MYC-amplified Cell Lines Research Articles

Combination drug screen identifies synergistic drug interaction of BCL-XL and class I histone deacetylase inhibitors in MYC-amplified medulloblastoma cells

PurposePatients with MYC-amplified Group 3 medulloblastoma (MB) (subtype II) show poor progression-free survival rates. Class I histone deacetylase inhibitors (HDACi) are highly effective for the treatment of MYC-amplified MB in vitro and in vivo. Drug combination regimens including class I HDACi may represent an urgently needed novel treatment approach for this high risk disease.MethodsA medium-throughput in vitro combination drug screen was performed in three MYC-amplified and one non-MYC-amplified MB cell line testing 75 clinically relevant drugs alone and in combination with entinostat. The drug sensitivity score (DSS) was calculated based on metabolic inhibition quantified by CellTiter-Glo. The six top synergistic combination hits were evaluated in a 5 × 5 combination matrix and a seven-ray design. Synergy was validated and characterized by cell counts, caspase-3-like-activity and poly-(ADP-ribose)-polymerase-(PARP)-cleavage. On-target activity of drugs was validated by immunoprecipitation and western blot. BCL-XL dependency of the observed effect was explored with siRNA mediated knockdown of BCL2L1, and selective inhibition with targeted compounds (A-1331852, A-1155463).Results20/75 drugs effectively reduced metabolic activity in combination with entinostat in all three MYC-amplified cell lines (DSS ≥ 10). The combination entinostat and navitoclax showed the strongest synergistic interaction across all MYC-amplified cell lines. siRNA mediated knockdown of BCL2L1, as well as targeted inhibition with selective inhibitors showed BCL-XL dependency of the observed effect. Increased cell death was associated with increased caspase-3-like-activity.ConclusionOur study identifies the combination of class I HDACi and BCL-XL inhibitors as a potential new approach for the treatment of MYC-amplified MB cells.Graphical abstractGraphical abstract created with BioRender.com, illustrating the workflow and summarizing main results.

Comprehensive Metabolic Profiling of MYC-Amplified Medulloblastoma Tumors Reveals Key Dependencies on Amino Acid, Tricarboxylic Acid and Hexosamine Pathways.

Simple SummaryThe oncogene MYC alters cellular metabolism. Medulloblastoma is the most common malignant pediatric brain tumor. MYC-amplified medulloblastoma has a poor prognosis, and the metabolism of MYC-amplified medulloblastoma is poorly understood. We performed comprehensive metabolic profiling of MYC-amplified medulloblastoma and found increased reliance on potentially targetable pathways. We also found that the metabolism of MYC-amplified cell lines differed from orthotopic brain tumors in vitro and in flank tumors, suggesting that analyses conducted in vitro or in flank tumors may miss key vulnerabilities.Reprograming of cellular metabolism is a hallmark of cancer. Altering metabolism allows cancer cells to overcome unfavorable microenvironment conditions and to proliferate and invade. Medulloblastoma is the most common malignant brain tumor of children. Genomic amplification of MYC defines a subset of poor-prognosis medulloblastoma. We performed comprehensive metabolic studies of human MYC-amplified medulloblastoma by comparing the metabolic profiles of tumor cells in three different conditions—in vitro, in flank xenografts and in orthotopic xenografts in the cerebellum. Principal component analysis showed that the metabolic profiles of brain and flank high-MYC medulloblastoma tumors clustered closely together and separated away from normal brain and in vitro MYC-amplified cells. Compared to normal brain, MYC-amplified medulloblastoma orthotopic xenograft tumors showed upregulation of the TCA cycle as well as the synthesis of nucleotides, hexosamines, amino acids and glutathione. There was significantly higher glucose uptake and usage in orthotopic xenograft tumors compared to flank xenograft tumors and cells in culture. In orthotopic tumors, glucose was the main carbon source for the de novo synthesis of glutamate, glutamine and glutathione through the TCA cycle. In vivo, the glutaminase II pathway was the main pathway utilizing glutamine. Glutathione was the most abundant upregulated metabolite in orthotopic tumors compared to normal brain. Glutamine-derived glutathione was synthesized through the glutamine transaminase K (GTK) enzyme in vivo. In conclusion, high MYC medulloblastoma cells have different metabolic profiles in vitro compared to in vivo, and key vulnerabilities may be missed by not performing in vivo metabolic analyses.

Cell-Based Drug Discovery: Identification and Optimization of Small Molecules that Reduce c-MYC Protein Levels in Cells.

Elevated expression of the c-MYC oncogene is one of the most common abnormalities in human cancers. Unfortunately, efforts to identify pharmacological inhibitors that directly target MYC have not yet yielded a drug-like molecule due to the lack of any known small molecule binding pocket in the protein, which could be exploited to disrupt MYC function. We have recently described a strategy to target MYC indirectly, where a screening effort designed to identify compounds that can rapidly decrease endogenous c-MYC protein levels in a MYC-amplified cell line led to the discovery of a compound series that phenocopies c-MYC knockdown by siRNA. Herein, we describe our medicinal chemistry program that led to the discovery of potent, orally bioavailable c-MYC-reducing compounds. The development of a minimum pharmacophore model based on empirical structure activity relationship as well as the property-based approach used to modulate pharmacokinetics properties will be highlighted.

