GluSph levels were increased in the anterior and posterior segments of the eye, suggesting that accumulation of lipids in the eye may contribute to degeneration in this compartment. We conclude that the Gba KI model provides robust and reproducible eye phenotypes which may be used to test for efficacy and establish biomarkers for GBA1-related therapies.
Asunto(s)
Enfermedad de Gaucher , Glaucoma de Ángulo Abierto , Glucosilceramidasa , Mutación Missense , Enfermedad de Parkinson , Sustitución de Aminoácidos , Animales , Modelos Animales de Enfermedad , Enfermedad de Gaucher/enzimología , Enfermedad de Gaucher/genética , Enfermedad de Gaucher/patología , Técnicas de Sustitución del Gen , Glaucoma de Ángulo Abierto/enzimología , Glaucoma de Ángulo Abierto/genética , Glaucoma de Ángulo Abierto/patología , Glucosilceramidasa/genética , Glucosilceramidasa/metabolismo , Humanos , Ratones , Ratones Transgénicos , Enfermedad de Parkinson/enzimología , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patologíaRESUMEN
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RESUMEN
Geographic atrophy (GA), the advanced form of dry age-related macular degeneration (AMD), is characterized by progressive loss of retinal pigment epithelium cells and photoreceptors in the setting of characteristic extracellular deposits and remains a serious unmet medical need. While genetic predisposition to AMD is dominated by polymorphisms in complement genes, it remains unclear how complement activation contributes to retinal atrophy. Here we demonstrate that complement is activated on photoreceptor outer segments (POS) in the retina peripheral to atrophic lesions associated with GA. When exposed to human serum following outer blood-retinal barrier breakdown, POS act as potent activators of the classical and alternative complement pathway. In mouse models of retinal degeneration, classical and alternative pathway complement activation on photoreceptors contributed to the loss of photoreceptor function. This was dependent on C5a-mediated recruitment of peripheral blood monocytes but independent of resident microglia. Genetic or pharmacologic inhibition of both classical and alternative complement C3 and C5 convertases was required to reduce progressive degeneration of photoreceptor rods and cones. Our study implicates systemic classical and alternative complement proteins and peripheral blood monocytes as critical effectors of localized retinal degeneration with potential relevance for the contribution of complement activation to GA.
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Activación de Complemento/genética , Atrofia Geográfica/fisiopatología , Células Fotorreceptoras Retinianas Bastones/metabolismo , Animales , Atrofia/patología , Activación de Complemento/fisiología , Complemento C3/genética , Complemento C3/fisiología , Complemento C4/genética , Complemento C4/fisiología , Atrofia Geográfica/genética , Humanos , Degeneración Macular/fisiopatología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/metabolismo , Células Fotorreceptoras/metabolismo , Retina/metabolismo , Degeneración Retiniana/patología , Epitelio Pigmentado de la Retina/metabolismoRESUMEN
Age-related macular degeneration (AMD), a leading cause of vision impairment in the ageing population, is characterized by irreversible loss of retinal pigment epithelial (RPE) cells and photoreceptors and can be associated with choroidal neovascularization. Mononuclear phagocytes are often present in AMD lesions, but the processes that direct myeloid cell recruitment remain unclear. Here, we identify IL-33 as a key regulator of inflammation and photoreceptor degeneration after retina stress or injury. IL-33(+) Müller cells were more abundant and IL-33 cytokine was elevated in advanced AMD cases compared with age-matched controls with no AMD. In rodents, retina stress resulted in release of bioactive IL-33 that in turn increased inflammatory chemokine and cytokine expression in activated Müller cells. Deletion of ST2, the IL-33 receptor α chain, or treatment with a soluble IL-33 decoy receptor significantly reduced release of inflammatory mediators from Müller cells, inhibited accumulation of mononuclear phagocytes in the outer retina, and protected photoreceptor rods and cones after a retina insult. This study demonstrates a central role for IL-33 in regulating mononuclear phagocyte recruitment to the photoreceptor layer and positions IL-33 signaling as a potential therapeutic target in macular degenerative diseases.
