bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3504: A chemical biology screen identifies a unique vulnerability of neuroendocrine cancer cells to SQLE inhibition
Authors: Mahoney Christopher , Pirman David , Chubukov Victor , Sleger Taryn , Padyana Anil , Gross Stefan , Hayes Sebastian , Fan Zi Peng , McDonald Gabrielle , Chen Yu , Murtie Joshua , Cianchetta Giovanni , Nagaraja Raj , Narayanaswamy Rohini , Choe Sung , Murray Stuart , Jin Shengfang , Biller Scott , Roddy Thomas , Smolen Gromoslaw A.
Published: August 2018

Numerous reports have described the differential metabolism of cancer cells as compared to their normal counterparts. However, only relatively few metabolic genes with cancer-specific mutations have been reported and the identification of cancer subsets with particular metabolic vulnerabilities remains a challenge.

To explore potential cancer-specific dependencies, we conducted a chemical biology screen utilizing a collection of small molecule inhibitors targeting diverse metabolic pathways in a large panel of cancer cell lines. A subset of neuroendocrine tumors, particularly small cell lung cancers (SCLC), displayed a striking dependence on squalene epoxygenase, SQLE, an enzyme in the cholesterol biosynthetic pathway. To develop further confidence in these findings, we have determined the first three-dimensional SQLE structure and further advanced a pharmacological toolbox for SQLE. Using these tools, we showed that the observed effects are on target and that the patterns of cellular sensitivity observed in vitro display excellent translation to in vivo xenografts studies. Interestingly, using a variety of orthogonal approaches, we demonstrated that SQLE sensitivity appears not to be related to overall inhibition of the cholesterol pathway but rather to specific and toxic accumulation of the SQLE substrate, squalene.

Collectively, these findings highlight the utility of chemical biology screens and identify SQLE as a potential therapeutic target in a subset of neuroendocrine tumors, particularly SCLC.

Citation Format: Christopher Mahoney, David Pirman, Victor Chubukov, Taryn Sleger, Anil Padyana, Stefan Gross, Sebastian Hayes, Zi Peng Fan, Gabrielle McDonald, Yu Chen, Joshua Murtie, Giovanni Cianchetta, Raj Nagaraja, Rohini Narayanaswamy, Sung Choe, Stuart Murray, Shengfang Jin, Scott Biller, Thomas Roddy, Gromoslaw A. Smolen. A chemical biology screen identifies a unique vulnerability of neuroendocrine cancer cells to SQLE inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3504.
Abstract 3507: Targeting the insulin-like growth factor-1 receptor in MTAP-deleted renal cell carcinomas
Authors: Xu Jihao , Chang Wen-Hsin , Huang Carissa , Yang David , Chen Ching-Hsien
Published: August 2018

Renal cell carcinoma (RCC) has emerged as a metabolic disease characterized by dysregulated expression of metabolic enzymes. Patients with metastatic RCC have an unusually poor prognosis and near-universal resistance to all current therapies. To ultimately improve cancer treatment and survival rate, there is an urgent need to reveal the mechanisms by which metabolic enzymes and aberrant pathways regulate oncogenic signaling. Through an integrated two-step analysis of RCC metabolic pathways, we previously identified dysregulated expression of methylthioadenosine phosphorylase (MTAP) in aggressive RCC. An accumulation of cellular methylthioadenosine has been observed in MTAP-deleted cancer cells. Interestingly, we found a decreased of protein-methylation level with concomitant with an increase in tyrosine phosphorylation after MTAP knockout. Next, we performed a phospho-kinase antibody array screen and identified the type 1 insulin-like growth factor-1 receptor (IGF1R) as the top-one candidate with upregulated tyrosine phosphorylation in response to MTAP loss. IGF1R phosphorylation acted upstream of Src and STAT3 signaling in MTAP-knockout RCC cells. Reduction of phospho-IGF1R by a selective inhibitor of IGF-1R, linsitinib, inhibited the cell migration and invasion capability of MTAP-deleted cells. Data from cell viability assays showed that MTAP loss enhanced linsitinib-mediated cytotoxicity in RCC cells. Our data suggest that IGF1R signaling is a driver pathway to confer aggrieves nature of MTAP-deleted renal cell carcinomas.

Citation Format: Jihao Xu, Wen-Hsin Chang, Carissa Huang, David Yang, Ching-Hsien Chen. Targeting the insulin-like growth factor-1 receptor in MTAP-deleted renal cell carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3507.
Abstract 3518: Piperlongumine, a novel treatment option to overcome BRAF inhibitor resistance in melanoma
Authors: Ramachandran Sharavan , Fofaria Neel , Srivastava Sanjay
Published: August 2018

