bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 2438: Identification of the mitochondrial binding site on the amino-terminal end of hexokinase II
Authors: Bryan Nadezda , Hwang Michelle M. , Raisch Kevin P. 
Published: August 2015

Abstract
Hexokinase II is responsible for the first step in the glycolysis pathway. Hexokinase II adds a phosphate onto the glucose molecule so it can proceed down the pathway to give the cancer cell energy for its continuous growth. Tumor cells overexpress the hexokinase II enzyme. In fact, it is the overexpression of the hexokinase II enzyme that makes the diagnosis of cancer possible when imaged by positron emission tomography (PET). Hexokinase II binds to the voltage-dependent anion channel (VDAC) on the mitochondrial membrane and when it is attached to the mitochondrial membrane it is blocking a major pathway for cell death. Thus, hexokinase II is responsible for two characteristics of cancer cells, rapid tumor growth and difficulty to kill. Therefore the overall goal of our laboratory is to identify novel compounds with the ability to displace or translocate hexokinase II from the mitochondrial membrane to the cytoplasm. To attain our goal, we propose to use the crystal structure of hexokinase II to create a molecular model of the enzyme. However the amino acids responsible for binding to VDAC are not known. Therefore, we made a series of truncations and point mutations to identify the binding site to VDAC. Truncations of the first 10 and 20 amino acids indicated that important amino acids for binding were located within the first 10 amino acids. We made a series of single, double and triple point mutations within the first 10 amino acids. Immunofluorescence and immunoblot data has confirmed that mutating the fifth amino acid from histidine to proline completely abolished binding to the mitochondrial membrane. To prepare hexokinase II for crystallization, we have cloned the enzyme downstream of a thrombin cleavage site and His-tag. We were able to purify hexokinase II using a cobalt column, then eluting hexokinase II using thrombin to cleave at the thrombin cleavage site. Purified hexokinase II is isolated after removal of the thrombin using Protein A beads. After the crystal structure of hexokinase II is obtained, we will use the University of Florida Structure-based Molecular Docking Core facility to identify compounds predicted to fit into the VDAC binding site. The compounds will be characterized to validate a mechanism-of-action of translocating hexokinase II from the mitochondrial membrane to the cytoplasm, blocking glycolysis and decreasing ATP synthesis. The positive compounds will be analyzed to determine the best strategy for moving our studies closer to clinical application in cancer patients.
Citation Format: Nadezda Bryan, Michelle M. Hwang, Kevin P. Raisch. Identification of the mitochondrial binding site on the amino-terminal end of hexokinase II. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2438. doi:10.1158/1538-7445.AM2015-2438
Abstract 2442: XPO1 is selinexor prime target: validation by mutating cysteine 528 on both XPO1 alleles using CRISPR/Cas9 genome editing
Authors: Neggers Jasper E. , Vercruysse Thomas , Jacquemyn Maarten , Vanstreels Els , Baloglu Erkan , Shacham Sharon , Crochiere Marsha , Senapedis William , Landesman Yosef , Daelemans Dirk 
Published: August 2015

