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Supplementary Table 1. CB cis-sQTL. Supplementary Table 2. SCG cis-sQTL. Supplementary Table 3. trans-sQTL. Supplementary Table 4. Genes differentially expressed on Chr10 in 95% CI. Supplementary Table 5. Genes differentially expressed on ChrX in 95% CI. Supplementary Table 6. Cis-sQTL Co-localizing with SCNVs. Supplementary Table 7. Genotyping markers used in this study. Supplementary Table 8. Oligonucleotides used in this study.

Supplementary Figure S1. DDX26B and RBMX2 Influence Alternative Splicing. Supplementary Figure S2. Astn2 possesses a cis-sQTL. Supplementary Figure S3. SCNVs Identified by WGS. Supplementary Figure S4. Fubp1 RT-PCR From Parental Mouse Strains. Supplementary Figure S5. FUBP1 Expression Correlates with Survival in Two Neuroblastoma Datasets. Supplementary Figure S6. Total Set of Identified Unique Splicing Motifs. Supplementary Figure S7. Motifs with Matches to an RNA Binding database. Supplementary Figure S8. Recurrent Somatic Mutations Occur in Intronic Splicing Motifs in GBM.

Supplementary Figure Legends, Table Legends

Supplementary Figure S1. PA2G4 is a MYCN transactivation target gene. A, Quantification of protein expression using anti-PA2G4 and anti-MYCN antibodies against whole cell protein lysates from BE(2)-C and Kelly cells, following MYCN siRNA knockdown for 48 hours. B, mRNA expression of PA2G4 and MYCN in SH-EP MYCN3 overexpression cells treated with 1µg/ml doxycycline for 24-96 hr. C, The effect of doxycycline-induced MYCN overexpression in SHEP-TRE-MYCN cells on c-MYC and PA2G4 protein expression. D, Chromatin immunoprecipitation (ChIP) assay in Kelly cells using an anti-MYCN antibody, and real-time PCR analysis with primers identifying the MYCN DNA binding sites in the PA2G4 gene promoter (500bP upstream of transcription start site [TSS]) or Intron 1a & 1b regions of the PA2G4 gene, with and without MYCN siRNA knockdown. ChIP and real-time PCR analysis using primers against a region 1200bp upstream of TSS was used as a negative control for MYCN chromatin binding. ChIP and real-time PCR analysis using primers against the ornithine decarboxylase (ODC1) gene promoter region was used as a positive control for MYCN chromatin binding. E, Immunoblot analysis of PA2G4, MYCN and MYC protein levels in a panel of human MYCN amplified and non-amplified neuroblastoma, and normal fibroblast, cell lines using antibodies recognising PA2G4, MYCN and MYC.

Supplementary Figure S1. F, Confocal microscopy of SHEP Tet21N cells using anti-PA2G4 (red) and anti-MYCN (green) antibodies. G, Immunoblot analysis of cytoplasmic and nuclear fractions from BE(2)-C and Kelly human neuroblastoma cell lines with antibodies recognizing PA2G4 and MYCN, or GAPDH and topoisomerase as loading controls. H, Confocal microscopy of neuroblastoma (BE(2)-C and Kelly) cells using anti-PA2G4 (red) and anti-MYCN (green) antibodies. Alexafluor 555 anti-rabbit (to detect PA2G4) and Alexafluor 488 anti-mouse (to detect MYCN) were used as the secondary antibodies. I, Real-time PCR mRNA expression of MYCN and PA2G4 in ganglia from homozygote TH-MYCN+/+ mice, compared to wild type littermate control mice, obtained at different postnatal age (weeks).

MYCN is a major driver for the childhood cancer, neuroblastoma, however, there are no inhibitors of this target. Enhanced MYCN protein stability is a key component of MYCN oncogenesis and is maintained by multiple feedforward expression loops involving MYCN transactivation target genes. Here, we reveal the oncogenic role of a novel MYCN target and binding protein, proliferation-associated 2AG4 (PA2G4). Chromatin immunoprecipitation studies demonstrated that MYCN occupies the PA2G4 gene promoter, stimulating transcription. Direct binding of PA2G4 to MYCN protein blocked proteolysis of MYCN and enhanced colony formation in a MYCN-dependent manner. Using molecular modeling, surface plasmon resonance, and mutagenesis studies, we mapped the MYCN–PA2G4 interaction site to a 14 amino acid MYCN sequence and a surface crevice of PA2G4. Competitive chemical inhibition of the MYCN–PA2G4 protein–protein interface had potent inhibitory effects on neuroblastoma tumorigenesis in vivo. Treated tumors showed reduced levels of both MYCN and PA2G4. Our findings demonstrate a critical role for PA2G4 as a cofactor in MYCN-driven neuroblastoma and highlight competitive inhibition of the PA2G4-MYCN protein binding as a novel therapeutic strategy in the disease.Significance:Competitive chemical inhibition of the PA2G4–MYCN protein interface provides a basis for drug design of small molecules targeting MYC and MYCN-binding partners in malignancies driven by MYC family oncoproteins.

Supplementary Figure S2. PA2G4 increases MYCN protein stability. A and B, Representative immunoblots from cycloheximide (CHX) chase assays measuring the half-life of MYCN protein after PA2G4 knockdown (A) or overexpression (B) in BE(2)-C and Kelly cells for 48 hours. Cells were then treated with 100 µg/µl CHX for up to 60 minutes followed by immunoblotting. C, Co-IP of total protein from BE(2)-C cells using IgG, anti-MYCN, anti-PA2G4 antibodies, followed by immunoblotting with anti-MYCN, anti-PA2G4, anti-AURKA, anti-Fbxw7 or anti-vinculin antibodies.

Supplementary Information

Supplementary Figure S6. PA2G4 increases neuroblastoma tumorigenicity. A, Representative images of athymic nude mice inoculated with neuroblastoma (SH-EP) cells stably transfected with either EV control or a PA2G4 expression vector at 10 weeks post-injection. B, Images of tumor formation after mice were culled 12 weeks post-injection. C, Real-time PCR analysis of PA2G4 mRNA expression level in tumors from mice injected with either SH-EP cells overexpressing PA2G4, or SH-EP EV control cells. β2-microglobulin was used as a reference gene for total RNA loading. D, Protein was extracted from SH-EP tumor xenografts overexpressing PA2G4 and control vectors, then analysed for PA2G4-Flag and MYCN expression by immunoblotting using anti-MYCN and anti-PA2G4 antibodies, using a Vinculin loading control. E, Immunoblots of three tumor samples from each siRNA-treated cohort showing the levels of PA2G4 and MYCN protein expression, using a Vinculin loading control. F, Real-time PCR analysis of PA2G4 and MYCN mRNA expression in tumor samples taken from tumour-bearing mice xenografted with BE(2)-C neuroblastoma cells, which had been treated with either nano-particle encapsulated siRNA control, PA2G4 siRNA or PA2G4-p48 siRNA. Data are shown as means and SD derived from 6 mice per group, P-values were calculated by t-test.

