bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3778: In vitro and in vivo characterization of E7090, a novel and selective FGFR inhibitor, for the treatment of endometrial cancer
Authors: Miyano Saori Watanabe , Yamamoto Yuji , Kodama Kotaro , Sugi Naoko Hata , Minoshima Yukinori , Matsui Junji , Iwata Masao
Published: July 2016

Genetic abnormalities (gene fusion, mutation and amplification) of Fibroblast Growth Factor Receptor (FGFR) family members are known to cause constitutive activation of their signaling pathways, which play an important role in proliferation, survival and migration of cancer cells, tumor angiogenesis, and drug resistance. FGFR2 is mutated in about 10% of endometrial cancer patients and is associated with a higher risk of recurrence. E7090, an oral available FGFR1, 2, and 3 inhibitor that the chemical-structure and basic kinase inhibitory activity of this compound have been presented at AACR2015, is currently under a first-in-human study (NCT02275910) in Japan. In this presentation, the efficacy of E7090 against human endometrial cancer was examined.

E7090 exhibited selective potent antiproliferative activity against 3 human endometrial cancer cell lines harboring FGFR2 mutation with IC50 values of approximately 10 nM among 8 endometrial cancer cell lines. These cell lines harbor S252W mutation (extracellular domain mutation; MFE280) or N549K mutation (kinase domain mutation; AN3CA and MFE296), that are the most common mutations of FGFR2 in endometrial cancer. E7090 also showed antiproliferative activity against Ba/F3 cells expressing mutated type of FGFR2, confirming the inhibitory activity of E7090 against mutated FGFR2. The antiproliferative activity of E7090 against these cell lines was based on inhibition of phosphorylated FRS2α and ERK1/2 as downstream molecules of FGF/FGFR signaling. Especially, the antiproliferative activities of E7090 against AN3CA and MFE296 harboring N550K mutation were more potent compared to these of other FGFR specific inhibitors. In addition, oral administration of E7090 resulted in significant tumor growth inhibition in MFE280, AN3CA and MFE296 xenograft models in mice. Especially, E7090 showed tumor regression in AN3CA xenograft model with a delta T/C value of -66% at a dose of 50 mg/kg.

These results indicated that E7090 showed potent inhibitory activity against mutated type of FGFR2 both in vitro and in vivo, and suggest that E7090 may provide therapeutic opportunities for endometrial cancer harboring FGFR2 mutations.

Citation Format: Saori Watanabe Miyano, Yuji Yamamoto, Kotaro Kodama, Naoko Hata Sugi, Yukinori Minoshima, Junji Matsui, Masao Iwata. In vitro and in vivo characterization of E7090, a novel and selective FGFR inhibitor, for the treatment of endometrial 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 3778.
Abstract 3797: Validation of a novel antibody against fibulin-3 for targeted therapy of glioblastoma
Authors: Nandhu Mohan Sobhana , Behera Prajna , Bhaskaran Vivek , Chiocca Ennio Antonio , Viapiano Mariano S.
Published: July 2016

Glioblastomas (GBM) are highly invasive brain tumors that resist cytotoxic and antiangiogenic therapies, resulting in a high rate of recurrence. Fibulin-3 is an extracellular matrix protein secreted by GBM cells but absent from normal brain. This protein activates Notch signaling to promote tumor invasion and resistance to apoptosis. Conditional knockdown of fibulin-3 reduces GBM growth, invasion and vascularization, thus extending survival. Here we report the initial validation of a function-blocking antibody against this unique GBM target. We first identified the Notch-activating motif of fibulin-3 and developed an immunizing peptide against a key sequence within this motif. A monoclonal antibody generated against this peptide (mAb428.2) showed high affinity for fibulin-3 (Kd 5 nM) and no cross-reactivity against highly homologous fibulin-4 or -5. Antibody mAb428.2 detected fibulin-3 in the stroma and capillaries of human GBM without cross-reactivity against normal brain. The antibody (50-250 μg/ml) blocked the activation of Notch induced by fibulin-3 in GBM cells and caused GBM cell cytotoxicity but had no effects on astrocytes or HEK293 cells. Treatment of mice carrying subcutaneous GBM stem-cell (GSC) derived tumors with mAb428.2 (30 mg/kg IV, q24h x 8 days) resulted in tumor slowdown and extended median survival (47% and 64% in two different GSC models). mAb428.2-treated tumors showed decreased BrdU uptake and increased inflammatory reaction in the tumor stroma (macrophage infiltration and cytokine levels). Mice bearing intracranial tumors did not respond to mAb428.2 when delivered by IV or IP routes, likely due to the inability of the antibody to cross the blood brain barrier. This deficiency was overcome by direct intraparenchymal delivery of the mAb428.2 using chronic infusion (0.3 mg of mAb in 200 μl delivered at 1 μl/h over 7d), which resulted in a 26% increase in median survival. These encouraging results suggest that targeted reagents against fibulin-3 may be of clinical importance for novel combination therapies against GBM.

