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Supplementary Figure S1. Neuroblastoma cell lines are sensitive to binimetinib. Supplementary Figure S2. Gene expression profiling of human neuroblastoma cell lines with regards to relative sensitivity to MEK1/2 and CDK4/6 inhibition Supplementary Figure S3. Combined MEK and CDK4/6 inhibition does not induce significant levels of apoptosis or senescence. Supplementary Figure S4. G1 arrest induced by MEK1/2 and CDK4/6 inhibition is reversible. Supplementary Figure S5. Gene expression profiling of human neuroblastoma cell lines with regards to the synergy observed from combined binimetinib-ribociclib treatment. Figure S6. Neuroblastoma xenografts are sensitive to combined binimetinib-ribociclib treatment.

Table S1. Specific mutations of genes listed in Table 1. Table S2. Foundation Medicine sequencing calls of cancer-specific gene panel. Table S3. Gene lists corresponding to microarray analysis described in Figures 1A and 1B: (A) binimetinib and (B) ribociclib. Significant genes (in bold type) are defined as having an adjusted p-value of 0.25 or less.

This document contains Supplementary information, including materials and methods as well as Figure Legends for the Supplementary Figures.

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.

Background Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype that lacks targeted therapies. Patients with TNBC have a very poor prognosis because the disease often metastasizes. New treatment approaches addressing drivers of metastasis and tumor growth are crucial to improving patient outcomes. Developing targeted gene therapy is thus a high priority for TNBC patients. PEA15 (phosphoprotein enriched in astrocytes, 15 kDa) is known to bind to ERK, preventing ERK from being translocated to the nucleus and hence blocking its activity. The biological function of PEA15 is tightly regulated by its phosphorylation at Ser104 and Ser116. However, the function and impact of phosphorylation status of PEA15 in the regulation of TNBC metastasis and in epithelial-to-mesenchymal transition (EMT) are not well understood. Methods We established stable cell lines overexpressing nonphosphorylatable (PEA15-AA) and phospho-mimetic (PEA15-DD) mutants. To dissect specific cellular mechanisms regulated by PEA15 phosphorylation status, we performed RT-PCR immune and metastasis arrays. In vivo mouse models were used to determine the effects of PEA15 phosphorylation on tumor growth and metastasis. Results We found that the nonphosphorylatable mutant PEA15-AA prevented formation of mammospheres and expression of EMT markers in vitro and decreased tumor growth and lung metastasis in in vivo experiments when compared to control, PEA15-WT and phosphomimetic PEA15-DD. However, phosphomimetic mutant PEA15-DD promoted migration, mesenchymal marker expression, tumorigenesis, and lung metastasis in the mouse model. PEA15-AA-mediated inhibition of breast cancer cell migratory capacity and tumorigenesis was the partial result of decreased expression of interleukin-8 (IL-8). Further, we identified that expression of IL-8 was possibly mediated through one of the ERK downstream molecules, Ets-1. Conclusions Our results show that PEA15 phosphorylation status serves as an important regulator for PEA15’s dual role as an oncogene or tumor suppressor and support the potential of PEA15-AA as a therapeutic strategy for treatment of TNBC.

Background: Hemodynamic monitoring, implemented by the placement of peripheral arterial catheters (PACs), is a characterizing aspect of the intensive care units. Peripheral arterial catheters can continually detect blood pressure and quickly conduct blood sampling. The use of PACs is generally considered safe, without serious complications. Currently, only 25% of the implanted catheters are actually subject to complications, including accidental removal, dislocation, occlusion, and infection. All of these complications arise from inadequate catheter stabilization at the level of the skin. This study aimed to summarize and describe the effectiveness and characteristics of dressings and securement devices for catheter stabilization. Methods: A systematic review of literature from the following databases was conducted: MEDLINE, CINAHL, Cochrane, EMBASE, and OvidSP. We used the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement to guide article selection and reporting. Results: Herein 626 articles were referred, with 5 directly related to the topic under discussion. We found 3 studies that describe PAC failure and 4 about dislodgement. We described 6 types of dressings or securement devices and classified them into 3 categories for classifying PAC dressings or securement devices. These were detected and grouped as adhesive tissues, sutureless devices, and transparent polyurethane dressings. Conclusions: Current research indicates that transparent polyurethane dressings offer the most effective catheter stabilization, but adhesive tissues may constitute a valid alternative. However, there are limited high-quality studies about effective dressings and securement devices for PACs.

The fate of coronary artery stenting in children several years after implantation is unknown. We previously reported the case of an 8-year-old child undergoing stent implantation for a total left main coronary artery occlusion after arterial switch operation. Six months later, she needed another stent implantation for in-stent restenosis. Here we report the angiographic, intravascular ultrasound and optical coherence tomography findings at 5-year follow-up. Despite nongrowth of the left main coronary artery inherent to the stents, luminal patency, adequate struts apposition, and the absence of in situ complications were confirmed. Résumé: On ne sait rien de ce qu’il advient des endoprothèses coronariennes plusieurs années après l’implantation chez les enfants. Nous avons déjà présenté le cas d’une fillette de huit ans chez qui une endoprothèse a été implantée pour corriger une occlusion totale de l’artère coronaire principale gauche après une intervention de détransposition artérielle. Six mois plus tard, la patiente avait besoin d’une autre endoprothèse en raison d’une resténose de l’endoprothèse. Nous rapportons ici les observations effectuées à l’angiographie, à l’échographie intravasculaire et à la tomographie par cohérence optique à l’évaluation de suivi à cinq ans. Malgré l’absence de croissance de l’artère coronaire principale gauche en raison de la présence des endoprothèses, la perméabilité luminale, l’apposition adéquate des entretoises et l’absence de complications in situ ont été confirmées.

Background: STAT3 is a transcription factor that regulates various tumorigenic processes, such as tumor proliferation, metastasis, and drug resistance. Its overexpression and aberrant activation characterize many cancers (including breast, lung, ovarian, liver and colon cancer). Metastasis and drug resistance are two main causes of patient mortality. Activation of the JAK-STAT3 and the FGFR1-STAT3 pathways in breast and ovarian cancer cells promotes tumor initiation, migration, and taxol resistance. STAT3 also contributes to epithelial-mesenchymal transition and promotes 5-FU resistance in colorectal cancer cells. Its role in numerous malignancies has made STAT3 an ideal cancer therapeutic target. BP1003 is a neutral DOPC liposome incorporated with a nuclease-resistant P-ethoxy antisense oligodeoxynucleotide targeting STAT3 mRNA. Here we determine the effects of BP1003 in combination with taxol and 5-FU on breast and ovarian cancer cells. Methods: Western blots determined the extent by which BP1003 reduced STAT3 protein levels. The effect of sequential taxol or 5-FU (5 h pre-treatment) and BP1003 treatments (96 h) on cell viability was analyzed with resazurin-based assays. Migration and colony formation assays were performed to assess the metastatic potential of treated cells. Cells pre-treated with BP1003 (96 h) and taxol (2 h) were placed in chemotaxis chambers and allowed to migrate for 5-18 h before quantification. Colony formation involved the treatment of a low number of cells with chemotherapeutic drugs for 24 h prior to the addition of BP1003 for 10-14 days. Tumor spheroids were used to mimic in vivo solid tumor growth. Three days after formation, spheroids were treated with chemotherapeutic drugs for 1 day before being treated with BP1003 for 3 additional days. Results: Treatment of BT549, SK-Br-3 and SK-Ov-3 cells with 250 μg/ml of BP1003 resulted in a 30%-50% decrease in STAT3 expression. Cell viability decreased by 20-35% with 200 μg/ml of BP1003 and by 45-60% in combination with taxol or 5-FU. BP1003 in combination with taxol also decreased the colony formation potential of BT549 cells by 20%. Migration of BT549 cells was reduced by 30% with 200 μg/ml BP1003 alone and by 50% in combination with taxol. The area of BT549 and SK-Ov-3 spheroids under BP1003 and taxol combination treatments was 40%-50% smaller than control spheroids and 20%-30% smaller than taxol mono-treatment. Conclusions: BP1003 efficiently reduces STAT3 expression and enhances the sensitivity of breast and ovarian cancer cells to taxol and 5-FU. These results are in line with previous work in which BP1003 + gemcitabine displayed enhanced anti-tumor activity in pancreatic ductal adenocarcinoma. Together these results strongly suggest that BP1003 combination therapy is a novel strategy for patients with advanced solid tumors. Citation Format: Maria Gagliardi, Ana Tari Ashizawa. Targeting STAT3 with novel liposome-incorporated antisense oligonucleotide technology enhances the efficacy of paclitaxel (taxol) and 5-fluorouracil (5-FU) in breast and ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3285.