EMBR-11. SYNERGISTIC DRUG COMBINATIONS FOR THE TREATMENT OF MYC AMPLIFIED GROUP 3 MEDULLOBLASTOMA

Abstract Background Medulloblastoma (MB) is a highly aggressive brain tumour in children. Patients with Group 3 MB harbouring a MYC-amplification (subtype II) show a particularly poor outcome despite high-intensity multimodal therapy. We and others have previously shown that MYC amplified Group 3 MB cells are highly susceptible towards treatment with class I histone deacetylase (HDAC) inhibitors such as entinostat. However, in clinical trials HDACi as a monotherapy show only modest efficacy in solid tumours. We propose to increase the efficacy of class I HDACi by drug combinations. Methods To identify synergistic drug combinations (entinostat + X) for the treatment of MYC amplified MB we performed a drug screen with a library of n=75 clinically available compounds as single agents and in combination with entinostat in n=3 MYC amplified vs. n=1 MYC-non amplified cell lines. Synergistic behaviour of the six most promising drug combinations was validated by metabolic activity assays, cell count experiments and gene expression profiling. Synergy was assessed by the Loewe additivity model using a combination of ray design and checkerboard matrix. Results The drug screen revealed n=20/75 drugs that were particularly effective (drug sensitivity score ≥10) in combination with entinostat treatment in all three MYC amplified cell lines. Synergy assessment of the top n=6 drugs confirmed strong synergistic activity with entinostat for n=2 drugs (navitoclax, irinotecan). The BCL-2 family inhibitor navitoclax showed the most robust synergy with entinostat in subsequent validation experiments. Conclusion Several drugs either clinically available or currently in clinical trials, including the BCL-2/Xl/w inhibitor navitoclax, show promising effects in a combination therapy with entinostat for the treatment of MYC amplified Group 3 MB.

EMBR-01. CLASS I HDAC INHIBITORS AND PLK1 INHIBITORS SYNERGIZE IN MYC-AMPLIFIED MEDULLOBLASTOMA

BackgroundMedulloblastoma (MB) is one of the most common malignant pediatric CNS tumors. Patients with Group 3 MBs harboring MYC amplification exhibit low survival rates. Surviving patients suffer from therapy-induced sequelae, which calls for new targeted therapy strategies. We and others have previously shown the sensitivity of MYC-amplified MB to class I histone deacetylase (HDAC) inhibition. After demonstrating that the MYC target gene PLK1 is significantly downregulated upon class I HDACi treatment, we hypothesized that inhibition of both HDACs and PLK1 could have synergistic effects. MethodsCell metabolic activity changes upon HDAC and PLK1 inhibitor treatment were measured in MYC-amplified and non-amplified MB cell lines, as well as in an additional MYC-inducible cell line. The interaction effect of both inhibitors was determined by computation of the combination index (CI) using the Chou-Talalay method. Results were validated assessing cell viability, cell cycle, and apoptosis induction. Transcription profile changes after combination treatment were evaluated. Results MYC-amplified MB cell lines were more sensitive than non-amplified cell lines to PLK1i treatment, showing IC50 in clinically achievable concentration ranges. Inhibition of class I HDACs and PLK1 synergistically reduced cell metabolic activity in lower concentrations in MYC-amplified compared to non-amplified MB cell lines. We also observed a significant loss of viability and cells in G1 phase, as well as induction of apoptosis after combination treatment in MYC-amplified cells. MYC target gene sets were significantly downregulated in the MYC-amplified cell line HD-MB03 after treatment with combination. We demonstrated reduction of MYC protein levels upon PLK1i treatment. In vivo evaluation of combination treatment using orthotopic Group 3 MYC-amplified MB PDX models is ongoing. ConclusionOur data suggest that MYC-amplification is a predictive marker for PLK1i treatment in MB. The combination of HDACi and PLKi could be a candidate therapy for future clinical trials for MYC-amplified group 3 MB.

Abstract A53: Synergistic interaction of HDACi and PLK1i in Group 3 MYC-amplified medulloblastoma

Abstract Medulloblastoma (MB) is one of the common malignant brain tumors in children. Patients with MYC-amplified Group 3 MBs exhibit particularly poor survival rates even after intensive therapy. Surviving patients often suffer from long-term side effects. This calls for new therapeutic strategies, such as targeted therapy options. The sensitivity of MYC-amplified MBs to class I histone deacetylase (HDAC) inhibition was previously shown. In order to elucidate potential combination partners, we have identified PLK1 as one of the top regulated genes following treatment of MYC-amplified Group 3 MB cells with class I HDACi entinostat. Our aim here is to investigate possible combination treatments with entinostat and several PLK1i (volasertib, GSK461364, onvansertib). The cell metabolic activity was evaluated using MYC-amplified and nonamplified MB, high-grade glioma (HGG) and neuroblastoma (NB) cell lines after single and combination treatments. The interaction effect was determined by combination index (CI) (Chou-Talalay CI calculation method). Results were validated assessing cell viability, cell cycle profile, and caspase activity upon treatment with single agents or combination. On-target activity was examined using immunoblotting for pTCTP and H3K27ac. We have confirmed our findings in an inducible MYC cell line. The gene expression profile was analyzed in HD-MB03 cell line after entinostat, volasertib, or combination treatment. We demonstrate that the MYC target gene PLK1 is significantly downregulated upon HDACi treatment. Based on this, we hypothesized that inhibition of both class I HDACs and PLK1 could have synergistic effects. MYC-amplified cell lines were more sensitive than nonamplified cell lines to treatment with all PLK1i investigated, showing 3 to 10 times lower IC50. PLK1i and entinostat interacted synergistically (CI below 0.9) at lower concentrations in MYC-amplified compared to non-amplified MB cell lines. Similar results were obtained for MYC or N-MYC-amplified HGG and NB cell lines. We also observed the loss of viability and loss of fraction of cells in G1 phase in MYC-amplified MB cells after treatment with entinostat and PLK1i. MYC target genes were significantly downregulated in the MYC-amplified MB cell line HD-MB03 after treatment with PLK1i and entinostat. Moreover, we demonstrated reduction of MYC levels and faster MYC degradation upon volasertib treatment. Our data suggest that MYC-amplification might serve as a predictive biomarker for PLK1i treatment in MB and other entities. The combination of entinostat and PLKi could be a candidate therapy for future clinical trials for MYC-amplified Group 3 MB, and possibly other tumors harboring MYC amplification. Citation Format: Gintvile Valinciute, Jonas Ecker, Thomas Hielscher, Christin Schmidt, Marc Remke, Gianluca Sigismondo, Jeroen Krijgsveld, Stefan M. Pfister, Olaf Witt, Till Milde. Synergistic interaction of HDACi and PLK1i in Group 3 MYC-amplified medulloblastoma [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 A53.

