bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3588: Identification of a potent, orally bioavailable and selective MCT4 Inhibitor for the treatment of solid Warburg tumors
Authors: Hambruch Nina , Herkert Barbara , Gege Christian , Mallinger Aurelie , Fabian Johannes , Kinzel Olaf , Wang Yansong , Fink Gero , Albers Michael , Funk Anette , Bittman-Waetzig Annika , Fleckenstein Tilly , Braun Floriane , Kremoser Claus
Published: August 2019

A key metabolic alteration in tumor cells is an elevated glycolysis rate, resulting in the production of increased amounts of protons and lactate leading to lactic acidosis within the tumor microenvironment. The tumor-promoting effects of lactic acidosis are versatile, ranging from stimulation of proliferation, angiogenesis and metastasis to the reshaping of the tumor immune environment to a tumor-permissive phenotype. In tumors lactate is transported across the plasma membrane by the monocarboxylate transporters 1 and 4 (MCT 1/4) in symport with a proton. While MCT1 is expressed almost ubiquitously at low levels, MCT4 expression is regulated by hypoxia-inducible factor 1α (Hif1α), the central player in the cellular response to hypoxia. Thus, MCT4 is present in hypoxic, highly glycolytic cells. Given their central role in prevention of intracellular acidification and lactate transport, MCTs appear to be indispensable for glycolytic tumor growth. Since MCT4 has the strongest implication on the development and aggressiveness of diverse cancer types it has emerged as a new target for the treatment of solid tumors.

We have identified MCT4 specific inhibitors structurally based on an internal compound library using cell-based lactate export assays. A subsequent extensive medicinal chemistry program was able to significantly improve activity and selectivity of the primary structures. Cellular target engagement was verified by cell-based assays like cellular thermal shift assay (CETSA). These efforts resulted in the identification of highly potent, orally bioavailable and selective MCT4 inhibitors (MCT4 IC50 < 20nM; MCT1 IC50 > 100µM) with good physicochemical properties and beneficial pharmacokinetics in mouse.

In vivo efficacy and tumor lactate modulation were evaluated in NCI-H358 xenograft and MC38 syngeneic mouse models. Thereby we explore the potential of our MCT4 inhibitor to act as a single anti-cancer agent or to synergize with other established therapies like angiogenesis inhibitors or cancer immunotherapeutics.

Citation Format: Nina Hambruch, Barbara Herkert, Christian Gege, Aurelie Mallinger, Johannes Fabian, Olaf Kinzel, Yansong Wang, Gero Fink, Michael Albers, Anette Funk, Annika Bittman-Waetzig, Tilly Fleckenstein, Floriane Braun, Claus Kremoser. Identification of a potent, orally bioavailable and selective MCT4 Inhibitor for the treatment of solid Warburg tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3588.
Abstract 3590: Mechanisms and inhibition RIOK2 for obesity-driven prostate cancer
Authors: Macias Everardo , Cho Jiyoon , Rosowicz Drew , Heidari Zahra , You Sungyong , Maravilla Erick J. , Chi Jen-Tsan , Freedland Stephen J.
Published: August 2019

Obesity is associated with greater risk of high-grade prostate cancer (PC), recurrence after therapy, metastases, and PC specific mortality. The fact that obesity is a PC risk factor creates an opportunity to identify and target the mechanisms underlying obesity-enhanced lethal PC. We exploited this link by performing an shRNA in vivo screen in obese mice targeting the entire kinome, which found Right Open Reading Frame Kinase 2 (RIOK2) as essential for PCs in obese mice. We found RIOK2 gene signature is correlated with a 56% increased risk of metastatic PC. Moreover, when we queried only men with a BMI ≥ 30 kg/m2 we found that high levels of RIOK2 gene signature increased the risk of metastasis by an additional 50%. RIOK2, a serine/threonine kinase, is critical for de novo ribosome maturation and assembly, which is essential to maintain the high proliferation rates in cancer cells. Indeed, we found that RIOK2 loss reduced de novo protein synthesis in PC cells and decreased cell viability. Conversely, RIOK2 overexpression enhanced PC cell growth. As our goal is to find actionable PC targets to reduce obesity effects on PC, we conducted a protein structure based compound screen in silico and identified a lead kinase inhibitor of RIOK2 (kiRIOK2). Our lead kiRIOK2 inhibits cell proliferation, protein synthesis, anchorage-independent growth and invasion of multiple PC lines in the µM range. SAR optimized kiRIOK2 have increased efficacy and bioavailability. Our studies are providing probes and mechanistic insights to further investigate the role of RIOK2 in PC biology. In the long-term, this may help mitigate the added risk of obesity on PC metastasis and PC-specific mortality.

