bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4460: Acquisition of stem cell-like properties during acquired resistance to epidermal growth factor receptor inhibitors.
Authors: Shien Kazuhiko , Toyooka Shinichi , Yamamoto Hiromasa , Soh Junichi , Thu Kelsie L. , Hashida Shinsuke , Maki Yuho , Ichihara Eiki , Asano Hiroaki , Tsukuda Kazunori , Takigawa Nagio , Kiura Katsuyuki , Gazdar Adi F. , Lam Wan L. , Miyoshi Shinichiro
Published: August 2014

Acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) is a critical issue in the treatment of lung cancer. While several mechanisms have been demonstrated to be responsible for acquired resistance, all mechanisms have not been uncovered. In this study, we investigated the molecular and cellular profiles of the acquired resistant cells to EGFR-TKI in EGFR mutant lung cancers. Four EGFR-mutant cell lines were exposed to gefitinib by stepwise escalation and high-concentration exposure methods, and resistant sublines to gefitinib were established. The molecular profiles and cellular phenotypes of these resistant sublines were characterized. Although previously reported alterations including secondary EGFR T790M mutation, MET amplification, and appearance of epithelial to mesenchymal transition (EMT) features were observed, these 2 drug-exposure methods revealed different resistance mechanisms. The resistant cells with EMT features exhibited down-regulation of microRNA-200c by DNA methylation. Furthermore, the HCC827-derived subline characterized by the high-concentration exposure method exhibited not only EMT features but also stem cell-like properties, including aldehyde dehydrogenase isoform 1 (ALDH1A1) overexpression, increase of side population, and self-renewal capability. Resistant sublines with stem cell-like properties were resistance to conventional chemotherapeutic agents but equally sensitive to histone deacetylase and proteasome inhibitors, compared with their parental cells. ALDH1A1 was up-regulated in clinical samples with acquired resistance to gefitinib. In conclusion, our study indicates that the manner of EGFR-TKI exposure influences the mechanism of acquired resistance and the appearance of stem cell-like property with EGFR-TKI treatment.

Citation Format: Kazuhiko Shien, Shinichi Toyooka, Hiromasa Yamamoto, Junichi Soh, Kelsie L. Thu, Shinsuke Hashida, Yuho Maki, Eiki Ichihara, Hiroaki Asano, Kazunori Tsukuda, Nagio Takigawa, Katsuyuki Kiura, Adi F. Gazdar, Wan L. Lam, Shinichiro Miyoshi. Acquisition of stem cell-like properties during acquired resistance to epidermal growth factor receptor inhibitors. [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 4460. doi:10.1158/1538-7445.AM2013-4460
Abstract 4486: 5-Hydroxymethyl-2’-deoxyuridine, but not temozolomide, enhances the selective synthetic lethality in BRCA1 and BRCA2-deficient cells caused by PARP inhibition.
Authors: Clement Jean , Nakamura Jun
Published: August 2014

Inhibition of poly(ADP-ribose) polymerase (PARP) has been determined as an attractive chemotherapeutic treatment for homologous recombination (HR)-deficient tumors. PARP is involved in repairing DNA single strand breaks (SSBs) via the base excision repair (BER) pathway; therefore, PARP inhibitor (PARPi) leads to an accumulation of DNA double strand breaks (DSBs) at the sites of SSBs during DNA replication. While the cytotoxic effect of PARPi was demonstrated to be potentiated by addition of genotoxic alkylating agent temozolomide (TMZ), it is not clear that this combined treatment further increases selectivity to cause cell death in HR-deficient cells, such as BRCA-deficient cells, over HR-proficient cells. TMZ randomly increases SSBs at scattered locations in the genome; in contrast, incorporation of 5-hydroxymethyl-2’-deoxyuridine (5-HmedU) in DNA has a potential to cause multiple SSBs within a short distance during BER. We hypothesized that a combination treatment of PARPi and 5-hmedU could increase selectivity of PARPi-mediated cytotoxicity in HR-deficient cells more so than the PARPi and TMZ combination. Here we show that in single chemical treatment experiments, the IC50 of 5-HmedU was eight times lower in BRCA1-/- and BRCA2-/- mutant cells, but in TMZ treatments the IC50 was only two times lower compared HR-proficient cells. Furthermore, the 5-HmedU and PARPi agent Olaparib combination enhanced a synergistic cell killing effect in BRCA1-/- and BRCA2-/- mutant cells with higher selectivity than the PARPi and TMZ combination. Advantages of the 5-HmedU and PARPi combination is as follows: 1] 5-HmedU causes DNA damage through vast genome incorporation in highly proliferating cells. Thus, 5-HmedU is more effective at selecting highly proliferating tumor cells; 2] 5-HmedU single treatment more selectively targeted BRCA1-/- and BRCA2-/- mutant cells than TMZ. This selectivity could be due to more efficient DSB formation by 5-HmedU through accumulation of clustered SSBs; and 3] combination treatment of 5-HmedU and PARPi produces a synergistic selectivity to kill HR-deficient BRCA1-/- and BRCA2-/- mutant cells whereas TMZ and PARPi combination does not. A possible mechanism of the selective cell killing effect by 5-HmedU and PARPi could result from an accumulation of closely located DSBs with PARP-DNA strand break complexes in HR-deficient cells.