Accumulation of protoporphyrin IX in medulloblastoma cell lines and sensitivity to subsequent photodynamic treatment

Medulloblastoma (MB) is the most common malignant primary brain tumor of childhood. High risk patients still have a poor outcome, and especially young patients suffer from standard therapy induced sequelae. Therefore, other therapeutic options need to be explored. In glioblastoma (GBM), application of 5-aminolaevulinic acid (5-ALA) results in selective accumulation of protoporphyrin IX (PPIX) in the tumor cells, which can be exploited during fluorescence-guided surgery to increase the extent of resection or for photodynamic therapy (PDT) induced phototoxicity. It is not entirely clear, whether MB cells accumulate PPIX and are sensitive to PDT. Human MYC-amplified (Med8A and D283) and non-amplified (UW228-2 and ONS76) MB cell lines were incubated for 2, 4 or 6 h with increasing doses (0-100 μg/ml) of 5-ALA, and PPIX accumulation was determined by flow cytometry. To assess sensitivity to 5-ALA/PDT, cells were incubated with 5-ALA and subsequently exposed to laser light of 635 nm wavelength (18.75 J/cm2). After an additional 24 h culture period, viability of cells was quantified using the WST-1 assay. Expression of ferrochelatase was detected by reverse transcription and quantitative polymerase chain reaction. Ferrochelatase activity was quantified by measuring the enzymatic conversion of PPIX to zinc-protoporphyrin. Expression of the ABCG2 transporter protein CD338 was detected by flow cytometry. All MB cell lines showed a time- and dose-dependent accumulation of PPIX after exposure to exogenous 5-ALA and became sensitive to 5-ALA/PDT-induced phototoxicity. PPIX accumulation was reduced compared to U373 GBM cells at shorter incubation periods and limiting 5-ALA doses. Moreover, not all MB cells became PPIX positive and overall phototoxicity was lower in the MB cell lines. Notably, the MYC-amplified MB cells demonstrated a more pronounced photosensitivity compared to their non-amplified counterparts. There was no difference in expression of ferrochelatase, but enzymatic activity appeared to be reduced in the MB cells compared to U373 GBM cells, whereas CD338 was expressed on the MB cells only. Medulloblastoma cell lines accumulate PPIX after application of 5-ALA and become sensitive to PDT, associated with low ferrochelatase expression and activity. Photosensitivity is more pronounced in MYC-amplified cell lines. In contrast to GBM cells, however, PPIX accumulation appears to be reduced, restricted to a subset of cells and associated with lower photosensitivity of the MB cell lines, possibly due to expression of the ABCG2 transporter protein CD338 on MB cells.

Abstract 5859: Enhanced therapy for MYC-driven medulloblastoma by targeting protein translation pathway

