Loss-of-function mutations in the retinoblastoma gene
are common in several treatment-refractory cancers such as small-cell lung cancer and triple-negative breast cancer. To identify drugs synthetic ...lethal with
mutation (
), we tested 36 cell-cycle inhibitors using a cancer cell panel profiling approach optimized to discern cytotoxic from cytostatic effects. Inhibitors of the Aurora kinases AURKA and AURKB showed the strongest
association in this assay. LY3295668, an AURKA inhibitor with over 1,000-fold selectivity versus AURKB, is distinguished by minimal toxicity to bone marrow cells at concentrations active against
cancer cells and leads to durable regression of
tumor xenografts at exposures that are well tolerated in rodents. Genetic suppression screens identified enforcers of the spindle-assembly checkpoint (SAC) as essential for LY3295668 cytotoxicity in RB1-deficient cancers and suggest a model in which a primed SAC creates a unique dependency on AURKA for mitotic exit and survival. SIGNIFICANCE: The identification of a synthetic lethal interaction between
and AURKA inhibition, and the discovery of a drug that can be dosed continuously to achieve uninterrupted inhibition of AURKA kinase activity without myelosuppression, suggest a new approach for the treatment of RB1-deficient malignancies, including patients progressing on CDK4/6 inhibitors.
.
.
Mechanisms driving resistance to cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) in hormone receptor-positive (HR
) breast cancer have not been clearly defined. Whole-exome sequencing of 59 tumors ...with CDK4/6i exposure revealed multiple candidate resistance mechanisms including
loss, activating alterations in
, and
, and loss of estrogen receptor expression.
experiments confirmed that these alterations conferred CDK4/6i resistance. Cancer cells cultured to resistance with CDK4/6i also acquired
, or
alterations, which conferred sensitivity to AURKA, ERK, or CHEK1 inhibition. Three of these activating alterations-in
, and
-have not, to our knowledge, been previously demonstrated as mechanisms of resistance to CDK4/6i in breast cancer preclinically or in patient samples. Together, these eight mechanisms were present in 66% of resistant tumors profiled and may define therapeutic opportunities in patients. SIGNIFICANCE: We identified eight distinct mechanisms of resistance to CDK4/6i present in 66% of resistant tumors profiled. Most of these have a therapeutic strategy to overcome or prevent resistance in these tumors. Taken together, these findings have critical implications related to the potential utility of precision-based approaches to overcome resistance in many patients with HR
metastatic breast cancer.
.
Assessing the pathogenicity of genetic variants can be a complex and challenging task. Spliceogenic variants, which alter mRNA splicing, may yield mature transcripts that encode non-functional ...protein products, an important predictor of Mendelian disease risk. However, most variant annotation tools do not adequately assess spliceogenicity outside the native splice site and thus the disease-causing potential of variants in other intronic and exonic regions is often overlooked. Here, we present a plugin for the Ensembl Variant Effect Predictor that packages MaxEntScan and extends its functionality to provide splice site predictions using a maximum entropy model. The plugin incorporates a sliding window algorithm to predict splice site loss or gain for any variant that overlaps a transcript feature. We also demonstrate the utility of the plugin by comparing our predictions to two mRNA splicing datasets containing several cancer-susceptibility genes.
Source code is freely available under the Apache License, Version 2.0: https://github.com/Ensembl/VEP_plugins.
Supplementary data are available at Bioinformatics online.
