bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 226: Optimization of folate receptor alpha-targeting tunable drug conjugates
Authors: Sigler Sara C. , Gupta Vinayak , Nguyen Hanh N. , Rood Michael K. , Wanner Jutta , Werner Doug S.
Published: August 2019

We have integrated a family of innovative technologies with an aligned set of properties into Tunable Drug Conjugates (TDCs). TDCs are made up of a targeting moiety (e.g. small molecules, peptides, antibodies or antibody fragments), combined with our proprietary Silicon-based linkers (SiLinkers) and novel Payload Cassette (PC) designs which allow for delivery of multiple payloads, preferentially Conditionally Active Payloads (CAPs). CAPs are cytotoxic moieties which cannot readily permeate into normal cells due to their charged state at physiological pH of 7.4; however, at the more acidic pH of the endosome or the tumor microenvironment, the charge is neutralized enabling the CAP to readily permeate into and accumulate within the cancer cells. Our goal is to establish a beneficial Therapeutic Index (TI) and dosing flexibility by creating TDCs around a rapid payload release/rapid systemic clearance approach. Therefore, SiLinker/CAP constructs are designed to concentrate payload in the targeted tumor cell and associated tumor microenvironment while minimizing systemic payload release and rapidly clearing the parent TDC. Folate Receptor alpha (FRα) is overexpressed in multiple cancers, most notably, ovarian, endometrial, and NSCLC. Our optimized FRα-targeting TDCs use folic acid as the ligand and deliver various triple- and hexa-CAP cassettes. The various CAPs were selected from different target classes in order to allow for optimized treatment of different cancer types. We will present comparative cellular data, in vivo xenograft studies in KB, OV90 and IGROV1 models, as well as DMPK parameters of our lead FRα-TDCs. Using our triple- and hexa-PCs, we have achieved cures in in vivo xenograft studies which were durable and lasted even after treatment was stopped. We also demonstrate that in vivo our hexa-PC shows superior efficacy compared to our triple-PC in the moderate FRα expressing models. Based on our data, we believe that the combined properties of our innovative CAP-TDCs and rapid payload release/rapid systemic clearance approach will further increase TI, differentiate from other Drug Conjugates, and ultimately provide a compelling addition to existing Drug Conjugate technologies.

Citation Format: Sara C. Sigler, Vinayak Gupta, Hanh N. Nguyen, Michael K. Rood, Jutta Wanner, Doug S. Werner. Optimization of folate receptor alpha-targeting tunable drug conjugates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 226.
Abstract 2263: Ceramide mimetics regulation of tumor cell response to immune checkpoint inhibitor immunotherapy
Authors: Redd Priscilla S. , Lu Chunwan , Klement John D. , Ibrahim Mohammed , Yang Dafeng , Payne Daniela , Lebedyeva Iryna , Liu Kebin
Published: August 2019

Programmed cell death-1 (PD-1) is an immunosuppressive receptor expressed on T cells upon activation. Once interacting with its ligand, programmed death ligand-1 (PD-L1), the PD-1:PD-L pathway curbs T cell activation and thereby serves as a natural protection mechanism against autoimmunity. However, this pathway has been hijacked by cancer cells in order to evade the immune system. This interface has already been recognized as an effective target for cancer therapy leading to the development of immune checkpoint inhibitor (ICI) immunotherapy. Blocking the PD-1:PD-L axis unleashes T cells from suppression to maintain their effector function against tumor cells. ICI has durable efficacy in many types of cancer; however, colorectal cancer, except for the approximately 4% microsatellite-instable (MSI) colorectal cancer, stands out as one of the few human cancers that does not respond to ICI immunotherapy. One potential mechanism that these tumor cells may be using is resisting cell death, making them insensitive to apoptosis induction by T cells. Ceramide, the central metabolite of the sphingolipid metabolism pathway, plays a critical role in many cell signaling processes, and one of its most prevalent roles can be seen in cell death. Our lab has developed five novel ceramide mimetics (IG4, IG7, IG14, IG17, and IG19) which function to sensitize tumor cells to apoptosis and enhance T cell-mediated cell death. We have previously shown that these ceramide mimetics significantly increase FasL-induced apoptosis by perforin-deficient cytotoxic T lymphocytes. Using the murine colon carcinoma cell line CT26, we show that the ceramide mimetics induce tumor cell death in a dose-dependent manner. Interestingly, the ceramide mimetics appear to be functioning by different mechanisms in vitro. Still, we report here that these five ceramide mimetics exhibit tumor suppression activity in a colon tumor mouse model in a dose-dependent manner in vivo. Also, ceramide mimetic IG19 significantly increased the efficacy of anti-PD-1 mAb immunotherapy in suppressing colon tumor development in vivo. Our data thus indicate that ceramide mimetics are potential effective enhancers for ICI immunotherapy in colon cancer.