Abstract
Human Exportin-1 (XPO1) is the key nuclear-cytoplasmic transport protein that exports a variety of different cargo proteins out of the cell's nucleus. Inhibition of XPO1-mediated protein export results in nuclear retention of cargo proteins including tumour suppressor proteins and is a novel therapeutic strategy for cancer. Selinexor, an orally bioavailable XPO1 inhibitor, effectively demonstrates potent activity against multiple types of cancer. Here we directly demonstrate the high selectivity of selinexor for XPO1 inside living cells.
Selinexor binds to the cargo-binding pocket of XPO1 and inhibits the formation of XPO1-cargo complex. The binding of selinexor to XPO1 involves a slowly reversible Michael addition type interaction of its acrylate moiety with cysteine 528 residue of XPO1. Using CRISPR/Cas9 genome editing we have now created a homozygous Jurkat T-ALL cell line carrying a Cys528Ser mutation in XPO1. This cell line was used to study the specificity of selinexor for XPO1 inside the cancer cell. Selinexor was highly toxic to wild-type Jurkat cells in the low nanomolar range while the mutant cells were resistant up to micromolar concentrations. These results were confirmed by resistance to induction of caspase dependent apoptosis as well as cell cycle arrest in mutant cells. The single Cys528Ser mutation thus confers resistance to selinexor. In addition, selinexor failed to accumulate cargo proteins in the nucleus of mutant cells. Moreover, the Cys528Ser substitution conferred resistance to the direct interaction of this drug to XPO1 in cells.
In conclusion, these results validate XPO1 as the selinexor's prime target and pinpoint its unique selectivity to the Cys528 residue of XPO1. Our findings also demonstrate the general utility of CRISPR/Cas9 genome editing for introducing point mutations in the genome of cancer cells for drug-target specificity validation.
Citation Format: Jasper E. Neggers, Thomas Vercruysse, Maarten Jacquemyn, Els Vanstreels, Erkan Baloglu, Sharon Shacham, Marsha Crochiere, William Senapedis, Yosef Landesman, Dirk Daelemans. XPO1 is selinexor prime target: validation by mutating cysteine 528 on both XPO1 alleles using CRISPR/Cas9 genome editing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2442. doi:10.1158/1538-7445.AM2015-2442
Abstract 2456: The transmembrane domain of prostate specific membrane antigen: Deciphering the role of the Small-XXX-Small motif in oligomerization
Authors: Berg Brianna S. , Cook Brandan M. , Hyder Jack R. , Godfroy James I. , Yin Hubert , Saludes Jonel P. 
Published: August 2015

Abstract
Prostate cancer (PCa) is the second leading cause of cancer-related deaths among males in the United States. The prevalence of this disease and the benefits associated with early detection and treatment call for closer investigation into potential biomarkers and therapeutics targets. The high expression of the integral membrane protein called prostate specific membrane antigen (PSMA, EC 3.4.17.21) is implicated in PCa invasiveness as well as neovasculature metastasis of nonprostatic solid tumors. Little is known about the exact role of PSMA in PCa progression albeit it is one of the most validated biomarker for the diagnosis and detection of PCa. We have demonstrated that the isolated PSMA transmembrane domain (TMD) is capable of oligomerization under reducing gel electrophoresis conditions, interacting independently of extracellular domain dimerization. Following this discovery, we proceeded to determine the factor(s) responsible for its oligomerization. Since it is known that certain motifs are critical for TMD oligomerization of many transmembrane proteins, we hypothesized that the Small-XXX-Small motif in PSMA TMD may play a role in the oligomerization and subsequent activation of PSMA. Thus, to gain insight into the assembly of PSMA and the factors responsible for this assembly, we investigated if mutations at this TM motif will disrupt TM oligomerization. Our initial studies revealed that PSMA TMD has two repeat units of the Small-XXX-Small motif. This motif is known to be crucial in inducing a strong peptide self-assembly, analogous to the bitopic protein Glycophorin A. We synthesized the PSMA TMD mutant peptides using Biotage Alstra microwave peptide synthesizer, purified by high performance liquid chromatography, and characterized by MALDI-TOF mass spectrometry to establish homogeneity. SDS-PAGE gel shift assays revealed a direct correlation of oligomerization propensity and the presence of the motif, while motif knockdown led to observed loss of this property. ToxR reporter assay that qualitatively assesses relative TMD interactions revealed that oligomerization of PSMA TMD is ∼150% stronger than Glycophorin A. These findings will serve as a road map in the design of tight-binding exogenous anti-TMD peptides that target this oligomerization hot spot for possible diagnostic and therapeutic applications.
Citation Format: Brianna S. Berg, Brandan M. Cook, Jack R. Hyder, James I. Godfroy, Hubert Yin, Jonel P. Saludes. The transmembrane domain of prostate specific membrane antigen: Deciphering the role of the Small-XXX-Small motif in oligomerization. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2456. doi:10.1158/1538-7445.AM2015-2456
Abstract 2483: Met degradation by SAIT301, a Met monoclonal antibody, reduces the invasion and migration of nasopharyngeal cancer cells via inhibition of EGR-1 expression
Authors: Lee Bok-Soon , Kang Sam , Kim Kyung-Ah , Song Yun-Jeong , Cheong Kwang Ho , Cha Hyun-Young , Kim Haeng-Jun , Hwang Hye Sook , Kim Yeon Soo , Kim Chul-Ho 
Published: August 2015

