bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3693: Oligomerization status of p53 serves as an indicator of sensistivity of p53 wildtype tumors to the therapeutic combination of DNA damaging agent and checkpoint inhibitor
Authors: Puli Pawan , Lipski Robert , Levesque Aime A.
Published: July 2016

It is well established that cell cycle arrest in response to treatment with DNA damaging agents can be abrogated in p53-defective cells by treatment with the Chk1 inhibitors, but our more recent studies have shown that some p53 wild-type tumors are also sensitive to checkpoint abrogation. Two possible explanations are cytoplasmic sequestration of p53 or a defect in p53 oligomerization. In this study, we investigated the DNA damage response and UCN-01 sensitivity of two p53 wildtype neuroblastoma cell lines: SK-N-SH, and SH-SY5Y. In order to determine the responses of these cells to DNA damage, the cells were treated with concentrations of SN38 ranging from 0-30 ng/ml. Both lines arrested in G2, S, or G1 depending upon SN38 concentration and displayed an increase in p53 levels with increasing SN38. Additionally, p53 was phosphorylated on serine 15 and serine 20 following SN38 treatment in all three cell lines, suggesting that p53 is active. In order to determine whether these cells are susceptible to UCN-01-mediated abrogation of cell cycle arrest, cell were treated with 3 ng/ml SN38 for 24 hours, followed by 25 nM UCN-01 for 6 and 24 hours. The SK-N-SH showed now sensitivity to UCN-01 treatment whereas the SH-SY5Y abrogated S arrest within 6 hours and abrogated G2 arrest within 24 hours. We also analyzed the oligomerization status of p53 using glutaraldehyde crosslinking. The SK-N-SH cells possessed levels of p53 dimers and tetramers similar to what has previously been reported in p53 wildtype MCF10A cells. The SH-SY5Y, however, had extremely low levels of dimers and tetramers. Consistent with this, only SK-N-SH showed activation of p21waf1 and repression of cyclin B in response to SN38 treatment. Previous studies have reported cytoplasmic sequestration as a mechanism of p53 inactivation in p53 wildtype neuroblastomas. In order to determine the sub-cellular distribution of p53, we prepared nuclear and cytoplasmic extracts. Both the SK-N-SH and SH-SY5Y had primarily nuclear p53. The results of this study suggest that oligomerization status may serve as an indicator of sensitivity of p53 wildtype tumors to the therapeutic combination of DNA damage agent and checkpoint inhibitor.

Citation Format: Pawan Puli, Robert Lipski, Aime A. Levesque. Oligomerization status of p53 serves as an indicator of sensistivity of p53 wildtype tumors to the therapeutic combination of DNA damaging agent and checkpoint inhibitor. [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 3693.
Abstract 3701: Transcriptomic consequences of MDMX (MDM4) oncoprotein expression knockdown in MDMX-amplified breast cancer cells
Authors: Ho Yi-hsuan , Hutton Claire , Newell Herbie , Lunec John
Published: July 2016

The tumour suppressor p53 is activated by cellular stress to induce cell cycle arrest and/or apoptosis. Although the TP53 gene is frequently mutated in cancer, approximately half of human cancers express wild type and functional p53. However, p53 activity is often suppressed by its negative regulators, MDM2 and MDMX. Small molecule antagonists have been developed to inhibit p53-MDM2 binding to release the growth inhibitory and/or pro-apoptotic functions of p53. Previous studies have indicated that MDMX amplification and/or increased protein expression may be associated with resistance to MDM2-p53 binding antagonists. However, specific inhibitors for MDMX have yet to be developed and an MDM2/MDMX-p53 co-inhibitor (RO-5963) does not show better efficacy against high MDMX expressing cells than single target MDM2 inhibitors. To better understand the cellular function and therapeutic potential of targeting MDMX, we have investigated the downstream transcriptomic consequences of knocking down MDMX expression.

Knockdown of MDMX protein expression, demonstrated by Western blot analysis, was achieved in MRK-nu-1 MDMX amplified and TP53 wild-type breast carcinoma cells using both lentiviral shRNA and siRNA. MRK-nu-1 cell growth suppression was observed after knockdown of MDMX. Affymetrix Human Transcriptome Array 2.0 was used to detect differences in the expression of full genes and alternatively spliced forms between negative control and MDMX knockdown groups. Transcriptome Analysis Console 3.0 software was used to analyse the microarray data to identify significant changes in gene expression, alternative splicing and associated functional pathways. Alterations in mRNA expression for a selected panel of genes was confirmed by qRT-PCR.

A greater degree of MDMX expression knockdown was achieved with the siRNA (>95%) than the lentiviral shRNA (>50%) This was reflected by increased suppression of growth and larger changes in gene expression patterns with the siRNA knockdown. Although a similar set of genes was affected by both the siRNA and shRNA knockdown, the magnitude of changes was greater following the more pronounced knockdown with siRNA. The expression of a number of p53 transcriptional target genes was found to be altered, including CDKN1A, MDM2, CCNG2, RRM2B, BTG2, ZMAT3 and FAS consistent with a role for MDMX in suppression of p53 function in these MDMX amplified cells.