To determine the characteristics of workplace violence towards emergency nurses in Campania, South Italy.In Italy, workplace violence is a severe and widespread problem. A national survey describes that, working in South Italy significantly increases the probability of being exposed. However, available data in Campania workplace violence towards nurse emergency department (ED) is lacking.We used a cross-sectional design, in two emergency department. Between April and May 2019, we distributed a questionnaire (QIN16VIPs). Three types of violence have been investigated: verbal/physical violence, verbal violence and physical violence. The data were analyzed using procedures of descriptive statistics.A total of 83 questionnaires (response rate 92.3%), 48.2% of emergency nurses experienced verbal violence, 21.7% both verbal and physical violence and only 28.9% denied having experienced either. Nurses feeling at risk in emergency setting were more exposed to workplace violence (p=0.001). Anxiety (p=0.023) and anger (p=0.001) were perceived feeling with significant repercussions on the degree of evidence working in ED (p=0.043) and on trust in management (p=0.001).Workplace violence turns out to be a serious and widespread problem that generates negative feelings in the attacked subject that affect the personal and work dimension. The promotion of simple and anonymous reporting systems would help nurses to be more aware of the importance of reporting the event, which is still in deficit today, thus preventing a true and proper estimate of the phenomenon. Future research should be focused for preventive measures could be drawn up to reduce the problem.Definire le dimensioni e le caratteristiche della violenza nei confronti degli infermieri del Dipartimento Emergenza Accettazione del contesto campano.In Italia, la violenza sul luogo di lavoro è considerato un fenomeno in forte aumento. Studi recenti hanno evidenziato che l’area geografica meridionale sembra risultare a maggior rischio di violenza sul posto di lavoro, tuttavia i dati sull’incidenza del fenomeno in Campania sono ancora scarsi.È stato condotto uno studio trasversale in due ospedali metropolitani campani. Tra Aprile e Maggio 2019 Ë stato somministrato a 90 infermieri il questionario QuIN16VIPs, speci- ficamente sviluppato per analizzare il fenomeno della violenza verso gli infermieri di Pronto Soccorso. » stata utilizzata una statistica descrittiva e correlazionale.In base agli 83 questionari ritirati (tasso di risposta del 92,3%) il 48,2% dei rispondenti riferisce di aver subito nell’ultimo anno di servizio episodi di violenza verbale, il 21,7% di aver subito entrambe le modalità di aggressione (sia verbale e che fisica), l’1,2% violenza fisica, mentre il 28,9% dichiara di non aver subito violenza. Gli infermieri che hanno subito un’aggressione risul- tano avere una percezione più alta del pericolo (0.001), innescando sentimenti di ansia (p=0.023) e rabbia (p=0.001) con ripercussioni significative sul grado di motivazione a lavorare in Pronto Soccorso (p=0.043) e sulla fiducia nella dirigenza (p=0.001).La violenza sul luogo risulta essere un problema serio e molto diffuso che genera nel soggetto aggredito sentimenti negativi che si ripercuotono sulla sfera personale e lavorativa. Ricerche future dovrebbero orientarsi su strategie di prevenzione del fenomeno.

Background: Several risk factors, such as age, alcohol abuse, dementia, and severe illness, can contribute to the development of delirium. However, limited information is available in the literature regarding the risk of delirium among surgical, trauma, neurological, and medical intensive care patients. Aims and objectives: To describe the prevalence of risk factors associated with delirium in intensive care units. Design: This study used an observational design. Methods: We enrolled 165 patients hospitalized in two intensive care units in Italy. Patients were first evaluated using the Prediction of Delirium model and were subsequently evaluated using the Intensive Care Delirium Screening Checklist; evaluation lasted a maximum of 5 days for each admitted patient after sedation. A logistic regression model was used to identify the prevalence and risk factors of delirium. Results: The average age of the patients was 57.6 (SD = 18.3) years, and the patients were predominantly male (65.0%). The majority of patients had been subjected to trauma (38.8%); 37.6% had undergone general surgical interventions, and 23.6% had undergone medical interventions. Delirium occurred in 55.8% of the 165 patients. The risk of delirium was independently associated with coma (odds ratio = 10.6; 95% confidence interval, 3.08-39.9) and the Acute Physiology and Chronic Health Evaluation II score (odds ratio = 4.27; 95% confidence interval, 1.58-11.53). Conclusions: This study confirmed that coma and the Acute Physiology and Chronic Health Evaluation II score were non-modifiable risk factors for delirium. Further studies could categorize the different types of coma. Proper delirium management could limit the impact on the recovery of these patients, their autonomy, and their reintegration into the social and professional world. Relevance to clinical practice: Delirium increases intensive care unit and hospital length of stay. Early identification and risk factor assessment by critical care nurses are considered the key factors in the treatment of delirium.

Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer characterized by metastasis, drug resistance and high rates of recurrence. With a lack or targeted therapies, TNBC is challenging to treat and carries a poor prognosis. Patients with TNBC tumors expressing high levels of ERK2 have a poorer prognosis than those with low ERK2-expressing tumors. The MAPK pathway is often found to be highly activated in TNBC, however the precise functions of the ERK isoforms (ERK1 and ERK2) in cancer progression have not been well defined. We hypothesized that ERK2, but not ERK1, promotes the cancer stem cell (CSC) phenotype and metastasis in TNBC. Stable knockdown clones of the ERK1 and ERK2 isoforms were generated in SUM149 and BT549 TNBC cells using shRNA lentiviral vectors. ERK2 knockdown significantly inhibited anchorage-independent colony formation and mammosphere formation, indicating compromised self-renewal capacity. This effect correlated with a reduction in migration and invasion. SCID-beige mice injected via the tail vein with ERK clones were employed to determine metastatic potential. SUM149 shERK2 cells had a significantly lower lung metastatic burden than control mice or mice injected with SUM149 shERK1 cells. The Affymetrix HGU133plus2 microarray platform was employed to identify gene expression changes in ERK isoform knockdown clones. Comparison of gene expression levels between SUM149 cells with ERK2 or ERK1 knockdown revealed differential and in some cases opposite effects on mRNA expression levels. Those changes associated with ERK2 knockdown predominantly altered regulation of CSCs and metastasis. Our findings indicate that ERK2 promotes metastasis and the CSC phenotype in TNBC.