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Inmunidad Innata , Interleucina-33/metabolismo , Degeneración Macular/inmunología , Anciano , Anciano de 80 o más Años , Animales , Estudios de Casos y Controles , Núcleo Celular/inmunología , Citocinas/metabolismo , Células Ependimogliales/inmunología , Células Ependimogliales/patología , Femenino , Humanos , Técnicas In Vitro , Proteína 1 Similar al Receptor de Interleucina-1 , Interleucina-33/química , Interleucina-33/deficiencia , Interleucina-33/genética , Mácula Lútea/inmunología , Mácula Lútea/patología , Degeneración Macular/genética , Degeneración Macular/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Procesamiento Proteico-Postraduccional , Ratas , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores de Interleucina/deficiencia , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Epitelio Pigmentado de la Retina/inmunología , Epitelio Pigmentado de la Retina/patologíaRESUMEN
We have recently reported a series of tetrahydroquinazoline (THQ) mTOR inhibitors that produced a clinical candidate 1 (GDC-0349). Through insightful design, we hoped to discover and synthesize a new series of small molecule inhibitors that could attenuate CYP3A4 time-dependent inhibition commonly observed with the THQ scaffold, maintain or improve aqueous solubility and oral absorption, reduce free drug clearance, and selectively increase mTOR potency. Through key in vitro and in vivo studies, we demonstrate that a pyrimidoaminotropane based core was able to address each of these goals. This effort culminated in the discovery of 20 (GNE-555), a highly potent, selective, metabolically stable, and efficacious mTOR inhibitor.
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Inhibidores Enzimáticos/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Tropanos/farmacología , Cromatografía Liquida , Inhibidores Enzimáticos/química , Humanos , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Tropanos/químicaRESUMEN
Aberrant activation of the PI3K-Akt-mTOR signaling pathway has been observed in human tumors and tumor cell lines, indicating that these protein kinases may be attractive therapeutic targets for treating cancer. Optimization of advanced lead 1 culminated in the discovery of clinical development candidate 8h, GDC-0349, a potent and selective ATP-competitive inhibitor of mTOR. GDC-0349 demonstrates pathway modulation and dose-dependent efficacy in mouse xenograft cancer models.
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Selective inhibitors of mammalian target of rapamycin (mTOR) kinase based upon saturated heterocycles fused to a pyrimidine core were designed and synthesized. Each series produced compounds with K(i) < 10 nM for the mTOR kinase and >500-fold selectivity over closely related PI3 kinases. This potency translated into strong pathway inhibition, as measured by phosphorylation of mTOR substrate proteins and antiproliferative activity in cell lines with a constitutively active PI3K pathway. Two compounds exhibiting suitable mouse PK were profiled in in vivo tumor models and were shown to suppress mTORC1 and mTORC2 signaling for over 12 h when dosed orally. Both compounds were additionally shown to suppress tumor growth in vivo in a PC3 prostate cancer model over a 14 day study.