Citation Format: Yi-hsuan Ho, Claire Hutton, Herbie Newell, John Lunec. Transcriptomic consequences of MDMX (MDM4) oncoprotein expression knockdown in MDMX-amplified breast 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 3701.
Abstract 3708: Targeting synthetic lethality with PARP inhibition in colorectal cancer
Authors: Augustine Titto , Maitra Radhashree , Zhang Jinghang , Nayak Jay , Goel Sanjay
Published: July 2016

Background:

Small molecule poly ADP-ribose (PAR) polymerase (PARP) inhibitors (PARPi) purportedly result in DNA damage and are promising agents in the treatment of cancer. In a quest to identify best “synthetic lethality” for treatment, we have screened various combination therapies in colorectal cancer (CRC) cell lines. In addition, we have interrogated if the presence of microsatellite instability (MSI) status favor susceptibility of cells to synthetic lethality and thus acts as a prognostic marker of combination therapy.

Methods:

Using the SRB assay, the 12 combinations of the PARPi, veliparib (V), rucaparib (R), olaparib (Ol) and PJ34, and the cytotoxic drugs, irinotecan (I), 5-fluorouracil (5-FU) and oxaliplatin (Ox) were screened in HCT116 and LIM2405 cell lines. We assessed the effect of the combination using the CalcuSyn software for combination index (CI). We next evaluated the effectiveness and sequence dependence of combination in an additional 2 cell lines that had MSI status (RKO and HCT15) and 2 that had microsatellite stable (MSS) status (HT29 and SW837) and in 7 HCT116 isogenic lines (mutants of Bax, p21, p53, PTEN, Kras and DNMT and wild type of PIK). The combination of I and R was confirmed by FACS analysis in HCT116 and LIM2405.

Results:

Ol and R showed synergy in combination with I, whereas, V and PJ34 showed antagonism with Ox, including in Ox-sensitive cells. Single agent cytotoxicity was observed at 200nM for R, whereas V required more than 30uM. Early PARP-1 protein upregulation was observed upon Ox administration; but the changes were not reversed with either R or V. Nevertheless, R at 200nM and V 1uM significantly reduced PAR formation in Ox-sensitive cell lines. FACS analysis showed S phase arrest in SW837 (MSS) (p = 0.03) whereas G2-M arrest was observed in LIM2405 (MSI) (non-significant) upon treatment with R 400nM and I 200nM. In addition, studies using isogenic cell lines revealed the importance of p21, p53 and PTEN in exerting synergistic effect while combining R and I. Administration of I prior to R (24h each) was more cytotoxic than vice versa and simultaneous treatment in MSI cell lines.

Conclusion:

Among the various combinations studied, R with I was the most synergistic. The effect appears to be more pronounced when I precedes R, particularly in MSI cell lines. Alteration in cell cycle is MSI status dependent. Further work to identify mechanism of synergy is ongoing.

Citation Format: Titto Augustine, Radhashree Maitra, Jinghang Zhang, Jay Nayak, Sanjay Goel. Targeting synthetic lethality with PARP inhibition in colorectal 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 3708.
Abstract 3715: Benzimidazolone sulphonamides - potent, selective and drug-like inhibitors of poly(ADP Ribose) Glycohydrolase (PARG)
Authors: Jordan Allan , Acton Ben , Hamilton Nicola , Hitchin James , Hutton Colin , James Dominic , Jones Cliff , Jones Stuart , McGonagle Alison , Small Helen , Smith Kate , Stowell Alex , Tucker Julie , Waddell Ian , Waszkowycz Bohdan , Ogilvie Donald
Published: July 2016

In recent years, many proteins involved in DNA repair, such as ATR, ATM and PARP, have received considerable attention as potential points of therapeutic intervention in cancer. Indeed, these efforts have recently delivered several agents into clinical evaluation or FDA regulatory approval. However, the DNA repair protein poly(ADP ribose) glycohydrolase (PARG), which plays an equally critical role in DNA single stand break repair, to successful drug discovery efforts.

Through our innovative collaboration with AstraZeneca, we have discovered a novel PARG-binding pharmacophore and have employed this information to discover drug-like chemotypes, facilitating the development of potent and selective inhibitors.

This poster will describe our emerging results in this area, where a novel benzimidazolone sulphonamide scaffold has been shown potently to inhibit PARG in both biochemical and cellular assays with potencies of 40 nM and 60 nM respectively. Moreover, these agents display pharmacology consistent with the anticipated mode of action, appropriate drug-like properties and are selective against PARP1 and the close glycohydrolase homologue ARH3. The medicinal chemistry optimisation of this scaffold will be described, alongside the recent biological results obtained. Ultimately, this work has helped deliver tool compounds which may help to elucidate the true pharmacology and roles of PARG in cancer and other disease settings.