Citation Format: Mohan Sobhana Nandhu, Prajna Behera, Vivek Bhaskaran, Ennio Antonio Chiocca, Mariano S. Viapiano. Validation of a novel antibody against fibulin-3 for targeted therapy of glioblastoma. [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 3797.
Abstract 3807: Histone deacetylase inhibitors repress hypoxia signaling through affecting Hsp90
Authors: Yang Chunzhang , Zhuang Zhengping
Published: July 2016

Histone deacetylase inhibitors (HDACis) are among the most promising recently developed anti-cancer agents. Recent work suggests that HDACis suppress both malignant cell metabolism and progression by interfering with the hypoxia-inducible factor (HIF) signaling, which plays a key role in tumor progression in both actual and falsely perceived cellular hypoxia (pseudohypoxia). However, the precise biochemical mechanism of HDACis repression of HIFs function remains unclear. In this study, we demonstrated that the class-2 histone deacetylase inhibitor SAHA potently inhibits hypoxia signaling via interfering with heat shock protein 90 (Hsp90). HDACis increase in the acetylation of Hsp90, resulting in less HIF-α recognition and nuclear translocation. Accumulated cytoplasmic HIF-α is not transcriptionally active and degradaded through proteosomal pathway. Finally, we demonstrated that SAHA remarkably reduces hypoxia signaling and decreases tumor growth in vivo. These findings provide insight into new possible therapeutic strategies for using HDACis in tumors with known HIF pathway aberrations.

Citation Format: Chunzhang Yang, Zhengping Zhuang. Histone deacetylase inhibitors repress hypoxia signaling through affecting Hsp90. [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 3807.
Abstract 3811: The mechanobiome of pancreatic ductal adenocarcinoma: a new, targetable drug space
Authors: Surcel Alexandra , Zhu Qingfeng , Schiffhauer Eric , Anders Robert A. , Robinson Douglas N.
Published: July 2016

Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer mortality, with 37,000 people dying annually in the US. Existing strategies for treating cancer primarily mainly focus on inhibiting cell growth through specific genetic pathways, which typically either fail to completely abolish the disease or which lead to compensatory regulatory changes, and hence, drug resistance. Targeting cell mechanics remains an under-used approach for drug development.

The direct driver of cell shape change events intrinsic to cellular functions, such migration and invasion, is the mechanobiome - a collection of cytoskeletal proteins which are the final determinants of a cell's mechanical attributes and which lie downstream of KRAS and other regulatory molecules. Targeting, and ultimately, inhibiting these processes is less likely to be subject to compensatory regulation by cancer cells. We determined via western blot analysis and immunohistochemistry of patient-derived samples that key players involved in mechanosensation-myosin IIA, IIC, α-actin-4, and filamin B -show increased expression in cancerous ductal epithelial over normal tissue, while non-mechanosensory, or variable mechanosensory, paralogs (myosin IIB, α-actin-1, and filamin A) show decreased expression. This upregulation of highly mechanosensory proteins has initiated an investigation into the necessity and sufficiency of the myosin II paralogs in PDAC metastasis through overexpression and knockdown of expression, coupled with mechanical assays. In addition to resolving the mechanobiome of PDAC, we have previously shown that targeting of myosin IIC by 4-hydroxyacetophenone affects PDAC mechanics. We are testing the in vivo efficacy of 4-HAP by conducting a murine multi-arm study of metastatically human derived pancreatic cancer cells. Preliminary results suggest a protective effect against the metastasis of human pancreatic cancer cells among mice treated with 4-HAP every other day.

Citation Format: Alexandra Surcel, Qingfeng Zhu, Eric Schiffhauer, Robert A. Anders, Douglas N. Robinson. The mechanobiome of pancreatic ductal adenocarcinoma: a new, targetable drug space. [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 3811.
Abstract 3816: Reactive oxygen species and ERK2 phosphorylation are required for NSC59984 to induce mutant p53 protein degradation and restore p53 signaling
Authors: Zhang Shengliang , Zhou Lanlan , Dicker David , El-Deiry Wafik S.
Published: July 2016

Tumor suppressor p53 is mutated in over 50% of human cancers. p53 mutation abolishes wild-type p53 function and also endows mutant p53 with a gain-of-function (GOF) that drives tumor growth and drug resistance. Targeting mutant p53 is an attractive strategy for cancer therapy. We recently reported (Zhang et al., Cancer Research, 2015) a small-molecule NSC59984 with dual capabilities to restore p53 signaling and destabilize mutant p53 protein (depleting GOF). We now demonstrate the role of reactive oxygen species (ROS) and ERK2 in the mechanism of action of NSC59984. We observe a sustained-phosphorylation of ERK2 in cancer cells treated with NSC59984. Blockade of ERK2 rescues mutant p53 from NSC59984-mediated degradation, and inhibits restoration of p53 signaling in mutant p53-expressing cells. Thus, sustained ERK2 phosphorylation is required for NSC59984-induced degradation of mutant p53 protein, and depletion of mutant p53 contributes to the restoration of p53 pathway. NSC59984 induces MDM2 phosphorylation that correlates with ERK2 phosphorylation. The effect of NSC59984 on MDM2 phosphorylation is blocked by U0126 and knockdown of ERK2. NSC59984-mediated mutant p53 protein degradation is inhibited by MDM2 knockdown, and enhanced by MDM2 overexpression. Thus, ERK2-dependent MDM2 phosphorylation is a major determinant of NSC59984-mediated mutant p53 degradation. We investigated the role of ROS in the effect of NSC59984 on ERK2 phosphorylation. ROS is induced by NSC59984 and a decrease in ROS by NAC inhibits NSC59984-induced ERK2 phosphorylation, and mutant p53 protein degradation. Increased ROS by BSO treatment enhances the NSC59984 effect on ERK2 phosphorylation, mutant p53 protein degradation and restoration of p53 signaling. We conclude that ROS is required for NSC59984 to sustain ERK2 phosphorylation, which, in turn, is required for NSC59984-induced mutant p53 protein degradation via MDM2. The combination of NSC59984 and BSO synergistically induces cell death in colorectal cancer cells. ROS and ERK2 are two important factors required for NSC59984 to degrade mutant p53 protein, restore p53 signaling and induce cell death. These results provide a rationale for clinical testing of NSC59984 in tumors with high ROS.

