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Supplementary Figure 2 - PDF file 55K, Neuroblastoma tissue microarray. A tissue microarray demonstrates expression of RB in neuroblastoma that is higher in high-risk and MYCN amplified samples compared to low-risk or non-amplified samples (p = 0.03)

Primer sequences and Neuroblastoma cell line mutations

Supplementary Figure 5 - PDF file 138K, CDK4/6 inhibition does not induce cytotoxicity in neuroblastoma. (A) Caspase 3/7 activation and (B) PARP cleavage were not observed in cell lines with demonstrated sensitivity to LEE011. SN38, a known cytotoxic agent, was used as a positive control

Supplementary Table 1 - PDF file 78K, Genomic aberrations in the Cyclin D/CDK4/CDK6/RB pathway occur frequently in neuroblastoma cell lines

Caspase 9 activity of shYAP model cells in serum starved conditions; Methylation status at HRK promoter region

Supplementary Figure 4 - PDF file 88K, Protein level expression of FOXM1 in neuroblastoma

Supplementary Figure 1 - PDF file 56K, Expression of CDK4, CDK6, CCND1, and RB1 in neuroblastoma patients. Expression analysis of neuroblastoma primary tumors demonstrates that CDK4 expression, and to a lesser extent RB1 expression, is higher in high-risk neuroblastoma patients than in low risk patients

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.

YAP protein expression in paired isogenic cell line; NGP pGAMA-YAP model; GSEA of stemness pathway genes

Cytotoxic therapy treatment of shYAP and YAP overexpression models

Supplementary Figure 6 - PDF file 75K, Statistical analysis of in vivo growth suppression. (A) Growth rates of BE2C, 1643, and EBC1 xenografts treated with 200 mg/kg LEE011 or vehicle, as determined by linear mixed effects analysis. (B) Summary table of statistics

Supplementary Table 2 - PDF file 48KB, Copy number variations within the Cyclin D/CDK4/CDK6/RB pathway occur in high-risk neuroblastoma patients

Following chemotherapy and relapse, high-risk neuroblastoma tumors harbor more genomic alterations than at diagnosis, including increased transcriptional activity of the Yes-associated protein (YAP), a key downstream component of the Hippo signaling network. Although YAP has been implicated in many cancer types, its functional role in the aggressive pediatric cancer neuroblastoma is not well-characterized. In this study, we performed genetic manipulation of YAP in human-derived neuroblastoma cell lines to investigate YAP function in key aspects of the malignant phenotype, including mesenchymal properties, tumor growth, chemotherapy response, and MEK inhibitor response. Standard cytotoxic therapy induced YAP expression and transcriptional activity in patient-derived xenografts treated in vivo, which may contribute to neuroblastoma recurrence. Moreover, YAP promoted a mesenchymal phenotype in high-risk neuroblastoma that modulated tumor growth and therapy resistance in vivo. Finally, the BH3-only protein, Harakiri (HRK), was identified as a novel target inhibited by YAP, which, when suppressed, prevented apoptosis in response to nutrient deprivation in vitro and promoted tumor aggression, chemotherapy resistance, and MEK inhibitor resistance in vivo. Collectively, these findings suggest that YAP inhibition may improve chemotherapy response in patients with neuroblastoma via its regulation of HRK, thus providing a critical strategic complement to MEK inhibitor therapy.Significance:This study identifies HRK as a novel tumor suppressor in neuroblastoma and suggests dual MEK and YAP inhibition as a potential therapeutic strategy in RAS-hyperactivated neuroblastomas.

Supplementary Figure 3 - PDF file 127K, MYCN expression is inversely correlated with LEE011 sensitivity. (A) High mRNA expression of MYCN correlates with low LEE011 IC50s (r = -0.55, p = 0.02). This correlation is further strengthened (r = -0.7, p = 0.003) by the removal of two outliers, SKNSH (IC50 = 148 nM) and its subclone, SY5Y (IC50 = 154 nM). (B) Sensitive cell lines also tend to express higher levels of MYCN protein than resistant cell lines

PDF file - 189K

Rhabdomyosarcoma (RMS) is an aggressive pediatric soft tissue sarcoma. There are two main subtypes of RMS, alveolar rhabdomyosarcoma (ARMS) and embryonal rhabdomyosarcoma. ARMS typically encompasses fusion‐positive rhabdomyosarcoma, which expresses either PAX3‐FOXO1 or PAX7‐FOXO1 fusion proteins. There are no targeted therapies for ARMS; however, recent studies have begun to illustrate the cooperation between epigenetic proteins and the PAX3‐FOXO1 fusion, indicating that epigenetic proteins may serve as targets in ARMS. Here, we investigate the contribution of BMI1, given the established role of this epigenetic regulator in sustaining aggression in cancer. We determined that BMI1 is expressed across ARMS tumors, patient‐derived xenografts, and cell lines. We depleted BMI1 using RNAi and inhibitors (PTC‐209 and PTC‐028) and found that this leads to a decrease in cell growth/increase in apoptosis in vitro, and delays tumor growth in vivo. Our data suggest that BMI1 inhibition activates the Hippo pathway via phosphorylation of LATS1/2 and subsequent reduction in YAP levels and YAP/TAZ target genes. These results identify BMI1 as a potential therapeutic vulnerability in ARMS and warrant further investigation of BMI1 in ARMS and other sarcomas., Treatment of alveolar rhabdomyosarcoma (ARMS) remains a major therapeutic challenge in pediatric oncology. Here, we demonstrate that genetic and pharmacologic inhibition of BMI1 reduces ARMS viability. We show that BMI1 inhibits the tumor suppressive Hippo pathway and, conversely, that BMI1 disruption upregulates Hippo signaling. Collectively, these findings provide an initial framework for targeting BMI1 in ARMS and additional sarcomas.