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Antineoplásicos/síntesis química , Complejos Multiproteicos/antagonistas & inhibidores , Pirimidinas/síntesis química , Pirroles/síntesis química , Quinazolinas/síntesis química , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Administración Oral , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Disponibilidad Biológica , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina , Diana Mecanicista del Complejo 2 de la Rapamicina , Ratones , Ratones Desnudos , Simulación del Acoplamiento Molecular , Trasplante de Neoplasias , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Neoplasias de la Próstata , Pirimidinas/química , Pirimidinas/farmacología , Pirroles/química , Pirroles/farmacología , Quinazolinas/química , Quinazolinas/farmacología , Relación Estructura-Actividad , Trasplante HeterólogoRESUMEN
Breast cancer has been redefined into three clinically relevant subclasses: (i) estrogen/progesterone receptor positive (ER+/PR+), (ii) HER2/ERRB2 positive, and (iii) those lacking expression of all three markers (triple negative or basal-like). While targeted therapies for ER+/PR+ and HER2+ tumors have revolutionized patient treatment and increased lifespan, an urgent need exists for identifying novel targets for triple-negative breast cancers. Here, we used integrative genomic analysis to identify candidate oncogenes in triple-negative breast tumors and assess their function through loss of function screening. Using this approach, we identify lactate dehydrogenase B (LDHB), a component of glycolytic metabolism, as an essential gene in triple-negative breast cancer. Loss of LDHB abrogated cell proliferation in vitro and arrested tumor growth in fully formed tumors in vivo. We find that LDHB and other related glycolysis genes are specifically upregulated in basal-like/triple-negative breast cancers as compared with other subtypes, suggesting that these tumors are distinctly glycolytic. Consistent with this, triple-negative breast cancer cell lines were more dependent on glycolysis for growth than luminal cell lines. Finally, we find that patients with breast cancer and high LDHB expression in their tumors had a poor clinical outcome. While previous studies have focused on the ubiquitous role of LDHA in tumor metabolism and growth, our data reveal that LDHB is upregulated and required only in certain cancer genotypes. These findings suggest that targeting LDHB or other components of lactate metabolism would be of clinical benefit in triple-negative breast cancer.
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Neoplasias de la Mama/genética , Lactato Deshidrogenasas/genética , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Procesos de Crecimiento Celular/genética , Línea Celular Tumoral , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Lactato Deshidrogenasas/biosíntesis , Células MCF-7 , Ratones , Ratones Desnudos , Pronóstico , Receptor ErbB-2/metabolismo , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/metabolismo , Trasplante HeterólogoRESUMEN
The mammalian target of rapamycin (mTOR) is a central node in a complex signaling network that is regulated by several pathways deregulated in human cancers, including the PI3K/Akt and MAPK pathways. Targeting mTOR therefore presents an opportunity for therapeutic intervention. However, mTOR inhibition with rapamycin analogs or kinase inhibitors reduces cell growth but does not induce apoptosis, and the clinical benefit of rapamycin analogs has been modest. In this study we show that mTOR kinase inhibitors can potentiate apoptosis when used in combination with upstream targeted agents such as PI3K and MEK inhibitors. This increased apoptosis is dependent on genetic background, and correlates with active growth factor survival pathways. In PI3K mutant tumors, mTOR inhibition leads to partial reactivation of Akt which allows cells to survive, whereas in KRAS mutant tumors, this same reactivation of Akt occurs but is not required for cell survival. These data suggest the use of selected rational combinations of mTOR kinase inhibitors with other targeted inhibitors in specific tumor genotypes to achieve the maximal cytotoxic response by inhibiting two nodes in the activated signaling network.
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Apoptosis/efectos de los fármacos , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Western Blotting , Línea Celular Tumoral , Ensayo de Inmunoadsorción Enzimática , HumanosRESUMEN
CDK8 is a cyclin-dependent kinase that mediates transcriptional control of pathways linked to both cancer and stem cells. In this study, we show that CDK8 is required for both tumor growth and maintenance of tumor dedifferentiation in vivo and uncover a common role for CDK8 in controlling cancer and stem cell function. Acute CDK8 loss in vivo strongly inhibited tumor growth and promoted differentiation. Transcriptional profiling identified a set of embryonic stem cell-related genes that are activated by CDK8 in cancer. Consistent with this, we found that CDK8 expression correlated to the embryonic stem cell pluripotency state and loss of CDK8 caused embryonic stem cells to differentiate. This effect was, at least partially, mediated by the ability of CDK8 to regulate MYC protein and downstream MYC target gene expression. Similar regulation of MYC target genes by CDK8 was observed in colon tumor cells, and increased expression of a CDK8-regulated, embryonic stem cell MYC target gene signature was associated with loss of differentiation and poor outcome in primary human colon cancers. Together, these observations reveal that CDK8 acts, at least in part, through MYC to maintain both tumors and embryonic stem cells in an undifferentiated state. This raises the intriguing possibility that targeting CDK8 therapeutically may specifically inhibit the stem-like properties of cancer cells.