Citation Format: Priscilla S. Redd, Chunwan Lu, John D. Klement, Mohammed Ibrahim, Dafeng Yang, Daniela Payne, Iryna Lebedyeva, Kebin Liu. Ceramide mimetics regulation of tumor cell response to immune checkpoint inhibitor immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2263.
Abstract 2309: Chimeric antigen receptor T-cell therapy targeting glypican-1 in pancreatic cancer
Authors: Li Nan , Li Dan , Ren Hua , Torres Madeline , Ho Mitchell
Published: August 2019

Pancreatic cancer is the fourth most common cause of death from cancer in the US, with a 5-year overall survival of 8.5%. While immunotherapy with chimeric antigen receptor (CAR) T cells has shown promise in certain hematological malignancies, their efficacy for solid tumors including pancreatic cancer remains elusive. Thus, laboratory research is urgently needed to identify and validate a new target of CAR T-cell therapy for patients with pancreatic cancer. In this study, we made CAR T cells that recognize glypican-1 (GPC1), a cell surface heparan sulfate proteoglycan protein that is overexpressed in pancreatic cancer. We isolated two GPC1-specific antibodies; the mouse monoclonal antibody HM2 that binds the C-lobe of GPC1 close to the cell surface, and the camel single domain antibody D4 that recognizes a conformational epitope in the protein core of GPC1. The transduction efficiencies of CAR (HM2) and CAR (D4) in primary T cells were 86% and 58%, respectively. GPC1-overexpressing KLM1 cells (2B9) and GPC1-overexpressing A431 cells (H8) were efficiently lysed by both CAR T cells in dose-dependent manners. By contrast, minimal cell lysis was observed in GPC1-negative cell line A431. In the presence of GPC1-positive tumor cells, greater amount of TNF-α, IFN-γ and IL-2 were produced by GPC1-specific CAR T cells than mock-transduced T cells. Although both CAR T cells showed similar cytolytic activity in vitro, CAR (D4) T cells induced 2-4 times more cytokines than CAR (HM2) T cells when co-cultured with GPC1-postive tumor cells. Notably, both GPC1-targeted CAR T cells demonstrated potent antitumor efficacy in a peritoneal dissemination xenograft mouse model. Our findings indicate that GPC1-targeted CAR T cell therapy could be a new approach for treating pancreatic cancer.

Citation Format: Nan Li, Dan Li, Hua Ren, Madeline Torres, Mitchell Ho. Chimeric antigen receptor T-cell therapy targeting glypican-1 in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2309.
Abstract 231: Optimizing lysosomal activation of antibody-drug conjugates (ADCs) by incorporation of novel cleavable dipeptide linkers
Authors: Salomon Paulin , Harris Luke , Reid Emily E. , Maloney Erin K. , Wilhelm Alan J. , Miller Michael L. , Chari Ravi V. , Keating Thomas A. , Singh Rajeeva
Published: August 2019

Peptides constitute a major linker class in antibody-drug conjugates (ADCs), designed to connect antibodies to cytotoxic drug molecules. An optimal linker for an ADC should be stable in circulation and be cleaved efficiently in lysosomes upon binding and internalization of the ADC in target cells, releasing drug molecules that exit lysosomes and inhibit key cellular functions. Previous studies have focused on a limited selection of peptide linkers and specific biochemical tools, such as a cathepsin B cleavage assay, leading to the selection of a valine-citrulline dipeptide linker with a p-aminobenzyloxycarbonyl (PABC) spacer, which is now often used in candidate ADCs.

In this study, we screened a panel of dipeptide linkers for efficient lysosomal proteolysis. Dipeptide linkers bearing different amino acids (typically both L, with D used as a control) were synthesized with a fluorogenic leaving group, 7-amino-4-methylcoumarin, which provided a convenient model system for screening of peptide cleavage using lysosomal extracts from cancer cells as well as several individual cathepsins. The linkers were also tested for stability in mouse, rat, cynomolgus, and human plasma.

Based on these screens, we identified several novel, previously unreported peptide linker designs and incorporated them into ADCs bearing a DNA-alkylating indolinobenzodiazepine (IGN) payload. These ADCs with different peptide linkers were assayed for in vitro cytotoxicity in multiple cancer cell lines, in vivo efficacy in human tumor xenograft models in mice, and ex vivo plasma stability. In addition, we measured the impact of peptide linkers on the kinetics of proteolytic processing of ADCs in cancer cell lines. We observed that several dipeptide linker designs were superior in rates of lysosomal processing compared to a reference standard L-Ala-L-Ala dipeptide linker.

Citation Format: Paulin Salomon, Luke Harris, Emily E. Reid, Erin K. Maloney, Alan J. Wilhelm, Michael L. Miller, Ravi V. Chari, Thomas A. Keating, Rajeeva Singh. Optimizing lysosomal activation of antibody-drug conjugates (ADCs) by incorporation of novel cleavable dipeptide linkers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 231.
Abstract 2315: Alteration of tumor metabolism by CD4+ T cells leads to TNF-α-dependent intensification of oxidative stress and tumor cell death
Authors: Ding Zhichun , Habtetsion Tsadik , Zhou Gang
Published: August 2019

The inhibitory effects of cancer on T cell metabolism have been well established, but the metabolic impact of immunotherapy on tumor cells is poorly understood. Here, we developed a CD4+ T cell-based adoptive immunotherapy protocol that was curative for mice with implanted colorectal tumors. By conducting metabolic profiling on tumors, we show that adoptive immunotherapy profoundly altered tumor metabolism, resulting in glutathionedepletion and accumulation of reactive oxygen species (ROS) in tumor cells. We further demonstrate that T cell-derived tumor necrosis factor alpha (TNF-α) can synergize with chemotherapy to intensify oxidative stress and tumor cell death in an NADPH (nicotinamide adenine dinucleotide phosphate hydrogen) oxidase-dependent manner. Reduction of oxidative stress, by preventing TNF-α-signaling in tumor cells or scavenging ROS, antagonized the therapeutic effects of adoptive immunotherapy. Conversely, provision of pro-oxidants after chemotherapy can partially recapitulate the antitumor effects of T cell transfer. These findings imply that reinforcing tumor oxidative stress represents an important mechanism underlying the efficacy of adoptive immunotherapy.

