bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5447: Phosphorylation status of Ascl1 regulates neuroblastoma self-renewal and differentiation.
Authors: Wylie Luke A. , Thiele Carol J. , Philpott Anna
Published: August 2014

Neuroblastoma (NB) is a tumor of infancy that accounts for 15% of all pediatric cancer mortality. NB bares striking similarity to undifferentiated neuroblasts of the sympathetic nervous system in gene expression profiles and histological appearance. In most neuroblastoma tumors, the cell cycle is hyperactive due to overexpression of cyclin D1 and amplification of MYCN. However, retinoic acid (RA) induces differentiation and cell cycle exit of some NB cell lines in vitro and provides significant benefit to patients in the clinic. As neuroblastoma appears to result from sustained proliferation and inhibition of differentiation, we sought to understand how cell cycle and differentiation are linked within NB. We focused our efforts on Ascl1, a proneural transcription factor that is both necessary and sufficient for neural differentiation of noradrenergic neurons and has been shown to be regulated by the cell cycle. Paradoxically, Ascl1 is both highly expressed and associated with poor prognosis in NB. However, we hypothesized that Ascl1 phosphostatus critically regulates its ability to induce differentiation.

We show that Ascl1 is highly expressed and phosphorylated across multiple NB cell lines and is largely phosphorylated by CDKs. Phosphorylated Ascl1 actively promotes the G1-S transition by upregulating E2f, Skp2, and Cdk2. These targets all promote CDK activity, further maintaining Ascl1 phosphorylation. However, a phosphomutant form of Ascl1, where putative CDK sites are mutated, arrests cells in the G1 phase by preferentially upregulating Ebf3 and p27. These targets inhibit CDK activity promoting Ascl1 dephosphorylation and further upregulation of Ebf3 and p27. We propose a model whereby Ascl1 sits between two positive feedback loops, both forming a critical link between cell cycle and differentiation, but also as an agent to commit cells to either a self-renewing undifferentiated state or a postmitotic differentiated state.

Given this model, we hypothesized that pharmacologic inhibition of CDK activity when combined with a pro-differentiation agent such as RA would synergistically cause differentiation in neuroblastoma. Indeed, CDK inhibitor and RA combination treatment causes increased differentiation in RA-susceptible cell lines and induces differentiation in RA-resistant cell lines. Given that most neuroblastoma tumors have an overactive cell cycle and also highly express Ascl1, combination therapy of CDK inhibitor and RA may provide generalized benefit to NB patients.

Citation Format: Luke A. Wylie, Carol J. Thiele, Anna Philpott. Phosphorylation status of Ascl1 regulates neuroblastoma self-renewal and differentiation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5447. doi:10.1158/1538-7445.AM2013-5447
Abstract 5458: Individual and combined activities of STAT1 and STAT3 have prognostic relevance for the progression of colorectal carcinoma.
Authors: Gordziel Claire , Nivarthi Harini , Knösel Thomas , Dolznig Helmut , Moriggl Richard , Friedrich Karlheinz
Published: August 2014

Aberrant activities of JAK/STAT signalling pathways have been implicated in the development and spread of various cancer entities, among them colorectal carcinoma (CRC). By immunohistochemical analysis of a tissue microarray covering more than 400 CRC biopsies from patients with full clinical documentation, the expression and activity status of STAT1 and STAT3 was determined. Results were correlated with clinical information. A significant association of high STAT1 activity (as indicated by nuclear location) with longer patient overall survival was observed. Unexpectedly, strong STAT3 expression in surgical specimens could be clearly correlated with an increase in median overall survival by about 30 months.

Interestingly, combined consideration of STAT1/3 expression and activity levels in CRC specimens revealed patterns that match the clinical outcome with statistical significance. For instance, tumors with concomitant absence of STAT1 and STAT3 have a worse prognosis, while high STAT1 activity along with low STAT3 activity proved associated with longer patient survival.

To address the signal transduction background of these findings, STAT1/3 expression was experimentally modified in

CRC cell lines and effects on the growth of xenograft tumors were studied. Mimicking

STAT1/3 patterns from patient biopsies in xenografts resulted in alterations in tumor growth well corresponding to clinical data. From these experiments we draw the conclusions that the ratio of nuclear STAT1/STAT3 is a determinant of colon carcinoma progression.

Supported by: Deutsche Krebshilfe, IZKF Jena, European Regional Development Fund ERDF

Citation Format: Claire Gordziel, Harini Nivarthi, Thomas Knösel, Helmut Dolznig, Richard Moriggl, Karlheinz Friedrich. Individual and combined activities of STAT1 and STAT3 have prognostic relevance for the progression of colorectal carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5458. doi:10.1158/1538-7445.AM2013-5458
Abstract 547: Inhibition of PI 3-K isoforms lead to reactivation of Akt signaling in a subset of breast cancer cell lines.
Authors: Collmann Emilie , Toker Alex
Published: August 2014

The aggressive behavior of malignant breast cancer cells is determined by a complex array of signaling pathways that regulate functions such as growth, survival, and migration. Production of phosphatidylinositol-3,4,5-P3 (PIP3) by phosphoinositide 3-kinase (PI 3-K) results in activation of downstream targets including the protein kinase Akt that plays critical roles in cell survival and growth. Recent studies have highlighted alternate, PI 3-K independent pathways often associated with malignancy that lead to Akt reactivation. The goal of our studies is to investigate the role of PI 3-K catalytic subunits in Akt reactivation in breast cancer.

