bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1921: The effect of Hoechst 33342 and Hoechst 33258 on side population (SP) cells or stem cell-like population
Authors: Tang Naimei , Zhang Crystal , Noraian Cyndi , Wali Anil , Pass Harvey , Harbut Michael , Zhang Xinbo
Published: May 2015

Hoechst 33342 side population (SP) analysis is widely used for identifying and sorting side population (SP) or stem cell-like population from a variety of tissues and species including cancer cell lines. However, Hoechst 33342, but not its derivative Hoechst 33258, is an apoptotic inducer. Our previous results showed that Hoechst 33342 induced apoptosis in seven mesothelioma cell lines through cytochrome C release, caspase activation and degradation of Poly (ADP-ribose) polymerase. We hypothesize Hoechst 33342 may cause SP cell death during Hoechst 33342 staining and Hoechst 33258 may be a safe indicator for identification and isolation of SP cells.

The aims of this study are 1) to detect the effect of H 33342 on SP cells, and 2) to identify if H33258 can be an indicator for stem cell isolation.

Procedure: The effect of Hoechst dyes on the tumorigenicity of H2373 mesothelioma cells was analyzed by soft agar clonogenic colony formation. MTT assay have been employed to estimate the effect of Hoechst dyes on cell growth in H2373 mesothelioma cells, and Hoechst 33342 and Hoechst 33258 SP analysis was performed on a FACSDiVa cell sorter.

Results: Our result demonstrated that Hoechst 33342 staining can causes SP cell damage and also decreases cell colony formation and cell proliferation after using the routine staining doses and intervals for SP cell analysis. In contrast, Hoechst 33258 has little effect on the cell colony formation and cell proliferation. We have found that verapamil, ABC transporter inhibitor, significantly inhibits the efflux of intracellular Hoechst 33258, which means Hoechst 33258 may be used as a marker in isolating ABC transporter positive cells, as Hoechst 33342 does.

Conclusion: 1) Hoechst 33342 can induce apoptosis of SP cells of mesothelioma cancer cells 2) Hoechst 33258 may be a safe indicator and replace Hoechst 33342 for identification and isolation of side population.

Future Studies: 1) More in vitro methods such as clonogenic assay, drug resistant assay, and invasion assay are required for further determination of stem cell-like properties of side population isolated by Hoechst 33258 when compared to that isolated by Hoechst 33342. 2) Xenograft (or animal) study is ultimately required to determine the capacity of in vivo tumor formation of the side population cells isolated by Hoechst 33258 after the completion of in vitro study

Note: This abstract was not presented at the meeting.

Citation Format: Naimei Tang, Crystal Zhang, Cyndi Noraian, Anil Wali, Harvey Pass, Michael Harbut, Xinbo Zhang. The effect of Hoechst 33342 and Hoechst 33258 on side population (SP) cells or stem cell-like population. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1921. doi:10.1158/1538-7445.AM2014-1921
Abstract 1934: Role of CDK1 in human stem cell biology
Authors: Wang Xiao Q. , Chen Lin , Norbury Chris
Published: May 2015

Self-renewal and pluripotency of stem cells (SC) is maintained by well-characterized transcriptional factors that assemble a core regulatory network, although less defined auto- and cross-regulatory signaling pathways are also critical. Accumulating evidence indicates that cell cycle regulators play an important role in control of pluripotency and differentiation that goes beyond their roles in cell cycle regulation. Cyclin-dependent kinase 1 (CDK1) is essential for cell cycle progression and embryonic development, and has recently been implicated in a signaling network with pluripotency proteins.

In this study, we investigated the potential role of CDK1 in regulation of pluripotency and differentiation. CDK1 expression is enriched in the pluripotent state of human embryonic stem cells (hESCs) and down regulated during differentiation. shRNA knockdown of CDK1 or a selective CDK1 inhibitor (RO-3306) promoted hESC differentiation, shown by the reduced expression of the pluripotency markers Oct4, Nanog, and Sox2, and the up-regulation of the mesendoderm transcripts Brachyury, Eomes, Goosecoid, and MixL1. Interestingly, down-regulation of CDK1 by both shRNA and RO-3306 could not block ES cells at G2/M or other phases of the cell cycle, suggesting that relatively milder knockdown of CDK1 did not induce hESC cell cycle arrest or impair overall proliferation but was enough to promote differentiation. Knockdown of CDK1 also decreased phosphorylation of Akt at Thr308 and Ser473, one of the key regulators of pluripotency signaling. Further, JNJ-770621, a dual inhibitor of CDK1/CDK2 and Aurora kinases but not Akt or Erk, induced human embryonal carcinoma cell (NCCIT) differentiation accompanied by reduced phosphorylation of Akt. Addition of UO126 and SB431542, which inhibit MEK/ERK and TGFβ/Smad2/ respectively (downstream of PI3K/Akt signaling), reversed the differentiation induced by shCDK1 or RO-3306 treatment. Moreover, phosphorylation of PDK1 (upstream of Akt) was notably reduced in CDK1 knockdown cells but not in serum-induced differentiation, indicating that the inhibition of activation of PDK1 or Akt could be the primary effect of CDK1 down-regulation. The results suggest that threshold levels of CDK1 might direct- or cross-regulate key signaling pathways, such as that operating through PI3K-PDK1-Akt.