Citation Format: Everardo Macias, Jiyoon Cho, Drew Rosowicz, Zahra Heidari, Sungyong You, Erick J. Maravilla, Jen-Tsan Chi, Stephen J. Freedland. Mechanisms and inhibition RIOK2 for obesity-driven prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3590.
Abstract 3593: PDK1 is a preferential metabolic target in glioblastoma
Authors: Guda Maheedhara R. , Asuthkar Swapna , Tsung Andrew J. , Velpula Kiran K.
Published: August 2019

Glioblastoma (GBM), the most common adult primary malignant brain tumor, carries a high morbidity and mortality despite therapeutic advances. GBM’s progression is associated with a transition from oxidative phosphorylation to aerobic glycolysis as its main source of energy. Our laboratory recently confirmed that overexpression of pyruvate dehydrogenase kinase isoform 1 (PDK1) positively contributes to GBM progression. Here we report a novel role of mitofusin 1 (MFN1), a mitochondrial outer membrane GTPase that is a key regulator of mitochondrial fusion associated with glycolysis. We have shown that both PDK1 and MFN1 are highly co-expressed in glioblastoma patient specimens. Data obtained from The Cancer Genome Atlas demonstrated a positive correlation between PDK1 and MFN1 mRNA levels and the survival of GBM patients. The data obtained from the subtyped glioma stem cells corroborated with the datamining studies. To directly explore the potential MFN1-PDK1 interplay, we used CRISPR/Cas9 to suppress MFN1 expression in proneural, classical and mesenchymal subtypes of GBM stem cells (CSC). In another experiment, using ChIP assay, we showed that both PDK1 and MFN1 are under the transcriptional control of Myc oncogene. We present evidence that silencing MFN1 regulates altered metabolism in GBM CSC by increasing ROS levels, altering mitochondrial membrane potential, reduced lactate metabolites via NMR spectroscopy. Taken together, our results indicate that combined targeting of PDK1 and MFN1 shift Warburg effect towards oxidative phosphorylation, and may have increased therapeutic potential for glioblastoma patients.

Citation Format: Maheedhara R. Guda, Swapna Asuthkar, Andrew J. Tsung, Kiran K. Velpula. PDK1 is a preferential metabolic target in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3593.
Abstract 3598: Phosphoglycerate mutase 1 inhibitor causes metabolic disadvantages in glioblastoma by rescue of FBP1 repression
Authors: Lee Sungmin , Kim Hyunwoo , Kang Hyunkoo , Youn BuHyun
Published: August 2019

Radiotherapy is a primary treatment for patients with glioblastoma (GBM). Despite high therapeutic efficacy, the tumor cells that survive after radiotherapy may cause side effects. In this study, we found that fructose 1,6-bisphosphatase 1 (FBP1) which is a rate-limiting enzyme in gluconeogenesis was downregulated upon treatment with ionizing radiation (IR). IR-induced infiltrating GBM has overexpressed Ets1 which was suggested to be a transcriptional repressor of FBP1. Besides, glucose uptake and extracellular acidification rates were increased upon FBP1 downregulation, which indicated an elevated glycolysis level. We found that emodin, an inhibitor of phosphoglycerate mutase 1 derived from natural substances, significantly suppressed the glycolysis rate and the IR-induced GBM aggressiveness in the in vivo orthotopic xenograft mouse models. We propose that the downregulation of FBP1 level reprogrammed the metabolic state of GBM, and thus, FBP1 is a promising therapeutic target regulating GBM metabolism following radiotherapy.

Note: This abstract was not presented at the meeting.

Citation Format: Sungmin Lee, Hyunwoo Kim, Hyunkoo Kang, BuHyun Youn. Phosphoglycerate mutase 1 inhibitor causes metabolic disadvantages in glioblastoma by rescue of FBP1 repression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3598.
Abstract 3600: Antibiotics suppress growth of breast cancer cells and synergize cytotoxicity of 2-Deoxy-D-glucose: Treating cancer like an infection
Authors: Jiang Xianpeng , Benoit Amanda , Jiang Toby , Elliott Robert L.
Published: August 2019

Mitochondria evolved from free-living bacteria via endocytosis within a eukaryotic host cell millions of year ago. We hypothesized that antibiotics might damage mitochondria of human cancer cells and lead to oxidative damage of cancer cells. We examined three classes of antibiotics, azithromycin, doxycycline and ciprofloxacin in the human breast cancer cell lines MCF-7 and MDA-MB-231. The combination of antibiotics and the anti-glycolytic agent, 2-Deoxy-D-glucose (2-DG) was also tested in the cells. Cell growth was measured by MTT [3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide] assay. Mitochondrial membrane potential and reactive oxygen species were tested by fluorescent microscopy. Gene expression and DNA damage were measured by real time polymerase chain reaction (qPCR) and ELISA. All antibiotics suppressed mitochondrial membrane potential and growth of MCF-7 and MDA-MB-231 cells in a dose dependent pattern. The IC50of all three antibiotics were less than clinical patient’s maximum serum concentration (Cmax) or concentrations in tissues. The IC50 of ciprofloxacin in MCF-7 (1.25-2.5µM) and MDA-MB-231 (2.5-3.5µM) are much lower than the Cmax 7.7µM while patients take 500mg every 12 hours orally in clinical practice. The DNA oxidative damage metabolite, 8-OHdG, in the media was significantly increased after cancer cells were cultured in the media containing antibiotics for 24 hours. Three antibiotics upregulated gene expression of glycolytic enzymes including hexokinase 2(HK2), phosphofructokinase 1 (PFKM) and pyruvate kinase muscle isozyme M2 (PKM2), lactate dehydrogenase A (LDHA), and glucose transporters 1 (SLC2A1) and 3 (SLC2A3). The antibiotics increased uptake of 3[H]-2-DG of MCF-7 and MDA-MB-231. These results suggest that cancer cells increase aerobic glycolysis to produce ATP after antibiotic treatment. Thus, we combined 2-DG and antibiotics to treat the breast cancer cell lines. The antibiotics and 2-DG synergistically inhibited growth of the breast cancer cells. MDA-MB-231, a chemo-drug resistant cell line, has similar sensitivity to antibiotics and 2-DG as the MCF-7, a drug sensitive cell line. In summary, azithromycin, doxycycline and ciprofloxacin suppress the growth of breast cancer cells and augment the cytotoxicity of 2-DG to breast cancer cells. These findings suggest we can treat cancer with antibiotics similar to treating an infection.

