bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3477: Graduate public health students as health educators: A pilot project to promote cancer health in Puerto Rico
Authors: Moreno Laura , Vélez Himilce , Jiménez Julio , Vadaparampil Susan , Muñoz-Antonia Teresita , Torres José , Quinn Gwendolyn
Published: July 2016

Introduction: Community health workers, known as promotores de salud in Spanish, have been recognized as an effective communication channel to promote health education among marginalized groups. However, cancer education programs often cease efforts when funding ends. This can be particularly problematic in Puerto Rico (PR), where cancer is the leading cause of death. In an effort to identify new ways to provide sustainable cancer health education programs, the NCI-funded PACHE partnership between Moffitt Cancer Center and Ponce Health Sciences University (PSHU) trained graduate public health students to disseminate cancer education to PR communities. This was achieved by developing a culturally adapted version of “Cancer 101: A Cancer Education and Training Program” (Cancer 101) to the PR community. A pilot study was conducted to test students’ ability to: I) deliver the educational intervention, charlas, in the community; II) utilize a specific module from the curriculum, “Cancer in PR”, which focused on current island data and statistics, and definitions of the cancer and its various types; and III) to measure community members’ cancer knowledge before and after the charla.

Methods: Students enrolled in the class Principles of Health Education in Practice (n = 15) participated in a two-day Cancer 101 training session. Upon completion, students were divided into 5 teams (n = 3 per team), each delivering the “Cancer in PR” charla to a community group of their selection. Cancer knowledge was assessed before and after the charla via a 10-item true or false questionnaire. Items were summed to create an overall knowledge score (range: 0-10).

Results: A total of 201 participants attended the community educational intervention at 10 different locations in PR (Salinas Elderly, Guancha, Hogar San Jose, etc.). Participants pre and post-test mean score (SD) were 4.54 (2.31) and 7.65 (2.28) respectively, with a 3.1 (2.79) point increase in overall cancer knowledge (p<.000). Students reported the Cancer 101 toolkit was feasible and appropriate for each of the communities in which they delivered the intervention.

Conclusion: Results from this pilot project suggest public health students are an innovative and sustainable way to improve cancer health communication and a novel strategy for reducing cancer health disparities.

Citation Format: Laura Moreno, Himilce Vélez, Julio Jiménez, Susan Vadaparampil, Teresita Muñoz-Antonia, José Torres, Gwendolyn Quinn. Graduate public health students as health educators: A pilot project to promote cancer health in Puerto Rico. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3477.
Abstract 348: Small molecule Axl Ig1-Gas6 inhibitors block Gas6-inducible Axl signaling and suppress tumorigenicity
Authors: Bansal Nitu , Kimani Stanley , Singh Kamlendra , Comollo Thomas , Kumar Sushil , Kholodovych Vladyslav , Peng Youyi , Welsh William , Joseph Bertino , Birge Raymond B.
Published: July 2016

TAM receptors (Tyro-3, Axl, and Mer) are a family of three homologous receptor tyrosine kinases (RTKs) expressed predominantly on myeloid-derived hematopoietic cells and epithelial cells which function as inhibitory receptors that dampen inflammatory responses and maintain tissue tolerance. Additionally, all three TAMs have been implicated in various human malignancies, where level of expression is often associated with more aggressive staging of the cancers, poorer predicted patient survival, as well as drug-resistant cancers. Over the past several years, considerable effort has been made to generate small molecule tyrosine kinase inhibitors and biological therapeutics (such as monoclonal antibodies and soluble extracellular receptor traps) targeting TAM receptors in human cancers. Early preclinical studies show promising anti-tumor activities using either kinase inhibitors or monoclonal antibodies against Axl and Mer, although cross-reactivity with other tyrosine kinase inhibitors is associated with off-target specificities. Here, we report the development and characterization of a series of small molecule inhibitors that target the interface between the Ig1 domain of Axl and Gas6, and inhibit native receptors and Axl reporter lines with sub-micro-molar affinities. Additionally, these compounds inhibit Gas6 inducible motility and invasion in Axl-expressing cell lines, and suppress tumor growth in xenograft models of non small cell lung cancer. Together, these observations demonstrate that Axl can be targeted by small molecules that bind to the Ig1 domain/Gas6 interaction, and validate Ig1 inhibitors as novel agents for treatment of cancer.

Citation Format: Nitu Bansal, Stanley Kimani, Kamlendra Singh, Thomas Comollo, Sushil Kumar, Vladyslav Kholodovych, Youyi Peng, William Welsh, Bertino Joseph, Raymond B. Birge. Small molecule Axl Ig1-Gas6 inhibitors block Gas6-inducible Axl signaling and suppress tumorigenicity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 348.
Abstract 3482: Inhibition of glucose metabolism in neuroendocrine tumors by lanreotide, sunitinib, and rapamycin
Authors: Rubinek Tami , Shabtay-Orbach Ayelet , Hesin Arkadi , Rubinstein Tammi , Wolf Ido
Published: July 2016

Background: Neuroendocrine tumors (NETs) represent a heterogeneous family of neoplasms, which may develop from different endocrine glands or endocrine cells dispersed throughout the digestive and respiratory tracts. Lanreotide is a somatostatin analog approved for the treatment of NETs. While Lanreotide extends survival of patients and slows tumor growth, controversy exists regarding its mechanism of action. We hypothesized that lanreotide can interfere with the metabolic activity of NETs, and compared its effect to that of rapamycin and sunitinib.