Tumor cell metabolism is a hallmark of chemoresistance and its role has been well-defined in BRAF inhibitor resistance in melanoma. Melanoma cells are highly proliferative and are shown to develop addiction towards the metabolic pathways like glycolysis and mitochondrial respiration to meet energy demands. Melanoma cells predominantly utilize cytosolic aerobic glycolysis or Warburg effect to develop resistance towards BRAF inhibitors (vemurafenib and dabrafenib). Our observations indicated that vemurafenib resistant cells possessed 1.5 fold higher extra-cellular acidification rate (ECAR), an indicator of glycolysis when compared to sensitive cells. We also observed that STAT3, a known regulator of glycolysis was up-regulated in A375 and SKMEL28 BRAF inhibitor resistant cells when compared to the parental cell line. In addition, LDH-A, a key glycolytic enzyme was significantly overexpressed in A375 vemurafenib resistant cells when compared to its parental counterpart. Consequently, we observed that vemurafenib and dabrafenib treatment induces the expression of STAT3 in A375 and SKMEL-28 melanoma cells. Our results showed that piperlongumine inhibits the survival of A375 vemurafenib resistant (A375VR) melanoma cells. Our observations indicated that the combination of piperlongumine with vemurafenib and dabrafenib shows synergistic effects by suppressing 75% - 85% of cell survival when compared to vemurafenib/dabrafenib and piperlongumine (25% - 45%) treatment alone in A375 and SKMEL-28 BRAF inhibitor resistant melanoma cells. Moreover, the combination of piperlongumine with vemurafenib or dabrafenib induced 2-8 fold apoptosis when compared to vemurafenib/dabrafenib and piperlongumine treatment alone in A375 and SKMEL-28 BRAF inhibitor resistant melanoma cells. Piperlongumine treatment reduced the ECAR (glycolysis) in a concentration dependent manner after 12h of treatment in A375VR cells and suppressed nearly 50% of ECAR parameters glycolysis and glycolytic capacity. Treatment of A375VR cells with piperlongumine for 48h inhibited the expression of LDH-A and its upstream regulators pSTAT3 (Y705), β-catenin, c-Myc and HIF-1α as evaluated by western blot. In addition, oral administration of piperlongumine and vemurafenib combination suppressed 86% of tumor growth when compared to vemurafenib treatment. Taken together, our study thus indicate that piperlongumine potentiates the effects of BRAF inhibitors by inhibiting Warburg effect and its upstream regulators. [Supported in part by R01 grant CA129038, awarded to (S.K.S) by the National Cancer Institute.

Citation Format: Sharavan Ramachandran, Neel Fofaria, Sanjay Srivastava. Piperlongumine, a novel treatment option to overcome BRAF inhibitor resistance in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3518.
Abstract 3521: Glutamine metabolic inhibition synergizes with L-asparaginase in MYCN-amplified neuroblastoma
Authors: Maxwell Micah J. , Poore Brad , Hanaford Allison , Alt Jesse , Rais Rana , Slusher Barbara S. , Eberhart Charles G. , Raabe Eric H.
Published: August 2018

Neuroblastoma is the most common extracranial solid tumor in children. Though it accounts for about 10% of pediatric cancers, it is disproportionately responsible for 15% of pediatric cancer deaths. MYCN is amplified in 20% of neuroblastomas and correlates with adverse outcome. MYCN is involved in the maintenance of cancer stem cells, as well as in driving tumor cell growth, proliferation and tumorigenesis. Attempts to inhibit MYCN directly have been largely unsuccessful. MYCN is known to drive tumor cell reliance on glutamine for cellular metabolism. 6-Diazo-5-oxo-L-norleucine (DON) is a well-characterized glutamine analogue that inhibits glutamine metabolism by irreversibly inactivating multiple glutamine-utilizing enzymes. DON was well tolerated in a previous phase I clinical trial in pediatric patients, but it has never been systematically tested in neuroblastoma patients. We show that MYCN-amplification confers sensitivity to DON therapy in in vitro models of neuroblastoma, and that DON administered by intraperitoneal injection twice weekly significantly reduces flank tumor volume in orthotopic mouse models of MYCN-amplified neuroblastoma (mean tumor volume 1715 mm3 vs. 207 mm3 in control animals, p-value = 0.00017 by t-test). We have also developed an orally bioavailable DON prodrug, JHU083, and we found that this drug administered orally three times weekly was similarly able to suppress neuroblastoma tumor growth in mice (mean tumor volume 1,115 mm3 vs. 217 mm3 in control animals, p-value = 0.0000088 by t-test). In metabolic flux experiments, tracing glutamine and glucose donation of 13C and 15N via liquid chromatography/mass spectrometry, DON prevents asparagine synthesis, depleting intracellular asparagine levels by 40% compared to control treated cells (p-value = 0.006). DON combined with L-asparaginase synergistically inhibits growth of MYCN-amplified neuroblastoma cell lines (CI = 0.25 by the Chou-Talalay method, indicating strong synergy; p-value = 0.00011). We conclude that DON depletes cellular pools of asparagine, and combination therapy with DON and L-asparaginase synergistically inhibits the growth of MYCN-amplified neuroblastoma. These studies provide the preclinical justification for potential clinical trials for the use of DON or DON prodrugs in combination with L-asparaginase as new therapeutic options for patients with MYCN-amplified neuroblastoma.

Citation Format: Micah J. Maxwell, Brad Poore, Allison Hanaford, Jesse Alt, Rana Rais, Barbara S. Slusher, Charles G. Eberhart, Eric H. Raabe. Glutamine metabolic inhibition synergizes with L-asparaginase in MYCN-amplified neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3521.
Abstract 3536: SENP1 is a p53 deSUMOylating enzyme and its depletion induces p53 activity
Authors: Dai Mushui , Chauhan Krishna , Chen Yingxiao , Sun Xiao-Xin
Published: August 2018

SUMOylation plays important roles in diverse cellular processes by regulating protein-protein interaction, protein localization, trafficking, stability and activity. p53 can by SUMOyalted at lysine (K) 384. Yet, how SUMOylation regulates p53 activity remain unclear. SUMOylation can be revered by a group of proteins called deSUMOylating enyzme including the SUMO specific protease (SENP) family proteins. How p53 is regulated by deSUMOylation is also not well understood. Here, we show that SENP1 directly interacts with and desumoylates p53 in cells and in vitro. Depletion of SENP1 by siRNA-mediated knockdown drastically increased the p53 transactivation activity without inducing p53 protein levels, resulting p53-depndent cell cycle arrast. Further, SENP1 knockdown significantly promoted p53 transactivation activity in cells following DNA damage. Additionally, we found that SENP1 binds to and deSUMOylates PML and aborageted the enhancing effect of PML on p53 SUMOylation. Thus, SNEP1 is novel p53 desumoylating enzyme that regulates p53 activity but not its stability.