Citation Format: Xianpeng Jiang, Amanda Benoit, Toby Jiang, Robert L. Elliott. Antibiotics suppress growth of breast cancer cells and synergize cytotoxicity of 2-Deoxy-D-glucose: Treating cancer like an infection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3600.
Abstract 3603: Sphingomyelin synthase inhibition in sarcomas results in ceramide accumulation and apoptosis
Authors: Barrott Jared J. , Meldrum Trevor , Afrin Farjana , Pashikanti Srinath
Published: August 2019

Sphingolipid metabolism is critical to cellular homeostasis especially as cells undergo mitosis. It is, therefore, unsurprising that the sphingolipid pathway is upregulated in a number of a cancers. Sphingoid bases such as sphingosine and ceramide are rapidly synthesized to provide the building blocks of other sphingolipids. Inhibition of downstream enzymes that convert sphingosine and ceramide into sphingolipids can lead to an increase of ceramide, which results in cellular toxicity and initiation of apoptosis. Using the marine natural product Jaspine B and chemically synthesized analogues, we investigated the potency of inhibiting sphingomyelin synthase in sarcoma cell lines. Sphingomyelin synthase is a downstream enzyme that converts ceramide into sphingomyelin by adding the polar head group phosphorylcholine to ceramide. Jaspine B and its analogues exhibited submicromolar potency against several human sarcoma cell lines. It was determined that the inhibition of sphingomyelin synthase resulted in an increase in ceramide and sphingosine levels and that sphingomyelinase enzymatic activity increased. While the mitochondria mass remained the same, the membrane potential of the mitochondria was significantly affected by the presence of the inhibitors. In conclusion, inhibitors of sphingolipid metabolism are viable therapeutic options with high untapped potential to treat cancer.

Citation Format: Jared J. Barrott, Trevor Meldrum, Farjana Afrin, Srinath Pashikanti. Sphingomyelin synthase inhibition in sarcomas results in ceramide accumulation and apoptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3603.
Abstract 3607: Inhibition of mutant IDH enzymes reduces production of 2-HG but does not restore wild type IDH activity in vitro or in vivo
Authors: Sellers Katie , Sleger Taryn , Ronseaux Sebastien , Narayanaswamy Rohini , Hudson Christine , Roddy Thomas , Nicolay Brandon
Published: August 2019

Mutations in isocitrate dehydrogenase (IDH) 1 and 2 occur in variety of malignancies, and notably >70% of low grade gliomas. These mutations lead to neomorphic enzymatic activity that results in the production of the oncometabolite (D)-2-hydroxyglutarate (2-HG) through the consumption of NADPH. IDH mutant cells and tumors have been shown to have heightened sensitivity to reactive oxygen species (ROS) and radiation, though it is unclear if this sensitivity is due to a loss of IDH wild type enzymatic NADPH production, the neoenzymatic activity of the mutant enzyme, or a combination of these activities. In support of the hypothesis that sensitivity to ROS and radiation is due to a reduced IDH wild type activity in IDH mutant cells, we previously demonstrated that combined treatment of mutant IDH mouse xenografts with AG-881, a potent orally available mutant IDH inhibitor, and radiation resulted in superior tumor growth inhibition than either modality alone. To extend this investigation, we have now developed a novel assay to read out the wild type activity of IDH1/2 enzymes in both cell-based assays and mouse xenograft models. Using an isotopic labeling approach that allows us to fully differentiate the activity of wild type IDH1/2 from the mutant enzymatic activity in the same cell or tumor, we observed that mutant IDH cells and tumors have a significantly reduced level of wild type IDH1/2 activity compared to normal tissue or IDH wild type tumors. Importantly, pharmacologic inhibition of mutant IDH1/2 that resulted in >90% reduction in 2-HG production in mutant cells and tumors did not restore the IDH1/2 wild type activity. These data support our previous observations and suggest that therapeutic strategies targeting the reduced activity of wild type IDH enzymes, such as modalities that induce ROS or treatment with radiation, would be effective when combined with mutant IDH inhibitors.

Citation Format: Katie Sellers, Taryn Sleger, Sebastien Ronseaux, Rohini Narayanaswamy, Christine Hudson, Thomas Roddy, Brandon Nicolay. Inhibition of mutant IDH enzymes reduces production of 2-HG but does not restore wild type IDH activity in vitro or in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3607.
Abstract 3610: Cellular fatty acid uptake is enhanced in tamoxifen-resistant breast cancer cells
Authors: Hwang Sewon , Lee Mi-Ock
Published: August 2019