Citation Format: Shengliang Zhang, Lanlan Zhou, David Dicker, Wafik S. El-Deiry. Reactive oxygen species and ERK2 phosphorylation are required for NSC59984 to induce mutant p53 protein degradation and restore p53 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 3816.
Abstract 3817: Recombinant stapled proteins for the treatment of chronic myeloid leukemia
Authors: Bruno Benjamin J. , Cornillie Sean P. , Cheatham Thomas E. , Chou Daniel H. , Lim Carol S.
Published: July 2016

The oncoprotein Bcr-Abl is the cause of chronic myeloid leukemia (CML). Current therapies target the tyrosine kinase domain of Bcr-Abl, but due to their non-curative nature, resistance to these drugs is common. Therefore, new treatments are needed for those patients refractive to available therapies. Bcr-Abl homo-oligomerization via its N-terminal coiled coil (CC) domain is required for tyrosine kinase activity. Our previous work has shown that it is possible to inhibit Bcr-Abl activity by targeting the CC domain with a peptidomemetic known as CCmut, delivered as a protein using a cell-penetrating peptide (CPP-CCmut). CPP-CCmut selectively enters leukemic cells and inhibits Bcr-Abl, as seen by Western blot, 7AAD, Annexin V, cell proliferation, and colony forming assays.

This leukemia cell-permeable CPP-CCmut protein domain is our lead compound for CML therapy, but to be translatable to the clinic it must be modified to improve proteolytic stability. For this reason, we created stapled versions of this CPP-CCmut. Stapled peptides are resistant to proteolysis, and are emerging as a viable strategy for disrupting protein-protein interactions in diseases including HIV and cancer. Until recently, peptide stapling required incorporation of unnatural amino acids; therefore these peptides were created via solid-state synthesis. However, the synthesis of the 81 amino acid CPP-CCmut is no trivial matter. The ability to staple a longer sequence such as our protein domain is now possible using thiol-ene coupling. This exciting new chemistry allows for the stapling of recombinant proteins at substituted cysteine residues (instead of stapling substituted unnatural amino acids incorporated into synthetically made peptides). Thiol-ene coupling is used here to staple recombinant CPP-CCmut, and provides a path for translation of our lead compound to the clinic.

Molecular modeling has identified optimal locations for staples on CPP-CCmut, which include 29/36, 36/43, 50/57, 57/64, as well as double staples 29/36-50/57 and 36/43-50/57. These staple locations still allow for dimerization of CPP-CCmut with Bcr-Abl, as indicated by free energy calculations and helicity. The staple locations have also been optimized for maximal coverage from proteolytic degradation.

This work acts as a proof-of-concept that proteins (not just peptides) can be stapled, thus greatly expanding the variety and complexity of stapled proteins for use in the exciting field of targeting protein-protein interfaces to treat disease.

Citation Format: Benjamin J. Bruno, Sean P. Cornillie, Thomas E. Cheatham, Daniel H. Chou, Carol S. Lim. Recombinant stapled proteins for the treatment of chronic myeloid leukemia. [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 3817.
Abstract 3818: Therapeutic potential of blocking CCR5 by maraviroc in breast cancer bone metastasis
Authors: Pervaiz Asim , Zepp Michael , Ali Doaa M. , Berger Martin R. , Adwan Hassan
Published: July 2016

Introduction:

Bone metastasis is a highly unfavorable condition observed in up to 70% of the patients with breast cancer. Treatment options for this condition are not curative, but palliative, only, which highlights the need for exploring new therapeutic options. In this study, we propose that targeting a chemokine receptor (CCR5), often upregulated in primary and secondary breast cancers, can contribute to a more successful treatment of breast cancer bone metastasis.

Methodology:

Following the blockage of CCR5 by maraviroc, cytotoxic effects were measured by MTT assay, while the migratory effects were determined by migration and scratch healing assays. Apoptosis related activities were investigated by nuclear staining and western blot analysis. Maraviroc mediated cytostatic changes were analyzed by a ready-made Human Cell Cycle Regulation Panel (Roche, Germany). In vivo experiments were performed by implanting MDA-MB-231 cells via the saphenous artery to the left hind limb of male nude rats (RNU strain) for inducing bone metastasis. Treatment with maraviroc was started from 2nd and 7th day of transplantation in two groups of rats (n = 6/group) designated as A and B, respectively, and was compared with an untreated control group (n = 8 rats).

Results:

CCR5 blockage by maraviroc (concentration > 100μM) induced concentration dependent cytotoxicity in MDA-MB-231 and MCF-7 cells. Maraviroc exposure also showed significant inhibition of migration of the cells, while nuclear staining indicated the condensation/fragmentation of nuclear content. Maraviroc exposure induced significant expression of cleaved caspase 7 and PARP, while real time RT-PCR showed pronounced inhibition of multiple genes (≥2fold) including cyclins, CDKs and their down-stream targets. Treatment of tumor bearing animals, with intra-peritoneal injections of maraviroc (25mg/kg, 3-4 weeks daily), reduced the tumor burden significantly (p < 0.05) in group A (50-75%), while the effects were minimal in group B (<25%). Concomitantly, no signs of toxicity were observed in rats of the two groups.

Conclusion:

CCR5 blockage by maraviroc induces significant anti-neoplastic effects in breast cancer cells. At mechanistic level, these effects included induction of apoptosis and alterations in cell cycle. Considerable inhibition in group A, but minimal inhibition of tumor burden in group B indicates that targeting CCR5 by maraviroc during early stages of breast cancer bone metastasis is a promising treatment option which should be further explored.

