Regulation of translation initiation plays a critical role in the control of cell growth and division in eukaryotic cells. Translation of many growth regulatory proteins including cyclins depends ...critically on translation initiation factors because their mRNAs are translated inefficiently. We report that clotrimazole, a potent antiproliferative agent both in vitro and in vivo, inhibits cell growth by interfering with translation initiation. In particular, clotrimazole causes a sustained depletion of intracellular Ca2+stores, which results in activation of PKR, phosphorylation of eIF2α , and thereby in inhibition of protein synthesis at the level of translation initiation. Consequently, clotrimazole preferentially decreases the expression of the growth promoting proteins cyclin A, E and D1, resulting in inhibition of cyclindependent kinase activity and blockage of cell cycle in G1.
The vast majority of ovarian cancer-related deaths are caused by metastatic dissemination of tumor cells, resulting in subsequent organ failure. However, despite our increased understanding of the ...physiological processes involved in tumor metastasis, there are no clinically approved drugs that have made a major impact in increasing the overall survival of patients with advanced, metastatic ovarian cancer. We identified prosaposin (psap) as a potent inhibitor of tumor metastasis, which acts via stimulation of p53 and the antitumorigenic protein thrombospondin-1 (TSP-1) in bone marrow-derived cells that are recruited to metastatic sites. We report that more than 97% of human serous ovarian tumors tested express CD36, the receptor that mediates the proapoptotic activity of TSP-1. Accordingly, we sought to determine whether a peptide derived from psap would be effective in treating this form of ovarian cancer. To that end, we developed a cyclic peptide with drug-like properties derived from the active sequence in psap. The cyclic psap peptide promoted tumor regression in a patient-derived tumor xenograft model of metastatic ovarian cancer. Thus, we hypothesize that a therapeutic agent based on this psap peptide would have efficacy in treating patients with metastatic ovarian cancer.
The clinical efficacy of epidermal growth factor receptor (EGFR)–targeted therapy in
-mutant non–small cell lung cancer is limited by the development of drug resistance. One mechanism of EGFR ...inhibitor resistance occurs through amplification of the human growth factor receptor (
) proto-oncogene, which bypasses EGFR to reactivate downstream signaling. Tumors exhibiting concurrent
mutation and
amplification are historically thought to be codependent on the activation of both oncogenes. Hence, patients whose tumors harbor both alterations are commonly treated with a combination of EGFR and MET tyrosine kinase inhibitors (TKIs). Here, we identify and characterize six patient-derived models of
-mutant,
-amplified lung cancer that have switched oncogene dependence to rely exclusively on MET activation for survival. We demonstrate in this MET-driven subset of EGFR TKI-refractory cancers that canonical EGFR downstream signaling was governed by MET, even in the presence of sustained mutant EGFR expression and activation. In these models, combined EGFR and MET inhibition did not result in greater efficacy in vitro or in vivo compared to single-agent MET inhibition. We further identified a reduced
mRNA expression stoichiometry as associated with
oncogene dependence and single-agent MET TKI sensitivity. Tumors from 10 of 11 EGFR inhibitor–resistant
-mutant,
-amplified patients also exhibited a reduced
mRNA ratio. Our findings reveal that a subset of
-mutant,
-amplified lung cancers develop dependence on MET activation alone, suggesting that such patients could be treated with a single-agent MET TKI rather than the current standard-of-care EGFR and MET inhibitor combination regimens.
The thiazolidinedione (TZD) class of peroxisome proliferator-activated receptor (PPAR) gamma ligands, known for their ability to induce adipocyte differentiation and increase insulin sensitivity, ...also exhibits anticancer properties. Currently, TZDs are being tested in clinical trials for treatment of human cancers expressing high levels of PPARgamma because it is assumed that activation of PPARgamma mediates their anticancer activity. Using PPARgamma(-/-) and PPARgamma(+/+) mouse embryonic stem cells, we report here that inhibition of cell proliferation and tumor growth by TZDs is independent of PPARgamma. Our studies demonstrate that these compounds block G(1)-S transition by inhibiting translation initiation. Inhibition of translation initiation is the consequence of partial depletion of intracellular calcium stores and the resulting activation of protein kinase R that phosphorylates the alpha subunit of eukaryotic initiation factor 2 (eIF2), thus rendering eIF2 inactive. PPARgamma-independent inhibition of translation initiation most likely accounts for the anticancer properties of thiazolidinediones.