Citation Format: Allan Jordan, Ben Acton, Nicola Hamilton, James Hitchin, Colin Hutton, Dominic James, Cliff Jones, Stuart Jones, Alison McGonagle, Helen Small, Kate Smith, Alex Stowell, Julie Tucker, Ian Waddell, Bohdan Waszkowycz, Donald Ogilvie. Benzimidazolone sulphonamides - potent, selective and drug-like inhibitors of poly(ADP Ribose) Glycohydrolase (PARG). [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 3715.
Abstract 3725: B-Raf inhibition-induced paradoxical activation of MEK/ERK signaling enhances DR5 expression and DR5 activation-induced apoptosis in Ras-mutant cancer cells
Authors: Oh You-Take , Deng Jiusheng , Yue Ping , Sun Shi-Yong
Published: July 2016

B-Raf inhibitors have been used for the treatment of some B-Raf-mutated cancers such as melanoma and thyroid cancer. They effectively inhibit B-Raf/MEK/ERK signaling in cancers harboring mutant B-Raf, but lead to a paradoxical activation of MEK/ERK signaling in Ras-mutant cancers. Death receptor 5 (DR5), a cell surface pro-apoptotic protein, triggers apoptosis upon ligation with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or aggregation. Given the critical role of MEK/ERK signaling in the positive regulation of DR5 expression as we have previously demonstrated, this study focuses on determining the impact of B-Raf inhibition on DR5 expression and DR5 activation-induced apoptosis in Ras-mutant cancer cells. Using chemical and genetic approaches, we have demonstrated that the B-Raf inhibitor PLX4032 induces DR5 upregulation exclusively in Ras-mutant cancer cells; this effect is dependent on Ras/c-Raf/MEK/ERK signaling activation. PLX4032 induces DR5 expression at transcriptional levels, largely due to enhancing CHOP/Elk1-mediated DR5 transcription. Pre-treatment of Ras-mutated cancer cells with PLX4032 sensitizes them to TRAIL-induced apoptosis; this is also a c-Raf/MEK/ERK-dependent event. Collectively our findings highlight a previously undiscovered effect of B-Raf inhibition on the induction of DR5 expression and the enhancement of DR5 activation-induced apoptosis in Ras-mutant cancer cells. Our results may suggest a novel therapeutic strategy against Ras-mutated cancer cells by driving their death due to DR5-dependent apoptosis through B-Raf inhibition. (This study was supported by Emory Winship Cancer Institute Robbins Scholar awards to YTO and to JD and Halpern Research Scholar award to SYS. SYS is a Georgia Research Alliance Distinguished Cancer Scientist and Halpern Research Scholar)

Citation Format: You-Take Oh, Jiusheng Deng, Ping Yue, Shi-Yong Sun. B-Raf inhibition-induced paradoxical activation of MEK/ERK signaling enhances DR5 expression and DR5 activation-induced apoptosis in Ras-mutant 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 3725.
Abstract 3760: XCE853 is a promising protein disulfide isomerase (PDI) inhibitor exhibiting a strong inhibitory activity in preclinical tumor models
Authors: Prevost Gregoire P. , Garrido Marine , Serova Maria , Briand Jean François , Gutmann Mathieu , Ladam Patrick , Carniato Denis , Tijeras-Raballand Annemilai , De Grammont Armand , Chauchereau Anne , Pitty Marc-Henry , Foster Paul
Published: July 2016

Protein disulfide isomerase (PDI) is a chaperone protein that regulates oxidative protein folding as well as cell viability. Increased PDI levels have been documented in a variety of human cancers, including ovarian, prostate, and lung cancers. Inhibition of PDI activity leads to apoptosis in cancer, suggesting that PDI is a promising druggable target.

XCE853 is a synthetic small molecule displaying an excellent docking with the human PDI. XCE853 inhibits in vitro recombinant PDI activity. In addition, the proliferation of a large panel of human tumor cells is blocked by XCE853 with IC50s in the nanomolar range through an irreversible cytolysis.

XCE853 is also active on a large panel of drug resistant human cancer cells. XCE853 induced an irreversible cytolysis of human tumor cells after a short in vitro exposure independently of efflux pumps leading to a tumor cell death by autophagy and particles release (vesicles or protein aggregates).

In addition, the ex-vivo approach using fresh human tumor explants cultivated in 3 dimensions with low concentrations of XCE853 has shown a strong decrease of the proliferation (KI-67 labeling) in several tumor types.

Finally, XCE853 displayed excellent oral bioavailability in mice and was able to block the growth of several human cancers using in vivo xenograft models leading to a complete tumor growth arrest even after the cessation of the treatment.

Altogether, these data support further efforts on this drug candidate to initiate the preclinical studies and to define the most relevant human tumor types.

Citation Format: Gregoire P. Prevost, Marine Garrido, Maria Serova, Jean François Briand, Mathieu Gutmann, Patrick Ladam, Denis Carniato, Annemilai Tijeras-Raballand, Armand De Grammont, Anne Chauchereau, Marc-Henry Pitty, Paul Foster. XCE853 is a promising protein disulfide isomerase (PDI) inhibitor exhibiting a strong inhibitory activity in preclinical tumor 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 3760.
Abstract 3769: Continuous, low intensity systemic dosing maximizes the therapeutic margin of Eg5/KSP inhibition in an orthotopic model of urothelial cell carcinoma
Authors: Williams Leigh , Ellston Rebecca , Trueman Dawn , Musgrove Helen , Ruston Linette , Aquila Brian , Pease Elizabeth , Klein Stephanie , Davies Barry R.
Published: July 2016

