bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 6303: CRISPR/Cas9 RNP mediated efficient gene editing in hard-to-transfect cells
Authors: Li Peixue , Li Zhao , Lin Qing
Published: September 2020

It has been challenging for efficient gene editing in cells which are hard to transfect, such as stem cells and primary T cells. Typical sgRNA and Cas9 intracellular delivery techniques are limited by their reliance on cell type and exogenous materials as well as their toxic effects on cells (for example, electroporation) or packaging capacity limitation by viral vectors. Here we established an effective approach for gene editing in different cell types and achieved successful genome editing with CRISPR RNP electroporation. Also, high efficient double knockout mutations could be obtained by electroporation of multiple sgRNAs at one shot. Furthermore, this method also could reduce the exposure time to Cas9 nuclease which may cause potential off-target effects. With the advantages of broad applicability across different cell types, particularly hard-to-transfect cells, and flexibility of application, these methods could potentially enable the development of the stem cell or T cell based therapies.

Citation Format: Peixue Li, Zhao Li, Qing Lin. CRISPR/Cas9 RNP mediated efficient gene editing in hard-to-transfect cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6303.
Abstract 6307: In vivo drug screening platform accelerates anti-hCD40 antibody drug discovery
Authors: Yang Yi , Li Zhihong , Zhu Yu , Xie Jingshu , Chen Yunyun
Published: September 2020

The CD40 (also known as TNFRSF5) and its ligand CD40L (also known as CD154) belong to the TNF/TNFR family. CD40 is expressed on antigen presenting cells (APCs), including B cell, monocytes, macrophages and dendritic cells. CD40-CD40L is a pair of costimulatory molecules that, after interacting at the cell surface, promotes intracellular signaling by recruiting TNFR-associated factor (TRAF) in the cytosol, thereby activating multiple downstream signaling pathways. Therapeutic targeting of CD40 by agonistic monoclonal antibodies (mAbs) aims to activate CD40+ APCs to effectively boost tumor-specific cytotoxic T cells to eliminate tumor cells. In principle it has the advantage of greater tumor specificity over other T cell activating approaches, such as checkpoint inhibition or indiscriminate T cell activation.

We are interested in developing a novel immune checkpoint monoclonal antibody that targets CD40 and can treat cancer patients. Using Biocytogen's antibody research platform, dozens of candidate antibodies were obtained by classical hybridoma technology as well as high-throughput flow cytometry screening. Next, these murine antibodies were screened directly in humanized mouse models (B-hCD40) bearing a syngeneic cancer cell line. We discovered candidates that have potent anti-tumor activity in vivo. These candidate antibodies were further humanized, and we found an antibody called YH003 with potent antitumor activity in the MC38 tumor model. Fortunately, YH003 was able to inhibit tumor growth in combination with Pembrolizumab in the double humanized model of h-CD40 and h-PD1 bearing a syngeneic B16F10 cancer cell line. In vitro studies demonstrated that YH003 bound specifically and potently to human and monkey CD40 proteins, we used cynomolgus monkeys to further explore the safety evaluation. In conclusion, we use Biocytogen's in vivo drug screening platform to obtain more novel CD40 antibodies with anti-tumor activity in addition to YH003, accelerating the progress of preclinical antibody discovery.

Citation Format: Yi Yang, Zhihong Li, Yu Zhu, Jingshu Xie, Yunyun Chen. In vivo drug screening platform accelerates anti-hCD40 antibody drug discovery [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6307.
Abstract 6321: BEND3 modulates sensitivity to the UBA1 inhibitor TAK-243 by regulating expression of the multidrug transporter BCRP
Authors: Barghout Samir H. , Aman Ahmed , Blatman Zachary , Arevalo Karen , Thomas Geethu , MacLean Neil , Wang Xiaoming , Hurren Rose , Ketela Troy , Abohawya Moustafa , Kiyota Taira , Al-Awar Rima , Schimmer Aaron D.
Published: September 2020

TAK-243 (MLN7243) is a first-in-class inhibitor of the ubiquitin-activating enzyme (UBA1) that catalyzes the first step in the ubiquitylation cascade whereby proteins are tagged with mono- or poly-ubiquitin to induce their degradation or modify their functions. Based on its preclinical efficacy and tolerability, TAK-243 has entered phase 1 clinical trials in advanced malignancies. However, the determinants of sensitivity to TAK-243 remain largely unknown. Therefore, we conducted a positive-selection, genome-wide CRISPR/Cas9 knockout screen in OCI-AML2 cells followed by selection with lethal TAK-243 concentrations to identify genes essential for TAK-243 action. We identified BEN domain-containing protein 3 (BEND3), a transcriptional repressor and a regulator of chromatin organization, as the top gene whose knockout conferred resistance to TAK-243 (FDR = 0.0012). BEND3-targeting gRNAs were enriched up to 10,000-fold after selection with the drug. To validate the screen results, we independently knocked out BEND3 in OCI-AML2 cells and confirmed the resistance phenotype. In vivo, tumors of BEND3-knockout cells were resistant to TAK243 (20 mg/kg twice weekly) as opposed to control tumors that showed dramatic reductions in tumor growth rate. As assessed by immunoblotting, BEND3 knockout dampened TAK-243 effects on ubiquitylation, proteotoxic stress and DNA damage response. Mechanistically, BEND3 knockout upregulated the ABC efflux transporter breast cancer resistance protein (BCRP; ABCG2), and decreased intracellular levels of TAK-243. It also conferred partial cross-resistance to pevonedistat and TAK-981–related selective inhibitors of the NEDD8-activating enzyme (NAE) and the SUMO-activating enzyme (SAE), respectively, as well as known substrates of BCRP (mitoxantrone and doxorubicin). Finally, TAK-243 sensitivity strongly correlated with BCRP expression in a panel of 30 cancer cell lines of different origin, and chemical inhibition of BCRP but not P-gp sensitized intrinsically resistant high-BCRP cells to TAK-243. Thus, our data demonstrate that BEND3 regulates the expression of BCRP for which TAK-243 is a substrate. Moreover, BCRP expression could serve as a predictor of TAK-243 sensitivity.