TPS279 Background: Amivantamab, a fully human EGFR and MET bispecific antibody, has shown clinical activity against tumors with primary activating EGFR mutations, EGFR resistance mutations, or MET pathway activation. Amivantamab has demonstrated activity in both EGFR- and MET-driven non-small cell lung cancer, with preclinical evidence demonstrating its ability to recruit immune effector cells. While two anti-EGFR antibodies are incorporated as part of the standard of care (SoC) for metastatic colorectal cancer (mCRC) patients, MET is highly expressed or amplified in subsets of mCRC and additionally plays a role in mediating resistance to anti-EGFR therapies; therefore, amivantamab may provide benefit in this setting. Methods: This open-label, multicenter, global Ph1b/2 study will assess the safety and anti-tumor activity of amivantamab as a monotherapy and characterize the safety and tolerability of amivantamab in addition to SoC chemotherapy in KRAS, NRAS, BRAF, and EGFR ectodomain wild type participants with advanced or metastatic CRC. The Ph2 amivantamab monotherapy Cohorts A and B will assess the anti-tumor activity in participants with left-sided CRC who have progressed on or after SoC fluoropyrimidine-, oxaliplatin-, and irinotecan-based chemotherapy and an anti-VEGF treatment, without (Cohort A) or with (Cohort B) prior exposure to anti-EGFR treatment. The Ph2 amivantamab monotherapy Cohort C will assess the antitumor activity in participants with right-sided CRC who have progressed on or after SoC fluoropyrimidine-, oxaliplatin-, and irinotecan-based chemotherapy and an anti-VEGF treatment, with or without an anti-EGFR treatment. The Ph1b dose confirmation cohorts (Ph1b-D and Ph1b-E) will assess the safety and confirm the recommended Ph2 combination dose (RP2CD) of amivantamab in addition to SoC chemotherapy regimens (mFOLFOX6 or FOLFIRI). Upon confirmation of the RP2CD, the Ph2 Cohorts D and E, which are distinct cohorts from Ph1b-D or Ph1b-E, will further characterize the safety, tolerability, and preliminary anti-tumor activity of amivantamab in addition to SoC mFOLFOX6 or FOLFIRI in mCRC patients who have progressed after front-line therapy. The primary objectives are to assess the anti-tumor activity of amivantamab as a monotherapy and characterize the safety of amivantamab when added to SoC chemotherapy in participants with mCRC (Ph2 cohorts), as well as to assess the RP2CD of amivantamab when added to SoC chemotherapy (Ph1b). The key secondary objectives are to characterize the safety of amivantamab as a monotherapy and to assess the anti-tumor activity of amivantamab when added to SoC chemotherapy in participants with mCRC. This study is currently enrolling (NCT05379595) as of August 2022 in 12 countries, with goal enrollment of 225 participants. Clinical trial information: NCT05379595 .

Following chemotherapy and relapse, high-risk neuroblastoma tumors harbor more genomic alterations than at diagnosis, including increased transcriptional activity of the Yes-associated protein (YAP), a key downstream component of the Hippo signaling network. Although YAP has been implicated in many cancer types, its functional role in the aggressive pediatric cancer neuroblastoma is not well-characterized. In this study, we performed genetic manipulation of YAP in human-derived neuroblastoma cell lines to investigate YAP function in key aspects of the malignant phenotype, including mesenchymal properties, tumor growth, chemotherapy response, and MEK inhibitor response. Standard cytotoxic therapy induced YAP expression and transcriptional activity in patient-derived xenografts treated in vivo, which may contribute to neuroblastoma recurrence. Moreover, YAP promoted a mesenchymal phenotype in high-risk neuroblastoma that modulated tumor growth and therapy resistance in vivo. Finally, the BH3-only protein, Harakiri (HRK), was identified as a novel target inhibited by YAP, which, when suppressed, prevented apoptosis in response to nutrient deprivation in vitro and promoted tumor aggression, chemotherapy resistance, and MEK inhibitor resistance in vivo. Collectively, these findings suggest that YAP inhibition may improve chemotherapy response in patients with neuroblastoma via its regulation of HRK, thus providing a critical strategic complement to MEK inhibitor therapy. Significance: This study identifies HRK as a novel tumor suppressor in neuroblastoma and suggests dual MEK and YAP inhibition as a potential therapeutic strategy in RAS-hyperactivated neuroblastomas.

Neuroblastoma is an aggressive pediatric malignancy of the neural crest with suboptimal cure rates and a striking predilection for widespread metastases, underscoring the need to identify novel therapeutic vulnerabilities. We recently identified the RNA binding protein LIN28B as a driver in high-risk neuroblastoma and demonstrated it promotes oncogenic cell proliferation by coordinating a RAN-Aurora kinase A network. Here, we demonstrate that LIN28B influences another key hallmark of cancer, metastatic dissemination. Using a murine xenograft model of neuroblastoma dissemination, we show that LIN28B promotes metastasis. We demonstrate that this is in part due to the effects of LIN28B on self-renewal and migration, providing an understanding of how LIN28B shapes the metastatic phenotype. Our studies reveal that the let-7 family, which LIN28B inhibits, decreases self-renewal and migration. Next, we identify PDZ Binding Kinase (PBK) as a novel LIN28B target. PBK is a serine/threonine kinase that promotes the proliferation and self-renewal of neural stem cells and serves as an oncogenic driver in multiple aggressive malignancies. We demonstrate that PBK is both a novel direct target of let-7i and that MYCN regulates PBK expression, thus elucidating two oncogenic drivers that converge on PBK. Functionally, PBK promotes self-renewal and migration, phenocopying LIN28B. Taken together, our findings define a role for LIN28B in neuroblastoma metastasis and define the targetable kinase PBK as a potential novel vulnerability in metastatic neuroblastoma.

