bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4821: The WEE1 inhibitor AZD-1775 has synergic activity with trabectedin or lurbinectedin in ovarian cancer cells
Authors: Uboldi Sarah , Carrassa Laura , Frapolli Roberta , Ubezio Paolo , Erba Eugenio , D’Incalci Maurizio
Published: July 2016

AZD-1775 is a small molecule that selectively inhibits the tyrosine kinase WEE1, with a reported antineoplastic activity in ovarian cancer. WEE1 is a tyrosine kinase key regulator of DNA damage surveillance pathways that controls G2/M transition and ensures faithful DNA replication. WEE1 is required during normal S phase to avoid deleterious DNA breakage and prevent loss of genome integrity in the absence of exogenous DNA damage.

Trabectedin is a drug approved in 2007 by EMA and 2015 by FDA for the treatment of soft tissue sarcomas and in 2009 by EMA for the treatment of ovarian cancer patients, who have relapsed after 6 months from Platinum-based therapy, in combination with pegylated liposomal doxorubicin. Lurbinectedin (PM01183) is a trabectedin analog with a similar mechanism of action and, according to ongoing clinical investigations, less toxic and thus suitable for combinations of potential clinical interest. Since both trabectedin and lurbinectedin cause a G2/M block and AZD-1775 is able to inhibit the WEE1 activity with consequent disruption of G2/M checkpoint, we hypothesized that the combined treatment with AZD-1775 could be synergistic in ovarian cancer cells.

The sensitivity of the ovarian cancer cell lines OVCAR-5, A2780 and IGROV-1 to trabectedin, lurbinectedin and AZD-1775 alone or in combination (72h of continous treatment) was evaluated by a cell proliferation assay. The cytotoxic effect induced by the simultaneous administration of trabectedin with AZD-1775 or lurbinectedin with AZD-1775 was evaluated by a combination index analysis.

The results obtained showed that the combination trabectedin with AZD-1775 or lurbinectedin with AZD-1775 is synergic in all the ovarian cancer cell lines analyzed (Combination Index <1). The combination lurbinectedin with AZD-1775 seems to be better than trabectedin with AZD-1775, in OVCAR5 cell line. The analysis of cell cycle and apoptosis are in progress in order to investigate the mechanism of the observed synergism. These positive results prompted us to study these combinations in vivo, in ovarian cancer xenografts derived from the cell lines used for the in vitro studies.

If the synergism observed in vitro will be confirmed in vivo, there will be a strong rationale to test these combinations in ovarian cancer patients.

Citation Format: Sarah Uboldi, Laura Carrassa, Roberta Frapolli, Paolo Ubezio, Eugenio Erba, Maurizio D’Incalci. The WEE1 inhibitor AZD-1775 has synergic activity with trabectedin or lurbinectedin in ovarian cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4821.
Abstract 4824: Synergistic interaction of PIM kinase inhibitor, JP_11646, and cytotoxic or targeted therapies in acute leukemia models
Authors: Pundt Krista , Baldino Camern , Caserta Justin , Adjei Alex , Lee Kelvin , Zhu Xu , Verone-Boyle Alissa , Engler Frank , Pitzonka Laura , Fetterly Gerald
Published: July 2016

Standard therapy for acute myeloid leukemia (AML) results in significant mortality and morbidity with an overall cure rate of only 25%. Implementation of novel therapeutic options is needed in order to significantly improve patient survival and outcome. PIM serine/threonine kinases are overexpressed in hematological malignancies and regulate cell cycle, survival and drug resistance pathways—thus providing viable therapeutic targets. We hypothesized that targeted or cytotoxic therapy will enhance anti-tumor activity in combination with JP-11646, a novel PIM2 inhibitor, in preclinical AML models.

Preclinical data has shown JP-11646 exerted potent inhibition of AML cells in vitro and in vivo in subcutaneous AML xenograft models. An in vitro MTT assay was used to determine the overall anti-proliferative effects of single and combination therapies with respect to the downstream effects of PIM kinase and mechanism of action of JP-11646 in leukemia. First, the single agent efficacy in human AML (HEL, and MV(4;11)) cell lines were examined after 72 hours of exposure to JP-11646, rapamycin, daunorubicin and cytarabine. Overall potency, IC50, (0.17 μM (0.15-0.19), 0.003 μM (0.002-0.004), 0.067 μM(0.065-0.0699) and 0.31 μM (0.294-0.345), respectively) and maximal inhibition, Imax, of the various therapies was determined. Potential synergistic effects were first tested by treating AML cell lines with JP-11646 and daunorubicin, cytarabine, or rapamycin (an inhibitor of a pathway that regulates downstream molecules of the PIM kinase pathway). To quantify the synergy or interaction of JP-11646 in combination, a simple inhibitory non-competitive interaction model with an interaction parameter (Ø) as well as a three dimensional interaction surface was employed (Ø<1 synergism, Ø>1 antagonism). The intensity of the interaction (Ø) of JP-11646 with rapamycin (0.819 (0.395-0.543)), daunorubicin (0.469 (0.665-0.975)) or cytarabine (0.173 (0.118-0.229)) demonstrated synergistic effects across all combinations.

Here, we demonstrate that novel drug targets and drugs for AML treatment alone, and in combination with pre-existing therapies, may lead to improved strategies for AML treatment and these findings support future development of JP-11646 in novel combinatorial regimens for AML therapy.