Citation Format: Mushui Dai, Krishna Chauhan, Yingxiao Chen, Xiao-Xin Sun. SENP1 is a p53 deSUMOylating enzyme and its depletion induces p53 activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3536.
Abstract 3539: Establishing ubiquitylation patterns in cells: Efficient monitoring of oncogenic ubiquitylation activity
Authors: Foote Peter K. , Lu Xiaolong , Singh Rajesh
Published: August 2018

Cells regulate their internal functions by the ubiquitylation of many key proteins, especially onco-proteins and tumor suppressors. As a result, ubiquitin proteasome system (UPS) substrates and the relevant UPS enzymes have been identified as attractive anti-cancer targets. However, traditional methods for monitoring ubiquitylation patterns, including by immunoprecipitation coupled with immunoblot analysis (IP/IB) or by mass spectrometry methods, are either insensitive, low throughput, or highly intensive. Here, we describe three Tandem Ubiquitin Binding Entity (TUBE)-based assays, UbiTest, HT-UbiTest, and UbiQuant S, which provide a platform to efficiently detect ubiquitylation of anti-cancer targets. UbiTest is an immunoblot-based assay, which can be used to easily identify ubiquitlyated proteins. Compared to traditional ubiquitin IP/IB, this assay avoids epitope masking of antibodies caused by ubiquitin modification. HT-UbiTest extends this concept using a TUBE pulldown followed by two-antibody detection in a 96-well plate, and allows high-throughput absolute quantification of a target of interest. UbiQuant S is a high-throughput assay to quantify ubiquitylation status in cells or tissue. Ubiquitylated proteins are identified by energy transfer between AlphaLISA bead-labeled TUBE and antibody against the endogenous protein of interest in a homogenous plate-based assay. Furthermore, we have extended these technologies to identify K63- and K48-linked poly-ubiquitylated proteins using poly-ubiquitin linkage-selective TUBEs. Several prominent therapeutic targets in oncology and immuno-oncology have been used to verify these assays, including Cbl-b and its substrates, p53, and USP7. The efficiency and sensitivity of these assays have wide applications in drug discovery targeting ubiquitylation-dependent signaling pathways. Examples using DUB and ubiquitin ligase inhibitors illustrate the power of this system. These novel assays are versatile tools for understanding the role of ubiquitylation in cancer treatment, and provide platforms to monitor disease biomarkers in response to drug action.

Citation Format: Peter K. Foote, Xiaolong Lu, Rajesh Singh. Establishing ubiquitylation patterns in cells: Efficient monitoring of oncogenic ubiquitylation activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3539.
Abstract 3542: Missense mutations in USE1 promote lung tumorigenesis
Authors: Lee Peter C. W.
Published: August 2018

The UBA6-USE1 enzyme cascade is a poorly characterized arm of the ubiquitin-proteasome system. We found that UBA6 and USE1 proteins are frequently overexpressed in lung cancer patients (83.01% and 92.45%, respectively; n = 106). Stable overexpression of UBA6 or USE1 significantly increased cell proliferation, migration, and invasiveness in lung cancer cells and xenograft models, whereas their knockdown significantly reduced cell proliferation, migration, and invasion. USE1 has a conserved D-box domain and the level of the protein was regulated by the anaphase-promoting complex through its interaction with CDC20 and CDH1. Furthermore, several missense mutations in USE1 identified in patients prolong the half-life and stability of the protein. Our findings reveal novel roles for USE1 in lung cancer and the possible use of USE1 as a novel biomarker and therapeutic target for lung cancer treatment.

Citation Format: Peter C. W. Lee. Missense mutations in USE1 promote lung tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3542.
Abstract 3543: USP7 inhibitor P5091 inhibits esophageal squamous cell carcinoma growth by inducing NOXA-mediated apoptosis
Authors: Hu Tao , Zhang Jing-Yang , Sha Bei-Bei , Li Miao-Miao , Liu Xing-Ge , Zhang Yi , Dong Zi-Ming , Li Pei , Chen Ping
Published: August 2018

Recently, deubiquitinase USP7/HAUSP was reported to be an attractive anti-cancer target. However, its expression and role in esophageal cancer remain elusive. Here, immunohistochemistry and western blot assay were used to detect the expression of USP7/HAUSP in esophageal squamous cell carcinoma (ESCC) tissues. Specific inhibitor P5091 was used to inhibit the activity of USP7 and explore the role and mechanism of USP7 in KYSE450, KYSE510 and KYSE30 ESCC cell lines.

Result: Staining-intensity analysis by immunohistochemistry demonstrated that USP7 was expressed higher in ESCC tissues compared with their corresponding adjacent tissues (P<0.01), which was confirmed by western blot analysis. CCK-8 assay, colony formation assay and tumor growth assay in subcutaneous transplantation tumor model indicated that P5091 could significantly inhibit ESCC cell growth in vitro and in vivo. Moreover, P5091 induced apoptosis by triggering unfolded protein reaction, accumulating the protein of ATF4 and thus transcriptionally upregulating the expression of NOXA.