9008 Background: Amivantamab, a fully human bispecific antibody targeting epidermal growth factor receptor (EGFR) and MET, is approved for the treatment of non-small cell lung cancer (NSCLC) with EGFR exon 20 insertion after prior platinum-based chemotherapy. Given its bispecific nature, amivantamab is being explored in patients (pts) with primary MET exon 14 skipping mutation (METex14) in the MET-2 cohort of the CHRYSALIS study. Methods: CHRYSALIS (NCT02609776) is an ongoing phase 1 dose escalation/dose expansion study of amivantamab in pts with advanced NSCLC. Pts with primary METex14 whose disease progressed on or who declined current standard of care therapy were treated with amivantamab 1050 mg (pts 54% regardless of prior treatment (Table). Median duration of response (DOR) was not reached (range, 2.1-12.2 months); 67% (8/13) had DOR ≥6 months. Ten of the 12 responders remain on treatment (6.0-14.4 months) with ongoing responses; 2 discontinued after 2 and 12 months, respectively. Safety profile was consistent with previously reported experience of amivantamab (Sabari 2021 JTO 16(3):S108-109). Treatment-related adverse events leading to dose reduction or discontinuation occurred in 3 pts, each. Conclusions: Amivantamab demonstrates anti-tumor activity in primary METex14 NSCLC including after prior MET inhibitor treatment. Enrollment is ongoing and updated data will be shown. Clinical trial information: NCT02609776. [Table: see text]

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.

Anti-GD2 antibody therapy significantly improved high-risk neuroblastoma (NB) patient survival yet is associated with toxicity due to GD2 expression on pain fibers. Also, GD2 may be downregulated in NB following chemotherapy and relapse. It is paramount to identify novel tumor-associated antigens that are upregulated on the cell surface throughout therapy with minimal normal tissue expression. We utilized a novel cell surface capturing technology to define the NB cell surfaceome before and after chemotherapy and nominate potential targets for cell-mediated immunotherapy. Two NB patient derived xenografts (PDXs) and one cell line xenograft (IMR5) were treated with topotecan/cyclophosphamide in vivo. Treated and untreated tumors were disaggregated and N-glycoproteins on intact cells were oxidized, selectively labeled with biocytin-hydrazide, lysed, proteins digested, and cell surface peptides enriched using NeutrAvidin beads, followed by LC-MS analysis. We prioritized cell surface proteins that were abundant on all NB models and did not change under the selective pressure of chemotherapy. Our top hit was confirmed for cell surface expression by flow, evaluated for primary tumor and normal cell expression using gene expression datasets, and evaluated for functional relevance using CRISPR/CAS9 and immunotherapy targeting via chimeric antigen receptor (CAR). In two replicates, 292 cell surface proteins were identified as abundant and unchanged following chemotherapy. 34/292 proteins had high abundance of ≥70 a.u. across all xenografts, including NB surface proteins ALK and NCAM1. One protein identified was PTK7, a member of the WNT signaling pathway that is overexpressed in adult tumors. PTK7 is highly expressed in primary NB tumors with very low normal tissue expression. PTK7 protein is present on the cell surface in 17/17 NB cell lines and PDXs. While high PTK7 expression in primary NB is associated with poor patient prognosis, CRISPR/CAS9 knockout of PTK7 shows no functional effect on cell survival, proliferation or chemotherapy response. We developed a codon-optimized, second-generation CAR construct specific to PTK7. Our CAR-engineered T cells specifically bound to recombinant human-PTK7 Fc chimera. PTK7 CAR Jurkat T cells became activated in the presence of PTK7-expressing KELLY, IMR-5, and NB-1643 NB cells, and activation was not seen in the presence of PTK7 negative CMK leukemia cells or NB-1643 PTK7 knockout cells, confirming activation is specific to the CAR recognizing PTK7. Primary T cell PTK7 CAR studies are ongoing to assess cytotoxicity against NB. Multiple surface proteins remain stable on NB tumors after chemotherapy treatment. PTK7 is a potential cell surface antigen that could be utilized for immunotherapy approaches in NB while minimizing damage to normal tissues. PTK7 is robustly expressed in other pediatric and adult cancers, suggesting broader clinical relevance. Citation Format: Arhanti Sadanand, Victor Maximov, Jasmine Lee, Suttipong Suttapitugsakul, Jenny Shim, Chris Doering, Ronghu Wu, Robert Schnepp, Trent Spencer, Kelly Goldsmith. Proteomic profiling identifies PTK7 as a novel immunotherapeutic candidate for neuroblastoma [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 3034.

