The functional variant rs34330 of CDKN1B is associated with risk of neuroblastoma.

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.

Preclinical Therapeutic Synergy of MEK1/2 and CDK4/6 Inhibition in Neuroblastoma.

Purpose: 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.Experimental Design: 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.Results: Sensitivity to binimetinib and ribociclib was inversely related (r = -0.58, P = 0.009). MYCN 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.Conclusions: 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. Clin Cancer Res; 23(7); 1785-96. ©2016 AACR.

CASC15-S Is a Tumor Suppressor lncRNA at the 6p22 Neuroblastoma Susceptibility Locus.

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.

Rare variants in TP53 and susceptibility to neuroblastoma.

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.

Dual CDK4/CDK6 inhibition induces cell-cycle arrest and senescence in neuroblastoma.

PURPOSE: Neuroblastoma is a pediatric cancer that continues to exact significant morbidity and mortality. Recently, a number of cell-cycle proteins, particularly those within the Cyclin D/CDK4/CDK6/RB network, have been shown to exert oncogenic roles in neuroblastoma, suggesting that their therapeutic exploitation might improve patient outcomes. EXPERIMENTAL PROCEDURES: We evaluated the effect of dual CDK4/CDK6 inhibition on neuroblastoma viability using LEE011 (Novartis Oncology), a highly specific CDK4/6 inhibitor. RESULTS: Treatment with LEE011 significantly reduced proliferation in 12 of 17 human neuroblastoma-derived cell lines by inducing cytostasis at nanomolar concentrations (mean IC50 = 307 ± 68 nmol/L in sensitive lines). LEE011 caused cell-cycle arrest and cellular senescence that was attributed to dose-dependent decreases in phosphorylated RB and FOXM1, respectively. In addition, responsiveness of neuroblastoma xenografts to LEE011 translated to the in vivo setting in that there was a direct correlation of in vitro IC50 values with degree of subcutaneous xenograft growth delay. Although our data indicate that neuroblastomas sensitive to LEE011 were more likely to contain genomic amplification of MYCN (P = 0.01), the identification of additional clinically accessible biomarkers is of high importance. CONCLUSIONS: Taken together, our data show that LEE011 is active in a large subset of neuroblastoma cell line and xenograft models, and supports the clinical development of this CDK4/6 inhibitor as a therapy for patients with this disease. Clin Cancer Res; 19(22); 6173-82. ©2013 AACR.

Interleukin-1ß promotes skeletal colonization and progression of metastatic prostate cancer cells with neuroendocrine features.

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-1ß (IL-1ß), 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-1ß by immunodetection in prostate tissue arrays. Our study also shows that the exogenous overexpression of IL-1ß 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-1ß 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-1ß and synaptophysin while concurrently lacking prostate-specific antigen (PSA, KLK3) expression. Collectively, these findings indicate that IL-1ß supports the skeletal colonization and metastatic progression of prostate cancer cells with an acquired neuroendocrine phenotype.

Combination therapy targeting the Chk1 and Wee1 kinases shows therapeutic efficacy in neuroblastoma.

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.

RNAi screen of the protein kinome identifies checkpoint kinase 1 (CHK1) as a therapeutic target in neuroblastoma.

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 IC(50) values of 564 nM and 548 nM, respectively. In contrast, the control lines had high micromolar IC(50) 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.

Targeting the {alpha} receptor for platelet-derived growth factor as a primary or combination therapy in a preclinical model of prostate cancer skeletal metastasis.