Supplementary Figure S5 F-H F), at 72 hours post-transfection of EV or a PA2G4 expression vector. G, Cell viability measured by the Alamar Blue assay and immunoblot analyses using an antibody identifying MYC-tagged PA2G4-p42 protein expression in Kelly and SH-SY5Y cells transfected with EV or the MYC-tagged PA2G4-p42 expression vector at 48 and 72 hours post-transfection. Vinculin was used as loading control. H, Colony formation in vitro by Kelly and SH-SY5Y cells following transfection with either EV or MYC-tagged PA2G4-p42.

Supplementary Figure S3 F-G F, Left panel: Histopathologic and immunohistochemical analyses of MYCN;GFP tumors treated with vehicle (left) or WS6 (right). Left Panel, Top to Bottom: Neuroblastoma tumour sections immunohistochemically stained for Haematoxylin & Eosin (H&E), Proliferating Cell Nuclear Antigen (pCNA), Neural Hu protein C (Hu-C), MYCN, PA2G4 and Tyrosine Hydroxylase. Scale bar, 50 μm. Right Panel: Histograms illustrating the staining intensity of cells of vehicle (left) or WS6 treated neuroblastoma tumours expressing either MYCN, PA2G4 or Tyrosine Hydroxylase as measured by Image J software. Sample means (horizontal bars) were compared by students t-test (two-tailed). *** represents p-value

Supplementary Figure S6 G-K G, Overall patient survival using Kaplan-Meier survival probability plots from the Cologne data set (http://r2.amc.nl) for PA2G4 mRNA expression subdivided around the median PA2G4 expression level among 477 neuroblastoma patients. H, Kaplan-Meier plot for event-free survival of 649 neuroblastoma patients from the kocak dataset (R2 microarray analysis and visualization platform, http://r2.amc.nl) I, Real-time PCR mRNA expression of PA2G4 among 40 neuroblastoma patient tumors treated at Sydney Children's Hospital, subdivided by MYCN amplification status. J, Multivariate event-free survival analysis using cox regression modelling. The p-values were obtained from the cox-regression analysis. K, Incidence of PA2G4 amplification among a range of different human cancer types within The Cancer Genome Atlas (TCGA). Results were generated using cBioportal for Cancer Genomics (https://cancergenome.nih.gov/). NEPC, neuroendocrine prostate cancer; CS, carcinosarcoma.

Amplification of MYCN is a poor prognostic feature in neuroblastoma (NBL) indicating aggressive disease. We and others have shown BET bromodomain inhibitors (BETi) target MYCN indirectly by downregulating its transcription. Here we sought to identify agents that synergize with BETi and to identify biomarkers of resistance. We previously performed a viability screen of ∼1,900 oncology-focused compounds combined with BET bromodomain inhibitors against MYCN-amplified NBL cell lines. Reanalysis of our screening results prominently identified inhibitors of aurora kinase A (AURKAi) to be highly synergistic with BETi. We confirmed the anti-proliferative effects of several BETi+AURKAi combinations in MYCN-amplified NBL cell lines. Compared to single agents, these combinations cooperated to decrease levels of N-myc. We treated both TP53-wild type and mutant, MYCN-amplified cell lines with the BETi JQ1 and the AURKAi Alisertib. The combination had improved efficacy in the TP53-WT context, notably driving apoptosis in both genetic backgrounds. JQ1+Alisertib combination treatment of a MYCN-amplified, TP53-null or TP53-restored genetically engineered mouse model of NBL prolonged survival better than either single agent. This was most profound with TP53 restored, with marked tumor shrinkage and apoptosis induction in response to combination JQ1+Alisertib. BETi+AURKAi in MYCN-amplified NBL, particularly in the context of functional TP53, provided anti-tumor benefits in preclinical models. This combination should be studied more closely in a pediatric clinical trial.

Purpose Radiation dose calculated on tumors for radiopharmaceutical therapy varies significantly from tumor to tumor and from patient to patient. Accurate estimation of radiation dose requires multiple time point measurements using radionuclide imaging modalities such as SPECT or PET. In this report, we show our technical development of reducing the number of scans needed for reasonable estimation of tumor and normal organ dose in our pretherapy imaging and dosimetry platform of 124 I-metaiodobenzylguanidine (MIBG) positron emission tomography/computed tomography (PET/CT) for 131 I-MIBG therapy of neuroblastoma. Methods We analyzed the simplest kinetic data, areas of two-time point data for five patients with neuroblastoma who underwent 3 or 4 times of 124 I-MIBG PET/CT scan prior to 131 I-MIBG therapy. The data for which we derived areas were percent of injected activity (%IA) and standardized uptake value of tumors. These areas were correlated with time-integrated activity coefficients (TIACs) from full data (3 or 4 time points). TIACs are direct correlates with radiation dose as long as the volume and the radionuclide are known. Results The areas of %IAs between data obtained from all the two-time points with time points 1 and 2 (day 0 and day 1), time points 2 and 3 (day 1 and day 2), and time points 1 and 3 (day 0 and day 2) showed reasonable correlation (Pearson's correlation coefficient |r| > 0.5) with not only tumor and organ TIACs but also tumor and organ absorbed doses. The tumor and organ doses calculated using %IA areas of time point 1 and time point 2 were our best fits at about 20% individual percent difference compared to doses calculated using 3 or 4 time points. Conclusions We could achieve reasonable accuracy of estimating tumor doses for subsequent radiopharmaceutical therapy using only the two-time point imaging sessions. Images obtained from these time points (within the 48-h after administration of radiopharmaceutical) were also viewed as useful for diagnostic reading. Although our analysis was specific to 124 I-MIBG PET/CT pretherapy imaging data for 131 I-MIBG therapy of neuroblastoma and the number of imaging datasets was not large, this feasible methodology would generally be applicable to other imaging and therapeutic radionuclides with an appropriate data analysis similar to our analysis to other imaging and therapeutic radiopharmaceuticals.