p38α mitogen-activated protein kinase (MAPK) is activated in cancer cells in response to environmental factors, oncogenic stress, radiation, and chemotherapy. p38α MAPK phosphorylates a number of ...substrates, including MAPKAP-K2 (MK2), and regulates the production of cytokines in the tumor microenvironment, such as TNF-α, interleukin-1β (IL-1β), IL-6, and CXCL8 (IL-8). p38α MAPK is highly expressed in human cancers and may play a role in tumor growth, invasion, metastasis, and drug resistance. LY2228820 dimesylate (hereafter LY2228820), a trisubstituted imidazole derivative, is a potent and selective, ATP-competitive inhibitor of the α- and β-isoforms of p38 MAPK in vitro (IC(50) = 5.3 and 3.2 nmol/L, respectively). In cell-based assays, LY2228820 potently and selectively inhibited phosphorylation of MK2 (Thr334) in anisomycin-stimulated HeLa cells (at 9.8 nmol/L by Western blot analysis) and anisomycin-induced mouse RAW264.7 macrophages (IC(50) = 35.3 nmol/L) with no changes in phosphorylation of p38α MAPK, JNK, ERK1/2, c-Jun, ATF2, or c-Myc ≤ 10 μmol/L. LY2228820 also reduced TNF-α secretion by lipopolysaccharide/IFN-γ-stimulated macrophages (IC(50) = 6.3 nmol/L). In mice transplanted with B16-F10 melanoma, tumor phospho-MK2 (p-MK2) was inhibited by LY2228820 in a dose-dependent manner threshold effective dose (TED)(70) = 11.2 mg/kg. Significant target inhibition (>40% reduction in p-MK2) was maintained for 4 to 8 hours following a single 10 mg/kg oral dose. LY2228820 produced significant tumor growth delay in multiple in vivo cancer models (melanoma, non-small cell lung cancer, ovarian, glioma, myeloma, breast). In summary, LY2228820 is a p38 MAPK inhibitor, which has been optimized for potency, selectivity, drug-like properties (such as oral bioavailability), and efficacy in animal models of human cancer.
Radial glial cells are presumptive neural stem cells (NSCs) in the developing nervous system. The direct requirement of radial glia for the generation of a diverse array of neuronal and glial ...subtypes, however, has not been tested. We employed two novel transgenic zebrafish lines and endogenous markers of NSCs and radial glia to show for the first time that radial glia are essential for neurogenesis during development. By using the gfap promoter to drive expression of nuclear localized mCherry we discerned two distinct radial glial‐derived cell types: a major nestin+/Sox2+ subtype with strong gfap promoter activity and a minor Sox2+ subtype lacking this activity. Fate mapping studies in this line indicate that gfap+ radial glia generate later‐born CoSA interneurons, secondary motorneurons, and oligodendroglia. In another transgenic line using the gfap promoter‐driven expression of the nitroreductase enzyme, we induced cell autonomous ablation of gfap+ radial glia and observed a reduction in their specific derived lineages, but not Blbp+ and Sox2+/gfap‐negative NSCs, which were retained and expanded at later larval stages. Moreover, we provide evidence supporting classical roles of radial glial in axon patterning, blood–brain barrier formation, and locomotion. Our results suggest that gfap+ radial glia represent the major NSC during late neurogenesis for specific lineages, and possess diverse roles to sustain the structure and function of the spinal cord. These new tools will both corroborate the predicted roles of astroglia and reveal novel roles related to development, physiology, and regeneration in the vertebrate nervous system. GLIA 2016;64:1170–1189
Main Points
Gfap+ radial glia are the major NSC and required for embryonic neurogenesis.
Two distinct cycling NSC types exist in the embryonic spinal cord.
Radial glial death is cleared by microglia and triggers a proliferative response by Sox2+ NSCs.
Elevated eukaryotic translation initiation factor 4E (eIF4E) function induces malignancy in experimental models by selectively enhancing translation of key malignancy-related mRNAs (c-myc and BCL-2). ...eIF4E activation may reflect increased eIF4E expression or phosphorylation of its inhibitory binding proteins (4E-BP). By immunohistochemical analyses of 148 tissues from 89 prostate cancer patients, we now show that both eIF4E expression and 4E-BP1 phosphorylation (p4E-BP1) are increased significantly, particularly in advanced prostate cancer versus benign prostatic hyperplasia tissues. Further, increased eIF4E and p4E-BP1 levels are significantly related to reduced patient survival, whereas uniform 4E-BP1 expression is significantly related to better patient survival. Both immunohistochemistry and Western blotting reveal that elevated eIF4E and p4E-BP1 are evident in the same prostate cancer tissues. In two distinct prostate cancer cell models, the progression to androgen independence also involves increased eIF4E activation. In these prostate cancer cells, reducing eIF4E expression with an eIF4E-specific antisense oligonucleotide currently in phase I clinical trials robustly induces apoptosis, regardless of cell cycle phase, and reduces expression of the eIF4E-regulated proteins BCL-2 and c-myc. Collectively, these data implicate eIF4E activation in prostate cancer and suggest that targeting eIF4E may be attractive for prostate cancer therapy.