Medulloblastoma (MB) is the most common pediatric malignant brain tumor and is currently divided into WNT, SHH, Group 3 and Group 4 subtypes. Even with multimodal chemotherapy, radiotherapy and surgery, many children with Group 3 MBs do not survive. While the molecular aberrations underlying WNT- and SHH-driven MBs are relatively well understood, the oncogenic drivers that lead to Group 3/4 MBs are poorly defined, limiting therapeutic progress. In addition to genetic mutations and alterations, cancers display dysregulated transcription and translation. RNA-binding proteins (RBPs) play key roles in both transcription and translation, and a subset of RBPs are differentially expressed in many different cancers. Indeed, we have previously demonstrated an oncogenic role for the RBP LIN28B in neuroblastoma and it is known to be upregulated in Wilms tumor, hepatoblastoma, germ cell tumors, leukemia among others. LIN28B is a key regulator of let-7 family miRNAs, which in turn inhibit LIN28B and other oncogenes. We hypothesize that LIN28B plays an important role in Group 3 MB and that a better understanding of LIN28B and LIN28B-driven networks will reveal novel therapeutic vulnerabilities. In support of our hypothesis we find that among the four subtypes, LIN28B levels are highest in Group 3 MB, and that overexpression is associated with significantly worse survival. Down-regulation of LIN28B results in significant reduction in cell proliferation by CellTiter-Glo and increased apoptosis by Caspase-Glo (as well as induction of cleaved PARP on immunoblots). In contrast overexpression of LIN28B increases Group 3 cell proliferation and tumor sphere formation. In addition we find that PDZ-binding kinase (PBK) a downstream target of LIN28B is downregulated when LIN28B is depleted. PBK knock down also leads to decreased proliferation of Group 3 MB cells. Finally, in order to robustly define the signaling networks downstream from LIN28B that are involved in Group 3 MB metastasis, we have performed who transcriptome RNA-seq profiling of two group 3 cell lines following LIN28B depletion and plan to interrogate a subset of these based on expression change and functional relevance to LIN28B-mediated Group 3 MB metastasis. This work will help define the role for LIN28B in Group 3 MB aggressiveness and pave the way for similar studies in other cancers. Citation Format: Shubin W. Shahab, Jo Lynne Rokita, Kyle Juraschka, Sachin Kumar, Michael Taylor, Robert W. Schnepp, Tobey J. MacDonald, Anna M. Kenney. Targeting the RNA binding protein LIN28B in Group 3 medulloblastoma decreases proliferation and promotes apoptosis [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 3024.

Medulloblastoma (MB) is the most common pediatric malignant brain tumor and is currently divided into WNT, SHH, Group 3 and Group 4 subtypes. Even with multimodal chemotherapy, radiotherapy and surgery, many children with Group 3 MBs do not survive. We have previously demonstrated an oncogenic role for the RNA-binding protein (RBP) LIN28B in neuroblastoma. LIN28B is a key regulator of let-7 family miRNAs, which in turn inhibit LIN28A/B and other oncogenes. LIN28B has also been found to be upregulated in Wilms tumor, hepatoblastoma, germ cell tumors, leukemia among others. We hypothesize that LIN28B plays an important role in Group 3 MB and that a better understanding of LIN28B and LIN28B-driven networks will reveal novel therapeutic vulnerabilities. LIN28B levels are highest in Group 3 MB patients, and its overexpression is associated with significantly worse survival. Here we demonstrate that down-regulation of LIN28B using shRNA results in significant reduction in cell proliferation by CellTiter-Glo and increased apoptosis by Caspase-Glo (as well as induction of cleaved PARP on immunoblots). In contrast overexpression of LIN28B increases Group 3 cell proliferation and tumor sphere formation The LIN28 inhibitor 1632 also leads to significant reduction in G3 MB cell proliferation. In addition, we find that PDZ-binding kinase (PBK) a downstream target of LIN28B is downregulated when LIN28B is depleted. PBK knock down also leads to decreased proliferation of Group 3 MB cells. Finally RNA-seq profiling following LIN28B depletion reveals additional components of the LIN28B pathway which may be amenable to therapeutic targeting. This work will help define the role for LIN28B in Group 3 MB aggressiveness and establish LIN28B and LIN28B-driven networks as novel therapeutic targets in these patients.

Neuroblastoma is an aggressive pediatric malignancy of the neural crest with suboptimal cure rates and a striking predilection for widespread metastases, underscoring the need to identify novel therapeutic vulnerabilities. We recently identified the RNA binding protein LIN28B as a driver in high-risk neuroblastoma and demonstrated it promotes oncogenic cell proliferation by coordinating a RAN-Aurora kinase A network. Here, we demonstrate that LIN28B influences another key hallmark of cancer, metastatic dissemination. Using a murine xenograft model of neuroblastoma dissemination, we show that LIN28B promotes metastasis. We demonstrate that this is in part due to the effects of LIN28B on self-renewal and migration, providing an understanding of how LIN28B shapes the metastatic phenotype. Our studies reveal that the let-7 family, which LIN28B inhibits, opposes the effects of LIN28B. Next, we identify PDZ Binding Kinase (PBK) as a novel LIN28B target. PBK is a serine/threonine kinase that promotes the proliferation and self-renewal of neural stem cells and serves as an oncogenic driver in multiple aggressive malignancies. We demonstrate that PBK is both a novel direct target of let-7 and that MYCN regulates PBK expression, thus elucidating two oncogenic drivers that converge on PBK. Functionally, PBK promotes self-renewal and migration, phenocopying LIN28B. Taken together, our findings define a role for LIN28B in neuroblastoma metastasis and define the targetable kinase PBK as a potential novel vulnerability in metastatic neuroblastoma.

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.