Mutant RAS is common in pancreatic carcinoma (PDAC), non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) and exists predominantly in the GTP-bound (RAS(ON)) state, leading to excessive downstream oncogenic signaling. KRASG12C(OFF) inhibitors have provided clinical proof of concept for targeting mutant KRAS. Preclinical data suggests inhibition of RAS(ON) may be a superior therapeutic strategy. In addition, KRASG12 mutations such as KRASG12D and KRASG12V remain unserved.RMC-6236 is a first-in-class, potent, oral tri-complex RASMULTI(ON) small molecule inhibitor designed to treat cancers driven by a variety of RAS mutations. RMC-6236 non-covalently binds to an abundant intracellular chaperone protein, cyclophilin A (CypA), resulting in a binary complex that engages RAS(ON) to form a high-affinity, RAS-selective tri-complex that sterically inhibits RAS binding to effectors. Exposure to RMC-6236 suppressed ERK phosphorylation and cell growth, and induced apoptosis in multiple human RAS-addicted cancer cell lines in vitro.RMC-6236 induced dose-dependent, deep, and durable suppression of RAS pathway activation up to 48 hours in preclinical xenograft models in vivo. Prolonged exposure in tumors was observed relative to blood and various healthy tissues, likely mediated by high affinity binding of RMC-6236 to tumor CypA.RMC-6236 at tolerable doses induced profound and durable tumor regressions in multiple cell line-derived (CDX) and patient-derived (PDX) RASMUT xenograft models, including NSCLC, CRC and PDAC. Anti-tumor activity was particularly notable in KRAS position 12 (G12X) mutant tumors, particularly KRASG12D, KRASG12V, and KRASG12R, with significant tumor regressions observed. Tumor growth inhibition was durable even in tumors that did not regress. Intermittent scheduling of RMC-6236 was active and permitted a higher dose intensity than daily dosing.RMC-6236 promoted anti-tumor immunity in vivo and was additive with anti-PD1 antibodies, driving durable complete responses and immunologic memory in a KRAS mutant CRC model. Furthermore, RMC-6236 treatment reversed oncogenic RAS-driven immune evasion mechanisms in a checkpoint blockade refractory KRAS mutant model, significantly transforming the tumor microenvironment in favor of anti-tumor immunity.These preclinical results support the inclusion of NSCLC, PDAC, and CRC patients in our planned clinical trial of RMC-6236 in patients with KRASG12X advanced solid tumors. Citation Format: Elena S. Koltun, Meghan A. Rice, W. Clay Gustafson, David Wilds, Jingjing Jiang, Bianca J. Lee, Zhengping Wang, Stephanie Chang, Mike Flagella, Yunming Mu, Nuntana Dinglasan, Nicole Nasholm, James W. Evans, Yingyun Wang, Kyle Seamon, Yang Liu, Cristina Blaj, John Knox, Rebecca Freilich, Elsa Quintana, Jim Cregg, Alun Bermingham, Adrian L. Gill, Jacqueline Am Smith, Mallika Singh. Direct targeting of KRASG12X mutant cancers with RMC-6236, a first-in-class, RAS-selective, orally bioavailable, tri-complex RASMULTI(ON) inhibitor [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 3597.

3098 Background: RMC-5552 is a potent bi-steric mTORC1-selective inhibitor that activates the downstream tumor suppressor 4EBP1, thereby inhibiting initiation of protein translation. This novel therapeutic moiety addresses a key limitation of rapalogs, which do not effectively inhibit phosphorylation of 4EBP1. RMC-5552 has previously demonstrated significant anti-tumor activity in preclinical models of human cancers with mTOR pathway activation. Additionally, mTOR signaling plays a key role in therapeutic response and resistance in RAS-addicted cancers, which represent a significant unmet medical need. Methods: We examined the combination of bi-steric mTORC1 inhibitors (RMC-5552 and the research tool compound RMC-6272) with direct inhibitors of active RAS (RAS(ON) inhibitors) in mutant KRAS-driven models. To enable the clinical testing of RMC-5552 as a companion inhibitor for RAS(ON) inhibitors, a Phase 1/1b dose-escalation trial of RMC-5552 monotherapy is currently testing a once-a-week IV schedule. Results: RMC-5552 and RMC-6272 demonstrated marked combinatorial anti-tumor activity with RAS(ON) inhibitors across a series of preclinical models of KRAS mutated non-small cell lung cancer. The combination enhanced tumor apoptosis and resulted in durable tumor regressions as compared to tumor growth inhibition resulting from single agents alone. As of 13 January 2022, a total of 14 patients with solid tumors have been evaluated in an ongoing Phase 1/1b trial over 5 dose levels ranging from 1.6 to 12 mg IV weekly. Median age was 62 years and the majority received ≥3 prior therapies. The most common (> 25%) drug-related adverse events were mucositis/stomatitis (43%) and decreased appetite (29%). The most common grade 3 drug-related adverse events were mucositis/stomatitis observed in 3 patients in dose levels ≥ 10 mg (21%) and were dose-limiting. The dose of 6 mg IV weekly was well tolerated. Plasma exposures of RMC-5552 were dose-proportionate at lower dose levels up to 6 mg but increased above dose proportionality with higher dose levels. Plasma exposures at 6 mg and above were consistent with those resulting in inhibition of tumor p4EBP1 in preclinical models. Of 5 patients evaluable for efficacy at doses of 6 mg and higher, one confirmed PR was observed in a patient with head and neck cancer with a pathogenic mutation in PTEN (ORR 20%) and 3 patients had a best response of SD. Dose-optimization is ongoing. Conclusions: RMC-5552 is clinically active in tumors with mTORC1 signaling activation at a tolerable dose and schedule and has the potential to be a companion inhibitor of choice for RAS(ON) inhibitors in RAS-addicted tumors. Clinical trial information: NCT04774952.

Effective treatment of pediatric solid tumors has been hampered by the predominance of currently “undruggable” driver transcription factors. Improving outcomes while decreasing the toxicity of treatment necessitates the development of novel agents that can directly inhibit or degrade these elusive targets. MYCN in pediatric neural-derived tumors, including neuroblastoma and medulloblastoma, is a paradigmatic example of this problem. Attempts to directly and specifically target MYCN have failed due to its similarity to MYC, the unstructured nature of MYC family proteins in their monomeric form, the lack of an understanding of MYCN-interacting proteins and ability to test their relevance in vivo, the inability to obtain structural information on MYCN protein complexes, and the challenges of using traditional small molecules to inhibit protein–protein or protein–DNA interactions. However, there is now promise for directly targeting MYCN based on scientific and technological advances on all of these fronts. Here, we discuss prior challenges and the reasons for renewed optimism in directly targeting this “undruggable” transcription factor, which we hope will lead to improved outcomes for patients with pediatric cancer and create a framework for targeting driver oncoproteins regulating gene transcription.