B-Raf represents an attractive target for anticancer therapy and the development of small molecule B-Raf inhibitors has delivered new therapies for metastatic melanoma patients. We have discovered a ...novel class of small molecules that inhibit mutant B-Raf(V600E) kinase activity both in vitro and in vivo. Investigations into the structure-activity relationships of the series are presented along with efforts to improve upon the cellular potency, solubility, and pharmacokinetic profile. Compounds selectively inhibited B-Raf(V600E) in vitro and showed preferential antiproliferative activity in mutant B-Raf(V600E) cell lines and exhibited selectivity in a kinase panel against other kinases. Examples from this series inhibit growth of a B-Raf(V600E) A375 xenograft in vivo at a well-tolerated dose. In addition, aminoquinazolines described herein were shown to display pERK elevation in nonmutant B-Raf cell lines in vitro.
An allosteric inhibitor, EAI045, is reported that is selective for certain drug-resistant EGFR mutants, but spares the wild-type receptor; combination therapy of EAI045 with ...EGFR-dimerization-blocking antibodies is effective in mouse models of lung cancer driven by mutant versions of EGFR that are resistant to all previously developed inhibitors.
EGFR tyrosine kinase inhibitors (TKIs) gefitinib, erlotinib and afatinib are approved treatments for non-small cell lung cancers harboring activating mutations in the EGFR kinase
1
,
2
, but ...resistance arises rapidly, most frequently due to the secondary T790M mutation within the ATP-site of the receptor.
3
,
4
Recently developed mutant-selective irreversible inhibitors are highly active against the T790M mutant
5
,
6
, but their efficacy can be compromised by acquired mutation of C797, the cysteine residue with which they form a key covalent bond
7
. All current EGFR TKIs target the ATP-site of the kinase, highlighting the need for therapeutic agents with alternate mechanisms of action. Here we describe rational discovery of EAI045, an allosteric inhibitor that targets selected drug-resistant EGFR mutants but spares the wild type receptor. A crystal structure shows that the compound binds an allosteric site created by the displacement of the regulatory C-helix in an inactive conformation of the kinase. The compound inhibits L858R/T790M-mutant EGFR with low-nanomolar potency in biochemical assays, but as a single agent is not effective in blocking EGFR-driven proliferation in cells due to differential potency on the two subunits of the dimeric receptor, which interact in an asymmetric manner in the active state
8
. We observe dramatic synergy of EAI045 with cetuximab, an antibody therapeutic that blocks EGFR dimerization
9
,
10
, rendering the kinase uniformly susceptible to the allosteric agent. EAI045 in combination with cetuximab is effective in mouse models of lung cancer driven by L858R/T790M EGFR and by L858R/T790M/C797S EGFR, a mutant that is resistant to all currently available EGFR TKIs. More generally, our findings illustrate the utility of purposefully targeting allosteric sites to obtain mutant-selective inhibitors.
Abstract 1692
Poster Board I-718
Transforming growth factor-b-activated kinase 1 (TAK1) is a key regulator of NF-kB activation. TAK1 can be activated by a variety of pro-inflammatory cytokines and T ...and B cell receptors. Recent experiments demonstrated that deletion of TAK1 results in inactivation of both JNK and NF-kB signaling resulting in massive apoptotic death of hematopoietic cells in mice. In this study, we examined the expression pattern of TAK1 and its role as a potential therapeutic target for lymphoma. First, we examined TAK1 expression in a panel of lymphoid cell lines by western blot, and found it to be highly expressed in mantle cell lymphoma cell lines (Mino, SP53, and Jeko-1). These lines expressed relatively low levels of the tumor suppressor protein A20. Mino and SP53 expressed high level of p-p38. Subsequently, we investigated the in vitro activity of the novel TAK1 small molecule inhibitor AZ-Tak1 in these cell lines. AZ-Tak1 is a potent and a relatively selective inhibitor of TAK1 kinase activity, with an IC50 of 0.009 mM. It also inhibits Jak2 but at a much higher concentration (IC50=0.18 mM). AZ-Tak1 treatment decreased the level of p38 and ERK in mantle cell lymphoma cells, and induced apoptosis in a dose and time dependent manner, with an IC50 of 0.1-0.5 mM. Using the annexin-V and PI staining and FACS analysis, After 48 hours of incubation, AZ-Tak1 (0.1 mM) induced apoptosis in 28%, 34% and 86% of Mino, SP53, and Jeko cells, respectively, which was increased to 32%, 42%, and 86% when 0.5 mM concentration was used. Similar activity was also observed when primary mantle cell lymphoma cells were examined. Using pathway-specific protein arrays focusing on apoptosis, kinases, and transcription factors, AZ-Tak1 (0.5 mM) altered the level of several proteins that regulate cell growth and survival, especially members of the inhibitors of apoptosis (IAP) family. Specifically, AZ-Tak1 decreased the level of SMAC/DIABOLO and cytochrome –C in the mitochondria, which was associated with a decrease in the level of the anti-apoptotic protein X-linked IAP (XIAP) and activation of the intrinsic apoptotic pathway as evident by activation of caspase 9, cleavage of caspase 3, and induction of apoptosis. Furthermore, and consistant with its ability to inhibit Jak2 activity, AZ-Tak1 reduced STAT2 and STAT6 levels. AZ-Tak1 demonstrated no significant effect on bcl-2 family members. Finally, co-treatment with HDAC inhibitors demonstrated synergistic effect with low concentrations of AZ-Tak1. Collectively, our data demonstrate that targeting TAK1 by the small molecule inhibitor AZ-Tak1 induces cell death in mantle cell lymphoma by activating the intrinsic apoptosis pathway, suggesting that targeting TAK1 may have a therapeutic value for the treatment of mantle cell lymphoma.