Citation Format: Samir H. Barghout, Ahmed Aman, Zachary Blatman, Karen Arevalo, Geethu Thomas, Neil MacLean, Xiaoming Wang, Rose Hurren, Troy Ketela, Moustafa Abohawya, Taira Kiyota, Rima Al-Awar, Aaron D. Schimmer. BEND3 modulates sensitivity to the UBA1 inhibitor TAK-243 by regulating expression of the multidrug transporter BCRP [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6321.
Abstract 6330: DPP4 modulates ACSL4 to promote lipid peroxidation and regulate ferroptosis in pancreatic cancer
Authors: Zhou Xiang , Wang Weiming , Zhang Baofu , Lin Zixia , Wang Yi , Chen Gang
Published: September 2020

BACKGROUND and AIMS: Pancreatic ductal adenocarcinoma (PDAC) is one of the highest mortality rate cancers with almost no curative chemotherapeutic treatment. Ferroptosis is a recently recognized regulated necrotic cell death and that has emerged to play an important role in cancer biology. Recent discoveries have highlighted the important role of Dipeptidyl-peptidase-4 (DPP4) in TP53-deficient cancer cells. PDAC Patients with a lower DPP4 level have a poorer postoperative survival. We investigated whether DPP4 contributes to ferroptosis in pancreatic cancer.

METHODS: DPP4 expression status was evaluated in PDAC tissues using tissue chip immunohistochemistry. The effects of DPP4 re-expression or knockdown on cell proliferation, the cell cycle and energy metabolism were determined. DPP4 interacting protein was identified by mass spectrometry and DPP4-related cancer pathways by RNA-seq analyses. The clinical impact of DPP4 in driving ferroptosis and enhancing gemcitabine sensitivity was assessed in patient-derived xenograft tumours (PDX), subcutaneous, and DPP4 knock-down (KD) or knock-in (KI) mice orthotopic xenograft models of PDAC.

RESULTS: DPP4 was commonly low expression in PDAC. DPP4 significantly inhibited cell proliferation by causing G1-S cell cycle arrest, inhibiting energy metabolism and promoting erastin-induced ferroptosis in vitro, and suppressed xenograft tumour growth in both subcutaneous and orthotopic xenograft mouse models (both p<0.001). Mechanistically, DPP4 facilitated ferroptosis by binding ACSL4 and the DPP4-ACSL4 interaction stabilized ACSL4 by inhibiting its ubiquitin-mediated degradation, and subsequent lipid peroxidation. This interaction inactivated their downstream key elements of NF2-YAP signalling cascades. Importantly, the DPP4-ACSL4 pathway dictated ferroptosis sensitivity and promoted the anticancer activity of gemcitabine. Activating of the DPP4-ACSL4 pathway enhanced gemcitabine sensitivity by disinhibiting ferroptosis in vitro and in PDX, subcutaneous, and DPP4 KD or KI orthotopic xenograft models of PDAC.

CONCLUSIONS: Collectively, these findings identify a novel role of DPP4 in ferroptosis and suggest a potential therapeutic strategy for overcoming gemcitabine resistance.

Citation Format: Xiang Zhou, Weiming Wang, Baofu Zhang, Zixia Lin, Yi Wang, Gang Chen. DPP4 modulates ACSL4 to promote lipid peroxidation and regulate ferroptosis in pancreatic cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6330.
Abstract 6332: Pim kinase inhibition promotes mitochondrial fission to combat chemoresistance in non-small cell lung cancer
Authors: Chauhan Shailender S. , Toth Rachel K. , Jensen Corbin C. , Kashatus David F. , Casillas Andrea L. , Warfel Noel A.
Published: September 2020

Background: Resistance to chemotherapy represents a major obstacle to the successful treatment of non-small cell lung cancer (NSCLC). A fused mitochondrial network is associated with therapeutic resistance, but the factors controlling mitochondrial dynamics in lung cancer are not fully understood. The goal of this study was to determine how PIM kinases impact mitochondrial dynamics, ROS production, and response to chemotherapy in lung cancer.