Expression of eukaryotic translation initiation factor 4E (eIF4E) is commonly elevated in human and experimental cancers, promoting angiogenesis and tumor growth. Elevated eIF4E levels selectively ...increase translation of growth factors important in malignancy (e.g., VEGF, cyclin D1) and is thereby an attractive anticancer therapeutic target. Yet to date, no eIF4E-specific therapy has been developed. Herein we report development of eIF4E-specific antisense oligonucleotides (ASOs) designed to have the necessary tissue stability and nuclease resistance required for systemic anticancer therapy. In mammalian cultured cells, these ASOs specifically targeted the eIF4E mRNA for destruction, repressing expression of eIF4E-regulated proteins (e.g., VEGF, cyclin D1, survivin, c-myc, Bcl-2), inducing apoptosis, and preventing endothelial cells from forming vessel-like structures. Most importantly, intravenous ASO administration selectively and significantly reduced eIF4E expression in human tumor xenografts, significantly suppressing tumor growth. Because these ASOs also target murine eIF4E, we assessed the impact of eIF4E reduction in normal tissues. Despite reducing eIF4E levels by 80% in mouse liver, eIF4E-specific ASO administration did not affect body weight, organ weight, or liver transaminase levels, thereby providing the first in vivo evidence that cancers may be more susceptible to eIF4E inhibition than normal tissues. These data have prompted eIF4E-specific ASO clinical trials for the treatment of human cancers.
The pattern of change in maternal bone turnover throughout pregnancy is poorly characterized.
We investigated changes across pregnancy in a marker of maternal bone resorption, urinary C-terminal ...telopeptide of type I collagen (CTX), the influence of gestational vitamin D supplementation, and associations between CTX and maternal postnatal bone indices.
MAVIDOS (the Maternal Vitamin D Osteoporosis Study) is a randomized, double-blind, placebo-controlled trial of 1000 IU cholecalciferol/d compared with placebo from 14 weeks of gestation to birth. Maternal second-void urinary α- and β-CTX were measured (ELISA) at 14 and 34 weeks of gestation; DXA was performed within 2 wk postpartum. The Mann–Whitney Rank Sum test, Spearman’s rank correlation, and linear regression were used to compare median CTX values within and between groups from early to late pregnancy, and associations with maternal bone outcomes.
In total, 372 women had CTX and 25-hydroxyvitamin D 25(OH)D measured in early and late pregnancy. CTX at 14 and 34 weeks of gestation were correlated in both placebo (r = 0.31) and cholecalciferol (r = 0.45) groups (P < 0.0001). Median CTX increased from 14 to 34 weeks of gestation in both groups (n = 372 total) placebo (n = 188): from 223.6 to 449.7 μg/mmol creatinine; cholecalciferol (n = 184): from 222.3 to 419.3 μg/mmol creatinine; P = 0.03 for placebo compared with cholecalciferol difference in CTX at 34 weeks of gestation. The conditional mean ± SD increase in CTX z-score (SD) from early to late pregnancy was greater in the placebo group (n = 188) than in the cholecalciferol group (n = 184) (placebo: 0.16 ± 0.92; cholecalciferol: −0.16 ± 1.06; P-difference < 0.01). Higher CTX at 34 weeks of gestation was associated, similarly in both groups, with lower maternal total hip and lumbar spine bone mineral content and bone mineral density (BMD) (e.g., lumbar spine BMD: β = −0.02 g · cm−2 · SD−1 increase in CTX; 95% CI: −0.027, −0.002 g · cm−2 · SD−1; P = 0.02, n = 283).
Maternal urinary CTX, a bone resorption marker, rises through pregnancy, although to a lesser degree with gestational cholecalciferol supplementation, and is inversely associated with maternal bone mass postpartum. This trial was registered at www.isrctn.com as ISRCTN 82927713 and eudract.ema.europa.eu as EudraCT 2007-001716-23.