Citation Format: Jean Clement, Jun Nakamura. 5-Hydroxymethyl-2’-deoxyuridine, but not temozolomide, enhances the selective synthetic lethality in BRCA1 and BRCA2-deficient cells caused by PARP inhibition. [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 4486. doi:10.1158/1538-7445.AM2013-4486
Abstract 4508: Novel multifunctional nanocarriers for drug delivery, photodynamic therapy, sonodynamic therapy, MRI and PET imaging.
Authors: Lam Kit S. , Li Yuanpei , Lin Tzu-yin , Zhang Hongyong , Feng Caihong , Luo Yan , Pan Chong-Xian , Xiao Kai , Xiao Wenwu , Lee Joyce , Liu Ruiwu
Published: August 2014

We have previously reported the development of a versatile nanocarrier platform comprised of PEG-cholic acid cluster telodendrimers that can self-assemble together with hydrophobic cytotoxic anti-cancer agents to form stable nanoparticle drugs. We have successfully loaded these nanoparticles with a number of different cancer drugs. These include paclitaxel, doxorubicin, vincristine, vinblastine, actinomycin D, bortezomib, temsirolimus, sarofinib, and lapatinib. Using electron paramagnetic resonance spectroscopy, we have demonstrated that the micelles can be disrupted by HDL, LDL, VLDL or chylomicron causing premature drug release. Such undesirable properties can be eliminated by cross-linking the telodendrimers with disulfide bonds or boronate-catechol linkages. Very recently, we have developed a series of novel highly versatile multi-functional nancarriers that not only allow efficient delivery of hydrophobic drugs, but can also (i) deliver proteins and nucleic acids to target cells, (ii) allow efficient photodynamic therapy and sonodynamic therapy, and (iii) function as sensitive MRI and PET imaging agents for the detection of cancers. Tumor cell surface binding ligands identified through one-bead-one-compound combinatorial chemistry can be easily incorporated into these nanocarriers for effective tumor targeting. On-demand releasable mechanism can also be built into such novel nanoplatforms for more efficacious therapy.

Citation Format: Kit S. Lam, Yuanpei Li, Tzu-yin Lin, Hongyong Zhang, Caihong Feng, Yan Luo, Chong-Xian Pan, Kai Xiao, Wenwu Xiao, Joyce Lee, Ruiwu Liu. Novel multifunctional nanocarriers for drug delivery, photodynamic therapy, sonodynamic therapy, MRI and PET imaging. [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 4508. doi:10.1158/1538-7445.AM2013-4508
Abstract 451: Smad2 is the TGF-β receptor-regulated Smad to suppress the immunogenicity of mouse and human dendritic cells.
Authors: Yoon Jeong-Hwan , Han Jin-Soo , Park Seok Hee , Weinstein Michael , Nakae Susumu , Yamashita Tadashi , Lee In-Kyu , Sumida Takayuki , Sudo Katsuko , Kuroda Masahiko , Mamura Mizuko
Published: August 2014

TGF-β plays pivotal roles in the regulation of DCs. However, the molecular mechanisms by which TGF-β regulates DCs are undetermined. Here, we show that one of the TGF-β receptor-regulated Smads (R-Smads), Smad2 is the predominant intermediary for TGF-β to suppress the immunogenicity of DCs. Notably, Smad2 remained expressed, whereas Smad3 was down-regulated in mouse BM-derived DCs (BMDCs) and human monocyte-derived DCs (MoDCs) upon differentiation by GM-CSF and IL-4. Smad2 mediated the inhibitory effect of TGF-β on the expression of co-stimulatory molecules (CD80, CD86, CD40) on BMDCs. One of the TGF-β superfamily cytokines, Activin-A did not affect their expression, although it shares Smad2 and Smad3 as R-Smads. Deletion of Smad2 up-regulated the dendrite formation, co-stimulatory molecule expression, and effector cytokine production by DCs, thereby efficiently activating allogeneic T cells. Furthermore, DC-specific deletion of Smad2 by Cd11cCre-loxP system suppressed the progression of a mouse EL4 lymphoma model by enhancing anti-lymphoma cytotoxic activity. Thus, our results suggest that TGF-β signaling through Smad2 in DCs could be a therapeutic target to enhance anti-tumor immunity.

Citation Format: Jeong-Hwan Yoon, Jin-Soo Han, Seok Hee Park, Michael Weinstein, Susumu Nakae, Tadashi Yamashita, In-Kyu Lee, Takayuki Sumida, Katsuko Sudo, Masahiko Kuroda, Mizuko Mamura. Smad2 is the TGF-β receptor-regulated Smad to suppress the immunogenicity of mouse and human dendritic cells. [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 451. doi:10.1158/1538-7445.AM2013-451
Abstract 4513: Engineering apoferritin nanocages for tumor imaging and therapy.
Authors: Xie Jin , Zhen Zipeng , Tang Wei
Published: August 2014