Citation Format: Zhichun Ding, Tsadik Habtetsion, Gang Zhou. Alteration of tumor metabolism by CD4+ T cells leads to TNF-α-dependent intensification of oxidative stress and tumor cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2315.
Abstract 2325: Long term time-course monitoring of NK cell-mediated ADCC using the Celigo Image Cytometer
Authors: Chan Leo L. , Andrade Lucas Ferrari De , Thomas Charles A. , Kuksin Dmitry , Wucherpfennig Kai
Published: August 2019

Antibody-dependent cell-mediated cytotoxicity (ADCC) assay has been widely performed for immuno-oncological research. Traditionally, ADCC assays are conducted by measuring the amount of released Chromium, calcein AM, or Lactate dehydrogenase (LDH) molecules after the target cancer cells are killed by effector/antibody pair. These methods can be inaccurate due to indirect measurement of supernatant at the end point of 4 hour, however, there is a need to characterize the effects of antibody-dependent cytotoxicity for longer than 72 hours. Previously, we have demonstrated an image-based ADCC detection method using the Celigo Image Cytometer, where the target cells are stained with calcein AM and other tracer dyes to monitor cell count or viability. However, the fluorescence of these dyes can significantly diminish within a 48 hour time frame. In this work, we demonstrated the time-course monitoring of NK cell-mediated ADCC of A375 cells in the presence or absence of IL2 and antibodies for 76 hours. First, A375-ZsGreen cells were seeded into a 96-well plate with and without target antibodies. Next, NK cells with and without IL2 were added to the wells at 10:1, 5:1, 2:1, and 1:1 E:T ratios. The co-culture was allowed to incubate for 76 hours and the image cytometer was used to scan and analyze at times 0, 3, 4, 6, 24, 27, 30, 51, 74, and 76 hours. The software was able to count individual cells as well as confluence % at each time point by segmenting highly fluorescent objects in the images. We were able to show time-dependent ADCC killing with decrease in ZsGreen positive cells as the read-out. In addition, the uniformity of cell killing can be observed in the Celigo obtained whole well images. One of the most important findings was that there was regrowth of target cells after the initial 30 hours of cell killing, indicating the activated NK cells without antibodies did not eradicate the cancer cells. Therefore, the ability to perform long term time-course monitoring of ADCC is highly important to better characterize the effects of target antibodies on cell killing function.

Citation Format: Leo L. Chan, Lucas Ferrari De Andrade, Charles A. Thomas, Dmitry Kuksin, Kai Wucherpfennig. Long term time-course monitoring of NK cell-mediated ADCC using the Celigo Image Cytometer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2325.
Abstract 2373: The mechanism study of propranolol in suppressing the genesis and development of gastrointestinal cancer through simultaneously activating autologous CD8+T cells and inhibiting tumor AKT/MAPK pathway
Authors: Liao Ping
Published: August 2019

Background: Propranolol may exert the anti-tumor effect in cutaneous and acral melanoma by suppressing AKT and MAPK pathways. Reduced tumor growth following propranolol results from enhanced CD8+T-cell-mediated antitumor immune response.

Objective: To clarify the efficacy and mechanism of propranolol in suppressing the genesis and development of gastrointestinal cancer through simultaneously activating autologous CD8+T cells and inhibiting tumor AKT/MAPK pathway.

Methods: MTS assay was performed to detect gastrointestinal cancer cells viability, TUNEL staining was used to measure apoptosis in cell lines. IHC was used to detect the expressions of Ki67/p-AKT/p-MEK/p-ERK in tumor tissue. Flow cytometry was performed to explore the immune status of the tumor microenvironment. Clinical study has been performed by Case-control design. Subjects who had not been received neoadjuvant were randomly divided into propranolol or placebo group. Then the primary foci of gastrointestinal cancer subjects were removed by D2 gastrectomy. IHC was used to observe the immune status, tumor proliferation in tumor tissue.

Results: We found propranolol reduced cell viability in CT26WT and MFC (mice derived melanoma cell line). Furthermore, propranolol could shrink the tumor size in CT26WT and MFC xenografts (P<0.0001). The frequency of CD3+CD8+ T cells in spleen was significantly elevated in propranolol-treated mice compared with controls. The IFNg and T-bet was significantly increased in the CD8+ T-cell population of propranolol treated mice tumor tissue. Propranolol reduced cell viability and induced apoptosis in AGS, HGC, SW480 and HCT116 cell line. But We did not observe differences in NCM460 cell line (human intestinal epithelial cells). Western Blot showed propranolol reduced the expression of p-AKT, p-MEK and p-ERK. In this prospective clinical study, the frequency of CD8+ was significantly elevated in tumor microenvironment. The expression of p-AKT, p-MEK and p-ERK was decreased in propranolol-treated subjects compared with controls.

Conclusion: The potential of propranolol to suppress the genesis and development of gastrointestinal cancer through simultaneously activating autologous CD8+T cells and inhibiting tumor AKT/MAPK pathway.