PURPOSE: Platelet-derived growth factor α (PDGFRα) is highly expressed in primary prostate cancer and associated skeletal metastases. Here, we tested whether targeting this receptor could impair metastatic colonization and progression, as well as prolong survival, either as primary or as combination therapy. EXPERIMENTAL DESIGN: We used a preclinical animal model of metastasis in which PC3-ML human prostate cancer cells are inoculated directly in the blood circulation. First, the humanized, monoclonal antibody IMC-3G3 was administered to mice bearing established skeletal metastases. Second, we targeted the stromal PDGFRα with IMC-1E10, an antibody specific for the murine receptor. Third, IMC-3G3 and the bisphosphonate zoledronic acid (ZA), administered separately or in combination, were tested on the progression of skeletal lesions and overall survival. In addition, the ability of IMC-3G3 and ZA to impair initial colonization of the bone marrow by prostate cancer cells was investigated. RESULTS: The blockade of PDGFRα on prostate cancer cells by IMC-3G3 reduces the size of established skeletal metastases, whereas the IMC-1E10 antibody directed against the stromal PDGFRα fails to inhibit metastatic progression. IMC-3G3 and ZA, either separately or in combination, significantly slow tumor growth and seem to prolong survival. Lastly, the blockade of PDGFRα by IMC-3G3 inhibits the initial phase of bone colonization, whereas ZA is ineffective at this stage. CONCLUSION: This study presents compelling evidence that targeting PDGFRα with IMC-3G3 delays the progression of early metastatic foci and reduces the size of more established lesions. In addition, IMC-3G3, either alone or in combination with ZA, prolongs survival in animal models.

The alpha-receptor for platelet-derived growth factor confers bone-metastatic potential to prostate cancer cells by ligand- and dimerization-independent mechanisms.

Prostate adenocarcinoma is the second leading cause of cancer death among men, due primarily to the fact that the majority of prostate cancers will eventually spread to the skeleton. Metastatic dissemination requires a complex series of coordinated events that result in cells that escape from the primary tumor into the circulation and eventually colonize a distant organ. The ability of these cells to evolve into macroscopic metastases depends strongly on their compatibility with, and ability to utilize, this new microenvironment. We previously showed that bone-metastatic prostate cancer cells exposed to human bone marrow respond by activation of cell survival pathways, such as phosphoinositide 3-kinase/Akt, and that these events are mediated by the alpha-receptor for platelet-derived growth factor (PDGFRalpha). Our studies and others have shown that PDGFRalpha may be activated by mechanisms independent of PDGF ligand binding. Here, we provide conclusive evidence that soluble components of human bone marrow can activate PDGFRalpha through a mechanism that does not require the canonical binding of PDGF ligand(s) to the receptor. In particular, we found that dimerization of PDGFRalpha monomers is not induced by human bone marrow, but this does not prevent receptor phosphorylation and downstream signaling from occurring. To establish the relevance of this phenomenon in vivo, we used a PDGFRalpha mutant lacking the extracellular ligand-binding domain. Our studies show that this truncated PDGFRalpha is able to restore bone-metastatic potential of prostate cancer cells as effectively as the full-length form of the receptor.

Human bone marrow activates the Akt pathway in metastatic prostate cells through transactivation of the alpha-platelet-derived growth factor receptor.

The factors regulating the bone tropism of disseminated prostate cancer cells are still vaguely defined. We report that prostate cancer cells that metastasize to the skeleton respond to human bone marrow with a robust stimulation of the phosphatidylinositol 3-kinase/Akt pathway, whereas prostate cells that lack bone-metastatic potential respond negligibly. The majority of this Akt activation is dependent on alpha-platelet-derived growth factor receptor (alpha-PDGFR) signaling, which was shown using the small-molecule inhibitor of PDGFR signaling AG1296. Low concentrations of PDGF-AA and PDGF-BB found in bone marrow aspirates, which were detected by ELISA, do not account for the high levels of alpha-PDGFR signaling. Additionally, neutralizing PDGF binding using a alpha-PDGFR-specific antibody (IMC-3G3) failed to produce a significant inhibition of bone marrow-induced Akt activation. However, the inhibitory effect of IMC-3G3 rivaled that of AG1296 when incubation was done under conditions that stimulated alpha-PDGFR internalization. We conclude that alpha-PDGFR is activated by multiple soluble factors contained within human bone marrow, in addition to its natural ligands, and this transactivation is dependent on receptor localization to the plasma membrane. Therefore, alpha-PDGFR expression may provide select prostate phenotypes with a growth advantage within the bone microenvironment.