Methods: Live cell imaging and microscopy were used to determine the effect of PIM on mitochondrial phenotype and ROS. Biochemical studies were performed to characterize the signaling axis linking PIM1 to Drp1 and mitochondrial fission in lung cancer. Xenograft models of lung cancer were used to evaluate the combination of PIM inhibition and chemotherapy. Immunohistochemistry and transmission electron microscopy were used to analyze mitochondrial phenotype, proliferation, and apoptosis in vivo.

Results: Inhibition of PIM kinases caused excessive mitochondrial fission and significant upregulation of mitochondrial superoxide, increasing intercellular ROS. PIM inhibition significantly increased the protein levels and mitochondrial localization of Drp1. An inverse correlation between PIM1 and Drp1 was confirmed in NSCLC patient samples. Inhibition of PIM sensitized NSCLC cells to chemotherapy and produced a synergistic anti-tumor response, characterized by significantly increased ROS and apoptosis.

Conclusion: PIM kinase expression is an important mechanism of intrinsic and acquired resistance to chemotherapy in NSCLC. PIM inhibition activates Drp1 to promote mitochondrial fission and superoxide production, sensitizing cancer cells to chemotherapy. These data improve our knowledge of how mitochondria are regulated in cancer and provide justification for combining PIM inhibition with chemotherapy in NSCLC.

Citation Format: Shailender S. Chauhan, Rachel K. Toth, Corbin C. Jensen, David F. Kashatus, Andrea L. Casillas, Noel A. Warfel. Pim kinase inhibition promotes mitochondrial fission to combat chemoresistance in non-small cell lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6332.
Abstract 6367: KPG-818, a novel cereblon modulator, inhibits hematological malignancies in preclinical models
Authors: Ge Chuansheng , Liao Baisong , Zhang Lei
Published: September 2020

The clinical use of lenalidomide in multiple myeloma (MM), mantle cell lymphoma (MCL) and myelodysplastic syndromes (MDS) represents a significant advance in the treatment of hematological malignancies and has altered the landscape of the blood cancer therapy. Yet, many hematological indications and refractory diseases are still without effective systemic and curative therapies. Here we present KPG-818, a small molecule compound with high affinity binding to cereblon of the E3 ubiquitin ligase (CRBN-CRL4) complex, significantly inhibits the production of the pre-inflammatory cytokines such as TNF-α and IL-6 in LPS-stimulated human PBMC cells, inhibits HUVEC tube formation, and exhibits potent anti-proliferation in multiple lymphoma and multiple myeloma cell lines in vitro. Consistent with the finding in that lenalidomide causes selective degradation of Aiolos and Ikaros, KPG-818 potently inhibits the level of the two lymphoid transcription factors in MM1.S cell line model and human PBMC cells. In in vivo pharmacology studies, KPG-818 significantly inhibits tumor growth in MM.1S multiple myeloma, WSU-DLCL-2 diffuse large B cell lymphoma, DOHH-2 follicular lymphoma, REC-1 mantle cell lymphoma xenograft models. Importantly, KPG-818 prolongs the survival of the mouse cohorts bearing systemic human H929 multiple myeloma in a dose-dependent manner. Moreover, KPG-818 shows favorable DMPK and toxicity profiles in pre-clinical IND enabling studies.

In summary, these immune modulation, anti-angiogenesis and antitumor results encourage the further clinical evaluation of KPG-818 in patients with Multiple Myeloma, Diffuse Large B Cell Lymphoma, Follicular Lymphoma, Mantle Cell Lymphoma and other hematological malignancies.

Citation Format: Chuansheng Ge, Baisong Liao, Lei Zhang. KPG-818, a novel cereblon modulator, inhibits hematological malignancies in preclinical models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6367.
Abstract 6378: CDC20 is a novel therapeutic target in triple-negative breast cancer
Authors: Sharma Jayesh K. , Alluri Prasanna , Udden Nashir
Published: September 2020

Background:

Approximately 15% of breast cancers lack expression of Estrogen Receptor (ER), Progesterone Receptor (PR) and Human Epidermal Growth Factor Receptor 2 (HER-2), and are referred to as Triple-Negative Breast Cancer (TNBC). Due to a lack of targeted therapies, clinical outcomes in patients with TNBC continue to be poor. Likewise, due to the inherently aggressive biology of the disease, patients are forced to deal with the toughest breast cancer while having the least treatment options. In this study, we identified Cell Division Cycle protein 20 (CDC20), a key regulatory protein that mediates sister chromatid separation in mitosis, as a novel therapeutic target in TNBC.

Methods:

To uncover new drivers of TNBC, we nominated genes that are significantly overexpressed in patients with TNBC, compared to non-TNBCs. After identifying CDC20 as a top hit in this analysis, we used RNAseq analyses, expression microarrays, and Western blotting to assess CDC20 expression levels in breast cancer patients and cell lines. Kaplan-Meier analyses were formed to study the correlation between clinical outcomes and CDC20 expression levels, including recurrence-free survival, metastasis-free survival and overall survival. shRNA and siRNA-mediated knockdown of CDC20 in TNBC cell lines were employed to study the role of CDC20 in TNBC proliferation, invasion, and migration.