Abstract Medulloblastoma is the most frequent pediatric brain tumor of neuroectodermal cerebellar origin. High-risk MB patients with MYC overexpression and amplification usually show an extremely poor clinical outcome responding poorly to current therapies. Recent preclinical studies have revealed that the aberrant activation and interactions of PI3K/AKT/mTOR signaling is frequently associated with MYC-driven MB therapy-resistance. In addition, it is evident that both MYC and mTOR components of the PI3K/AKT/mTOR signaling axis can directly control and share a common translation pathway in increasing protein synthesis by regulating the expression of multiple components of the protein synthesis machinery, including ribosomal proteins and initiation factors of translation. Thus, inhibiting enhanced protein synthesis may represent a highly relevant strategy for the treatment of MYC-driven MB. Therefore, in this study, we investigated the single agents and combined anti-MB efficacies of a recently well-established BRD4 (bromodomain-containing protein 4)/MYC inhibitor JQ1 and PI3K-mTOR dual inhibitor BEZ235. Using four MB cell lines including three MYC amplified cell lines, the in vitro efficacy of this combined approach on cell growth/apoptosis and cell cycle along with associated molecular mechanism(s) were investigated. We used an MTT assay to measure survival and proliferation, flow-cytometric-based propidium-iodide staining to determine the effects of these inhibitors on cell-cycle, Annexin-V analyses to examine the cells undergoing apoptosis following treatments and western blot analyses to determine the expression levels of target molecules. Our results showed that both inhibitors as single agents, significantly decreased MB cell growth and induced apoptosis by targeting the key molecules of the associated pathways. Combined treatment of JQ1 and BEZ235 significantly decreased MB cell growth/survival in a dose-dependent fashion compared to single agent activity. Moreover, JQ1 and BEZ235 alone or combined significantly induced G1 cell-cycle arrest which coincided with decreased expression of cyclin-D1 and increased expression of p21 proteins in MYC-driven MB cells. Mechanistically, the co-treatment of JQ1 and BEZ235 significantly downregulated the expression levels of phosphorylated 4EBP1/p70-S6K (mTOR components) and BRD4/MYC proteins. Together, our findings demonstrate that the combination of JQ1 and BEZ235 showed significant synergistic efficacy against MYC-driven MB, by enhancing the inhibition of protein synthesis (translation) pathway. Initial evidence from this targeted approach, suggests that additional preclinical evaluation is warranted to determine likely clinical utility of targeted therapy for high-risk MB patients. Citation Format: Nagendra K. Chaturvedi, Don W. Coulter, Matthew J. Kling, Sutapa Ray, Timothy R. McGuire, Shantaram S. Joshi, J. Graham Sharp. Enhanced therapy for MYC-driven medulloblastoma by targeting protein translation pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5859.

Improved therapy for medulloblastoma: targeting hedgehog and PI3K-mTOR signaling pathways in combination with chemotherapy.

Aberrant activation and interactions of hedgehog (HH) and PI3K/AKT/mTOR signaling pathways are frequently associated with high-risk medulloblastoma (MB). Thus, combined targeting of the HH and PI3K/AKT/mTOR pathways could be a viable therapeutic strategy to treat high-risk patients. Therefore, we investigated the anti-MB efficacies of combined HH inhibitor Vismodegib and PI3K-mTOR dual-inhibitor BEZ235 together or combined individually with cisplatin against high-risk MB. Using non-MYC- and MYC-amplified cell lines, and a xenograft mouse model, the in vitro and in vivo efficacies of these therapies on cell growth/survival and associated molecular mechanism(s) were investigated. Results showed that combined treatment of Vismodegib and BEZ235 together, or with cisplatin, significantly decreased MB cell growth/survival in a dose-dependent-fashion. Corresponding changes in the expression of targeted molecules following therapy were observed. Results demonstrated that inhibitors not only suppressed MB cell growth/survival when combined, but also significantly enhanced cisplatin-mediated cytotoxicity. Of these combinations, BEZ235 exhibited a significantly greater efficacy in enhancing cisplatin-mediated MB cytotoxicity. Results also demonstrated that the MYC-amplified MB lines showed a higher sensitivity to combined therapies compared to non-MYC-amplified cell lines. Therefore, we tested the efficacy of combined approaches against MYC-amplified MB growing in NSG mice. In vivo results showed that combination of Vismodegib and BEZ235 or their combination with cisplatin, significantly delayed MB tumor growth and increased survival of xenografted mice by targeting HH and mTOR pathways. Thus, our studies lay a foundation for translating these combined therapeutic strategies to the clinical setting to determine their efficacies in high-risk MB patients.

Abstract 161: Medulloblastoma therapy targeting Hedgehog and PI3K-mTOR signaling pathways in combination with chemotherapy

Abstract Medulloblastoma (MB) is the most frequent malignant brain tumor in children. MB patients with high-risk disease have poorly understood biology and few targeted therapies available. Preclinical studies and molecular profiling of MB have revealed that the aberrant activation and interaction of the SHH and PI3K/AKT/mTOR signaling networks are frequently associated with poor prognosis MB cases. Emerging evidence also demonstrate the key role of activated PI3K/AKT/mTOR pathway components in SHH-driven MB therapy-resistance, thus combined targeting of the SHH and PI3K/AKT/mTOR pathways may be a viable therapeutic strategy to treat high-risk patients. Therefore, we investigated the combined efficacy of SHH inhibitor vismodegib and PI3K-mTOR dual inhibitor BEZ235 or their combination, individually with the chemotherapeutic drug cisplatin against high-risk MB. Using four MB cell lines, including non-MYC and MYC amplified cell lines, and a xenograft mouse model, the in vitro and in vivo efficacies of the proposed therapies on cell growth/survival along with associated molecular mechanism(s) were investigated. Our results showed that both inhibitors as single agents significantly decreased MB cell growth and induced apoptosis by targeting the key molecules of the associated pathways in vitro. BEZ235 as single agent showed a greater anti-MB efficacy compared to vismodegib. Combined treatment of vismodegib and BEZ235 together or with cisplatin significantly decreased MB cell growth/survival and anchorage-independent growth in a dose-dependent fashion compared to single agent activity. Corresponding changes in the expression of the targeted molecules following therapy were observed. Results from the combined approach suggested that the inhibitors not only suppressed MB cell growth/survival when combined, but also significantly enhanced the cytotoxic effects of cisplatin. Of these combinations, BEZ235 exhibited a significantly greater efficacy in enhancing the cisplatin-mediated MB cytotoxicity. In vitro studies also demonstrated that the MYC amplified MB cell lines showed a higher sensitivity to these combined therapies compared to relatively non-MYC amplified cell lines. Therefore, as a next logical step, we tested the efficacy of above combined approaches against MYC-amplified MB in vivo using NSG mice. Our in vivo results showed that the combination of vismodegib and BEZ235 or their combinations individually with cisplatin significantly delayed tumor growth and increased survival of xenograft mice compared to single agent activity. These combination not only significantly reduced tumor growth and increased survival of the mice but also significantly enhanced anti-MB efficacy by targeting SHH and mTOR pathways in MB in vivo. Thus, our studies lay a foundation for translating these combined therapeutic strategies to the clinical setting to determine their efficacies in high-risk MB patients. Citation Format: Nagendra K. Chaturvedi, Don W. Coulter, Timothy R. McGuire, Matthew J. Kling, Sutapa Ray, Shantaram S. Joshi, John G. Sharp. Medulloblastoma therapy targeting Hedgehog and PI3K-mTOR signaling pathways in combination with chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 161. doi:10.1158/1538-7445.AM2017-161

