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.
Full text
Available for:
BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
The RAS/RAF/MEK pathway is activated in more than 30% of human cancers, most commonly via mutation in the K-ras oncogene and also via mutations in BRAF. Several allosteric mitogen-activated ...protein/extracellular signal-regulated kinase (MEK) inhibitors, aimed at treating tumors with RAS/RAF pathway alterations, are in clinical development. However, acquired resistance to these inhibitors has been documented both in preclinical and clinical samples. To identify strategies to overcome this resistance, we have derived three independent MEK inhibitor-resistant cell lines. Resistance to allosteric MEK inhibitors in these cell lines was consistently linked to acquired mutations in the allosteric binding pocket of MEK. In one cell line, concurrent amplification of mutant K-ras was observed in conjunction with MEK allosteric pocket mutations. Clonal analysis showed that both resistance mechanisms occur in the same cell and contribute to enhanced resistance. Importantly, in all cases the MEK-resistant cell lines retained their addiction to the mitogen-activated protein kinase (MAPK) pathway, as evidenced by their sensitivity to a selective inhibitor of the ERK1/2 kinases. These data suggest that tumors with acquired MEK inhibitor resistance remain dependent on the MAPK pathway and are therefore sensitive to inhibitors that act downstream of the mutated MEK target. Importantly, we show that dual inhibition of MEK and ERK by small molecule inhibitors was synergistic and acted to both inhibit the emergence of resistance, as well as to overcome acquired resistance to MEK inhibitors. Therefore, our data provide a rationale for cotargeting multiple nodes within the MAPK signaling cascade in K-ras mutant tumors to maximize therapeutic benefit for patients.
Docetaxel is a front-line standard-of-care chemotherapeutic drug for the treatment of breast cancer. Phosphoinositide 3-kinases (PI3K) are lipid kinases that regulate breast tumor cell growth, ...migration, and survival. The current study was intended to determine whether GDC-0941, an orally bioavailable class I selective PI3K inhibitor, enhances the antitumor activity of docetaxel in human breast cancer models in vitro and in vivo.
A panel of 25 breast tumor cell lines representing HER2+, luminal, and basal subtypes were treated with GDC-0941, docetaxel, or the combination of both drugs and assayed for cellular viability, modulation of PI3K pathway markers, and apoptosis induction. Drug combination effects on cellular viability were also assessed in nontransformed MCF10A human mammary epithelial cells. Human xenografts of breast cancer cell lines and patient-derived tumors were used to assess efficacy of GDC-0941 and docetaxel in vivo.
Combination of GDC-0941 and docetaxel decreased the cellular viability of breast tumor cell lines in vitro but to variable degrees of drug synergy. Compared with nontransformed MCF10A cells, the addition of both drugs resulted in stronger synergistic effects in a subset of tumor cell lines that were not predicted by breast cancer subtype. In xenograft models, GDC-0941 enhanced the antitumor activity of docetaxel with maximum combination efficacy observed within 1 hour of administering both drugs. GDC-0941 increased the rate of apoptosis in cells arrested in mitosis upon cotreatment with docetaxel.
GDC-0941 augments the efficacy of docetaxel by increasing drug-induced apoptosis in breast cancer models.
Abstract
Immunotherapeutic agents have shown dramatic success in oncology in recent years and several have been approved in melanoma and lung cancer. One strategy for extending the benefit of ...immunotherapy is to evaluate these agents in combination with standard of care chemotherapy, with the goal of bringing the benefit of immunotherapy into earlier lines of treatment.
We have carried out a systematic evaluation of the effects of different classes of chemotherapeutic agents (including alkylating agents, platinum based agents, and taxanes) on the tumor immune microenvironment of syngeneic murine tumor models (CT26 and MC38) in two different strains of mice (BALB/c and C57BL/6). We found that different chemotherapeutic agents caused markedly different changes in the composition and cell phenotype of the tumor immune environment. The majority of chemotherapeutic agents resulted in a mild to moderate increase in CD8+ T cells in the tumor. Some agents also altered the activation state of these CD8+ cells as measured by the level of PD-1 and IFN-gamma. A subset of the chemotherapeutic agents resulted in profound changes in the number of CD4+ cells, including regulatory T cells, as well as changes in myeloid subpopulations including monocytes, neutrophils, and macrophages. In addition to single agents studies, we carried out combination studies of selected chemo agents with simultaneous addition of anti-PD-L1. Several of these combinations caused pronounced increases in efficacy above single agent treatment. We will present the combination pharmacodynamic marker changes and efficacy of these treatments.
Our findings demonstrate that chemotherapeutic agents can stimulate the tumor immune environment, at least transiently, through multiple mechanisms. Different subsets of chemotherapeutic agents display unique changes in the tumor immune infiltrate, and may require different immunotherapeutic approaches. These findings provide a rationale for testing immunotherapy-chemotherapy combinations in the clinic.