Results:

CDC20 was significantly overexpressed in TNBCs (compared to non-TNBCs) patients and is a predictor of poor clinical outcomes and radiation resistance. CDC20 expression was also significantly higher in TNBC cell lines compared to non-TNBC cell lines, reflecting what is seen in clinical data sets, such as in The Cancer Genome Atlas. Sh and siRNA-mediated knockdown of CDC20 expression resulted in decreased proliferation, invasion and migration in TNBC cell lines.

Conclusions and future directions:

Our findings support the development of pharmacologic strategies to inhibit CDC20 function as a potential targeted therapy against TNBC. Efforts are underway to validate our findings in vivo in xenograft models of TNBC in mice.

Citation Format: Jayesh K. Sharma, Prasanna Alluri, Nashir Udden. CDC20 is a novel therapeutic target in triple-negative breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6378.
Abstract 6384: TEAD inhibition reduces tumor proliferation in Merlin null meningioma and schwannoma
Authors: Laraba Liyam , de Guibert Julio Grimm , Tang Tracy T. , Post Leonard , Parkinson David B.
Published: September 2020

Introduction:

Meningiomas are the most common intracranial tumor and arise from the arachnoidal cell layer of the meninges. Schwannomas develop from Schwann cells which provide myelin and trophic support in the peripheral nerves. The tumor suppressor Merlin is deleted in approximately 50-60% of meningiomas and 70% of schwannomas. Loss of Merlin results in dysregulation of the Hippo pathway and consequent aberrant activation of the transcriptional coactivators YAP and TAZ. Nuclear localisation of YAP and TAZ has been shown to drive tumor phenotypes in many cancers. The TEAD family of transcription factors are major targets of YAP and TAZ-dependent signaling. TEAD family members undergo autopalmitoylation and this modification is required for transcriptional activation by YAP or TAZ. Vivace therapeutics have identified small molecule inhibitors of autopalmitoylation that block the interaction of TEAD family members with YAP and TAZ. This study seeks to establish the efficacy of small molecule inhibitors of TEAD activity in meningioma and schwannoma tumor cells.

Experimental Procedures:

Meningioma cell lines, primary human meningioma and schwannoma cells, as well as a schwannoma mouse model (PerisotinCRE /NF2 fl/fl) were treated with Vivace inhibitors to assess their effects upon tumor cell proliferation and TEAD driven transcription.

Results:

TEAD inhibition significantly reduced the proliferation of the BenMen-1 meningioma cell line, as well as in primary human meningioma cells. In schwannoma, inhibitors reduced proliferation and also reduced the expression of CTGF, a target of TEAD activation in both primary human meningioma and schwannoma cells. These novel inhibitors are also currently being tested in the Periostin CRE-NF2fl/fl schwannoma mouse model to evaluate their efficacy in vivo.

Conclusions:

The use of these novel compounds to inhibit TEAD function shows great potential as an effective treatment for reducing the rate of cell proliferation in meningioma and schwannoma, two clinically important tumor types.

Citation Format: Liyam Laraba, Julio Grimm de Guibert, Tracy T. Tang, Leonard Post, David B. Parkinson. TEAD inhibition reduces tumor proliferation in Merlin null meningioma and schwannoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6384.
Abstract 6465: Clinicopathological features of TROP-2 and pTROP-2 in gastric cancer
Authors: Kushiyama Syuhei , Yashiro Masakazu , Nishimura Sadaaki , Togano Shingo , Kuroda Kanji , Sugimoto Atsushi , Sera Tomohiro , Yamamoto Yurie , Okuno Tomohisa , Miki Yuichiro , Nakada Hiroshi , Ohira Masaichi
Published: September 2020

Introduction: The human trophoblast cell-surface antigen TROP-2 is a cell surface glycoprotein receptor involved in tight junctions at the epithelial barrier. Although TROP-2 is reportedly overexpressed in various epithelial tumors and its expression correlates with aggressive tumor behavior, there is no report of the clinicopathological features of gastric cancer (GC) with TROP-2 and phosphorylated TROP-2 (pTROP-2) expression. We evaluated the clinicopathologic significance of TROP-2 and pTROP-2 expression in GC patients.

Materials and methods: We retrospectively analyzed the cases of 720 GC patients who underwent resection for GC. The expressions of TROP-2 and pTROP-2 in each tumor were evaluated by immunohistochemistry. We then divided the patients into the TROP-2 high group, TROP-2 low group, pTROP-2 high group, and pTROP-2 low group. The association between TROP-2 or pTROP-2 expression and clinicopathological variables were assessed by the chi-square test. Overall survival was analyzed by Kaplan-Meier method, and compared by log-rank test. The Cox proportional hazards model was used for multivariate analysis.