Background The treatment of high-risk neuroblastoma continues to present a formidable challenge to pediatric oncology. Previous studies have shown that Bromodomain and extraterminal (BET) inhibitors can inhibit MYCN expression and suppress MYCN-amplified neuroblastoma in vivo. Furthermore, alterations within RAS-MAPK (mitogen-activated protein kinase) signaling play significant roles in neuroblastoma initiation, maintenance, and relapse, and mitogen-activated extracellular signal-regulated kinase (MEK) inhibitors demonstrate efficacy in subsets of neuroblastoma preclinical models. Finally, hyperactivation of RAS-MAPK signaling has been shown to promote resistance to BET inhibitors. Therefore, we examined the antitumor efficacy of combined BET/MEK inhibition utilizing I-BET726 or I-BET762 and trametinib in high-risk neuroblastoma. Procedure Utilizing a panel of genomically annotated neuroblastoma cell line models, we investigated the in vitro effects of combined BET/MEK inhibition on cell proliferation and apoptosis. Furthermore, we evaluated the effects of combined inhibition in neuroblastoma xenograft models. Results Combined BET and MEK inhibition demonstrated synergistic effects on the growth and survival of a large panel of neuroblastoma cell lines through augmentation of apoptosis. A combination therapy slowed tumor growth in a non-MYCN-amplified, NRAS-mutated neuroblastoma xenograft model, but had no efficacy in an MYCN-amplified model harboring a loss-of-function mutation in NF1. Conclusions Combinatorial BET and MEK inhibition was synergistic in the vast majority of neuroblastoma cell lines in the in vitro setting but showed limited antitumor activity in vivo. Collectively, these data do not support clinical development of this combination in high-risk neuroblastoma.

Background: B-cell lymphoma 2 (Bcl-2) is an oncoprotein that promotes tumor cell survival and drug resistance. Elevated Bcl-2 expression in numerous human cancers has made it an ideal drug target. Venetoclax, the first FDA approved Bcl-2 inhibitor, is indicated for hematologic malignancies. Accompanying its promising clinical results, however, is the emergence of venetoclax resistance. A recent study found that AML patients who had relapsed from frontline venetoclax-based treatment were refractory to salvage therapy and had a median survival of less than 3 months. Thus, novel treatment approaches for such patients are urgently needed. BP1002, a non-toxic neutral dioleoylphosphatidylcholine liposome incorporating a nuclease-resistant P-ethoxy antisense oligonucleotide (oligo), was developed to target Bcl-2 mRNA. The safety and efficacy of BP1002 is being investigated in patients with advanced lymphoid malignancies. Venetoclax in combination with decitabine is an approved treatment for elderly AML patients. Here we determine the effects of BP1002 plus decitabine in venetoclax-resistant cells. Methods: Cell lines: Venetoclax-resistant hematologic cell lines, MV-4-11- R, SU-DHL-2-R, SU-DHL-6-R, were established from the parental cells by exposing the cells to increasing doses of venetoclax for 10-12 weeks.BP1002: A Bcl-2 antisense oligo, which targets the translation initiation site of human Bcl-2 mRNA, was incorporated in BP1002. Combination study: Parental and venetoclax-resistant cells were treated with decitabine for 2 days followed by the addition of BP1002 for another 4 days of incubation. Resazurin-based cell viability assays and Western blots were conducted to determine the inhibitory effects of BP1002 in parental and resistant cells. Results: The IC50 values of venetoclax in resistant cells were 3 to 40 times higher than those in parental cells. MV-4-11-R, SU-DHL-6-R, SU-DHL-2-R cells are resistant up to 0.1 μM, 0.8 μM, and 7 μM venetoclax, respectively. Compared to parental cells, venetoclax-resistant cells displayed 2- to 3-fold elevated levels of Bcl-2 protein and other anti-apoptotic proteins such as Mcl-1. BP1002 in combination with decitabine decreased the viability of parental cells. BP1002 reduced Bcl-2 levels in venetoclax-resistant cells. Also, the BP1002 plus decitabine combination was more potent than the venetoclax plus decitabine combination in reducing the viability of the resistant cells, by about 50-70%. Conclusions: Our data indicate that, despite expressing higher levels of anti-apoptotic proteins, venetoclax-resistant cells are sensitive to the BP1002 plus decitabine combination. These promising preclinical results suggest that the BP1002 plus decitabine combination is a potential treatment for patients who have relapsed from frontline venetoclax-based therapies. Citation Format: Maria Gagliardi, Ana Tari Ashizawa. The combination of liposomal Bcl-2 antisense oligonucleotide (BP1002) with decitabine is efficacious in venetoclax-resistant cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 939.

Termed ‘master gene regulators’ long ncRNAs (lncRNAs) have emerged as the true vanguard of the ‘noncoding revolution’. Functioning at a molecular level, in most if not all cellular processes, lncRNAs exert their effects systemically. Thus, it is not surprising that lncRNAs have emerged as important players in human pathophysiology. As our body's first line of defense upon infection or injury, inflammation has been implicated in the etiology of several human diseases. At the center of the acute inflammatory response, as well as several pathologies, is the pleiotropic transcription factor NF-κβ. In this review, we attempt to capture a summary of lncRNAs directly involved in regulating innate immunity at various arms of the NF-κβ pathway that have also been validated in human disease. We also highlight the fundamental concepts required as lncRNAs enter a new era of diagnostic and therapeutic significance.

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.

Background: Triple-negative breast cancer (TNBC) is an aggressive disease associated with recurrence, metastasis, and resistance to chemotherapy and radiation; effective targeted therapies are needed. Although the MEK inhibitors (MEKi) selumetinib (AZD6244) and pimasertib (AS703026) showed therapeutic efficacy in preclinical models, they failed as effective single agents in the clinic due to development of intrinsic resistance. To identify potential mediators of this resistance, we performed a synthetic lethal siRNA screen and identified myeloid cell leukemia-1 (MCL1) as a potential mediator of selumetinib resistance. Mcl-1 is an anti-apoptotic protein that is highly amplified in numerous human cancers and associated with cell immortalization and chemoresistance. We hypothesized that Mcl-1 promotes MEKi resistance in TNBC, and sought to confirm this and discern of mechanisms. Results: We established selumetinib- and pimasertib-resistant clones of TNBC cells SUM-149 and MDA-MB-231 to a final concentration of 15µM selumetinib and 11µM pimasertib by continuous exposure to increasing concentrations over a 6-month period. MEKi-resistant cells showed a significantly higher proliferation rate than the parental cells. More colonies were observed among the MEKi-resistant cells than in the parental cells in a colony formation assay (40%, p=0.05) and a similar outcome was observed in a mammosphere assay (65%, p=0.01), suggesting a higher fraction of tumor-initiating cells. To determine if the Mcl-1-specific inhibitor S63845 could sensitize the MEKi-resistant cells, we treated parental and resistant cells with either selumetinib or pimasertib together with S63845, a highly specific Mcl-1 inhibitor. After treatment with the Mcl-1 inhibitor, the resistant SUM-149 and MDA-MB-231 cells had similar cell proliferation rates to those of their parental counterparts. To extrapolate the mechanism of MEKi resistance, we performed an RT2 Profiler PCR Human Mitochondria Array; mitochondrial uncoupling proteins UCP4 and UCP2 were upregulated in the SUM149 and MDA-MB-231 MEKi-resistant cells, respectively. MDA-MB-231 MEKi-resistant cells had higher levels of glycolysis than the parental control cells, suggesting that the MEKi-resistant cells had undergone a metabolic shift to enhance glycolysis. This is consistent with a observation that metabolic alterations play a part in drug resistance. In addition, the Warbug effect, in which tumor cells adopt aerobic glycolysis, is considered the major adaptive mechanism and increased glycolysis is commonly displayed in resistant cells. Furthermore, knockdown of UCP2 in selumetinib-resistant MDA-MB-231 cells made them ~20% more sensitive to selumetinib. Inhibition of glycolysis using 2-deoxy-D-glucose in combination with MEKi increased drug sensitivity by 20-30%. Conclusion: We found preliminary evidence of a link between Mcl-1 and glycolytic metabolism that may be involved in MEK resistance. We will also design combinational studies of MEKi and Mcl-1 inhibitors in vivo in TNBC models. Citation Format: Maria Gagliardi, Moises Tacam, Lakesla Iles, Yuan Qi, Lajos Pusztai, Debu Tripathy, Geoffrey Bartholomeusz, Chandra Bartholomeusz. Mechanism of MEK inhibitor resistance in triple negative breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1897.