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Desdiferenciación Celular/fisiología , Quinasa 8 Dependiente de Ciclina/metabolismo , Células Madre Embrionarias/enzimología , Neoplasias Experimentales/enzimología , Neoplasias Experimentales/patología , Células Madre Pluripotentes/enzimología , Animales , Western Blotting , Línea Celular Tumoral , Separación Celular , Células Madre Embrionarias/citología , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica/fisiología , Genes myc , Humanos , Inmunohistoquímica , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Células Madre Pluripotentes/citología , TransfecciónRESUMEN
A series of inhibitors of mTOR kinase based on a quaternary-substituted dihydrofuropyrimidine was designed and synthesized. The most potent compounds in this series inhibited mTOR kinase with K(i) < 1.0 nM and were highly (>100×) selective for mTOR over the closely related PI3 kinases. Compounds in this series showed inhibition of the pathway and antiproliferative activity in cell-based assays. Furthermore, these compounds had excellent mouse PK, and showed a robust PK-PD relationship in a mouse model of cancer.
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Antineoplásicos/síntesis química , Furanos/síntesis química , Pirimidinas/síntesis química , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Administración Oral , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Disponibilidad Biológica , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Estabilidad de Medicamentos , Furanos/farmacocinética , Furanos/farmacología , Humanos , Ratones , Ratones Desnudos , Modelos Moleculares , Trasplante de Neoplasias , Inhibidores de las Quinasa Fosfoinosítidos-3 , Pirimidinas/farmacocinética , Pirimidinas/farmacología , Ratas , Especificidad de la Especie , Estereoisomerismo , Relación Estructura-Actividad , Trasplante HeterólogoRESUMEN
p21-activated kinases (PAKs) are serine/threonine protein kinases that serve as important mediators of Rac and Cdc42 GTPase function as well as pathways required for Ras-driven tumorigenesis. PAK1 has been implicated in signaling by growth factor receptors and morphogenetic processes that control cell polarity, invasion, and actin cytoskeleton organization. To better understand the role of PAK1 in tumorigenesis, PAK1 genomic copy number and expression were determined for a large panel of breast, lung, and head and neck tumors. PAK1 genomic amplification at 11q13 was prevalent in luminal breast cancer, and PAK1 protein expression was associated with lymph node metastasis. Breast cancer cells with PAK1 genomic amplification rapidly underwent apoptosis after inhibition of this kinase. Strong nuclear and cytoplasmic PAK1 expression was also prevalent in squamous nonsmall cell lung carcinomas (NSCLCs), and selective PAK1 inhibition was associated with delayed cell-cycle progression in vitro and in vivo. NSCLC cells were profiled using a library of pathway-targeted small-molecule inhibitors, and several synergistic combination therapies, including combination with antagonists of inhibitor of apoptosis proteins, were revealed for PAK1. Dual inhibition of PAK1 and X chromosome-linked inhibitor of apoptosis efficiently increased effector caspase activation and apoptosis of NSCLC cells. Together, our results provide evidence for dysregulation of PAK1 in breast and squamous NSCLCs and a role for PAK1 in cellular survival and proliferation in these indications.