Urothelial Cell Carcinoma (UCC) is the fifth most common cancer, but no new generation molecularly targeted therapies have been registered to treat this disease. Non muscle-invasive bladder cancer can be treated by systemic or intravesical dosing routes. However, the urothelium is one of the most formidable permeability barriers in nature, and may limit the penetration of small molecules into tumour tissue from the intravesical location. Inhibitors of mitosis, including spindle proteins such as Eg5, are attractive targets for cancer therapy, but their efficacy when dosed systemically is severely limited by bone marrow toxicities such as neutropenia and thrombocytopenia. AZ9814, a very potent and selective small molecule inhibitor of Eg5, induces apoptosis and inhibits proliferation of UCC cells in culture with a GI50 of < 1 nM. We have developed an orthotopic model of bladder cancer in the nude rat coupled with a programmable mini-pump system to continuously infuse AZ9814 into the bladder, and compared the therapeutic index of this delivery route with systemic dosing schedules using intraperitoneal injection and continuous subcutaneous infusion using an osmotic mini-pump. Continuous intravesical infusion resulted in severe bladder toxicity including hydropic degeneration and ulceration of urothelium, mucosal erosion and bilateral hydronephrosis, with limited penetration of compound into the tumour, and no significant impact on tumour pharmacodynamics and growth. In contrast, continuous low intensity systemic dosing resulted in significant tumour pharamacodynamic activity and control of tumour growth, albeit with significant but non-lethal effects on bone marrow toxicity. Daily intraperitoneal injection of AZ9814 resulted in very modest anti-tumour effects which were limited by bone marrow toxicity. It is concluded that Eg5 inhibitors have potential to treat UCC, but continuous systemic infusion is the best dosing schedule to maximize therapeutic index.

Citation Format: Leigh Williams, Rebecca Ellston, Dawn Trueman, Helen Musgrove, Linette Ruston, Brian Aquila, Elizabeth Pease, Stephanie Klein, Barry R. Davies. Continuous, low intensity systemic dosing maximizes the therapeutic margin of Eg5/KSP inhibition in an orthotopic model of urothelial cell 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 3769.
Abstract 3770: HEXIM1 as a pharmacodynamic marker for monitoring target engagement of ABBV-075
Authors: Huang Xiaoli , Lin Xiaoyu , Li Leiming , Wang Rongqi , Roberts Lisa , Hessler Paul , Uziel Tamar , Lam Lloyd , Magoc Terry , Albert Daniel H. , Elmore Steven W. , Fang Guowei , Rosenberg Saul H. , McDaniel Keith , Kati Warren , Shen Yu
Published: July 2016

Bromodomain and extra-terminal (BET) family are dual bromodomain-containing proteins that play an important role in transcription regulation. ABBV-075 is a small molecule BET bromodomain inhibitor currently in phase 1 clinical trials. Here we report the identification of Hexim1 and other BET-responsive genes as robust pharmacodynamic markers for monitoring ABBV-075 target engagement in tumors and in surrogate tissues such as whole blood and skin. Transcription profiling of cancer cell lines for their responses to BET inhibitors identified a set of 9 BET-regulated genes, including Hexim1, that responded to BET inhibition across cancer cell lines and in xenograft tumors. Further characterization of BET-responsive genes in surrogate tissues such as mouse skin, mouse/human PBMCs and whole blood revealed that the mRNA level of Hexim1 exhibited the best response to BETi treatment across different settings. A dose-dependent increase of Hexim1 expression was detected in the whole blood of ABBV-075 treated tumor bearing mice, and the Hexim1 response was closely correlated with the plasma drug concentration and largely reflected the anti-tumor efficacy at various dose levels of ABBV-075. Hexim1 is part of a protein/RNA complex that sequesters pTEFb and prevents its recruitment by BRD4. It also reportedly mediates the anti-proliferative activity of BRD4 in AML. Taken together, Hexim1 could serve as a functional relevant pharmacodynamic marker for monitoring ABBV-075 target engagement in animal models and in the clinical setting.

Citation Format: Xiaoli Huang, Xiaoyu Lin, Leiming Li, Rongqi Wang, Lisa Roberts, Paul Hessler, Tamar Uziel, Lloyd Lam, Terry Magoc, Daniel H. Albert, Steven W. Elmore, Guowei Fang, Saul H. Rosenberg, Keith McDaniel, Warren Kati, Yu Shen. HEXIM1 as a pharmacodynamic marker for monitoring target engagement of ABBV-075. [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 3770.
Abstract 3794: Novel therapeutic targets in head and neck cancer
Authors: Kondratyev Maria , Pesic Aleksandra , Marastoni Stephano , Ketela Troy , Moffat Jason , Virtanen Carl , Sayad Azin , Bashkurov Mikhail , Dati Alessandro , Ailles Laurie , Grenman Reidar , Koritzinsky Marianne , Wouters Brad
Published: July 2016

HNSCC is 6th most common malignancy in the world. Despite advances in diagnosis and treatment, the survival rates remain low due to frequent recurrences the biology of which remains unclear. Using functional genomic technologies we identified new therapeutic targets for metastatic disease in HNSCC. Whole genome shRNA screens were conducted in matched sets of cell lines derived from primary tumors and respective metastatic sites, identifying genes essential for cell survival only following metastasis. To test if knockdown of selected targets inhibits metastasis in a therapeutic setting, we established orthotropic model of HNSCC that metastasize to lymph nodes in the mouse. Components of Notch pathway were identified as essential for survival of cells derived from metastatic sites. Whole exome sequencing identified a novel mutation in one of the EGF domains of Notch3 that was acquired only in the metastatic line. Mutations in EGF domains have been reported to influence interaction with specific ligands, dictating which ligand can activate Notch signaling. Our data indicate that metastatic, but not primary tumor cells, undergo apoptosis upon knockdown of Notch3 and that a distinct set of target genes is induced upon interaction between Notch3 and Jag2 ligand. Furthermore, our results indicate that suppression of Notch3 improves survival in mice bearing orthotropic tumors derived from the metastatic HNSCC lines. Our data demonstrate that metastatic cells from head and neck tumors acquire dependency on Notch3 signaling. Novel treatments targeting components of this pathway may prove effective in targeting metastatic cells alone or in combination with conventional therapies.