Citation Format: Marcia P. Belvin, Shiuh-Ming Luoh, Jeanne Cheung, Erin McNamara, Rafael Cubas, Jeong Kim. Effects of chemotherapeutic agents on the tumor immune microenvironment. abstract. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4898.
Nicotinamide adenine dinucleotide (NAD) is a cofactor involved in a wide range of cellular metabolic processes and is a key metabolite required for tumor growth. NAMPT, nicotinamide ...phosphoribosyltransferase, which converts nicotinamide (NAM) to nicotinamide mononucleotide (NMN), the immediate precursor of NAD, is an attractive therapeutic target as inhibition of NAMPT reduces cellular NAD levels and inhibits tumor growth in vivo. However, there is limited understanding of the metabolic response to NAD depletion across cancer cell lines and whether all cell lines respond in a uniform manner. To explore this we selected two non-small cell lung carcinoma cell lines that are sensitive to the NAMPT inhibitor GNE-617 (A549, NCI-H1334), one that shows intermediate sensitivity (NCI-H441), and one that is insensitive (LC-KJ). Even though NAD was reduced in all cell lines there was surprising heterogeneity in their metabolic response. Both sensitive cell lines reduced glycolysis and levels of di- and tri-nucleotides and modestly increased oxidative phosphorylation, but they differed in their ability to combat oxidative stress. H1334 cells activated the stress kinase AMPK, whereas A549 cells were unable to activate AMPK as they contain a mutation in LKB1, which prevents activation of AMPK. However, A549 cells increased utilization of the Pentose Phosphate pathway (PPP) and had lower reactive oxygen species (ROS) levels than H1334 cells, indicating that A549 cells are better able to modulate an increase in oxidative stress. Inherent resistance of LC-KJ cells is associated with higher baseline levels of NADPH and a delayed reduction of NAD upon NAMPT inhibition. Our data reveals that cell lines show heterogeneous response to NAD depletion and that the underlying molecular and genetic framework in cells can influence the metabolic response to NAMPT inhibition.
Full text
Available for:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Targeted therapeutics that block signal transduction through the RAS–RAF–MEK and PI3K–AKT–mTOR pathways offer significant promise for the treatment of human malignancies. Dual inhibition of MAP/ERK ...kinase (MEK) and phosphatidylinositol 3-kinase (PI3K) with the potent and selective small-molecule inhibitors GDC-0973 and GDC-0941 has been shown to trigger tumor cell death in preclinical models. Here we have used phosphomotif antibodies and mass spectrometry (MS) to investigate the effects of MEK/PI3K dual inhibition during the period immediately preceding cell death. Upon treatment, melanoma cell lines responded by dramatically increasing phosphorylation on proteins containing a canonical DNA damage-response (DDR) motif, as defined by a phosphorylated serine or threonine residue adjacent to glutamine, s/tQ. In total, >2,000 s/tQ phosphorylation sites on >850 proteins were identified by LC-MS/MS, including an extensive network of DDR proteins. Linear mixed-effects modeling revealed 101 proteins in which s/tQ phosphorylation was altered significantly in response to GDC-0973/GDC-0941. Among the most dramatic changes, we observed rapid and sustained phosphorylation of sites within the ABCDE cluster of DNA-dependent protein kinase. Preincubation of cells with the inhibitors of the DDR kinases DNA-dependent protein kinase or ataxia-telangiectasia mutated enhanced GDC-0973/GDC-0941–mediated cell death. Network analysis revealed specific enrichment of proteins involved in RNA metabolism along with canonical DDR proteins and suggested a prominent role for this pathway in the response to MEK/PI3K dual inhibition.
Full text
Available for:
BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
GDC-0973 is a potent and selective mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor. Pharmacokinetic-pharmacodynamic (PK-PD) modeling was used to ...relate GDC-0973 plasma and tumor concentrations, tumor pharmacodynamics and antitumor efficacy to establish pharmacokinetic endpoints and predict active doses in the clinic.
A PK-PD model was used to characterize GDC-0973 tumor disposition and in vivo potency in WM-266-4 xenograft mice. Simulations were conducted using the PK-PD model along with human pharmacokinetics to identify a target plasma concentration and predict active doses. In vivo potency and antitumor efficacy were characterized in A375 melanoma xenograft mice, and a population-based integrated PK-PD-efficacy model was used to relate tumor pharmacodynamics (%pERK decrease) to antitumor activity.