Methods: The pancreatic carcinoid BON-1 cells and pulmonary carcinoid NCI-H727 cells were used. Viability was tested using colony formation assay. mRNA levels of the glycolytic enzymes hexokinase 2 (HK2), lactate dehydrogenase (LDH) and pyruvate kinase M2 (PKM2) was tested using real-time PCR. Phosphorylation levels of AMP-activated kinase (AMPK) and acetyl CoA carboxylase (ACC) were tested using Western blot. The activity of HK2 was tested using HK enzymatic assay. Lactate secretion was tested in the routine biochemistry laboratory of our institution.

Results: Lanreotide, rapamycin and sunitinib significantly decreased colony formation. They also reduced relative expression levels of HK2, LDH and PKM2 24 and 48h after treatment and decreased HK2 activity after 24h. An increase in the phosphorylation levels of AMPK and ACC was observed shortly after treating the cells with lanreotide and rapamycin. All three agents also decreased lactate concentration in the media.

Conclusions: These data indicate tumor metabolism as an attractive target for therapies against NET, and a novel mechanism of action of somatostatin analogs, namely, alteration of tumor metabolism.

Citation Format: Tami Rubinek, Ayelet Shabtay-Orbach, Arkadi Hesin, Tammi Rubinstein, Ido Wolf. Inhibition of glucose metabolism in neuroendocrine tumors by lanreotide, sunitinib, and rapamycin. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3482.
Abstract 3485: Estrogen receptor ligands and their responses in de novo and tamoxifen resistant cell models
Authors: Gutgesell Lauren M. , Thatcher Gregory R. J. , Patel Hitisha , Xiong Rui
Published: July 2016

Approximately 75% of breast cancer incidences are Estrogen Receptor positive (ER+). Tamoxifen, a selective estrogen receptor modulator (SERM), is the standard of care for many of these ER+ breast cancer patients. Unfortunately, tamoxifen resistance occurs in almost 50% of patients within 5 years of treatment, and endocrine-independence accompanying resistance negates the effects of aromatase inhibitors. Paradoxically, estradiol (E2) has shown clinical efficacy in patients with resistant breast cancer. Understanding the antiproliferative role of E2 and ER signaling in resistant ER+ cell lines is essential to gain a better understanding of paradoxical clinical efficacy and for the appropriate biomarker-assisted selection of endocrine therapy for various stages of ER+ breast cancer. We have discovered ER ligands, based upon a single chemical scaffold with a diverse set of pharmacological responses, which can be used to better understand the role of ER signaling in resistance and therapy: selective ER modulators (SERMs), selective ER downregulators (SERDs), selective estrogen mimics (SEMs), and selective human ER partial agonists (ShERPAs). These compounds were initially classified using an ERE luciferase reporter assay and affinity for ER confirmed by biochemical assays. The effects of these novel ER-directed chemical probes on cell viability were further examined in multiple 2D and 3D spheroid models of tamoxifen resistance. Finally, ERα localization upon administration of these ligands in tamoxifen-sensitive and tamoxifen-resistant cells was studied. While SERDs and SEMs showed growth inhibition in tamoxifen resistant cell lines, both molecules had different responses and mechanisms of growth inhibition. ERα was localized to extranuclear sites upon administration of E2, SEMs, and ShERPAs, an observation specific to the resistant phenotype and mechanistically associated with spheroid disintegration. SERDs inhibited the antiproliferative actions of E2, but were antiproliferative in resistant cell lines. Further dissection of the role of ER in resistance and survival is needed to define the appropriate ER-directed, endocrine therapy in ER+ breast cancer.

Citation Format: Lauren M. Gutgesell, Gregory R. J. Thatcher, Hitisha Patel, Rui Xiong. Estrogen receptor ligands and their responses in de novo and tamoxifen resistant cell models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3485.
Abstract 3492: Differential sensitivity of normal liver and hepatocarcinoma tissues to tBid induced apoptosis is determined by the abundance of VDAC2
Authors: Naghdi Shamim , Roy Soumya Sinha , Walter Ludivine , Hajnoczky Gyorgy
Published: July 2016

A conserved step in extrinsic apoptosis is the outer mitochondrial membrane (OMM) permeabilization by truncated Bid (tBid) that is generated from Bid upon cleavage by Caspase-8 activated at death receptors. tBid directly or indirectly activate proapoptotic proteins i.e. Bak in OMM and/ or Bax in cytosol which is followed by a change in their conformation and oligomerization, leading to a pore formation in the OMM. To better understand the mitochondrial phase of this pathway we applied tBid to semi-permeabilized cells and isolated mitochondria. We monitored cytochrome c release, and the ensuing mitochondrial membrane potential collapse, using immunoblotting and fluorimetry as the markers for mitochondrial cell death. We found that normal liver tissue and normal hepatocytes (Rat, Mouse or Human) are unexpectedly resistant to tBid, whereas hepatocellular carcinoma (HCC) tissues and cell lines (e.g. PLC, Huh7) are greatly sensitized. Considering that the effect of tBid is most effectively mediated through Bak, its abundance in the mitochondria was tested. Bak level is significantly higher in tumor tissue/cell lines, whereas OMM-bound tBid and Bax levels are similar in both cancerous and normal tissue. Since we have recently found that voltage dependent anion channel 2 (V2), a pore forming OMM protein, is specifically required for Bak targeting to OMM, the abundance of V2 was also measured. V2 showed significant upregulation in HCC tissue/cell lines. Rescue studies using V2 sensitized normal hepatocytes and liver mitochondria to tBid. We propose that V2 might be the main regulator for the differential tBid sensitivity in normal liver and hepatocellular carcinoma.