Citation Format: Asim Pervaiz, Michael Zepp, Doaa M. Ali, Martin R. Berger, Hassan Adwan. Therapeutic potential of blocking CCR5 by maraviroc in breast cancer bone metastasis. [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 3818.
Abstract 382: Concomitant administration of acid reducing agents can impact the pharmacokinetics of the balanced pan-PI3K and mTOR inhibitor PQR309
Authors: Jorga Karin , Huehn Eva , Fraczkiewicz Grace , Schmitz Debora , Fabbro Doriano , Dimitrijevic Sasa
Published: July 2016

Introduction

Acid reducing agents (ARAs) - such as proton pump inhibitors, H2-antagonists or antacids - are commonly prescribed medications to reduce stomach acidity in cancer patients. Increased gastric pH can significantly impair the dissolution of agents that exhibit pH-dependent solubility, thus possibly reducing their absorption and influence their pharmacokinetics (PK).

PQR309 is a novel oral balanced dual pan-PI3K and mTOR inhibitor currently in clinical development for treatment of solid tumors and hematological malignancies. PQR309 is a weak base and exhibits pH-dependent solubility. Therefore, the assessment of the potential for drug-drug interactions with ARAs is essential to avoid treatment failure in cancer patients.

Methods

GastroPlusTM v.9.0 (Simulations Plus, Inc.) was used to build a physiologically-based absorption and pharmacokinetic (PBPK) model of PQR309. A baseline model was built with the physicochemical properties of the compound and pre-clinical PK data after iv and po administration. The model was then extended using PK data from cancer patients. Volume of distribution and renal clearance were calculated using the Lukacova and fup*GFR methods, respectively. Hepatic clearance was estimated from in vitro clearance obtained in hepatocytes and the intestinal absorption was calculated by the Advanced Compartmental Absorption and Transit (ACATTM) Model.

Results

The parameter sensitivity analysis showed that at fasted stomach pH of ∼1.5, PQR309 is fully dissolved and rapidly absorbed in the upper part of gastrointestinal tract leading to a distinct maximum plasma concentration (Cmax) and a predicted bioavailability of almost 100%. At a stomach pH of 4.0 dissolution is delayed and the absorption shifted towards distal parts of the gastrointestinal tract. The predicted bioavailability is still estimated to be good (> 75%), but the observed plasma concentration time profile is much shallower and the expected Cmax considerably reduced.

Conclusions

Our model predicts that concomitant administration of ARAs can impact PQR309 absorption and change its pharmacokinetic profile. The clinical relevance of this potential drug-drug interaction is unknown. In currently ongoing clinical studies co-medication of PQR309 with ARAs should be avoided until further clinical investigations confirm the model predictions and mitigation strategies are explored.

Citation Format: Karin Jorga, Eva Huehn, Grace Fraczkiewicz, Debora Schmitz, Doriano Fabbro, Sasa Dimitrijevic. Concomitant administration of acid reducing agents can impact the pharmacokinetics of the balanced pan-PI3K and mTOR inhibitor PQR309. [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 382.
Abstract 383: In vitro effects of PI3K/mTOR inhibition in canine hemangiosarcoma
Authors: Pyuen Alex A. , Thamm Douglas H.
Published: July 2016

While extremely rare in humans, hemangiosarcoma (HSA) accounts for nearly 2% of canine neoplasia, and is characterized by both aggressive local growth/invasion and a high rate of metastasis. Both canine and human HSA exhibit sustained aberrant PI3K/Akt/mTOR pathway signaling. The purpose of this study was to examine the in vitro effects of a novel dual PI3K/mTOR inhibitor, VDC-597, in canine HSA cells. Three canine HSA cell lines (DEN-HSA, CIN-HSA, and SB-HSA) were employed in multiple in vitro assays. Western analysis evaluated activation (phosphorylation) of key downstream pro-survival proteins in the PI3K/mTOR pathway. Changes in tumor cell growth/apoptosis were assessed using both bioreductive assays (Alamar Blue) and in vitro live-cell imaging (IncuCyte), in the presence and absence of doxorubicin, a standard-of-care cytotoxic drug for canine and human sarcomas, as well as in the presence and absence of U-0126, an inhibitor of the MEK pathway. Migration was assessed using both Boyden chamber and scratch assays, via live-cell imaging (IncuCyte). Matrigel invasion was assessed using traditional Boyden chambers. Finally, ELISA was utilized to quantify relative expression of vascular endothelial growth factor (VEGF). VDC-597 suppressed activation of both Akt and 4eBP1 in canine HSA cells in a dose- and time-dependent fashion, with an IC50 of approximately 0.3 uM, a concentration predicted to be clinically achievable based on preliminary early-phase canine and human studies. VDC-597 dose-dependently reduced proliferation, migration, invasion, and VEGF expression in HSA cells, while promoting tumor cell apoptosis. VDC-597 demonstrated additive antiproliferative effects when combined with doxorubicin and U-0126. Together, these results suggest that inhibitors of the PI3K/Akt/mTOR pathway may act against multiple components of the neoplastic process, including proliferation/apoptosis, chemosensitivity, invasion/migration and angiogenesis, and justify the evaluation of PI3K/mTOR inhibitors in canine, and eventually human, HSA. Experiments to examine the effect of VDC-597 in reducing tumor burden and metastasis in a rodent model are ongoing.