Surprisingly, knock-down of the catalytic subunit of class I PI 3-K PIK3CA actually enhances phosphorylation of Akt, as well as the downstream targets S6K1 and GSK3β in a subset of triple negative breast cancer (TNBC) cell lines. The resulting increase in Akt phosphorylation is mTORC2-dependent and is insensitive to the class I PI 3-K inhibitors. Rescue experiments with wild type PIK3CA alleles reveal a further enhancement of Akt phosphorylation, when compared to PIK3CA knock-down alone. Moreover, silencing of distinct class I subunits PIK3CB and PIK3CD also enhanced Akt phosphorylation in TNBC. However, this effect is not universal and is not observed in numerous other breast cancer cell lines, or using specific PIK3CA inhibitors. This reactivation of Akt signaling suggests the interruption of a feedback loop similar to the mTOR-S6K-IRS1 negative feedback loop. It also indicates specific roles for individual PI 3-K isoforms in breast cancer and dependent on the mutational status of PTEN and PIK3CA.

This research is supported by Susan G. Komen for the Cure postdoctoral fellowship KG111139 to EC and AT.

Citation Format: Emilie Collmann, Alex Toker. Inhibition of PI 3-K isoforms lead to reactivation of Akt signaling in a subset of breast cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 547. doi:10.1158/1538-7445.AM2013-547
Abstract 5472: Targeting chronic lymphocytic leukemia cells with a humanized monoclonal antibody specific for CD44.
Authors: Zhang Suping , Wu Christina , Farrah-Fecteau Jessie , Cui Bing , Chen Liguang , Zhang Ling , Wu Rongrong , Rassenti Laura , Lao Fitzgerald S. , Weigand Stefan , Kipps Thomas J.
Published: August 2014

Chronic lymphocytic leukemia (CLL) cells express high levels of CD44, a cell-surface glycoprotein receptor of hyaluronic acid (HA). We evaluated the cytotoxic potential of a humanized anti-CD44 mAb (RG7356), which currently is undergoing clinical evaluation for the treatment of patients with solid-tumor malignancies. We found that the mAb was directly cytotoxic for leukemia B cells, but had little effect on normal B cells. Moreover, independent of compliment or cytotoxic effector cells, RG7356 could induce leukemia cells to undergo caspase-dependent apoptosis, particularly those CLL cells that expressed the zeta-associated protein of 70 kD (ZAP-70). The cytotoxic effect of this mAb was not mitigated when the CLL cells were co-cultured with mesenchymal stromal cells (MSC) or HA, or stimulated via ligation of the B-cell receptor (BCR) with anti-μ. RG7356 induced rapid internalization of CD44 on CLL cells at 37oC, resulting in reduced expression of ZAP-70, which we found complexed with CD44. Administration of 1 mg/kg of this mAb to immune-deficient mice engrafted with human CLL cells resulted in complete clearance of engrafted leukemia cells. These studies indicate that this mAb might have therapeutic activity, particularly in patients with CLL that express ZAP-70.

Citation Format: Suping Zhang, Christina Wu, Jessie Farrah-Fecteau, Bing Cui, Liguang Chen, Ling Zhang, Rongrong Wu, Laura Rassenti, Fitzgerald S. Lao, Stefan Weigand, Thomas J. Kipps. Targeting chronic lymphocytic leukemia cells with a humanized monoclonal antibody specific for CD44. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5472. doi:10.1158/1538-7445.AM2013-5472
Abstract 5477: Engineered toxin body demonstrating CD20-specific binding and cell kill in B-cell non-Hodgkin's lymphoma cells.
Authors: Wirth Rachel , Rajagopalan Sangeetha , Willert Erin , Null William , Erdman Jennifer , Liu Jen-Sing , Higgins Jack P.
Published: August 2014

Non-Hodgkin's lymphoma (NHL) offers an attractive area for therapeutic biologics. The most successful example of this targeted approach is rituximab, which is approved for front and second line therapy of NHL. Rituximab targets the CD20 antigen expressed on 90% of B-cell NHL cells. However, rituximab has limited direct cell-kill effects, acting instead primarily through indirect immune responses induced by antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC). Direct-cell kill approaches to CD20 have been limited to radioimmunotherapy because CD20 does not efficiently internalize. Despite improved clinical efficacy seen over Rituximab with both 131 tositumomab and ibritumomab tiuxetan, challenges around the administration of radioimmunotherapies have severely curtailed therapeutic use. We have developed an engineered toxin body (ETB) comprising a CD20 scFv and a modified Shiga-like Toxin A (3F7) capable of specifically recognizing and killing CD20 expressing cells. Utilizing a flow cytometry based binding affinity assay, 3F7 demonstrated selective binding to CD20 positive Raji cells whereas no significant binding was observed in CD20 negative BC-1 cells. Further, direct competition of 3F7 binding to Raji cells was observed in the presence of rituximab or the parent monoclonal antibody. 3F7 demonstrated CD20-specific direct cell kill of Raji, Daudi and Ramos cells with minimal cell kill of CD20 negative BC-1 or Jurkat cells. Initial dose finding studies in mice found that a therapeutic dosing schedule was well tolerated. Raji xenograft studies in mice are ongoing. 3F7 is a novel therapeutic approach with direct cell-kill activity against CD20-expressing cells which could expand treatment options for B-cell lymphoma.