The metaiodobenzylguanidine (MIBG) scan is one of the most sensitive noninvasive lesion detection modalities for neuroblastoma. Unlike (123)I-MIBG, (124)I-MIBG allows high-resolution PET. We evaluated (124)I-MIBG PET/CT for its diagnostic performance as directly compared with paired (123)I-MIBG scans. Methods: Before (131)I-MIBG therapy, standard (123)I-MIBG imaging (5.2 MBq/kg) was performed on 7 patients, including whole-body (anterior–posterior) planar imaging, focused-field-of-view SPECT/CT, and whole-body (124)I-MIBG PET/CT (1.05 MBq/kg). After therapy, 2 of 7 patients also completed (124)I-MIBG PET/CT as well as paired (123)I-MIBG planar imaging and SPECT/CT. One patient underwent (124)I-MIBG PET/CT only after therapy. We evaluated all 8 patients who showed at least 1 (123)I-MIBG–positive lesion with a total of 10 scans. In 8 pairs, (123)I-MIBG and (124)I-MIBG were performed within 1 mo of each other. The locations of identified lesions, the number of total lesions, and the curie scores were recorded for the (123)I-MIBG and (124)I-MIBG scans. Finally, for 5 patients who completed at least 3 PET/CT scans after administration of (124)I-MIBG, we estimated the effective dose of (124)I-MIBG. Results: (123)I-MIBG whole-body planar scans, focused-field-of-view SPECT/CT scans, and whole-body (124)I-MIBG PET scans found 25, 32, and 87 total lesions, respectively. There was a statistically significant difference in lesion detection for (124)I-MIBG PET/CT versus (123)I-MIBG planar imaging (P < 0.0001) and (123)I-MIBG SPECT/CT (P < 0.0001). The curie scores were also higher for (124)I-MIBG PET/CT than for (123)I-MIBG planar imaging and SPECT/CT in 6 of 10 patients. (124)I-MIBG PET/CT demonstrated better detection of lesions throughout the body, including the chest, spine, head and neck, and extremities. The effective dose estimated for patient-specific (124)I-MIBG was approximately 10 times that of (123)I-MIBG; however, given that we administered a very low activity of (124)I-MIBG (1.05 MBq/kg), the effective dose was only approximately twice that of (123)I-MIBG despite the large difference in half-lives (100 vs. 13.2 h). Conclusion: The first-in-humans use of low-dose (124)I-MIBG PET for monitoring disease burden demonstrated tumor detection capability superior to that of (123)I-MIBG planar imaging and SPECT/CT.

591 Background: RAS proteins (such as KRAS, NRAS, HRAS) are small GTPases that drive cell proliferation and survival when bound to GTP. Mutant RAS proteins exist predominantly in the GTP-bound (RAS(ON)) state, leading to excessive downstream signaling via interaction with effectors such as RAF kinases. Oncogenic KRAS is required for the initiation, progression, and maintenance of pancreatic ductal adenocarcinoma (PDAC) (Hezel et al, 2006, Ying et al 2012). Although extinction of KRAS expression as well as pharmacological inhibition of RAS effectors clearly abrogate the growth of human PDAC models, clinical trials of drugs targeting key components of the RAS pathway have remained largely unsuccessful. Several factors contribute to these failures including redundancy in signaling surrogates downstream of KRAS and/or tumor complexity driven by co-occurring genomic alterations and intra-tumoral heterogeneity. Methods: RMC-6236 is a small molecule that binds to an intracellular chaperone protein, Cyclophilin A (CypA), resulting in an inhibitory binary complex that binds active, GTP-bound RAS to form a tri-complex and suppresses RAS signaling by disrupting interactions with effectors such as RAF kinases. Results: Here, we demonstrate that single agent RMC-6236, a first-in-class, orally bioavailable, RAS-selective tri-complex inhibitor of multiple RAS mutations and wild-type RAS (RASMULTI inhibitor) is highly efficacious in preclinical models of KRAS mutant PDAC (with marked activity in RAS-mutant colorectal cancer models described in Koltun et al, AACR 2021). RMC-6236 suppresses phosphorylation of ERK kinases, downstream effectors of RAS involved in cell proliferation, and induces growth suppression and apoptosis in multiple human cancer cell lines in vitro. Oral administration of RMC-6236 produces deep, durable, and dose-dependent suppression of tumor RAS pathway activation in vivo. An extended duration of tumor pharmacodynamic activity, relative to plasma exposure, is observed that likely reflects retention of RMC-6236 in tumor tissue due to high affinity binding to CypA. Daily dosing of RMC-6236 drives profound and durable tumor regressions in multiple cell line derived (CDX) and patient derived (PDX) xenograft models of KRAS mutant PDAC at doses that are well-tolerated. Conclusions: These results indicate that direct targeting of mutant and possibly wild-type RAS in PDAC, without inhibition of signaling nodes outside the canonical RAS pathway, has the potential to translate into clinical benefit for patients with pancreatic cancer harboring mutations in KRAS that may be superior to therapies aimed at upstream or downstream signaling elements within the RAS pathway. Our preclinical data strongly support the inclusion of PDAC patients in our planned clinical trial of RMC-6236 in patients with advanced solid tumors.

Progenitor heterogeneity and identities underlying tumor initiation and relapse in medulloblastomas remain elusive. Utilizing single-cell transcriptomic analysis, we demonstrated a developmental hierarchy of progenitor pools in Sonic Hedgehog (SHH) medulloblastomas, and identified OLIG2-expressing glial progenitors as transit-amplifying cells at the tumorigenic onset. Although OLIG2+ progenitors become quiescent stem-like cells in full-blown tumors, they are highly enriched in therapy-resistant and recurrent medulloblastomas. Depletion of mitotic Olig2+ progenitors or Olig2 ablation impeded tumor initiation. Genomic profiling revealed that OLIG2 modulates chromatin landscapes and activates oncogenic networks including HIPPO-YAP/TAZ and AURORA-A/MYCN pathways. Co-targeting these oncogenic pathways induced tumor growth arrest. Together, our results indicate that glial lineage-associated OLIG2+ progenitors are tumor-initiating cells during medulloblastoma tumorigenesis and relapse, suggesting OLIG2-driven oncogenic networks as potential therapeutic targets.

MYCN is a major driver for the childhood cancer, neuroblastoma, however, there are no inhibitors of this target. Enhanced MYCN protein stability is a key component of MYCN oncogenesis and is maintained by multiple feedforward expression loops involving MYCN transactivation target genes. Here, we reveal the oncogenic role of a novel MYCN target and binding protein, proliferation-associated 2AG4 (PA2G4). Chromatin immunoprecipitation studies demonstrated that MYCN occupies the PA2G4 gene promoter, stimulating transcription. Direct binding of PA2G4 to MYCN protein blocked proteolysis of MYCN and enhanced colony formation in a MYCN-dependent manner. Using molecular modeling, surface plasmon resonance, and mutagenesis studies, we mapped the MYCN–PA2G4 interaction site to a 14 amino acid MYCN sequence and a surface crevice of PA2G4. Competitive chemical inhibition of the MYCN–PA2G4 protein–protein interface had potent inhibitory effects on neuroblastoma tumorigenesis in vivo. Treated tumors showed reduced levels of both MYCN and PA2G4. Our findings demonstrate a critical role for PA2G4 as a cofactor in MYCN-driven neuroblastoma and highlight competitive inhibition of the PA2G4-MYCN protein binding as a novel therapeutic strategy in the disease. Significance: Competitive chemical inhibition of the PA2G4–MYCN protein interface provides a basis for drug design of small molecules targeting MYC and MYCN-binding partners in malignancies driven by MYC family oncoproteins.