Conclusion: These results showed that P5091 effectively inhibited ESCC cell growth in vitro and in vivo and supported the clinical investigation of P5091 for the treatment of ESCC.

Keywords: P5091, esophageal squamous cell carcinoma, ATF4, apoptosis, NOXA

* T H and J-Y Z contributed to this work equally.

Citation Format: Tao Hu, Jing-Yang Zhang, Bei-Bei Sha, Miao-Miao Li, Xing-Ge Liu, Yi Zhang, Zi-Ming Dong, Pei Li, Ping Chen. USP7 inhibitor P5091 inhibits esophageal squamous cell carcinoma growth by inducing NOXA-mediated apoptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3543.
Abstract 3548: EPAC-RAP1, the alternative cyclic AMP signaling pathway, regulates cell cycle progression in primary melanoma cells
Authors: Krishnan Aishwarya , Prabhakar Kirthana , Ndiaye Mary , Ahmad Nihal , Rodriguez Carlos Ivan , Setaluri Vijayasaradhi
Published: August 2018

Cyclic AMP (cAMP) is an important second messenger that acts as a key mediator in a wide range of cellular pathways. In melanocytes, the G-Protein Coupled Receptor (GPCR) Melanocortin-1-Receptor (MC1R), an important regulator of melanocyte proliferation and melanogenesis, activates cAMP signaling. Polymorphisms in MC1R lead to cAMP signaling impairments which are accompanied by higher risk for melanoma development. Despite the importance of cAMP signaling in melanocytes, very little is known about its role in melanoma. Exchange Protein directly Activated by cAMP (EPAC) constitutes an alternative pathway to cAMP signaling and is known to mediate the crosstalk between the cAMP and MAPK pathways. In this study, we show that pharmacological inhibition of EPAC, delays cell cycle progression and inhibits the growth of human primary melanoma cells. Flow cytometry and western blot analysis of cell cycle proteins showed that inhibition of EPAC arrests cells in G1-S and delays their mitotic progression. Paradoxically, inhibition of EPAC also caused an increase in AKT phosphorylation, suggesting a cell survival response elicited upon the cell cycle arrest caused by inhibition of EPAC allows continued survival of primary melanoma cells. Inhibition of EPAC in metastatic melanoma cells, on the other hand, stimulated their growth. These data show differential role of EPAC in primary and metastatic melanoma cells and provide a basis for targeting EPAC for prevention of cutaneous melanoma progression.

Citation Format: Aishwarya Krishnan, Kirthana Prabhakar, Mary Ndiaye, Nihal Ahmad, Carlos Ivan Rodriguez, Vijayasaradhi Setaluri. EPAC-RAP1, the alternative cyclic AMP signaling pathway, regulates cell cycle progression in primary melanoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3548.
Abstract 3551: Lipid raft integrity is essential for metastasis and chemoresistance properties of CD133+ cancer stem cells in pancreatic cancer
Authors: Gupta Vineet K. , Sharma Nikita S. , Kesh Kousik , Dauer Patrcia , Nomura Alice , Giri Bhuwan , Dudeja Vikas , Saluja Ashok , Banerjee Sulagna
Published: August 2018

Pancreatic cancer remains a significant health burden associated with limited patient survival and poses a major therapeutic challenge. Among cancer cells, Cancer stem cells (CSCs) are instrumental in inducing chemoresistance and metastasis in pancreatic cancer. CD133 has been identified as a CSC surface marker in several malignancies including pancreatic cancer. However, the functional role of CD133 in CSCs still not clear. The present study investigates the role of caveolar lipid raft integrity in functional properties of CD133+ CSCs. Current study showed first time that CD133 localizes to caveolar lipid rafts in pancreatic cancer cells, and associates with Caveolin1 (Cav-1) and cholesterol to form an integral signaling complex which drives the downstream processes of chemoresistance and metastasis. Further analysis showed that the integrity of the lipid-raft is crucial for maintenance of the functional properties of pancreatic CSCs, and its disruption leads to increased chemo-sensitivity to chemotherapeutic compound paclitaxel both in vitro as well as in vivo. Additionally, disruption of lipid raft results in decreased invasiveness and metastatic ability of the cells by deregulation of FAK signaling. The study also reveals that pancreatic cancer cells that lack the CSC population marker, CD133, did not respond to lipid raft disruption. Our results indicated that targeting the lipid-raft integrity in pancreatic cancer can specifically eradicate the CD133+ CSCs in pancreatic tumors, leading to a better prognosis for the disease.