The most accepted therapeutic regimen for treating ER-alpha breast cancer is Tamoxifen. Yet over 30% of patients gain resistance. Recently, lipid metabolism has been rising as a new mode of cancer drug resistance. In order for a rapid growth, cancer cells show a strong avidity for lipid which they obtain by de novo lipogenesis or increasing the uptake of exogenous lipids. Lipid metabolism may confer drug resistance, but little is known about its association with endocrine resistance. Thus, we aimed to investigate the association of lipid metabolism in the context of tamoxifen resistance. First, fatty acid translocase and membrane-associated fatty acid binding protein, both of which are responsible for fatty acid uptake, were overexpressed in MCF7/TAMR-8 and T47D/TR-1, TR-2 cell lines. Also the mRNA level of fatty acid activating enzyme, long-chain fatty-acid-coenzyme A, were elevated as well. Next, through Oil-red-O staining and Nile red staining followed by FACS analysis, we examined whether tamoxifen-resistant cells actually increase fatty acid uptake. When supplemented with free fatty acids, tamoxifen-resistant cell lines showed higher efficiency in fatty acid uptake compared to the control cell lines. Subsequently when the amount of triglyceride accumulation was measured, more TG were present in resistant cells at the basal level and after free fatty acid treatment. Taken together, these results suggest lipid uptake by cancer cells may play a role in driving tamoxifen-resistance in breast cancer.

Citation Format: Sewon Hwang, Mi-Ock Lee. Cellular fatty acid uptake is enhanced in tamoxifen-resistant breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3610.
Abstract 3612: Decreased vitamin C uptake mediated by SLC2A3 promotes leukemia progression and impedes Tet2 restoration
Authors: Liu Jun , Hong Junshik , Ahn Kwangsung , Yoon Sungsoo
Published: August 2019

Background: Two new studies in Nature and Cell, from Agathocleous et al. (2017) and Cimmino et al. (2017), respectively, showed that vitamin C regulates HSC function and suppresses leukemogenesis by modulating Tet2 activity. However, little is known about the relationship between vitamin C transporter and hematological malignancies.

Purpose: In this study, we investigated the role of SLC2A3, a gene that encode GLUT3 which mainly transport glucose and oxidized form vitamin C, in hematological malignancies. Through this research we identified a set of biomarkers which can predict the effect of vitamin C treatment and develop stagey to improve this putative innovative treatment.

Results: We analyzed gene expression patterns of the major GLUT family genes in primary AML blast cells using previously published microarray datasets (GSE37307) and (GSE9476) and determined that SLC2A3 gene expression was significantly decreased in blast cells compared with normal hematopoietic cells. By contrast, expression of SLC2A1, SLC2A2 and SLC23A2 was not significant different in between primary AML cells and normal hematopoietic cells. Next, we analyzed the relevance of SLC2A3 expression to the survival of AML patients from TCGA and TARGET database and found that below-median SLC2A3 expression was associated with inferior overall survival. Subsequently, we detected the SLC2A3 mRNA expression level on 4 AML cell lines and 3 DLBCL cell lines, there were very low expression level on OCI-AML-3 and OCI-LY1. To check whether decreased expression of SLC2A3 has an impact on the Vitamin C effect, we treated the 7 cell lines with dose dependent vitamin C, up to 500 μM, 48 hours. The cell viability assay showed that OCI-AML-3 and OCI-LY1 have no response to Vitamin C, however the proliferation of NB4, HEL, HL60, OCI-LY19 and Toledo was suppressed by 250 μM Vitamin C.

Conclusions: SLC2A3 expression was decreased in AML blast cell and down regulation of SLC2A3 was closely associated with the poor outcomes of AML patients. In vitro study showed that SLC2A3 was essential for the effect of Vitamin C. Therefore, SLC2A3 could be a potential biomarker to predict the effect of Vitamin C treatment. In addition, through targeting SLC2A3, efficacy of Vitamin C treatment could be improved.

Citation Format: Jun Liu, Junshik Hong, Kwangsung Ahn, Sungsoo Yoon. Decreased vitamin C uptake mediated by SLC2A3 promotes leukemia progression and impedes Tet2 restoration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3612.
Abstract 3617: Trastuzumab-conjugated gold nanoparticles as novel HER2-targeted therapeutics against trastuzumab-resistant gastric cancer
Authors: Kumon Kento , Kubota Tetsushi , Kuroda Shinji , Kanaya Nobuhiko , Kakiuchi Yoshihiko , Tsumura Tomoko , Kikuchi Satoru , Kagawa Shunsuke , Tazawa Hiroshi , Fujiwara Toshiyoshi
Published: August 2019