Note: This abstract was not presented at the meeting.

Citation Format: Ping Liao. The mechanism study of propranolol in suppressing the genesis and development of gastrointestinal cancer through simultaneously activating autologous CD8+T cells and inhibiting tumor AKT/MAPK pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2373.
Abstract 2377: Development and characterization of scFvs against TIP1/PDZ protein for cancer targeting
Authors: Singh Abhay , Kapoor Vaishali , Diebolder Philipp , Rhee Aaron , Rogers Buck , Thotala Dinesh , Hallahan Dennis
Published: August 2019

We identified TIP1 as a potential target to treat various cancers. TIP1 is over-expressed in glioblastoma, lung, head and neck and breast cancer. It is also a radiation inducible neo-antigen. TIP1consists of a single PDZ domain that binds the c-terminus of various cellular proteins like β-catenin, Rho, FAS, etc. It plays an important role in cell signaling, cancer development, and progression. TIP-1’s involvement in various survival pathways makes it an attractive target for anticancer therapeutics. Cancer can be targeted by different agents like antibodies (monoclonal, scFv, bi-specific, diabodies), peptides and small molecules. We will be targeting TIP1 using single-chain variable fragment (scFv) antibodies. scFv is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a peptide linker. We generated a phage-displayed scFv library (WASHU 2) from patient samples. For library screening, we cloned and purified the recombinant TIP1 protein. We used the TIP1 protein as bait for biopanning to pulldown the TIP1-binding scFvs from the library. Three rounds of biopanning were performed to enrich the specific TIP1 binding scFvs. Phage ELISA was performed to confirm the binding of the identified clones to the TIP1 protein. Approximately fifty-one clones showing positive binding to TIP1 protein were identified.We have sequenced the identified clones to verify the full length of scFv sequence. Unique clones are being characterized and developed further as targeting agents of various cancers. We found cancer specific binding of our lead scFvs in mouse models of cancer. The lead scFvs will be expressed as scFv-Fc to enhance their biological activity. We will characterize the scFv-Fcs for their binding affinity to TIP1, cancer cell surface binding, in vivo tumor targeting and tissue cross-reactivity. The scFvs having the potential to internalize will be developed as immunotoxins.

Citation Format: Abhay Singh, Vaishali Kapoor, Philipp Diebolder, Aaron Rhee, Buck Rogers, Dinesh Thotala, Dennis Hallahan. Development and characterization of scFvs against TIP1/PDZ protein for cancer targeting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2377.
Abstract 2384: TAK-169, an exceptionally potent CD38 targeted engineered toxin body, as a novel direct cell kill approach for the treatment of multiple myeloma
Authors: Willert Erin K. , Robinson Garrett L. , Higgins Jack P. , Liu Jensing , Lee Janice , Syed Sakeena , Zhang Yuhong , Tavares Dan , Lublinsky Anya , Chattopadhyay Nibedita , Wang Haiqing , Packer Laura , Shi Pu , Harbison Carole , Patel Sanjay , Newcomb John
Published: August 2019

Advances in the treatment of multiple myeloma have been driven by the development of new modalities, including proteasome inhibitors, immunomodulatory agents and monoclonal antibodies. TAK-169 is an engineered toxin body (ETB) comprising a proprietarily engineered, deimmunized form of the ribosome inactivating Shiga-like toxin A-subunit (SLTA) genetically fused to an antibody fragment that specifically targets the CD38 cell surface receptor. Although CD38 is a poorly internalizing receptor, TAK-169 is able to efficiently internalize and directly kill CD38-expressing cells through the enzymatic and irreversible inhibition of protein synthesis. This novel mechanism of action may be relevant in patients who have progressed after or are unlikely to respond to CD38-targeted antibody therapy. A library of unique CD38-targeting antibodies fused to deimmunized SLTA was screened in a variety of efficacy and safety models to identify TAK-169. TAK-169 demonstrated potent cytotoxicity across a range of myeloma cell lines with a range of CD38 expression in vitro as well as in patient-derived samples including with previous exposure to daratumumab. TAK-169 retains activity in whole blood and PBMCs, indicating that the presence of red blood cells is not restrictive to cytotoxicity. Furthermore, TAK-169 retains activity in the presence of excess approved, CD38 targeted therapeutic daratumumab. TAK-169 is efficacious in several myeloma xenograft models, both subcutaneous and disseminated. Complete regressions were observed using both a once-weekly and bi-weekly schedule. In addition, a syngeneic model cell line expressing human CD38 was shown to be responsive to TAK-169 treatment in an immunocompetent mouse model, suggesting the possibility of immune mediated cell death induced by TAK-169. Tolerability studies in non-human primates demonstrate that repeat administration is tolerated at doses expected to be efficacious. Takeda Pharmaceuticals and Molecular Templates jointly discovered TAK-169 and are co-developing the molecule for the treatment of multiple myeloma. TAK-169 is expected to enter clinical studies in 2019.