Ferritin is a major iron storage protein found in human and most living organisms. Each ferritin is comprised of 24 subunits, which self-assemble to form a cage-like nanostructure. Apoferritins (FRTs) refer to those that are without iron encapsulated. FRT is an interesting nanoplatform and holds great potential in cancer imaging and drug delivery for several reasons. 1) Biosafety. Compared to many other biomaterials, FRTs have fewer concerns in toxicity and immunogenicity. 2) Unique nanostructure. Despite the rigidness under physiological conditions, the FRT nanoarchitecture can be broken down into FRT subunits in an acidic environment (pH = 2). Interestingly, such a process is reversible. When the pH is tuned back to neutral, the FRT subunits will be reconstituted into a nanocage structure, and almost in an intact fashion. This feature can be harnessed to form hybrid nanostructures and to allow for efficient drug loading. 3) Easy surface modification. As a protein, FRT nanocages can be genetically modified to present a peptide sequence on the surface. In addition, FRTs have multiple lysine and cysteine groups on the surface that can be easily linked with functional molecules, such as a fluorescence dye or a targeting peptide. 4) High loading capacity. In addition to iron and transition metals, many small molecules are able to be encapsulated into FRT interiors efficiently. Making use of these unique characteristics, we recently developed several multifunctional FRT-based probes for tumor imaging. Very recently, we found that doxorubicin can be internalized into RGD modified FRTs (RFRTs) with high efficiency (up to 70 wt%). The Dox-loaded RFRTs kept integrin selectivity, which was confirmed by both in vitro and in vivo imaging. Therapeutic studies on U87MG subcutaneous models observed a good tumor growth inhibition rate (89.6%) and reduced cardiotoxicity.

Citation Format: Jin Xie, Zipeng Zhen, Wei Tang. Engineering apoferritin nanocages for tumor imaging and therapy. [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 4513. doi:10.1158/1538-7445.AM2013-4513
Abstract 4531: Nanoscale Artificial Antigen Presenting Cells for T Cell Immunotherapy.
Authors: Perica Karlo , Bieler Joan G. , De León Medero Andrés , Chiu Yen-Ling , Durai Malarvizhi , Niemöller Michaela , Assenmacher Mario , Richter Anne , Oelke Mathias , Schneck Jonathan
Published: August 2014

Artificial antigen presenting cells (aAPC), which deliver stimulatory signals to cytotoxic lymphocytes, are a powerful tool for adoptive immunotherapy and in vivo T cell stimulation. Thus far, bead-based aAPC have been synthesized by coupling T cell activating proteins such as CD3 or MHC-peptide to beads several microns in diameter. By reducing the size of aAPC, we anticipated more efficient delivery of these stimulatory signals to tumor and lymph nodes where antigen-specific T cells reside. We thus manufactured aAPC based on two nanoscale particle platforms: biocompatible iron-dextran paramagnetic particles, 50-100 nm in diameter, and avidin-coated quantum dot nanocrystals, less than 20 nm in diameter. Nanoscale aAPC induced antigen-specific T cell proliferation from both TCR transgenic mouse splenocytes and human, polyclonal peripheral blood T cells. In a subcutaneous mouse melanoma model, both iron-dextran particles and quantum dot nanocrystals mediated tumor rejection when injected in vivo. To our knowledge, this is the first description of a nanoscale, particle-based aAPC that induces T cell proliferation in vitro and leads to effective T cell stimulation and tumor rejection in vivo. Furthermore, nano-aAPC represent a novel platform for studying receptor-ligand interactions at the membrane-nanoparticle interface.

Citation Format: Karlo Perica, Joan G. Bieler, Andrés De León Medero, Yen-Ling Chiu, Malarvizhi Durai, Michaela Niemöller, Mario Assenmacher, Anne Richter, Mathias Oelke, Jonathan Schneck. Nanoscale Artificial Antigen Presenting Cells for T Cell Immunotherapy. [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 4531. doi:10.1158/1538-7445.AM2013-4531
Abstract 4545: Identifying neuroblastoma-specific ligands for cancer targeted therapy using a one-bead one-compound combinatorial approach.
Authors: Iskandar Andy , Duong Connie , Chen Cathy , Sanchez Eduardo , Olivos David , Lam Kit , Satake Noriko
Published: August 2014

Neuroblastoma is the most common extra-cranial tumor in children, with around 700 new diagnoses each year. High-risk patients have poor outcomes and only 30% survive despite the most intensive therapies available, which can also harm healthy cells. Thus, we are developing novel targeted therapies for neuroblastoma to increase treatment efficacy and limit damage to healthy cells.

We utilized the One-Bead One-Compound (OBOC) combinatorial split-mix method developed by Kit Lam (Lam et al., 1991) to identify unique ligands against neuroblastoma cells for therapeutic delivery. The OBOC libraries consist of hundreds to millions of 90 μm Tentagel beads, and each bead displays 1013 copies of a peptide on its surface. Thus, each bead has one unique compound on its exterior.

Twelve random OBOC libraries with varying peptide lengths (5 to 16 amino acids) were screened against three human neuroblastoma cell lines (SK-N-BE, SK-N-DZ, and IMR-32) to determine a library for more stringent screening of ligands. 37,500 beads from each library were screened against each cell line and the libraries averaged 2 positive candidates per 18,750 beads within 2 hours of incubation. The results also suggested preference for longer peptide libraries, but an intermediate length random decamer library was chosen for further screening due to both a good yield of positive beads and convenient sequencing.

Each neuroblastoma line was screened against 150,000 beads from the decamer library to identify potential ligands. Positive beads were selected, retested three times, and decoded using Edman degradation. Several cancer cell lines (A549 lung cancer, and Reh and Jurkat leukemia cell lines) were used as negative controls. Ten beads per cell line displaying high binding affinity were selected. Candidate beads with positive binding to all three neuroblastoma cell lines are currently being sequenced and the ligands will be synthesized. We plan to characterize the ligands for high affinity and specificity, as well as internalization capacity, and to identify their receptors.