Citation Format: Rachel Wirth, Sangeetha Rajagopalan, Erin Willert, William Null, Jennifer Erdman, Jen-Sing Liu, Jack P. Higgins. Engineered toxin body demonstrating CD20-specific binding and cell kill in B-cell non-Hodgkin's lymphoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5477. doi:10.1158/1538-7445.AM2013-5477
Abstract 5480: Characterization of anti-human APRIL monoclonal antibodies capable of inhibiting APRIL-dependent B cell function and hyperproliferation.
Authors: Guadagnoli Marco , Cameron Katherine , Veraar Elise , Kimberley Fiona , van Elsas Andrea , van Eenennaam Hans , Medema Jan Paul
Published: August 2014

A Proliferation Inducing Ligand (APRIL) is a TNF ligand that, via its receptors TACI and BCMA, is involved in both B cell physiology as well as in proliferation and survival of malignant B cells. To target APRIL-dependent stimulation of B cell cancers, we recently produced and characterized two monoclonal antagonistic anti-human APRIL antibodies called humanAPRIL.01A (hA.01A) and humanAPRIL.03A (hA.03A). In a first biochemical assay to validate their blocking activity, hA.01A was shown to fully prevent APRIL from binding to its receptors, whereas a substantial difference was detected for hA.03A, which inhibited APRIL binding to BCMA less efficiently than hA.01A. Epitope mapping subsequently revealed that hA.01A and hA.03A bind distinct sites on APRIL, which provided a structural rationale of their different blocking activities. Importantly, this differential inhibition profile can be used to functionally dissect BCMA and TACI-dependent signals and indicated that B cell survival and IgA production are regulated differently by these receptors. Primary CLL cultures were shown to be sensitive to APRIL neutralization using hA.01A. hA.01A also inhibited hyperplasia in a transgenic mouse model reminiscent of B-CLL. Altogether, these data indicate that hA.01A is a novel tool potentially useful for the targeted treatment of B cell-derived cancers such as B-CLL and Multiple Myeloma.

Citation Format: Marco Guadagnoli, Katherine Cameron, Elise Veraar, Fiona Kimberley, Andrea van Elsas, Hans van Eenennaam, Jan Paul Medema. Characterization of anti-human APRIL monoclonal antibodies capable of inhibiting APRIL-dependent B cell function and hyperproliferation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5480. doi:10.1158/1538-7445.AM2013-5480
Abstract 5481: Therapy of human LN229 glioblastoma growing in the brain of nude mice by Macitentan, a dual endothelin receptor A and B antagonist, combined with Temozolomide.
Authors: Kim Sun Jin , Kim Seung Wook , Choi Hyun Jin , Lee Ho Jeong , Lehembre Francois , He Junqin , Regenass Urs , Conrad Charles A , Yung W.K. , Fidler Isaiah J.
Published: August 2014

Purpose: We determined whether the blockade of endothelin receptor pathways can reverse the chemoprotection mediated by astrocytes and produce therapy of Human LN 229 Glioblastoma cells growing in the brain of nude mice treated with the dual endothelin receptor antagonist, Macitentan and Temozolomide (TMZ).

Materials and Methods: In the first set of experiments, murine astrocytes were cultured with tumor cells in the presence or absence of Macitentan and TMZ. For therapy studies the leuciferas labeled LN229 cells were implanted (stereotactically) into the brains of nude mice. The mice were ear tagged and imaged by IVIS spectrometer at multiple times. On day 24 the mice were randomized to receive vehicle, TMZ (p.o., 7.5mg/kg, one week on and two weeks off), Macitentan (p.o., 10mg/kg, daily) and Macitentan plus TMZ. Survival of mice was compared by Kaplan-Meyer curve (log-rank test) and immunohistochemical analyses were carried out for multiple markers.

Results: Simultaneous blocking of endothelin receptor A and B pathways reversed the in culture protection induced by astrocytes against TMZ (p<0.05). Therapy with Macitentan and TMZ significantly prolonged the survival of mice (p<0.0001) .The blockade of endothelin receptor A and B significantly decreased the level of expression of phosphorylated AKT and MAPK, and down-regulated the expression of BCL2L1, TWIST1 and GSTA5 in LN 229 cells growing in the brain. Proliferation of tumor cells and tumor-associated endothelial cells was significantly decreased. Moreover the apoptosis of LN229 cells was significantly increased in the brain of the combination treatment group.

Conclusion: Blockade of endothelin receptor A and B pathways by Macitentan combined with TMZ provides a new approach for the treatment of patients with Glioblastoma.

Citation Format: Sun Jin Kim, Seung Wook Kim, Hyun Jin Choi, Ho Jeong Lee, Francois Lehembre, Junqin He, Urs Regenass, Charles A Conrad, W.K. Yung, Isaiah J. Fidler. Therapy of human LN229 glioblastoma growing in the brain of nude mice by Macitentan, a dual endothelin receptor A and B antagonist, combined with Temozolomide. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5481. doi:10.1158/1538-7445.AM2013-5481
Abstract 5485: Apoptosis-targeted cell and drug delivery to tumor.
Authors: Chi Lianhua , Bonaparte Napoleon , Park Rang-woon , Kim In-san , Lee Byung-heon
Published: August 2014