Citation Format: Erin K. Willert, Garrett L. Robinson, Jack P. Higgins, Jensing Liu, Janice Lee, Sakeena Syed, Yuhong Zhang, Dan Tavares, Anya Lublinsky, Nibedita Chattopadhyay, Haiqing Wang, Laura Packer, Pu Shi, Carole Harbison, Sanjay Patel, John Newcomb. TAK-169, an exceptionally potent CD38 targeted engineered toxin body, as a novel direct cell kill approach for the treatment of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2384.
Abstract 2396: Development of a new generation of dimeric, highly cytotoxic fusion proteins containing active GrB and VEGF targeting tumor neovasculature: Incorporating IgG heavy chain Fc fragments improves cytotoxicity, stability and pharmacokinetics
Authors: Mohamedali Khalid A. , Cheung Lawrence H. , Alvarez-Cienfuegos Ana , Rosenblum Michael G.
Published: August 2019

VEGF121 is a naturally-occurring splice variant that binds to VEGF receptors R-1 and R-2, which are over-expressed on the endothelium of tumor vasculature but not normal vasculature. The serine protease granzyme B (GrB) is capable of inducing intense cellular apoptosis through both caspase-dependent and caspase-independent multiple-cascade mechanisms. We have previously reported the expression of a fusion protein composed of Granzyme B and VEGF121 (GrB/VEGF121) and its subsequent in vitro and in vivo characterization. Here we report on the development and characterization of GrB-Fc-VEGF, which fuses the two domains via an IgG heavy chain Fc-fragment. We hypothesized that this modification, resulting in a relatively high molecular weight (133 kDa vs 80 kDa) would result in improved in vivo circulation and efficacy. VEGF inter-chain dimerization domains were modified such that dimerization occurred only through the Fc domain. GrB-Fc-VEGF121was expressed in HEK-293E cells under serum-free conditions and purified from the media by immobilized metal affinity chromatography (25 mg/L). Western blotting confirmed incorporation of both VEGF121 and GrB moieties into the construct. Comparison of GrB-Fc-VEGF121 with its expected molecular weight suggests significant glycosylation when produced in HEK293E cells. The enzymatic activity of GrB in GrB-Fc-VEGF121 was comparable to that of human GrB. In vitro studies of GrB-Fc-VEGF121 suggests improved cytotoxicity against cell lines expressing high levels of VEGFR-1 or VEGFR-2, unlike GrB/VEGF121, which targeted VEGFR-2+ cells more efficiently. Thus, the GrB-Fc-VEGF121 produced in HEK293E cells is active and has significant potential as a targeted therapeutic. In vitro cytotoxicity against an expanded panel of cells, internalization, and in vivo pharmacokinetics and efficacy against various tumor models are currently ongoing and will be presented. Research conducted, in part, by the Clayton Foundation for Research.

Citation Format: Khalid A. Mohamedali, Lawrence H. Cheung, Ana Alvarez-Cienfuegos, Michael G. Rosenblum. Development of a new generation of dimeric, highly cytotoxic fusion proteins containing active GrB and VEGF targeting tumor neovasculature: Incorporating IgG heavy chain Fc fragments improves cytotoxicity, stability and pharmacokinetics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2396.
Abstract 2421: Factors associated with abnormal cervical cancer screening results among screening compliant women in the United States
Authors: Chido-Amajuoyi Onyema Greg , Shete Sanjay
Published: August 2019

Introduction:

Formal guidelines recommend routine cervical cancer screening using the Papanicolaou (Pap) test and / or screening for high risk Human Papillomavirus (HPV). This study aims to describe the factors associated with abnormal cervical cancer screening outcomes.

Methods:

We analyzed data from 7,716 women, aged 30 to 65 years, who were respondents of the 2015 Cancer Control Supplement (CCS) of the National Health Interview Survey (NHIS). Weighted multinomial logistic regression was used to examine the factors associated with i) abnormal Pap test, ii) positive high-risk HPV test, as well as iii) abnormal Pap and positive high-risk HPV tests.

Results:

Overall, 16.8% [95% CI, 15.6 to 18.0] of women who screened for cervical cancer in the last 3 years, had an abnormal result. Risk of abnormal cervical cancer screening results decreased as age increased. Compared to the younger age group (30-45 years), those aged 56-65 years had lower odds of an abnormal Pap test (aOR: 0.71, 95% CI: 0.55, 0.92), positive high-risk HPV test (aOR: 0.51, 95% CI: 0.27, 0.97) or abnormal Pap and positive high-risk HPV tests (aOR: 0.55, 95% CI: 0.35, 0.88). Similarly, women who were married or living with a partner were less likely to have an abnormal Pap test (aOR: 0.83, 95% CI: 0.69, 1.00), than unmarried women. On the contrary, geographic region, race and cigarette smoking were found increase the odds of an abnormal cervical cancer screening result. Blacks were more likely to have abnormal screening results compared to Whites. Current smokers had increased odds of abnormal Pap test (aOR: 1.33, 95% CI: 1.02, 1.73) and positive high-risk HPV test (aOR: 1.94, 95% CI: 1.13, 3.32), compared to never smokers.

Discussion:

Study findings call for continued surveillance of cancer screening outcomes. More so there is a need for further studies to examine factors promoting an increased likelihood of abnormal cervical cancer screening results in populations identified in this study.

Note: This abstract was not presented at the meeting.