Currently, there are very few targeted therapies for neuroblastoma. We believe our ligand discoveries can be developed for future novel targeted therapies in neuroblastoma.

Citation Format: Andy Iskandar, Connie Duong, Cathy Chen, Eduardo Sanchez, David Olivos, Kit Lam, Noriko Satake. Identifying neuroblastoma-specific ligands for cancer targeted therapy using a one-bead one-compound combinatorial approach. [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 4545. doi:10.1158/1538-7445.AM2013-4545
Abstract 4547: High-throughput screen to identify compounds targeted to embryonal rhabdomyosarcoma harboring mutant HRAS using cells derived from a pediatric Costello syndrome patient.
Authors: Cartledge Donna M. , Drake Katherine M. , Gripp Karen W. , Sol-Church Katia , Kolb E. Anders , Napper Andrew D.
Published: August 2014

Costello syndrome (CS) is a disorder caused by HRAS gene mutations. Substitutions in glycine residues at positions 12 or 13 of the HRAS gene lead to aberrant activation of the guanine-nucleotide binding protein, HRAS. Homozygous expression of mutant HRAS predisposes pediatric patients to early onset of cancer, especially embryonal rhabdomyosarcoma (ERMS), while heterozygosity at this locus results in non-malignant abnormalities. Since the mid 1970’s there has been only a modest increase in the 5-year survival rate for ERMS in the United States to no more than 70%, so new treatment strategies are needed. To address this need, we have acquired, propagated, and banked patient-derived ERMS cells harboring a homozygous p.G12A substitution and have used these cells to develop a high-throughput screening (HTS) assay to discover compounds that selectively kill cells bearing homozygous HRAS mutation. The assay employs luminescent detection of ATP using CellTiter-Glo to quantify cell viability. Cell number, growth conditions, and compound delivery were optimized to obtain a robust 72-hr cell growth assay. The potent cytotoxic agent dactinomycin was used to ensure reproducible sensitivity of the assay. The assay was validated for HTS by pilot screening of a library of 2,000 known drugs and bioactive natural products, and hits were confirmed by retesting in dose-response. Confirmed active compounds were tested for selectivity for ERMS harboring homozygous mutant HRAS in a panel of patient-derived and purchased cell lines: RD, an ERMS line not driven by HRAS mutation; RH36, a mutant HRAS (p.Q61K) ERMS line that is not derived from CS; fibroblasts derived from CS patients with heterozygous HRAS mutations (CS242, p.G12A and CS066, p.G12S), and normal fibroblasts. In future work, compounds identified by HTS as displaying selectivity for homozygous mutant HRAS-expressing ERMS cells will be evaluated in pairwise combinations and also in combination with currently used chemotherapeutics. Targeting mutant HRAS-activated cell growth could direct the design of a novel therapeutic strategy for treatment of children with ERMS.

Citation Format: Donna M. Cartledge, Katherine M. Drake, Karen W. Gripp, Katia Sol-Church, E. Anders Kolb, Andrew D. Napper. High-throughput screen to identify compounds targeted to embryonal rhabdomyosarcoma harboring mutant HRAS using cells derived from a pediatric Costello syndrome patient. [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 4547. doi:10.1158/1538-7445.AM2013-4547
Abstract 4556: NOSH-aspirin is a potent inhibitor of colon cancer cell growth: effects of positional isomerism.
Authors: Eschbach Erin K. , MacKessack-Leitch Andrew C. , Vannini Federica , Kodela Ravinder , Chattopadhyay Mitali , Kashfi Khosrow
Published: August 2014

Background: We recently reported the synthesis of NOSH-aspirin, a novel hybrid capable of releasing both nitric oxide (NO) and hydrogen sulfide (H2S). In NOSH-aspirin, the two moieties that release NO and H2S are covalently linked at the 1, 2 positions of acetyl salicylic acid, ie ortho-NOSH-aspirin. Here we compared the cell growth inhibitory properties of ortho-NOSH-aspirin to that of the 1,3 (meta-NOSH-aspirin) and the 1,4 (para-NOSH-aspirin) positional isomers. We also incorporated electron donating/withdrawing groups about the bezyl ring in order to evaluate the effect of substitution on stability and biological activity of these novel compounds. Methods: Cell line: HT-29 and HCT 15 human colon adenocarcimoa; Cell growth: MTT; Cell cycle phase distribution: Flow cytometry; Apoptosis: subdiploid (sub-G0/G1) peak in DNA content histograms; Proliferation: PCNA; ROS: measured hydrogen peroxide and super oxide by flow cytometry using DCFDA and DHE dyes. Results: All positional isomers of NOSH-aspirin were potent growth inhibitors of both cell lines. The IC50s for growth inhibition in nM at 24h were, HT-29: ortho-NOSH-ASA (0.04 ± 0.011), meta-NOSH-ASA (0.24 ± 0.11), para-NOSH-ASA (0.46 ± 0.17); significance between the groups were: o vs m P>0.05, o vs p P<0.01, m vs p P>0.05; HCT 15: ortho-NOSH-ASA (0.062 ± 0.006), meta-NOSH-ASA (0.092 ± 0.004), para-NOSH-ASA (0.37 ± 0.04); significance between the groups were: o vs m P>0.05, o vs p P<0.001, m vs p P<0.001. Electron donating/withdrawing groups significantly affected these IC50s. All positional isomers qualitatively had similar effects on proliferation, apoptosis, and caused G0/G1 cell cycle arrest in both colon cancer cell lines. The underlying mechanism for these observations appeared to be mediated through generation of intracellular H2O2 and superoxide anions as pretreatment of cells with N-acetylcysteine, partially blocked these effects. Conclusions: Positional isomerism affects the potency of NOSH-aspirin and offers a platform for drug design. Further, NOSH-aspirin merits further evaluation as an anti-cancer agent.