Targeting the interaction of HDAC2 and MYC in Group 3 medulloblastoma

Patients with high-risk subgroup medulloblastoma (MB) show poor overall survival rates despite aggressive treatment while survivors suffer from important treatment-associated morbidity. Particularly poor outcome is observed in patients with tumors of the molecular subgroup Group 3 with an amplification and high levels of the transcription factor MYC. We have previously shown that Group 3 MB cell lines with MYC amplification are highly susceptible to pan- and class I-histone deacetylase (HDAC) inhibitor treatment. We here explore the molecular interaction of the well known oncogenic driver MYC and class I HDAC2. Six established MB cell lines were used for experiments in cell culture, including MYC amplified cell lines. The functional interaction between MYC and HDAC2 was investigated by enzymatic inhibition of class I HDACs using class I-selective HDAC inhibitors (HDACi) and by siRNA-mediated knockdown of HDAC2. Observed protein degradation was rescued with proteasome inhibition using small molecule proteasome inhibitor MG132. The direct interaction of HDAC2 and MYC was analyzed in co-immunoprecipitation and mass spectrometry studies. The pharmacological inhibition of class I HDACs including HDAC2 as well as artificial HDAC2 protein depletion lead to reduced MYC protein levels, most likely due to increased proteasomal degradation. MYC and HDAC2 are localized in a protein complex together with other potential drug targets. Our data suggest that class I HDAC inhibition leads to destabilization of MYC protein depriving the cells from one of their main oncogenic drivers. The MYC/HDAC2 multi-protein complex represents an attractive target for novel combinatorial treatment approaches. Funding: MYCNet (BMBF Germany), Verein fur krebskranke Kinder Odenwald e.V.

Abstract 3502: Selective targeting of CTNBB1-, KRAS- or MYC-driven cell growth by combinations of existing drugs

Abstract Purpose: Combination therapy plays an important role in achieving durable responses in anticancer therapy. Ideally, compound combinations work synergistically in cancer cells and not in non-malignant cells. Here we present a rational approach for identifying pairs of selective inhibitors that show a synergistic interaction only in specific genetic backgrounds, i.e. mutated in the CTNNB1 (β-catenin) or KRAS genes, or expressing amplified MYC. Experimental Procedures: We have established a panel of sixty six genetically well-characterized cell lines that represents the most frequently occurring oncogenic drivers in cancer (1). A collection of more than 100 inhibitors targeting all important oncogenic signaling pathways, including many kinase and epigenetic regulators, was profiled in a subset of forty four cell lines, generating highly reproducible and high-quality cell proliferation data. The inhibitor response was correlated to the genetic background of the cell lines by Anova analysis (1). Compounds that targeted similar subsets of cell lines, were tested in combination for synergistic interaction, using in vitro proliferation assays with equipotent mixtures, followed by curve shift analysis, isobolograms and combination index scoring. The approach was validated by showing that the MEK inhibitor trametinib and the BRAF inhibitor dabrafenib, which are targeted to BRAF-mutant cell lines, show synergistic interaction in this subpopulation, and not in other cell line types. Results: For all inhibitors, the drug response in cell line panel (Oncolines™) was analyzed. Notably, the Wnt-pathway inhibitor ICG-001 and the MEK inhibitor trametinib were shown to preferentially inhibit CTNNB1-mutated cancer cell lines. Neratinib, a spectrum selective EGFR inhibitor, and GSK-1070916, an Aurora kinase inhibitor, were shown to target MYC-amplified cell lines. The ERBB2 inhibitor TAK-165 and trametinib targeted KRAS mutant cell lines. These compounds thus show a pharmacological synthetic lethal effect. Subsequent combination studies showed synergy on cell proliferation in a high number of cases. ICG-001 works synergistically with trametinib in a CTNNB1-mutant colon cell line, and not in CTNNB1-wild type colon cell lines. Neratinib and GSK-1070916 work synergistically in MYC-amplified cell lines and not in non-MYC-amplified lines. Conclusions: Pharmacogenomic analysis of single agent responses identified compounds that preferentially inhibit the growth of cell lines harbouring mutant CTNNB1 or KRAS, or amplified MYC. Synergy studies in the pertinent genetic background identified new combinations of existing drugs with enhanced targeting of CTNNB1-mutated or MYC-amplified cancer cell lines. Our approach can efficiently discover novel drug combinations that target cancer genes more selectively and more potently.