Abstract
Nasopharyngeal carcinoma (NPC) is a common malignant tumor with high invasive and metastatic potential. The hepatocyte growth factor (HGF)-Met signaling pathway has a critical role in mediating the invasive growth of many different types of cancer, including head and neck squamous cell carcinoma. HGF also stimulates NPC cell growth and invasion in the cell line model.
In this study, we determined the inhibitory effect of Met, using a Met-targeting monoclonal antibody (SAIT301), on the invasive and growth potential of NPC cell lines. Met inhibition by SAIT301 resulted in highly significant inhibition of cell migration and invasion in both the HONE1 and HNE1 cell lines. In addition, we also found that co-treatment of SAIT301 and HGF decreased the anchorage-independent growth induced by HGF in HNE1 cell lines. After SAIT301 treatment, Met, together with its downstream signaling proteins, showed downregulation of p-Met and p-ERK, but not p-AKT, in both HONE1 and HNE1 cell lines. Interestingly, we found that HGF treatment of NPC cell lines induced early growth response protein (EGR-1) expression, which is involved in cell migration and invasion. In addition, co-treatment with SAIT301 and HGF inhibited the HGF-induced expression of EGR-1. Next, knockdown of EGR-1 using small-interfering RNA inhibited HGF-induced cell invasion in NPC cell lines, suggesting that the expression level of EGR-1 is important in HGF-induced cell invasion of NPC cells. Therefore, the results support that SAIT301 inhibited Met activation as well as the downstream EGR-1 expression and could have therapeutic potential in NPC. Taken together, we suggest that Met is an anticancer therapeutic target for NPC that warrants further investigation and clinical trials and SAIT301 may be a promising tool for NPC therapy.
Citation Format: Bok-Soon Lee, Sam Kang, Kyung-Ah Kim, Yun-Jeong Song, Kwang Ho Cheong, Hyun-Young Cha, Haeng-Jun Kim, Hye Sook Hwang, Yeon Soo Kim, Chul-Ho Kim. Met degradation by SAIT301, a Met monoclonal antibody, reduces the invasion and migration of nasopharyngeal cancer cells via inhibition of EGR-1 expression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2483. doi:10.1158/1538-7445.AM2015-2483
Abstract 2485: Novel monoclonal antibody targeting cancer stem cells
Authors: Holderness Jeff , Turner Amy , dolezal Sam , Wolpe Steve , Pierce Michael , Gelber Cohava 
Published: August 2015

Abstract
Caerus Therapeutics (Caerus) is developing a monoclonal antibody directed to a cancer specific epitope on CD66c (CEACAM6) designated 109.12. CD66c has been shown in preclinical studies to be expressed on the colorectal cancer stem cell population but not on normal intestinal stem cells, making this a potential target for a therapeutic antibody. Previous attempts by other companies to develop anti-CD66c antibodies were unsuccessful due to cross-reactivity with CD66c on normal tissues. Caerus has developed a monoclonal antibody to a cancer-specific, glycan epitope that is only expressed in the adult during malignant transformation, enabling the targeting of CD66c on both cancer stem cells (CSCs) as well as bulk tumor without cross-reactivity with CD66c on normal cells. The glycan epitope recognized by the antibody has a novel structure not previously described that is highly protein-specific and site-specific. The specificity is due to a unique dual-recognition determinant for the site-specific glycosylation.
In addition to its expression on colorectal tumorspheres, the epitope recognized by the 109.12 antibody is expressed by a wide variety of cancers including colon, pancreatic, lung stomach and a subtype of breast cancer. The biological role of CD66c in malignancy and tumor dissemination is well documented; the specificity of the antibody for a CD66c epitope only expressed in malignant cells offers multiple opportunities for immunotherapy and diagnosis of a diverse spectrum of cancers.
Citation Format: Jeff Holderness, Amy Turner, Sam dolezal, Steve Wolpe, Michael Pierce, Cohava Gelber. Novel monoclonal antibody targeting cancer stem cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2485. doi:10.1158/1538-7445.AM2015-2485
Abstract 2492: Eradication of breast cancer with bone metastasis by autologous formalin- fixed tumor vaccine (AFTV) and radiation
Authors: Kuranishi Fumito , Uemae Yoji , Ohno Tadao , Sumi Yusuke 
Published: August 2015