Citation Format: Erin K. Eschbach, Andrew C. MacKessack-Leitch, Federica Vannini, Ravinder Kodela, Mitali Chattopadhyay, Khosrow Kashfi. NOSH-aspirin is a potent inhibitor of colon cancer cell growth: effects of positional isomerism. [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 4556. doi:10.1158/1538-7445.AM2013-4556
Abstract 4568: Rapid assessment of TORC1 suppression predicts responsiveness to RAF and MEK inhibition in BRAF mutant melanoma.
Authors: Corcoran Ryan B. , Rothenberg Stephen M. , Hata Aaron N. , Faber Anthony C. , Winokur Daniel , Piris Adriano , Nazarian Rosalynn M. , Brown Ronald D. , Godfrey Jason T. , Mino-Kenudson Mari , Settleman Jeffrey , Wargo Jennifer A. , Flaherty Keith T. , Haber Daniel A. , Engelman Jeffrey A.
Published: August 2014

The clinical development and potent efficacy of selective RAF and MEK inhibitors has transformed the treatment of the ∼50% of melanoma patients whose tumors harbor BRAF mutations. However, a substantial percentage of these patients fail to respond to therapy, and all who respond eventually develop resistance. We found that suppression of TORC1 activity, as measured by decreases in ribosomal protein S6 phosphorylation (P-S6) in response to RAF or MEK inhibitors, was a highly effective predictor of sensitivity in BRAF mutant melanoma cell lines in vitro and in mouse xenografts. TORC1 and P-S6 were suppressed in response to RAF or MEK inhibitors in sensitive but not resistant melanoma cells, despite robust suppression of the MAPK pathway by these inhibitors. Notably, suppression of P-S6 levels, as assessed by immunohistochemistry in paired biopsies obtained from BRAF mutant melanoma patients before and during treatment, was associated with significantly improved progression-free survival. Finally, we show that changes in P-S6 levels specifically in tumor cells can be readily monitored by multiplexed, quantitative immunofluorescence microscopy of fine needle aspiration biopsies obtained from patients before and during the first two weeks of BRAF inhibitor therapy. This provides a minimally invasive means for monitoring the efficacy of treatment in real time, before changes in tumor volume are apparent on traditional radiographs. Together, these results establish changes in P-S6 levels with therapy as a robust biomarker to guide the treatment of patients with BRAF mutant melanoma, and present a powerful methodology for monitoring changes in potentially any signaling pathway in response to therapy in patients.

Citation Format: Ryan B. Corcoran, Stephen M. Rothenberg, Aaron N. Hata, Anthony C. Faber, Daniel Winokur, Adriano Piris, Rosalynn M. Nazarian, Ronald D. Brown, Jason T. Godfrey, Mari Mino-Kenudson, Jeffrey Settleman, Jennifer A. Wargo, Keith T. Flaherty, Daniel A. Haber, Jeffrey A. Engelman. Rapid assessment of TORC1 suppression predicts responsiveness to RAF and MEK inhibition in BRAF mutant melanoma. [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 4568. doi:10.1158/1538-7445.AM2013-4568
Abstract 4595: Activating mutations in the NT5C2 nucleotidase gene drive chemotherapy resistance in relapsed ALL.
Authors: Tzoneva Gannie , Perez-Garcia Arianne , Carpenter Zachary , Khiabanian Hossein , Tosello Valeria , Allegretta Maddalena , Paietta Elisabeth , Racevkis Janis , Rowe Jakob M. , Tallman Martin S. , Paganin Maddalena , Hoff Jana , Kirschner-Schwabe Renate , Palomero Teresa , Rabadan Raul , Ferrando Adolfo
Published: August 2014

Acute lymphoblastic leukemia (ALL) is an aggressive hematological tumor resulting from the malignant transformation of lymphoid progenitors. Despite intensive chemotherapy, 20% of pediatric and over 50% of adult ALL patients fail to achieve a complete remission or relapse after intensified chemotherapy, making disease relapse and resistance to therapy the most significant challenge in the treatment of this disease. Using whole exome sequencing, here we identify mutations in NT5C2, a 5’-nucleotidase enzyme responsible for inactivation of nucleoside analog chemotherapy drugs, in 20/103 (19%) relapse T-ALLs and in 1/35 (3%) relapse B-precursor ALLs analyzed. NT5C2 mutant proteins show increased nucleotidase activity in vitro and conferred resistance to chemotherapy with 6-mercaptopurine and 6-thioguanine when expressed in ALL lymphoblasts. These results support a prominent role for activating mutations in NT5C2 and increased nucleoside analog metabolism in disease progression and chemotherapy resistance in ALL.