Abstract 2023: MYC amplification and overexpression in metastatic anaplastic thyroid cancer dictates response to therapy

Abstract Anaplastic thyroid cancer (ATC) is a very aggressive and lethal cancer with few months of survival from diagnosis. Although less than 2% of all thyroid carcinomas are ATC, it accounts for up to 40% of thyroid carcinoma deaths. TRP53 inactivating mutations and PI3K activation through loss of PTEN, or PIK3CA mutations and amplification are common in ATC. In fact, targeted deletion of Pten and Trp53 in the mouse thyroid results in ATC development. Using this mouse model, we found that Myc is amplified in 38% (11/29) of [Pten,Trp53]thyr-/− ATCs with distant metastasis, but only in 10% (3/30) non-metastatic ATCs. Conversely Myc was not amplified in any of 15 metastatic follicular thyroid carcinomas developed by Ptenthyr-/− mice. Analysis of human ATCs showed Myc amplification in 17% (3/18) tumors and protein overexpression in 25% of samples in a tissue microarray. Myc amplification has been shown to be associated with resistance to PI3K targeted therapies in different tumor types. We evaluated the role of Myc in ATC growth and response to therapy using cell lines derived from our [Pten,p53]thyr-/− mouse model. Myc-amplified cell lines grew faster than cell lines from tumors without Myc amplification both in full medium and under serum deprivation. Myc knockdown resulted in a dramatic decrease in growth rate, especially under serum starvations, suggesting that tumors with Myc amplification are less dependent on growth factors present in the serum. Myc knockdown in Myc-amplified cells completely impaired tumor formation in syngeneic mice. To evaluate the effect of Myc amplification on drug sensitivity, we screened a panel of drugs on cultured cells. Myc knockdown sensitized ATC cells to PLK1 inhibition with BI6727, PI3K inhibition with BKM120, as well as to doxorubicin and etoposide. Conversely, Myc-amplified cells were more sensitive to carboplatin. Our results show that Myc amplification is a biomarker for more aggressive and resistant ATCs and that MYC-targeting strategies could be employed to sensitize these tumors to conventional and targeted therapy. Citation Format: Emrullah Yilmaz, Marika A. Russo, Arturo Orlacchio, Toni Forde, Antonio Di Cristofano. MYC amplification and overexpression in metastatic anaplastic thyroid cancer dictates response to therapy. [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 2023. doi:10.1158/1538-7445.AM2015-2023

Direct and indirect targeting of MYC to treat acute myeloid leukemia.

PurposeAcute myeloid leukemia (AML) is the most common acute leukemia in adults and is often resistant to conventional therapies. The MYC oncogene is commonly overexpressed in AML but has remained an elusive target. We aimed to examine the consequences of targeting MYC both directly and indirectly in AML overexpressing MYC/Myc due to trisomy 8/15 (human/mouse), FLT3-ITD mutation, or gene amplification.MethodsWe performed in vivo knockdown of Myc (shRNAs) and both in vitro and in vivo experiments using four drugs with indirect anti-MYC activity: VX-680, GDC-0941, artemisinin, and JQ1.ResultsshRNA knockdown of Myc in mice prolonged survival, regardless of the mechanism underlying MYC overexpression. VX-680, an aurora kinase inhibitor, demonstrated in vitro efficacy against human MYC-overexpressing AMLs regardless of the mechanism of MYC overexpression, but was weakest against a MYC-amplified cell line. GDC-0941, a PI3-kinase inhibitor, demonstrated efficacy against several MYC-overexpressing AMLs, although only in vitro. Artemisinin, an antimalarial, did not demonstrate consistent efficacy against any of the human AMLs tested. JQ1, a bromodomain and extra-terminal bromodomain inhibitor, demonstrated both in vitro and in vivo efficacy against several MYC-overexpressing AMLs. We also confirmed a decrease in MYC levels at growth inhibitory doses for JQ1, and importantly, sensitivity of AML cell lines to JQ1 appeared independent of the mechanism of MYC overexpression.ConclusionsOur data support growing evidence that JQ1 and related compounds may have clinical efficacy in AML treatment regardless of the genetic abnormalities underlying MYC deregulation.Electronic supplementary materialThe online version of this article (doi:10.1007/s00280-015-2766-z) contains supplementary material, which is available to authorized users.

Targeting class I histone deacetylase 2 in MYC amplified group 3 medulloblastoma.

IntroductionMedulloblastoma (MB) is the most frequent malignant brain tumor in children. Four subgroups with distinct genetic, epigenetic and clinical characteristics have been identified. Survival remains particularly poor in patients with Group 3 tumors harbouring a MYC amplification. We herein explore the molecular mechanisms and translational implications of class I histone deacetylase (HDAC) inhibition in MYC driven MBs.Material and MethodsExpression of HDACs in primary MB subgroups was compared to normal brain tissue. A panel of MB cell lines, including Group 3 MYC amplified cell lines, were used as model systems. Cells were treated with HDAC inhibitors (HDACi) selectively targeting class I or IIa HDACs. Depletion of HDAC2 was performed. Intracellular HDAC activity, cellular viability, metabolic activity, caspase activity, cell cycle progression, RNA and protein expression were analyzed.ResultsHDAC2 was found to be overexpressed in MB subgroups with poor prognosis (SHH, Group 3 and Group 4) compared to normal brain and the WNT subgroup. Inhibition of the enzymatic activity of the class I HDACs reduced metabolic activity, cell number, and viability in contrast to inhibition of class IIa HDACs. Increased sensitivity to HDACi was specifically observed in MYC amplified cells. Depletion of HDAC2 increased H4 acetylation and induced cell death. Simulation of clinical pharmacokinetics showed time-dependent on target activity that correlated with binding kinetics of HDACi compounds.ConclusionsWe conclude that HDAC2 is a valid drug target in patients with MYC amplified MB. HDACi should cover HDAC2 in their inhibitory profile and timing and dosing regimen in clinical trials should take binding kinetics of compounds into consideration.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-015-0201-7) contains supplementary material, which is available to authorized users.