Citation Format: Onyema Greg Chido-Amajuoyi, Sanjay Shete. Factors associated with abnormal cervical cancer screening results among screening compliant women in the United States [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2421.
Abstract 2440: The microRNA-mRNA interactions in ALK-rearranged lung adenocarcinoma
Authors: Subat Sophia , Inamura Kentaro , Ninomiya Hironori , Nagano Hiroko , Okumura Sakae , Ishikawa Yuichi
Published: August 2019

Background: ALK-rearranged lung adenocarcinoma, which is responsive to ALK inhibitors, exhibits distinct clinicopathological and molecular features. In ALK-rearranged lung cancer, miRNA/mRNA expression signatures or individual miRNA-mRNA interactions have been reported. However, no study has performed integrative analysis to identify comprehensive miRNA-mRNA interactive network in this type of tumor. Therefore, we conducted this study to reveal a landscape of miRNA-mRNA interactions present in ALK-rearranged lung adenocarcinoma.

Methods: To identify differentially expressed miRNAs and mRNAs according to ALK-rearranged status, microarray expression profiling was conducted using 77 surgically resected specimens of lung adenocarcinoma with a known ALK status (42 ALK-positive and 35 ALK-negative cases). We made a multistep bioinformatics approach to build an miRNA-mRNA regulatory network observed in ALK-rearranged lung tumors.

Results: Seventy-three miRNAs (comprising 56 downregulated and 17 upregulated miRNAs) were differentially expressed according to ALK-rearranged status. The 56 miRNAs included miR-19a-3p, miR-362-5p, and miR-340-5p, all of which have been reported to be downregulated in ALK-rearranged lung tumors. On the basis of miRNA expression data, we identified 80 distinct biological processes in ALK-positive tumors, which included “proteoglycans in cancer”, “FoxO signaling pathway”, “pathways in cancer”, “mTOR signaling pathway”, and “HIF-1 signaling pathway”. The mRNA expression signature of ALK-rearranged tumors included ALK, ETV1, and CX3CL1, all of which have been

associated with lung tumorigenesis or tumor progression. Integrative analyses of miRNA and mRNA expression data revealed a refined list of putative miRNA-mRNA correlations. Of 136 putative miRNA-mRNA interactions, we identified 101 distinctive interactions with potential involvement in oncogenic machinery in ALK-positive tumors, such as miR-19a-3p/TLR2, miR-362-5p/SLC34A2, miR-1260b/ETV1, and miR-4481/CX3CL1 pairs. Network structural analysis provided a comprehensive view of complex miRNA-mRNA interactions in ALK-rearranged lung adenocarcinoma.

Conclusion: Overall, this observational study provides insight into the unique miRNA-mRNA regulatory network present in ALK-rearranged lung adenocarcinoma. Our findings, if validated, would inform future research examining the interplay of miRNAs and mRNAs in ALK-rearranged tumors.

Citation Format: Sophia Subat, Kentaro Inamura, Hironori Ninomiya, Hiroko Nagano, Sakae Okumura, Yuichi Ishikawa. The microRNA-mRNA interactions in ALK-rearranged lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2440.
Abstract 2463: Prototype open-access biomarker knowledgebase for genetic tests for breast cancer
Authors: Baker Dara , Abrams Natalie Fedorova , Bell Amanda , Colbert Maureen , Dingerdissen Hayley , Holmes Evan , Gupta Samir , Kahsay Robel , Kincaid Heather , Liu David , Mahmood A. S. M. Ashique , Bastian Frédéric B. , Robinson-Rechavi Marc , Schwartz Elena , Vijay-Shanker K. , Crichton Daniel , Mazumder Raja
Published: August 2019

Cancer biomarkers have become integral components of many clinical protocols and research studies. Nonetheless there still is an urgent need for more accurate, less invasive, and cost-effective biomarker tests to advance precision oncology. Despite considerable success in the preclinical setting, most candidate biomarker tests have yet to receive regulatory clearance or approval and gain market acceptance. To better understand the challenges facing modern biomarker research and facilitate access to biomarker-related information, this study aims to delineate the paths to approval of genetic tests for breast cancer. Public databases were searched to identify approved tests and respective biomarkers. A prototype format for unified biomarker information was developed iteratively due to the highly variable presentation of this data. A knowledgebase of FDA-approved genetic biomarkers for breast cancer was built as a pilot implementation of this format. This model is extensible to a larger database of biomarkers to be hosted in OncoMX (https://oncomx.org), which integrates cancer mutation, differential expression, literature mining, and pathway information. Fields in the prototype database were populated based on findings from the FDA website, PubMed, and other open-source databases. The resulting dataset is structured with the corresponding readme following the BioCompute Object (BCO) model. This BCO-compliant approach enables provenance capture and transparency of data processing, both of which are critical to large-scale data integration efforts. The adherence to BCO standards is expected to enhance the usability of the resulting data and to streamline the subsequent integration of multiple biomarker datasets. In the rapidly evolving field of oncologic biomarker research, this open-source biomarker knowledgebase offers clinicians and researchers a streamlined access to genetic tests information including corresponding genes, guides to evidence, clinical trial data, links to archival databases, and context for the behavior of the implicated gene in other disease and normal processes.