Citation Format: Gannie Tzoneva, Arianne Perez-Garcia, Zachary Carpenter, Hossein Khiabanian, Valeria Tosello, Maddalena Allegretta, Elisabeth Paietta, Janis Racevkis, Jakob M. Rowe, Martin S. Tallman, Maddalena Paganin, Jana Hoff, Renate Kirschner-Schwabe, Teresa Palomero, Raul Rabadan, Adolfo Ferrando. Activating mutations in the NT5C2 nucleotidase gene drive chemotherapy resistance in relapsed ALL. [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 4595. doi:10.1158/1538-7445.AM2013-4595
Abstract 4610: Small molecule inhibitors of membrane-bound MCL-1 and BCL-2.
Authors: Patel Luv G. , Paranal Ronald M. , Ryan Jeremy , Sarosiek Kristopher , Bradner James E. , Letai Anthony
Published: August 2014

Small molecule inhibitors of membrane-bound MCL-1 and BCL-2

Mcl-1 and Bcl-2 are anti-apoptotic members of the Bcl-2 family of proteins. These proteins sequester activators of Bax-Bak oligomerization and inhibit the subsequent mitochondrial depolarization. Knockdown of anti-apoptotic Bcl-2 family proteins has induced apoptosis in a variety of cancers, signifying that these malignancies are dependent on these proteins for survival. Thus, their inhibition by small molecules is an emerging strategy in cancer therapy. While Bcl-2 specific and pan-inhibitors have emerged, there remains a dearth of molecules that effectively inhibit Mcl-1. In fact, increased Mcl-1 expression is a common mechanism of resistance to these therapies.

Previous efforts to identify small molecules that inhibit MCL-1 or BCL-2 function have exploited assays using soluble, truncated, bacterially-expressed recombinant proteins. Since in vivo MCL-1 and BCL-2 exist at full length and are membrane bound, we thought we could improve the identification of biologically active small molecules by screening in a more physiologically relevant environment. It is likely that membrane association affects the structure and access of binding sites. We have therefore developed a screening assay to assess inhibition of full-length Mcl-1 and Bcl-2 in a membrane-bound state. The screen identified compounds that selectively permeabilize membranes whose integrity depends on either MCL-1 or BCL-2. The screen was optimized for high-throughput 384-well format. Positive controls for screening were peptides representing the BH3 domains of Bad and Noxa, known to be selective inhibitors of Bcl-2 and Mcl-1, respectively. We screened >70,000 compound wells. These compounds include an assortment of known bioactives, natural product extract mixtures, and commercial library compounds. We have identified several lead compounds from the primary screen and have validated their performance in biochemical and biological assays.

Citation Format: Luv G. Patel, Ronald M. Paranal, Jeremy Ryan, Kristopher Sarosiek, James E. Bradner, Anthony Letai. Small molecule inhibitors of membrane-bound MCL-1 and BCL-2. [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 4610. doi:10.1158/1538-7445.AM2013-4610
Abstract 4616: Oncogenic c- and N-Myc disrupt circadian rhythm.
Authors: Altman Brian J. , Hsieh Annie , Gouw Arvin , Venkataraman Anand , Li Bo , Bellovin David , Simon M. Celeste , Felsher Dean , Hogenesch John , Dang Chi V.
Published: August 2014

Circadian rhythms are regulated by feedback loops comprising a network of factors that regulate Clock-associated genes. Chronotherapy seeks to take advantage of altered circadian rhythms in some cancers to better time administration of treatments to increase efficacy and reduce toxicity. Taking advantage of cancers that have substantially different circadian rhythms, or are ‘out of phase’, with normal tissues, could open a wide therapeutic window to make them vulnerable to chemotherapy or targeted drugs at different times than normal tissue. However, there is currently no basis to identify which cancers have disrupted circadian rhythms and would be amenable to chronotherapy. c- and N-Myc are oncogenic transcription factors translocated or amplified in many cancers. While the role of Myc in circadian rhythm is currently unknown, it may affect circadian rhythm by binding to the same E-box promoter regions used by the central regulators of circadian rhythm, Clock/Bmal1. Additionally, Myc increases NAD+ levels through upregulation of NAMPT, and NAD+ is a crucial cofactor in the activity of the circadian regulator Sirt1. Thus, we hypothesized that Myc may disrupt circadian rhythm through two mechanisms: inappropriate engagement of E-box promoters and also upregulation of NAMPT leading to dysregulated Sirt1 activity.

Here we show in neuroblastoma, osteosarcoma, and hepatocellular carcinoma cells that overexpressed Myc specifically upregulated the negative circadian regulator Rev-erbα, which in turn decreased expression of Bmal1. Inhibition of NAMPT downstream of Myc upregulation also led to major perturbations in circadian gene expression, suggesting a role for NAD modulation downstream of Myc in disruption of circadian rhythm. Importantly, My-expressing cells showed dramatically disrupted circadian oscillations, which could be partially rescued by inhibiting expression of Rev-erbα. Together, these data suggest that Myc-driven cancers have altered circadian oscillation due to upregulation of Rev-erbα and NAMPT, and that cancers driven by Myc may thus be good candidates for chronotherapy.