Enzastaurin (LY317615.HCl) is currently in a phase III registration trial for diffuse large B-Cell lymphoma and numerous phase II clinical trials. Enzastaurin suppresses angiogenesis and induces ...apoptosis in multiple human tumor cell lines by inhibiting protein kinase C (PKC) and phosphoinositide 3-kinase (PI3K)/AKT pathway signaling. PI3K/AKT pathway signaling liberates eukaryotic translation initiation factor 4E (eIF4E) through the hierarchical phosphorylation of eIF4E binding proteins (4E-BP). When hypophosphorylated, 4E-BPs associate with eIF4E, preventing eIF4E from binding eIF4G, blocking the formation of the eIF4F translation initiation complex. Herein, we show that enzastaurin treatment impacts signaling throughout the AKT/mTOR pathway leading to hypophosphorylation of 4E-BP1 in cancer cells of diverse lineages (glioblastoma, colon carcinoma, and B-cell lymphoma). Accordingly, enzastaurin treatment increases the amount of eIF4E bound to 4E-BP1 and decreases association of eIF4E with eIF4G, thereby reducing eIF4F translation initiation complex levels. We therefore chose to evaluate whether this effect on 4E-BP1 was involved in enzastaurin-induced apoptosis. Remarkably, enzastaurin-induced apoptosis was blocked in cancer cells depleted of 4E-BP1 by siRNAs, or in 4EBP1/2 knockout murine embryonic fibroblasts cells. Furthermore, eIF4E expression was increased and 4E-BP1 expression was decreased in cancer cells selected for reduced sensitivity to enzastaurin-induced apoptosis. These data highlight the importance of modulating 4E-BP1 function, and eIF4F complex levels, in the direct antitumor effect of enzastaurin and suggest that 4E-BP1 function may serve as a promising determinant of enzastaurin activity.
The
DNA base excision repair pathway is responsible for the repair of
alkylation and oxidative DNA damage. A crucial step in the base
excision repair pathway involves the cleavage of an
...apurinic/apyrimidinic (AP) site in DNA by an AP endonuclease (APE). The
major AP endonuclease in mammalian cells is APE/ref-1, a
multifunctional enzyme that acts not only as an AP endonuclease but as
a redox-modifying factor for a variety of transcription factors. The
purpose of this study was to determine the expression of APE/redox
factor-1 (ref-1) in ovarian tissues, particularly ovarian cancers.
Formalin-fixed, paraffin-embedded specimens of ovarian tissues (normal,
various benign conditions, and epithelial cancers) were studied using
both polyclonal and monoclonal antibodies to APE/ref-1. The
relationship between APE/ref-1 protein levels and DNA repair activity
was studied in ovarian Hey and Hey-C2 cell lines using Western blot and
a specific AP-site oligonucleotide cleavage assay. Hey and Hey-C2 cells
were fractionated, and the nuclear and cytoplasmic extracts were
quantitated for protein levels and assessed for APE/ref-1 with Western
blot. Normal ovarian tissues consistently demonstrated strong nuclear
staining of the surface epithelium, epithelial inclusions, corpora
lutea and albicantia, and stroma. Cytoplasmic staining was absent. A
similar pattern was seen for benign conditions including endometriosis.
Low malignant potential ovarian cancers stained in a pattern similar to
normal ovarian and nonneoplastic tissues; however, two specimens also
had areas of cytoplasmic staining. Epithelial ovarian cancers were
remarkably different from all other ovarian tissues studied. Both
nuclear and cytoplasmic staining of the malignant epithelium were seen
and ranged from strong to weak, often with considerable staining
heterogeneity within the same tumor. The AP-site oligonucleotide
cleavage assay indicated that APE/ref-1 protein levels correlate well
with DNA repair activity. The increased levels of APE/ref-1 in the
Hey-C2 cells was mainly attributable to increased cytoplasmic enzyme.
APE/ref-1 immunoreactivity is altered in malignant ovarian tumors.
Further studies will determine whether the altered expression and
subcellular location reflect changes in redox regulatory functions.
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