When tumor cells undergo apoptosis in response to chemotherapy, the levels of apoptotic biomarkers such as phosphatidylserine and histone H1 are increased at the cell surface. Here we hypothesized that the chemotherapy-induced apoptosis would amplify in situ apoptotic biomarkers (homing signals) for apoptosis-targeted drug carriers and enhance drug delivery. To examine this possibility, we employed a phage display-identified CQRPPR peptide (ApoPep-1) as a targeting moiety, which was able to recognize apoptotic cells by binding to histone H1 on the surface of apoptotic cells. When injected into tumor-bearing mice, ApoPep-1-labeled, fluorescent liposomes containing doxorubicin inhibited tumor growth more efficiently than untargeted or folate-labeled liposomes. Moreover, in vivo fluorescence imaging could enable monitoring of tumor response during the chemotherapy. The imaging signals at tumor were increased by the homing of apoptosis-targeted liposomes, which was correlated with the increase of apoptosis and the amount of doxorubicin (payloads) at the tumor and, conversely, with the decrease of tumor volume. Next, we harnessed the chemotherapy-induced apoptosis of tumor cells as a homing signal for the delivery of apoptosis-targeted

T cells to tumor. When labeled with ApoPep-1 using an oleyl acid-derived membrane anchor, targeted T cells preferentially bound to apoptotic tumor cells over living cells. In vivo imaging showed higher levels of tumor homing of targeted, fluorescent T cells in mice treated with chemotherapy more than those of untargeted T cells. These results demonstrate that the apoptosis-targeted delivery can efficiently enhance the delivery of cells or drugs to tumor and, when combined with imaging of apoptosis, provides a real-time monitoring of tumor response for cancer theragnosis.

Citation Format: Lianhua Chi, Napoleon Bonaparte, Rang-woon Park, In-san Kim, Byung-heon Lee. Apoptosis-targeted cell and drug delivery to tumor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5485. doi:10.1158/1538-7445.AM2013-5485
Abstract 5486: Cancer therapeutics and the βGBP cytokine. Activation of death pathways prior to and after PI3K inhibition, and efficacy in vivo.
Authors: Mallucci Livio , Wells Valerie
Published: August 2014

Phosphatidylinositol-3-OH kinase (PI3K) signaling is crucial to the viability of cancers and represents a selective target for therapeutic intervention. Our strategy against cancer is based on the use of a newly identified cytokine (βGBP), a natural inhibitor of both class IA and class IB PI3K that in cancer cells, but not in normal cells, is a potent activator of cell death: a novel therapeutic concept which carries no chemotherapeutic disadvantages and that can be tested and utilised in clinical trials.

Cell death by Hu-r-βGBP follows two modes:

1) Apoptosis consequent to downregulation of signaling downstream of PI3K where inhibition of PI3K activity by Hu-r-βGBP has two major outcomes: suppression of Ras-GTP loading leading to block of ERK activation, and negation of akt gene expression leading to loss of Akt, conditions that either by blocking the ability of cancer cells to proliferate or by impairing their ability to survive erase oncogenicity in many cancer cells including those from prostate, HER2-driven and triple negative breast cancer.

2) Apoptosis consequent to induction of protein-protein interactions from βGBP receptor to PI3K where conformational changes conveyed by allosteric modulation from the βGBP receptor to p110 lead to changes in PIP2 spatial distribution at the plasma membrane, loss of PIP2-actin linkages, increased macromolecular mobility within the plane of the plasma membrane, clustering of death receptors and devastating death in Kras-mutant cancer cells, therapeutically reproducible in vivo.

Citation Format: Livio Mallucci, Valerie Wells. Cancer therapeutics and the βGBP cytokine. Activation of death pathways prior to and after PI3K inhibition, and efficacy in vivo. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5486. doi:10.1158/1538-7445.AM2013-5486
Abstract 5487: Inhibitory effect of soluble EP2 receptor on ovarian tumor growth in nude mice and usefulness of TMPRSS4 as a molecular target for synergistic efficacy.
Authors: Takahashi Tetsuyuki , Uehara Hisanori , Izumi Keisuke
Published: August 2014

Prostaglandin E2 (PGE2) plays an important role in cancer cell growth, progression, and metastasis and exerts its effects by binding to E-prostanoid receptor (EP). In the previous study, we showed that soluble fragment of EP2 receptor containing the second extracellular region (FuEP2/Ex2) has neutralizing activity to PGE2 and inhibits the growth of prostate and endometrial cancer cells. Here, we established a stable transfectant expressing FuEP2/Ex2 from human ovarian cancer SKOV/ip cells (SKOV/ip-FuEP2/Ex2) and SKOV/ip-FuEP2/Ex2 and vector-control cells were injected into nude mice intraperitoneally. Tumor weight and ascites volume in SKOV/ip-FuEP2/Ex2-injected mice decreased significantly compared to control cell-injected mice. Furthermore, microvessel density and numbers of M2-poralized macrophage in tumor lesion were significantly decreased in SKOV/ip-FuEP2/Ex2-injected mice. By using RNAs from tumor lesion in each group, the cDNA microarray analysis was also conducted. Six of upregulated genes and eight of downregulated genes in SKOV/ip-FuEP2/Ex2-derived tumor were identified. In upregulated genes, MMP-7, TMPRSS4, and CYP1B1, which is involved in cancer progression and metastasis, were included. Treatment with AEBSF, an inhibitor of TMPRSS4, or siRNA for TMPRSS4 further reduced cell growth of SKOV/ip-FuEP2/Ex2 cells in vitro and in vivo. Our results suggest that FuEP2/Ex2 has a suppressive effect in peritoneal metastasis model of ovarian cancer and that MMP-7, TMPRSS4, and CYP1B1 may act as a survival factor under the condition in which EP-mediated signaling are inhibited. Moreover, targeting of TMPRSS4 may enhance the inhibitory effect of FuEP2/Ex2 on ovarian cancer metastasis.