Citation Format: Krista Pundt, Camern Baldino, Justin Caserta, Alex Adjei, Kelvin Lee, Xu Zhu, Alissa Verone-Boyle, Frank Engler, Laura Pitzonka, Gerald Fetterly. Synergistic interaction of PIM kinase inhibitor, JP_11646, and cytotoxic or targeted therapies in acute leukemia models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4824.
Abstract 4834: Preclinical analysis of the Notch gamma secretase inhibitor BMS-906024 in combination with chemotherapy in the treatment of lung adenocarcinoma
Authors: Morgan Katherine M. , Lee Francis , Michaud Erin , Fischer Bruce S. , Pine Sharon R.
Published: July 2016

Notch signaling is aberrantly activated in approximately one third of non-small cell lung cancer (NSCLC) cases, primarily through loss of the endogenous Notch inhibitor, Numb, or via gain-of-function mutations in the Notch1 receptor. Notch activity is associated with poor overall survival among NSCLC patients whose tumors are wildtype for TP53. Here, we characterized the interaction between BMS-906024, a clinically relevant gamma secretase inhibitor (GSI) that inhibits Notch activation, and front-line chemotherapy in preclinical models of NSCLC. MTS drug synergy assays consisting of treatment with BMS-906024, cisplatin or paclitaxel, or the combination of GSI and chemotherapy were performed on a panel of human NSCLC cell lines, most of which were derived from adenocarcinomas. Analysis of the drug effects with CalcuSyn yielded significantly lower CI values for the GSI BMS-906024 combined with paclitaxel than with cisplatin (average CI = 0.54 vs 0.85, respectively; P = 0.001). The synergy between BMS-906024 and paclitaxel was significantly greater in Kras-wildtype than Kras-mutant cells (average CI = 0.39 vs 0.68, respectively; P = 0.009), while there was no correlation with EGFR or TP53 status. Treatment of lung adenocarcinoma xenografts in NOD scid gamma mice confirmed enhanced antitumor activity for the combination treatment of BMS-906024 and paclitaxel by mechanisms currently under investigation. These results are a step toward identification of the optimal combination of the GSI BMS-906024 with standard chemotherapies, as well as potential biomarkers that could be used to predict patient response to Notch-targeted therapy.

Citation Format: Katherine M. Morgan, Francis Lee, Erin Michaud, Bruce S. Fischer, Sharon R. Pine. Preclinical analysis of the Notch gamma secretase inhibitor BMS-906024 in combination with chemotherapy in the treatment of lung adenocarcinoma. [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 4834.
Abstract 4853: BMVC specifically binds the major G-quadruplex structure formed in the c-MYC promoter to lower c-MYC levels
Authors: Liu Wenting , Wu Guanhui , Lin Clement , Onel Buket , Chen Ding , Chang Ta-Chau , Yang Danzhou
Published: July 2016

The c-MYC proto-oncogene is one of the most deregulated genes in human cancers. Transcriptional repression of c-MYC is considered as one of the attractive strategies in targeting c-MYC. Previous studies revealed that the c-MYC promoter nuclease hypersensitive element (NHE) III1 region, which regulates 80-95% of total c-MYC transcription, can form DNA G-quadruplex (G4) and that stabilization of the c-MYC G4 could repress c-MYC gene expression. We have previously determined the molecular structure of the major G-quadruplex formed in the c-MYC promoter NHE (MycG4), which is an intramolecular parallel-stranded structure. BMVC, a carbazole derivative, was designed as a fluorescent probe for recognizing telomeric G4. However, we found that BMVC binds to MycG4 with much greater affinity and specificity. Cellular studies showed that BMVC was enriched in the nucleus at 48 hr after treatment, leading to markedly reduced c-MYC expression levels. Promoter-driven luciferase assays revealed that BMVC could enhance the inhibitory effects of MycG4, suggesting BMVC can repress c-MYC expression in vivo through stabilization of MycG4. To understand the molecular interactions between BMVC and MycG4, we have determined the molecular structure of the complexes of BMVC with MycG4 by NMR. BMVC appears to bind the 5’-end of the MycG4 with very high affinity, as shown by the slow-exchange binding on the NMR timescale. The structure of the 5’-end complex of BMVC and MycG4 shows that BMVC is paired to the flanking adenine with specific hydrogen bonding interactions, while the BMVC-adenine plane stacks nicely on the 5’-end G-tetrad of MycG4. Compared to the previous MycG4 complex with a quindoline compound, the tighter and more specific binding of BMVC appears to be imparted by the novel arched shape of BMVC and specific pairing recognition of BMVC with the flanking bases of MycG4. BMVC can also bind to the 3’-end of MycG4 with lower affinity to form a second complex. However, the 3’-end complex shows much more dynamic conformation and less specific interactions. Our results indicated that the cellular targets of BMVC include not only telomeric G4, but also the c-MYC promoter G4. Because of the inherent fluorescence and novel binding mode of BMVC, our study provides useful information for future design of improved carbazole-based anticancer agents or specific cellular fluorescent probes, as well as insights into specific recognition of MycG4 by small molecules.