Results: TROP-2 high group was found to be overexpressed in 330 (46.7%) tumor samples. Significantly higher expression of TROP-2 was significantly correlated with intestinal type, tumor invasion depth, lymph node metastasis, lymphatic invasion, and venous invasion. pTROP-2 high group was found to be overexpressed in 306 (43.4%) tumor samples. Significantly higher expression of pTROP-2 was correlated with intestinal type, low tumor invasion depth, no lymph node metastasis, and no lymphatic invasion. TROP2 overexpression was predictive for poor overall survival of patients with GC (p = 0.0002, log rank test), while multivariate analysis to overall survival revealed that TROP-2 overexpression was not an independent prognostic marker (p=0.06). In contrast, pTROP-2 overexpression was predictive for good overall survival of patients with GC (p = 0.004, log rank test).

Conclusion: TROP-2 might be associated with metastatic ability of gastric cancer cells, resulting poor prognosis of patients with GC.

Citation Format: Syuhei Kushiyama, Masakazu Yashiro, Sadaaki Nishimura, Shingo Togano, Kanji Kuroda, Atsushi Sugimoto, Tomohiro Sera, Yurie Yamamoto, Tomohisa Okuno, Yuichiro Miki, Hiroshi Nakada, Masaichi Ohira. Clinicopathological features of TROP-2 and pTROP-2 in gastric cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6465.
Abstract 6487: Circulating tumor cells, tumor- and endothelium- derived extracellular vesicles, but not circulating endothelial cells associate with poor prognosis in colorectal cancer
Authors: Nanou Afroditi , Mol Linda , Koopman Miriam , Punt Cornelis J. , Terstappen Leon W.
Published: September 2020

Elevated tumor-derived EV (tdEV) and Circulating Tumor Cell (CTC) load in patients with metastatic cancer is associated with poor clinical outcome. In this study, we investigated whether endothelium-derived extracellular vesicles (edEVs) can be detected in the blood of metastatic colorectal cancer (mCRC) patients and whether there is an association with prognosis.

The open-source ACCEPT software was used to enumerate edEVs, tdEVs and other objects from digitally stored CellSearch® images obtained after Circulating Endothelial Cell (CEC) and CTC enrichment from blood of 395 mCRC patients, before the initiation of a new therapy. Patients had participated in the prospective phase III CAIRO2 study. ACCEPT classified objects, CTC and CEC counts and patient information were used in univariable and multivariable analysis to determine their relation with Progression-Free Survival (PFS) and Overall Survival (OS).

edEVs are present in blood of mCRC patients at 5-10 fold higher frequencies than CECs. A significantly shorter PFS and OS was observed for patients with ≥ 3 CTCs/ 7.5 mL, ≥ 40 tdEVs/ 7.5 mL and ≥ 287 edEVs/ 4.0 mL. The Hazard Ratio HR (95% CI) of PFS for increased CTCs, tdEVs and edEVs was 1.4 (1.1-1.9), 2.0 (1.5-2.6) and 1.7 (1.2-2.5), respectively. Similarly, the HR of OS for increased CTCs, tdEVs and edEVs was 2.2 (1.7-3.0), 2.7 (2.0-3.5) and 2.1 (1.5-2.8), respectively. There was no cut-off value for CECs leading to a dichotomization of patients with a significant HR. Both tdEVs and edEVs remained significant predictors of OS in multivariable analysis.

edEVs and tdEVs are independent prognostic factors for OS in mCRC patients.

Funded by the NWO Applied and Engineering Sciences Cancer-ID project #14190.

Citation Format: Afroditi Nanou, Linda Mol, Miriam Koopman, Cornelis J. Punt, Leon W. Terstappen. Circulating tumor cells, tumor- and endothelium- derived extracellular vesicles, but not circulating endothelial cells associate with poor prognosis in colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6487.
Abstract 6530: Old target, but new approach: Targeting cancer-associated MUC1 with ADC and Car-T
Authors: Jung Jin G , Kim Min-Seok , Jung Hwa-Chul , Choi Hoil , Moon Kyung-Duk
Published: September 2020

Purpose: The characteristics of aberrant overexpression and distinct features of cancer-associated (CA)-MUC1 has been often considered as a promising target in most of the solid cancers. MUC1 is composed of α and β subunits to form a full length of MUC1, and α subunit is released and circulated into the blood in cancer, which is used as biomarkers such as CA 15-3 and CA 27-29. As those α subunit-targeting strategies were failed in the numerous clinical trials due to, at least in part, the diminished α subunit of MUC1 in cancer, we have developed a humanized Ab, PAb001 targeting the residual β subunit of MUC1, called as an onco-tethered (OT)-MUC1. To prove the concept of this strategy, an antibody-drug conjugate (ADC) and chimeric antigen receptor (CAR) T cells were applied to assess its efficacies.

Methods: We produced a monoclonal antibody (mAb) against OT-MUC1 from mouse and sequentially generated its derivative humanized antibody, PAb001. Then, the target product profiles (TPP) were determined by SPR, ELISA, FACS, immunohistochemical analysis, pilot tissue cross-reactivity, and pharmacokinetics (PK) analysis. Then, to evaluate anti-cancer efficacies, we either conjugated PAb001 with Monomethyl auristatin E (MMAE) or generated the second-generation CAR T cells and then sequentially tested in vitro and in vivo cancer models.