Citation Format: Vineet K. Gupta, Nikita S. Sharma, Kousik Kesh, Patrcia Dauer, Alice Nomura, Bhuwan Giri, Vikas Dudeja, Ashok Saluja, Sulagna Banerjee. Lipid raft integrity is essential for metastasis and chemoresistance properties of CD133+ cancer stem cells in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3551.
Abstract 3565: T cell receptor signaling-responsive single chain IL-12 and IL-15 superagonist nanogel “backpacks” to enhance adoptive cell therapy in solid tumors
Authors: Fichter Michael
Published: August 2018

Adoptive cell therapy (ACT) using patient-derived tumor-specific T cells is a promising approach for cancer treatment especially for the treatment of hematological malignancies. However, therapeutic approaches using ACT in patients with solid tumors, such as hepatocellular carcinoma (HCC), has not shown substantially improved clinical outcomes in particular due to immunosuppressive tumor microenvironments. Therefore, strategies to enhance ACT T cell functionality in vivo and to overcome the immunosuppressive microenvironments in solid tumors are strongly needed. We developed a strategy combining nanomedicine with ACT, based on the chemical conjugation of cytokine-loaded nanoparticles (NPs) as synthetic “backpacks” to the surfaces of live lymphocytes for adoptive therapy. Novel protein nanoparticles were synthesized by crosslinking of single chain IL-12 (scIL-12)- or IL-15 superagonist through disulfide-containing crosslinkers, forming nanogels that when bound to the surface of T cells, release the supporting cytokines in response to antigen receptor signaling-mediated changes in cell surface redox state. This TCR-mediated drug release allowed drug delivery to the T cells to be focused in tumors and tumor-draining lymph nodes. Coupling of cytokine nanogels to T cell membranes induced significant cell activation in terms of interferon-γ production or proliferation in a concentration-dependent manner. Antigen-specific CD8+ T cells “backpacked” with scIL-12 or IL-15SA nanogels were capable of substantial tumor cell killing in a B16F10 melanoma cancer model in vivo, while avoiding lethal systemic toxicities triggered by systemically-administered immunomodulatory cytokines. Ongoing studies are seeking to apply this approach in HCC, and this approach bears the potential for the development of successful immunotherapeutic strategies against this and other solid tumors where effective treatment options are still lacking.

Citation Format: Michael Fichter. T cell receptor signaling-responsive single chain IL-12 and IL-15 superagonist nanogel “backpacks” to enhance adoptive cell therapy in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3565.
Abstract 3580: Enhancing adoptive cell therapies through exogenous regulation
Authors: Richardson Celeste , Elpek Kutlu , Ols Michelle , Kassum Tariq , Shamah Steve
Published: August 2018

Adoptive cell therapy with chimeric antigen receptor (CAR) modified T cells has demonstrated remarkable clinical efficacy in the treatment of certain B cell malignancies and more recently in multiple myeloma. However, in solid tumors, CAR-T therapy has been far less successful, likely due to the lack of robust CAR-T cell expansion, the immunosuppressive microenvironment, and clonal heterogeneity within these tumors. The trafficking of CAR-T cells to tumor sites provides an opportunity for selective delivery of cargo that can enhance CAR-T cell activity at the site of the tumor. Interleukin-12 (IL12) and membrane tethered interleukin-15 (mbIL15) have previously been shown to enhance CAR-T activity, yet the unregulated expression of either compromises safety and/or efficacy. The Obsidian Therapeutics platform equips engineered immune cells with new functionalities whose activity can be regulated via the administration of FDA-approved small-molecule drugs, putting dosing control back in the hands of the treating physician. We utilize small, fully human protein sequences called destabilizing domains (DDs) that confer reversible destabilization to a fused target protein. In the absence of ligand the fusion protein is degraded, whereas the presence of small molecule ligand restores expression and functionality. Furthermore, stabilization is titratable with small molecule ligand dose, providing finely tuned control over target protein expression and function. We have applied our technology to create DD-IL12 and DD-mbIL15 “cassettes” that provide exogenous regulation of cytokine activity when transduced into CAR-Ts for enhanced cellular function. We describe here the successful implementation of the DD technology in engineered primary human T cells and show small-molecule ligand controlled regulation of DD-IL12 and DD-mbIL15 protein expression in vivo in mouse models. These data demonstrate the feasibility of exogenous control over protein expression in primary human T cells for the development of next-generation CAR-T cell products with enhanced efficacy and more favorable safety profiles.

Citation Format: Celeste Richardson, Kutlu Elpek, Michelle Ols, Tariq Kassum, Steve Shamah. Enhancing adoptive cell therapies through exogenous regulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3580.
Abstract 3589: Preclinical evaluation of mesothelin-specific T cell receptor (TCR) fusion constructs (TRuC™s) utilizing the signaling power of the complete TCR complex: A new opportunity for solid tumor therapy
Authors: Ding Jian , Patel Ekta , Tavares Patrick , Quinn Justin , Choudhary Rashmi , Le Bonnie , Kiner Olga , Weiler Solly , Getts Daniel R. , Baeuerle Patrick , Hofmeister Robert
Published: August 2018

Despite success in treating hematological malignancies, T cells expressing chimeric antigen receptors (CARs) have shown poor efficacy in solid tumor indications. The poor utility of CARs is thought to be due to a number of factors including T cell exhaustion and a lack of persistence. Utilizing only the CD3ζ chain of six distinct T cell receptor subunits in combination with a costimulatory domain, the CARs bypass the natural TCR signaling complex. The failure to initiate and harness a complete TCR response is arguably a primary underlying factor preventing CAR-T cell success in solid tumor indications. Here, we present a novel T cell engineering platform: T Cell Receptor Fusion Constructs (TRuC™s), which target tumors in a MHC non-restricted fashion. Unlike CARs, the constructs integrate into the TCR complex, harnessing the full potential of natural T cell activation, effector function and regulation. Here we demonstrate preclinical evidence underscoring the efficacy of TRuC™-T cells re-programmed to target the solid tumor antigen mesothelin (TC-210). TC-210 showed robust anti-tumor activity in vitro and in vivo in mesothelioma, lung and ovarian tumor models. Surprisingly, this activity was associated with less proinflammatory cytokine production compared to CAR-T cells with the same binder and was supported by strong TRuC™-T cell persistence. Taken together, mesothelin targeting TRuC™-T cells have potent anti-tumor activity and present a promising approach for treating mesothelin-expressing solid tumors.