Citation Format: Wenting Liu, Guanhui Wu, Clement Lin, Buket Onel, Ding Chen, Ta-Chau Chang, Danzhou Yang. BMVC specifically binds the major G-quadruplex structure formed in the c-MYC promoter to lower c-MYC levels. [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 4853.
Abstract 4854: Bazedoxifene inhibits ESR1 somatic mutants with improved potency compared to tamoxifene and raloxifene
Authors: Fanning Sean W. , Dharmarajan Venkat , Mayne Christopher G. , Toy Weiyi , Carlson Kathryn E. , Martin Teresa A. , Nowak Jason , Nwachukwu Jerome , Hosfield David J. , Tajkhorshid Emad , Chandarlapaty Sarat , Griffin Patrick , Shen Yang , Katzenellenbogen John A. , Greene Geoffrey L.
Published: July 2016

Despite continued administration of antiestrogen therapies, approximately 50% of all estrogen receptor alpha (ERalpha) positive breast cancers will present new metastatic lesions. The acquisition of secondary hormone-resistant metastatic breast cancers represents a significant clinical barrier towards life-long disease free survival for the patient. Somatic mutations to the ERalpha gene (ESR1) Y537S and D538G represent a novel mechanism of acquired antiestrogen resistance because they confer hormone-free transcriptional activity and reduced selective estrogen receptor modulator (SERM) and selective estrogen receptor degrader (SERD) potency. Fulvestrant, a SERD, was the only molecule that could completely ablate mutant ERalpha activity. Unfortunately, fulvestrant possesses poor pharmacologic profiles that limit its therapeutic utility. Bazedoxifene (BZA) is a potent mixed SERM/SERD and has improved pharmacokinetics and oral bioavailability compared to fulvestrant. We show that BZA inhibits Y537S and D538G ESR1 somatic mutation transcriptional activity with a greater potency than the SERMs 4-hydroxytamoxifen (TOT) and raloxifene (RAL). Further investigations into the biophysical and structural basis for BZA action suggest that BZA increases the conformational dynamics of helix 12, a key molecular switch that governs ERalpha action resulting in SERD-like properties and improved potency against the somatic mutations compared to TOT and RAL.

Citation Format: Sean W. Fanning, Venkat Dharmarajan, Christopher G. Mayne, Weiyi Toy, Kathryn E. Carlson, Teresa A. Martin, Jason Nowak, Jerome Nwachukwu, David J. Hosfield, Emad Tajkhorshid, Sarat Chandarlapaty, Patrick Griffin, Yang Shen, John A. Katzenellenbogen, Geoffrey L. Greene. Bazedoxifene inhibits ESR1 somatic mutants with improved potency compared to tamoxifene and raloxifene. [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 4854.
Abstract 4857: Design and biological evaluation of substituted pyrrolo[3,2-d]pyrimidines as dual acting RTK and microtubule targeting agents
Authors: Gangjee Aleem , Shah Khushbu , Dybdal-Hargreaves Nicholas , Mooberry Susan , Bastian Anja , Ihnat Michael
Published: July 2016

Combination chemotherapy with antiangiogenic (cytostatic) and microtubule targeting (cytotoxic) agents (MTA) have shown significant promise and several studies with such combinations have progressed to the clinic. Single agents with both antiangiogenic activities as well as cytotoxicity would circumvent pharmacokinetic problems of multiple agents, might be effective at lower doses to alleviate toxicity, and delay or prevent tumor cell resistance. We have designed, synthesized and evaluated single agents that are novel pyrrolo[3,2-d]pyrimidine MTAs that also inhibit vascular endothelial growth factor receptor-2 (VEGFR-2), epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor â (PDGFRâ). Sixteen pyrrolo[3,2-d]pyrimidine analogs were synthesized with variations on the C-2 and the 4-anilino position. The importance of various substituents was evaluated based on their abilities to cause cellular microtubule depolymerization and inhibit proliferation of MDA-MB-435 cancer cells. Compounds with N5-Me were more potent than the N5-H analogs indicating that a conformational restriction induced by N5-Me could mimic the bioactive conformation. In addition, all C2-Cl compounds afforded 2 to 10-fold more potentIC50 values than their C2-Me counterparts as illustrated by C2-Cl analog AG336 (IC50 = 18.3 ± 5.0 nM) compared to its C2-Me analog AG85 (IC50 = 193 ± 39 nM). The most active antitubulin agents were evaluated against RTKs and demonstrated good inhibition for EGFR (IC50 = 29.5 - 91.5 nM), VEGFR-2 (182.3 - 543.6 nM) and PDGFRâ (IC50 = 204.4 - 450.2 nM), thus demonstrating dual-targeting ability. Preclinical assessment of AG165 at 75 mg/kg in mice in the NCI/ADR resistant tumor model demonstrated significant reduction of tumor volume compared to paclitaxel (10 mg/kg) treated mice [p = 0.0333], indicating antitumor activity in a multidrug resistant tumor model, in addition to excellent antitumor activity in MDA-MB-435 and MDA-MB-231 xenograft models. These results demonstrate the viability of these multitargeted single agents as potential clinically active antitumor agents.

Citation Format: Aleem Gangjee, Khushbu Shah, Nicholas Dybdal-Hargreaves, Susan Mooberry, Anja Bastian, Michael Ihnat. Design and biological evaluation of substituted pyrrolo[3,2-d]pyrimidines as dual acting RTK and microtubule targeting agents. [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 4857.
Abstract 4865: Metformin improves multiple cytokine producing ability of exhausted peripheral CD8+ T cells of cancer patients
Authors: Watanabe Mototsugu , Eikawa Shingo , Uehara Takenori , Kunisada Yuki , Toyooka Shinichi , Miyoshi Shinichiro , Udono Heiichiro
Published: July 2016

Background: We have recently shown that metformin, a prescribed drug for type 2 diabetes, can increase the number of CD8+ tumor infiltrating lymphocytes (CD8+ TILs) that can produce triple cytokines, thus, IFN-ã, IL-2 and TNF-á in mouse models. Increased numbers of such multifunctional CD8+ TILs have well correlated with tumor retardation, suggesting an underlying mechanism of metformin in tumor immunity. In the current study, we examined the effects of direct exposure of metformin in vitro to CD8+ T cells of cancer patients.