SummaryA more complete understanding of aberrant oncogenic signaling in neuroblastoma, a malignancy of the developing sympathetic nervous system, is paramount to improving patient outcomes. Recently, we identified LIN28B as an oncogenic driver in high-risk neuroblastoma. Here, we identify the oncogene RAN as a LIN28B target and show regional gain of chromosome 12q24 as an additional somatic alteration resulting in increased RAN expression. We show that LIN28B influences RAN expression by promoting RAN Binding Protein 2 expression and by directly binding RAN mRNA. Further, we demonstrate a convergence of LIN28B and RAN signaling on Aurora kinase A activity. Collectively, these findings demonstrate that LIN28B-RAN-AURKA signaling drives neuroblastoma oncogenesis, suggesting that this pathway may be amenable to therapeutic targeting.

The morphological approach in the reading of the Territorial City Maria Gagliardi Escuela Tecnica Superior de Arquitectura de Barcelona, UPC. Av. Diagonal, 649. 08028 Barcelona E-mail: bissolati27@hotmail.it Keywords (3-5): Territorial city, territorial grid, accumulation process, relief of innovative elements. Conference topics and scale: City transformations The crisis of the traditional city, with its morphological characteristics, has been largely debated. The new urban phenomenon has almost always been described as antithetical; by contrary this article interrogates the possibility of reading a continuity between the traditional and the contemporary city. The article brings forth the concept that the territory can be described as an urban phenomenon result of a change of scale of the city, and offers the definition of the contemporary city as Territorial City (Citta-Territorio). The article recovers this term introduced in the 70’s by the Italian morphological school, to emphasize the continuity with the European traditional morphological schools, taking advantage of its learnings, to compare the traditional city, with the contemporary city. The relevance of a morphological approach in the study of the contemporary Territorial City is discussed through an excursus of the main European schools’ contributions, with a particular schematization due to geographical areas. From the synthesis of these references emerges a basis for the definition of a Taxonomy organised around tree main elements that constitute the urban grid. Moreover, the article proposes a methodology for the analysis of the Territorial City that use the Taxonomy as reading tool; this method, illustrated through his experimentation on the Metropolitan Region of Barcelona, is of particular relevance towards the interpretation of the dynamics that have characterised the morphology of the current urban phenomenon, describing its aggregation logics and decomposing the historical-procedural continuum. References Caniggia, G., & Maffei, G. L. (1979). Composizione architettonica e tipologia edilizia - Lettura dell’edilizia di base . (Venezia: Marsilio). Conzen, M. R. G. (1960). Alnwick, Northumberland: A Study in Town Plan Analysis . (London: Institute of British Geographers). Hillier, B., & Hanson, J. (1984). The Social logic of space . (Cambridge: Cambridge University Press). Gagliardi, M. (2016). La Nuova Trama Territoriale: morfogenesi e logiche aggregative. Il caso di Barcellona (settore della Regione Metropolitana tra Sant Marti e Mataro) . (Doctoral dissertation) Universitat Politecnica de Catalunya - La Sapienza Universita di Roma. Dir. Font Arellano, A.; Bianchi, G.

Background: Triple-negative breast cancer (TNBC), which affects over 170 000 women worldwide every year, is considered the most arduous to treat subtype of breast cancer. With no targeted therapy, high rates of drug resistance and rapid metastasis, TNBC carries a poor prognosis. The MEK-ERK-MAPK signaling cascade is known to play a role in numerous cancers. Despite the lack of activating Ras/MAPK mutations in breast cancer, transcriptional signatures of this pathway are prevalent in TNBC. Our previous work showed that TNBC patients with tumors overexpressing ERK2 had a lower overall survival rate than did patients with low-ERK2-expressing tumors. MEK inhibitors selumetinib (AZD6244) and pimasertib (AS703026) are active in preclinical models, but not as single agents in the clinic. Using a synthetic lethal siRNA screen, we identified myeloid cell leukemia-1 (MCL1) as a potential contributor to selumetinib resistance. Mcl-1 is an anti-apoptotic protein that is highly amplified in numerous human cancers. It is associated with cell immortalization, transformation, and chemoresistance. Patients with TNBC tumors expressing high levels of Mcl-1 have lower overall survival and distant-metastasis-free survival rates. We hypothesized that Mcl-1 promotes MEK inhibitor resistance in TNBC. Methods/Results: To model MEK inhibitor resistance, we established selumetinib- and pimasertib-resistant clones of SUM-149 and MDA-MB-231 TNBC cells by continuous exposure to increasing concentrations of inhibitors over a six month period. We confirmed the onset of MEK resistance by demonstrating that resistant cells, in comparison to the parental cells, exhibited no change in cell proliferation upon treatment with the MEK inhibitors. Resistant cells also displayed more effective cell migration and mammosphere formation than parental cells, suggesting a higher fraction of tumor-initiating cells. We found Mcl-1 to be highly expressed in 83% (15 of 18) of TNBC cell lines but only 30% (3 of 10) of other breast cancer cell lines. Resistant cells had higher levels of Mcl-1 than did parental cells. To determine whether Mcl-1 is required for MEK sensitivity, we treated parental and resistant cells with either selumetinib or pimasertib together with S63845, a highly specific Mcl-1 inhibitor. The Mcl-1 inhibitor restored MEK sensitivity in both resistant cell lines. After treatment with the Mcl-1 inhibitor, the resistant SUM-149 and MDA-MB-231 cells had similar cell proliferation rates to those of their parental counterparts. Similar studies were done using an siRNA against Mcl-1. Conclusion: Our data demonstrate that Mcl-1 may promote TNBC resistance to MEK inhibitors and that Mcl-1 is a promising target for combination therapy. We will continue to explore the mechanisms of MEK inhibitor resistance by screening for additional genes/pathways involved. Our long-term goal is to design rational combination approaches to counteract the emergence of resistance by using novel molecularly targeted therapeutics. Citation Format: Gagliardi M, Chauhan G, Pitner MK, Iles L, Qi Y, Pusztai L, Tripathy D, Bartholomeusz G, Bartholomeusz C. Overcoming MEK inhibitor resistance in triple-negative breast cancer by targeting myeloid cell leukemia-1 (MCL1), an anti-apoptotic protein [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-03-06.