Citation Format: Alex A. Pyuen, Douglas H. Thamm. In vitro effects of PI3K/mTOR inhibition in canine hemangiosarcoma. [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 383.
Abstract 3830: Synthesis and anticancer activity of novel prodigiosin analogs
Authors: Tian Xiaobing , Zhang Shengliang , El-Deiry Wafik S.
Published: July 2016

We recently reported that Prodigiosin is a potent p53 pathway restoring small molecule that acts through p73 and by interfering with p73:mutant p53 protein interaction (Hong et al., Cancer Research, 2014). Prodigiosin is the parent member of the tripyrrole alkaloid family of natural products that shows potent anti-cancer activity against tumors with mutated p53. To improve pharmacological and medicinal properties of Prodigiosin including p73 induction and restoration of the p53 pathway in tumors with mutated p53, two new series of analogs were synthesized. We introduced carboxylic group substituents either directly on the C-ring or on the side-chain of the C-ring. Carboxylic group allowed for further derivatization of Prodigiosin. A number of new analogs have been prepared and tested for their anticancer activity. Preliminary biological activity assay showed that new compounds inhibited cancer cell proliferation from 0.1 μM to 10 μM both in p53 mutated SW480 and wild-type HCT116 colorectal cancer cell lines. Some of the newly synthesized compounds can induce p53 transcriptional reporter activity in p53 mutated SW480 colorectal cancer cells. Our ongoing experiments are focused on the signaling mechanism of apoptosis induced by the new analogs, identification of molecular targets of the compounds, and in vivo studies of Prodigiosin analogues as single agents or in combination with chemotherapy or targeted therapy.

Citation Format: Xiaobing Tian, Shengliang Zhang, Wafik S. El-Deiry. Synthesis and anticancer activity of novel prodigiosin analogs. [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 3830.
Abstract 3839: Vitamin E delta-tocotrienol targets human colon cancer stem cells and inhibits colon cancer metastasis and induces apoptosis
Authors: Husain Kazim , Coppola Domenico , Sebti Said M. , Malafa Mokenge P.
Published: July 2016

Background: Colon cancer is the most commonly diagnosed cancer both in men and women and cause of cancer related deaths worldwide. Treatment for the recurrent and metastatic colon cancer remains a center of clinical attention. Cancer stem cells (CSCs) are suggested to be the only cells within tumor with tumorigenic capacity and implicated in metastasis. We have shown that vitamin E delta-tocotrienol (VEDT) is the most bioactive tocotrienols against cancers. This study aimed to evaluate the effect of VEDT on colon cancer growth, metastatic epithelial to mesenchymal transition (EMT), migration/ invasion/angiogenesis and colon CSCs growth and apoptosis.

Methods: Human colon cancer cells HCT-116 and metastatic colon cancer cells SW-620 and colon CSCs were treated with VEDT (10-100 μM) for 72 h and cell viability recorded. VEDT (50 μM) was used for malignant transformation; migration/invasion, CSCs spheroid formation, and pluripotency were determined. Azoxymethane treated Fisher 344 rats were used as colon carcinogenesis model. They were treated with 1) vehicle control and 2) VEDT (200 mg/kg, orally twice a day) for 20 weeks. After 20 weeks the polyps and tumor formation were recorded.

Results: VEDT significantly inhibited the anchorage-dependent and independent cancer cell growth, migration/invasion and induced apoptosis compared to control. VEDT inhibited the CSC spheroid formation EMT (E-cadherin to vimentin), metastasis (MMP9), angiogenesis (VEGF), CSCs transcription factors (Nanog, Oct4, Sox2 and KLF4) compared to control.

Conclusion: Vitamin E delta-tocotrienol inhibited colon cancer growth and metastasis through inhibition of cancer stem cell pluripotency, EMT, migration, invasion, angiogenesis and induction of apoptosis.

Citation Format: Kazim Husain, Domenico Coppola, Said M. Sebti, Mokenge P. Malafa. Vitamin E delta-tocotrienol targets human colon cancer stem cells and inhibits colon cancer metastasis and induces apoptosis. [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 3839.
Abstract 3844: A novel liposomal Clodronate depletes tumor-associated macrophages in primary and metastatic melanoma: anti-angiogenic and anti-tumor effects
Authors: Brignole Chiara , Perri Patrizia , Piaggio Francesca , Pastorino Fabio , Di Paolo Daniela , Emionite Laura , Daga Antonio , Kondylis Vangelis , Pasparakis Manolis , Ribatti Domenico , Ponzoni Mirco
Published: July 2016

The depletion of tumor-associated macrophages (TAMs), involved in different stages of cancer development and progression, is an appealing strategy in cancer therapy.

We developed novel Clodronate-containing liposomes (Clo-Lipo-DOTAP) presenting good physicochemical properties (size distribution, polidispersity index and Z-potential).

In vitro, Clo-Lipo-DOTAP inhibited proliferation, reduced viability and induced apoptosis of a macrophage-like cell line in a dose- and time-dependent manner.

In proof of functionality experiments, Clo-Lipo-DOTAP depleted macrophages in a genetic mouse model of chronic hepatitis and hepatocellular carcinoma leading to a significant reduction of F4/80-positive cells in the liver and spleen of treated mice compared to PBS-treated controls. The number of granulocytes, B and T lymphocytes was not affected.

In B16/F10 subcutaneous melanoma-bearing mice, Clo-Lipo-DOTAP significantly reduced the volume of primary tumors (P < 0.001). Within the tumors, the expression F4/80 and α-SMA was significantly lowered. Plasma levels of IL-10, Mo KC, TNF-α, VEGF and PDGF-bb were statistically decreased. In B16/F10 lung metastatic melanoma model, treatment with Clo-Lipo-DOTAP significantly reduced the number of pulmonary nodules (P < 0.05). F4/80-positive cells and microvessel density were statistically decreased.