GDC-0973 showed a sustained tumor pharmacodynamic response due to longer residence in tumor than in plasma. Following single doses of GDC-0973, estimated in vivo IC(50) values of %pERK decrease based on tumor concentrations in xenograft mice were 0.78 (WM-266-4) and 0.52 μmol/L (A375). Following multiple doses of GDC-0973, the estimated in vivo IC(50) value in WM-266-4 increased (3.89 μmol/L). Human simulations predicted a minimum target plasma concentration of 83 nmol/L and an active dose range of 28 to 112 mg. The steep relationship between tumor pharmacodynamics (%pERK decrease) and antitumor efficacy suggests a pathway modulation threshold beyond which antitumor efficacy switches on.
Clinical observations of %pERK decrease and antitumor activity were consistent with model predictions. This article illustrates how PK-PD modeling can improve the translation of preclinical data to humans by providing a means to integrate preclinical and early clinical data.
Abstract
Immunotherapeutic agents have shown great promise in the clinic in recent years and this has led to their approval as single agents or as immune doublet combinations in melanoma and lung ...cancer. In order to increase the extent of benefit from these agents and to extend immunotherapies to additional patients, combinations are being evaluated of immunotherapeutic agents with chemotherapy and targeted agents.
Inhibitors of the mitogen-activated kinase protein kinase (MAPK) pathway, including BRAF and MEK inhibitors, have been approved in melanoma, and are being evaluated in additional indications. We evaluated the effects of MEK and ERK inhibitors on human T cells cultured in vitro as well as on the tumor microenvironment in preclinical syngeneic mouse models. In studies with human T cells in vitro, we find that MEK or ERK inhibition reduced proliferation of naïve T cells, but had little effect on the proliferation of central memory cells, suggesting a differential requirement on MAPK signaling for proliferation of these T cell subsets. In vivo, MEK and ERK inhibition resulted in an increase in CD8+ infiltration in the tumor, as well as a decrease of markers of T cell exhaustion such as PD-1 and EOMES. The combination of MEK or ERK inhibitors plus anti-PD-L1 resulted in increased CD8+ effector function as measured by increased interferon gamma (IFNg) levels. In some cases, the MEK and ERK inhibitors showed differences in their pharmacodynamic effects as single agents; however, in combination with anti-PD-L1, the effects were similar. For example, single agent MEK inhibition but not single agent ERK inhibition increased the number of CD4+ helper and regulatory T cells (Tregs) in the tumor. However, both of these CD4+ subsets, as well as CD4+ IFNg levels, were increased with MEK or ERK inhibitors combined with anti-PD-L1. Similarly, single agent MEK inhibition decreased the number of infiltrating CD11b+Ly6G+ myeloid cells whereas single agent ERK inhibition increased the number of infiltrating CD11b+Ly6C+ myeloid cells. However, in combination with anti-PD-L1, these changes in myeloid populations were less apparent.
Taken together, these data show that MAPK pathway suppression can modulate multiple immune cell subtypes within the tumor, with many of these changes expected to activate the immune infiltrate. Despite the increase in Tregs in response to ERK and MEK inhibition in combination with anti-PD-L1, these combinations were efficacious. This suggests that the other immune stimulatory effects of these treatments are sufficient to drive efficacy despite the presence of increased Tregs. Our data suggest that the combination of MAPK inhibition plus anti-PD-L1 inhibition, such as with atezolizumab, is a promising hypothesis to test in the clinic.
Citation Format: Marcia P. Belvin, Erin Williams, Shiuh-Ming Luoh, Jeanne Cheung, Christine Orr, Emily Chan, Peter Ebert, Ira Mellman, Jeong Kim, Mark Merchant. Effects of MAPK pathway inhibitors in the tumor immune microenvironment. abstract. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4905.
The Toll-Dorsal pathway in Drosophila and the interleukin-1 receptor (IL-1R)-NF-kappa B pathway in mammals are homologous signal transduction pathways that mediate several different biological ...responses. In Drosophila, genetic analysis of dorsal-ventral patterning of the embryo has defined the series of genes that mediate the Toll-Dorsal pathway. Binding of extracellular ligand activates the transmembrane receptor Toll, which requires the novel protein Tube to activate the cytoplasmic serine/threonine kinase Pelle. Pelle activity controls the degradation of the Cactus protein, which is present in a cytoplasmic complex with the Dorsal protein. Once Cactus is degraded in response to signal, Dorsal is free to move into the nucleus where it regulates transcription of specific target genes. The Toll, tube, pelle, cactus, and dorsal genes also appear to be involved in Drosophila immune response. Because the IL-1R-NF-kappa B pathway plays a role in vertebrate innate immunity and because plant homologues of the Toll-Dorsal pathway are important in plant disease resistance, it is likely that this pathway arose before the divergence of plants and animals as a defense against pathogens.
Full text
Available for:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
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 μmol/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.
PDF