Summary Wnt signaling is a key regulator of development that is often associated with cancer. Wingless, a Drosophila Wnt homolog, has been reported to be a survival factor in wing imaginal discs. However, we found that prospective wing cells survive in the absence of Wingless as long as they are not surrounded by Wingless-responding cells. Moreover, local autonomous overactivation of Wg signaling (as a result of a mutation in APC or axin) leads to the elimination of surrounding normal cells. Therefore, relative differences in Wingless signaling lead to competitive cell interactions. This process does not involve Myc, a well-established cell competition factor. It does, however, require Notum, a conserved secreted feedback inhibitor of Wnt signaling. We suggest that Notum could amplify local differences in Wingless signaling, thus serving as an early trigger of Wg signaling-dependent competition., Graphical Abstract Highlights ► Wingless-blind cells survive within large groups but die as part of small clones ► Patches of APC or axin mutant cells trigger apoptosis in surrounding normal cells ► The competitive nature of axin mutant cells is not mediated by Myc ► Tissue takeover by axin cells requires Notum, a secreted inhibitor of Wg signaling

Combretastatin-A4 (CA-4) is a natural derivative of the African willow tree Combretum caffrum. CA-4 is one of the most potent antimitotic components of natural origin, but it is, however, intrinsically unstable. A novel series of CA-4 analogs incorporating a 3,4-diaryl-2-azetidinone (β-lactam) ring were designed and synthesized with the objective to prevent cis-trans isomerization and improve the intrinsic stability without altering the biological activity of CA-4. Evaluation of selected -lactam CA-4 analogs demonstrated potent antitubulin, antiproliferative, and antimitotic effects in human leukemia cells. A lead -lactam analog, CA-432, displayed comparable antiproliferative activities with CA-4. CA-432 induced rapid apoptosis in HL-60 acute myeloid leukemia cells, which was accompanied by depolymerization of the microtubular network, poly(ADPribose) polymerase cleavage, caspase-3 activation, and Bcl-2 cleavage. A prolonged G2M cell cycle arrest accompanied by a sustained phosphorylation of mitotic spindle checkpoint protein, BubR1, and the antiapoptotic proteins Bcl-2 and Bcl-xL preceded apoptotic events in K562 chronic myeloid leukemia (CML) cells. Molecular docking studies in conjunction with comprehensive cell line data rule out CA-4 and β-lactam derivatives as P-glycoprotein substrates. Furthermore, both CA-4 and CA-432 induced significantly more apoptosis compared with imatinib mesylate in ex vivo samples from patients with CML, including those positive for the T315I mutation displaying resistance to imatinib mesylate and dasatinib. In summary, synthetic intrinsically stable analogs of CA-4 that display significant clinical potential as antileukemic agents have been designed and synthesized. Refereed/Peer-reviewed

Background The primary aim of surgical treatment for falcotentorial meningiomas is gross total excision. The vital surrounding brain structures make this a complex task. Seeking elements that might facilitate presurgical planning we reviewed our surgical records of falcotentorial meningiomas treated during the past 20 years. Methods Between 1975 and 1996, in the Neurosurgical Unit at the University of Rome, “La Sapienza,” 13 consecutive patients underwent surgery for falcotentorial meningiomas that had been localized on preoperative imaging and confirmed by histology. The surgical approach varied according to the site of the tumor. Results Nine meningiomas were totally removed and 4 subtotally. Three patients (23.0%), all treated early in the series, died after the operation. Ten patients (76.9%) survived: 3 (23.0%) had postoperative neurologic complications necessitating reintervention, and 7 patients (53.8%) had an uneventful postoperative course. Two of the 4 patients who had subtotal resections had regrowth at 1 year that responded to radiosurgery. Conclusions The ideal surgical approach to falcotentorial meningiomas should allow gross total removal and minimum brain retraction while safeguarding the galenic system and other vital neighboring structures. Toward achieving this aim we propose detailed preoperative imaging studies to classify falcotentorial meningiomas according to their site and direction of growth—craniocaudal or anteroposterior—in relation to the cerebellar tentorium.

A key step in the canonical Wnt signalling pathway is the inhibition of GSK3β, which results in the accumulation of nuclear β-catenin (also known as CTNNB1), and hence regulation of target genes. Evidence suggests that endocytosis is required for signalling, yet its role and the molecular understanding remains unclear. A recent and controversial model suggests that endocytosis contributes to Wnt signalling by causing the sequestration of the ligand–receptor complex, including LRP6 and GSK3 to multivesicular bodies (MVBs), thus preventing GSK3β from accessing β-catenin. Here, we use specific inhibitors (Dynasore and Dyngo-4a) to confirm the essential role of endocytosis in Wnt/Wingless signalling in human and Drosophila cells. However, we find no evidence that, in Drosophila cells or wing imaginal discs, LRP6/Arrow traffics to MVBs or that MVBs are required for Wnt/Wingless signalling. Moreover, we show that activation of signalling through chemical blockade of GSK3β is prevented by endocytosis inhibitors, suggesting that endocytosis impacts on Wnt/Wingless signalling downstream of the ligand–receptor complex. We propose that, through an unknown mechanism, endocytosis boosts the resting pool of β-catenin upon which GSK3β normally acts.

Brain metastases from thyroid carcinoma is unusual, with a frequency of 1%. We report twelve patients, with single brain metastases and with a karnofsky performance scale score >60 at admission. No metastasis was seen during the uptake of iodine-131, even in the cases from differentiated thyroid carcinoma, suggesting absence of differentiation between primary and metastasic disease. The histopathology of thyroid carcinomas was anaplastic in five cases, differentiated in six, and medullary in one. Only in four patients, brain was the unique site of metastatic spread; in others, bones and lungs were also involved. All metastases were surgically removed, and all patients were treated with radiotherapy (45 Gy) in the postoperative course. The survival average was 19.8 months, and the quality of life was satisfactory in all patients. One patient remained alive till 5 years. Anaplastic histopathology and size of the primitive, and also bone involvement of thyroid disease were significant risk factors in our cases (p < 0.05). According to the literature, surgery is the best therapeutical choice. Alternative strategies in the management of brain metastasis, such as iodine-131 therapy, are discussed, paying particular attention to the relevant side effects.

We performed a clinical and genetic study of patients affected by cavernous angiomas (CA) of the nervous system. We examined initial signs and symptoms in sporadic and familial cases. We obtained clinical, neuroimaging and genetic data on 15 Italian patients with CA of the nervous system with positive, doubtful or apparently negative family history. Genetic markers surrounding three different gene regions (7q, 3q and 7p) were analysed. In one small family, genetic linkage was consistent with all chromosome loci. In another family with the unusual association of cerebral and spinal CA, linkage with chromosome 7q and, likely, 7p was excluded, while linkage with locus 3q was possible. Our results indicate that Italian families with CA may show genetic heterogeneity. Non-specific and subtle onset symptoms hide the presence of CA within families. Patients with multiple CA may have silent cerebral lesions confirming the low penetrance of clinical signs in spite of radiological ones.

Summary The authors report 11 patients with cerebral glioblastoma who lived at least 5 years after their initial diagnosis. There were 6 female and 5 male; the mean age was 39 years (range 24‐55 years). All patients were treated surgically and postoperatively received whole-brain radiotherapy and chemotherapy. Five patients (45%) presented local recurrences after an average interval of 3.9 years from treatment. At average follow-up of 9 years (range 5‐14 years), 7 patients (64%) were alive after an average interval of 8.1 years; 4 patients (36%) died from local relapse. Survival was influenced by patient age and, to a lesser degree, by treatment. A review of the literature, together with our own series, suggest that death from recurrence disease is unusual in glioblastoma patients who survive more than 5 years.