Methods: Peripheral blood mononuclear cells (PBMC) from cancer patients and healthy volunteers were obtained for the analysis. We cultured the PBMC with or without metformin overnight, followed by stimulation with phorbol 12-myristate 13-acetate (PMA) / ionomycin in vitro in various glucose concentrations for 6 hours. Expressions of markers for immune-check points (PD-1 and Tim-3) were examined together with accumulated intracellular cytokines, IFN-ã, IL-2 and TNF-á by flow cytometry analysis.

Results: PD-1 and Tim-3 expressions of cancer patients’ PBMC were apparently higher than those of healthy volunteers’. Increased production of multiple cytokines was observed in CD8+ T cells of almost all cancer patients by metformin pretreatment, whereas no changes were detected in the samples from healthy volunteers. Moreover, deprivation of glucose clearly limited the production of cytokines on CD8 + T cells in a cancer patient. These results suggested patient’ CD8 + T cells showed lower activity of glycolysis compared with a healthy volunteer and metformin might stimulate glycolysis followed by reversion of mitochondrial respiration in exhausted CD8 + T cells.

Conclusion: Metformin treatment might be useful in reversion of exhausted CD8+T cells in cancer bearing patients.

Citation Format: Mototsugu Watanabe, Shingo Eikawa, Takenori Uehara, Yuki Kunisada, Shinichi Toyooka, Shinichiro Miyoshi, Heiichiro Udono. Metformin improves multiple cytokine producing ability of exhausted peripheral CD8+ T cells of cancer patients. [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 4865.
Abstract 4893: Molecular detection of ALL in the peripheral blood is more sensitive than flow cytometric analysis of the bone marrow in patients with treatment-related hypocellularity
Authors: Smithers Hannah , Finney Olivia , Kirsch Ilan , Crossley Beryl , Jensen Michael , Gardner Rebecca
Published: July 2016

Measurable residual disease detection (MRD) is an important tool for early detection and monitoring relapse. Traditionally, bone marrow is collected and analyzed by flow cytometry (FC) to evaluate MRD status in acute lymphoblastic leukemia patients (ALL). More recently, highly sensitive molecular methods such as deep sequencing allow for higher sensitivity detection of MRD. Although the bone marrow is considered to be more representative of tumor burden than sampling the blood, bone marrow collection is more invasive, disruptive, and resource intensive than peripheral blood draws. ALL patients enrolled in the Seattle Children's Pediatric Leukemia Adoptive Therapy (PLAT-02) trial undergo MRD assessment pre and post CD19 CAR T cell infusion using FC analysis of a bone marrow sample. We have used high throughput sequencing (HTS) in paired blood and bone marrow samples from these patients to evaluate the complete B cell repertoire and assess MRD up to 63 days post-infusion. To-date, we have obtained 38 paired samples. Using HTS alone we have observed a significant correlation in both the frequency of total reads and the number of malignant cells per million in paired peripheral blood and bone marrow samples. 26/38 of the bone marrow samples were determined either aparticulate or hypocellular. Importantly, 8/14 of patients that were MRD negative in the bone marrow by FC were MRD positive in the blood by HTS. These data suggest, that, in cases where the bone marrow is hypocellular or aparticulate, peripheral blood samples evaluated by HTS are more sensitive than traditional flow cytometric analysis in the bone marrow for detection of MRD. The impact of B cell aplasia may be important in increasing the sensitivity of HTS in the detection of MRD. The ability to detect an ALL B cell clone in the blood through HTS has many clinical implications; blood can be sampled more often, is less of a burden to patients, and can provide earlier prediction of relapse.

Citation Format: Hannah Smithers, Olivia Finney, Ilan Kirsch, Beryl Crossley, Michael Jensen, Rebecca Gardner. Molecular detection of ALL in the peripheral blood is more sensitive than flow cytometric analysis of the bone marrow in patients with treatment-related hypocellularity. [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 4893.
Abstract 4896: A novel method for evaluating antibody-dependent cell-mediated cytotoxicity by flow cytometry using cryopreserved human peripheral blood mononuclear cells
Authors: Kitano Shigehisa , Yamashita Makiko , Aikawa Hiroaki , Kuchiba Aya , Hayashi Mitsuhiro , Yamamoto Noboru , Tamura Kenji , Hamada Akinobu
Published: July 2016

For predicting the clinical efficacy of antibody-dependent cellular cytotoxicity (ADCC) -dependent antibody therapy, analyzing the cytotoxic functions of effector cells such as NK cells against target cancer cells is thought to be necessary. The 51Cr release assay has long been the most widely used method for quantification of ADCC activity. However, the reproducibilities of these release assays are not adequate, and they do not allow evaluation of the lysis susceptibilities of distinct cell types within the target cell population. In this study, we established a novel method for evaluating cytotoxicity, which involves the detection and quantification of dead target cells using flowcytometry. CFSE (carboxyfluorescein succinimidyl ester) was used as a dye to specifically stain and thereby label the target cell population, allowing living and dead cells, as well as also target and effector cells, to be quantitatively distinguished. Furthermore, with our new approach, ADCC activity was more reproducibly, sensitively, and specifically detectable in not only freshly isolated but also in frozen human peripheral blood mononuclear cells (PBMCs) compared to the calcein-AM release assay. This assay, validated herein, is expected to be a standard assay to evaluate ADCC activity and finally contribute clinical development of ADCC dependent-antibody therapies.  