Citation Format: Dara Baker, Natalie Fedorova Abrams, Amanda Bell, Maureen Colbert, Hayley Dingerdissen, Evan Holmes, Samir Gupta, Robel Kahsay, Heather Kincaid, David Liu, A. S. M. Ashique Mahmood, Frédéric B. Bastian, Marc Robinson-Rechavi, Elena Schwartz, K. Vijay-Shanker, Daniel Crichton, Raja Mazumder. Prototype open-access biomarker knowledgebase for genetic tests for breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2463.
Abstract 2475: FLT3-Explorer: A bioinformatics pipeline for detecting low signal in FLT3 internal tandem duplications in acute myeloid leukemia
Authors: Laing Christian , Shi Wenge , Pollner Reinhold
Published: August 2019

Introduction. Acute Myeloid Leukemia (AML) is the most common acute Leukemia among adults with a poor overall prognosis. With over 20,000 cases in the US alone, about a third of AML patients pertain to an important oncogenic driver composed of internal tandem duplication (ITD) somatic mutations in exons 14 and 15 of the FMS-like tyrosine kinase 3 (FLT3) gene. Detecting the presence of measurable residual disease in patients with AML who are in morphologic remission has been shown to be a powerful predictor of eventual relapse. Yet, despite its importance, the ability to detect FLT3-ITD mutations is hampered by the limited sensitivity of conventional PCR-based assays in general and in Next Generation Sequencing-based assays, in part due to the difficulty to align ITD-containing reads to the reference genome.

Methods. In order to facilitate the analysis of AML samples, we developed FLT3-Explorer, a comprehensive bioinformatics pipeline composed of open source software, combined with in-house algorithms to accurately detect low levels of FLT3-ITD mutations. The input of the pipeline is unmapped reads in fastq format. The pipeline then searches for either ITD sequences previously observed in our extensive database of historical data, or a de-novo detection procedure is used to determine mutation sequences of variable lengths within the FLT3 gene. To assess the performance of FLT3-Explorer, we considered a cohort of clinical samples from bone marrow and peripheral blood, as well as cell lines. For sample collection, genomic DNA was isolated, and polymerase chain reaction was performed using primers flanking exons 13 and 15 to amplify the FLT3 gene. DNA was then sequenced using NGS Illumina sequencing. Results were validated using fragment size analysis by Capillary Electrophoresis assays. Also, to determine the limits of detection, various dilutions were performed on cell lines. Furthermore, to assess the performance of our pipeline, we performed a comparison with other FLT3-ITD programs publicly available.

Summary. Results from our analysis showed that our method identifies FLT3-ITD mutations of variant allele fraction as low as 0.003%. Also, we found that our pipeline can detect as many FLT3-ITD mutants as other methods, or more, when the mutant signal is considerably low.

Conclusions. FLT3-Explorer constitutes a powerful solution for low FLT3-ITD detection signal of mutant sequences, of diverse lengths, that can be used on FLT3-ITD AML patients to track disease progression and relapse.

Citation Format: Christian Laing, Wenge Shi, Reinhold Pollner. FLT3-Explorer: A bioinformatics pipeline for detecting low signal in FLT3 internal tandem duplications in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2475.
Abstract 2480: MutSpliceDB: A database of splice sites mutations effects
Authors: Palmisano Alida , Vural Suleyman , Zhao Yingdong , Sonkin Dmitriy
Published: August 2019

Splice site mutations are one of the well-known classes of genetic alterations playing an important role in biology. Splice site mutations in cancer are most frequently observed as inactivating alterations in tumor suppressor genes (for example, TP53 or RB1) and to a lesser degree as activating alterations in oncogenes (for example MET). Splice site mutations may lead to alterations in mRNA transcripts, causing for example exon(s) inclusion/exclusion or, intron retention. Interpreting the consequences of a specific splice site mutation is not straightforward, especially if the mutation is located outside of the canonical splice sites. Accurate interpretation of the impact a splice site mutation has can further our understanding of biology, influence patient treatment, and in case of germline splice site mutations, may also have relevance to familial disease predisposition. To facilitate the interpretation of splice site mutation effects, we developed MutSpliceDB: https://brb.nci.nih.gov/splicing a public resource of splice sites mutations effects, documenting mutation effect(s) on splicing based on manually reviewed RNA-seq BAM files from sample(s) with particular splice site mutations.

Citation Format: Alida Palmisano, Suleyman Vural, Yingdong Zhao, Dmitriy Sonkin. MutSpliceDB: A database of splice sites mutations effects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2480.
Abstract 2488: CellMinerCDB and CellMiner web-applications for genomics and pharmacogenomics analyses of cancer cell lines
Authors: Reinhold William C. , Varma Sudir , Wang Yang-Hsin , Elloumi Fathi , Pommier Yves
Published: August 2019

The CellMiner (http://discover.nci.nih.gov/cellminer)and CellMinerCDB (https://discover.nci.nih.gov/cellminercdb/) web-applications provide complimentary functionality and datatsets that may be used for comparisons of genomic, molecular and pharmacological data within the NCI-60 cancerous cell lines, Cancer Cell Line Encyclopedia (CCLE), Genomics of Drug Sensitivity in Cancer (GDSC), Cancer Therapeutics Response Portal (CTRP), NCI/DTP small cell lung cancer (SCLC), and NCI Almanac cell line sets. CellMiner contains data for the NCI-60, including the most extensive sets of molecular and drug activity data (generated by the NCI Developmental Therapeutics Program https://dtp.cancer.gov),found for any of the databases. CellMinerCDB contains all the above mentioned cell line sets, including the substantially increased cell line numbers and tissue of origin types found in the CCLE, GDSC, and CTRP. The two web-applications have separate but complimentary functionalities. Each cell line set has some variable number of data types, some of which measure the same parameters, and some that do not. The number and make up of cell lines also varies, from 60 for the NCI-60, 69 for the SCLC, and ~1000 for the CCLE, GDSC, and CTRP. As there are partial overlaps of cell lines between many of these cell line sets, one may fill in some data type gaps by merging data from two sources, as well as do quality control by comparisons of the same data from multiple institutions. This rich set of data and functions facilitates the exploration of the relationships between and among molecular alterations and pharmacological responses in cancer cell lines from the omic perspective.