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Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Proteínas de Neoplasias/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacocinética , Quinasas p21 Activadas/antagonistas & inhibidores , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Caspasas/metabolismo , Supervivencia Celular/efectos de los fármacos , Sistemas de Liberación de Medicamentos , Activación Enzimática/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ganglios Linfáticos/enzimología , Ganglios Linfáticos/patología , Metástasis Linfática , Masculino , Ratones , Ratones Desnudos , Proteínas de Neoplasias/metabolismo , Neoplasias/enzimología , Neoplasias/patología , Quinasas p21 Activadas/metabolismoRESUMEN
PURPOSE: The pathways underlying basal-like breast cancer are poorly understood, and as yet, there is no approved targeted therapy for this disease. We investigated the role of mitogen-activated protein kinase kinase (MEK) and phosphatidylinositol 3-kinase (PI3K) inhibitors as targeted therapies for basal-like breast cancer. EXPERIMENTAL DESIGN: We used pharmacogenomic analysis of a large panel of breast cancer cell lines with detailed accompanying molecular information to identify molecular predictors of response to a potent and selective inhibitor of MEK and also to define molecular mechanisms underlying combined MEK and PI3K targeting in basal-like breast cancer. Hypotheses were confirmed by testing in multiple tumor xenograft models. RESULTS: We found that basal-like breast cancer models have an activated RAS-like transcriptional program and show greater sensitivity to a selective inhibitor of MEK compared with models representative of other breast cancer subtypes. We also showed that loss of PTEN is a negative predictor of response to MEK inhibition, that treatment with a selective MEK inhibitor caused up-regulation of PI3K pathway signaling, and that dual blockade of both PI3K and MEK/extracellular signal-regulated kinase signaling synergized to potently impair the growth of basal-like breast cancer models in vitro and in vivo. CONCLUSIONS: Our studies suggest that single-agent MEK inhibition is a promising therapeutic modality for basal-like breast cancers with intact PTEN, and also provide a basis for rational combination of MEK and PI3K inhibitors in basal-like cancers with both intact and deleted PTEN.
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Neoplasias de la Mama/tratamiento farmacológico , Inhibidores Enzimáticos/farmacología , MAP Quinasa Quinasa 1/antagonistas & inhibidores , Inhibidores de las Quinasa Fosfoinosítidos-3 , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Análisis por Conglomerados , Relación Dosis-Respuesta a Droga , Femenino , Citometría de Flujo , Perfilación de la Expresión Génica , Humanos , Immunoblotting , MAP Quinasa Quinasa 1/genética , MAP Quinasa Quinasa 1/metabolismo , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos , Ratones Desnudos , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismoRESUMEN
PURPOSE: Oncogenic activation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway is prevalent in breast cancer and has been associated with resistance to HER2 inhibitors in the clinic. We therefore investigated the combinatorial activity of GDC-0941, a novel class I PI3K inhibitor, with standard-of-care therapies for HER2-amplified breast cancer. EXPERIMENTAL DESIGN: Three-dimensional laminin-rich extracellular matrix cultures of human breast cancer cells were utilized to provide a physiologically relevant approach to analyze the efficacy and molecular mechanism of combination therapies ex vivo. Combination studies were done using GDC-0941 with trastuzumab (Herceptin), pertuzumab, lapatinib (Tykerb), and docetaxel, the principal therapeutic agents that are either approved or being evaluated for treatment of early HER2-positive breast cancer. RESULTS: Significant GDC-0941 activity (EC(50) <1 micromol/L) was observed for >70% of breast cancer cell lines that were examined in three-dimensional laminin-rich extracellular matrix culture. Differential responsiveness to GDC-0941 as a single agent was observed for luminal breast cancer cells upon stimulation with the HER3 ligand, heregulin. Combined treatment of GDC-0941, trastuzumab, and pertuzumab resulted in growth inhibition, altered acinar morphology, and suppression of AKT mitogen-activated protein kinase (MAPK) / extracellular signed-regulated kinase (ERK) kinase and MEK effector signaling pathways for HER2-amplified cells in both normal and heregulin-supplemented media. The GDC-0941 and lapatinib combination further showed that inhibition of HER2 activity was essential for maximum combinatorial efficacy. PI3K inhibition also rendered HER2-amplified BT-474M1 cells and tumor xenografts more sensitive to docetaxel. CONCLUSIONS: GDC-0941 is efficacious in preclinical models of breast cancer. The addition of GDC-0941 to HER2-directed treatment could augment clinical benefit in breast cancer patients.