TP53 is the most frequently mutated gene in human malignancies; however, de novo somatic mutations in childhood embryonal cancers such as neuroblastoma are rare. We report on the analysis of three ...independent case-control cohorts comprising 10290 individuals and demonstrate that rs78378222 and rs35850753, rare germline variants in linkage disequilibrium that map to the 3' untranslated region (UTR) of TP53 and 5' UTR of the Δ133 isoform of TP53, respectively, are robustly associated with neuroblastoma (rs35850753: odds ratio OR = 2.7, 95% confidence interval CI = 2.0 to 3.6, P combined = 3.43×10(-12); rs78378222: OR = 2.3, 95% CI = 1.8 to 2.9, P combined = 2.03×10(-11)). All statistical tests were two-sided. These findings add neuroblastoma to the complex repertoire of human cancers influenced by the rs78378222 hypomorphic allele, which impairs proper termination and polyadenylation of TP53 transcripts. Future studies using whole-genome sequencing data are likely to reveal additional rare variants with large effect sizes contributing to neuroblastoma tumorigenesis.
Neuroblastoma is uniquely sensitive to single-agent inhibition of the DNA damage checkpoint kinase Chk1, leading us to examine downstream effectors of this pathway and identify mitotic regulator Wee1 ...as an additional therapeutic target in this disease. Wee1 was overexpressed in both neuroblastoma cell lines and high-risk patient tumors. Genetic or pharmacologic abrogation of Wee1 signaling results in marked cytotoxicity in 10 of 11 neuroblastoma cell lines with a median IC(50) of 300 nmol/L for the Wee1-selective small-molecule inhibitor MK-1775. Murine tumor lines derived from mice that were either heterozygous or homozygous for MycN were particularly sensitive to single-agent inhibition of Wee1 (IC(50)s of 160 and 62 nmol/L, respectively). Simultaneous pharmacologic inhibition of Chk1 and Wee1 acted in a synergistic fashion to further impede neuroblastoma cell growth in vitro, in a manner greater than the individual inhibitors either alone or combined with chemotherapy. Combination Chk1 and Wee1 inhibition also revealed in vivo efficacy in neuroblastoma xenografts. Taken together, our results show that neuroblastoma cells depend on Wee1 activity for growth and that inhibition of this kinase may serve as a therapeutic for patients with neuroblastoma.
Chromosome 6p22 was identified recently as a neuroblastoma susceptibility locus, but its mechanistic contributions to tumorigenesis are as yet undefined. Here we report that the most highly ...significant single-nucleotide polymorphism (SNP) associations reside within CASC15, a long noncoding RNA that we define as a tumor suppressor at 6p22. Low-level expression of a short CASC15 isoform (CASC15-S) associated highly with advanced neuroblastoma and poor patient survival. In human neuroblastoma cells, attenuating CASC15-S increased cellular growth and migratory capacity. Gene expression analysis revealed downregulation of neuroblastoma-specific markers in cells with attenuated CASC15-S, with concomitant increases in cell adhesion and extracellular matrix transcripts. Altogether, our results point to CASC15-S as a mediator of neural growth and differentiation, which impacts neuroblastoma initiation and progression.
Neuroblastoma is a childhood cancer that is often fatal despite intense multimodality therapy. In an effort to identify therapeutic targets for this disease, we performed a comprehensive ...loss-of-function screen of the protein kinome. Thirty kinases showed significant cellular cytotoxicity when depleted, with loss of the cell cycle checkpoint kinase 1 (CHK1/CHEK1) being the most potent. CHK1 mRNA expression was higher in MYC-Neuroblastoma-related (MYCN)-amplified (P < 0.0001) and high-risk (P = 0.03) tumors. Western blotting revealed that CHK1 was constitutively phosphorylated at the ataxia telangiectasia response kinase target site Ser345 and the autophosphorylation site Ser296 in neuroblastoma cell lines. This pattern was also seen in six of eight high-risk primary tumors but not in control nonneuroblastoma cell lines or in seven of eight low-risk primary tumors. Neuroblastoma cells were sensitive to the two CHK1 inhibitors SB21807 and TCS2312, with median IC₅₀ values of 564 nM and 548 nM, respectively. In contrast, the control lines had high micromolar IC₅₀ values, indicating a strong correlation between CHK1 phosphorylation and CHK1 inhibitor sensitivity (P = 0.0004). Furthermore, cell cycle analysis revealed that CHK1 inhibition in neuroblastoma cells caused apoptosis during S-phase, consistent with its role in replication fork progression. CHK1 inhibitor sensitivity correlated with total MYC(N) protein levels, and inducing MYCN in retinal pigmented epithelial cells resulted in CHK1 phosphorylation, which caused growth inhibition when inhibited. These data show the power of a functional RNAi screen to identify tractable therapeutical targets in neuroblastoma and support CHK1 inhibition strategies in this disease.