Abstract
INTRODUCTION) Bone-metastatic mammary carcinoma patients show the downhill course to a tragic ending despite of intensive chemo-radiation therapy, except an our case concomitantly treated with AFTV (AACR2013, World J Surg Oncol 2013; 11:127). We here report, as the second successful case, eradication of a large bone metastasis of advanced breast cancer after treatment with AFTV and radiation. (CASE) A 47-years old female with stage II breast cancer was operated in July, 2000. Left axillar recurrence was found 13 years later by ultrasonography and FNBC. PET scan revealed a huge bone metastasis in sternum. She was treated with radiation and AFTV (J Neurosurgery 2014;4:1) in Oct. - Nov. 2013. Follow-up PET (Sep. 2014) showed no hot spot, and we diagnosed the patient, complete remission.
(CONCLUSION) Complete remission of bone metastasis of mammary carcinoma is very rare. The initial successful case beared triple-negative breast cancer. Treatment with AFTV and radiation without intensive chemotherapy like the present case will be acceptable to any patient with bone-metastatic mammary carcinoma, though a large scale clinical trial should be required to confirm the therapeutic efficacy of AFTV.
Citation Format: Fumito Kuranishi, Yoji Uemae, Tadao Ohno, Yusuke Sumi. Eradication of breast cancer with bone metastasis by autologous formalin- fixed tumor vaccine (AFTV) and radiation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2492. doi:10.1158/1538-7445.AM2015-2492
Abstract 2494: Vaccine-induced TNF alpha producing T cells synergize with cisplatin in tumor eradication
Authors: Melief Cornelis J. , van der Sluis Tetje C. , van der Burg Sjoerd H. 
Published: August 2015

Abstract
Previously we have shown that Synthetic Long Peptide (SLP) vaccination against Human Papilloma Virus (HPV) oncogenic proteins is safe and induces functional T-cell responses in mice and humans. Although vaccination induces potent clinical responses in mice and in patients with premalignant lesions, no clinical responses were observed in patients with cervical cancer. Here we show in a preclinical mouse model for HPV induced malignancies that SLP vaccination can be safely combined with clinically relevant chemotherapeutics to eradicate tumors. In mice that received either peptide vaccination or chemotherapy, only a temporary regression in tumor size was observed. Importantly, combined chemo-immunotherapy induced complete and sustained tumor eradication in nearly all mice. The chemotherapeutic agent cisplatin displayed the strongest synergy with SLP vaccination. Long term regression of tumors occurred at both the maximal tolerated dose of cisplatin and at 40% of that dose when combined with SLP vaccination, but at the later dose side effects such as body weight loss were absent. Upon vaccination tumors were highly infiltrated with Tumor Necrosis Factor alpha (TNFα) and Interferon gamma (IFN-γ) producing CD8+ CTLs. Tumor cells incubated with these cytokines and cisplatin in vitro strongly enhance their chemokine expression, compatible with the abundant leukocyte infiltration into the tumor upon combined chemo-immunotherapy. Accordingly, when combined with cisplatin treatment, SLP vaccine-induced CTLs appeared to migrate earlier from the tumor rim into the tumor beds. Moreover, analysis of the tumor cells in vivo showed that combined treatment significantly decreased the proliferative capacity of tumor cells compared to single treated or untreated tumors. Furthermore, we showed that TNFα enhanced cisplatin- induced, JNK dependent tumor cell apoptosis. This cell death was accompanied by an increased expression of pro-apoptotic molecules. The synergy between cisplatin and SLP vaccination was largely abolished by in vivo treatment with monoclonal antibody against TNFα. Together, our data show that combined peptide treatment with cisplatin leads to superior tumor eradication in the absence of T cell immunosuppression.
Citation Format: Cornelis J. Melief, Tetje C. van der Sluis, Sjoerd H. van der Burg. Vaccine-induced TNF alpha producing T cells synergize with cisplatin in tumor eradication. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2494. doi:10.1158/1538-7445.AM2015-2494
Abstract 251: Combining Ibrutinib with immune checkpoint blockade to induce therapeutic antitumor immune response in solid tumors
Authors: Sagiv-Barfi Idit , Kohrt Holbrook , Czerwinski Debra , Ng Patrick , Chang Betty , Levy Ronald 
Published: August 2015