Results: Based on the TPP criteria, PAb001 antibody was chosen over a hundred of candidate antibodies tested. Next, it was conjugated with MMAE, treated and measured its cytotoxicity in ovarian and breast cancer cell lines, including triple-negative breast cancers (TNBC). As a result, PAb001-ADC selectively inhibited the growth of cancer cell lines, depending on the expression level of MUC1. Also, it showed 80-100 % tumor growth inhibition rates (TGI) in the cell lines- and patient-derived xenograft models. Lastly, OT-MUC1 Car-T cells showed significant and selective growth inhibition of cancer cells.

Conclusion: Taken together, the preclinical studies of PAb001-ADC and Car-T indicate the high therapeutic potentials targeting OT-MUC1 in cancer, which further provided the rationale for clinical development.

Citation Format: Jin G Jung, Min-Seok Kim, Hwa-Chul Jung, Hoil Choi, Kyung-Duk Moon. Old target, but new approach: Targeting cancer-associated MUC1 with ADC and Car-T [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6530.
Abstract 6550: WD105, a novel decursin derivative, showed greater growth inhibition and androgen receptor suppression in LNCaP human prostate cancer cells
Authors: Wu Wei , Ding Huaiwei , Zhang Huiyong
Published: September 2020

Androgen receptor (AR) is a primary target for treatment of prostate cancer (PCa). AR signaling plays a critical function in all stages of PCa. Our previous research demonstrated that decursin and its isomer decursinol angelate, a pair of natural compounds from Angelica gigas Nakai, inhibit AR signaling and suppressed the growth of prostate cancer cells in vitro and in vivo. In this study, we have designed a series of pro-drug conjugates of decursin, based on acid-esterified modification, in order to achieve higher water solubility, stronger cytocidal effect and better AR suppression activity. Twelve novel esterified derivatives of decursin were synthesized from decursinol and characterized by 1H-, 13C-NMR and HR-MS techniques. Among them, WD105 displayed the best solubility. Initial structure-activity relationship (SAR) studies were carried out with a cell growth (CCK8) assay and identifıed WD105 and WD106 as lead compounds with more potency at suppressing LNCaP cell growth than decursin judged by IC50 value. In addition, both WD105 and WD106 induced caspase-mediated cell apoptosis (48h), evidenced by cleavage of PARP, in a concentration-dependent manner. Furthermore, WD105 decreased the transcriptional activity of AR and androgen-induced expression of PSA better than WD106 and decursin when assessed by Western Blot and ELISA. Taken together, these data suggest that WD105 has greater in vitro activity than decursin as an AR targeting lead compound. Future studies will characterize the PK metrics and, if warranted, determine the in vivo antitumor effect of WD105 on the growth of LNCaP-AR xenografts.

Citation Format: Wei Wu, Huaiwei Ding, Huiyong Zhang. WD105, a novel decursin derivative, showed greater growth inhibition and androgen receptor suppression in LNCaP human prostate cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6550.
Abstract 6562: Design, synthesis and anticancer properties of trans-cinnamaldehyde and curcumin based scaffolds compounds
Authors: Omar Abdel-Sattar M. , Al-Abd Ahmed M.
Published: September 2020

Cinnamaldehyde and curcumin scaffolds are known for their anticancer potential via reactive oxygen species (ROS) generation and cellular damage. Herein, we synthesized a set of heterocyclic compounds build on the naturally-occurring trans-cinnamaldehyde and curcumin scaffolds and assessed their antiproliferative/cytotoxic properties against hepatocellular carcinoma (HepG2), breast adenocarcinoma (MCF-7) and colorectal cancer cells (HCT-116). The general structure of the synthesized compounds comprised 2-(4-fluorophenyl)-4-imidazolone nucleus attached to a 2-phenylpropylidene moiety. The imidazolone's N1 diversity point was attached to a variety of groups that are different in steric as well as electronic properties. Compounds FC01, FC09, FC11, FC12, FC14, FC30 and FC37 (groups attached to N1 diversity point are phenyl, methyl, 4-methylpiperazinyl, n-propyl, 4-hydroxyphenyl, 2-furylmethyl, and 4-methoxyphenyl, respectively) showed considerable cytotoxicity with IC50 ranging from 0.1 to 11.9 µM against MCF-7, HepG2, and HCT-116 cells. Within one hour of exposure to the IC50's of compounds FC09, FC12 and FC31, total ROS was significantly increased to 143.3±1.2%, 158.2±8.6% and 138±9.3% of control. After 2 hours of exposure, compounds FC09, FC12, FC14, FC30 and FC31 significantly raised the total ROS by 148.3±2.1%, 144.2±5.9%, 112±3.1%, 134.8±10.3% and 140.7±5.7%, respectively. Interestingly, all compounds under investigation significantly raised the intracellular ROS within 1.5 h of exposure. After 24 h of exposure, only compounds FC01, FC07, FC30, and FC31 kept the intracellular level of ROS significantly higher than control cells (111.1±3.6, 113.8±4.3%, 110.4±2.2%, and 117.7±7.5%, respectively). On the other hand, the ROS level within cells treated with the IC50 of compounds FC09 and FC12 for 24 h was significantly lower than control cells (85.7±1.3 and 68.4±4.7, respectively). Apoptosis/necrosis differential assessment using annexin-V/FITC with PI as a counterstain coupled with flow cytometric analysis, showed that compounds FC01, FC09, and FC12 induced significant apoptosis as well as necrosis by 2-5 folds compared to control cells. Compound FC14 failed to induce any significant apoptosis or necrosis. In conclusion, trans-cinnamaldehyde and curcumin scaffold-based compounds under investigation, particularly FC01, FC09 and FC12 might represent promising candidates in anticancer molecules for further optimization.