Citation Format: Maria Kondratyev, Aleksandra Pesic, Stephano Marastoni, Troy Ketela, Jason Moffat, Carl Virtanen, Azin Sayad, Mikhail Bashkurov, Alessandro Dati, Laurie Ailles, Reidar Grenman, Marianne Koritzinsky, Brad Wouters. Novel therapeutic targets in head and neck 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 3794.
Abstract 38: The clinical significance of GLUT1 expression in gastrointestinal stromal tumor
Authors: Kuroda Daisuke , Kurashige Junji , Iwatsuki Masaaki , Eto Tsugio , Tamaoki Yuka , Ohuchi Mayuko , Nakamura Kenichi , Tokunaga Ryuma , Shigaki Hironobu , Hayashi Hiromitsu , Ishimoto Takatsugu , Baba Yoshifumi , Sakamoto Yasuo , Yoshida Naoya , Baba Hideo
Published: July 2016

Background: Gastrointestinal stromal tumor (GIST) is, though it is as rare as its incidence is about 7 to 20 cases per million population per year, one of the most common non-epithelial malignant tumors of the gastrointestinal tract. Recently it was reported that Fluorine-18 fluorodeoxyglucose (18F-FDG) positron emission tomography-computed tomography (PET/CT) is useful in diagnosis and evaluation of malignant grade of GIST. At the same time, expression of glucose transporter 1 (GLUT1), which is a membranous protein facilitates the transport of glucose across the plasma membranes into cells, was reported to be correlated with maximum standardized uptake value (SUVmax) in PET/CT and malignant grade of various kinds of cancers. In this study, the correlation of GLUT1 expression with malignant grade in GIST was investigated.

Methods: We evaluated the protein expression of GLUT1 in immunohistochemistry in the surgically resected specimens in 67 GIST cases, and analyzed the correlation of the other clinicopathological factors.

Results: The number of cases in which the expression of GLUT1 judged to be confirmed in immunohistochemistry was 45 (73.7%). GLUT1 expression group had significantly higher preoperative SUVmax in PET/CT than GLUT1 non-expression group (p = 0.03), had larger tumors in diameter (p = 0.015), and was more malignant by Fletcher classification (p<0.01). Additionally, tumor rupture rate tended to be higher in GLUT1 expression group, though there is no tendency of the correlation with tumor necrosis. Eventually, relapse free survival, as not significantly, tend to be shorter in GLUT1 expression group.

Conclusion: In our study, expression of GLUT1 is correlated with malignant grade in GIST. GLUT1 expression might be a phase of higher metabolic and malignant activity of GIST cells. GLUT1 might be a useful prognostic marker in GIST cases.

Citation Format: Daisuke Kuroda, Junji Kurashige, Masaaki Iwatsuki, Tsugio Eto, Yuka Tamaoki, Mayuko Ohuchi, Kenichi Nakamura, Ryuma Tokunaga, Hironobu Shigaki, Hiromitsu Hayashi, Takatsugu Ishimoto, Yoshifumi Baba, Yasuo Sakamoto, Naoya Yoshida, Hideo Baba. The clinical significance of GLUT1 expression in gastrointestinal stromal tumor. [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 38.
Abstract 3812: FAK inhibition resensitizes platinum-resistant serous ovarian cancer
Authors: Bean Lisa M. , Sulzmaier Florian J. , Tancioni Isabelle , Uryu Sean , Jean Christine , Lawson Christine , Chen Xiao Lei , Kleinschmidt Elizabeth G. , Anderson Kristen M. , Cordasco Edward A. , Axelrod Joshua , Kolev Vihren N. , Pachter Jonathan A. , Stupack Dwayne G. , Schlaepfer David D.
Published: July 2016

Platinum and taxol administration is standard of care chemotherapy for serous ovarian cancer. Tumor recurrence occurs in a high percentage of patients, and this is directly related to poor overall survival. Ovarian cancer stem cell (CSC) resistance to chemotherapy treatment can give rise to tumor recurrence. Focal adhesion kinase (FAK), an intracellular tyrosine kinase, has been linked to mesothelioma and breast CSC survival. Here, we find that FAK activation is elevated in platinum (CP)-resistant ovarian cancer cells and that FAK tyrosine phosphorylation is increased after CP treatment of CP-sensitive ovarian cancer cells. Nanomolar levels of FAK inhibitor (VS-4718) selectively blocked CP-resistant ovarian carcinoma methylcellulose colony growth via cell cycle inhibition but not apoptosis. Oral VS-4718 administration to mice reduces CP-resistant orthotopic tumor burden with a concomitant decrease in tumor-associated aldehyde dehydrogenase (ALDH) activity, a marker of ovarian CSCs. Residual ovarian tumor cells from VS-4718-treated mice exhibit reduced ALDH activity and secondary tumor initiating capacity. CRISPR-mediated FAK knockout or VS-4718 treated ovarian carcinoma cells exhibit diminished Oct-4 transcription factor and ALDH-1A1 CSC-associated protein marker expression. Co-administration of VS-4718 with CP-taxol chemotherapy reduced CP-resistant tumor burden and exhibited additive inhibitory effects on ovarian carcinoma spheroid growth. As we find that CP activates FAK and that FAK activity sustains ovarian carcinoma CSC phenotypes, our results support the testing of FAK inhibitors in combination with CP to prevent recurrent and chemo-resistant ovarian cancer.