Neuroblastoma is treated with aggressive multimodal therapy, yet more than 50% of patients experience relapse. We recently showed that relapsed neuroblastomas frequently harbor mutations leading to ...hyperactivated ERK signaling and sensitivity to MEK inhibition therapy. Here we sought to define a synergistic therapeutic partner to potentiate MEK inhibition.
We first surveyed 22 genetically annotated human neuroblastoma-derived cell lines (from 20 unique patients) for sensitivity to the MEK inhibitor binimetinib. After noting an inverse correlation with sensitivity to ribociclib (CDK4/6 inhibitor), we studied the combinatorial effect of these two agents using proliferation assays, cell-cycle analysis, Ki67 immunostaining, time-lapse microscopy, and xenograft studies.
Sensitivity to binimetinib and ribociclib was inversely related (
= -0.58,
= 0.009).
amplification status and expression were associated with ribociclib sensitivity and binimetinib resistance, whereas increased MAPK signaling was the main determinant of binimetinib sensitivity and ribociclib resistance. Treatment with both compounds resulted in synergistic or additive cellular growth inhibition in all lines tested and significant inhibition of tumor growth in three of four xenograft models of neuroblastoma. The augmented growth inhibition was attributed to diminished cell-cycle progression that was reversible upon removal of drugs.
Here we demonstrate that combined binimetinib and ribociclib treatment shows therapeutic synergy across a broad panel of high-risk neuroblastoma preclinical models. These data support testing this combination therapy in relapsed high-risk neuroblastoma patients, with focus on cases with hyperactivated RAS-MAPK signaling.
.
The genetic aetiology of sporadic neuroblastoma is still largely unknown. We have identified diverse neuroblastoma susceptibility loci by genomewide association studies (GWASs); however, additional ...SNPs that likely contribute to neuroblastoma susceptibility prompted this investigation for identification of additional variants that are likely hidden among signals discarded by the multiple testing corrections used in the analysis of genomewide data. There is evidence suggesting the CDKN1B, coding for the cycle inhibitor p27Kip1, is involved in neuroblastoma. We thus assess whether genetic variants of CDKN1B are associated with neuroblastoma. We imputed all possible genotypes across CDKN1B locus on a discovery case series of 2101 neuroblastoma patients and 4202 genetically matched controls of European ancestry. The most significantly associated rs34330 was analysed in an independent Italian cohort of 311 cases and 709 controls. In vitro functional analysis was carried out in HEK293T and in neuroblastoma cell line SHEP‐2, both transfected with pGL3‐CDKN1B‐CC or pGL3‐CDKN1B‐TT constructs. We identified an association of the rs34330 T allele (‐79C/T) with the neuroblastoma risk (Pcombined = 0.002; OR = 1.17). The risk allele (T) of this single nucleotide polymorphism led to a lower transcription rate in cells transfected with a luciferase reporter driven by the polymorphic p27Kip1 promoter (P < 0.05). Three independent sets of neuroblastoma tumours carrying ‐79TT genotype showed a tendency towards lower CDKN1B mRNA levels. Our study shows that a functional variant, associated with a reduced CDKN1B gene transcription, influences neuroblastoma susceptibility.