Citation Format: Jian Ding, Ekta Patel, Patrick Tavares, Justin Quinn, Rashmi Choudhary, Bonnie Le, Olga Kiner, Solly Weiler, Daniel R. Getts, Patrick Baeuerle, Robert Hofmeister. Preclinical evaluation of mesothelin-specific T cell receptor (TCR) fusion constructs (TRuC™s) utilizing the signaling power of the complete TCR complex: A new opportunity for solid tumor therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3589.
Abstract 3597: Xenobiotic detoxification enzymes and cancer risk: A multiplex immunofluorescence detection of AE1/AE3, GSTA1 and GSTA3 in the tissue microarrays of gastrointestinal tract
Authors: Ma Victor Wan-San , Cheung Goretti Hoi-Yan , Lau Eunice Yuen-Ting , Cho William Chi-Shing
Published: August 2018

Background: Glutathione S-transferases (GSTs) are an enzyme family responsible for the metabolism of toxins and carcinogens in the phase II detoxification pathway. Among the 5 alpha class isoforms, GSTA1 and GSTA3 are involved in metabolizing several active carcinogenic substrates generated from benzo-[a]-pyrene mainly found in grilled meats and burnt coal. The expression of GSTA1 and GSTA3 in gastrointestinal (GI) tract may be implicated with cancer development associated with the intake of xenobiotic substances.

Methods: Formalin-fixed and paraffin-embedded tissue cores of esophagus (190), stomach (180), small intestine (372), colon (120) and rectum (142) were stained with a multiplex tyramide-immunofluorescence (T-IMF) technique for the simultaneous demonstration of cytokeratin AE1/AE3, GSTA1 and GSTA3. The tissue types range from normal to cancer progression spectrum. All tissue cores were screened for AE1/AE3 expression to confirm the epithelial cell lineage before scoring for GSTA1 and GSTA3 positivity. A p-value <0.05 by Chi-squared test is considered significant.

Results: T-IMF is useful in studying the interaction of multi-markers in a single slide. This technique is superior to chromogenic immunohistochemical application in multi-color presentation. This highly sensitive and handy technique will save multiple sectionings and precious tissues. We found a significant down-regulation of two alpha class GSTs in the malignancies of GI tract (Table 1).

Table 1. Positive percentages of glutathione S-transferases markersMarkerEsophagusStomachSmall intestineColonRectumNMNMNMNMNMGSTA141%24%99%40%90%15%22%3%43%21%p-value0.0603<0.0001<0.00010.0010.0194GSTA378%35%99%54%96%64%100%39%88%40%p-value<0.0001<0.0001<0.0001<0.0001<0.0001Abbreviation: N= normal; M= malignant

Conclusion: The differential expression of these detoxification enzymes in normal epithelium vs GI cancer may shed some light on their role in protecting normal tissue from cancer development. This finding may provide a potential target for the development of intervention strategies against GI cancer. Further investigations will be conducted to decipher the mechanism underlying this observation.

Citation Format: Victor Wan-San Ma, Goretti Hoi-Yan Cheung, Eunice Yuen-Ting Lau, William Chi-Shing Cho. Xenobiotic detoxification enzymes and cancer risk: A multiplex immunofluorescence detection of AE1/AE3, GSTA1 and GSTA3 in the tissue microarrays of gastrointestinal tract [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3597.
Abstract 361: Identification of a novel dependence on the SWI/SNF chromatin remodeling complex in uveal melanoma
Authors: Rago Florencia , Xiang Xiaoqin , Desplat Aurore , Niemezyk Emilie , Ruddy David , Kerr Grainne , Robles Eusebio Manchado , Castelletti Deborah , Jagani Zainab
Published: August 2018

Uveal melanoma is a rare and aggressive cancer that originates in the pigmented tissues of the eye, which include the iris, the choroid and the ciliary body. To date, no targeted therapies have been approved for this cancer, and much remains to be described about the underlying biology of the disease. The most common driver mutation is an activating mutation in either the GNAQ or GNA11 GTPase, which in turns leads to hyperactivation of the MAPK pathway, but the precise downstream effectors driving tumorigenesis are poorly understood. Using pooled shRNA screening, we discovered that the SWI/SNF complex represents a novel genetic vulnerability in uveal melanoma, and further interrogated this dependency by a more targeted analysis across various uveal melanoma cell lines. While we observed the dropout of multiple SWI/SNF complex subunits in the screens, we defined the ATPase activity of SWI/SNF as central to the observed phenotype, implicating the chromatin remodeling function of the SWI/SNF complex in this phenotype. The regulation of gene expression by transcription factors can involve coordinated activity of SWI/SNF directed chromatin remodeling activity, so we began to investigate mechanistic links between SWI/SNF and other transcription factors such as MITF, which is known to play important roles in the differentiation of melanocytes. We observed that depletion of SWI/SNF affected transcription of certain MITF target genes, and are further investigating the precise pathways that are altered with SWI/SNF modulation. Taken together, these data suggest that the SWI/SNF complex plays an important role in maintaining tumor cell survival in uveal melanoma, and may represent a novel therapeutic target.