Citation Format: Tetsuyuki Takahashi, Hisanori Uehara, Keisuke Izumi. Inhibitory effect of soluble EP2 receptor on ovarian tumor growth in nude mice and usefulness of TMPRSS4 as a molecular target for synergistic efficacy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5487. doi:10.1158/1538-7445.AM2013-5487
Abstract 549: MAGI3-Akt3, a novel fusion protein in the PI 3-K pathway in cancer.
Authors: Brown Kristin , Toker Alex
Published: August 2014

Aberrant activation of the PI3-K/Akt pathway is found in approximately 70% of breast cancers. We recently identified a recurrent rearrangement that produces an in-frame fusion protein containing Akt3 and the scaffolding protein MAGI3 (1). MAGI3-Akt3 is the first fusion protein that has been identified in the PI 3-K pathway. We propose that MAGI3-Akt3 is a novel oncogene in breast cancer and that the oncogenic activity of the fusion protein results from disruption of the PI 3-K/Akt pathway. Within the fusion, Akt3 has a disrupted PH domain prior to glutamate 17 (E17). As a result the fusion protein is constitutively phosphorylated in the Akt3 kinase domain and expression of MAGI3-Akt3 in breast cancer cells enhances Akt substrate phosphorylation. Constitutive MAGI3-Akt3 kinase activity can be inhibited by ATP-competitive small molecule Akt inhibitors but is resistant to the allosteric Akt inhibitor MK2206 which is currently in phase II clinical trials. Our results indicate that ATP competitive Akt inhibitors should be evaluated in clinical trials for the treatment of fusion-positive breast cancers.

The fusion protein also contains a severely truncated portion of the scaffolding protein MAGI3. In particular, the fusion lacks the second PDZ domain of MAGI3 that has been reported to bind to PTEN and be required for the inhibitory effect of PTEN on PI 3-K pathway activity. We have demonstrated that disruption of MAGI3 in breast cancer cell lines does indeed promote PI 3-K pathway hyperactivity in a PTEN-dependent manner. In addition, the fusion lacks the fifth PDZ domain of MAGI3 and our studies indicate that this disrupts the interaction of MAGI3 with PHLPP2, a phosphatase that directly dephosphorylates Akt at pS473.

Our studies therefore demonstrate that the MAGI3-Akt3 translocation significantly perturbs PI 3-K pathway activity via activation of an oncogene (Akt3) and dysregulation of two tumor suppressors (PTEN and PHLPP2). We are currently continuing studies to elucidate the oncogenic mechanism(s) of MAGI3-Akt3 in breast cancer.

Citation Format: Kristin Brown, Alex Toker. MAGI3-Akt3, a novel fusion protein in the PI 3-K pathway in cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 549. doi:10.1158/1538-7445.AM2013-549
Abstract 5490: The cytotoxic activity of the anti-mesothelin immunotoxin SS1P is enhanced by the PKC inhibitor enzastaurin.
Authors: Mattoo Abid R. , Pastan Ira , Fitzgerald David
Published: August 2014

The activation of protein kinase C (PKC) contributes to tumor survival and proliferation provoking the development of inhibitory agents that may function as cancer therapeutics. Pseudomonas exotoxin-based immunotoxins are antibody-based recombinant proteins that employ an ADP-ribosylation domain for the enzymatic inhibition of protein synthesis of target cancer cells. Combining PKC inhibitors with immunotoxins was undertaken to explore possible synergy. Enzastaurin but not two other PKC inhibitors (Sortastaurin and G06976) combined with immunotoxins directed to surface mesothelin or the transferrin receptor to produce synergistic or additive cell death via apoptosis. Enhancement of killing was noted with enzastaurin concentrations of 5 uM or greater. Mechanistic insights of the combined immunotoxin-enzastaurin killing centered on the rapid complete loss of the prosurvival Bcl2 protein, Mcl-1, and the activation of caspase 3/7. Moreover, efficient cell killing was associated with loss of Akt and the BH3-only protein Bad. Enzastaurin-mediated enhanced cell killing was specific to Pseudomonas exotoxin-based immunotoxins as there was no enhancement with other agents that inhibit protein synthesis such as cycloheximide and diphtheria toxin. Immunotoxin-enzastaurin combinations could be useful for the treatment of human cancers that express mesothelin and are not efficiently killed by SS1P alone.

Citation Format: Abid R. Mattoo, Ira Pastan, David Fitzgerald. The cytotoxic activity of the anti-mesothelin immunotoxin SS1P is enhanced by the PKC inhibitor enzastaurin. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5490. doi:10.1158/1538-7445.AM2013-5490
Abstract 5496: ‘Salan’ titanium(IV) complexes: A new class of anti-cancer agents.
Authors: Braitbard Ori , Meker Sigalit , Stolarov Maya , Hochman Jacob , Tshuva Edit
Published: August 2014