// Kieuhoa T. Vo 1 , Erin E. Karski 1 , Nicole M. Nasholm 1 , Shelly Allen 1 , Fabienne Hollinger 1 , W. Clay Gustafson 1 , Janel R. Long-Boyle 2 , Stephen Shiboski 3 , Katherine K. Matthay 1 and Steven G. DuBois 1 1 Department of Pediatrics, UCSF Benioff Children’s Hospital, University of California, San Francisco School of Medicine, San Francisco, CA, USA 2 Department of Clinical Pharmacy, UCSF Benioff Children’s Hospital, University of California, San Francisco School of Medicine, San Francisco, CA, USA 3 Department of Epidemiology and Biostatistics, UCSF Benioff Children’s Hospital, University of California, San Francisco School of Medicine, San Francisco, CA, USA Correspondence to: Kieuhoa T. Vo, email: // Keywords : phase 1, mTOR, sirolimus, topotecan, cyclophosphamide Received : August 23, 2016 Accepted : October 13, 2016 Published : October 25, 2016 Abstract Purpose: To determine the maximum tolerated dose (MTD), toxicities, and pharmacodynamics effects of sirolimus combined with oral metronomic topotecan and cyclophosphamide in a pediatric population. Materials and Methods: Patients who were 1 to 30 years of age with relapsed/refractory solid tumors (including CNS) were eligible. Patients received daily oral sirolimus and cyclophosphamide (25-50 mg/m 2 /dose) on days 1-21 and oral topotecan (0.8 mg/m 2 /dose) on days 1-14 in 28-day cycles. Sirolimus steady-state plasma trough concentrations of 3-7.9 ng/mL and 8-12.0 ng/mL were evaluated, with dose escalation based on a 3+3 phase 1 design. Biomarkers of angiogenesis were also evaluated. Results: Twenty-one patients were treated (median age 18 years; range 9-30). Dose-limiting toxicities included myelosuppression, ALT elevation, stomatitis, and hypertriglyceridemia. The MTD was sirolimus with trough goal of 8-12.0 ng/mL; cyclophosphamide 25 mg/m 2 /dose; and topotecan 0.8 mg/m 2 /dose. No objective responses were observed. Four patients had prolonged stable disease > 4 cycles (range 4-12). Correlative biomarker analyses demonstrated reductions in thrombospondin-1 ( p =0.043) and soluble vascular endothelial growth factor receptor-2 plasma concentrations at 21 days compared to baseline. Conclusions: The combination of oral sirolimus, topotecan, and cyclophosphamide was well tolerated and biomarker studies demonstrated modulation of angiogenic pathways with this regimen.

MYCN amplification and overexpression are common in neuroendocrine prostate cancer (NEPC). However, the impact of aberrant N-Myc expression in prostate tumorigenesis and the cellular origin of NEPC have not been established. We define N-Myc and activated AKT1 as oncogenic components sufficient to transform human prostate epithelial cells to prostate adenocarcinoma and NEPC with phenotypic and molecular features of aggressive, late-stage human disease. We directly show that prostate adenocarcinoma and NEPC can arise from a common epithelial clone. Further, N-Myc is required for tumor maintenance, and destabilization of N-Myc through Aurora A kinase inhibition reduces tumor burden. Our findings establish N-Myc as a driver of NEPC and a target for therapeutic intervention.

INTRODUCTION: Oncogenic, driver BRAF-V600E mutations are frequently found in pediatric gliomas. While small molecule inhibitors of BRAF and/or MEK kinases are showing early clinical efficacy against these tumors, no experimental strategies thus far have focused on the degradation of the mutant BRAF protein itself. Members of our team have recently discovered that in contrast to wildtype BRAF protein, BRAF-V600E protein binds to aurora kinase A. We therefore hypothesized that CD532, a novel small molecule allosteric aurora kinase A inhibitor, would preferentially degrade BRAF-V600E over wildtype BRAF, and lead to decreased cell growth in BRAF-V600E gliomas. METHODS: We used an isogenic system of 3T3 fibroblasts transduced with empty vector, wildtype BRAF, KIAA1549:BRAF fusion, and BRAF-V600E, as well as established glioma cell lines AM38 (BRAF-V600E homozygous), DBTRG (BRAF-V600E heterozygous), U87 (BRAF wildtype), LN229 (BRAF wildtype with NF1 deficiency) and immortalized normal human astrocytes (NHA, BRAF wildtype). We performed cell cycle and viability assays, as well as immunoblot analyses for wildtype BRAF and BRAF-V600E. Cells were treated with CD532 at doses of 0.5 and 1 uM. RESULTS: The OncoPPi network mapping revealed that aurora kinase A binds to only BRAF-V600E but not wildtype BRAF protein. BRAF-V600E mutant cell lines showed a dramatic decrease in BRAF-V600E expression level after CD532 treatment. This was accompanied by a significant decrease in growth rate. Cells containing only wildtype BRAF or KIAA1549:BRAF did not show any changes in BRAF expression and had less inhibited growth after treatment with CD532. CONCLUSIONS: Degradation of BRAF-V600E mutant protein through inhibition of aurora kinase A shows preclinical efficacy against BRAF –V600e gliomas.

Combining anti-GD2 ganglioside monoclonal antibody with immune-activating cytokines improves survival in high-risk neuroblastoma, a breakthrough for immunotherapy in this disease. However, approaches such as immune checkpoint blockade, anti-GD2 CAR-T cells, and tumor vaccines have thus far shown limited success. While a better understanding of the tumor microenvironment (TME) promises to improve the success of immunotherapy strategies, few immunocompetent models exist for high-risk neuroblastoma. We used a 30-parameter mouse-specific CyTOF mass cytometetry panel to characterize the TME of neuroblastoma arising in TH-MYCN transgenic mice. The TME was dominated by tumor-associated macrophages (TAMs) and devoid of T, B or NK cells; recapitulating the TME of human high-risk neuroblastoma. The TH-MYCN model is highly penetrant only in the 129/SvJ strain however, a challenge to leveraging immunological tools available mainly in the C57/BL6 background. We therefore created a non-germline GEM model in which primary trunk neural crest cells isolated from C57BL6/J embryos were modified genetically by transducing MYCN (MYCN-nGEMM). Orthotopic transplantation in renal capsules resulted in neuroblastoma with 50% penetrance. Co-expression of ALK(F1174L)with MYCN resulted in 80% penetrance and decreased latency. Cell lines could be derived robustly from these tumors and serially transplanted with 100% penetrance. CyTOF analysis of MYCN-nGEMM tumors also revealed a highly immunosuppressive TME similar to TH-MYCN tumors, with 80% of immune cells being TAMs, and only 5% T-cells. MYCN-nGEMM tumors were then treated with various immunotherapies, including anti-GD2 antibody, checkpoint inhibitors, and a combination of both, and changes in the TME were analyzed by CyTOF. In summary, we have characterized an immune-intact model for MYCN-driven neuroblastoma that can leverage modern methods to rapidly modify the murine genome, enabling customization of this mouse model for immunological research. We propose this model to understand the immunosuppressive mechanisms of MYCN-driven neuroblastoma tumors, and evaluate the potential of combining MYCN-directed therapies with immunotherapy.