In summary, CDK1 is required for maintenance of stem cell pluripotency, in a manner that is distinct from its role in regulation of cell cycle progression, potentially through direct or indirect cross-regulation of fundamental pluripotency signaling of PI3K/PDK1/Akt-ERK-GSK3β and TGFβ/Smad.

Citation Format: Xiao Qi Wang, Lin Chen, Chris Norbury. Role of CDK1 in human stem cell biology. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1934. doi:10.1158/1538-7445.AM2014-1934
Abstract 1944: Divergent effects of BRAF activation in neural stem and progenitor-like glioblastoma cells
Authors: Lerner Robin G. , Ihara Yuichiro , Lewis Kate , Griveau Amelie , Reichhold Brian , Qu Dian , McMahon Martin , Rowitch David , James Charles D. , Petritsch Claudia
Published: May 2015

Glioblastoma multiforme (GBM) are heterogeneous tumors that are refractory to radiation and traditional chemotherapeutics. Separate studies have identified populations of neural stem and progenitor -like GBM cells, however the contribution of these cells types to tumor progression and the response to targeted therapy are poorly understood. Small molecules (such as vemurafenib) targeting the most common MAPK-activating mutation, BRAFV600E, are currently entering clinical trials for treatment of high-grade glioma. Understanding the contribution of different cell types to treatment responses will be necessary for the design of effective adjuvant therapeutic strategies.

We show that activating MAPK pathway gene mutations in GBM correlate with elevated expression levels of oligodendrocyte precursor (OPC) and neural stem cell (NSC)-associated genes. Cells expressing CD133 and NG2 coexist in human GBM and BRafV600E expressing mouse neurosphere cultures, and maintain some of their respective neural stem and progenitor-like properties.

In isogenic mouse cells expressing BRafV600E, NG2+ cells expand rapidly and symmetrically, while rates of asymmetric division are unaffected in slow-proliferating CD133+ cells. Tracing of proliferation rate with the membrane-associated dye PKH-26 reveals heterogeneity in the proliferation rate of CD133+ cells, but not proliferative NG2+ cells. BRAF-targeted inhibition of human GBM cells with PLX4720, the tool compound of vemurafenib, reduces S-phase entry of NG2+ cells, yet CD133+ cells are less affected. Interestingly targeted inhibition of components of the asymmetric division machinery selectively disrupts G2-M progression in CD133+ cells. The functionally divergent populations of CD133-expressing NSC-like cells will likely be therapeutically problematic with mono-therapeutic approaches. These data point to a differential regulation of key cell cycle processes between CD133+ stem-like cells and NG2+ OPC-like cells, that will inform the intelligent design of compound treatment strategies.

Note: This abstract was not presented at the meeting.

Citation Format: Robin G. Lerner, Yuichiro Ihara, Kate Lewis, Amelie Griveau, Brian Reichhold, Dian Qu, Martin McMahon, David Rowitch, Charles D. James, Claudia Petritsch. Divergent effects of BRAF activation in neural stem and progenitor-like glioblastoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1944. doi:10.1158/1538-7445.AM2014-1944
Abstract 1946: Inhibition of IKK/NF-κB impairs glioma stem cell function
Authors: Rinkenbaugh Amanda L. , Cogswell Patricia C. , Baldwin Albert S.
Published: May 2015

Glioma, the most frequently occurring primary brain tumor, has a median survival of less than 15 months, despite multimodal treatment combining surgery, radiation, and chemotherapy. It has been suggested that the presence of cancer stem cells (CSCs) within these tumors are responsible for resistance to treatment and high probability of recurrence. As a result, there is increasing interest in specifically targeting CSCs with new therapies to improve treatment of this disease. NF-κB has been implicated in numerous forms of cancer, as its target genes regulate hallmarks of cancer such as cell proliferation, survival, and invasion. Both glioma cell lines and tumor samples show high levels of phosphorylated p65, a marker of NF-κB activation. When comparing populations from tumor explants, NF-κB is preferentially activated in the CSC subset. We are examining the effects of IKK/NF-κB inhibition on both heterogeneous glioma explants and purified (CD133+) CSCs from these explants. Inhibition of NF-κB through either genetic or pharmacological means abrogates neurosphere formation, an in vitro measure of stem cell function. Additionally, real-time PCR analysis indicates that inhibition of NF-κB leads to a decrease in target genes implicated in glioma CSC biology, such as A20 and IL-6. We have also studied our glioma explants in both subcutaneous and intracranial xenografts to examine the role of NF-κB in vivo. We are utilizing microarrays to define the NF-κB-dependent gene expression signature in CD133+ cells. Our studies indicate a significant role for the NF-κB pathway in the biology of glioma CSCs, with possible parallels in CSCs of other tumor types.