Citation Format: Shigehisa Kitano, Makiko Yamashita, Hiroaki Aikawa, Aya Kuchiba, Mitsuhiro Hayashi, Noboru Yamamoto, Kenji Tamura, Akinobu Hamada. A novel method for evaluating antibody-dependent cell-mediated cytotoxicity by flow cytometry using cryopreserved human peripheral blood mononuclear cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4896.
Abstract 4909: The oncolytic peptide LTX-315 enhances tumor-specific immune responses and tumor regression in murine 4T1 breast cancer when combined with doxorubicin
Authors: Wang Meng Yu , Kristian Alexander , Camilio Ketil , Sveinbjornsson Baldur , Mælandsmo Gunhild Mari , Kvalheim Gunnar , Rekdal Øystein
Published: July 2016

The oncolytic effect of LTX-315 involves perturbation of the plasma membrane and distortion of the mitochondrial membrane with subsequent release of DAMPs (Damage-Associated Molecular Pattern molecules) such as ATP, cytochrome C and HMGB1 (1,2). Also multidomain proteins from the BCL-2 family seem to be partially involved in LTX-315 mediated killing (3). In addition, LTX-315 induces the hallmarks of immunogenic cell death (4). Preclinical studies have demonstrated that LTX-315 is able to induce complete tumour regression after intra-tumoral treatment (5,6). In this study the effect of combining intratumoral administration of LTX-315 and systemic treatment with Doxorubicin was investigated in the murine 4T1 breast cancer model. The combination treatment was investigated both in s.c. tumors and in tumors established in the mammary fat pad. The 4T1 cell were labeled with mcherry reporter gene in order to assess tumor growth by whole body imaging. LTX-315 (1 mg in 50 ìl) was injected intratumorally for three consecutive days. Doxorubicin (8mg/kg) was injected into the tail vein as a single dose together with the first injection of LTX-315. Tumor growth was assessed by caliper measurements and whole body luminescence. The combination of local administration of LTX-315 and systemic Doxorubicin, demonstrated significant synergy in both s.c. tumors and in tumors growing in the mammary fat pad. Furthermore, the combination induced a systemic tumour specific immune response since tumors did not develop in cured animals when re-challenged with tumor cells. The tolerable safety profile and novel mode of action of LTX-315 indicate a rationale for combining LTX-315 with other immunotherapies, targeted agents, and chemotherapy.

References:

1) Forveille et al., Cell Cycle (2015)

2) Eike et al., Oncotarget (2015)

3) Zhou et al., Oncotarget (2015)

4) Zhou et al. Cell Death & Differentiation (In rev)

5) Camilio et al., Cancer Immunol Immunother (2014)

6) Camilio et al., Oncoimmunology (2014)

Citation Format: Meng Yu Wang, Alexander Kristian, Ketil Camilio, Baldur Sveinbjornsson, Gunhild Mari Mælandsmo, Gunnar Kvalheim, Øystein Rekdal. The oncolytic peptide LTX-315 enhances tumor-specific immune responses and tumor regression in murine 4T1 breast cancer when combined with doxorubicin. [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 4909.
Abstract 4923: Expression of class III beta-tubulin (TUBB3) in 3580 colorectal cancers (CRCs) and correlation with clinico-pathological and molecular features
Authors: Xiu Joanne , Reddy Sandeep , El-Deiry Wafik S.
Published: July 2016

Class III beta-tubulin plays a crucial role in intracellular transport, meiosis and mitosis. High expression of TUBB3 has been shown to associate with poor prognosis and taxane resistance in various cancer types. CRC is known to be generally resistant to taxane therapy. We investigated expression of TUBB3 in 3580 CRCs and made correlations with clinicopathological and molecular parameters. 3580 CRC samples were evaluated by tumor profiling (Caris Life Sciences, Phoenix, AZ). Tests included Sanger or next generation sequencing (NGS), protein expression by immunohistochemistry (IHC) and gene amplification by in situ hybridization (ISH). TUBB3 expression was evaluated by immunohistochemistry (Ab: POLY, Covance) and expression higher than 2+, 30% was scored as positive. TUBB3 positive expression was observed in 37% (1320/3580) of the complete CRC cohort, specifically 27% in mucinous histology (164/617) and 17% in signet ring histology (29/171). While the expression was not associated with average patient age (59 years old) or gender (53% vs. 50% male), TUBB3 was more frequently expressed in tumors that originated from the left colon (370/1016 or 36%) than from the right colon (235/790 or 30%, p = 0.003). In the 1847 tumors taken from metastatic sites, 40% (746) overexpressed TUBB3 while in 1629 CRCs taken from primary tumors, only 34% (547) overexpressed TUBB3 (p<0.0001). Interestingly, in tumors that overexpressed TUBB3, 65% also overexpressed cMET (828/1275) and 33% also overexpressed TLE3 (431/1299). This compares to 50% cMET expression (1096/2207, p<0.0001) and 23% TLE3 expression (517/2225, p<0.0001) in tumors that were negative for TUBB3. Microsatellite instability detected by fragment analysis was more prevalent in the TUBB3-negative cohort than the TUBB3-positive cohort (7.4% or 77/1046 vs. 2.9% or 16/558, p = 0.0002). Mutations in APC (62% vs. 56%, p = 0.0003) and KRAS (55% vs. 46%, p<0.0001) were more frequent in TUBB3-positive tumors, while GNAS (2% vs. 5%, p<0.0001) and SMAD4 (10.1% vs. 14.6%, p = 0.0005) mutations were significantly more frequent in tumors that were negative for TUBB3 expression. Thus, high expression of TUBB3 was found in 37% of CRCs, and was significantly associated with tumors that originated from the left colon and with tumors taken from metastatic sites. Distinct biomarker features detected by IHC and sequencing suggest that TUBB3 expression may carry theranostic importance which warrants further investigation in clinical trials.