Palakurthi:Astra Zeneca: Employment. Byth:Astra Zeneca : Employment.
BackgroundTumor orchestrated metabolic changes in the microenvironment limit generation of anti-tumor immune responses. Availability of arginine, a semi-essential amino acid, is critical for ...lymphocyte proliferation and function. Levels of arginine are regulated by the enzymes arginase 1,2 and nitric oxide synthase (NOS). However, the role of arginase activity in lung tumor maintenance has not been investigated in clinically relevant orthotopic tumor models.MethodsRNA sequencing (RNA-seq) of sorted cell populations from mouse lung adenocarcinomas derived from immunocompetent genetically engineered mouse models (GEMM)s was performed. To complement mouse studies, a patient tissue microarray consisting of 150 lung adenocarcinomas, 103 squamous tumors, and 54 matched normal tissue were stained for arginase, CD3, and CD66b by multiplex immunohistochemistry. Efficacy of a novel arginase inhibitor compound 9 in reversing arginase mediated T cell suppression was determined in splenocyte ex vivo assays. Additionally, the anti-tumor activity of this compound was determined in vitro and in an autochthonous immunocompetent KrasG12D GEMM of lung adenocarcinoma model.ResultsAnalysis of RNA-seq of sorted myeloid cells suggested that arginase expression is elevated in myeloid cells in the tumor as compared to the normal lung tissue. Accordingly, in the patient samples arginase 1 expression was mainly localized in the granulocytic myeloid cells and significantly elevated in both lung adenocarcinoma and squamous tumors as compared to the controls. Our ex vivo analysis demonstrated that myeloid derived suppressor cell (MDSC)s cause T cell suppression by arginine depletion, and suppression of arginase activity by a novel ARG1/2 inhibitor, compound 9, led to restoration of T cell function by increasing arginine. Treatment of KrasG12D GEMM of lung cancer model with compound 9 led to a significant tumor regression associated with increased T cell numbers and function, while it had no activity across several murine and human non-small cell (NSCLC) lung cancer lines in vitro.ConclusionsWe show that arginase expression is elevated in mouse and patient lung tumors. In a KRASG12D GEMM arginase inhibition diminished growth of established tumors. Our data suggest arginase as an immunomodulatory target that should further be investigated in lung tumors with high arginase activity.
Most patients with late-stage high-grade serous ovarian cancer (HGSOC) initially respond to chemotherapy but inevitably relapse and develop resistance, highlighting the need for novel therapies to ...improve patient outcomes. The MEK/ERK pathway is activated in a large subset of HGSOC, making it an attractive therapeutic target. Here, we systematically evaluated the extent of MEK/ERK pathway activation and efficacy of pathway inhibition in a large panel of well-annotated HGSOC patient-derived xenograft models. The vast majority of models were nonresponsive to the MEK inhibitor cobimetinib (GDC-0973) despite effective pathway inhibition. Proteomic analyses of adaptive responses to GDC-0973 revealed that GDC-0973 upregulated the proapoptotic protein BIM, thus priming the cells for apoptosis regulated by BCL2-family proteins. Indeed, combination of both MEK inhibitor and dual BCL-2/X
inhibitor (ABT-263) significantly reduced cell number, increased cell death, and displayed synergy
in most models.
, GDC-0973 and ABT-263 combination was well tolerated and resulted in greater tumor growth inhibition than single agents. Detailed proteomic and correlation analyses identified two subsets of responsive models-those with high BIM at baseline that was increased with MEK inhibition and those with low basal BIM and high pERK levels. Models with low BIM and low pERK were nonresponsive. Our findings demonstrate that combined MEK and BCL-2/X
inhibition has therapeutic activity in HGSOC models and provide a mechanistic rationale for the clinical evaluation of this drug combination as well as the assessment of the extent to which BIM and/or pERK levels predict drug combination effectiveness in chemoresistant HGSOC.
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