The authors report 8 cases of central neurocytoma with a minimum follow-up of 3 years. Five patients were males and 3 females with average age of 21 years. All patients underwent surgical removal of tumor, and radiotherapy was delivered to 2 of the 3 patients in whom removal had been partial. At an average follow-up of 5 years, all patients are alive and none of them has presented signs of recurrence or progression of the disease. Some typical clinical and histological features of this tumor are pointed out.

Contents/Sommaire/Sumario Articles/Articles/Articulos Paper n. 1, pp. 1 - 46 Alessandra Arcuri and Sara Poli What price for the community enforcement of WTO dispute settlement body’s rulings? Essays/Essais/Ensayos Paper n. 2, pp. 1 - 20 Cristina Amato Financial Contracts And ‘Junk Bonds’ Purchases in the Italian Legal System: A Matter of (In)Correct Disclosure Conference Proceedings/Rapports des conferences/Reportes des conferencias Paper n. 3, pp. 1 - 16 Vittoria Barsotti and Vincenzo Varano Legal Education In Italy Paper n. 4, pp. 1 - 35 Onofrio Troiano, Diana Cerini, Giovanni Comande and Maria Gagliardi Italian report on Insurance law between business law and consumer law News/Annonces /Noticias Comparative Health Care Liability and Regulation: A Technology-Based Approach – International Summer School Pisa, Italy 24th – 4th June 2010 New Frontiers of Pharmaceutical Law - Young Researchers Workshop Lecce, Italy, 6th and 7th of May 2010

This paper is the Italian report, sent to the 18th international congress on comparative law - Washington 2010, on the specific subject “Insurance law between business law and consumer law”. It consists of detailed answers to the questionnaire, on several topics among which economic aspects, academic perception of the field, procedural aspects, legislation, “consumer” and “commercial” risks, substantive aspects.

Deterioration of water distribution networks causes a progressive increase of main failures with both a growth of maintenance cost and a general reduction of service performance indicators. For this reason, maintenance activities represent one of the most important items for a water utility budget and therefore, any decision on possible alternative actions, such as pipes repair, rehabilitation or renewal, must be based on economical and technical issues that must be taken into account in both short and long-term planning. Cost of such actions are really considerable and thus any strategy must be planned on the basis of a careful analysis of failures data. This paper presents a comparison between the statistical distributions of water main breakage data recorded within some districts of the distribution network of an Italian municipality recently subjected to a complete pipes replacement action within a large area. The analysis has been performed considering the breakage patterns over time in relation to several factors such as pipe length, material, age, water pressure, operational conditions, spatial location, type of failure, etc.

Endocytosis, with subsequent targeting to lysosomes for degradation, is traditionally seen as a way for cells to terminate signalling. However, in a few instances, endocytosis has been demonstrated to contribute positively to signalling. Here we review recent work on the role of endocytosis in Wnt signalling. Biochemical evidence suggests that the branch of Wnt signalling that controls planar cell polarity (PCP) does require endocytosis, although how endocytosis of Frizzled receptors is translated into PCP in vivo remains unknown. With respect to the main signalling branch (called the canonical or beta-catenin pathway), the literature is divided as to whether endocytosis is required. Results of in vivo experiments are inconclusive because of the toxic side-effects of blocking endocytosis. Some results with cultured cells suggest the need for endocytosis in canonical signalling; however, it remains unclear whether the ligand-receptor complex must enter the cell by clathrin-mediated or caveolae-mediated endocytosis in order to signal. Means of specifically altering Wnt trafficking as well as of tracking the internalization route in different cell types are needed.

Children with neuroblastoma exhibit marked variability in outcome based on age at diagnosis, disease stage and tumor biologic characteristics. Genomic amplification of MYCN is a potent oncogenic driver and negative prognostic marker in neuroblastoma patients. Therefore, there is a pressing need to identify therapeutic agents that target MYCN gene expression. The bromodomain and extra-terminal (BET) family of proteins are epigenetic regulators known to control expression of genes involved in cell growth and oncogenesis. Previous reports demonstrate that BET inhibition (BETi) significantly attenuates cellular proliferation in numerous cancer models, perhaps via modulation of the MYC and/or MYCN oncogenes. Here, we show that potent BET inhibition induces anti-tumor effects in preclinical neuroblastoma models. Specifically, GSK726 and GSK762 (GlaxoSmithKline) were used for in vitro cytotoxicity and in vivo therapeutic studies, respectively. Neuroblastoma cell lines (n=22) were treated with GSK726 to calculate IC50 values in a luminescence-based cell viability assay and differential sensitivity was observed with an IC50 range of 27 nM to 9.5 µM. In sensitive cell lines, cell cycle distribution and induction of apoptosis were also measured. In these cell lines, GSK726 treatment resulted in MYCN depletion, G1 arrest within 24 hours, and apoptosis as measured by cleaved-PARP. To assess in vivo efficacy, GSK762 was subcutaneously administered in xenograft models and in genetically engineered neuroblastoma mouse models overexpressing MYCN and MYCN/ALK F1174L in the neural crest. In both models, GSK762 treatment resulted in tumor growth delay. Further assessment of results seen in vitro and in vivo indicated that MYCN amplification status did not fully predict sensitivity to GSK726 or GSK762. Thus, to determine additional biomarkers of sensitivity, we examined baseline gene expression data at the extremes of IC50 values by comparing sensitive (n=6; IC50940 nM) neuroblastoma cell lines. Gene expression data from the neuroblastoma cell lines generated on HuGene1.0ST expression microarrays (Affymetrix) were utilized, and data were analyzed using the Limma package in R/Bioconductor. Univariate analysis, with a false discovery rate less than 0.25, revealed 6 genes (PTER, PCDHB14, RFTN1, JAK2, MYCBP, and DACH1) that were differentially expressed between sensitive and resistant cell lines in the MYCN amplified setting. While all 6 genes predicted BETi sensitivity in the MYCN-amplified subset of cell lines, only DACH1 expression predicted sensitivity to BET inhibition irrespective of MYCN amplification status (p=7.92x10-7). In addition, high DACH1 expression was correlated with poor patient outcome (p=1.74x10-5). As a result, high DACH1 expression serves as a candidate biomarker for future studies with respect to sensitivity to BETi. Ultimately, these studies will help to optimize the clinical utility of BETi in neuroblastoma and perhaps other MYC-driven malignancies. Citation Format: Jason R. Healy, Robert W. Schnepp, Lori S. Hart, Pichai Raman, Laura Danielson, Michael Russell, Priya Khurana, Maria Gagliardi, Ryan M. Kinsey, Anastasia Wyce, Olena Barbash, Peter J. Tummino, Louis Chesler, John M. Maris. Antitumor activity and sensitivity evaluation of novel BET inhibitors in neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B34.