Citation Format: Amanda L. Rinkenbaugh, Patricia C. Cogswell, Albert S. Baldwin. Inhibition of IKK/NF-κB impairs glioma stem cell function. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1946. doi:10.1158/1538-7445.AM2014-1946
Abstract 1953: The role of NR5a2 in pancreas development and carcinogenesis
Authors: Nissim Sahar , Wucherpfennig Julia , Wang Xiao-Xu , Kimmelman Alec , Goessling Wolfram
Published: May 2015

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal cancers today. A recent genome-wide association study (GWAS) has implicated the nuclear receptor NR5A2 in modulating risk for pancreatic cancer. While a role for this gene in pancreatic cancer has not been previously recognized, it is known to have an important role in stem cell pluripotency and metabolism. Using the zebrafish model organism, we demonstrate that knockdown of nr5a2 results in absence of exocrine pancreas, while leaving endocrine pancreas unaffected, indicating an essential role in pancreas organ formation. Further, we localize expression of nr5a2 to the endodermal bud that gives rise to the exocrine pancreas, consistent with a role for nr5a2 in regulating differentiation of pancreas progenitors. In resected human PDAC specimens and in vitro cell lines, we find that NR5A2 expression is altered compared to normal ductal epithelium. Moreover, shRNA knockdown of NR5A2 dramatically alters proliferation of PDAC cell lines, supporting the hypothesis that NR5A2 is involved in pancreatic carcinogenesis. These data support a model that NR5a2 regulates the proliferation and differentiation of exocrine pancreas progenitor cells during development. Consistent with this model, we find that signaling through nr5a2 interacts with notch activity. We postulate that aberrant expression of NR5a2 results in dysregulation of these processes in the adult pancreas by driving ductal epithelial cells to acquire properties of progenitor cells, and therefore is an important step in pancreas oncogenesis.

Note: This abstract was not presented at the meeting.

Citation Format: Sahar Nissim, Julia Wucherpfennig, Xiao-Xu Wang, Alec Kimmelman, Wolfram Goessling. The role of NR5a2 in pancreas development and carcinogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1953. doi:10.1158/1538-7445.AM2014-1953
Abstract 1980: Role of lysyl oxidase and lysyl oxidase-like 2 in gastric carcinoma
Authors: Kasashima Hiroaki , Yashiro Masakazu , Go Yukie , Masuda Go , Kinoshita Haruhito , Tokumoto Mao , Morisaki Tamami , Fukuoka Tatsunari , Toyokawa Takahiro , Kubo Naoshi , Tanaka Hiroaki , Muguruma Kazuya , Ohira Masaichi , Hirakawa Kosei
Published: May 2015

Object: Lysyl oxidase (LOX) family is the extracellular matrix-modifying enzymes comprising of LOX and LOX-like (LOXL) 1-4. These enzymes have intracellular functions involved in the regulation of motility, migration, differentiation, and transcription in various types of cells. LOX is associated with mesenchymal reconstruction that is essential for cancer invasion and metastasis. LOX overexpression is crucial to promoting tumor growth and metastasis in several cancers such as breast cancer and colorectal cancer, however, role of LOX in gastric cancer remain unknown. Here, we investigated the clinicopathological significance of LOX and LOXL2 in gastric cancer.

Materials and Methods: A total of 549 patients who had undergone surgery for gastric cancer at Osaka-City University were enrolled. LOX and LOXL2 expressions were evaluated by intensity of staining and percentage of stained tumor cells at the invading tumor front. This study was approved by the Osaka City University ethics committee. Informed consent was obtained from all patients.

Results: LOX and LOXL2 expression were positive in 265 (48.3%) and 342 (58.5%) of the 549 gastric tumors. Both LOX and LOXL2 expression in cancer cells were significantly associated with macroscopic type (p<0.01), histological type (p<0.01), depth of invasion (p<0.01), lymph node status (p<0.01), vessel invasion (p<0.01). The prognosis of patients with LOX-positive tumors or LOXL2-positive tumors was significantly (p<0.001 or p<0.001, log-rank) worse than that of patients with LOX-negative or LOXL2-negative tumors, respectively. A multivariate analysis indicated LOX and LOXL2 expression in cancer cells to be an independent prognostic factor (p=0.016 or p<0.001).

Conclusions: LOX and LOXL2 expression might be associated with metastatic activity of gastric cancer cells. LOX and LOXL2 might be useful predictive prognostic factors for patients with gastric cancer.

Citation Format: Hiroaki Kasashima, Masakazu Yashiro, Yukie Go, Go Masuda, Haruhito Kinoshita, Mao Tokumoto, Tamami Morisaki, Tatsunari Fukuoka, Takahiro Toyokawa, Naoshi Kubo, Hiroaki Tanaka, Kazuya Muguruma, Masaichi Ohira, Kosei Hirakawa. Role of lysyl oxidase and lysyl oxidase-like 2 in gastric carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1980. doi:10.1158/1538-7445.AM2014-1980
Abstract 1995: The Functional analyses of Oct4 in metastasis of human hepatocellular carcinoma
Authors: Dong Qiongzhu , Dai Chun , Jia Huliang , Qin Lunxiu
Published: May 2015

There is an increasing evidence that in a malignant bulk tumor, a small proportion of ‘cancer stem cells’ exist, leading to tumorigenesis,metastasis or recurrence by their plasticity. However, the functional status and significance of stem cell factors in hepatocellular carcinogenesis is unknown. In this study, we analyzed the expression of 3 stem cell factors: Oct4, Sox2 and Nanog in HCC patients, and found that Oct4 and Sox2, 3 stem cell factors, were expressed in HCCs. Expression of Oct4 correlated with HCC recurrence and metastasis. Through gain- and loss-of-function studies, Oct4 was demonstrated to significantly promote in vitro cell proliferation, invasion, transformation. In addition, Oct4 reduced chemosensitivity of HCC cells. Importantly, Oct4 enhanced in vivo tumor growth and metastasis in nude mice models bearing human HCC. Moreover, Oct4 dramatically stimulated expression of HCC stem cell markers, including Epcam,ELF, KLF4,CD44 and CD133 as well as stem cell signaling pathway (IL-6/surviving, TGF-beta and Wnt / beta-catenin passway). Conclusion: Oct4 plays a critical role in HCC development and progression and its deregulated signaling could be a promising new molecular target for designing novel preventive and therapeutic strategies to control this malignancy

Note: This abstract was not presented at the meeting.