Citation Format: Shamim Naghdi, Soumya Sinha Roy, Ludivine Walter, Gyorgy Hajnoczky. Differential sensitivity of normal liver and hepatocarcinoma tissues to tBid induced apoptosis is determined by the abundance of VDAC2. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3492.
Abstract 3504: Arginylated BiP/oxidized DJ-1 complex mediates crosstalk between the endoplasmic reticulum and mitochondria
Authors: Lee. Dae-Hee
Published: July 2016

In cell death induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), although much is known about signal pathways involving external signals through cell surface receptors or internal signals through mitochondrial dysfunction, little is known about signal pathways activated by endoplasmic reticulum (ER) stress during TRAIL treatment. Here, we report that TRAIL-induced caspase activation is a prerequisite for TRAIL-induced ER stress response. During TRAIL treatment, ER chaperon BiP dissociates from ER stress sensors such as PERK and IRE1α and the activation of the PERK-eIF2α-ATF4-CHOP apoptotic signal transduction pathway occurs. Meanwhile ER-stress induces BiP release to the cytosol and ATE1-encoded Arg-tRNA transferase arginylates BiP. TRAIL also induces loss of ΔΨm and redox sensor DJ-1 detects mitochondria-derived ROS. Oxidized DJ-1 binds to arginylated BiP (R-BiP) in the cytosol. DJ-1 binding activity was abrogated in C106A and C53A but not C46A mutant. R-BiP/oxidized DJ-1 complex may mediate crosstalk between the ER and mitochondria.

Key words: TRAIL; endoplasmic reticulum; apoptosis; colorectal cancer

Citation Format: Dae-Hee Lee. Arginylated BiP/oxidized DJ-1 complex mediates crosstalk between the endoplasmic reticulum and mitochondria. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3504.
Abstract 3510: Tolfenamic acid induces apoptosis and growth inhibition in anaplastic thyroid cancer: involvement of nonsteroidal anti-inflammatory drug-activated gene-1 expression and intracellular reactive oxygen species generation
Authors: Kim Yeon Soo , Chang Jae Won , Kang SungUn , Choi JaeWon , Shin YooSeob , Baek Seung Joon , Lee Seong-Ho , Oh Kyoung Ho , Kim Chul-Ho
Published: July 2016

Nonsteroidal anti-inflammatory drugs (NSAIDs) are usually used for the treatment of inflammatory diseases. However, certain NSAIDs also have antitumor activities in various cancers, including head and neck cancer, through cyclooxygenase-dependent or independent pathways. Nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1), a TGF-β superfamily protein, is induced by NSAIDs and has been shown to be induced by several antitumorigenic compounds and to exhibit proapoptotic and antitumorigenic activities. In this report, we demonstrate for the first time that tolfenamic acid (TA) transcriptionally induced the expression of NAG-1 during TA-induced apoptosis of anaplastic thyroid cancer (ATC) cells. TA reduced the viability of ATC cells in a dose-dependent manner and induced apoptosis, findings that were coincident with NAG-1 expression. Overexpression of the NAG-1 gene using cDNA enhanced the apoptotic effect of TA, whereas suppression of NAG-1 expression by small interfering RNA attenuated TA-induced apoptosis. Subsequently, we found that intracellular ROS generation plays an important role in activating the proapoptotic protein NAG-1. Then, we confirmed antitumorigenic effects of TA in a nude mouse orthotopic ATC model, and this result accompanied the augmentation of NAG-1 expression and ROS generation in tumor tissue. Taken together, these results demonstrate that TA induces apoptosis via NAG-1 expression and ROS generation in in vitro and in vivo ATC models, providing a novel mechanistic explanation and indicating a potential chemotherapeutic approach for treatment of ATC.

Citation Format: Yeon Soo Kim, Jae Won Chang, SungUn Kang, JaeWon Choi, YooSeob Shin, Seung Joon Baek, Seong-Ho Lee, Kyoung Ho Oh, Chul-Ho Kim. Tolfenamic acid induces apoptosis and growth inhibition in anaplastic thyroid cancer: involvement of nonsteroidal anti-inflammatory drug-activated gene-1 expression and intracellular reactive oxygen species generation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3510.
Abstract 3514: Inhibition of PI-3 kinase signaling potentiates the apoptotic response to mitotic arrest
Authors: Kassim Roaa M. , Shuster Charles B.
Published: July 2016