Citation Format: Abdel-Sattar M. Omar, Ahmed M. Al-Abd. Design, synthesis and anticancer properties of trans-cinnamaldehyde and curcumin based scaffolds compounds [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6562.
Abstract 6565: Impact of TIM-3 modulation on the sensitivity of anti-PD-1
Authors: Zhu Jian , Zhu Rebecca L.
Published: September 2020

Concurrent inhibition of TIM-3 and PD-1 may have enhanced anti-tumor effects. In this study, computational models were established to identify factors involved in the interactions between these two immunotherapeutic targets. Abstracts published on TIM-3, PD-1, PD-L1, anti-PD-1/PD-L1 were downloaded from PubMed, and analyzed by natural language processing and text mining. Interactions among genes, compound/therapies, cell/animal models, pathway/disease, and clinical studies were retrieved from the text. Networks were constructed to simulate effects of TIM-3 inhibition on anti-PD-1. The TIM-3/anti-PD-1 network consists of 57 nodes, 175 interactions with 11,626 possible regulatory paths from TIM-3 to anti-PD-1. Overall, 6142 paths are associated with the agonistic interactions between TIM-3 inhibition and anti-PD-1, while 5484 are antagonistic. Factors associated with the enhancement of anti-PD-1 by TIM-3 inhibition include up-regulation of IL-2, IFN-gamma, Th1 cells/cytokines, CD8+ T cells or NK cells, and down-regulation of IL-10, NFkB-p65, COX2 or EMT. Treatments with either EGFR inhibitors (e.g. gefitinib), anti-CTLA-4, IL-2 agonist or radiation therapy may enhance the combination efficacy of TIM-3 and PD-1 inhibitors. On the other hand, CTLA-4, galectin-9, Wnt/beta-catenin or Tregs combined with TIM-3 over-expression may confer resistance to anti-PD-1. PD-L1 is a validated biomarker for anti-PD-1 response and its expression can be correlated with TIM-3. Similar text-mining approach was adopted to construct the TIM-3/PD-L1 network which consists of 65 nodes, 165 interactions and 1372 paths from TIM-3 to PD-L1. Up-regulation of either galectin-9, WNT/beta-catenin or Tregs by TIM-3 results in the induction of PD-L1. As the network analyses revealed, interactions between TIM-3 and PD-L1 are context dependent, and assessment of the effect of TIM-3 inhibition on anti-PD-1 requires profiling of multiple factors along the regulatory and signaling pathways. The TIM-3/PD-L1 and TIM-3/anti-PD-1 networks prove to be useful tools to facilitate the therapeutic development of TIM-3 and PD-1 inhibitors, especially for selecting patients with potential agonistic effects of this promising drug combination.

Citation Format: Jian Zhu, Rebecca L. Zhu. Impact of TIM-3 modulation on the sensitivity of anti-PD-1 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6565.
Abstract 6588: Omics integration provides insight into esophageal cancer inhibitory mechanisms of a cranberry extract
Authors: Weh Katherine M. , Howard Connor L. , Tripp Bridget A. , Clarke Jennifer L. , Howell Amy B. , Kresty Laura A.
Published: September 2020

Our laboratory studies chemoprevention of esophageal adenocarcinoma (EAC) through utilization of the rat esophagogastroduodenal anastomosis (EGDA) surgical model of reflux-induced EAC. Specifically, we evaluated mechanisms by which cranberry proanthocyanidins (C-PAC) inhibit reflux-induced EAC by utilizing multi-omics integrative approaches employing multiple program platforms. Herein, we investigated whether pathway-based integration could be used to examine cross-talk between genes, metabolites and the microbial profiles. For this analysis, we utilized transcriptomic and untargeted metabolomic data from rat esophagi of animals that received water or C-PAC in the drinking water alone or combined with reflux inducing EGDA surgery. Additionally, we isolated DNA from fecal pellets and performed 16S rRNA sequencing to assess gut microbiome composition and functionality. Each omics dataset was analyzed for significant pathway enrichment and network generation using Metacore, DAVID and Metabolync. PICRUSt was utilized to predict microbiota functionality. Analysis of transcriptomic and metabolomic data suggest that EGDA upregulates inflammatory, NF-kB, DNA damage, cell cycle and immune function pathways as well as metabolic pathways related to eicosanoids, amino acids and primary and secondary bile acids, while C-PAC mitigates these alterations. Preliminary microbiome data analysis also suggests that bacteria related to inflammation, metabolite transport and DNA damage are increased in abundance in EGDA, with a decrease in abundance following C-PAC treatment. Analysis of omics datasets can provide insightful relationships into the cross-talk between different processes regulated by transcription, metabolism and the microbiome. Single dataset and integrative analysis showed similarities in altered pathways for vehicle and C-PAC treated animals in the context of reflux. Integration is limited by the infancy of multi-omics analysis programs, as well as the limited amount of research involving prediction of bacterial function and integration of microbiome data with other omics datasets. Further research and development of these omics programs is needed to determine accuracy of predicted results as well as continued investigation of identified pathways.