Citation Format: William C. Reinhold, Sudir Varma, Yang-Hsin Wang, Fathi Elloumi, Yves Pommier. CellMinerCDB and CellMiner web-applications for genomics and pharmacogenomics analyses of cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2488.
Abstract 249: Combination of ONC201 with radiation exhibits synergistic efficacy in high grade gliomas and other advanced cancers
Authors: Tarapore Rohinton S. , Jhawar Sachin , Stein Mark , Zloza Andrew , Mueller Sabine , Zhang Jie , Amoroso Francesca , Mills Ian , Oster Wolfgang , Allen Joshua
Published: August 2019

Background: Radiation is widely administered in many treatment settings for solid tumors. ONC201 is the first small molecule DRD2 antagonist for oncology that is being investigated in advanced cancer clinical trials as a single agent. ONC201 has exhibited preclinical and clinical anti-tumor activity in high grade gliomas and its immunostimulatory activity involving natural killer (NK) cells has been recently reported. Given that ONC201 exhibits broad synergy with anti-cancer drugs, excellent safety, and single activity in tumor types where radiation is used routinely, we evaluated the combination of ONC201 with radiation.

Methods: Cell viability was evaluated in human and/or mouse breast, prostate and high grade glioma cell lines in response to ONC201 (1 - 10uM), radiation (2- 10 Gy), or the combination. Incubation times ranged from 24 to 96 hours and the sequence of the two agents in combination was varied.

Results: Cell viability assays for ONC201 in combination with radiation in breast or prostate cancer cell lines revealed a cytotoxic response to the combination that was superior to either single agent. Western blot analysis of PC3 cells showed a synergistic induction of CHOP and ATF6 that are components of the integrated stress response. In glioblastoma or diffuse intrinsic pontine glioma (DIPG) cell lines, combination indices computed from cell viability experiments indicated modest synergy (~0.7 CI) for the combination ranging between 1-5 µM ONC201 and 2-10 Gy. In MDA-MB-468 breast cancer cells, Western blot analysis demonstrated a striking induction of PARP cleavage, a marker of caspase-mediated apoptosis, with 2 µM ONC201 in combination with 2 Gy radiation, whereas either single agent produced minimal PARP cleavage. In 4T1 murine triple-negative breast cancer subcutaneous tumors, the combination of oral ONC201 and radiation produced antitumor effects at subtherapeutic doses that required CD4+ T cells and NK cells.

Conclusions: ONC201 combined with radiation produced additive or synergistic anti-cancer effects in high grade glioma, breast and prostate cancer in vitro and increased anti-tumor efficacy in an in vivo model of breast cancer that involved immune cells.

Citation Format: Rohinton S. Tarapore, Sachin Jhawar, Mark Stein, Andrew Zloza, Sabine Mueller, Jie Zhang, Francesca Amoroso, Ian Mills, Wolfgang Oster, Joshua Allen. Combination of ONC201 with radiation exhibits synergistic efficacy in high grade gliomas and other advanced cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 249.
Abstract 2494: Emodin inhibits colon cancer by altering BCL-2 family proteins and cell survival pathways
Authors: Saunders Ian T. , Kapur Neeraj , Mir Hina , Singh Shailesh
Published: August 2019

The therapeutic outcome of colon cancer (CoCa) is often compromised due to the development of chemoresistance and toxicities associated with current therapeutics. Hence, identification of less toxic and efficacious agent is needed to treat CoCa. In this study, we tested the effect of natural agent, Emodin (6-methyl-1,3,8-trihydroxyanthraquinone) isolated from Asian plant rhubarb, on CoCa cell death and survival. Effects of Emodin on CoCa (DLD-1 and COLO-201) cell viability and death was determined by MTT and Annexin-V assays, respectively. Apoptosis-specific protein antibody array was used to define the Emodin induced molecular mechanisms involved in cell death vs. survival and results were confirmed by western blot analysis. Mitochondrial membrane potential and the expression/localization of Bcl-2 family protein were assessed via flow cytometry and WB, respectively. Our data show Emodin decreased the viability of CoCa cells and induced apoptotic cell death in a time and dose-dependent manner. Emodin-induced apoptosis resulted in increased death receptor expression, modulated caspase activation, and an imbalance in the Bax/Bcl-2 ratio. Changes in Bcl-2 family protein expression and localization correlated with loss in mitochondrial membrane potential. Emodin also negatively impacted MAPK/JNK, PI3K/AKT, NF-kB and STAT signaling pathways, which are associated with cell growth, differentiation, and Bcl-2 family expression or function. Modulation of cell survival and apoptotic pathways by Emodin and specifically the impact on Bcl-2 projects Emodin significance as promising therapeutic agent for CoCa treatment.

Citation Format: Ian T. Saunders, Neeraj Kapur, Hina Mir, Shailesh Singh. Emodin inhibits colon cancer by altering BCL-2 family proteins and cell survival pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2494.