In conclusion, the depletion of TAMs in primary and metastatic melanoma presents anti-tumor efficacy via inhibition of angiogenesis and modulation of inflammation related cytokines.

Citation Format: Chiara Brignole, Patrizia Perri, Francesca Piaggio, Fabio Pastorino, Daniela Di Paolo, Laura Emionite, Antonio Daga, Vangelis Kondylis, Manolis Pasparakis, Domenico Ribatti, Mirco Ponzoni. A novel liposomal Clodronate depletes tumor-associated macrophages in primary and metastatic melanoma: anti-angiogenic and anti-tumor effects. [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 3844.
Abstract 3846: Sensitivity of NOTCH1 mutant T-ALL to ABT-263
Authors: Dastur Anahita , Costa Carlotta , Faber Anthony , Benes Cyril
Published: July 2016

Effective targeted therapies are lacking for refractory and relapsed T-cell Acute Lymphoblastic Leukemia (T-ALL). Analysis of a large cell line screen revealed that NOTCH1 mutant T-ALL cells are sensitive to the Bcl-2/xL inhibitor ABT-263. We show that NOTCH1 inhibits the mTOR complex 1, resulting in low MCL-1 levels and sensitization to ABT-263. Further suppression of mTORC1 using small molecule inhibitors in combination with ABT-263 results in very high levels of apoptosis and tumor regressions in vivo. This suggests new therapeutic opportunities for NOTCH1 mutant T-ALL, including in the setting of gamma-secretase inhibitor (GSI) resistance.

Citation Format: Anahita Dastur, Carlotta Costa, Anthony Faber, Cyril Benes. Sensitivity of NOTCH1 mutant T-ALL to ABT-263. [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 3846.
Abstract 3860: The repurposed anthelmintic mebendazole in combination with trametinib suppresses refractory NRASQ61K melanoma
Authors: Simbulan-Rosenthal Cynthia M. , Dakshanamurthy Sivanesan , Gaur Anirudh , Chen You-Shin , Shimizu Rena , AbdusSumad Maryam , Zhou Hengbo , Zapas John , Calvert Valerie , Petricoin Emaneul F. , Atkins Michael B. , Byers Stephen W. , Rosenthal Dean S.
Published: July 2016

Structure-based drug repositioning in addition to random chemical screening is now a viable route to rapid drug development. Proteochemometric computational methods coupled with kinase assays showed that MBZ binds and inhibits kinases important in cancer, especially both BRAFWT and BRAFV600E. We find that MBZ synergizes with the MEK inhibitor trametinib to inhibit growth of dabrafenib-resistant BRAFWT-NRASQ61K melanoma cells in culture and in xenografts, and markedly decreased MEK and ERK phosphorylation in vivo. RPPA and immunoblot analyses show that both trametinib and MBZ inhibit the MAPK pathway, and cluster analysis revealed a protein cluster showing strong MBZ+trametinib - inhibited phosphorylation of MEK and ERK within 10 minutes, and its direct and indirect downstream targets related to stress response and translation, including ElK1 and RSKs within 30 minutes. Downstream ERK targets for cell cycle, including cMYC, were downregulated, consistent with S phase suppression by MBZ+trametinib, while apoptosis markers, including cleaved caspase-3, cleaved PARP and a sub-G1 population, were all increased with time. These data suggest that MBZ, an off-patent approved drug with no major side effects, should be considered as a therapeutic option in combination with trametinib, for patients with NRASQ61mut or other non-V600E BRAF mutant melanomas.

Citation Format: Cynthia M. Simbulan-Rosenthal, Sivanesan Dakshanamurthy, Anirudh Gaur, You-Shin Chen, Rena Shimizu, Maryam AbdusSumad, Hengbo Zhou, John Zapas, Valerie Calvert, Emaneul F. Petricoin, Michael B. Atkins, Stephen W. Byers, Dean S. Rosenthal. The repurposed anthelmintic mebendazole in combination with trametinib suppresses refractory NRASQ61K melanoma. [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 3860.
Abstract 3861: Ruxolitinib inhibits STAT-3 activation in glioblastoma
Authors: Fehling Samuel , McFarland Braden , Benveniste Etty
Published: July 2016

Glioblastoma (GBM), a primary Grade IV astrocytoma, is the most aggressive primary brain cancer in humans. Despite combined radiotherapy and chemotherapy, the median survival is 14 months. We and others have validated that levels of phosphorylated JAK1/2 and STAT-3 are increased in GBM tissues. When stimulated with IL-6 family members, JAK1/2 recruits and phosphorylates STAT-3. Phosphorylated STAT-3 dimerizes and translocates to the nucleus where it regulates genes involved in proliferation, angiogenesis, anti-apoptotic activity and immunosuppression. Elevated JAK/STAT activation is generated through an increase in IL-6 family cytokines as well as a decrease in JAK/STAT-3 negative regulators. To therapeutically inhibit the JAK/STAT-3 pathway in GBM we employed ruxolitinib, an FDA approved JAK1/JAK2 inhibitor. Human GBM cell lines U87-MG and U251-MG, as well as patient derived GBM xenograft lines JX6 and JX12, were treated with ruxolitinib in doses ranging from 0.01 to 10 μM. Ruxolitinib was observed to inhibit STAT-3 activation in a dose-dependent manner. Pre-treatment of U87-MG and U251-MG cells with ruxolitinib inhibited stimulus-induced STAT-3 activation and downstream gene expression in a dose-dependent manner. Future studies will examine ruxolitinib effects on migration, invasion, apoptosis and proliferation in vitro. Collectively, these data indicate the potential for ruxolitinib therapy for GBM patients.