Anti-mitotic drugs represent a common strategy for cancer chemotherapy by inducing a prolonged prometaphase delay and eventual cell death by apoptosis. Kinesin Spindle Protein (KSP) inhibitors arrest cells in mitosis by blocking bipolar spindle assembly, and these drugs have demonstrated limited side effects in clinical trials. However, they are not as effective as Taxol, whose efficacy is limited by the frequent development of resistance as well as the deleterious effects on non-dividing cells. Recent studies suggest that the ability of cells to maintain energy levels during mitosis could account for the differential responses of cancer cells to mitotic arrest. To determine if manipulating cellular metabolism could potentiate the efficacy of KSP inhibitors, we arrested cells in mitosis in the absence or presence of the PI3-kinase LY294002, and probed for the induction of apoptosis. Because tumor cells respond differently to mitotic arrest, we employed two cell lines that either survive mitotic arrest (MCF7) or undergo apoptosis shortly following mitotic slippage (HeLa). In both cases, simultaneous inhibition of KSP and PI3-kinase induced apoptosis more effectively than mitotic arrest or PI3-K inhibition alone. In the case of HeLa cells, there also appeared to be an acceleration of cell death such that arrested cells underwent apoptosis prior to mitotic slippage. Similar effects were observed using Akt inhibitors, providing additional evidence that PI3-kinase signaling was critical for surviving mitotic arrest. Lastly, we found that increased glucose had a protective effect, further supporting the notion that the ability to survive mitotic arrest depended on the ability to maintain energy levels. If confirmed, this may represent a novel approach for potentiating the anti-tumor activity of KSP inhibitors in the clinic.

Citation Format: Roaa M. Kassim, Charles B. Shuster. Inhibition of PI-3 kinase signaling potentiates the apoptotic response to mitotic arrest. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3514.
Abstract 3517: Expression studies of RBBP6 in breast and cervical cancer suggests a role in apoptosis activation
Authors: Choene Mpho , Moela Pontsho , Motadi Lesetja
Published: July 2016

Ubiquitin-like DWNN domain and RING finger-like domain presence on Retinoblastoma Binding protein 6 (RBBP6), is linked to its function as cancer promoting gene through p53 degradation via its p53-binding domain. RBBP6 bind and regulate the expression of p53 and pRB in the cell machinery implicates RBBP6 in cell proliferation during cancer development (Moela et al 2014). Studies indicate that overexpression of the mouse spliced variants led to apoptosis and cell cycle arrest whilst deregulation in mice led to embryonic lethality followed by p53 accumulation and a widespread apoptosis (Li et al., 2007). In this study, focuses on determining the expression pattern of RBBP6 in human tissue models of breast and cervical cancer. The study further aims to analyze apoptosis and cell cycle following either knockdown or overexpression of RBBP6 coupled to treatment with potential anticancer compounds, camptothecin and GABA. Quantitative analysis by qPCR in HeLa, SiHa, MCF-7and MDA-MB-231 cell lines shows that RBBP6 expression is fairly high in cancer cells when compared to normal lung fibroblast. Following co-treatment with GABA and cDNA or siRNA, in cDNA treated there was a reduction in both apoptosis as measured by flow cytometer and TP53 expression while in siRNA there was increased expression of TP53 and those of Bax and Bak1 which seemed to account for increased apoptosis. Cell cycle presented increase in G0/G1 and the G2/M following treatment with siRNA and GABA. GABA alone could not induce a significant increase in apoptotic cells whereas when combined with siRNA it was able to induce over 30% apoptosis. These results suggest that RBBP6 might be a cancer promoting gene.

Citation Format: Mpho Choene, Pontsho Moela, Lesetja Motadi. Expression studies of RBBP6 in breast and cervical cancer suggests a role in apoptosis activation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3517.
Abstract 3518: Flow cytometry as a single platform tool to evaluate multiple mechanisms of actions of therapeutic antibodies
Authors: Chadderton Antony R. , Harvey Shane , Strake Brandy , Giles-Komar Jill , Capocasale Renold J. , Raju T. Shantha
Published: July 2016

The unique ability of flow cytometry to simultaneously examine intricate details of multiple cell subsets is unparalleled and the platform is now a dominant tool for measuring antibody mediated effector functions on cancer cells. The objective of this study is to illustrate the utility of flow cytometry as a single platform for concurrently measuring the multiple mechanisms of actions of therapeutic antibodies such as ADCC, ADCP, Apoptosis, CDC and Trogocytosis on human cancer cells and immune functioning cells. Rituximab® was tested with the Burkett's Lymphoma cell line (Daudi) for all assays between the ranges of 0.01 and 100ug/mL. Apoptosis was evaluated by measuring Caspase 3/7 staining. ADCC assays were established with Rituximab®/CFSE labeled Daudi cells and human PBMC’s. ADCP was assessed with Rituximab®/ CFSE labeled Daudi cells and CD14+/CD11b+ in vitro generated phagocytes using a 4 hour exposure. CDC assays were established with Daudi cells/ 10% pooled human serum using 7-AAD as the cytotoxic indicator in a 4 hour assay. Trogocytosis was measured up to 4 hours with Rituximab®/ Daudi and PBMC's and CD19/20 and CD14. All assays were analyzed using a FACS ARIA III flow cytometer and replicates of 3 were evaluated for statistical relevance. Results demonstrated that Rituximab® induced effects were observed in all 5 assays using flow cytometry. Apoptosis was induced when 0.01ug/mL Rituximab® was present. ADCP occurred at levels as low as 0.01ug/mL Rituximab®. ADCC and CDC occurred at levels as low as 0.1ug/mL Rituximab®. Trogocytosis, indicated by the transfer of CD19+ B cells to CD14+ monocytes, occurred at 0.1ug/ml Rituximab®. In summary, we demonstrated the clinical applicability of flow cytometry as a single platform to simultaneously evaluate five different mechanisms of actions that can occur when therapeutic antibodies are used to treat cancer cells.