Citation Format: Joanne Xiu, Sandeep Reddy, Wafik S. El-Deiry. Expression of class III beta-tubulin (TUBB3) in 3580 colorectal cancers (CRCs) and correlation with clinico-pathological and molecular features. [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 4923.
Abstract 4932: Stratification markers for the risk of recurrence after curative resection of stage II or III gastric cancer and potential clinical applications
Authors: Oshima Takashi , Kimura Yayoi , Sakamaki Kentaro , Miyagi Yohei , Yoshikawa Takaki , Rino Yasushi , Sentani Kazuhiro , Oue Naohide , Yasui Wataru , Imada Toshio , Munetaka Munetaka
Published: July 2016

Objective: Standard treatment for stage II or III gastric cancer is curative resection followed by adjuvant chemotherapy. Treatment outcomes are expected to be further improved by individualized therapy based on biomarker analysis. We extracted mRNA and protein from frozen specimens of gastric cancer and analyzed markers for stratifying the risk of recurrence after curative resection of stage II or III gastric cancer. We report our currently available findings.

Methods: Candidate markers for stratification of recurrence risk were identified by quantitative real-time polymerase chain reaction (Q-PCR) at the gene level and liquid chromatography-tandem mass spectrometry (LC-MS/MS) at the protein level. The study group for quantitative real-time PCR comprised 255 patients with stage II or III gastric cancer (training set, 145 patients; validation set, 110 patients). The relative expression levels of 127 genes were measured in gastric cancer tissue. Genes identified be independent predictors of poor outcomes in the training set were verified in the validation set. The study group for exploratory proteomics analysis LC-MS/MS comprised 24 patients with T4a Nx M0, Stage III gastric cancer (recurrence and death, 12 patients; relapse-free survival >5 years, 12 patients). Proteins with max fold change >2 and Anova (P) <0.001 were searched.

Results: On Q-PCR, 5 genes; SPARC, HER2, CXCR4, INHBA, and VSNL1 were identified as candidate markers. On exploratory proteomics analysis by LC-MS-MS, 4 proteins were identified as candidate markers for recurrence risk stratification.

Conclusions: Promising candidate markers for stratification of the risk of recurrence after curative resection of stage II or III gastric cancer were identified at the gene and protein levels. These candidate markers are now being analyzed by tissue microarray, using specimens from about 500 patients each at two high-volume centers, with the goal of developing clinical applications.

Citation Format: Takashi Oshima, Yayoi Kimura, Kentaro Sakamaki, Yohei Miyagi, Takaki Yoshikawa, Yasushi Rino, Kazuhiro Sentani, Naohide Oue, Wataru Yasui, Toshio Imada, Munetaka Munetaka. Stratification markers for the risk of recurrence after curative resection of stage II or III gastric cancer and potential clinical applications. [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 4932.
Abstract 4934: Detection of HBV-host junction DNA sequences in urine of patients with hepatocellular carcinoma
Authors: Lin Selena , Trauger Evan R. , Song Benjamin P. , Lan Ling , Jongeneel Patrick M. , Thompson Emilie G. C. , Hoffman Malcolm C. , Jain Surbhi , Chang Ting-Tsung , Block Timothy M. , Song Wei , Su Ying-Hsiu
Published: July 2016

Human urine has been shown to contain DNA from circulation. In this study, we aim to provide unambiguous evidence that urine contains hepatocellular carcinoma (HCC)-derived DNA, and thus can be used for detecting HCC-associated DNA markers. We used novel hepatitis B virus (HBV)-host junction sequences (HBV-JSs), created by viral integration in the human genome, as unique markers to track the HBV-integrated DNA from tumor tissues to their corresponding urine samples. An HBV DR1-2 enriched Next-Generation sequencing (NGS) assay was developed to identify the major HBV-JSs in 15 HBV-HCC tissues. Upon validation of the major HBV-JSs by Sanger sequencing, a short-amplicon PCR assay tailed for each HBV-JS was developed to test the junctions’ existence in the corresponding urine DNA. 15 major HBV-JSs were identified by the HBV DR1-2 enriched NGS assay from 15 HBV related HCC (HBV-HCC) tissues, 13 of which were validated by Sanger sequencing. By using HBV-JS specific short-amplicon junction PCR assays, we detected and confirmed six of nine HBV-JSs for which there were matching urine samples. Urine contains detectable major HBV-JSs derived from HCC, and thus can be used for liquid biopsies to study not only the complexities of HBV-JS species during chronic HBV infection and carcinogenesis, but also other HCC-related DNA modifications for the early detection of HCC and disease management.