Background: Neuroblastoma, an aggressive cancer of the developing sympathetic nervous system, continues to cause significant morbidity and mortality, highlighting the need to identify novel therapeutic vulnerabilities. We previously demonstrated that the RNA binding protein (RBP) LIN28B is an oncogenic driver and induces neuroblastoma proliferation, in part by regulating a RAN GTPase-Aurora kinase A axis. LIN28B blocks the processing of the let-7 family of tumor suppressors and binds mRNAs directly. In addition to alterations in cell cycle/apoptosis, metastatic dissemination is a hallmark of cancer that is incompletely understood, and neuroblastoma exhibits a striking proclivity for widespread metastases. In these studies, we investigated how LIN28B shapes neuroblastoma metastasis. Methods: We generated GFP-luciferase expressing neuroblastoma cell line models in which LIN28B levels were manipulated, injected these models into the tail veins of NSG mice, and tracked dissemination using an IVIS Spectrum system. We used gain- and loss-of-function approaches (siRNAs, shRNAs, microRNA mimetics) to manipulate transcripts of interest in neuroblastoma cells and measured effects on self-renewal, invasion, and downstream signaling. To discover LIN28B-associated pathways, we assessed clinically annotated mRNA expression datasets. Results: Mice injected with LIN28B-depleted neuroblastoma cells exhibit a delayed onset of tumor metastasis, reduced tumor burden, and extended survival (103 days vs. 50 days, p Conclusions: Taken together, our findings suggest that LIN28B/let-7 shapes neuroblastoma metastasis, in part through influencing PBK, a kinase not previously implicated in the pathogenesis of aggressive pediatric solid tumors. Current studies are defining whether PBK, a therapeutically tractable target for which clinically relevant inhibitors exist, represents a novel therapeutic vulnerability in metastatic neuroblastoma. Citation Format: Dongdong Chen, Julie Cox, Jayabhargav Annam, Melanie Weingart, Grace Essien, Komal Rathi, Priya Khurana, Selma Cuya, Jo Lynne Harenza, Kristopher Bosse, Adeiye Pilgrim, John M. Maris, Robert W. Schnepp. A LIN28B-PDZ kinase axis promotes neuroblastoma metastasis [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B48.

Background: Despite intense efforts within pediatric oncology, novel, effective therapy for alveolar rhabdomyosarcoma, known as fusion-positive rhabdomyosarcoma (FP RMS) given the PAX-FOXO1 fusions characteristic of the disease, remains unrealized. Like many pediatric tumors, FP RMS displays a quiet genomic landscape, when focusing on the coding genome. However, the epigenome plays key roles in shaping tumor aggression, and previous studies have demonstrated that FP RMS is specifically enriched for methylation of Polycomb target genes, suggesting that Polycomb complexes may be deregulated and could constitute novel therapeutic targets. We hypothesized that BMI1, a key member of the Polycomb family and a tractable therapeutic target, represents a novel therapeutic vulnerability in FP RMS. Methods: We analyzed RNA and protein expression in FP RMS cell lines, patient-derived xenografts (PDXs), and human tumor specimens. We used genetic and pharmacologic approaches to manipulate BMI1 in FP RMS cells and measured effects on proliferation, cell cycle, apoptosis, and signal transduction. To examine the effect of in vivo inhibition of BMI1, we utilized xenograft models of FP RMS. Results: We examined RNA-Seq tumor datasets and tumor microarrays and demonstrated that BMI1 is robustly expressed in FP RMS tumors, PDXs, and cell line models. Next, in 2 cell line models, we depleted BMI-1 using shRNAs and siRNAs, and found that this led to striking (~70%) decreases in cell growth secondary to both G1/S phase arrest and apoptosis. Given these findings, we asked whether small-molecule inhibitors of BMI-1 mediated similar effects. We treated 4 independent FP RMS cell line models with both PTC-209 (first-generation inhibitor) and PTC-028 (orally available second-generation inhibitor with higher potency). Both compounds inhibited BMI-1 function and greatly reduced cell proliferation in FP rhabdomyosarcoma cell line models. Similar to genetically mediated depletion, pharmacologic inhibition of BMI1 led to G1/S phase arrest and apoptosis, as demonstrated by Annexin V staining and PARP cleavage. Finally, in a xenograft model of an aggressive FP RMS, PTC-028 treatment decreased tumor growth (p=0.0005) and significantly prolonged survival by 17 days (p=0.0002). Importantly, treatment was well tolerated without evidence of toxicity or weight loss. Conclusions: BMI1 is robustly expressed in FP RMS and both genetic and pharmacologic inhibition of BMI1 lead to striking decreases in cell proliferation, with concomitant cell cycle arrest and apoptosis. BMI1 inhibition significantly decreases tumor growth, prolongs survival, and is well tolerated. Currently, we are further investigating combining BMI1 inhibition with standard chemotherapy and novel agents, as well as defining molecular mechanisms by which BMI1 exerts molecular functions in FP RMS. Targeting BMI-1 could provide a novel therapeutic option for patients with FP RMS, with potential broader implications for additional aggressive sarcomas. Citation Format: Cara E. Shields, Selma M. Cuya, Sarah Chappell, Komal Rathi, Shiv Patel, Sindhu Potlapalli, Robert W. Schnepp. BMI1 constitutes a novel therapeutic vulnerability in fusion-positive rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A47.