Citation Format: Antony R. Chadderton, Shane Harvey, Brandy Strake, Jill Giles-Komar, Renold J. Capocasale, T. Shantha Raju. Flow cytometry as a single platform tool to evaluate multiple mechanisms of actions of therapeutic antibodies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3518.
Abstract 3531: Autophagy facilitates leukemogenesis in a murine model of MLL-AF9-driven AML
Authors: Liu Qiang , Chen Longgui , Wang Hong-Gang
Published: July 2016

Acute myeloid leukemia (AML) is a hematopoietic malignancy characterized by the clonal expansion of immature myeloid cells. There is an urgent and unmet need for the development of novel therapeutics against AML. Autophagy is a lysosomal catabolic process that has been implicated in leukemia cell survival and chemotherapy response. However, the biological role of autophagy in AML leukemogenesis remains poorly defined. In this study, we genetically ablated ATG5, a gene that is essential for autophagy, in AML cells during leukemogenesis in a murine model of MLL-AF9-driven AML. The homozygous deletion of ATG5 in AML cells significantly delayed MLL-AF9-induced leukemogenesis and progression in vivo. Likewise, the in vitro deletion of ATG5 in MLL-AF9 driven AML cells demonstrated decreased growth as well as reduced colony forming units. Interestingly, ATG5 deficiency demonstrated an altered differentiation hierarchy of MLL-AF9-induced AML cells in vivo that could be linked to decreased proliferation and suppressed leukemogenesis. Overall, this study provides insight at how autophagy can facilitate AML progression. A more comprehensive understanding on autophagy in AML is needed prior to the deployment of autophagy modulators for the treatment of AML.

Citation Format: Qiang Liu, Longgui Chen, Hong-Gang Wang. Autophagy facilitates leukemogenesis in a murine model of MLL-AF9-driven AML. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3531.
Abstract 3532: Compound Tc inhibits ATG4 to attenuate autophagic flux and sensitize cancer cells to chemotherapy
Authors: Shu. Chih-Wen
Published: July 2016

Autophagy is induced to protect cancer cells against stresses, including starvation, hypoxia and chemotherapy. Antimalarial drugs chloroquine and its derivative hydroxychloroquine are used to inhibit autophagy and being investigated with clinical trials for certain types of cancer. However, the tumor suppressive effects of the drugs might be autophagy independent, which require more specific inhibitors target autophagy components for clinical uses. Here, we screened FDA-approved drug library with both biochemical and cellular ATG4 reporter assays and found compound Tc blocked proteolysis activity of ATG4, essential proteases in autophagy machinery. Docking and MD simulation results appeared compound Tc was directly associated with active site of ATG4. Further, compound Tc obstructed the binding between ATG4 and MAP1LC3 and accumulated autophagosomes, which in turn reduce autophagic flux in cells. Moreover, compound Tc suppressed tumor viability and sensitized chemotherapy efficacy in two-dimensional and sphere culture model. Silencing ATG4 or overexpression of ATG4 catalytic mutant had no additional effects on tumor suppression compared to compound Tc alone, indicating compound Tc inhibited ATG4s activity to diminish tumor viability. In addition, compound Tc enhanced genotoxic stress-induced apoptosis to synergize chemotherapy efficacy in vitro and in vivo. Thus, we report that the clinical drug compound Tc is a potent ATG4 inhibitor, which could interrogate the role of autophagy in clinical setting and provide compound Tc as an anticancer drug or chemosensitizer.

Citation Format: Chih-Wen Shu. Compound Tc inhibits ATG4 to attenuate autophagic flux and sensitize cancer cells to chemotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3532.
Abstract 3534: Inhibition of melanoma cell survival through p62/LC3B autophagic signaling
Authors: Hambright Heather G. , Kumar Addanki P. , Ghosh Rita
Published: July 2016

The primary objectives of this study were i) systematic evaluation of the differences in basal autophagic activity between melanocytes and malignant melanoma cells to determine whether these differences may be selectively targeted using the ROS inducer, Nexrutine (NX); ii) determination of NX selectivity in inhibiting melanoma cell survival and iii) specific involvement of autophagy signaling protein, SQSTM1/p62 in NX-mediated autophagic cell survival. NX is obtained from the bark of the cork tree, Phellodendron amurense. A panel of melanocyte and melanoma cells was evaluated for basal autophagy level and effect of NX treatment, including protein levels and turnover of p62 and LC3B (+/- CQ), and number of autophagic puncta per cell. Trypan blue, MTT, and Annexin V-APC were used to evaluate the effect of NX on cell death, proliferation and apoptosis. Genetic approach using siRNA for p62 and overexpression using HA-p62 followed by survival, proliferation, and autophagy measurements were used to further evaluate the possibility of p62 as a molecular target for inhibition of autophagy seen using NX. We found that i) melanoma cells displayed high autophagic flux compared with melanocytes, ii) NX treatment a) inhibited autophagy in melanoma cells, b) inhibited melanoma cell survival, and c) induced apoptosis in melanoma cells, iii) knockdown of p62 resulted in a lesser inhibition of cell viability and autophagy after NX treatment in high p62-expressing melanoma cells, and iv) overexpression of p62 sensitized low p62-expressing normal melanocytes and melanoma cells to NX-mediated cell death and autophagy inhibition, suggesting that p62 is a bonafide molecular target of NX. Overall, we show that Nexrutine is able to inhibit the inherent high level of pro-survival autophagy in melanoma cells, leading to loss of cell viability and induction of apoptosis, while melanocytes remain unaffected by NX at the same doses. Further, SQSTM1/p62 was found to be a critical mediator of the NX-mediated inhibition of autophagy and cell viability. This study demonstrates that NX maybe a promising novel, non-toxic agent for melanoma.