While trastuzumab (Tmab), a monoclonal antibody that targets the human epidermal growth factor receptor 2 (HER2), has greatly contributed to improve survival outcomes in metastatic gastric cancer, an issue of concern is that no current HER2-targeted therapeutic agent is effective against Tmab-resistant gastric cancer. Nanotechnology, which has progressed rapidly in recent years, greatly contributes to progress in medical fields including cancer therapy, and especially, gold nanoparticles (AuNPs) are attention-grabbing nanomaterials that are promising drug carriers with unique properties of high in vivo stability and a large surface area available for attachment of materials such as antibodies. Methods: We created HER2-targeted AuNPs by conjugating Tmab onto their surface (T-AuNPs) and examined their therapeutic efficacy and cytotoxic mechanisms using HER2-postive Tmab-resistant (MKN7) or Tmab-sensitive (NCI-N87) gastric cancer cell lines in vitro and in vivo. We also examined T-AuNPs on HER2-negative MKN74 gastric cancer cells with adenoviral vector-mediated overexpression of the HER2 extracellular domain (HER2-ECD). Results: The size of T-AuNPs was 85.39 ± 0.68 nm and the surface was negatively charged with 39.43 ± 0.85 mV. In in vitro assays, T-AuNPs showed 6-fold higher cytotoxic activity than Tmab against NCI-N87 cells. Then, T-AuNPs showed significantly stronger cytotoxic effects than controls against both MKN7 and NCI-N87 cells although Tmab had no effect on MKN7 cells. Autophagy was proven to play an important role in T-AuNPs cytotoxic mechanisms. This autophagy was considered to be induced by HER2-dependent internalization of T-AuNPs, some of which were interestingly located in the cytoplasm independent of lysosomal encapsulation. Although T-AuNPs were not cytotoxic towards MKN74 cells, they became cytotoxic following HER2-ECD overexpression. Finally, Intratumoral injection of T-AuNPs showed potent antitumor effects, which were mediated by autophagy, against NCI-N87 and MKN7 subcutaneous tumors in vivo mouse models. Conclusion: HER2-targeted AuNPs with conjugated Tmab can be a promising strategy for the development of novel therapeutic agents to overcome Tmab resistance in gastric cancer.

Citation Format: Kento Kumon, Tetsushi Kubota, Shinji Kuroda, Nobuhiko Kanaya, Yoshihiko Kakiuchi, Tomoko Tsumura, Satoru Kikuchi, Shunsuke Kagawa, Hiroshi Tazawa, Toshiyoshi Fujiwara. Trastuzumab-conjugated gold nanoparticles as novel HER2-targeted therapeutics against trastuzumab-resistant gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3617.
Abstract 3623: Mesenchymal stem cells engineered with TAT peptide functionalized nanoparticles improve therapeutic efficacy of paclitaxel in an orthotopic lung tumor model
Authors: Prabha Swayam , Moku Gopikrishna , Layek Buddhadev , Panyam Jayanth
Published: August 2019

Tumors are characterized by uneven vascular perfusion with near normal blood flow in the outer most regions, while the inner regions can be avascular. In addition, elevated interstitial fluid pressure and rigid extracellular matrix compromise intra-tumoral solute transport and hence limited drug efficacy. Mesenchymal stem cells (MSCs) have the unique advantage of being able to migrate specifically to both primary tumors and metastases following systemic administration. However, poor payload capacity of MSCs limits their use as drug delivery carriers. To address this issue, we investigated polymeric nanoparticles that were functionalized with transactivator of transcription (TAT) peptide. Paclitaxel loaded PLGA nanoparticles (15-16 % w/w paclitaxel; diameter of 225 ± 7 nm; and zeta potential of −15 ± 4 mV) were prepared by emulsion-solvent evaporation method, followed by TAT-conjugation to the surface of nanoparticles via maleimide-thiol chemistry. Nanoengineered MSCs were generated by incubating MSCs in suspension with 100 µg/mL paclitaxel loaded nanoparticles for 4 hours at 37°C. Our studies demonstrated that TAT functionalization improved the paclitaxel loading in MSCs. In addition, engineering MSCs with TAT functionalized nanoparticles did not affect the differentiation and migration properties of MSCs. Further, MSCs engineered with TAT peptide functionalized nanoparticles resulted in significant inhibition of tumor growth and overall improved survival in a mouse orthotopic model of lung cancer compared to that with free drug treatment. In summary, our results demonstrated that MSCs nanoengineered using TAT functionalized nanoparticles can serve as an efficient carrier for tumor specific delivery of anticancer drugs, resulting in greatly improved therapeutic efficacy.

Citation Format: Swayam Prabha, Gopikrishna Moku, Buddhadev Layek, Jayanth Panyam. Mesenchymal stem cells engineered with TAT peptide functionalized nanoparticles improve therapeutic efficacy of paclitaxel in an orthotopic lung tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3623.
Abstract 3628: Comparison of CD44 expression in pancreatic cancer, pancreatic stem and normal pancreatic cells: Development of CLENs for tumor targeting and therapy using cell models
Authors: Adegunle Dotun D. , Ansah Eugene Boakye , Nguyen Danny , Sanghvi Kanchi , Goodrich Drew , Campbell Robert
Published: August 2019

Purpose: The incidence of drug resistance in pancreatic cancer correlates with poor prognosis, increased disease progression and unfavorable treatment outcomes. CD44, a surface membrane proteoglycan has been identified to be directly involved in pancreatic cancer drug resistance and metastasis. As such, the aim of this research was to screen various pancreatic cells for their expression of CD44, and to begin the early design and development of cell membrane lipid-extracted nanoliposmes (CLENs) for enhanced targeting and therapy.

Methods: Three pancreatic cancer cells- Panc-1, MiaPaCa 2, MS1 VEGF and a normal cell line- hTERT- were employed for this study. Stem cell, Mia PaCa-2, served as a positive control for screening purposes. Four liposome compositions were prepared using various ratios of DOPC, Chol, DPPE-PEG5000, and cellular membrane lipid-extracted (LE) was derived from target cell, Panc 1. To evaluate CD44 glycoprotein expression, cells were seeded in 48-well plates and incubated overnight. Fluorescence measurements were determined at excitation wavelength of 485/25 and emission wavelength at 528/20 nm. G44-26 monoclonal antibody was used for CLENs antibody conjugation studies. A Sephadex G-25 column was used for separation of bound and unbound antibody material, and fluorescence detection was used for cell culture analysis. Fluorescence detection was performed at excitation and emission wavelengths of 530/25 and 590/25, respectively.