Background: Rhabdomyosarcoma (RMS) is an extremely aggressive soft tissue sarcoma which affects mainly children. There are two subtypes: alveolar rhabdomyosarcoma (ARMS) and embryonal rhabdomyosarcoma (ERMS). ARMS is characterized by PAX-FOXO1 fusion proteins, whereas subsets of ERMS harbor alterations within RAS and TP53 pathways. Currently, the outcomes for ARMS (especially when metastatic) remain dismal, thus underscoring the urgent need to identify novel targets for this cancer. The genomic landscape of many pediatric cancers, including ARMS, is relatively sparse. This led us to ask whether key epigenetic factors are driving tumor aggressiveness and could constitute novel approaches for treating ARMS. Notably, the epigenetic complexes PRC1 and PRC2 are overexpressed in a variety of sarcomas and are associated with worse overall survival. We took a hypothesis-based approach and focused on PRC1. We discovered that B lymphoma Mo-MLV insertion region 1 (BMI-1), a protein member of PRC1, is overexpressed in ARMS cells. BMI-1 is a known oncogene in other cancers, but its potential oncogenic role in ARMS and other pediatric malignancies has not yet been interrogated; thus we aim to study it within this context. Methods: To analyze the function of BMI-1 in ARMS, we depleted the protein in ARMS cell line models by both shRNA/siRNA knockdown and measured expression, cell proliferation and apoptosis. We utilized two small molecule inhibitors, PTC-209 and PTC-028, to obtain IC50s in these cell lines, then determined effects on cell proliferation and apoptosis. Results: We examined RNA-Seq tumor datasets and determined that BMI1 is robustly expressed in ARMS tumors. Additionally, we confirmed that BMI-1 is also overexpressed in ARMS cell lines at the levels of RNA and protein. Next, we depleted BMI-1 using multiple shRNAs and siRNAS and found that this led to striking (~70%) decreases in cell growth. We also observed increased levels of apoptosis within knockdown cells. Given these results, we asked whether small molecule inhibitors of BMI-1 mediated similar phenotypes, and so we used the inhibitors PTC-209 and PTC-028. PTC-209 is a first-generation BMI-1 inhibitor, while PTC-028 is a second-generation orally available inhibitor with higher potency. Both compounds inhibited BMI-1 function and greatly reduced cell proliferation in ARMS cell lines within the nanomolar range; however, as expected, PTC-028 showed a more pronounced effect compared to PTC-209. Conclusions: BMI1 supports proliferation and survival in cell line models of ARMS. Both chemical and pharmacologic inhibition of BMI1 led to striking decreases in cell proliferation. Currently, we are further investigating the molecular impact of BMI1 inhibition, with plans to investigate its effectiveness within an in vivo ARMS model. Targeting BMI-1 pharmacologically could provide a novel therapeutic option for patients with ARMS and may apply more broadly to other sarcomas. Citation Format: Cara E. Shields, Selma M. Cuya, Sarah K. Chappell, Komal Rathi, Shiv Patel, Sindhu Potlapalli, Robert W. Schnepp. Targeting epigenetic regulator BMI-1 in alveolar rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3838.

LIN28B is an RNA binding protein that plays key roles in normal development and, when deregulated, oncogenesis; mechanistically, it blocks the processing of the let-7 family of tumor suppressors and binds mRNAs directly. We previously demonstrated that LIN28B induces neuroblastoma proliferation, in part by regulating the expression of RAN GTPase and Aurora kinase A (AURKA). However, given the widespread metastases seen within neuroblastoma, we speculated that LIN28B might also influence neuroblastoma dissemination. We used gain and loss of function approaches to genetically manipulate transcripts of interest in neuroblastoma cells and measured effects on self-renewal, invasion, and downstream signaling. To examine the impact of LIN28B on dissemination, we generated GFP-luciferase expressing neuroblastoma cell line models in which LIN28B levels were manipulated, injected these lines into the tail veins of NSG mice, and tracked dissemination using an IVIS Spectrum system. Results show that depletion of LIN28B significantly delayed the onset of tumor metastasis, reduced tumor burden, and extended mouse survival (104 days versus 50 days, p Citation Format: Dongdong Chen, Julie Cox, Jayabhargav Annam, Melanie Weingart, Grace Essien, Komal Rathi, Priya Khurana, Selma M. Cuya, Robert W. Schnepp. A LIN28B-PBK Axis promotes neuroblastoma dissemination and aggression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3668.

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.

Multiple endocrine neoplasia type 1 (MEN1) is a familial tumor syndrome linked to mutation of the MEN1 gene, which encodes a tumor suppressor, menin. We previously reported that menin upregulates the caspase 8 expression and promotes TNF-α-induced apoptosis. However, it remains unclear how menin upregulates caspase 8 expression and whether menin-mediated caspase 8 expression plays a role in repressing MEN1 development. Here we show that menin binds the 5’-untranslated region (5’-UTR) of the Caspase 8 locus in vivo and activates transcription of a reporter gene through the 5’-UTR. Menin directly binds the 5’-UTR in a sequence-independent manner in vitro. Moreover, Men1 ablation in cells reduces acetylation of histones H3 and H4 at the 5’-UTR of the caspase 8 locus bound by menin in vivo. Notably, the MEN1-derived menin point mutants lose their ability to bind the caspase 8 locus and fail to induce caspase 8 expression and TNF-α-mediated apoptosis. Consistent with these observations, the expression level of caspase 8 is markedly reduced in insulinomas from Men1+/− mice. Together, our results indicate that menin enhances the caspase 8 expression by binding the caspase 8 locus, and these results also suggest that menin suppresses MEN1 tumorigenesis, at least in part, by upregulating caspase 8 expression.

Precision medicine, the individualization of health care based on unique patient-specific variables, is not new, especially within oncology. Historically, there has been a “depersonalization” of cancer care by defining histotype-specific standard-of-care treatments for the majority of malignancies, although in contrast oncologists have been adept at individualizing therapy, especially when confronted with disease relapse. Practicing oncologists seek evidence-based approaches to improve patient outcomes, but much of the current personalization of care remains largely empirical. Nevertheless, over the past decade there has been increasing enthusiasm for using genomic data to more precisely diagnose cancer, predict outcomes, and prescribe “targeted” therapies. Although substantial progress has been made, both anticipated and unanticipated barriers exist in integrating sequencing technologies into the care of patients with cancer. Clearly, for multiple reasons, many challenges remain to prove that personalized genomic medicine can substantively improve outcomes for patients with cancer. These challenges are further accentuated with childhood cancers.