Citation Format: Samuel Fehling, Braden McFarland, Etty Benveniste. Ruxolitinib inhibits STAT-3 activation in glioblastoma. [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 3861.
Abstract 3863: Cancer immunotherapy: Could TK1 be used as a target for colon cancer
Authors: Townsend Michelle H. , Weagel Evita G. , Meng Wei , Chandler Craig , Robison Richard A. , O’Neill. Kim L.
Published: July 2016

The aim of this study is to determine whether Thymidine Kinase 1 (TK1) would be a suitable target for future immunotherapeutic treatment of colon cancer. The salvage pathway enzyme TK1 has been shown to be upregulated in cancer patients due to active proliferation of cancer cells and the resulting high demand for nucleotides. Present in both the cytosol and the serum of cancer patients, TK1 levels can be used to not only predict cancer occurrence, but also to predict tumor aggressiveness and future remission. While serum levels of TK1 have been established, surface expression of the enzyme have not been fully characterized. In colon cancer cells, TK1 has been shown to strongly associate with the plasma membrane. In this project we explored the presence of TK1 on the surface of colon cancer cells (HT29 and SW620 cell lines) using flow cytometry, confocal microscopy, scanning electron microscopy, membrane separation, and cytoplasmic staining. Using fluorescent antibodies conjugated to FITC, expression levels as high as 19% are observed in HT29's and 12% in SW620's in comparison to sodium potassium ATPase surface levels of 11% and 6% respectively. Confocal microscopy images revealed direct overlap between cells stained with a rhodamine red membrane dye and with a FITC conjugated anti-TK1 antibody which indicates a direct relationship between TK1 and the cellular surface of cancer cells. To further confirm surface expression, TK1 levels were assessed using gold labeling and scanning electron microscopy. Gold levels indicate the physical location of the TK1 on the surface of the colon cancer cells and their relative abundance. TK1 surface levels in colon cancer cells corresponded with the surface expression levels of sodium potassium ATPase and share relatively the same abundance. These results strongly indicate a direct relationship between TK1 and the surface of colon cancer cells, and suggests TK1 as a potential target for future immunotherapeutic treatment.

Citation Format: Michelle H. Townsend, Evita G. Weagel, Wei Meng, Craig Chandler, Richard A. Robison, Kim L. O’Neill. Cancer immunotherapy: Could TK1 be used as a target for colon 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 3863.
Abstract 3868: Aggressiveness niche within BRCA1-IRIS/TNBC tumors is supported by reciprocal interactions with the microenvironment
Authors: Sinha Abhilasha , ElShamy Wael M.
Published: July 2016

Introduction: Bi-directional interactions with stromal cells promote metastasis from breast tumors. We aimed at determining the role these bi-directional interactions between BRCA1-IRIS-overexpression (IRISOE) tumor cells and stromal cells in generating aggressive triple negative breast cancers.

Experimental Procedures: We used co-culturing assay, immunohistochemistry, in vitro invasion assay with or without cytokines neutralization, ELISA, western blot analysis, exposure to hypoxic conditions, cytokines receptor inactivation, in vivo orthotopic tumor formation assay, analysis of publically available data sets for the association of selected factors with overall survival, distant metastasis free survival and recurrent free survival to define the role of these bi-directional interactions in enhancing IRISOE tumors aggressiveness.

Results: Here we show formation of an aggressiveness niche within IRIS overexpressing triple negative breast cancers (IRISOE/TNBCs) defined as the necrotic/hypoxic core. To this niche, metabolically stressed IRISOE/TNBC cells attract mesenchymal stem cells (MSCs) to the aggressiveness niche through secreting high levels of IL-1β. IL-1R-negative MSCs are induced to express the receptor upon exposure to medium conditioned by IRISOE/TNBCs. Activated by IL-1β, IL-1R activates AKT, MAPK and NF-κB signaling in MSCs to which they respond by releasing inflammatory cytokines, such as CXCL1. Inhibiting IL-1β signaling utilizing neutralizing antibody attenuates MSCs recruitment and CXCL1 secretion, in vitro and in vivo. CXCL1 receptor; CXCR2 is expressed on IRISOE/TNBCs only and thus CXCL1 acts in a paracrine fashion to entrain IRISOE/TNBCs. Mechanistically, MSCs-entrained IRISOE/TNBCs secrete CCL2 and VEGF that activate and attract tumor associated macrophages (TAMs) and endothelial cells (ECs), respectively to the vicinity of IRISOE/TNBCs in the aggressiveness niche. S100A8 and IL-8 secreted from IRISOE/TNBCs-activated TAMs and ECs, respectively in concert with CXCL1 enhance IRISOE/TNBCs aggressiveness. In pre-clinical model, IRISOE cells co-injected with MSCs (10:1 ratio) developed faster and bigger orthotopic mammary tumors containing more TAMs and ECs compared to IRISOE/TNBCs injected alone. These tumors progressed to develop spontaneous lymph-node or distant metastases (e.g., bone, lung and/or brain).

Conclusion: We propose that in concert with an anti-BRCA1-IRIS drug, inhibitors to the reciprocal interactions between IRISOE/TNBCs and their surrounding stromal cells could be exploited as a viable therapy for IRISOE/TNBCs and their metastases.