Supported by R21 CA125719 & ACRCF (RG); NIDCR T32 DE14318/COSTAR (HGH)

Citation Format: Heather G. Hambright, Addanki P. Kumar, Rita Ghosh. Inhibition of melanoma cell survival through p62/LC3B autophagic signaling. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3534.
Abstract 354: Physiologically relevant target engagement using CETSA
Authors: Karén Jakob , Sioberg Catrine , Niekrasz Pawel , Neumuller Magnus , Martinez Molina Daniel
Published: July 2016

Targeted therapies are dependent on adequate occupancy and target engagement to reach their efficacy measure. Yet, more than half of novel candidate drugs entering clinical trial stage fail before proof of concept is achieved. Inadequate target engagement is often at the root cause of these failures. Therefore we have developed a versatile label-free method to quantify the target engagement of drugs on their native physiological relevant targets. The method is validated in multiple sample matrices from bench to bedside: in living cells and tissue from rodents and man.

CETSA is a patented biophysical method measuring the thermal stability of drug target proteins and the shift induced by their ligands. By quantifying the melting temperature and shift induced by the ligand we can quantify the potency of target engagement. This potency determination then allows filtering of candidate drugs by their ability to engage the target in its physiologically relevant form, in relevant sample formats such as intact cells or tissue.

We present a number of applications and readouts of the CETSA method quantifying target engagement of drugs used in the clinic and in development. Furthermore, we show that the target engagement of cyclin dependent kinase inhibitors correlated both to viability and to a dose dependent decrease of a cell cycle activity marker in cellular sample matrices. The biomarker, thymidine kinase activity, was analyzed with the commercially available test DiviTum®. By combining CETSA with DiviTum® in the same samples, it was thus possible to correlate CETSA outcomes to pharmacodynamic effects of a drug.

The utility and value of the CETSA method enabling preclinical development of novel drugs is substantiated by the examples included here. Combining quantifications of efficacy with the molecular target engagement of the drug yields an orthogonal dataset, which has a potential to validate the target-mediated efficacy. Such datasets can be generated from early in vitro systems to clinical studies and may reduce the high failure rate in drug development by facilitating the discovery, development and evaluation of novel drugs.

Citation Format: Jakob Karén, Catrine Sioberg, Pawel Niekrasz, Magnus Neumuller, Daniel Martinez Molina. Physiologically relevant target engagement using CETSA. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 354.
Abstract 3540: Exploring novel treatment agent in GIST by using connectivity map
Authors: Yen Chueh-Chuan , Chen Li-Tzong , Li Chien-Feng
Published: July 2016

Gastrointestinal stromal tumors (GIST) is gastrointestinal tract sarcoma which commonly contains a mutation in the KIT or PDGFRA. Imatinib mesylate, a specific tyrosine kinase inhibitor of the KIT and PDGFRA, causes regression or stabilization of disease in most of the patients with metastatic GIST. However, acquired resistance to imatinib is inevitable. Recently, E26 variant 1 (ETV1) pathway was found to be a key downstream effector of KIT. In this study, we explored the role of ETVI and its downstream effectors pathway in GIST by bioinformatics analysis, and identified potential novel agents that target ETV1 pathway of GISTs by using expression profile and a pattern-matching software “Connectivity Map” (CMAP), and confirmed the efficacy of novel agents by using GIST cell line in vitro assay. By using CMAP, four drugs with potential ETV1-inhibition effect were identified: suberanilohydroxamic acid (SAHA,Vorinostat) and trichostatin, two histone deacetylase inhibitors (HDACI), and trifluoperazine (TFP) and thioridazine (TDZ), two drugs of phenothiazine class. All four drugs were found to have ETV1-down regulating effect by immunoblotting. Phenothiazine could induce apoptosis and autophagy in GIST. Treatment of phenothiazine was found paradoxically up-regulate MEK activity. Combination of MEK inhibitor and phenothiazine showed great synergistic effect in exerting cytotoxicity against GIST cell lines. Our study established phenothiazine as a new class of agent with therapeutic effect in GIST, and combination of phenothiazine and MEK inhibitor showed great potential in GIST treatment.

Citation Format: Chueh-Chuan Yen, Li-Tzong Chen, Chien-Feng Li. Exploring novel treatment agent in GIST by using connectivity map. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3540.
Abstract 3549: The role of programed necrosis in oncolytic adenovirus-induced cell death in ovarian cancer
Authors: Weigert Melanie , Binks Alexander , Tait Stephen , McNeish Iain
Published: July 2016

Introduction.

Oncolytic viruses preferentially replicate within and kill cancer cells and are a promising novel cancer treatment. However, the mechanisms by which oncolytic adenoviruses induce death in cancer remains unclear. It was long thought that DNA viruses trigger classical apoptosis but there is now evidence that cell death induced by both adenovirus and vaccinia displays features of necrosis-like programmed cell death.