Multiple endocrine neoplasia type 1 (MEN1), an inherited tumor syndrome affecting endocrine organs including pancreatic islets, results from mutation of the tumor suppressor gene Men1 that encodes protein menin. Although menin is known to be involved in regulating cell proliferation in vitro, it is not clear how menin regulates cell cycle and whether mutation of Men1 acutely promotes pancreatic islet cell proliferation in vivo. Here we show that excision of the floxed Men1 in mouse embryonic fibroblasts (MEF) accelerates G0/G1 to S phase entry. This accelerated S-phase entry is accompanied by increased cyclin-dependent kinase 2 (CDK2) activity as well as decreased expression of CDK inhibitors p18Ink4c and p27Kip1. Moreover, Men1 excision results in decreased expression of p18Ink4c and p27Kip1 in the pancreas. Furthermore, complementation of menin-null cells with wild-type menin represses S-phase entry. To extend the role of menin in repressing cell cycle in cultured cells to in vivo pancreatic islets, we generated a system in which floxed Men1 alleles can be excised in a temporally controllable manner. As early as 7 days following Men1 excision, pancreatic islet cells display increased proliferation, leading to detectable enlargement of pancreatic islets 14 days after Men1 excision. These observations are consistent with the notion that an acute effect of Men1 mutation is accelerated S-phase entry and enhanced cell proliferation in pancreatic islets. Together, these results suggest a molecular mechanism whereby menin suppresses MEN1 tumorigenesis at least partly through repression of G0/G1 to S transition. (Cancer Res 2006; 66(10): 5707-15)

Mutations in the MEN1 gene are associated with the multiple endocrine neoplasia syndrome type 1 (MEN1), which is characterized by parathyroid hyperplasia and tumors of the pituitary and pancreatic islets. The mechanism by which MEN1 acts as a tumor suppressor is unclear. We have recently shown that menin, the MEN1 protein product, interacts with mixed lineage leukemia (MLL) family proteins in a histone methyltransferase complex including Ash2, Rbbp5, and WDR5. Here, we show that menin directly regulates expression of the cyclin-dependent kinase inhibitors p27 Kip1 and p18 Ink4c . Menin activates transcription by means of a mechanism involving recruitment of MLL to the p27 Kip1 and p18 Ink4c promoters and coding regions. Loss of function of either MLL or menin results in down-regulation of p27 Kip1 and p18 Ink4c expression and deregulated cell growth. These findings suggest that regulation of cyclin-dependent kinase inhibitor transcription by cooperative interaction between menin and MLL plays a central role in menin's activity as a tumor suppressor.

Menin is a tumor suppressor that is mutated in patients with multiple endocrine neoplasia type I (MEN1), an inherited tumor-prone syndrome. Since there is no obvious conserved structural domain in menin that suggests a function, little is known as to how menin suppresses tumorigenisis. Although menin interacts with a variety of nuclear proteins including transcription factors, it is unknown whether menin itself can directly bind DNA. Here we show that menin directly binds to double stranded DNA in a sequence-independent manner. It also binds a variety of DNA structures, including Y-structures, branched structures, and 4-way junction structures. The C-terminus of menin mediates binding to DNA, but MEN1 disease-derived mutations in the C-terminus abolish the ability of menin to bind DNA. Importantly, these MEN1 disease-related menin mutants also fail to repress cell proliferation as well as cell cycle progression at G2/M phase. Furthermore, detailed mutagenesis studies indicate that positively charged residues in two nuclear localization signals mediate direct DNA binding as well as repression of cell proliferation. Collectively, these results demonstrate, for the first time, a novel biochemical activity of menin, binding to DNA, and link its DNA binding to the regulation of cell proliferation.

Multiple endocrine neoplasia type I (MEN1) is an inherited tumor syndrome characterized by development of tumors in multiple endocrine organs. The gene mutated in MEN1 patients, Men1, encodes a nuclear protein, menin. Menin interacts with several transcription factors and inhibits their activities. However, it is unclear whether menin is essential for the repression of the expression of endogenous genes. Here, using menin-null cells, we show that menin is essential for repression of the endogenous IGFBP-2, a gene that can regulate cell proliferation. Additionally, complementation of menin-null cells with wild-type menin, but not with a MEN1 disease-related point mutant, restores the function of menin in repressing IGFBP-2. Consistent with this, the promoter of IGFBP-2 is repressed by wild-type menin, but not by a MEN1-related point mutant. Menin also alters the structure of the chromatin surrounding the promoter of the IGFBP-2 gene, as demonstrated by the deoxyribonuclease I hypersensitivity assay. Furthermore, nuclear localization signals in menin are crucial for repressing the expression of IGFBP-2. Together, these results suggest that menin regulates the expression of the endogenous IGFBP-2 gene at least in part through the promoter of IGFBP-2.