Citation Format: Abhilasha Sinha, Wael M. ElShamy. Aggressiveness niche within BRCA1-IRIS/TNBC tumors is supported by reciprocal interactions with the microenvironment. [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 3868.
Abstract 3872: Quantification of mutant SPOP proteins in prostate cancer using targeted proteomics
Authors: Wang Hui , Barbieri Christopher , He Jintang , Gao Yuqian , Wu Chaochao , Schepmoes Athena , Fillmore Thomas , Shi Tujin , Chae Sung-Suk , Huang Dennis , Mosquera Juan Miguel , Qian Wei-Jun , Smith Richard , Srivastava Sudhir , Kagan Jacob , Camp David , Rodland Karin , Rubin Mark , Liu Tao
Published: July 2016

Speckle-type POZ protein (SPOP) is an E3 ubiquitin ligase adaptor protein that functions as a potential tumor suppressor, and SPOP mutations have been identified in ∼10% of human prostate cancers. However, it remains unclear if mutant SPOP proteins can be utilized as biomarkers for diagnosis, prognosis or targeted therapy of prostate cancer. To address this issue, selected reaction monitoring (SRM) and PRISM (high-pressure, high-resolution separations coupled with intelligent selection and multiplexing) -SRM mass spectrometry assays have been developed for quantifying mutant SPOP proteins.

SRM assays for wild-type SPOP protein and 11 prostate cancer-derived mutations were developed. The presence of multiple lysine residues in the mutation regions precludes the use of conventional tryptic digestion. Arg-C was selected instead due to its superior performance in generating mutation site(s) containing SPOP peptides that are more suitable for SRM analysis comparing to other proteases (e.g., Asp-N). Although the resulting Arg-C peptides are longer and more hydrophobic than typical tryptic peptides, all the SRM assays showed a linear dynamic range of more than two orders of magnitude. The limits of quantification for the mutation site(s) containing peptides range from 10 to 100 fmol/μg of total protein in the cell lysate. Applying these SRM assays to analyze 293T cells with and without expression of the three most frequent SPOP mutations in prostate cancer (Y87N, F102C or F133V) led to confident detection of all three SPOP mutations in corresponding positive cell lines but not in the negative cell lines. Expression of the F133V mutation and wild-type SPOP was at much lower levels compared to that of F102C and Y87N mutations, which agrees with RT-PCR results. It is unknown if this is related to activity of the SPOP protein. PRISM-SRM assays have shown further improvement in sensitivity.

SRM enables multiplexed, isoform-specific detection of mutant SPOP proteins in cell lysates, which holds great potential in biomarker development for prostate cancer.

Citation Format: Hui Wang, Christopher Barbieri, Jintang He, Yuqian Gao, Chaochao Wu, Athena Schepmoes, Thomas Fillmore, Tujin Shi, Sung-Suk Chae, Dennis Huang, Juan Miguel Mosquera, Wei-Jun Qian, Richard Smith, Sudhir Srivastava, Jacob Kagan, David Camp, Karin Rodland, Mark Rubin, Tao Liu. Quantification of mutant SPOP proteins in prostate cancer using targeted proteomics. [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 3872.
Abstract 3878: Proteomic approach to identify biomarkers for invasive cervix cancer: a prospective pilot study
Authors: Mysona David P. , Pyrzak Adam , Allen Jennifer , Sharma Ashok , Weinberger Paul , Rungruang Bunja , She Jin-Xiong , Ghamande Sharad
Published: July 2016

Objectives: Cervical cancer is the second most common cancer of women worldwide. In this prospective study, we attempted to identify a set of serum biomarkers, which could identify patients who have invasive cervical cancer and possibly help monitor disease status (recurrence, remission).

Methods: Luminex bead array was used to measure serum protein concentrations of 19 different, promising proteins in cervical cancer patients (n = 23) compared to women with precancerous lesions (CIN2-3) (n = 20), and patients with normal cervical cytology (n = 20) in this IRB approved prospective study. Cervical cancer (adenocarcinoma and squamous carcinoma) patients had blood samples drawn within 30 days of diagnosis and repeat blood samples taken on follow up visits after completing therapy. These results were correlated to clinical data and outcomes. Protein expression was confirmed by immunohistochemistry in patient samples.

Results: CIN and controls have very similar levels of serum proteins. Six proteins (CA153, OPG, CEA, IGFBP7, IGFBP4, and MMP-7) had significantly different levels in cervical cancer patients compared to controls and CIN (p < 0.05) with Area Under the Curve values of 0.74, 0.77, 0.79, 0.83, 0.84, and 0.98 respectively. IGFBP4 had a sensitivity of 75% at 95% specificity for correctly identifying invasive disease. MMP-7 had a sensitivity of 92% at 95% specificity. MMP-7 was confirmed by IHC in patient specific cervical cancer tissue. ProteinAUCP valueSensitivity at 95% SpecificityMMP70.98 (0.96-0.99)5.29E-1392.11IGFBP40.84 (0.8-0.89)2.36E-0775IGFBP70.83 (0.78-0.87)6.23E-0757.89CEA0.79 (0.74-0.84)1.30E-0542.11OPG0.77 (0.72-0.82)3.80E-0535.14CA1530.74 (0.69-0.8)2.50E-047.89

Conclusions: Several serum proteins are significantly altered in cervical cancer patients compared to controls and CIN, and are worth studying for further clinical utility. MMP-7 may prove to be an important mediator of cervical cancer.

Citation Format: David P. Mysona, Adam Pyrzak, Jennifer Allen, Ashok Sharma, Paul Weinberger, Bunja Rungruang, Jin-Xiong She, Sharad Ghamande. Proteomic approach to identify biomarkers for invasive cervix cancer: a prospective pilot study. [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 3878.