Citation Format: Selena Lin, Evan R. Trauger, Benjamin P. Song, Ling Lan, Patrick M. Jongeneel, Emilie G. C. Thompson, Malcolm C. Hoffman, Surbhi Jain, Ting-Tsung Chang, Timothy M. Block, Wei Song, Ying-Hsiu Su. Detection of HBV-host junction DNA sequences in urine of patients with hepatocellular carcinoma. [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 4934.
Abstract 4942: Towards a screening blood test for esophageal adenocarcinoma
Authors: Shah Alok K. , Joshi Virendra , Le Cao Kim-Anh , Whiteman David C. , Barbour Andrew P. , Hill Michelle M.
Published: July 2016

BACKGROUND: The incidence of esophageal adenocarcinoma (EAC) has been rapidly increasing globally. The majority of EAC cases are diagnosed at late stages leading to poor 5-year survival of <20%. Monitoring programs for patients with the metaplastic condition, Barrett's esophagus (BE) involves endoscopy with biopsy. However, the cost-effectiveness is limited by the low rate of progression to cancer and expense of the method. Our research program aims to improve the cost-effectiveness of screening by developing a blood test to select patients for endoscopy/biopsy. Using our integrated lectin magnetic bead array (LeMBA) glycoprotein biomarker pipeline [1, 2] with Australian cohorts for the discovery and qualification phases, we identified serum lectin-glycoprotein biomarker candidates which can distinguish EAC from BE and healthy controls [3]. Here, we report the performance of these biomarker candidates in an independent patient cohort from the Ochsner Health System, New Orleans, USA.

APPROACH & METHODOLOGY: Serum samples were collected from consenting patients undergoing endoscopy (Control - 16, BE - 13, and EAC - 10). The control group consisted of all patients not classified as BE with or without dysplasia, or EAC. LeMBA-MRM-MS was performed using 4 different lectins with different glycan specificities; AAL (Fucoseα1-2, -3, -6 linked glycan), EPHA (bisecting GlcNAc), JAC (Galα1-6GalNAc and Galβ1-3GalNAc), NPL (Manα1-6Man). Statistical analysis was performed using Shiny MixOmics as previously described [3].

RESULTS & DISCUSSION: Several lectin-protein candidate biomarkers cross-validated in the USA cohort. Most notably, differentially glycosylated complement component C9 is highly diagnostic in both cohorts, achieving area under the receiver operating curve (AUROC) of 0.74 (AAL-C9) to 0.90 (JAC-C9) as a single marker. Complement C9 binding to all four monitored lectins were increased 1.4 to 1.8 fold in EAC, compared to either BE or controls. Another consistent biomarker is gelsolin, with its binding to EPHA and NPL lectin significantly decreased in EAC, compared to either BE or controls. Hence, glycosylated C9 and gelsolin show promise as potential serum biomarkers for EAC screening, and will be further evaluated in prospective cohorts.

REFERENCES: [1] Loo et al., J Proteome Res 9, 5496-5500 (2010); [2] Choi et al., Electrophoresis 32, 3564-3575 (2011); [3] Shah et al., Molecular & Cellular Proteomics 14:3023-39 (2015).

Citation Format: Alok K. Shah, Virendra Joshi, Kim-Anh Le Cao, David C. Whiteman, Andrew P. Barbour, Michelle M. Hill. Towards a screening blood test for esophageal adenocarcinoma. [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 4942.
Abstract 4944: Detection of functional P-selectin ligands expressed on colon cancer tissue using a novel flow-based assay
Authors: Martin Eric W. , Malgor Ramiro , Resto Vicente A. , Goetz Douglas J. , Burdick Monica M.
Published: July 2016

The presence of functional P-selectin ligands is well-documented for human colon cancer cell lines, but not in situ on human colon carcinoma tissue. Presently, immunostaining with antibodies is used to detect critical components of selectin ligands, e.g., sialofucosylated moieties. However, this static biochemical tissue analysis (SBTA) cannot ascertain if a potential selectin ligand is able to mediate (rolling) adhesion. Due to the immense difficulty in detecting functional selectin ligands using traditional methods, we have developed a flow-based assay known as dynamic biochemical tissue analysis (DBTA) for detecting functional selectin ligands expressed on human tissue. DBTA using P-selectin microspheres was performed on colon cancer tissue sections from multiple cases, in conjunction with SBTA using P-selectin, antibodies against purported selectin ligand carbohydrate moieties sLeX and sLeA (HECA-452, CSLEX-1, and KM-231), and antibodies against peptide structures of putative P-selectin ligands (CD24, CD44, and PSGL-1). Examination of serial sections, in the same regions of tissue displaying DBTA probe adhesion, revealed significant detection inconsistencies with SBTA. Subsequently, due to the well-documented force-dependency of selectin ligands, DBTA was conducted with microspheres coated with either HECA-452, CSLEX-1, or KM-231 to determine the effect of applied force on the detection capabilities of these antibodies. Analysis of signet ring cell colon carcinoma tissue revealed microspheres coated with HECA-452, CSLEX-1, or KM-231 antibodies all displayed significantly lower amounts of adhesion than the DBTA P-selectin microspheres. Interestingly, although microspheres coated with these antibodies adhered to signet ring cell carcinoma tissue, these DBTA probes did not interact with all regions of tissue that displayed adhesion with P-selectin microspheres. Specificity of interaction was validated using corresponding isotype control coated microspheres. Taken together, these results show DBTA with P-selectin coated microspheres is able to unequivocally detect functional P-selectin ligands, in contrast to SBTA (immunostaining) and DBTA using microspheres coated with antibodies. In summary, DBTA using P-selectin coated microspheres is able to detect functional P-selectin ligands expressed on colon cancer tissue, data that may provide valuable diagnostic and prognostic information for malignant tumors.