Citation Format: Qiongzhu Dong, Chun Dai, Huliang Jia, Lunxiu Qin. The Functional analyses of Oct4 in metastasis of human hepatocellular carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1995. doi:10.1158/1538-7445.AM2014-1995
Abstract 1998: Deficient expression of oncogenic Wip1 (PPM1D) negatively regulates melanoma progression and metastasis
Authors: Moon Bo-Hyun , Suman Shubhankar , Li Henghong , Yang Qian , Strawn Steven J. , LoBello Janine , Mazur Sharlyn J. , Appella Ettore , Chen Suzie , Fornace Albert J.
Published: May 2015

PPM1D belongs to the magnesium-dependent serine/threonine phosphatase family. It is well demonstrated that PPM1D amplification and overexpression is a frequent event in many human cancer types; however, its role in melanoma development and metastasis is not yet defined. The objectives of this study were to evaluate the role of Wip1 in melanoma progression and to evaluate correlations between Wip1 expression and clinical pathological parameters, such as grade and metastasis. In order to investigate the role of Wip1 in melanoma progression, we crossed Grm1 transgenic (TG3) mice, a well-characterized mouse model for melanoma, with Wip1 null mice (PPM1D-/-) to generate TG3:PPM1D-/- mice, and observed the rate of spontaneous melanoma formation. Compared with TG3 mice, TG3:PPM1D-/- mice exhibited dramatic reductions in melanoma incidence and metastasis to lymph nodes, lungs, liver and spleen. In addition, in-vitro down-regulation of Wip1 in Mass20 melanoma cells significantly decreased cellular proliferation and migration. Further, human melanoma tissue microarrays (TMAs) were used to determine PPM1D mRNA and protein expression levels in human tumors at different stages of progression. In situ staining of PPM1D mRNA and its corresponding protein Wip1 in human melanoma TMAs revealed higher expression levels in at least ∼50% of the metastatic tumors analyzed.

Overall, our results demonstrate abundant expression of Wip1 in late stage/high grade melanoma and suggest that therapeutic strategies targeting Wip1 in individuals with select tumor subtypes might improve the survival advantage in Wip1-positive melanoma patients. However, further studies are required to elucidate the interactions between Wip1 signaling and established pathways of melanoma progression.

Note: This abstract was not presented at the meeting.

Citation Format: Bo-Hyun Moon, Shubhankar Suman, Henghong Li, Qian Yang, Steven J. Strawn, Janine LoBello, Sharlyn J. Mazur, Ettore Appella, Suzie Chen, Albert J. Fornace. Deficient expression of oncogenic Wip1 (PPM1D) negatively regulates melanoma progression and metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1998. doi:10.1158/1538-7445.AM2014-1998
Abstract 2080: Plasma membrane fluidity drives metastasis in breast cancer
Authors: Zhao Weina , Tisza Michael , Sjol Jessie , Mani Sendurai , Chang Jeffrey T.
Published: May 2015

New therapeutic targets that can inhibit cancer metastasis are desperately needed. To address this issue, we have leveraged a computational approach and performed an unbiased screen for therapies that can inhibit an epithelial-to-mesenchymal transition (EMT) gene expression profile, a phenotype that is believed to promote metastases. This yielded predictions of drugs that can both promote and inhibit an EMT transcriptional profile. Following up on these predictions, we confirmed that these drugs can induce or repress an EMT in a range of breast cancer cell lines. Furthermore, we discovered that they do so by altering the fluidity of the plasma membrane. An increase in plasma membrane fluidity is necessary and sufficient to induce and maintain an EMT state, increased motility, and increased mammosphere forming ability. Finally, we found that human cancers can regulate their membrane fluidity through cholesterol homeostasis pathways. Human breast tumors with altered cholesterol profiles have a significantly shorter time to metastasis. These data demonstrate that plasma membrane fluidity is a cellular attribute that is leveraged by human tumors to increase metastatic capacity, and that it can, in principle, be targeted by therapeutics to prevent metastasis.

Citation Format: Weina Zhao, Michael Tisza, Jessie Sjol, Sendurai Mani, Jeffrey T. Chang. Plasma membrane fluidity drives metastasis in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2080. doi:10.1158/1538-7445.AM2014-2080
Abstract 2083: WNT5a/ROR1 axis in triple-negative breast cancer progression and potential therapy
Authors: Borcherding Nicholas , Ameka Magdalene , Kolb Ryan , Xie Qing , Zhang Weizhou
Published: May 2015