Hop is a small, divergent homeodomain protein that lacks certain conserved residues required for DNA binding. Hop gene expression initiates early in cardiogenesis and continues in cardiomyocytes throughout embryonic and postnatal development. Genetic and biochemical data indicate that Hop functions directly downstream of Nkx2-5. Inactivation of Hop in mice by homologous recombination results in a partially penetrant embryonic lethal phenotype with severe developmental cardiac defects involving the myocardium. Inhibition of Hop activity in zebrafish embryos likewise disrupts cardiac development and results in severely impaired cardiac function. Hop physically interacts with serum response factor (SRF) and inhibits activation of SRF-dependent transcription by inhibiting SRF binding to DNA. Hop encodes an unusual homeodomain protein that modulates SRF-dependent cardiac-specific gene expression and cardiac development.

Bromodomain inhibition comprises a promising therapeutic strategy in cancer, particularly for hematologic malignancies. To date, however, genomic biomarkers to direct clinical translation have been lacking. We conducted a cell-based screen of genetically-defined cancer cell lines using a prototypical inhibitor of BET bromodomains. Integration of genetic features with chemosensitivity data revealed a robust correlation between MYCN amplification and sensitivity to bromodomain inhibition. We characterized the mechanistic and translational significance of this finding in neuroblastoma, a childhood cancer with frequent amplification of MYCN. Genome-wide expression analysis demonstrated downregulation of the MYCN transcriptional program accompanied by suppression of MYCN transcription. Functionally, bromodomain-mediated inhibition of MYCN impaired growth and induced apoptosis in neuroblastoma. BRD4 knock-down phenocopied these effects, establishing BET bromodomains as transcriptional regulators of MYCN. BET inhibition conferred a significant survival advantage in three in vivo neuroblastoma models, providing a compelling rationale for developing BET bromodomain inhibitors in patients with neuroblastoma.

The mechanisms underlying genetic susceptibility at loci discovered by genome-wide association study (GWAS) approaches in human cancer remain largely undefined. In this study, we characterized the high-risk neuroblastoma association at the BRCA1-related locus, BARD1, showing that disease-associated variations correlate with increased expression of the oncogenically activated isoform, BARD1β. In neuroblastoma cells, silencing of BARD1β showed genotype-specific cytotoxic effects, including decreased substrate-adherence, anchorage-independence, and foci growth. In established murine fibroblasts, overexpression of BARD1β was sufficient for neoplastic transformation. BARD1β stabilized the Aurora family of kinases in neuroblastoma cells, suggesting both a mechanism for the observed effect and a potential therapeutic strategy. Together, our findings identify BARD1β as an oncogenic driver of high-risk neuroblastoma tumorigenesis, and more generally, they illustrate how robust GWAS signals offer genomic landmarks to identify molecular mechanisms involved in both tumor initiation and malignant progression. The interaction of BARD1β with the Aurora family of kinases lends strong support to the ongoing work to develop Aurora kinase inhibitors for clinically aggressive neuroblastoma. Cancer Res; 72(8); 2068–78. ©2012 AACR.

Neuroblastoma is a cancer of the sympathetic nervous system that accounts for approximately 10% of all pediatric oncology deaths. Here, we report a genome-wide association study of 2,817 neuroblastoma cases and 7,473 controls. We identified two new associations at 6q16, the first within HACE1 (rs4336470; combined P=2.7×10(-11); odds ratio 1.26, 95% confidence interval (CI) 1.18-1.35) and the second within LIN28B (rs17065417; combined P=1.2×10(-8); odds ratio 1.38, 95% CI 1.23-1.54). Expression of LIN28B and let-7 miRNA correlated with rs17065417 genotype in neuroblastoma cell lines, and we observed significant growth inhibition upon depletion of LIN28B, specifically in neuroblastoma cells that were homozygous for the risk allele. Low HACE1 and high LIN28B expression in diagnostic primary neuroblastomas were associated with worse overall survival (P=0.008 and 0.014, respectively). Taken together, these data show that common variants in HACE1 and LIN28B influence neuroblastoma susceptibility and indicate that both genes likely have a role in disease progression.

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.

A genome-wide association study (GWAS) has shown that single nucleotide variants within the LMO1 locus are associated with inherited susceptibility to neuroblastoma, a childhood cancer of the sympathetic nervous system. LMO1 encodes a transcriptional regulator previously linked to cancers. Acquired structural variation in the same locus is common in patients with neuroblastoma, suggesting that loci identified through GWAS approaches might also be prone to somatic alterations that influence tumour progression. Such studies could help to identify potential therapy targets and/or biomarkers of cancer aggressiveness. Here, single nucleotide variants within the LMO1 locus are shown to be associated with inherited susceptibility to neuroblastoma, a childhood cancer of the sympathetic nervous system. Acquired structural variation in the same locus was also frequently found in neuroblastoma patients, leading to the suggestion that loci identified through genome-wide association studies might be also prone to somatic alterations and therefore identify potential therapy targets and/or biomarkers of tumour aggressiveness. Neuroblastoma is a childhood cancer of the sympathetic nervous system that accounts for approximately 10% of all paediatric oncology deaths1,2. To identify genetic risk factors for neuroblastoma, we performed a genome-wide association study (GWAS) on 2,251 patients and 6,097 control subjects of European ancestry from four case series. Here we report a significant association within LIM domain only 1 (LMO1) at 11p15.4 (rs110419, combined P = 5.2 × 10−16, odds ratio of risk allele = 1.34 (95% confidence interval 1.25–1.44)). The signal was enriched in the subset of patients with the most aggressive form of the disease. LMO1 encodes a cysteine-rich transcriptional regulator, and its paralogues (LMO2, LMO3 and LMO4) have each been previously implicated in cancer. In parallel, we analysed genome-wide DNA copy number alterations in 701 primary tumours. We found that the LMO1 locus was aberrant in 12.4% through a duplication event, and that this event was associated with more advanced disease (P

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