Citation Format: Eric W. Martin, Ramiro Malgor, Vicente A. Resto, Douglas J. Goetz, Monica M. Burdick. Detection of functional P-selectin ligands expressed on colon cancer tissue using a novel flow-based assay. [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 4944.
Abstract 4979: Cabozantinib eradicates advanced murine prostate cancer by activating neutrophil-mediated anti-tumor innate immunity
Authors: Patnaik Akash , Swanson Kenneth D. , Csizmadia Eva , Gehring Marina P. , Helenius Katja , Pyzer Athalia R. , Wang Lily C. , Novak Jesse , Elemento Olivier , Thornley Thomas B. , Asara John M. , Clohessy John G. , Kelly Kathy , Pandolfi Pier Paolo , Rosenblatt Jacalyn M. , Avigan David E. , Ye Huihui , Signoretti Sabina , Balk Steven P. , Cantley Lewis C.
Published: July 2016

Several kinase inhibitors targeting aberrant signaling pathways in tumor cells have been deployed in cancer therapy. However, their impact on the tumor immune microenvironment remains poorly understood. The tyrosine kinase inhibitor cabozantinib showed striking responses in cancer clinical trial patients across several malignancies. Here we show that cabozantinib rapidly eradicates invasive, poorly-differentiated PTEN/p53 deficient murine prostate cancer. This was associated with enhanced release of neutrophil chemotactic factors from tumor cells, including CXCL12 and HMGB1, resulting in robust infiltration of neutrophils into the tumor. Critically, cabozantinib-induced tumor clearance in mice was abolished by antibody-mediated granulocyte depletion or HMGB1 neutralization or blockade of neutrophil chemotaxis with the CXCR4 inhibitor, plerixafor. Collectively, these data demonstrate that cabozantinib triggers a neutrophil-mediated anti-tumor innate immune response, resulting in rapid tumor clearance.

Citation Format: Akash Patnaik, Kenneth D. Swanson, Eva Csizmadia, Marina P. Gehring, Katja Helenius, Athalia R. Pyzer, Lily C. Wang, Jesse Novak, Olivier Elemento, Thomas B. Thornley, John M. Asara, John G. Clohessy, Kathy Kelly, Pier Paolo Pandolfi, Jacalyn M. Rosenblatt, David E. Avigan, Huihui Ye, Sabina Signoretti, Steven P. Balk, Lewis C. Cantley. Cabozantinib eradicates advanced murine prostate cancer by activating neutrophil-mediated anti-tumor innate immunity. [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 4979.
Abstract 5001: Quantitative systems pharmacology modeling and analysis provides biological insights into anti-PD-1 dosing and predicts optimal PD-1 x TIM-3 therapeutic properties for bispecifics and fixed dose combinations in immuno-oncology
Authors: Apgar Joshua , Wong Jamie , Phennicie Ryan , Mabry Robert , Novobrantseva Tatiana , Briskin Michael , Burke John M.
Published: July 2016

The goal of this collaboration was to enable early quantitative thought experiments and risk assessments by developing and interrogating a quantitative systems pharmacology (QSP) model of the co-modulation inhibitory receptors PD-1 and TIM-3 in immuno-oncology. The QSP model was to: (1) provide predictions of best-in-class profiles for a PD-1 and TIM-3 fixed dose combination (FDC) and dual antagonist platforms, and (2) provide biological insights.

The QSP model was based on first principles as a system of elementary mass-action, mechanistic PKPD, ordinary differential equations. The model parameters and reactions were based on biophysics, and are interpretable. The model reactions include protein synthesis and elimination, ligand-receptor and drug-target formation and turnover, and drug administration and first order clearance. There were four versions of the model: PD-1 monospecific, TIM-3 monospecific, PD-1 x TIM-3 bispecific and fixed dose combination (FDC) targeting PD-1 and TIM-3. The monospecific models were then benchmarked against published data such that model parameter values were set to known values and unknown parameters were estimated. Once benchmarked, the FDC and bispecific models were analyzed by systematically investigating how tuning the model parameters (e.g., affinity, avidity, dose, half-life, target expression, etc.) impacted target inhibition, and to simulate patient variability.

The model predictions were in good agreement with published clinical data from Nivolumab and Pembrolizumab, providing a hypothesis for the apparent inconsistencies in similar clinical doses for therapeutics with affinities differing by several orders of magnitude, and data from RMT3-23 in the TIM-3 driven mouse model. QSP model analysis predicted: (1) there would be diminishing returns on very tight binding biologics due to Target Mediated Drug Disposition (TMDD) that offsets potency, and (2) there is no advantage between FDC, 2-2 bispecific, and 2-1 bispecific formats, which are predicted to be roughly equivalent.

Citation Format: Joshua Apgar, Jamie Wong, Ryan Phennicie, Robert Mabry, Tatiana Novobrantseva, Michael Briskin, John M. Burke. Quantitative systems pharmacology modeling and analysis provides biological insights into anti-PD-1 dosing and predicts optimal PD-1 x TIM-3 therapeutic properties for bispecifics and fixed dose combinations in immuno-oncology. [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 5001.