Triple negative breast cancer (TNBC) accounts for 15-20% of total invasive breast cancer. These tumors are more aggressive and have a worse prognosis relative to other types of breast cancer, partially due to the lack of FDA-approved targeted therapy. Utilizing the RNA-seq data for invasive breast cancer specimens deposited in the Cancer Genome Atlas (TCGA), we found that the mRNA expression of receptor tyrosine kinase-like orphan receptor 1 (ROR1) decreased in the luminal A (n=231), luminal B (n=128) and Her2+ (n=58) subtypes relative to their corresponding normal controls. In contrast, ROR1 expression increased in all the pathologic stages of TNBC samples relative to normal specimens and ROR1 expression correlates positively with progression of TNBC (n=78). ROR1 is a receptor tyrosine kinase that is involved in embryonic patterning, but lacks expression in adult tissues. We undertook a series of mechanistic studies to understand the role of ROR1 in TNBC and evaluate ROR1 as a potential target for TNBC therapy. Using complex bioinformatics analysis, we found that ROR1 expression correlates with signature genes of TNBC, cancer stem cells, and TGFβ/SMAD pathways, which, in addition to recent observation of potential crosstalk between WNT5A/ROR1 signaling with TGFβ/SMAD, prompted us to examine if this crosstalk contributes to TNBC. Indeed, we found that WNT5a promotes SMAD2/3 activation in a ROR1- and TGFβR1-dependent manner. Inhibiting ROR1 by neutralizing antibody shifted cancer stem cells of TNBC to a more luminal-type, indicating a critical of ROR1 in self-renewal of cancer stem cells. SNAI1/2, master regulators of epithelial to mesenchymal transition (EMT), are potentially involved in the WNT5a/ROR1-mediated self-renewal of cancer stem cells. In agreement with these findings, TNBC cell lines with the highest surface expression of ROR1 had a greater cancer stem cell (CSC) population (CD24-CD44high). In collaboration with Speed Biosystems LLC, we developed an anti-ROR1 immunotoxin that specifically kills ROR1-high TNBC cells but not ROR1-low breast cancer cells or normal epithelial cells. Our findings suggest a role of WNT5a/ROR1 signaling in the progression of TNBC, through its role in potentiating TGFβ/SMAD/SNAI1/2 pathway thus promoting EMT and stem-like phenotype. ROR1 represents a novel therapeutic target for TNBC, especially for late-stage diseases.

Citation Format: Nicholas Borcherding, Magdalene Ameka, Ryan Kolb, Qing Xie, Weizhou Zhang. WNT5a/ROR1 axis in triple-negative breast cancer progression and potential therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2083. doi:10.1158/1538-7445.AM2014-2083
Abstract 2510: A porphodimethene chemical inhibitor of uroporphyrinogen decarboxylase
Authors: Yip Kenneth W. , Zhang Zhan , Sakemura-Nakatsugawa Noriko , Huang Jui-Wen , Yue Shijun , Jitkova Yulia , To Terence , Pai Emil , Schimmer Aaron , Lovell Jonathan , Sessler Jonathan , Liu Fei-Fei
Published: May 2015

Uroporphyrinogen decarboxylase (UROD), a heme biosynthetic pathway enzyme that converts uroporphyrinogen to coproporphyrinogen, was recently identified as a potential anticancer target. UROD inhibition leads to an increase in reactive oxygen species (ROS), likely mediated by the Fenton reaction, which decreases cancer cell viability and sensitizes cells to cisplatin and radiation. Because there is no known chemical UROD inhibitor suitable for translational research, we aimed to design, synthesize, and characterize such a compound. In silico design and docking was used to identify a potential porphyrin analogue. Subsequent synthesis produced a porphodimethene (named PI-16) that inhibited UROD in an enzymatic assay (IC50 = 9.9 µM). PI-16 did not affect porphobilinogen deaminase (at 62.5 µM), thereby exhibiting some specificity. In cellular assays, PI-16 reduced FaDu and ME-180 cancer cell viability with half maximal effective concentrations of 22.7 µM and 26.9 µM, respectively, whereas normal oral epithelial (NOE) cells were only minimally affected. PI-16 combined effectively with cisplatin and radiation, with potent synergy being observed with cisplatin (Chou-Talalay combination index < 1). This work presents the first known synthetic UROD inhibitor, and sets the foundation for the design, synthesis, and characterization of higher affinity and more effective compounds.

Citation Format: Kenneth W. Yip, Zhan Zhang, Noriko Sakemura-Nakatsugawa, Jui-Wen Huang, Shijun Yue, Yulia Jitkova, Terence To, Emil Pai, Aaron Schimmer, Jonathan Lovell, Jonathan Sessler, Fei-Fei Liu. A porphodimethene chemical inhibitor of uroporphyrinogen decarboxylase. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2510. doi:10.1158/1538-7445.AM2014-2510
Abstract 2515: Synthesis of substituted chromone-2-phenylcarboxamides as anti-breast cancer agents
Authors: Redda Kinfe Ken , Gangapuram Madhavi , Ghaffari Mohammad A. , Eyunni Suresh
Published: May 2015