Citation Format: Lisa M. Bean, Florian J. Sulzmaier, Isabelle Tancioni, Sean Uryu, Christine Jean, Christine Lawson, Xiao Lei Chen, Elizabeth G. Kleinschmidt, Kristen M. Anderson, Edward A. Cordasco, Joshua Axelrod, Vihren N. Kolev, Jonathan A. Pachter, Dwayne G. Stupack, David D. Schlaepfer. FAK inhibition resensitizes platinum-resistant serous ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3812.
Abstract 3848: Targeting hyaluronidase reduces stromal cell protection of chronic myeloid leukemia to imatinib therapy
Authors: Hostetler Bryan , Uchakina Olga , McKallip. Robert
Published: July 2016

An estimated 20-30% of CML patients will become resistant to tyrosine kinase inhibitors (TKIs) including imatinib. Recent reports suggest that CML resistance to TKI's is driven by interactions with protective microenvironment niches that influence their survival and self-renewal capacity. Hyaluronic acid (HA) has emerged as a key contributor in this phenomenon of CML resistance to TKI’s. HA influences cell viability through molecular weight dependent interactions with cell-surface receptors CD44 and RHAMM. Low molecular weight (LMW) HA, is typically found in association with many cancers including CML and promotes apoptotic resistance, proliferation, and migration of cancer cells. High molecular weight (HMW) HA is primarily produced by fibroblasts and its catabolism to LMW HA is mediated by hyaluronidase (HYAL) activity. HYAL activity may provide a potential therapeutic target in combination with current TKI treatments. Using a co-culture model, we investigated the role of HA produced by bone marrow stromal fibroblasts on CML viability when subject to imatinib (IM) treatment. Furthermore, we investigated the possibility of targeting hyaluronidase as an adjuvant in combination with IM for the treatment of CML. Co-culture with stromal fibroblasts protected CML cells from proliferation inhibition by IM. HA production and its catabolic products including LMW HA were elevated in the supernatant of co-cultures exposed to IM. The expression of genes encoding for HA metabolic proteins including hyaluronic acid synthases and HYALs, were significantly increased in co-cultures subject to IM stress. Additionally, HA treatment of CML mono-culture protected against chemotherapy-induced apoptosis. Treatment of CML co-culture with HYAL inhibitor, glycrrhizic acid (GA), inhibited HYAL activity, greatly reduced proliferation and increased apoptosis of CML cells alone and in combination with IM. This investigation provides additional evidence of the importance of stromal cell support in CML pathology and that resistance to TKI therapy may be mediated in part through upregulation of total HA production and its catabolism. Furthermore, we establish the potent therapeutic effects of GA on CML cells alone and in combination with current IM treatment strategies as a prospective method of circumventing TKI-resistant CML. For the first time we demonstrate GA's effectiveness as a HYAL inhibitor in culture. HYALs are an attractive target not only in CML resistance but in many cancers due to the reported role of its product, LMW HA, in inflammation, proliferation, and apoptotic resistance.

Citation Format: Bryan Hostetler, Olga Uchakina, Robert McKallip. Targeting hyaluronidase reduces stromal cell protection of chronic myeloid leukemia to imatinib therapy. [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 3848.
Abstract 3851: Synergy between statins and BCL-2 inhibitors in blood cancers
Authors: Lee Jong-Hoon S. , Vo Thanh-Trang T. , Fruman David A.
Published: July 2016

BCL-2 is a key pro-survival protein that is highly expressed in many leukemias and lymphomas. ABT-199 (venetoclax) is a small molecule inhibitor of BCL-2 that has demonstrated promising clinical potential in chronic lymphocytic leukemia (CLL). However, other hematologic malignancies are less responsive to ABT-199 as a single agent, suggesting that combinations of targeted therapies may be required to elicit more promising responses. We have investigated the potential of combining ABT-199 with HMG-CoA reductase (HMGCR) inhibitors (statins), which have known anti-cancer potential in hematologic malignancies. Using multiple chemically distinct statin compounds, we observed profound synergistic induction of apoptosis when combined with ABT-199 in both human diffuse large B cell lymphoma (DLBCL) as well as acute myeloid leukemia (AML) cell lines. This synergy was also seen in primary murine B lymphoma cells over-expressing MYC and BCL-2. Importantly, addition of exogenous mevalonate completely rescued cells from the combination, confirming on-target efficacy of HMGCR inhibition. Using BH3 profiling, we found that simvastatin significantly primed lymphoma cells for undergoing apoptosis (termed mitochondrial priming). Notably, the degree of priming correlated with its ability to synergize with ABT-199, suggesting that this method may be used to predict patient responses. Strikingly, the combination did not synergize to kill normal human peripheral blood mononuclear cells from healthy donors, suggesting that statins may selectively prime cancer cells for apoptosis. Mechanistic studies support the hypothesis that statins synergize with ABT-199 by suppressing protein prenylation, particularly protein geranylgeranylation. In support, the addition of exogenous geranylgeranyl pyrophosphate (GGPP) completely rescued cells from the effects of simvastatin. Furthermore, selective inhibition of protein geranylgeranyl transferase (GGT) was sufficient to recapitulate the effects of simvastatin in combination with ABT-199. Lastly, we have identified Rap1A de-prenylation as a marker of pharmacodynamic response to statins in vivo. Thus, this project highlights a novel combination for use in aggressive lymphomas, establishes its efficacy and tolerability using preclinical models, and provides proof-of-concept to warrant investigation of its clinical potential.