Early attempts at using titanium compounds (derivatives of titanocene dichloride and budotitane) as anti-cancer treatment were essentially unsuccessful. The main reason was their rapid hydrolysis in biological media, leading to a variety of unidentified products that hampered the elucidation of mechanistic aspects. Lately, we have introduced a new class of Ti(IV) complexes of salan type diamino bis(phenolato) ligands. These compounds demonstrate substantially enhanced hydrolytic stability, as well as cytotoxic activity towards a variety of cancer cell lines in vitro. The IC50 of different derivatives of these compounds towards murine mammary carcinoma, lymphoma, multi-drug resistant lymphoma, as well as human leukemia, melanoma and pancreatic cancer cell lines, is in the range of 0.6 - 5.7μM, as compared to 70-100μM of the older titanium compounds and to 50-60μM of cisplatin (Manna C.M. et al. ChemMedChem, 7, 703 (2012)). Exposure of murine T-69 and human OVCAR-1 and HT-29 cell lines to one of the salan Ti(IV) compounds resulted in increased levels of p53 and growth arrest in G-1 of the cell cycle implying activation of the apoptotic pathway in response to Ti(IV) complexes.The well-identified and highly stable hydrolysis products of the titanium compounds, which had previously been formulated into nanoparticles to enhance solubility and assist cellular penetration (Meker, S. et al. Angew. Chem. Int. Ed. 51,10515 (2012)), were active both in vitro (IC50 -0.45μM) in murine lymphoma and in vivo (50% tumor growth inhibition) of the same lymphoma cells following their introduction into syngeneic mice. These findings suggest the potential use of solubilized active titanium compound derivatives as a new class of anti-cancer agents.

Citation Format: Ori Braitbard, Sigalit Meker, Maya Stolarov, Jacob Hochman, Edit Tshuva. ‘Salan’ titanium(IV) complexes: A new class of anti-cancer agents. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5496. doi:10.1158/1538-7445.AM2013-5496
Abstract 5504: Combination of gemcitabine and docetaxel for the treatment of Korean children and adolescents with recurrent or refractory osteosarcoma.
Authors: Lee Jun Ah , Ko Yunmi , Kim Dong Ho , Yoo Ji Young , Park Eun Sil
Published: August 2014

Background: The combination of gemcitabine and docetaxel (GEMDOX) has demonstrated promise in adult sarcomas. Still, its efficacy in children and adolescents with recurrent or refractory osteosarcoma is controversial, due to a relatively small number of cases. We evaluated the efficacy of this combination in Korean pediatric patients with recurrent or refractory osteosarcoma.

Methods: A retrospective case review of 23 patients (15 male, 8 female) with recurrent or refractory osteosarcoma who received gemcitabine (675 – 900 mg/m2 intravenously on days 1 and 8) and docetaxel (100 mg/m2 intravenously on day 8) at the Korea Cancer Center Hospital was undertaken.

Results: The patients (ages 7.8 – 12.4 years) received a total of 78 courses of GEMDOX chemotherapy (median 3 courses; range 1 – 8 courses). For the 23 patients, 13 patients received GEMDOX as adjuvant chemotherapy, and the remaining 10 cases were eligible for response evaluation; there were 3 partial response (PR) and 1 stable disease (SD). The overall objective response was 30% and disease control rate was 40%. Median duration of response was 6.5 months (range, 3 – 13 months). GEMDOX chemotherapy was well tolerated and no treatment related death was observed. The most common toxicity was myelosuppression, however, no infectious complication have occurred.

Conclusion: The poor prognosis of patients with recurrent or refractory osteosarcoma, lack of effective salvage chemotherapy regimen, and case series reporting its activity, including ours, suggest that GEMDOX chemotherapy is beneficial in palliative setting and need to be considered for large scale clinical trials.

Citation Format: Jun Ah Lee, Yunmi Ko, Dong Ho Kim, Ji Young Yoo, Eun Sil Park. Combination of gemcitabine and docetaxel for the treatment of Korean children and adolescents with recurrent or refractory osteosarcoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5504. doi:10.1158/1538-7445.AM2013-5504
Abstract 5510: Tranilast inhibits growth of skin tumor cells in a xenograft mouse model of tuberous sclerosis complex.
Authors: Li Shaowei , Wang Ji-an , Thangapazham Rajesh L. , Klover Peter , Moss Joel , Darling Thomas N.
Published: August 2014

Pharmacological approaches for treating tumors in patients with tuberous sclerosis complex (TSC) are inadequate. Although mTORC1 inhibitors such as rapamycin represent a major new avenue for treatment, we and others have shown that rapamycin improves but does not eradicate TSC skin tumors. We tested the effects of tranilast, an antiallergic and antifibrotic drug with antiproliferative effects, on fibroblasts grown from angiofibromas, forehead plaques, periungual fibromas, and normal-appearing skin from 5 patients with TSC. Tumor cells showed loss of TSC2 expression and hyperactivation of the mTORC1 signaling pathway. Tranilast (5, 10, 20, 40 μM) inhibited the viability of TSC skin tumor cells from five patients in an MTT assay. These effects on cell viability appeared to be independent of mTORC1 activity, since tranilast did not inhibit phosphorylation of ribosomal protein S6. Four paired samples of RNA from tumor and normal cells, treated with or without tranilast, were studied using gene expression arrays, revealing that tranilast significantly altered the expression of at least 36 genes, of which 12 are involved in transcription. In a xenograft model in which dermal-epidermal constructs comprising normal human keratinocytes with either TSC angiofibroma cells or TSC normal fibroblasts from a TSC patient were grafted to nude mice (6-8 mice per group), tranilast treatment (300 mg/kg/day) reduced the number of skin tumor cells compared to vehicle control group (P< 0.05). Tranilast treatment did not block angiogenesis or lymphangiogenesis in the xenograft model. These results indicate that tranilast has antiproliferative effects independent of mTORC1 and suggest that tranilast may be useful as an adjunctive agent for the treatment of TSC skin tumors.