Abstract
The ability of T-cell responses to effectively attack tumors is restricted by multiple negative checkpoint regulators. Monoclonal antibodies blocking negative regulators of T -cells, such as CTLA-4 and PD-L1 can amplify pre-existing immune responses and evoke new therapeutic responses. Ibrutinib (Imbruvica®), a covalent inhibitor of BTK has shown therapeutic efficacy in several blood cancers, by a mechanism thought to be a direct effect on a critical signaling pathway intrinsic to the malignant B cell. Since BTK is predominantly expressed in hematopoietic cells, solid tumors have not been an obvious target of this drug. An additional target of ibrutinib is the inhibition of ITK, a key enzyme in the survival of Th2 T cells. It has been reported that ibrutinib can alter the balance of Th1 and Th2 T cells leading to enhanced immune responses.
Here we report that the combination of ibrutinib and a-PD-L1 antibody suppresses tumor growth, reduces lung metastasis and improves survival of mice bearing solid tumors such as 4T1-triple negative breast cancer and CT26-colon cancer. Moreover, the combination of ibrutinib and a-PD-L1 was able to overcome ibrutinib resistance in mouse models of lymphoma and myeloma. We present evidence that the synergistic therapeutic activity of Ibrutinib with anti PD1 blockade results in enhanced T cell anti-tumor immune responses.
In preclinical models, by combining inhibitory checkpoint blockade while shifting the immune response towards CD4 Th1 with ibrutinib, immune tolerance to tumors can be overcome. These results provide rationale for several newly planned clinical trials combining ibrutinib with PD-1 blockade in solid tumor and exploration of such a combination in hematologic malignancies.
Citation Format: Idit Sagiv-Barfi, Holbrook Kohrt, Debra Czerwinski, Patrick Ng, Betty Chang, Ronald Levy. Combining Ibrutinib with immune checkpoint blockade to induce therapeutic antitumor immune response in solid tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 251. doi:10.1158/1538-7445.AM2015-251
Abstract 2511: Therapeutic dendritic cell targeting MIP3α-gp100 DNA vaccination with immunomodulatory αIL-10 and αPD-1 antibodies significantly enhances survival in a mouse melanoma model system
Authors: Gordy James , Markham Richard B. 
Published: August 2015

Abstract
The current study demonstrates that the combination of a DNA vaccine encoding the chemokine MIP-3α fused to melanoma antigen gp100 with immunomodulatory antibodies prolongs survival in a melanoma mouse model. The current studies utilize the B16F10 mouse spontaneous melanoma syngeneic transplantable mouse model system. Previous published work with this vaccine platform has indicated the MIP-3α component targets nascent peptides to immature dendritic cells leading to processing by class I and class II MHC pathways. It has further been shown in this tumor system that prophylactic vaccination with the DNA construct alone delayed tumor growth. Other studies have provided evidence that αPD-1 or αIL-10 neutralizing antibodies enhance immunological melanoma therapies by modulating the tolerogenic tumor microenvironment. First, this study expanded previous results by finding a significant decrease in tumor burden and increase in survival by prophylactic intramuscular electroporation [IM EP] of the DNA vaccine construct. Adding three therapeutic doses of αIL-10 given subcutaneously [subQ] at the tumor site to the treatment further increased mouse survival. The development of cell-mediated and humoral immune responses were documented for the first time in this system by Elispot and in-cell ELISA assays. Secondly, a novel therapeutic vaccination protocol was developed by initiating vaccination and antibody therapies after tumor challenge. With this therapeutic protocol, we demonstrated for the first time that vaccine significantly delays tumor burden and increases median survival from 17 to 21 days (p = 0.006). Addition of αIL-10 (subQ) and αPD-1 (intra-peritoneally) antibodies led to further highly significant decreases in tumor burden and increased median survival from 17 to 25 days (p = 0.0005). Efficient targeting of antigen to immature dendritic cells with a chemokine fusion vaccine offers a potential alternative approach to the ex vivo dendritic cell antigen loading protocols currently undergoing clinical investigation. Combining this approach with agents able to modulate the tolerogenic tumor microenvironment offers promise as a novel melanoma therapy.
Citation Format: James Gordy, Richard B. Markham. Therapeutic dendritic cell targeting MIP3α-gp100 DNA vaccination with immunomodulatory αIL-10 and αPD-1 antibodies significantly enhances survival in a mouse melanoma model system. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2511. doi:10.1158/1538-7445.AM2015-2511