Results: The relative level of CD44 expression for each of the cell lines was as follows. Panc1>Mia PaCa-2 = hTERT>MS1-VEGF. There was a statistically significant difference between Panc-1 and Mia Paca 2 (P<0.001), between hTERT and MS1 VEGF (P<0.05), and between Panc-1 and hTERT (P<0.0001). The inclusion of Panc1-LE in combination with additional liposome components did not increase cellular uptake significantly. The conjugation of G44-26 monoclonal antibody to the liposomes increased average size of all 4 preparations. Average size for CLENs and G44-26 monoclonal antibody -CLENs increased from 140.75 ± 3.22 nm to 250.23 ± 15.31 nm.

Conclusion: Future studies are required to demonstrate the effect of G44-26 monoclonal antibody liposome conjugates to further exploit the optimal expression of CD44 for pancreatic drug targeting.

Citation Format: Dotun D. Adegunle, Eugene Boakye Ansah, Danny Nguyen, Kanchi Sanghvi, Drew Goodrich, Robert Campbell. Comparison of CD44 expression in pancreatic cancer, pancreatic stem and normal pancreatic cells: Development of CLENs for tumor targeting and therapy using cell models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3628.
Abstract 3631: Development of targeted nanoformulation of talazoparib for combined chemoradiation therapy in lung cancer
Authors: Singh Bijay , Abdelhalim Mostafa , Warrington Stephanee , Sridhar Srinivas
Published: August 2019

Poly (ADP-ribose) polymerase (PARP) inhibitor (PARPi) therapy exploits a synthetic lethality strategy in cancers with inherent damage in DNA repair or transcription pathways. Talazoparib is a potent PARPi that is currently indicated for oral inhibitor therapy in several cancer clinical trials. Oral administration of these inhibitors typically results in poor bioavailability and tumor accumulation. In contrast, nanoparticle formulation provides a safe vehicle for parenteral administration of therapeutic drugs with sustainable release reducing systemic toxicity and also protect from surveillance of immune cells, thereby increasing the bioavailability of the drugs in vivo. Moreover, targeting strategy for the nanoparticles will improve the accumulation of drugs in the tumors. Here, we developed a targeted formulation of Talazoparib (NanoTLZ) which was decorated with anti-EGFR antibody as a ligand to ameliorate the cellular uptake of NanoTLZ via EGFR-mediated endocytosis. In vitro tests showed that NanoTLZ is more effective in cell growth inhibition than free Talazoparib. When combined with radiation with different doses from 2-10Gy, NanoTLZ showed a strong radiosensitization effect as evidenced with almost no colonies formation at 6 Gy of radiation dose. The high therapeutic efficacy of combined chemoradiation therapy in Calu 6 cell line can be attributed to the higher accumulation of NanoTLZ in the cancer cells as compared to the free Talazoparib which is also marred with efflux of the drug from the cells. The slow and sustained release of Talazoparib from nanoparticle formulation inside the cells lead enhanced inhibition of DNA repair pathways. These studies provide very encouraging results to evaluate the efficacy of these nanoparticles in lung cancer animal models.

Citation Format: Bijay Singh, Mostafa Abdelhalim, Stephanee Warrington, Srinivas Sridhar. Development of targeted nanoformulation of talazoparib for combined chemoradiation therapy in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3631.
Abstract 3639: EGCG-gold nanoparticles exhibit greater anti-tumor activity over conventional gold nanoparticles or EGCG due to potential synergistic interactions
Authors: Chavva Suhash Reddy , Deshmukh Sachin Kumar , Kanchanapally Rajashekhar , Tyagi Nikhil , Coym Jason W. , Singh Ajay P. , Singh Seema
Published: August 2019

Epigallocatechin gallate (EGCG) possesses significant anti-tumor activity and binds to laminin receptor over-expressed on cancer cells with high affinity. However, the use of EGCG in therapeutics is limited due to its poor bioavailability and limited stability. Gold nanoparticles (GNPs) serve as excellent drug carriers and protect the conjugated drug from enzymatic metabolization. Here, we investigated if EGCG-formulated GNPs (E-GNPs) would demonstrate superior anti-cancer activity compared to EGCG or conventionally-synthesized citrate-GNPs (C-GNPs) due to their synergistic interactions. Cell viability studies showed greater growth inhibition by E-GNPs, compared to EGCG, or C-GNPs. Cellular uptake studies revealed that, unlike C-GNPs, E-GNPs were taken-up more efficiently by cancerous cells than noncancerous cells. Furthermore, data showed that E-GNPs induced more apoptosis in cancer cells, compared to EGCG, and C-GNPs. From the mechanistic standpoint, we observed that E-GNPs inhibited the nuclear translocation and transcriptional activity of NF-κB with greater potency than EGCG, whereas C-GNPs were only minimally effective. Altogether, our data suggest that E-GNPs can serve as effective anti-cancer agents.