Citation Format: Shaowei Li, Ji-an Wang, Rajesh L. Thangapazham, Peter Klover, Joel Moss, Thomas N. Darling. Tranilast inhibits growth of skin tumor cells in a xenograft mouse model of tuberous sclerosis complex. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5510. doi:10.1158/1538-7445.AM2013-5510
Abstract 5516: (R,R’)-4’-methoxy-1-naphthylfenoterol Inhibits GPR55 signaling and the modulation of motility in human cancer cells.
Authors: Paul Rajib K. , Wnorowski Artur , Gonzalez-Mariscal Isabel , Moaddel Ruin , Indig Fred E. , Wainer Irving W. , Bernier Michel
Published: August 2014

(R,R’)-4’-methoxy-1-naphthylfenoterol (MNF) promotes growth inhibition and apoptosis of human HepG2 hepatocarcinoma cells via cannabinoid receptor (CBR) activation. The synthetic CB1R inverse agonist, AM251, has been shown to block the anti-mitogenic effect of MNF in these cells; however, AM251 is also an agonist of the recently deorphanized, lipid-sensing receptor, GPR55, whose upregulation contributes to carcinogenesis. Here, we investigated the role of GPR55 in MNF signaling in human HepG2 and PANC-1 cancer cell lines in culture, with a focus on internalization of the fluorescent ligand Tocrifluor 1117 (T1117), reorganization of actin cytoskeleton, and cell motility as measured by scratch assay. Control experiments indicated that GPR55 knockdown by RNA interference markedly reduced cellular uptake of T1117, a process that was sensitive to MNF inhibition. GPR55 internalization mediated by the atypical cannabinoid O-1602 was blocked by MNF in GPR55-expressing HEK293 cells. Pretreatment of HepG2 and PANC-1 cells with MNF significantly abrogated the induction of ERK phosphorylation in response to AM251, O-1602 and fetal bovine serum, known to contain bioactive lipids. Moreover, MNF exerted a coordinated regulation of AM251 and O-1602 inducible processes, including cell motility. This study shows for the first time that MNF impairs GPR55-mediated signaling and, therefore, may have therapeutic potential for the management of cancer. This work was supported entirely by the Intramural Research Program of the NIA, NIH.

Citation Format: Rajib K. Paul, Artur Wnorowski, Isabel Gonzalez-Mariscal, Ruin Moaddel, Fred E. Indig, Irving W. Wainer, Michel Bernier. (R,R’)-4’-methoxy-1-naphthylfenoterol Inhibits GPR55 signaling and the modulation of motility in human cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5516. doi:10.1158/1538-7445.AM2013-5516
Abstract 5519: In vivo fluorescence lifetime imaging differentiates the bound versus unbound status of a cell surface receptor targeted ligand.
Authors: JeuneSmith Yoli , Cohen Allison S. , Tafreshi Narges K. , Vagner Josef , Gillies Robert , Morse David
Published: August 2014

In vivo fluorescence lifetime imaging allows for the isolation of probe-specific fluorescence lifetime from surrounding tissue auto-fluorescence. Fluorescence lifetime is not affected by agent concentration or depth, or image acquisition parameters. However, fluorescence lifetime can be affected by the in vivo tissue microenvironment, e.g. pH or hypoxia. In this work, fluorescence lifetime is used to characterize the presence and fate of a high-affinity peptidyl delta opioid receptor (δOR) ligand conjugated to the near-infrared fluorescent IRDye800CW (Li-Cor).

HCT116 colorectal cancer cells engineered to over-express the δOR and parental non-expressing HCT116 cells were bilaterally xenografted subcutaneously into the flanks of athymic nude mice. At a series of time-points following tail vein injection of the ligand, in vivo fluorescence images images were acquired using the Optix MX3 scanner (Advanced Research Technologies).

For 24 h, the ligand fluoresence lifetime was significantly different (p<0.05) in the δOR positive tumors relative to the negative tumors. At 48 hours, the fluorescence lifetime in the negative tumor decreased and was no longer different. We hypothesize that the early difference in fluorescence lifetime is attributed to the bound state of the ligand on receptor positive tumors relative to the unbound state in negative tumors. Over time, unbound ligand clears from the negative tumor leaving only a low concentration of ligand retained by non-specific interactions.

In vitro studies demonstrated that the fluoresecence lifetime of this ligand is not altered within the biological pH range. However, the tumor microenvironment is complex and multiple factors may be influencing the observed differences. Future plans include studies of 3D cell cultures using varied microenvironmental conditions and studies on δOR endogenous cell lines.

Citation Format: Yoli JeuneSmith, Allison S. Cohen, Narges K. Tafreshi, Josef Vagner, Robert Gillies, David Morse. In vivo fluorescence lifetime imaging differentiates the bound versus unbound status of a cell surface receptor targeted ligand. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5519. doi:10.1158/1538-7445.AM2013-5519
Abstract 5520: Novel therapy NKP-1339 is active in osteosarcoma.
Authors: Park Amy Y. , Sowers Rebecca , Thein Sajida , Baerga Rebecca , Gorlick Richard
Published: August 2014