We thank the following funding sources: NIH R01CA051497, R01CA57341, LLS 636311

Citation Format: Brian J. Altman, Annie Hsieh, Arvin Gouw, Anand Venkataraman, Bo Li, David Bellovin, M. Celeste Simon, Dean Felsher, John Hogenesch, Chi V. Dang. Oncogenic c- and N-Myc disrupt circadian rhythm. [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 4616. doi:10.1158/1538-7445.AM2013-4616
Abstract 4621: The histone methyltransferase EZH2 catalytic activity is required for cell growth in diffuse large B-cell lymphoma.
Authors: Audia James E. , Trojer Patrick , Garapaty Shivani , Lan Fei , Balasubramanian Vidya , Chan Eric , Hatton Charles , Campbell Robert , Cummings Richard , Normant Emmanuel , Bryant Barbara , Albrecht Brian , Harmange Jean Christophe , Dakin Les , Gehling Victor , Nasveschuk Chris , Vaswani Rishi , Cook Andrew
Published: August 2014

Lysine methyltransferases and demethylases were identified as transcriptional co-regulators functioning by either preserving particular chromatin methylation states or by controlling placement and removal of histone lysine methylation marks to promote dynamic changes in gene expression. The development of small molecule methyltransferase and demethylase inhibitors provides a novel approach to affect the regulation of transcription, and thus potentially allowing interference with aberrant transcriptional programs as observed, for instance, in cancer.

Enhancer of Zeste Homolog 2 (EZH2), the major histone H3 lysine 27 (K27) methyltransferase, is widely implicated in tumor progression. The presence of a recurrent mutation of a single tyrosine residue in the EZH2 catalytic domain in germinal center B-cell like diffuse large B-cell lymphoma (GCB-DLBCL) and follicular lymphoma suggests that these cancers might be dependent on altered EZH2 molecular function, the mutation facilitating the conversion of an H3K27 di-methylated to a tri-methylated state. Inhibition of the EZH2 catalytic activity will provide a new therapeutic approach to treat human cancers, especially lymphomas carrying activating mutations.

Constellation has identified, characterized and optimized potent, selective and reversible EZH2 small molecule inhibitors as well as studied the biological impact of such inhibition. We find that pharmacological inhibition of EZH2 causes selective cell viability defects with cell lines harboring EZH2 mutations being the most sensitive. Our EZH2 inhibitors potently engage the target in vivo and exhibit efficacy in lymphoma xenograft models. Genome-wide mapping of EZH2 and H3K27me3 sites in the absence and presence of the compound revealed that the EZH2 inhibitor caused significant changes to the local chromatin modification landscape, however only a subset of these alterations translated into gene expression changes. The discovery of additional contexts that define sensitivity to EZH2 inhibitors will be discussed.

Citation Format: James E. Audia, Patrick Trojer, Shivani Garapaty, Fei Lan, Vidya Balasubramanian, Eric Chan, Charles Hatton, Robert Campbell, Richard Cummings, Emmanuel Normant, Barbara Bryant, Brian Albrecht, Jean Christophe Harmange, Les Dakin, Victor Gehling, Chris Nasveschuk, Rishi Vaswani, Andrew Cook. The histone methyltransferase EZH2 catalytic activity is required for cell growth in diffuse large B-cell lymphoma. [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 4621. doi:10.1158/1538-7445.AM2013-4621
Abstract 4622: Targeting MYCN in Neuroblastoma by BET Bromodomain Inhibition.
Authors: Puissant Alexandre , Frumm Stacey M. , Alexe Gabriela , Bassil Christopher F. , Qi Jun , Chanthery Yvan H. , Nekritz Erin A. , Zeid Rhamy , Gustafson W. Clay , Greninger Patricia , Garnett Matthew J. , McDermott Ultan , Benes Cyril H. , Kung Andrew L. , Weiss William A. , Bradner James E. , Stegmaier Kimberly
Published: August 2014

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

Citation Format: Alexandre Puissant, Stacey M. Frumm, Gabriela Alexe, Christopher F. Bassil, Jun Qi, Yvan H. Chanthery, Erin A. Nekritz, Rhamy Zeid, W. Clay Gustafson, Patricia Greninger, Matthew J. Garnett, Ultan McDermott, Cyril H. Benes, Andrew L. Kung, William A. Weiss, James E. Bradner, Kimberly Stegmaier. Targeting MYCN in Neuroblastoma by BET Bromodomain Inhibition. [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 4622. doi:10.1158/1538-7445.AM2013-4622
Abstract 4630: Feedback dependent suppression of mitogenic signaling and its effect on RAF inhibition in BRAFV600E melanomas.
Authors: Lito Piro , Pratilas Christine , Joseph Eric , Tadi Madhavi , Halilovic Ensar , Zubrowski Matthew , Huang Alan , de Stanchina Elisa , Chandarlapaty Sarat , Poulikakos Poulikos , Rosen Neal
Published: August 2014

RAF inhibitors have remarkable activity in melanomas harboring BRAFV600E mutations. We previously found that active Ras induces RAF dimers in tumors with wild-type RAF, and, that RAF inhibitors transactivate these dimers. Because BRAFV600E melanomas have enhanced ERK signaling, they are hypothesized to have feedback-mediated suppression of Ras in order to maintain monomeric and inhibitor-sensitive BRAFV600E. To test this hypothesis we evaluated the response of these tumors to RAF inhibitors over time. We found that in the treatment-naive state, BRAFV600E melanomas had suppressed RTK signaling and maintained a low level of Ras-GTP. This occurred through a multifactorial negative feedback process driven by active ERK. In this baseline state, BRAFV600E was sensitive to RAF inhibitors. After treatment with RAF inhibitors, however, inhibition of ERK signaling lead to loss of feedback, a process that restored signaling from RTKs and resulted in Ras activation. In turn, this diminished the effect of RAF inhibitors and lead to a partial, but sustained, reactivation of ERK signaling. In this post-treatment state, ERK signaling was insensitive to RAF inhibitors but sensitive to MEK inhibitors. Targeting the regulatory components of this new steady state, either through co-administration of a MEK inhibitor or a RTK inhibitor along with a RAF inhibitor resulted in improved antitumor activity. These results highlight the need to understand the adaptive response to ERK pathway inhibition in order to design better combinatorial therapies and achieve maximal antitumor effects.