Abstract A37: Cross-cancer evaluation of MYC synthetic lethal targets in The Cancer Genome Atlas

Abstract Overview: Synthetic lethality in cancer has been investigated for the last decade, but only recently do we have the convergence of high throughput RNA interference screening capabilities with large genomic and molecular data sets derived from primary tumor samples that allows for more comprehensive characterization of putative therapeutic targets against multiple cancers. Accurately relating cancer cell line RNA interference synthetic lethal screening data to primary human tumors has proven to be challenging, yet critically important, for improving validation rates and therapeutic efficacy. To this end, we have combined siRNA synthetic lethal screening hits in MYC amplified cell lines [Toyoshima et al. PNAS 2012] with genomic and molecular data from twelve tumor types in The Cancer Genome Atlas (TCGA) to identify and prioritize cross-cancer therapeutic targets with direct evidence for synthetic lethal relationships in both cell line and primary tumor samples. Approach: In order to relate TCGA data to synthetic lethal siRNA screening in cell lines, we began by integrating all data types produced by TCGA, including: clinical data, sample characteristics, somatic mutations, gene expression, DNA methylation, miRNA expression, reverse-phase protein array data, and somatic copy number variation. From these data, feature matrices have been constructed which combine all available information regarding patients and samples for each given tumor type. For each such heterogeneous feature matrix, we have performed statistical analysis to infer associations in the data between MYC amplification and all other molecular features. Hits from MYC synthetic lethal cell line screening were evaluated for statistical significance in pairwise analysis of twelve TCGA tumor types, classes of pairwise data-derived associations as well as properties of network-level topologies were characterized, and relationships between genomic data and clinical features were investigated where possible. Results: Evidence for synthetic lethal relationships between MYC amplification and molecular features (e.g., gene expression and DNA methylation) for numerous experimentally validates hits was present across multiple TCGA tumor types. For example, statistically significant associations between MYC amplification and decreased CSNK1E methylation, indicative of increased expression, were present in head and neck squamous cell carcinoma, cutaneous melanoma, and stomach adenocarcinoma, corroborating the experimental findings of Toyoshima et al. Similarly, PES1 gene expression is increased in breast carcinoma, colon adenocarcinoma, and head and neck squamous cell carcinoma samples with MYC amplification. Synthetic lethal hits had differential expression profiles reflective of both increases and decreases in correlation with MYC amplification. Based on these preliminary findings, the intersection of siRNA cell line screening hits with accompanying statistical associations in primary tumor sample data may enable prioritization of preclinical models of MYC driven cancers. While the present approach has been applied to MYC amplification, synthetic lethal relationships are present for a multitude of significantly mutated, copy number aberrated, or epigenetically silenced genes in TCGA. Citation Format: Brady Bernard, Silvia Cermelli, Carla Grandori, Ilya Shmulevich. Cross-cancer evaluation of MYC synthetic lethal targets in The Cancer Genome Atlas. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr A37.

Abstract 5192: MYC amplification and overexpression Is associated With metastasis and drug resistance in a mouse model of anaplastic thyroid carcinoma.

Abstract Anaplastic thyroid carcinoma (ATC) is the most aggressive form of thyroid cancer. Despite a relatively low prevalence, it accounts for a disproportionate number of thyroid cancer-related deaths, due to its resistance to every therapeutic approach. We have generated a mouse model in which simultaneous selective activation of the PI3K pathway and inactivation of p53 in thyroid epithelial cells leads to the development of ATC. These tumors display all the typical features of their human counterpart and metastasize to the lungs in over 25% of cases (20 out of 76). Recent data have indicated that mammary tumors driven by PI3K activation quickly become resistant to PI3K inhibitors through mechanisms that include Myc amplification. We have used our model to test the hypothesis that Myc overexpression and/or amplification might contribute the aggressive features of PI3K-driven ATC. Analysis of 46 primary tumors developed by [Pten,p53]thyr-/− mice using a high-sensitivity real-time PCR approach demonstrates that the Myc locus is amplified (copy number ≥4) in about 25% of cases. Conversely, none of 20 metastatic follicular thyroid carcinomas developed by Ptenthyr-/− mice displayed Myc amplification. More strikingly, Myc is amplified in almost 50% (7/15) of ATCs with distant metastases, suggesting that Myc amplification confers more aggressive features to PI3K-driven anaplastic tumors, even in the absence of drug mediated selective pressure. Furthermore, when injected in syngeneic mice, cell lines established from Myc-amplified tumors grow faster than cell lines derived from tumors without Myc amplification. Myc-amplified cell lines are more resistant than Myc-diploid cells to the PI3K inhibitor BKM120, as well as to paclitaxel and doxorubicin treatment, and this increased resistance can be abrogated by shRNA-mediated Myc downregulation. These data support a model in which Myc cooperates with PI3K activation to induce more aggressive features, including drug resistance and enhanced metastatic behavior, in advanced thyroid carcinomas. Furthermore, these data lay the basis for new studies aimed at increasing therapeutic efficacy by interfering with Myc and Myc-dependent pathways. Citation Format: Marika A. Russo, Antonio Di Cristofano. MYC amplification and overexpression Is associated With metastasis and drug resistance in a mouse model of anaplastic thyroid carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5192. doi:10.1158/1538-7445.AM2013-5192