Citation Format: Suhash Reddy Chavva, Sachin Kumar Deshmukh, Rajashekhar Kanchanapally, Nikhil Tyagi, Jason W. Coym, Ajay P. Singh, Seema Singh. EGCG-gold nanoparticles exhibit greater anti-tumor activity over conventional gold nanoparticles or EGCG due to potential synergistic interactions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3639.
Abstract 3645: Cooperation between canonical Wnt and TGF-beta pathways promotes sarcoma angiogenesis
Authors: Hawkins Allegra G. , Pedersen Elisabeth A. , Jiang Wei , Treichel Sydney , Sperring Colin , Read Jay , Magnuson Brian , Patel Rajiv M. , Thomas Dafydd , Chugh Rashmi , Lawlor Elizabeth R.
Published: August 2019

Local and metastatic progression of solid tumors depends on crosstalk between tumor cells and the tumor microenvironment (TME), including both stromal cells and the extracellular matrix (ECM). We recently showed that high Wnt/beta-catenin activity in Ewing sarcoma correlates with diminished patient survival and that canonical Wnt signaling alters the tumor secretome, influencing ECM protein composition. In light of this, we investigated the hypothesis that Wnt/beta-catenin supports tumor progression by modulating tumor: TME crosstalk. Our results reveal that, in discrete tumor cell sub-populations, beta-catenin activation sensitizes cells to TGF-beta ligands through derepression of the TGF-beta receptor, TGFBR2, resulting in canonical Wnt-induced, TGF-beta-dependent upregulation of TGF-beta targets. Significantly, these Wnt/TGF-beta responsive targets include multiple AngioMatrix genes that are known to alter the TME to promote angiogenesis, including tenascin-C and collagens. Studies of Ewing sarcoma models, in vitro and in vivo, as well as in two independent patient cohorts, confirm that a direct relationship exists between beta-catenin and TGF-beta activation in tumor cells and angiogenesis in the local TME. Mechanistically, this is due, in part, to tenascin C-mediated promotion of endothelial cell proliferation. Thus, functional cooperation between canonical Wnt and TGF-beta signaling in Ewing cells induces secretion of pro-angiogenic factors. This study reveals a novel link between Wnt and TGF-beta signaling in Ewing sarcoma and illustrates the critical contribution of tumor cell heterogeneity and tumor: TME crosstalk to sarcoma progression.

Citation Format: Allegra G. Hawkins, Elisabeth A. Pedersen, Wei Jiang, Sydney Treichel, Colin Sperring, Jay Read, Brian Magnuson, Rajiv M. Patel, Dafydd Thomas, Rashmi Chugh, Elizabeth R. Lawlor. Cooperation between canonical Wnt and TGF-beta pathways promotes sarcoma angiogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3645.
Abstract 3657: Defining the role of the RNA-binding protein MSI2 in neuroblastoma
Authors: Pilgrim Adeiye , Cuya Selma , Chen Dongdong , Schnepp Robert
Published: August 2019

Neuroblastoma (NB) is the most common extracranial solid tumor of childhood with a 5-year survival rate approaching 50% in high-risk patients and like other pediatric cancers, lacks many targetable mutations. Thus, novel therapeutic approaches are needed. Previous research has shown that the RNA-binding protein (RBP), LIN28B, enhances MYCN expression and induces NB formation. Additionally, we previously demonstrated that LIN28B-RAN-AURKA signaling drives NB oncogenesis. These results highlight the role of RBPs in modulating NB tumorigenesis.

The Musashi family includes RBPs that have been previously shown to be associated with worse prognosis in adult malignancies, including colon cancer and pancreatic cancer, among others. Thus, we speculated that the Musashi family might influence aggressive phenotypes in pediatric tumors, such as neuroblastoma. MSI2 is robustly expressed in neuroblastoma tumors, and, indeed, increased levels of Musashi-2 (MSI2) correlate with worse NB prognosis. In order to begin to elucidate its role in NB tumorigenesis, we knocked-down MSI2 in human NB cell lines using multiple independent short hairpin RNAs (shRNA) and small interfering RNAs (siRNA) and examined the impact on proliferation, survival, and downstream target genes relative to scrambled control RNA-treated cells. From these experiments, we observed decreased colony formation in clonogenic assays, increased apoptosis and decreased proliferation. To gain an initial understanding of the downstream targets that MSI2 influences, we performed Nanostring analysis in 2 human NB cell lines, Kelly and SK-N-DZ and found that knocking down MSI2 led to a downregulation of many cancer-related genes including IDH2 and other metabolic targets. IDH2 is a mitochondrial enzyme and its mutant is known to be involved in gliomagenesis. Changes in IDH2 expression have also been linked to epigenetic alterations. Finally, wild-type IDH2is upregulated in lung cancer and contributes to tumor growth. Therefore, we are currently examining the functional relationship between MSI2 and IDH2 in NB.