Breast cancer is one of the most commonly diagnosed cancers and causes the second leading cancer deaths in women. The overall survival rate of breast cancer patients has substantially increased during the last decades due to early tumor detection. Unfortunately, the recovery rate of advanced breast cancer by current available drug treatment is still unacceptably low. Although chemotherapy is the main stay of cancer treatment at present, most of the drugs cause general toxicity to any proliferating cells and tend to severely limit the therapeutic values of these drugs. In an attempt to overcome these problems, the synthesis of new anti-breast cancer agents with unique mechanisms of action was developed. Chromones are benzannulated oxygen containing heterocyclic compounds widely distributed in nature that have a -pyrone ring. The chemical core of chromone has been classifies as a privileged structures in drug discovery, due to its uses in a wide variety of pharmacologically active compounds such as anti cancer, anti-HIV, antibacterial and anti-inflammatory agents. Based on these considerations, we attempted the synthesis of novel substituted chromone-2-phenylcarboxamides as anti-breast cancer agents. The starting material chromones-2-carboxylic acid was obtained by the hydrolysis reaction of ethyl esters with the mixture of glacial acetic acid and concentrated HCl (2:1v/v) and the mixture was heated to reflux for 12 h. The acids were obtained in 70-80% yields. A suspension of substituted chromone-2-carboxylic acid in DMF was added to substituted anilines, in the presence of diphenylphosphorylazide and triethylamine. The reaction mixture was stirred at room temperature for 24 h, which furnished the target compounds i.e., substituted chromone-2-phenylcarboxamides. These compounds were evaluated for their cytotoxic effects on MCF-7 ER+ve breast cancer cells, MDA-MB-231 ER-ve breast cancer cell lines and Ishikawa cells using the CellTiter-Glo luminescent cell viability assay. N-(4-hydroxyphenyl)-5,7-dimethoxy-4-oxo-4H-chromene-2-carboxamide showed the most potent cytotoxicity with an IC50 values of 29.5 and 25.7 µM on MCF-7 and Ishikawa cell lines, respectively.

This research was supported by National Center for Research Resources and the National Institute of Minority Health and Health Disparities of the National Institutes of Health through Grant Number 8 G12MD007582-28.

Citation Format: Kinfe Ken Redda, Madhavi Gangapuram, Mohammad A. Ghaffari, Suresh Eyunni. Synthesis of substituted chromone-2-phenylcarboxamides as anti-breast cancer agents. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2515. doi:10.1158/1538-7445.AM2014-2515
Abstract 2534: High-affinity small molecule inhibitors of the menin-MLL interaction reverse oncogenic transformation mediated by MLL fusion proteins in leukemia
Authors: Grembecka Jolanta E. , He Shihan , Senter Timothy J. , Borkin Dmitry , Pollock Jonathan , Han Changho , Upadhyay Sunil Kumar , Purohit Trupta , Miao Hongzhi , Gogliotti Rocco D. Gogliotti D. , Lindsley Craig W. , Cierpicki Tomasz , Stauffer Shaun R.
Published: May 2015

The protein-protein interaction (PPI) between menin and Mixed Lineage Leukemia (MLL) plays a critical role in acute leukemias with translocations of MLL gene, and inhibition of this interaction with small molecules represents a new potential therapeutic strategy for the MLL leukemia patients. We identified novel small molecule inhibitors of the menin-MLL interaction with distinct molecular scaffolds by performing a High Throughput Screening (HTS) of over 300,000 compounds. Extensive medicinal chemistry efforts performed for two lead classes to improve inhibitory activity of these compounds resulted in menin-MLL inhibitors with low nanomolar binding affinities. The menin-inhibitor co-crystal structures revealed that these compounds directly bind to menin and closely mimic the key interactions of MLL with menin, resulting in their high binding affinity. Interestingly, the hydroxymethylpiperidine class of the menin-MLL inhibitors extends beyond the MLL binding region on menin, providing additional opportunity for their optimization. We combined extensive crystallography studies with structure-based design to perform rational optimization and scaffold modification to rapidly improve activity and modulate physicochemical properties of the menin-MLL inhibitors. Treatment of MLL leukemia cells with the most potent menin-MLL inhibitors we developed resulted in very effective and selective inhibition of cell proliferation, induced apoptosis and differentiation of these cells. These effects were associated with downregulation of Hoxa9 and Meis1 expression, the downstream targets of MLL fusion proteins required for their leukemogenicity, demonstrating a very specific mechanism of action for these newly developed menin-MLL inhibitors. In vivo studies are currently undergoing to assess the effect of these compounds on leukemia progression in animal models of MLL leukemia. Our studies provide a novel and very attractive scaffolds for further development as a new potential therapeutic approach for the MLL leukemia patients.

Citation Format: Jolanta E. Grembecka, Shihan He, Timothy J. Senter, Dmitry Borkin, Jonathan Pollock, Changho Han, Sunil Kumar Upadhyay, Trupta Purohit, Hongzhi Miao, Rocco D. Gogliotti D. Gogliotti, Craig W. Lindsley, Tomasz Cierpicki, Shaun R. Stauffer. High-affinity small molecule inhibitors of the menin-MLL interaction reverse oncogenic transformation mediated by MLL fusion proteins in leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2534. doi:10.1158/1538-7445.AM2014-2534
Abstract 2562: Evaluate in vivo efficacy of anti-tumor immuno-therapeutics using MixenoTM mouse models
Authors: Zhang Juan , Qiu Junzhuan , Sun Ziyong , Dong Xin , Jiping Zha , Shi Qian
Published: May 2015

The past few years have witnessed a renaissance in the field of cancer immunotherapy, relating largely to the clinical advances associated with the development of immunomodulatory agents, e.g. monoclonal antibodies targeting the immune inhibitory pathways (CTLA-4 and PD-1/PD-L1). Often, the preclinical efficacy assessments are based on the evaluation of surrogate anti-mouse target antibodies using mouse syngenic tumor models, because of the need for T cell activation in an immune competent system. However, this strategy is limited due to the fact that the immune systems in human and mouse are different, and a surrogate molecule needs to be tested in those syngenic models. Here we set out to validate mouse models that harbor human immune cells by pre-engrafting the immuno-deficient mice with human PBMC (the MixenoTM model), and use them for efficacy evaluation of the humanized anti-PD-1 antibody. PD-L1 high-expression human tumor cell lines are selected for these in vivo models. T cell activation, infiltration and the cytokine production were analyzed to illustrate the immune cell involvement. In conclusion, the MixenoTM models are useful tools in immunotherapeutic antibody development, and may greatly increase the clinical translatability of animal studies.