Methods and Results.

In order to investigate the role of necrosis in cell death following oncolytic adenovirus infection, a panel of ovarian cancer cells with varying sensitivities was infected with the E1A CR2-deleted adenoviral mutant dl922-947. By electron microscopy, dl922-947 infection induces key morphological features of necrotic death, including membrane rupture, nuclear swelling and cytoplasmic vacuolation. Using specific necrosis inhibitors (necrostatin-1, necrosulfonamide, GSK’840B, GSK’872B and GSK’843A) as well as RNAi-mediated knockdown of receptor interacting protein kinase 1 (RIPK1), RIPK3 and mixed lineage kinase domain-like (MLKL), we show that adenovirus-infected ovarian cancer cells undergo RIPK3-dependent necrosis and that blockage of the downstream effector MLKL significantly attenuates cell death. Co-immunoprecipitation of caspase 8 shows formation of a complex containing RIPK1, FADD and caspase 8 during adenovirus infection. However, unlike Tumour Necrosis Factor-α (TNF-α)-induced programmed necrosis, which relies on the RHIM-dependent interaction of RIPK1 and RIPK3, RIPK1 appears redundant in adenovirus-induced death. Furthermore, the addition of a TNF-α blocking antibody to virus-infected cells has no effect on either cell death or overall cell survival.

In a RIPK3 overexpression model, we show a direct correlation between adenovirus-induced cell death and the extent of RIPK3 expression. Moreover, RIPK3 expression has no effect on infectivity, viral protein expression or infectious virus production. Finally, expression of RIPK3 increases necrosis induction in vivo following direct intra-tumoural dl922-947 injection, and significantly improves anti-tumour efficacy.

Conclusions.

Our data suggest that cell death induced by oncolytic adenoviruses differs from TNF-induced programmed necrosis but still relies on the kinase RIPK3 and its downstream component MLKL, making these two proteins possible targets for future oncolytic virotherapies.

Citation Format: Melanie Weigert, Alexander Binks, Stephen Tait, Iain McNeish. The role of programed necrosis in oncolytic adenovirus-induced cell death in ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3549.
Abstract 355: HT flow cytometry platform enabling measurement of immune signaling in whole blood and T-cells for drug discovery
Authors: fereshteh mark , Zhang Litao , Li Xin , Li Sha , Fan Yi , Schieven Gary
Published: July 2016

Oral agents targeting Janus-associated kinases (JAKs) are among the most promising new agents in clinical development. To better understand the relationship between JAK inhibition and clinical outcome, compounds targeting JAKs are required to be analyzed in peripheral whole blood. To date, this type of analysis has been low throughput and is associated with high cost. Here, we developed a robust 384-well flow based assay approach to screen small molecules for inhibition of JAK/STAT signaling in whole blood using high-throughput flow cytometry. The platform provides a highly sensitive analysis of signaling events in low volumes of blood facilitating a robust target engagement assay to measure inhibition of proximal signaling by selective JAK inhibitors. Further, automation technologies and processes optimization were established to overcome bottlenecks in sample integrity, handling and multiparametric data analysis without affecting assay performance. The result was dramatically increased sample throughput when compared to conventional manual flow cytometric approaches enabling the identification of novel selective JAK/STAT inhibitors enabling the investigation of IO and Immunology targets earlier in drug discovery.

Citation Format: mark fereshteh, Litao Zhang, Xin Li, Sha Li, Yi Fan, Gary Schieven. HT flow cytometry platform enabling measurement of immune signaling in whole blood and T-cells for drug discovery. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 355.
Abstract 3551: Discovery of orally bioavailable novel Mcl-1 inhibitors that exhibit selective anti-proliferative activity in Mcl-1 sensitive cancer cell lines
Authors: Lee Taekyu , Bian Zhiguo , Belmar Johannes , Shaw Subrata , Tarr James C. , Zhao Bin , Pelz Nick , Camper DeMarco , Goodwin Craig M. , Arnold Allison L. , Sensintaffar John L. , Browning Carrie F. , Rossanese Olivia W. , Olejniczak Edward T. , Fesik Stephen W.
Published: July 2016

Myeloid cell leukemia-1 (Mcl-1) is a member of the Bcl-2 family of proteins that regulate apoptosis. Amplification of Mcl-1 is found in various cancers, which causes the evasion of apoptosis and is one of the major resistance mechanisms for many chemotherapies. Mcl-1 mediates its effects primarily through high affinity interactions with pro-apoptotic BH3 containing proteins, Bak and Bax. Thus targeting Mcl-1 with small molecule inhibitors is a promising strategy but a very challenging task. Using fragment-based methods and structure-based design, we discovered a novel class of potent Mcl-1 inhibitors that exhibit selective anti-proliferative activity. New leads containing a tricyclic indole lactam scaffold exhibited dissociation constants of <0.5 nM with >1000-fold selectivity for Mcl-1 over Bcl-xL and Bcl-2. They also promoted apoptosis only in Mcl-1 sensitive cancer cell lines by activating caspases in a dose-dependent manner. These results provide a strong proof of concept for a selective inhibition of Mcl-1 function as an effective anti-cancer therapy. Finally, our leads also possess desirable pharmaceutical properties including in vivo oral bioavailability and represent an ideal starting point for developing clinically useful Mcl-1 inhibitors for the treatment of a wide variety of cancers.