Background: Neuroblastoma, a childhood cancer of the sympathetic nervous system, accounts for approximately 10-15% of pediatric oncology deaths. The genetic basis of neuroblastoma has grown clearer, with genome-wide association studies performed by our laboratory uncovering CASC15, BARD1, NBPF23, LMO1, HACE1, TP53 and LIN28B as susceptibility genes, with many (if not all) playing a major role in tumorigenesis. Here we focus on LIN28B, which binds mRNAs directly and is a master regulator of the let-7 family of tumor suppressor microRNAs, as we previously showed that high LIN28B expression is associated with advanced stage disease and worse patient outcome. Methods: To discover LIN28B-associated pathways in neuroblastoma, we performed gene set enrichment analysis (GSEA) on mRNA expression datasets and analyzed SNP-array based DNA copy number datasets. We used siRNAs, shRNAs, and microRNA mimetics to perturb transcripts of interest in neuroblastoma cells and measured effects on downstream signaling, protein-protein interactions, and proliferation. Results: We applied GSEA to mRNA expression profiles from 250 neuroblastoma tumors and found LIN28B expression to be robustly correlated with several biologically relevant gene sets, including “RAN signaling.” We focused on RAN signaling as RAN is a member of the Ras family of GTPases implicated in the pathogenesis of several malignancies and we demonstrated a strong positive correlation between LIN28B and RAN expression, most strikingly in the MYCN-amplified context (p = 2.2×10−10). We next analyzed 374 high-risk neuroblastoma tumors and found that 28% of them displayed recurrent somatic copy number gain of chromosome 12q24, the genomic location of RAN, which was associated with increased RAN expression (p = 0.0004) and was inversely related to MYCN amplification (p = 0.0021). Increased RAN expression was associated with stage 4 disease (p = 0.0047) and decreased overall survival (p = 0.0002). To further dissect the LIN28B-RAN relationship, we depleted LIN28B using shRNAs, showing that it reduced RAN RNA and protein levels. LIN28B directly bound RAN mRNA, likely enhancing its translation. As RAN promotes the phosphorylation and activation of Aurora kinase A (AURKA), we then demonstrated that LIN28B leads to AURKA activation via RAN. Moreover, we demonstrated that AURKA is a direct let-7 target, defining a separate mechanism by which LIN28B/let-7 influences AURKA expression. Finally, we showed that RAN depletion resulted in decreased neuroblastoma proliferation, phenocopying LIN28B depletion. Conclusions: These results demonstrate that enhanced LIN28B expression and chromosome 12q24 gain each independently promote RAN expression and that LIN28B and RAN signaling further converge on AURKA. Collectively, our studies support LIN28B as a master regulator of multiple oncogenes implicated in neuroblastoma pathogenesis, nominating it as a candidate for therapeutic targeting. Citation Format: Robert W. Schnepp, Priya Khurana, Edward F. Attiyeh, Sara Chodosh, Pichai Raman, Derek A. Oldridge, Maria E. Gagliardi, Karina Conkrite, Shahab Asgharzadeh, Robert C. Seeger, Blair Madison, Anil Rustgi, John M. Maris, Sharon J. Diskin. A LIN28B/RAN/AURKA signaling network promotes neuroblastoma tumorigenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4734. doi:10.1158/1538-7445.AM2015-4734

The authors report 2 cases of cerebral meningiomas following high-dose radiotherapy in elderly patients treated for skull basaloma. These two are the first cases over 65 years old cases reported in the literature. The pathological and clinical characteristics of high-dose radio-induced meningiomas are the following: 1) children appear particularly sensitive to the development of this tumor; 2) there is a female patient predominance, in contrast to the male dominance observed in low-dose meningioma, 3) these tumors present a peak frequency in third decade of life; 4) frequently, these tumors are atypical and recur.

Brain metastases from renal carcinoma may appear even a long time after surgical treatment of the primary tumor. The authors present 2 series of patients, one of which has already been published and the other new, for a total of 4 cases of brain metastasis from renal carcinoma with late onset, which occurred 13, 17, 26 and 12 years after primary surgical treatment. The other cases described in the literature were also critically reviewed.

Background: MYCN amplification is the most frequent somatically acquired genomic alteration in neuroblastoma and is a potent oncogenic driver. Targeting MYCN therapeutically has been complicated by the challenges inherent in targeting transcription factors. Recently, several groups have shown that inhibiting bromodomain and extra-terminal (BET) proteins (BRD2, BRD3, BRD4, and BRDT) and their ability to bind to acetylated lysine residues within histone tails resulted in silencing of MYC family protein expression and demonstrated therapeutic efficacy in preclinical cancer models. We hypothesized that potent inhibition of bromodomain-chromatin interactions would show anti-tumor activity in preclinical models of neuroblastoma and that reliable biomarkers of activity could be discovered. Methods: GlaxoSmithKline compounds GSK726 and GSK762 potently inhibit BET proteins (BETi) and were used for in vitro cytotoxicity and in vivo therapeutic studies, respectively. We exposed a panel of 15 cell lines to a 4-log dose range of GSK726 to determine IC50s in a luminescence-based cell viability assay. We also measured cell cycle changes by flow cytometry and apoptosis by immunoblotting for cleaved PARP. We tested the in vivo efficacy of the GSK762 in subcutaneous xenograft models (3 sensitive and 2 resistant cell lines) and in genetically engineered neuroblastoma mouse models (overexpressing MYCN and MYCN/ALK F1174L in the neural crest). For biomarker discovery, gene expression data from the neuroblastoma cell lines generated on HuGene1.0ST expression microarrays (Affymetrix) were utilized, and data were analyzed using the Limma package in R/Bioconductor. Results: Neuroblastoma cell lines (N=15) were differentially sensitive to GSK726, with a median IC50 of 128nM and a range of 17nM to >10uM. In sensitive cell lines, GSK726 treatment resulted in MYCN depletion, G1 arrest within 24 hours, and apoptosis. In subcutaneous xenograft models and genetically engineered neuroblastoma mouse models, GSK762 treatment resulted in tumor growth delay. Importantly, MYCN amplification status did not fully predict sensitivity to GSK726 or GSK762. Thus, to determine additional biomarkers of sensitivity, we examined baseline gene expression data at the extremes of IC50s by comparing sensitive (N=6; IC50940 nM) neuroblastoma cell lines. Univariate analysis, with an FDR < 0.25, revealed 6 genes (PTER, PCDHB14, RFTN1, JAK2, MYCBP, and DACH1) differentially expressed between sensitive and resistant cell lines in the MYCN amplified setting. While all 6 genes predicted BETi sensitivity in the MYCN-amplified subset of cell lines, only DACH1 expression predicted sensitivity to BET inhibition irrespective of MYCN amplification status (7.92x10-7). Moreover, DACH1 levels were heterogeneously expressed in a panel of 88 primary neuroblastoma tumors (Versteeg-88) and high expression was correlated with poor patient outcome (1.74x10-5). Conclusions: BET inhibitors demonstrated significant anti-tumor activity in a subset of neuroblastoma preclinical models. Although MYCN protein levels decreased in neuroblastoma cell lines that were sensitive to BETi, MYCN amplification status alone did not fully explain BETi sensitivity. DACH1 expression serves as a candidate biomarker for sensitivity to BET inhibition. These studies will help to optimize the clinical utility of BETi in neuroblastoma and perhaps other MYC-driven malignancies. Citation Format: Robert Schnepp, Lori Hart, Pichai Raman, Laura Danielson, Maria Gagliardi, Ryan Kinsey, Anastasia Wyce, Olena Barbash, Peter Tummino, Louis Chesler, John Maris. Defining the antitumor activity and sensitivity profiles of BET inhibitors in neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B75.