Citation Format: Jong-Hoon S. Lee, Thanh-Trang T. Vo, David A. Fruman. Synergy between statins and BCL-2 inhibitors in blood cancers. [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 3851.
Abstract 3852: U3-1784, a human anti-FGFR4 antibody for the treatment of cancer
Authors: Bartz Rene , Fukuchi Keisuke , Lange Tanja , Gruner Katrin , Ohtsuka Toshiaki , Watanabe Ichiro , Hayashi Shinko , Redondo-Müller Mauricio , Takahashi Mizuki , Agatsuma Toshinori , Bange Johannes , Abraham Reimar
Published: July 2016

Fibroblast Growth Factor Receptor 4 (FGFR4) is the fourth member of the Fibroblast Growth Factor Receptor (FGFR) family of receptor tyrosine kinases. All of the FGFR's have been implicated in cancer development due to increased activation of their enzymatic activity either by gene mutation, over-expression or inadvertent ligand-mediated stimulation. One important alteration that may lead to FGFR4 activation in cancer is the overexpression of its ligand FGF19 in 20-40% of primary liver cancer.

Here, we report the development of U3-1784, a phage display-derived fully human antibody that specifically binds to FGFR4 but not to isoforms of FGFR1-3. The antibody binds to an epitope in the putative ligand binding domain of the receptor and consequently inhibits ligand binding and downstream signaling. In a panel of 10 tumor models derived from hepatocellular carcinoma, U3-1784 significantly inhibits the growth of FGF19-expressing models up to 90% whereas models without FGF19 expression are insensitive. These results strongly suggest that the FGFR4/FGF19 axis is an oncogenic driver in hepatocellular carcinoma. U3-1784 is currently in phase I clinical trials.

Citation Format: Rene Bartz, Keisuke Fukuchi, Tanja Lange, Katrin Gruner, Toshiaki Ohtsuka, Ichiro Watanabe, Shinko Hayashi, Mauricio Redondo-Müller, Mizuki Takahashi, Toshinori Agatsuma, Johannes Bange, Reimar Abraham. U3-1784, a human anti-FGFR4 antibody for the treatment of 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 3852.
Abstract 3883: Variant peptide identification system for bottom-up proteomics: Finding hidden sequence alterations in MS data
Authors: Hajduch Marian , Hruska Miroslav , Varanasi Lakshman , Voller Jiri , Dzubak Petr
Published: July 2016

In database search, acquired spectra are matched with theoretical spectra of database peptides. Peptides from large databases are likely to match to more than one spectrum, but only a small proportion of peptide interpretations are correct. Incorrect interpretations are likely to arise for two reasons: random matches and non-random matches to homologous peptides. The former is often eliminated by selection of an appropriate similarity threshold or by utilization of the statistical significance of a spectral match. The match to homologous peptide poses a challenge as there is often large similarity in their fragmentation spectra as well. Non-random, but incorrect matches occur surprisingly often in the identification of peptide sequence variants. For this reason, a high identification score does not imply correctness of interpretation. Increasing spectral score threshold enhances specificity in general, but results in significant loss of sensitivity. Therefore, a single spectral criterion is inadequate for distinguishing between homologous peptides.

In this study, we reconceive the system for elimination of incorrect interpretations. We propose a system that uses multidimensional LC/MS information in combination with a priori knowledge of sample content to filter out unlikely interpretations. The system calculates retention time of candidate peptides, performs isotopic analysis of precursors and constructs preliminary protein assembly to increase separation between correct and incorrect matches. The approach is used for reevaluation of database search results and is generally applicable to the analysis of standard bottom-up proteomic data.

The system was used for identification of alterations in MS2 proteomics data and validated against RNA-Seq data from the same sample. The results demonstrate that non-spectral data can be used to efficiently eliminate peptide interpretations that have no correspondence in the RNA and as such are likely false positives. The approach is sensitive and yields a large proportion of altered peptides that have RNA-Seq support. The method can potentially help overcome the problem of the large database search present, e.g., in proteogenomic studies, particularly important in cancer research and diagnostics. In summary, general prior knowledge of sample content and the use of LC and MS1 data improve on the MS2-based identification of peptides.

Citation Format: Marian Hajduch, Miroslav Hruska, Lakshman Varanasi, Jiri Voller, Petr Dzubak. Variant peptide identification system for bottom-up proteomics: Finding hidden sequence alterations in MS data. [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 3883.
Abstract 3888: Systematic characterization of aberrant signaling induced by oncogenic fusions in non-small cell lung cancer
Authors: Wagner Sebastian A. , Beli Petra , Serve Hubert
Published: July 2016

Recent studies have greatly expanded the knowledge about genetic alterations in non-small cell lung cancer (NSCLC). Gene fusions involving the tyrosine kinases ALK, ROS and RET are found in 5 - 10% of NSCLC patients and are considered as oncogenic drivers. However, the cellular signaling activated downstream of oncogenic fusions has not been systematically investigated and it remains unclear if different fusions induce specific targetable signaling patterns.