Pharmacologically difficult targets, such as MYC transcription factors, represent a major challenge in cancer therapy. For the childhood cancer neuroblastoma, amplification of the oncogene MYCN is associated with high-risk disease and poor prognosis. Here, we deployed genome-scale CRISPR-Cas9 screening of MYCN-amplified neuroblastoma and found a preferential dependency on genes encoding the polycomb repressive complex 2 (PRC2) components EZH2, EED, and SUZ12. Genetic and pharmacological suppression of EZH2 inhibited neuroblastoma growth in vitro and in vivo. Moreover, compared with neuroblastomas without MYCN amplification, MYCN-amplified neuroblastomas expressed higher levels of EZH2. ChIP analysis showed that MYCN binds at the EZH2 promoter, thereby directly driving expression. Transcriptomic and epigenetic analysis, as well as genetic rescue experiments, revealed that EZH2 represses neuronal differentiation in neuroblastoma in a PRC2-dependent manner. Moreover, MYCN-amplified and high-risk primary tumors from patients with neuroblastoma exhibited strong repression of EZH2-regulated genes. Additionally, overexpression of IGFBP3, a direct EZH2 target, suppressed neuroblastoma growth in vitro and in vivo. We further observed strong synergy between histone deacetylase inhibitors and EZH2 inhibitors. Together, these observations demonstrate that MYCN upregulates EZH2, leading to inactivation of a tumor suppressor program in neuroblastoma, and support testing EZH2 inhibitors in patients with MYCN-amplified neuroblastoma.

PurposeVesicular monoamine transporters 1 and 2 (VMAT1 and VMAT2) are thought to mediate MIBG uptake in adult neuroendocrine tumors. In neuroblastoma, the norepinephrine transporter (NET) has been investigated as the principal MIBG uptake protein, though some tumors without NET expression concentrate MIBG. We investigated VMAT expression in neuroblastoma and correlated expression with MIBG uptake and clinical features.MethodsWe evaluated VMAT1 and VMAT2 expression by immunohistochemistry (IHC) in neuroblastoma tumors from 76 patients with high-risk metastatic disease treated in a uniform cooperative group trial (COG A3973). All patients had baseline MIBG diagnostic scans centrally reviewed. IHC results were scored as the product of intensity grading (0 - 3+) and percent of tumor cells expressing the protein of interest. The association between VMAT1 and VMAT2 scores and clinical and biological features was tested using Wilcoxon rank-sum tests.ResultsPatient characteristics were typical of high-risk neuroblastoma, though the cohort was intentionally enriched in patients with MIBG-nonavid tumors (n = 20). VMAT1 and VMAT2 were expressed in 62% and 75% of neuroblastoma tumors, respectively. VMAT1 and VMAT2 scores were both significantly lower in MYCN amplified tumors and in tumors with high mitotic karyorrhectic index. MIBG-avid tumors had significantly higher VMAT2 scores than MIBG-nonavid tumors (median 216 vs. 45; p = 0.04). VMAT1 expression did not correlate with MIBG avidity.ConclusionVMAT1 and VMAT2 are expressed in the majority of neuroblastomas. Expression correlates with other biological features. The expression level of VMAT2 but not that of VMAT1 correlates with avidity for MIBG.

The MYCN oncoprotein has remained an elusive target for decades. We recently reported a new class of kinase inhibitors designed to disrupt the conformation of Aurora kinase A enough to block its kinase-independent interaction with MYCN, resulting in potent degradation of MYCN. These studies provide proof-of-principle for a new method of targeting enzyme activity-independent functions of kinases and other enzymes.

Chronic myelogenous leukemia (CML) results from a t(9,22) translocation, producing the p210(BCR-ABL) oncoprotein, a tyrosine kinase that causes transformation and chemotherapy resistance. To further understand mechanisms mediating chemotherapy resistance, we identified 556 differentially regulated genes in HL-60 cells stably expressing p210(BCR-ABL) versus those expressing an empty vector using cDNA macro- and oligonucleotide microarrays. These BCR-ABL-regulated gene products play diverse roles in cellular function including apoptosis, cell cycle regulation, intracellular signaling, transcription, and cellular adhesion. In particular, we identified up-regulation of the inducible form of heat shock protein 70 (Hsp70), and further explored the mechanism for its up-regulation. In HL-60/BCR-ABL and K562 cells (expressing p210(BCR-ABL)), abundant cytoplasmic Hsp70 expression was detected by immunoblot analysis. Moreover, cells isolated from bone marrow aspirates of patients in different stages of CML (chronic, aggressive, and blast crisis) express Hsp70. Expression of p210(BCR-ABL) in BCR-ABL negative cells induced transcription of the proximal Hsp70 promoter. Mutational analysis mapped the major p210(BCR-ABL) responsive element to a high affinity 5'(A/T)GATA(A/G)-3' "GATA" response element (GATA-RE) that binds GATA-1 in CML cells. The GATA-RE was sufficient to confer p210(BCR-ABL)- and p185(BCR-ABL)-mediated trans-activation to an inert promoter. Short interfering RNA mediated "knockdown" of Hsp70 expression in K562 cells induced marked sensitivity to paclitaxel-induced apoptosis. Together these findings indicate that BCR-ABL confers chemotherapeutic resistance through intracellular signaling to the GATA-RE element found in the promoter region of the anti-apoptotic Hsp70 protein. We suggest that down-regulation of the GATA-Hsp70 pathway may be useful in the treatment of chemotherapy-resistant CML.

Regulation of mRNA splicing, a critical and tightly regulated cellular function, underlies the majority of proteomic diversity and is frequently disrupted in disease. Using an integrative genomics approach, we combined both genomic data and exon-level transcriptome data in two somatic tissues (cerebella and peripheral ganglia) from a transgenic mouse model of neuroblastoma, a tumor that arises from the peripheral neural crest. Here, we describe splicing quantitative trait loci associated with differential splicing across the genome that we use to identify genes with previously unknown functions within the splicing pathway and to define de novo intronic splicing motifs that influence splicing from hundreds of bases away. Our results show that these splicing motifs represent sites for functional recurrent mutations and highlight novel candidate genes in human cancers, including childhood neuroblastoma. Significance: Somatic mutations with predictable downstream effects are largely relegated to coding regions, which comprise less than 2% of the human genome. Using an unbiased in vivo analysis of a mouse model of neuroblastoma, we have identified intronic splicing motifs that translate into sites for recurrent somatic mutations in human cancers. Cancer Discov; 5(4); 380–95. ©2015 AACR. This article is highlighted in the In This Issue feature, p. 333

Back with a Vengeance After surgery, gliomas (a type of brain tumor) recur in nearly all patients and often in a more aggressive form. Johnson et al. (p. 189 , published online 12 December 2013) used exome sequencing to explore whether recurrent tumors harbor different mutations than the primary tumors and whether the mutational profile in the recurrences is influenced by postsurgical treatment of patients with temozolomide (TMZ), a chemotherapeutic drug known to damage DNA. In more than 40% of cases, at least half of the mutations in the initial glioma were undetected at recurrence. The recurrent tumors in many of the TMZ-treated patients bore the signature of TMZ-induced mutagenesis and appeared to follow an evolutionary path to high-grade glioma distinct from that in untreated patients.