Background: Neuroblastoma (NB) is a radiosensitive malignancy accounting for 10% of childhood cancer mortality. NB cells frequently express the norepinephrine transporter (NET) providing a specific mechanism for uptake of NET-ligands. Meta-[131I]iodobenzylguanidine ([131I]MIBG) is a NET-ligand radiotherapeutic that shows single-agent response rates in refractory NB of 40-50%. However, due to the long path lengths of 131 I beta (β)-emission, and low biological effectiveness compared to alpha (α)-emitting radionuclides, [131I]MIBG is generally not curative, perhaps due to non-targeting of isolated circulating tumor cells. Here we report our efforts to optimize NET-targeted radiotherapy by developing relevant preclinical models of refractory NB for α-particle therapeutic [211At] MABG therapy. Methods: We first determined NET (SLC6A2) mRNA and protein expression in 35 human NB cell lines using quantitative RT-PCR and western blotting. We then chose 5 lines with absent to intermediate levels of native NET expression (NB1691, SKNSH, IMR5, NLF and SKNBE2) for dual forced overexpression of human NET and luciferase cDNAs. We used [125I]MIBG for cell-based uptake assays in all isogenic pairs and biodistribution experiments in athymic mice bearing three separate NET-transduced xenografts (N=5 per cell line). These cell lines were also treated with [131I]MIBG and/or external beam radiation (XRT) followed by multi-log cytotoxicity assays. Therapeutic trials of [131I]MIBG (25 mCi/kg) in NB1691 subcutaneous xenograft and metastatic mouse models were also conducted. In parallel, [211At] MABG was synthesized by: (i) cyclotron-production of 211 At via 209 Bi(α,2n)211At reaction (ii) distillation of 211 At from the target, and (iii) solid phase no-carrier-added synthesis of [211At] MABG by radioastato-destannylation. [211At] MABG uptake studies were performed in isogenic NB cell lines. Results: Unlike primary human NBs, NET expression was low in the majority of 35 cell-lines studied (median normalized expression value = 0.145; range 0.000-1.005), but all transduced lines showed significant overexpression (0.860-1.107) comparable to human primary tumors. Transduced lines showed 4-10 fold higher uptake of [125I]MIBG than non-transduced isogenic parental cell lines in vitro, and demonstrated significant tumor-specific uptake and retention in vivo with tumor-muscle ratios ranging from 13.80 to 29.48. In vitro cytotoxicity experiments using [131I]MIBG showed NET-expressing cell lines to be more susceptible to treatment compared to non-NET expressing pairs (IC50 of 2.937nCi vs. 15.99 nCi). Treatment of mice bearing NB1691-NET xenografts with [131I]MIBG showed tumor growth delay (p=0.0065), but no significant impact on survival, likely due to de novo radioresistance (1200 cGy of XRT had no impact on NB1691 proliferation; IMR-05 showed 97% decreased cell viability). Lastly, we successfully synthesized [211At] MABG, with radiochemical yields of ∼20% and showed NET specific uptake of [211At] MABG into 1691 NET transfected cells. Conclusions: Development of targeted radiotherapy for neuroblastoma has been limited by the lack of preclinical models and alternative therapeutics. Our development of multiple isogenic pairs with varying NET expression, documentation of de novo radiation sensitivity, and the production of [211At] MABG, will allow for rapid assessment of targeted radiotherapeutic strategies (including combination approaches) to support clinical development of alpha-particle therapeutics in a childhood cancer. Citation Format: V Batra, AM Chacko, M Gagliardi, C Hou, J L. Mikitsh, R H. Freifelder, A Kachur, B C. LeGeyt, A Schmitz, L Toto, G Vaidyanathan, M R. Zalutsky, K K. Matthay, W A. Weiss, W C. Gustafson, D Pryma, J M. Maris. Preclinical development of meta-[211At] astatobenzylguanidine ([211At] MABG) targeted radiotherapy for neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B48.

Background: Despite an aggressive treatment regime, 40% of patients with high-risk neuroblastoma die of their disease. The mitogen-activated protein kinase (MAPK) signaling cascade is hyperactive in many cancers, including a subset of high-risk neuroblastomas. We, therefore, sought to determine the efficacy of MEK inhibition in neuroblastoma and develop a responder hypothesis of anti-tumor activity based on molecular genetic features. Methods: MEK162 (Novartis Pharmaceuticals), an orally available MEK1/2 inhibitor, is currently in adult clinical trials for advanced malignancies harboring RAS/RAF mutations. In determining MEK162 IC50 values, we employed the RT-CES cell impedance assay as it measures both cytostatic and cytotoxic responses in a temporal manner. Additional in vitro analysis of MEK162 activity included immunoblotting, cell cycle analysis with flow cytometric methods, and phosphorylation arrays across a panel of 23 neuroblastoma cell lines. Four of these cell lines (2 sensitive and 2 resistant) were also tested in vivo in subcutaneous xenograft models with tumor volume endpoints. The cell line IC50 ranking was cross-referenced with HuGene1.0ST expression microarrays (Affymetrix), phosphorylation arrays (Full Moon Biosystems), and exome sequencing in order to identify the genetic and proteomic underpinnings predictive of response to MEK inhibition. Microarray analysis was performed using the Limma package (Bioconductor/R), unsupervised clustering, and gene set enrichment (GSEA). Results: Neuroblastoma cell lines (N=23) showed a wide range of sensitivity to MEK162 across a 4-log dose range (median IC50 = 771 nM, range 5 nM-10 μM). Sensitive cell lines demonstrated G1 arrest within 24 hours of exposure to MEK162. In vivo subcutaneous xenograft experiments recapitulated the in vitro response of MEK162 sensitivity in each of the cell lines tested. In order to identify determinants of sensitivity, we profiled the genomic signature of 23 cell lines in the exponential growth phase prior to confluence. Of the ten most sensitive cell lines, six possessed mutations predicted to be damaging and indicative of RAS/MAPK pathway hyperactivation (NRAS-Q61K, NF1 copy number loss, KRAS-G12D). No resistant lines possessed genetic evidence of RAS hyperactivation. However, baseline phosphorylated-ERK status was not robustly predictive of drug activity. For the purpose of biomarker discovery, we considered a total of 4 cells lines as sensitive (IC50< 200 nM) and 4 resistant (IC50> 3,000 nM). Unsupervised clustering and GSEA of the most differentially expressed genes derived from comparing sensitive (N=4) and resistant (N=4) cell lines confirmed increased baseline MAPK pathway activity in the sensitive cell lines. In addition, resistance to MEK162 was found to be associated with MYCN expression (R=.70/p=0.00043), suggesting that MYCN may serve as a biomarker of MEK162 resistance. Independently, we have demonstrated a correlation between MYCN-amplification and sensitivity to CDK4/6 inhibition (p=0.0227), suggesting the combination of MEK and CDK4/6 inhibition may synergize in the inhibition of neuroblastoma growth. Conclusions: MEK inhibition is effective in a definable subset of human-derived neuroblastoma preclinical models. Canonical activation of the RAS/MAPK pathway via RAS mutation and/or NF1 inactivation partially account for sensitivity to MEK inhibition, however, biomarkers predictive of sensitivity to MEK inhibition remain to be identified. We demonstrate that both MYCN expression and CDK4/6 activity are associated with MEK resistance, providing a basis for defining MEK162 combination strategies in the treatment of neuroblastoma. Citation Format: Lori S. Hart, Vandana Batra, Pichai Raman, Maria Gagliardi, JulieAnn Rader, Lucy Chen, Christine Fritsch, Giordano Caponigro, Malte Peters, Markus Boehm, John M. Maris. MEK 1/2 inhibition and biomarkers of response in preclinical models of neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B82.