In this study we have employed quantitative mass spectrometry (MS)-based phosphoproteomics to systematically characterize the signaling induced by oncogenic fusions frequently found in NSCLC. To this end, cDNA sequences of oncogenic fusions containing the tyrosine kinases ALK, ROS and RET and different fusion partners were constructed and ectopically expressed in HEK 293 and lung cancer cell lines. Subsequently, tyrosine phosphorylated peptides were enriched and identified by LC-MS. Stable isotope labeling with amino acids in cell culture was employed to precisely quantify the abundance of phosphorylation sites in cells expressing different oncogenic fusions.

Our data demonstrate that all investigated oncogenic fusions induce massive tyrosine phosphorylation of proteins and lead to increased phosphorylation of the majority of all quantified phospho-tyrosine sites. For instance, we quantified ∼1300 unique phosphotyrosine sites from HEK 293 cells expressing EML4-ALK. Of those, 952 increased more than 4-fold in abundance. Comparative analyses of the signaling patterns allowed defining kinase-specific phosphorylation signatures. In addition, comparison of signaling patterns induced by different fusions permitted us to investigate the contribution of the non-kinase fusion partner to the downstream signaling. We were able to identify several phosphorylation events that were associated with specific non-kinase fusion partners. For example, kinase fusions containing the kinesin-1 heavy chain (KIF5B) specifically led to phosphorylation of cytoskeletal proteins, including cytoskeleton-associated protein 5 (CKAP5). We are currently investigating the functional role of these phosphorylation events for the oncogenic transformation.

In summary, we demonstrate that MS-based phosphoproteomics is a powerful tool to investigate aberrant signaling induced by oncogenic fusions in non-small cell lung cancer. Importantly, we find that the kinases involved in the oncogenic fusions largely define the downstream signaling and that the investigated fusion partners only exhibited a minor effect on the downstream signaling.

Citation Format: Sebastian A. Wagner, Petra Beli, Hubert Serve. Systematic characterization of aberrant signaling induced by oncogenic fusions in non-small cell lung 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 3888.
Abstract 3920: Polymorphisms of DNA-repair pathways and its association with radiotherapy-induced acute skin toxicity among tri-racial breast cancer populations
Authors: Eum Sung Yong , Lee Eunkyung , Wright Jean L. , Takita Christiane , Martin Eden R , Slifer Susan , Hu Jennifer J.
Published: July 2016

Radiotherapy-induced skin toxicity is one of the critical quality of life issues in breast cancer patients receiving adjuvant radiation therapy (RT). Finding a biomarker which can predict the development of acute RT-induced toxicity prior to RT is critical to improve precision medicine in radiation oncology. This study evaluated the association of single nucleotide polymorphisms (SNPs) in DNA-repair genes in 268 breast cancer undergoing adjuvant RT. We analyzed 3,747 polymorphisms in 119 DNA repair-related genes; 15 genes in base excision repair (BER), 11 in mismatch repair (MMR), 26 in nucleotide excision repair (NER), 15 in homologous recombination, 5 in non-homologous end-joining (NHEJ), 15 in DNA polymerases, and 47 in other DNA repair-related pathways. Skin toxicity was assessed at post-RT using the modified NCI's Common Toxicity Criteria for Adverse Events (CTCAE) and multiple logistic regression analyses were conducted to assess the associations between SNPs and grade ≥ 4 skin toxicity n = 115). After controlling for covariates, 221 SNPs were significant at p <0.05 and 34 at p < 0.01. Significant associations (p<0.01) were found in 7 SNPs in PAPD5 gene, 1 in POLD1, POLE, and POLI in DNA polymerase; 5 in RAD51L1 and 1 in RAD54L in HR; 4 in MMS19 in NER; 2 in MSH4 and 1 in PMS1 in MMR; 2 in RPA3 in BER; 2 in RAD18, and 1 in UBE2V2 in Rad6 pathway; 2 in CHEK2 in DNA damage response pathway; 1 in MGMT in direct reversal of DNA damage; and 1 in BLM in diseases associated with sensitivity to DNA damaging agents pathways. Among these, most significant association was found in BLM variant (s16944918) which showed that carrying at least one minor T allele was associated with increased risk of RT-induced skin toxicity (odds ratio [OR] = 3.36; 95% confidence interval (CI) = 1.62-6.97, p = 0.001). For RPA3 (rs11978293), carriers with at least one minor G allele were more likely to develop RT-induced skin toxicity (OR = 2.17, 95% CI = 1.34-3.52, p = 0.002). The current study suggests that genetic variations in multiple DNA-repair genes/pathways may contribute to inter-individual variation in RT-induced normal tissue toxicity in breast cancer patients and suggests the possibility of potential predictive values of genetic polymorphisms in RT sensitivity. Further validation studies with larger sample size are warranted.

Citation Format: Sung Yong Eum, Eunkyung Lee, Jean L. Wright, Christiane Takita, Eden R, Martin, Susan Slifer, Jennifer J. Hu. Polymorphisms of DNA-repair pathways and its association with radiotherapy-induced acute skin toxicity among tri-racial breast cancer populations. [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 3920.