Protein kinase C betaII (PKC betaII) has been implicated in proliferation of the intestinal epithelium. To investigate PKC betaII function in vivo, we generated transgenic mice that overexpress PKC betaII in the intestinal epithelium. Transgenic PKC betaII mice exhibit hyperproliferation of the colonic epithelium and an increased susceptibility to azoxymethane-induced aberrant crypt foci, preneoplastic lesions in the colon. Furthermore, transgenic PKC betaII mice exhibit elevated colonic beta-catenin levels and decreased glycogen synthase kinase 3beta activity, indicating that PKC betaII stimulates the Wnt/adenomatous polyposis coli (APC)/beta-catenin proliferative signaling pathway in vivo. These data demonstrate a direct role for PKC betaII in colonic epithelial cell proliferation and colon carcinogenesis, possibly through activation of the APC/beta-catenin signaling pathway.

Malignant glioma, the most common primary brain tumor, is generally incurable. Although phosphatidylinositol-3-kinase (PI3K) signaling features prominently in glioma, inhibitors generally block proliferation rather than induce apoptosis. Starting with an inhibitor of both lipid and protein kinases that induced prominent apoptosis and that failed early clinical development because of its broad target profile and overall toxicity, we identified protein kinase targets, the blockade of which showed selective synthetic lethality when combined with PI3K inhibitors. Prioritizing protein kinase targets for which there are clinical inhibitors, we demonstrate that cyclin-dependent kinase (CDK)1/2 inhibitors, siRNAs against CDK1/2, and the clinical CDK1/2 inhibitor roscovitine all cooperated with the PI3K inhibitor PIK-90, blocking the antiapoptotic protein Survivin and driving cell death. In addition, overexpression of CDKs partially blocked some of the apoptosis caused by PIK-75. Roscovitine and PIK-90, in combination, were well tolerated in vivo and acted in a synthetic-lethal manner to induce apoptosis in human glioblastoma xenografts. We also tested clinical Akt and CDK inhibitors, demonstrating induction of apoptosis in vitro and providing a preclinical rationale to test this combination therapy in patients.

[(124)I]m-iodobenzylguanidine ((124)I-mIBG) provides a quantitative tool for pretherapy tumor imaging and dosimetry when performed before [(131)I]m-iodobenzylguanidine ((131)I-mIBG) targeted radionuclide therapy of neuroblastoma. (124)I (T (1/2) = 4.2 days) has a comparable half-life to that of (131)I (T (1/2) = 8.02 days) and can be imaged by positron emission tomography (PET) for accurate quantification of the radiotracer distribution. We estimated expected radiation dose in tumors from (131)I-mIBG therapy using (124)I-mIBG microPET/CT imaging data in a murine xenograft model of neuroblastoma transduced to express high levels of the human norepinephrine transporter (hNET).In order to enhance mIBG uptake for in vivo imaging and therapy, NB 1691-luciferase (NB1691) human neuroblastoma cells were engineered to express high levels of hNET protein by lentiviral transduction (NB1691-hNET). Both NB1691 and NB1691-hNET cells were implanted subcutaneously and into renal capsules in athymic mice. (124)I-mIBG (4.2-6.5 MBq) was administered intravenously for microPET/CT imaging at 5 time points over 95 h (0.5, 3-5, 24, 48, and 93-95 h median time points). In vivo biodistribution data in normal organs, tumors, and whole-body were collected from reconstructed PET images corrected for photon attenuation using the CT-based attenuation map. Organ and tumor dosimetry were determined for (124)I-mIBG. Dose estimates for (131)I-mIBG were made, assuming the same in vivo biodistribution as (124)I-mIBG.All NB1691-hNET tumors had significant uptake and retention of (124)I-mIBG, whereas unmodified NB1691 tumors did not demonstrate quantifiable mIBG uptake in vivo, despite in vitro uptake. (124)I-mIBG with microPET/CT provided an accurate three-dimensional tool for estimating the radiation dose that would be delivered with (131)I-mIBG therapy. For example, in our model system, we estimated that the administration of (131)I-mIBG in the range of 52.8-206 MBq would deliver 20 Gy to tumors.The overexpression of hNET was found to be critical for (124)I-mIBG uptake and retention in vivo. The quantitative (124)I-mIBG PET/CT is a promising new tool to predict tumor radiation doses with (131)I-mIBG therapy of neuroblastoma. This methodology may be applied to tumor dosimetry of (131)I-mIBG therapy in human subjects using (124)I-mIBG pretherapy PET/CT data.

Neuroblastoma, a tumor of peripheral neural crest origin, numbers among the most common childhood cancers. Both amplification of the proto-oncogene MYCN and increased neoangiogenesis mark high-risk disease. Because angiogenesis is regulated by phosphatidylinositol 3-kinase (PI3K), we tested a clinical PI3K inhibitor, NVP-BEZ235, in MYCN-dependent neuroblastoma. NVP-BEZ235 decreased angiogenesis and improved survival in both primary human (highly pretreated recurrent MYCN-amplified orthotopic xenograft) and transgenic mouse models for MYCN-driven neuroblastoma. Using both gain- and loss-of-function approaches, we demonstrated that the anti-angiogenic efficacy of NVP-BEZ235 depended critically on MYCN in vitro and in vivo. Thus, clinical PI3K/mammalian target of rapamycin inhibitors drove degradation of MYCN in tumor cells, with secondary paracrine blockade of angiogenesis. Our data demonstrated significantly improved survival in treated animals and suggest that NVP-BEZ235 should be tested in children with high-risk, MYCN-amplified neuroblastoma.

As a neuroendocrine tumor, neuroblastoma expresses various gastrointestinal (GI) hormones, such as vasoactive intestinal peptide, gastrin-releasing peptide (GRP), neurotensin, and somatostatin, which exert diverse cellular functions in neuroblastoma. In particular, we have recently found that GRP and its cell surface receptor, GRP-R, are abundantly expressed in neuroblastomas. Moreover, more advanced-stage neuroblastomas demonstrated an increased level of GRP-R, suggesting an important role of GRP in aggressive tumor behavior. This review describes the role of several GI hormones commonly expressed in neuroblastoma and discusses in depth the mitogenic actions of GRP in neuroblastoma. In addition, the molecular mechanisms involved in the GRP-induced stimulation of neuroblastoma cell growth are discussed. Our study results demonstrate a role of GRP as an autocrine/paracrine growth factor and elucidate involvement of specific intracellular signaling, the phosphatidylinositol 3-kinase pathway, in the growth regulation of neuroblastoma.