Citation Format: Piro Lito, Christine Pratilas, Eric Joseph, Madhavi Tadi, Ensar Halilovic, Matthew Zubrowski, Alan Huang, Elisa de Stanchina, Sarat Chandarlapaty, Poulikos Poulikakos, Neal Rosen. Feedback dependent suppression of mitogenic signaling and its effect on RAF inhibition in BRAFV600E melanomas. [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 4630. doi:10.1158/1538-7445.AM2013-4630
Abstract 4668: Antitumor efficacy of AZD1480 in head and neck squamous cell carcinoma.
Authors: Sen Malabika , Black John , Wheeler Sarah , Freilino Maria L. , Joyce Sonali , Lui Vivian , Zeng Yan , Marshall Korey , Grandis Jennifer R.
Published: August 2014

Aberrant activation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway has been implicated in cancer cell proliferation and survival. AZD1480, a pharmacologic inhibitor of JAK2, has been tested in several cancer models including glioblastoma, lung, renal, prostate, breast, and ovarian cancer. However JAK inhibitors, including AZD1480 has not been evaluated in head and neck squamous cell cancer (HNSCC). In the present study, the in vitro and in vivo efficacy of AZD1480 was evaluated in HNSCC preclinical models. AZD1480 decreased HNSCC proliferation in cell lines with varying levels of pJAK2 and pSTAT3 with an IC50 ranging from 0.9μM - 4μM. Further evaluation demonstrated downmodulation of pJAK2 and pSTAT3 in HNSCC cell lines by AZD1480. Furthermore, in vivo antitumor efficacy of AZD1480 was tested in heterotopic xenograft tumor models derived from 2 HNSCC patient tumors. AZD1480 at a dose of 30mg/kg, was given twice daily via oral gavage and compared with vehicle (0.5% Hypromellose) control. Tumors treated with AZD1480 exhibited significant growth inhibition relative to the tumors treated with vehicle (p=0.0037). In addition, significant decrease in pSTAT3Tyr705 expression was observed in both the patient tumors treated with AZD1480 compared to vehicle alone indicating that AZD1480 targets pSTAT3 and may be considered for study in the treatment of HNSCC patients.

Citation Format: Malabika Sen, John Black, Sarah Wheeler, Maria L. Freilino, Sonali Joyce, Vivian Lui, Yan Zeng, Korey Marshall, Jennifer R. Grandis. Antitumor efficacy of AZD1480 in head and neck squamous cell 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 4668. doi:10.1158/1538-7445.AM2013-4668
Abstract 468: Phenotyping TILs in situ: Automated enumeration of intra- and extra-follicular Foxp3+ regulatory T cells in follicular lymphoma.
Authors: Mansfield James , Nelson Lilli , van der Loos Chris , Byers Richard
Published: August 2014

Background. In many cancers, tumor-infiltrating lymphocytes (TILs) indicate levels of tumor immunogenicity and are a strong predictor of survival. In particular, increased levels of regulatory T cells (Tregs) are associated with poorer prognosis in some cancers. An understanding of the phenotype and spatial distribution of TILs in situ within tumor regions would be advantageous. However, visual TIL assessment cannot easily determine the type of lymphocyte in situ and multimarker quantitation is difficult with standard methods. Here we present a multi-marker, computer-aided event-counting method for determining the phenotypes of lymphocytes in follicular lymphoma sections using a multispectral imaging (MSI) and automated tissue segmentation and counting approach.

Design. A single section of a tissue microarray containing 70 follicular lymphoma cores from [42 male, 28 female, 17 alive and 53 dead at the end of followup, survival range 2-171 months (median 55 months)] was stained for CD3, Foxp3 and hematoxylin. Each core was imaged using MSI and the individual staining of each marker separated from each other using spectral unmixing. The images were analyzed using software which had been trained to recognize the follicular areas based on the tissue morphology. Then the Foxp3 status of each CD3+ TIL was then determined and the number of each Treg (Foxp3+ T cell) counted for both the intra- and extra-follicular tissue compartments.

Results. Results indicate that machine-learning software can be trained to accurately recognize follicular and non-follicular regions within each core. MSI enabled the accurate quantitation of two immunostains in the sample without crosstalk. The number of Tregs were determined for each core and ranged from 0 to 453 per core (median 58, stddev 97.5).

Conclusion. Understanding the number and location (intra- and extra-follicular) of Tregs is an assay with potentially important clinical prognostic implications. Thus study shows that an automated method for counting Tregs can be developed for follicular lymphoma. This multimarker phenotyping and counting approach shows the potential for broad applicability in the enumeration of a wide range of specifically phenotyped TILs in situ in many solid tumors.

Citation Format: James Mansfield, Lilli Nelson, Chris van der Loos, Richard Byers. Phenotyping TILs in situ: Automated enumeration of intra- and extra-follicular Foxp3+ regulatory T cells in follicular lymphoma. [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 468. doi:10.1158/1538-7445.AM2013-468