Osteosarcoma (OS) is the most common primary malignant bone tumor in children and young adults. Despite significant advancements in the diagnosis and treatment of OS, overall survival trends have remained stagnant with conventional chemotherapy. New chemotherapeutic agents and combination regimens are being studied to target pathways that are believed to be active in OS. NKP-1339 is a novel small molecule anti-cancer compound that targets the GRP78 pathway. A NKP-1339 single agents Phase I has been completed [1]. GRP78 is a master regulator of endoplasmic reticulum stress. High GRP78 levels are found in a wide variety of cancer types and have been correlated with tumor proliferation, metastasis, angiogenesis and tumor cell survival. Treatment with anti-cancer agents, both cytotoxic and targeted agents, has been shown to give rise to further induction of GRP78, and such elevation been correlated to resistance. NKP-1339 down-regulates GRP78 levels selectively in tumor cells leading to tumor cell death. In this study, we explore NKP-1339 activity against OS cell lines. In vitro cytotoxicity assays were performed with NKP-1339, as a single agent, on both standard and patient derived OS cell lines. Three out of four standard cell lines (75%) and two out of four patient derived cell lines (50%) showed IC50 of 80-100 uM. These results, in terms of uM range and activity against tumor lines that are resistant to other agents, are consistent with previous in vitro studies of NKP-1339 with other tumor types. The IC50 uM levels of NKP-1339 are achievable in patient plasma. In vitro combination studies are underway to determine the synergistic anti-tumor effects of NKP-1339 with standard chemotherapeutic agents used in OS. Future studies will include treatment of OS xenograft models with NKP-1339 as a single agent and in combination, and if promising, an OS clinical trial will be initiated.

[1] Thompson et al. ASCO 2012 abstract #3033

Citation Format: Amy Y. Park, Rebecca Sowers, Sajida Thein, Rebecca Baerga, Richard Gorlick. Novel therapy NKP-1339 is active in osteosarcoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5520. doi:10.1158/1538-7445.AM2013-5520
Abstract 5531: A bioluminescent cell viability assay optimized for 3D microtissues.
Authors: Valley Michael P. , Zimprich Chad , Cali James J. , Lazar Dan F.
Published: August 2014

It has been well demonstrated that the morphology, gene expression, and overall biological response of cells assessed in 3-dimensional (3D) culture models are frequently more physiologically relevant than that of cells studied in standard 2D culture formats. In spite of rapidly growing interest, one hindrance to the use of 3D models for drug discovery and other research efforts is the lack of convenient and effective assays explicitly validated for application to 3D microtissues. Here we report on a bioluminescent ATP-detection assay comprised of both an improved formulation and an optimized protocol specifically designed to measure the viability of cells grown in 3D culture. The ability of the 3D-optimized reagent to effectively lyse cells from throughout the full thickness of 3D microtissues, including cancer cell spheroids, was clearly demonstrated by use of confocal laser fluorescent microscopy in conjunction with a fluorogenic, membrane-impermeant DNA-binding dye. Importantly, the assay's ability to accurately report the ATP content, and therefore viability, of various 3D microtissues (including a range of cancer cell line spheroids) was demonstrated by comparison to a trichloroacetic acid ATP extraction method. In scaffold-free as well as scaffold-dependent 3D models, recovery and detection of greater than 80% of ATP present was routinely observed. In contrast, another frequently used ATP-detection assay was found to have greatly reduced lytic and ATP detection properties (<50% recovery). This new, homogeneous 3D-assay format requires minimal sample manipulation, a brief incubation period (generally less than 30 min), and exhibits excellent sensitivity. It should be amenable to both low- and high-throughput applications and accurately report drug sensitivities of cells assayed in both 2D and 3D culture models.

Citation Format: Michael P. Valley, Chad Zimprich, James J. Cali, Dan F. Lazar. A bioluminescent cell viability assay optimized for 3D microtissues. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5531. doi:10.1158/1538-7445.AM2013-5531
Abstract 5536: NAD+ Kinase inhibition synergizes with ROS-inducing chemotherapies.
Authors: Tedeschi Philip M. , Kerrigan John , Banerjee Debabrata , Bertino Joseph R. , Abali Emine E.
Published: August 2014

Background: NAD+ kinase (NADK) is at present the only known enzyme that converts NAD+ to NADP+, which is subsequently reduced to NADPH. The demand for NADPH in cancer cells is elevated as reducing equivalents are required for the high levels of nucleotide, protein and fatty acid synthesis found in proliferating cells, as well as for neutralizing high levels of reactive oxygen species (ROS) present in these cells. The aim of this study was to determine whether inhibitors of NADK synergize with chemotherapeutic drugs known to induce ROS.

Methods: Using a NADK inhibitor our group previously identified, thionicotinamide adenine dinucleotide phosphate (NADPS), (Mol Pharmacol., in press), we explored combinations of NADPS with various classes of drugs known to induce ROS: tubulin inhibitors (docetaxel, paclitaxel, vinblastine), a nucleoside analog (gemcitabine), topoisomerase I and II inhibitors (irinotecan and doxorubicin, respectively), and a DNA crosslinking agent (cisplatin). Fixed ratios of NADPS and the combination drug based on their EC50 values were tested in a colon cancer cell line (C85) using a MTS assay. The combination index (CI) was determined by the Chou-Talalay method for drug combination based on the median effect analysis.

Results: All drugs were found to synergize with NADPS. Knockdown of NADK with siRNA or inhibition of NADK by NADPS in combination with MTX revealed equivalent efficacy. Quantification of intracellular NADPH and ROS levels are currently underway.

Conclusions: NADK inhibitors along with drugs that increase ROS- mediated stress may represent an efficacious antitumor combination and should be explored further against in vivo tumor models.

Citation Format: Philip M. Tedeschi, John Kerrigan, Debabrata Banerjee, Joseph R. Bertino, Emine E. Abali. NAD+ Kinase inhibition synergizes with ROS-inducing chemotherapies. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5536. doi:10.1158/1538-7445.AM2013-5536