Citation Format: Juan Zhang, Junzhuan Qiu, Ziyong Sun, Xin Dong, Zha Jiping, Qian Shi. Evaluate in vivo efficacy of anti-tumor immuno-therapeutics using MixenoTM mouse models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2562. doi:10.1158/1538-7445.AM2014-2562
Abstract 2565: Conformation overrides base sequence - insights from a novel class TLR-9 agonists
Authors: Kapp Kerstin , Kleuss Christiane , Schroff Matthias , Wittig Burghardt
Published: May 2015

Introduction

DNA-based TLR-9 agonists are potent activators of immune cell populations and the immune system. Preclinical and ongoing clinical studies support the use of TLR-9 agonists as immunomodulators and/or adjuvants, and proof their anti-tumor effect by enhancing both the humoral and cellular responses. From the 4 classes of known activators with distinct immunomodulation profiles all members except one comprise of linear DNA molecules. The exception is dSLIM®, the active ingredient of MGN1703 in several clinical trials. dSLIM® establishes a new class of TLR-9 agonists, represented by covalently closed dumbbell-like DNA molecules consisting entirely of natural DNA with two single-stranded CG-containing loops separated by a double-stranded stem.

Results

By cytokine secretion and cellular activation of relevant cells in human PBMC we show that CG-motifs - such as in ODN2006, a class B TLR9 agonist - if incorporated in dSLIM® conformation reveal an improved immunomodulatory potential compared to their corresponding linear CpG oligonucleotides. Increased cytokine secretion encompasses the central anti-cancer cytokine IFN-alpha, the potent angiostatic cytokine IP-10, or IFN-gamma the key activator of NK-, NKT-, and cytotoxic T-cell responses. After 24-48h exposure to dSLIM® TLR-9 agonists, also activation surface markers of relevant cell subpopulations of PBMC are up-regulated including CD80 of pDCs, CD86 of B-cells, CD86 and CD169 of monocytes, CD69 of NK, NKT as well as T cells. Consequently, dSLIM® triggers an explicit specific cytotoxicity against Jurkat target cells while ODN2006 is without significant effect in this readout. All these effects are considered important and relevant cancer-fighting features of dSLIM®.

Conclusions

Differences in immunomodulation profiles between classes of TLR-9 agonists appear to be more closely related to their conformation than their individual nucleotide sequences. We show individual loop sequences in dSLIM® that differentiate the immunomodulation profile of molecules only to a moderate extent. Inherent to the dumbbell-like structure of dSLIM® TLR-9 agonists is their potential to dimerize loop-binding TLR-9 molecules in the membrane of immune-competent cells. Thereby, dSLIM® TLR-9 agonists most probably not only bind and activate but also cluster TLR-9 molecules thereby changing the TLR-9 triggered immune response in a qualitative way. Stabilizing chemical modifications in linear CpG ODN also impact the immunomodulatory profile of TLR-9 agonists and are discussed.

Citation Format: Kerstin Kapp, Christiane Kleuss, Matthias Schroff, Burghardt Wittig. Conformation overrides base sequence - insights from a novel class TLR-9 agonists. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2565. doi:10.1158/1538-7445.AM2014-2565
Abstract 258: Characterizing cancer gene mutations in non-small cell lung cancer from African American patients
Authors: Bollig-Fischer Aliccia , Gadgeel Shirish , Chen Wei , Cote Michele , Schwartz Ann G. , Bepler Gerold
Published: May 2015

Background and Hypothesis: Lung cancer is the leading cause of cancer-related deaths in the United States. The reasons for higher incidence and inferior survival rates among African American lung cancer patients have not been adequately defined. We hypothesized that molecular differences, specifically differential incidence of somatic cancer gene mutations, may be a contributing factor to these population-based disparities. Large-scale, high-throughput genomic studies of the major subtype of lung cancer, non-small cell lung cancer (NSCLC), have so far neglected to adequately represent African American patients. Such information holds potential to transform treatment decisions to improve care for subsets of patients and address disparities.

Methods: We tested our hypothesis using a MALDI-TOF mass spectrometry approach to analyze tumor DNA for 214 coding mutations in 26 cancer genes previously linked to NSCLC, including oncogenes and tumor suppressor genes. The sample sets investigated included NSCLC specimens from 335 patients of European ancestry and 137 African Americans. For 299 of these 472, DNA from normal matched specimens was available and included in the study, thus totaling 771 samples.

Results: There was no significant difference in frequencies for somatic cancer gene coding mutations across populations and gene mutation frequencies were within reported ranges, e.g., KRAS (11%), EGFR (10%) and PIK3CA (2%). However, this study also uncovered a previously unrecognized germ line variant in the DDR2 oncogene. In addition, we found that the frequencies of single nucleotide polymorphisms (SNPs) in STK11, cMet, and NOTCH differ between African American and European ancestry patients.