Citation Format: Adeiye Pilgrim, Selma Cuya, Dongdong Chen, Robert Schnepp. Defining the role of the RNA-binding protein MSI2 in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3657.
Abstract 3659: Alyref is a novel binding partner and co-factor for MYCN-driven oncogenesis in neuroblastoma
Authors: Nagy Zsuzsanna , Kanikevich Maxwell , Koach Jessica , Mayoh Chelsea , Carter Daniel , Liu Tao , Du Yanhua , Jiang Cizhong , Haber Michelle , Norris Murray , Cheung Belamy , Marshall Glenn
Published: August 2019

MYCN amplification is a poor prognostic indicator in neuroblastoma associated with high-risk disease. Therapies that directly repress the MYCN oncogenic signal in neuroblastoma are limited. We and others have shown that MYCN requires multiple cofactors to increase its protein stability in neuroblastoma cells, so that the very high MYCN levels required to drive tumourigenesis can be achieved. Here, we have identified ALYREF, a nuclear molecular chaperone protein, as a novel regulator of MYCN function in neuroblastoma. High expression of ALYREF predicted poor neuroblastoma patient survival and substantially correlated with MYCN levels in a large dataset (n=649) of human neuroblastoma tumour samples. ALYREF mRNA expression was also significantly increased in ganglia cells from the homozygous TH-MYCN neuroblastoma mouse in comparison to ganglia from wild-type littermates. Using co-immunoprecipitation and mass spectrometry, we identified ALYREF, as a direct binding partner of nuclear MYCN protein. Chromatin immunoprecipitation showed that MYCN bound the ALYREF gene promoter, and knockdown MYCN by MYCN siRNAs decreased ALYREF expression. A set of overexpression and knockdown experiments in MYCN-amplified neuroblastoma cells revealed that MYCN and ALYREF form a positive forward feedback expression loop. Overexpression of ALYREF further increased MYCN expression and protein stability in MYCN-amplified neuroblastoma cells. We found that ALYREF had a critical function in regulating the turnover of MYCN protein through transcriptional repression of the E3 protein ubiquitin ligase, NEDD4. The stabilized N-Myc oncoprotein enhanced ALYREF expression and stimulated cell growth of neuroblastoma. Additionally, we demonstrated that ALYREF plays a significant role in maintaining cell viability and proliferation of MYCN-amplified neuroblastoma cells. Taken together, our findings demonstrate a crucial role for ALYREF in regulating MYCN function and suggest that ALYREF-mediated stabilization of MYCN protein contributes to the development and progression of the disease. Thus inhibition of ALYREF activity using small molecules is a potential therapy for MYCN-amplified tumours.

Citation Format: Zsuzsanna Nagy, Maxwell Kanikevich, Jessica Koach, Chelsea Mayoh, Daniel Carter, Tao Liu, Yanhua Du, Cizhong Jiang, Michelle Haber, Murray Norris, Belamy Cheung, Glenn Marshall. Alyref is a novel binding partner and co-factor for MYCN-driven oncogenesis in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3659.
Abstract 3661: GLI1 contributes to proliferation, survival and drug-resistance of Ewing sarcoma (EWS) cells
Authors: Yoon Joon Won , Lamm Marilyn , Iannaccone Philip , Walterhouse David
Published: August 2019

Ewing sarcoma (EWS) is characterized by TET-ETS fusions, most commonly the EWSR1-FLI1 fusion. GLI1, a transcription factor in the Hedgehog (HH) signal transduction pathway and target of canonical HH signaling, is a direct transcriptional target of EWSR1-FLI1. A role for the HH signaling pathway and GLI1 in acquisition of a multidrug resistance phenotype has been previously observed in several human cancers. The role of the HH signal transduction pathway and GLI1 in the biology of EWS remains incompletely understood. We hypothesize that the majority of EWS cell lines express GLI1 based on non-canonical up-regulation by EWSR1-FLI1 and that further up-regulation of GLI1 may be associated with the development of drug resistance. Here, we show that EWS cell lines (CHLA-9, CHLA-10, CHLA-258, TC-32 and TC-71) express canonical HH pathway components (PTCH1, SMO, GLI1 and GLI3) and that cells lines established at the time of recurrence have higher GLI1 expression (CHLA-258 and TC-71) than those established at the time of diagnosis (CHLA-9, CHLA-10 and TC-32). Exposure to Sonic HH ligand in order to activate canonical HH signaling did not affect cell viability (MTT assay), proliferation (BrdU assay), or apoptosis (caspase 3/7 assay). However, inhibition of GLI1 activity with GANT61 reduced GLI1 expression (qRT PCR), cell viability (MTT assay) and cell proliferation (BrdU assay) but increased apoptosis (caspase 3/7 assay) and sensitivity of the cells to vincristine (VCR) (MTT assay), suggesting GLI1 plays roles in each of these processes. We then established VCR-resistant EWS cell lines (TC-71 [45-fold increase in the IC-50] and TC-32 [27-fold increase in the IC-50]) by exposing cells to serially increasing concentrations of VCR. We used an 86-gene cancer drug resistance PCR array (Qiagen) to characterize gene expression differences between untreated vs. recurrent EWS cell lines and between parental vs. VCR-resistant EWS cell lines. Among the changes in gene expression, we showed higher GLI1 expression in the recurrent EWS cell lines (CHLA-258 cells [4.8-fold] and TC-71 [3.4-fold]) vs. untreated CHLA-9 cells, and in VCR-resistant TC-71 cells (2.2-fold) vs. parental TC-71 cells. Down-regulation of GLI1 by GANT61 or GLI1 siRNA enhanced sensitivity of the cells to VCR. Our results suggest that GLI1 expression contributes to proliferation, survival and drug-resistance of EWS cell lines. Strategies to inhibit GLI1 may have therapeutic benefit in EWS.

Citation Format: Joon Won Yoon, Marilyn Lamm, Philip Iannaccone, David Walterhouse. GLI1 contributes to proliferation, survival and drug-resistance of Ewing sarcoma (EWS) cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3661.