Citation Format: Florencia Rago, Xiaoqin Xiang, Aurore Desplat, Emilie Niemezyk, David Ruddy, Grainne Kerr, Eusebio Manchado Robles, Deborah Castelletti, Zainab Jagani. Identification of a novel dependence on the SWI/SNF chromatin remodeling complex in uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 361.
Abstract 3626: A genome scale screen to identify regulators of PD-L1 in HNSCC
Authors: Mann Jacqueline , Ludwig Megan , Kulkarni Aditi , Kafelghazal Judy , Eisenberg Julia , Hoesli Rebecca , Nesvizhskii Alexey , Brenner Chad
Published: August 2018

The immune checkpoint inhibitor Pembrolizumab was recently approved for the treatment of metastatic and recurrent HNSCC, but only 18% of patients in the initial trial responded to therapy, and it remains difficult to predict patients likely to experience benefit. We expect a broader understanding of checkpoint regulation in HNSCC to lead to improved response rates and identification of potential responders. To this end, we sought to identify targetable genetic factors modulating PD-L1, a molecule that serves to dampen the anti-tumor immune response. We developed a high throughput screen employing a Genome-scale CRISPR Knock-Out (GeCKO) library in HNSCC cell lines to identify targetable genes and pathways that may regulate PD-L1 expression. In UM-SCC-49, stable knockout pools with representation of approximately 300 gRNAs for each of over 20,000 target genes were expanded and serially sorted to create stable sub-populations with enhanced or diminished PD-L1 expression. These sub-populations were sequenced to identify gRNAs that increased or reduced PD-L1 expression, indicating that their genetic targets may function to regulate PD-L1. We observed 70% library coverage (44,900 gRNAs) in the initial population and 15% and 11% coverage in the sub-populations selected for high and low PD-L1 expression, respectively. We prioritized for validation 61 gRNAs that were enriched at least 2 fold in High or Low PDL1 pool over the control pool. Future experiments focus on validation of these targets in UM-SCC-49 and other cell lines. As we expand this screen and validate hits across multiple genetic backgrounds, we expect to reveal signaling networks promoting immune evasion and potential targets for combination immunotherapeutic protocols.

Citation Format: Jacqueline Mann, Megan Ludwig, Aditi Kulkarni, Judy Kafelghazal, Julia Eisenberg, Rebecca Hoesli, Alexey Nesvizhskii, Chad Brenner. A genome scale screen to identify regulators of PD-L1 in HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3626.
Abstract 3647: Prior DNA repair improves the read quality in next-generation sequencing of cell-free tumor DNA
Authors: Yin Hong , Greer Adam H. , Mills Glenn
Published: August 2018

The cell-free tumor DNA (ctDNA) in patient's plasma is a noninvasive tumor biomarker. The detection of ctDNA is helpful to evaluate tumor progression, tumor response to the therapy, and subtyping of tumor cells. Targeting next-generation sequencing (NGS) technology provides a sensitive and specific technique for ctDNA detection. It can detect multiple tumor-related gene mutations. However, collection of high quality of cell-free DNA (cfDNA) may not be available in some cases. The quality of cfDNA in plasma is impacted by multiple factors, such as plasma collection and storage conditions, DNase activity in the blood, and the apoptotic and necrotic status of the tumor. These factors affect the integrity of cfDNA, leading to full or partial degradation of cfDNA, including double-strand DNA breaks and single strand DNA nicking. Lesions on cfDNA decrease the usability of ctDNA by digital PCR and targeted NGS. Due to amount limitation of cfDNA in some cases, the repair of damaged cfDNA could be a necessary rescue step in producing viable libraries for NGS.

CfDNA was extracted from the plasma with Zymo Research's Quick-cfDNA Serum and Plasma kit and was quantified by realtime PCR with Alu primer sets. The size of cfDNA was about 160 base pairs, as estimated by Agilent Tapestation 2200 with the High Sensitivity DNA kit. The DNA repair of cfDNA was conducted with PreCR Repair Mix of New England BioLabs. The sequencing library was prepared with Accel-Amplicon 56G Oncology Panel of Swift Biosciences and sequenced in Illumina's MiSeq. The quality of sequencing data was determined with FastQC. Compared to PreCR Repair Mix treated cfDNA samples, fastQ data from some of untreated cfDNA samples showed failed per base sequencing quality, about 50% of sequencing cycles with the mean quality score below 30. The per sequencing quality score of these cfDNA samples failed also to pass the quality filter. The read size distribution analysis indicated that about 60 to 70% of read sized between 30 to 45 bases and about 75 to 80% of read was smaller than 75 bases. However, sequencing data from same cfDNA samples treated with PreCR Repair Mix show a greatly improved sequencing quality. Both per base sequencing quality and per sequencing quality score passed quality filter. The above 97% of reads sized over 92 bases, complying with the requirement of 56G Oncology Panel.

In conclusion, the DNA repair process before library preparation is necessary to rescue damaged cfDNA for amplicon-based NGS.