Conclusion: This body of work is among the largest reported on to date comparing somatic cancer gene mutations in African American and European-ancestry patients. The outcomes show that across a broad spectrum of coding mutations, genomic tumor testing could equally benefit both populations.

Citation Format: Aliccia Bollig-Fischer, Shirish Gadgeel, Wei Chen, Michele Cote, Ann G. Schwartz, Gerold Bepler. Characterizing cancer gene mutations in non-small cell lung cancer from African American patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 258. doi:10.1158/1538-7445.AM2014-258
Abstract 2583: Intratumoral treatment of one tumor lesion with LTX-315 induces complete tumor regression and long-term specific protective immune responses in a metastatic rodent tumor model
Authors: Rekdal Øystein , Nestvold Janne , Wang Meng Y. , Camilio Ketil André , Areffard Ali , Sveinbjørnsson Baldur , Kvalheim Gunnar
Published: May 2015

Host defense peptides are naturally occurring peptides that have an important function in innate immune responses in almost every life form. Recently it has been documented that several host defense peptides have anticancer activity. Based on a naturally occurring host defense peptide we have do novo designed the short chemically modified peptide LTX-315. We have demonstrated that LTX-315 induces an immunogenic type of cell death with subsequent release of danger signals (e.g. HMBG1, ATP and Cytochrome C) and tumor associated antigens (TAA`s). In addition LTX-315 also has the ability to directly modulate immune-responses.

In a novel rat mescenhymal sarcoma model (rTMSC) we demonstrate that LTX-315 induces a complete tumor regression by intratumoral (i.t.) injection. Studies on treated tumor tissue confirmed massive necrosis and infiltration of immune cells. Successfully treated animals were protected against re-challenge with the tumor cell type treated, but not against other types of tumor cells. Moreover, tumor resistance could be adoptively transferred by spleen cells from LTX-315-treated animals. The resistance was abrogated by depletion of T-lymphocytes.

To clarify whether intratumoral injection of LTX-315 in one tumor lesion can have an effect on metastatic disease, intraperitoneal tumor and two subcutaneous tumors were established in the animals. Thereafter, LTX-315 was injected into one of the subcutaneous lesion and tumor growth assessed by living imaging. The results showed that LTX-315 eradicated all three lesions and the animal went into durable complete remission.

We propose that by targeting tumor locally LTX-315 can be used for individualized therapeutic in situ vaccination against cancer.

Citation Format: Øystein Rekdal, Janne Nestvold, Meng Yu Wang, Ketil André Camilio, Ali Areffard, Baldur Sveinbjørnsson, Gunnar Kvalheim. Intratumoral treatment of one tumor lesion with LTX-315 induces complete tumor regression and long-term specific protective immune responses in a metastatic rodent tumor model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2583. doi:10.1158/1538-7445.AM2014-2583
Abstract 2586: Bortezomib reduces preexisting antibodies to recombinant immunotoxins in mice
Authors: Manning Michael L. , Mason-Osann Emily , Onda Masanori , Pastan Ira
Published: May 2015

Recombinant immunotoxin (RIT) therapy is limited in patients by neutralizing antibody responses. Most patients with normal immune systems make neutralizing antibodies after one cycle (three doses) of RIT, preventing repeated dosing. Furthermore, some patients have preexisting antibodies from environmental exposure to Pseudomonas exotoxin (PE), the component of the RIT that elicits the neutralizing antibody response. Bortezomib is an FDA-approved proteasome inhibitor which selectively targets and kills plasma cells which are necessary for a neutralizing antibody response. Bortezomib, in combination with methotrexate, rituximab, and cyclophosphamide, was shown to reduce antibody levels >64-fold following enzyme replacement therapy in the clinical setting of Pompe disease. We hypothesized bortezomib may abrogate the neutralizing antibody levels, making dosing of RIT possible in mice already immune to RIT.

We immunized BALB/c mice with multiple doses of SS1P, a RIT whose antibody portion targets mesothelin. Antibody titers were determined by immune-capture ELISA. Mice with elevated levels were separated into groups to receive bortezomib (1mg/kg i.v., twice a week for 5.5 weeks) or saline control. Bortezomib treatment lowered the starting antibody titer in mice previously immunized with RIT by an average of 50%, while antibody titers in control mice increased by an average of 20%. Despite success in lowering titers, bortezomib did not abrogate titer in any mice.

To further diminish preexisting antibodies, we performed a separate experiment in which mice with preexisting anti-SS1P antibodies received bortezomib with concomitant pentostatin and cyclophosphamide (P/C) therapy. P/C is an immune-depleting regimen already shown to be effective in preventing the onset of new neutralizing antibodies. Combination treatment lowered the starting antibody titer in mice previously immunized with RIT by an average of 73%, while antibody titers in control mice increased by an average of 80%.

We have shown that bortezomib reduces, but does not eliminate, antibody titer in mice with preexisting antibodies to RIT. Addition of the immune-depleting P/C regimen further diminishes antibody titers. We are investigating remaining plasma cell populations as a potential source of remaining antibody. Ongoing experiments also include incorporating other FDA-approved agents into our study.

Citation Format: Michael L. Manning, Emily Mason-Osann, Masanori Onda, Ira Pastan. Bortezomib reduces preexisting antibodies to recombinant immunotoxins in mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2586. doi:10.1158/1538-7445.AM2014-2586