The peroxisome proliferator-activated receptors (PPARs) are dietary lipid sensors that regulate fatty acid and carbohydrate metabolism. The hypolipidemic effects of the fibrate drugs and the ...antidiabetic effects of the glitazone drugs in humans are due to activation of the α (NR1C1) and γ (NR1C3) subtypes, respectively. By contrast, the therapeutic potential of the δ (NR1C2) subtype is unknown, due in part to the lack of selective ligands. We have used combinatorial chemistry and structure-based drug design to develop a potent and subtype-selective PPARδ agonist, GW501516. In macrophages, fibroblasts, and intestinal cells, GW501516 increases expression of the reverse cholesterol transporter ATP-binding cassette A1 and induces apolipoprotein A1-specific cholesterol efflux. When dosed to insulin-resistant middle-aged obese rhesus monkeys, GW501516 causes a dramatic dose-dependent rise in serum high density lipoprotein cholesterol while lowering the levels of small-dense low density lipoprotein, fasting triglycerides, and fasting insulin. Our results suggest that PPARδ agonists may be effective drugs to increase reverse cholesterol transport and decrease cardiovascular disease associated with the metabolic syndrome X.
Despite the progress made in the early detection and treatment of prostate adenocarcinoma, the metastatic lesions from this tumor are incurable. We used genome-wide expression analysis of human ...prostate cancer cells with different metastatic behavior in animal models to reveal that bone-tropic phenotypes upregulate three genes encoding for the cytokine interleukin-1β (IL-1β), the chemokine CXCL6 (GCP-2), and the protease inhibitor elafin (PI3). The Oncomine database revealed that these three genes are significantly upregulated in human prostate cancer versus normal tissue and correlate with Gleason scores ≥7. This correlation was further validated for IL-1β by immunodetection in prostate tissue arrays. Our study also shows that the exogenous overexpression of IL-1β in nonmetastatic cancer cells promotes their growth into large skeletal lesions in mice, whereas its knockdown significantly impairs the bone progression of highly metastatic cells. In addition, IL-1β secreted by metastatic cells induced the overexpression of COX-2 (PTGS2) in human bone mesenchymal cells treated with conditioned media from bone metastatic prostate cancer cells. Finally, we inspected human tissue specimens from skeletal metastases and detected prostate cancer cells positive for both IL-1β and synaptophysin while concurrently lacking prostate-specific antigen (PSA, KLK3) expression. Collectively, these findings indicate that IL-1β supports the skeletal colonization and metastatic progression of prostate cancer cells with an acquired neuroendocrine phenotype.
The Rey Auditory Verbal Learning Test RAVLT; Rey, A. (1941). L’examen psychologique dans les cas d’encéphalopathie traumatique.
Archives de Psychologie,
28, 21 is a commonly used neuropsychological ...measure that assesses verbal learning and memory. Normative data have been compiled Schmidt, M. (1996).
Rey Auditory and Verbal Learning Test: A handbook. Los Angeles, CA: Western Psychological Services. When assessing an individual suspected of neurological dysfunction, useful comparisons include the extent that the patient deviates from healthy peers and also how closely the subject's performance matches those with known brain injury. This study provides the means and S.D.'s of 392 individuals with documented neurological dysfunction closed head TBI (
n
=
68), neoplasms (
n
=
57), stroke (
n
=
47), Dementia of the Alzheimer's type (
n
=
158), and presurgical epilepsy left seizure focus (
n
=
28), presurgical epilepsy right seizure focus (
n
=
34) and 122 patients with no known neurological dysfunction and psychiatric complaints. Patients were stratified into three age groups, 16–35, 36–59, and 60–88. Data were provided for trials I–V, List B, immediate recall, 30-min delayed recall, and recognition. Classification characteristics of the RAVLT using Schmidt, M. (1996).
Rey Auditory and Verbal Learning Test: A handbook. Los Angeles, CA: Western Psychological Services meta-norms found the RAVLT to best distinguish patients suspected of Alzheimer's disease from the psychiatric comparison group.
PDF