Citation Format: Hong Yin, Adam H. Greer, Glenn Mills. Prior DNA repair improves the read quality in next-generation sequencing of cell-free tumor DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3647.
Abstract 3683: Reciprocal expressions of TFF-1 and TTF-1 in lung adenocarcinomas
Authors: Matsubara Daisuke , Soda Manabu , Yoshimoto Taichiro , Amano Yusuke , Kihara Atsushi , Sakuma Yuji , Endo Shunsuke , Hagiwara Koichi , Fukayama Masashi , Mano Hiroyuki , Niki Toshiro
Published: August 2018

The molecular targeted therapies against EGFR, ALK etc. have improved progression-free survival of patients with lung adenocarcinomas. However, targetable driver mutations have mainly been found in TTF-1-positive terminal respiratory unit (TRU)-type, but have yet been undiscovered in non-TRU type despite the advances in genomics technologies. In order to find the molecular candidate characterizing major subtype of non-TRU type adenocarcinomas, we compared TTF-1-positive/CDH1-positive adenocarcinoma cell lines (“TRU-type” cell lines) (n=11) and TTF-1-negative/CDH1-positive adenocarcinoma cell lines (“non-TRU-type” cell lines) (n=8), and found that TFF-1 (trefoil factor family-1), a gastric mucosal protective factor, was characteristically highly expressed in “non-TRU-type” cell lines. Immunohistochemical analysis of primary lung adenocarcinomas (n=70) revealed that TFF-1 expression was inversely correlated with TTF-1 expression (p=0.0116) and was very rare in primary lung adenocarcinoma cases (5.7%:4/70). So we focused on the non-TRU type adenocarcinomas on the basis of histologic pattern (acinar, solid or mucinous morphology predominant tumors without non-mucinous lepidic growth components) (n=43). Among them, TFF-1 was frequently expressed (62%:27/43), and significantly correlated with the expressions of MUC5AC, HNF4A and CK20 (p<0.0001, p=0.0001, and p=0.0007, respectively), and inversely correlated with TTF-1 expression (p=00039). Next generation sequencing analysis of the 43 cases revealed that KRAS gene mutations and Inactivating mutations and hypermethylation of the TTF-1 gene were frequent in TFF-1 positive cases. The analysis of publicly available data of 442 lung adenocarcinoma cases revealed that high expression of TFF-1 correlated with low expression of TTF-1 and worse prognosis. We confirmed that shRNA against TFF-1 inhibited the cell proliferation and the colony formation, and induced the apoptosis, in the TFF-1 positive “non-TRU-type” cell lines. These results indicate that TFF-1 is not only the marker but also can possibly be the molecular target of a subset of non-TRU-type lung adenocarcinomas.

Citation Format: Daisuke Matsubara, Manabu Soda, Taichiro Yoshimoto, Yusuke Amano, Atsushi Kihara, Yuji Sakuma, Shunsuke Endo, Koichi Hagiwara, Masashi Fukayama, Hiroyuki Mano, Toshiro Niki. Reciprocal expressions of TFF-1 and TTF-1 in lung adenocarcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3683.
Abstract 3706: Novel therapeutic strategy for pancreatic cancer with lectin drug conjugate (LDC): The efficacy and pilot safety test
Authors: Shimomura Osamu , Oda Tatsuya , Tateno Hiroaki , Ozawa Yusuke , Kimura Sota , Hirabayashi Jun , Ohkohchi Nobuhiro
Published: August 2018

Background: We recently developed lectin-drug conjugate (LDC) for targeting pancreatic cancer specific glycan and showed potent anti-cancer effect both in vitro and in vivo (Osamu Shimomura et al., Molecular Cancer Therapeutics 2017). The additional evaluation of anti-cancer effects and the toxicity studies of LDC need to be clarified.

Methods: LDC, rBC2LC-N lectin fused with 38 kDa of pseudomonas exotoxin (PE) A, was used in this study. We confirmed of the anti-cancer effect of LDC in orthotopic mouse models and developed dissemination mouse models to make sure of the survival benefits in LDC by both intra-peritoneally and intra-venously injection by log-rank test. We checked the damage of all organs pathologically after injection of the minimal or maximal tolerance doses and performed the blood sample analyses.

Results: We have already validated the cytotoxic effect of LDC for PDAC cell lines in vitro i.e., IC50= 1.04 pico g/ml. Intraperitoneal and intravenous injection of LDC reduced tumor weight from 390 mg in normal saline group to 130.8 mg and 203 mg, respectively in the orthotopic models (P=0.001 and P=0.007, respectively), and improved median survival duration from 62 days to 105 and 90 days in the peritoneal dissemination models, respectively (P<0.0001 and P<0.0001, respectively). Intravenous injection of LDC conferred comparable therapeutic effects to intraperitoneal injection. Inconsistent with the apprehension that all lectins mediate harmful hemagglutination, the rBC2LC-N never caused hemagglutination with human erythrocytes in every blood types. The laboratory test after injection of therapeutic dose (1µg/mouse) of LDC showed no obvious abnormality except slightly elevation of aspartate and alanine aminotransferase. Surprisingly, no adverse event were observed after administration of rBC2LC-N lectins without PE up to 15 µg/mouse in wild type mice. Although some epithelial desquamations were revealed in the mucosa of the gut after fatal dose of LDC injection, we could not figure out the organ damage after the therapeutic dose of LDC injection pathologically.

Conclusions: We show the successful in vivo application of a lectin for cancer treatment. Lectin itself did not cause severe adverse events and could be a potential candidate of a drug carrier, providing a new insight into anti-cancer drug development.

Citation Format: Osamu Shimomura, Tatsuya Oda, Hiroaki Tateno, Yusuke Ozawa, Sota Kimura, Jun Hirabayashi, Nobuhiro Ohkohchi. Novel therapeutic strategy for pancreatic cancer with lectin drug conjugate (LDC): The efficacy and pilot safety test [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3706.