Ras inhibits endoplasmic reticulum stress in human cancer cells with amplified Myc

In neuroblastoma LAN-1 cells harboring an amplified MycN gene, disruption of cooperation between Ras and MycN proteins by the Ras inhibitor farnesylthiosalicylic acid (FTS, Salirasib) reportedly arrests cell growth. Our aim was to establish whether this is a general phenomenon. We examined the effects of FTS on gene-expression profiles, growth and death of NCIH929 myeloma cells and K562 leukemia cells, which-like LAN-1 cells-exhibit Myc gene amplification and harbor active Ras. Under specified conditions, FTS reduced Ras and Myc and induced cell growth arrest and death in all Myc-amplified cell lines but not in SHEP, a neuroblastoma cell line without Myc gene amplification. Gene-expression analysis revealed a common pattern of FTS-induced endoplasmic reticulum (ER) stress, known as the unfolded protein response (UPR), in Myc-amplified cells, but not in SHEP. Thus, Ras negatively regulates ER stress in cells with amplified Myc. ER stress was also inducible by dominant-negative (DN)-Ras or shRNA to Ras isoforms, all of which induced an increase in BIP (the master regulator of ER stress) and its downstream targets Nrf2 and eIF2alpha, both regulated by active p-PERK. FTS also induced an increase in p-PERK, while small interfering RNA to PERK reduced Nrf2 and ATF4 and rescued cells from FTS-induced death. BIP and its downstream targets were also increased by inhibitors of MAPK p38 and MEK. Ras, acting through MAPK p38 and MEK, negatively regulates the ER stress cascades BIP/PERK/Nrf2 and eIF2alpha/ATF4/ATF3. These findings can explain the Ras-dependent protection of Myc-amplified cells from ER stress-associated death.

N- myc oncogene amplification is correlated to trace metal concentrations in neuroblastoma cultured cells

N- myc oncogene amplification is a powerful predictor of aggressive behavior of neuroblastoma (NB), the most common solid tumor of the early childhood. Since N- myc overexpression – subsequent to amplification – determines a phenotype of invasiveness and metastatic spreading, it is assumed that N- myc amplified neuroblasts synthesize zinc metalloenzymes leading to tumor invasion and formation of metastases. In order to test a possible relation between N- myc oncogene amplification and trace metal contents in human NB cells, Fe, Cu and Zn concentrations have been measured by nuclear microprobe analysis in three human neuroblastoma cell lines with various degrees of N- myc amplification. Elemental determinations show uniform distribution of trace metals within the cells, but variations of intracellular trace metal concentrations with respect to the degree of N- myc amplification are highly dependent on the nature of the element. Zinc concentration is higher in both N- myc amplified cell lines (IMR-32 and IGR-N-91) than in the non-amplified cells (SK–N–SH). In contrast, intracellular iron content is particularly low in N- myc amplified cell lines. Moreover, copper concentrations showed an increase with the degree of N- myc amplification. These results indicate that a relationship exists between intracellular trace metals and N- myc oncogene amplification. They further suggest that trace metals very probably play a determinant role in mechanisms of the neuroblastoma invasiveness.

Development of a polyclonal antibody with defined specificity against synthetic peptides from the N-myc oncoprotein using multiple antigen peptide and hemocyanin conjugation methods

Background/Purpose: The importance of determining N-myc oncoprotein rather than genomic N- myc amplification has been emphasized in neuroblastoma, especially in an international project to register biological risk factors in all neuroblastomas. A method to raise a specific polyclonal antibody against the N-myc oncoprotein in large quantities was sought using the synthetic antigen peptide and the multiple antigen peptide (MAP) method. Methods: Two sets of peptides, HGRGPPTAGSTAQSPG and GVAPPRPGGROTSGGDH, conserved in the N-myc oncoprotein were synthesized. The hemocyanin-conjugated peptides and the lysine core-conjugated (multiple antigen peptide method) peptides were injected into rabbits with adjuvant. IgG fractions precipitated from the sera were purified on an affinity column coupled with these peptides, and the potency and specificity of the purified IgGs were examined by immunoblotting and immunohistochemistry in small cell lung cancer cell lines with known positivity-negativity of amplification and expression of N- myc, c- myc, and L- myc. Results: Peptides conjugated to the lysine core raised more potent antibodies than those conjugated to hemocyanin. Purified IgG against GVAPPRPGGRQTSGGDH reacted positively with an N- myc-amplified lung cancer cell line, but not with N- myc-unamplified and c- myc/L- myc-amplified cell lines on either immunoblotting or immunostaining. This IgG strongly stained the nuclei of cells in a series of surgical specimens and cell lines of neuroblastoma with N- myc amplification. Conclusion: A polyclonal antibody specific for a synthetic peptide from the N-myc oncoprotein was thus obtained and will find wide international use.