Chronic myelogenous leukemia (CML) results from a t(9,22) translocation, producing the p210(BCR-ABL) oncoprotein, a tyrosine kinase that causes transformation and chemotherapy resistance. To further understand mechanisms mediating chemotherapy resistance, we identified 556 differentially regulated genes in HL-60 cells stably expressing p210(BCR-ABL) versus those expressing an empty vector using cDNA macro- and oligonucleotide microarrays. These BCR-ABL-regulated gene products play diverse roles in cellular function including apoptosis, cell cycle regulation, intracellular signaling, transcription, and cellular adhesion. In particular, we identified up-regulation of the inducible form of heat shock protein 70 (Hsp70), and further explored the mechanism for its up-regulation. In HL-60/BCR-ABL and K562 cells (expressing p210(BCR-ABL)), abundant cytoplasmic Hsp70 expression was detected by immunoblot analysis. Moreover, cells isolated from bone marrow aspirates of patients in different stages of CML (chronic, aggressive, and blast crisis) express Hsp70. Expression of p210(BCR-ABL) in BCR-ABL negative cells induced transcription of the proximal Hsp70 promoter. Mutational analysis mapped the major p210(BCR-ABL) responsive element to a high affinity 5'(A/T)GATA(A/G)-3' "GATA" response element (GATA-RE) that binds GATA-1 in CML cells. The GATA-RE was sufficient to confer p210(BCR-ABL)- and p185(BCR-ABL)-mediated trans-activation to an inert promoter. Short interfering RNA mediated "knockdown" of Hsp70 expression in K562 cells induced marked sensitivity to paclitaxel-induced apoptosis. Together these findings indicate that BCR-ABL confers chemotherapeutic resistance through intracellular signaling to the GATA-RE element found in the promoter region of the anti-apoptotic Hsp70 protein. We suggest that down-regulation of the GATA-Hsp70 pathway may be useful in the treatment of chemotherapy-resistant CML.

The protein kinase C (PKC) family of serine/threonine kinases plays an important role in numerous cancer signaling pathways, including those downstream of the bcr-abl oncogene. We demonstrated previously that atypical PKCiota is required for Bcr-Abl-mediated resistance of human K562 chronic myelogenous leukemia (CML) cells to Taxol-induced apoptosis. Here, we report that the pattern of PKC isozyme expression characteristic of CML cells is regulated by Bcr-Abl. When Bcr-Abl was expressed in Bcr-Abl-negative HL-60 promyelocytic leukemia cells, expression of the PKCbetaI, PKCbetaII, and PKCiota genes was induced, whereas expression of the PKCdelta gene was reduced to levels similar to those found in CML cells. Given the importance of PKCiota in Bcr-Abl-mediated transformation, we characterized the mechanism by which Bcr-Abl regulates PKCiota expression. A 1200-bp PKCiota promoter construct isolated from genomic DNA was highly active in Bcr-Abl-positive K562 cells and was activated when Bcr-Abl-negative cells were transfected with Bcr-Abl. Bcr-Abl-mediated induction of the PKCiota promoter was dependent upon MEK1/2 activity, but not phosphatidylinositol 3-kinase or p38 MAPK activity. Mutational analysis of the PKCiota promoter revealed a region between 97 and 114 bp upstream of the transcriptional start site that is responsible for Bcr-Abl-mediated regulation. Mutation of a consensus Elk1-binding site within this region abolished Bcr-Abl-mediated regulation. We conclude that Bcr-Abl regulates PKCiota expression through the MEK-dependent activation of an Elk1 element within the proximal PKCiota promoter. Our results indicate that Bcr-Abl-mediated transformation involves transcriptional activation of the PKCiota gene, which in turn is required for Bcr-Abl-mediated chemoresistance.

MYC family proteins represent a critical node in oncogenesis across a wide range of pediatric and adult cancers. This is particularly true in neuroblastoma, where amplification of MYCN confers an especially poor prognosis. Proteolytic degradation of MYCN protein is regulated in part by a kinase-independent function of Aurora Kinase A. We describe a class of allosteric inhibitors that disrupts the native conformation of Aurora A and causes degradation of MYCN protein across MYCN-expressing neuroblastoma cell lines. We compare co-crystal structures with structure-activity relationships across multiple inhibitors and chemotypes to delineate an Aurora A conformation-specific effect on proteolytic degradation of MYCN protein, which is driven markedly by conformation disrupting inhibitors, and impacted more modestly by current clinical Aurora Kinase A inhibitors, which disrupt the kinase structure less efficiently. Using functional assays of apoptosis, cell cycle, and proliferation we further define a distinct MYCN-specific effect distinguishing it from simple inhibition of mitosis by conventional Aurora kinase inhibitors. This new class of conformation disrupting inhibitors, which block stabilizing interactions between Aurora A and MYCN, represents a novel strategy to target MYCN-driven cancers. This abstract is also presented as Poster B46. Citation Format: W. Clay Gustafson, Justin G. Meyerowitz, Erin A. Nekritz, Elise Charron, Katherine K. Matthay, Nicholas T. Hertz, Martin Eilers, Kevan M. Shokat, William A. Weiss. Drugging MYCN protein stability through an allosteric transition in Aurora kinase A. [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 PR08.

Amplification of MYCN is associated with high-risk SHH-driven and group 4 medulloblastoma, while mis-expression and amplification of MYC is associated with aggressive group 3 tumors. The biology of these tumors, particularly the group 3/4 tumors, is poorly understood, with no effective targeted therapies. We have recently developed a model of group 4 medulloblastoma in which high levels of mutationally-stabilized MYCN protein drive oncogenesis. We have shown in other tumor types that modulation of the translational apparatus downstream of the mammalian target of rapamycin (mTOR) is required for MYC-driven oncogenesis and that there is a synergistic relationship between MYC and the mTOR axis to promote tumor formation. mTOR signals through two primary outputs, rpS6 kinase (S6K) and the translation initiation factor eIF4E. A new class of clinical mTOR active site inhibitors disrupts signaling through both effectors, whereas the allosteric binder rapamycin disrupts only S6K. We describe genetic and therapeutic studies aimed at distinguishing the effects of S6K and eIF4E in both tumor formation and in therapy. These mechanistically distinct activities have enormous therapeutic implications, as in our medulloblastoma model, active site inhibitors of mTOR, but not rapamycin, demonstrate efficacy. Taken together, this supports the idea that signaling through S6K is dispensable, whereas eIF4E is required for MYC-driven oncogenesis. Citation Format: Justin G. Meyerowitz, W. Clay Gustafson, Erin Nekritz, Fredrik Swartling, Kevan M. Shokat, Davide Ruggero, William A. Weiss. Targeting the translational apparatus in MYCN-driven medulloblastoma. [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 B78.

Basal, "insulin-independent" glucose uptake into skeletal muscle is provided by glucose transporters positioned at the plasma membrane. The relative amount of the three glucose transporters expressed in muscle has not been previously quantified. Using a combination of qualitative and quantitative ribonuclease protection assay (RPA) methods, we found in normal human muscle that GLUT1, GLUT3, and GLUT4 mRNA were expressed at 90 +/- 10, 46 +/- 4, and 156 +/- 12 copies/ng RNA, respectively. Muscle was fractionated by DNase digestion and differential sedimentation into membrane fractions enriched in plasma membranes (PM) or low-density microsomes (LDM). GLUT1 and GLUT4 proteins were distributed 57% to 67% in LDM, whereas GLUT3 protein was at least 88% in the PM-enriched fractions. These data suggest that basal glucose uptake into resting human muscle could be provided in part by each of these three isoforms.