Citation Format: Taekyu Lee, Zhiguo Bian, Johannes Belmar, Subrata Shaw, James C. Tarr, Bin Zhao, Nick Pelz, DeMarco Camper, Craig M. Goodwin, Allison L. Arnold, John L. Sensintaffar, Carrie F. Browning, Olivia W. Rossanese, Edward T. Olejniczak, Stephen W. Fesik. Discovery of orally bioavailable novel Mcl-1 inhibitors that exhibit selective anti-proliferative activity in Mcl-1 sensitive cancer cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3551.
Abstract 3561: Role of bcl-2 family proteins in phorbol ester-induced apoptosis of acute myeloid leukemia cells
Authors: Holbert Cassandra , Forrester Jeffrey , Roberts Michael
Published: July 2016

Human acute myeloid cell lines undergo genetic re-programming in response to differentiation inducers. The M3 AML cell line, HL-60, has served as a model system for studying phorbol ester-induced myeloid differentiation into macrophage-like cells. Macrophage differentiation is preceded by cell cycle arrest and followed by apoptosis. Therefore, the system elucidates the changes in the leukemia cell gene expression program necessary to override the genetic alterations driving continuous cell division, preventing myeloid differentiation, and blocking programmed cell death. We hypothesize that defining the evolving transcriptome as AML cells reverse their proliferative, undifferentiated, and pro-survival phenotype will reveal genes regulating these processes and possibly identify new targets for AML treatment. Whole genome exon DNA microarray analyses identifies 1260 genes whose RNA expression levels change significantly during the reprogramming. Among these genes are members of the BCL-2 family that encode both pro- and anti-apoptotic proteins (BBC3, PMAIP1, BID and BCL2, MCL1, BCL2A1, respectively). Myeloid cell leukemia 1 (MCL1) is a BCL-2 family protein defined as an anti-apoptotic protein, however MCL1 increases in expression following phorbol ester treatment in HL-60 cells, as well as other AML lines including THP-1, Kasumi-1, and KG-1. The MCL1 gene can be alternatively spliced to generate a transcript encoding a BH3-only variant (MCL1S) known to exhibit pro-apoptotic function. Western blotting demonstrates the accumulation of MCL1S protein prior to the initiation of apoptosis in phorbol ester treated cells. Here we present evidence for splice variant switching and explore the possibility that reduction in the pro-survival form, MCL1L, and accumulation of MCL1S mediates the p53-independent initiation of apoptosis. Currently, we are evaluating the effects on the leukemic phenotype and apoptotic response to phorbol ester in HL-60 cell lines overexpressing MCL1L, MCL1S, and PMAIP1, as well as HL-60 lines containing CRISPR-Cas9 mediated knockouts of MCL1 and PMAIP1.

Citation Format: Cassandra Holbert, Jeffrey Forrester, Michael Roberts. Role of bcl-2 family proteins in phorbol ester-induced apoptosis of acute myeloid leukemia cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3561.
Abstract 3564: MUC13 protects colorectal cancer cells from death by activating the NF-κb pathway and is a potential therapeutic target
Authors: Sheng Yong H. , He Yaowu , hasnain sumaira Z. , Wang Ran , Tong Hui , Clarke Daniel T. , Lourie Rohan , Oancea Iulia , wong kuanyau , Lumley John W. , Florin Timothy H. , Sutton Philip , Hooper John. D. , Mcmillan Nigel A. , Mcguckin Michael A.
Published: July 2016

MUC13 is a transmembrane mucin glycoprotein that is overexpressed by many cancers, although its functions are not fully understood. NF-κB is a key transcription factor promoting cancer cell survival, but therapeutically targeting this pathway has proved difficult because NF-κB has pleiotropic functions. Here, we report that MUC13 prevents colorectal cancer cell death by promoting two distinct pathways of NF-kB activation, consequently up-regulating BCL-XL. MUC13 promoted TNF-induced NF-κB activation by interacting with TNFR1 and the E3 ligase, cIAP1, to increase ubiquitination of RIPK1. MUC13 also promoted genotoxin-induced NF-κB activation by increasing phosphorylation of ATM and SUMOylation of NEMO. Moreover, elevated expression of cytoplasmic MUC13 and NF-κB correlated with colorectal cancer progression and metastases. Our demonstration that MUC13 enhances NF-κB signalling in response to both TNF and DNA damaging agents provides a new molecular target for specific inhibition of NF-κB activation. As proof of principle, silencing MUC13 sensitized colorectal cancer cells to death in response to cytotoxic drugs and inflammatory signals and abolished chemotherapy-induced enrichment of CD133+ CD44+ cancer stem cells, slowed xenograft growth in mice, and synergized with 5-fluourouracil to induce tumor regression. Therefore, these data indicate that combining chemotherapy and MUC13 antagonism could improve the treatment of metastatic cancers.

Citation Format: Yong H. Sheng, Yaowu He, sumaira Z. hasnain, Ran Wang, Hui Tong, Daniel T. Clarke, Rohan Lourie, Iulia Oancea, kuanyau wong, John W. Lumley, Timothy H. Florin, Philip Sutton, John. D. Hooper, Nigel A. Mcmillan, Michael A. Mcguckin. MUC13 protects colorectal cancer cells from death by activating the NF-κb pathway and is a potential therapeutic target. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3564.