Citation Format: Katherine M. Weh, Connor L. Howard, Bridget A. Tripp, Jennifer L. Clarke, Amy B. Howell, Laura A. Kresty. Omics integration provides insight into esophageal cancer inhibitory mechanisms of a cranberry extract [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6588.
Abstract 6611: Functional characterization of a novel gene in the antigen presentation pathway allows specific immunotherapeutic targeting of glioblastoma cancer stem cells
Authors: Kidwell Reilly L. , Li Xiqing , Rich Jeremy N.
Published: September 2020

Glioblastoma is the most prevalent and aggressive primary brain tumor in adults, and conventional treatment strategies have made minimal progress in prolonging overall survival. Cancer stem cells are critical for tumor initiation, growth, and maintenance, and are able to co-opt complex host mechanisms to evade immune recognition. Immune checkpoint inhibition has proven revolutionary for the treatment of several types of solid tumors, but has largely failed in attempts to improve outcomes in glioblastoma. Developing a deeper understanding of the functional immunomodulatory role of genes unique to glioma stem cells (GSCs) may help to elucidate this enigma, and inform more specific immunotherapeutic targeting. To better understand the ability of GSCs to promote immunosuppression and self-tolerance in the tumor niche, we interrogated RNA-seq data from GSCs and differentiated glioma cells (DGCs) and identified a novel gene in the antigen presentation pathway (APP). In vitro targeting of this gene increased sensitivity to CD8+ T cell killing and reduced expression of other T-cell inhibitory ligands in GSCs alone, decreasing tumor-mediated immunosuppression. Compared to DGCs, GSCs were found to be key promoters of immune self-tolerance and evasion, with unaltered GSCs exhibiting minimal peptide binding to MHC Class I and, therefore, decreased antigen presentation. Specific targeting of the identified APP gene led to a reversal of this interaction, with increased binding capacity and, correspondingly, tumor antigen recognition. In vivo administration of a specific APP gene targeting agent in human GSC xenograft mice displayed decreased tumor burden and a striking prolongation of overall survival compared to both controls and mice treated with known immune checkpoint inhibitors. High expression of the APP gene is associated with poor clinical prognosis, supporting the clinical relevance of this discovery. Thus, novel targeting of the antigen-presenting pathway may provide a therapeutic vulnerability unique to glioblastoma cancer stem cells.

Citation Format: Reilly L. Kidwell, Xiqing Li, Jeremy N. Rich. Functional characterization of a novel gene in the antigen presentation pathway allows specific immunotherapeutic targeting of glioblastoma cancer stem cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6611.
Abstract 6644: A comprehensive in vitro and in vivo system to evaluate STING agonist efficacy in cancer therapeutics
Authors: Shi Xiaohe , Fan Feifei , Wei Ruicheng , Zuo Xiangyang , Lu Xiutao , Sun Quanhong
Published: September 2020

A number of stimulator of interferon genes protein (STING) agonists has recently been developed to treat cancer and infectious diseases. To investigate the binding of ligand to STING and subsequent activation of the STING pathway, we used the protein thermal shift assay, a high-throughput screening method for intermolecular interaction involving a protein of interest, to identify previously unknown compounds that bind to STING. To determine the functional consequence of ligand binding, we constructed IFN-β-Luc THP1 reporter cell line, to evaluate the pathway activation together with IFNβ secretion. In addition, we incubated human peripheral blood mononuclear cells (PBMCs) with STING agonist and measured phosphorylation of STING, phosphorylation of IRF3 and the secretion of cytokines such as IFN-β by ELISA or Luminex. To determine the in vivo therapeutic effects of STING agonists, we performed efficacy studies in syngeneic mouse models of melanoma (B16F10 cell line), colorectal cancer (CT-26 cell line) or breast cancer (4T1 cell line) with intratumoral delivery of test compounds. Test Treatment with STING agonists resulted in significant tumor growth inhibition as measured by tumor volume and significantly improved survival. Tumor-reactive IFN-γ secreting CD8+ T cells were harvested from spleens, purified by immunomagnetic sorting, and immediately used in ELISPOT assays using the mouse IFN-γ ELISPOT Kit. Tumor-infiltrating lymphocytes (TILs) have also been assessed in harvested melanoma (B16F10) tumors. We found that treatment with STING agonists resulted in increased CD69 percentage in CD8+ T cells in TILs, and decreased MHCII levels in macrophages and DCs in TILs.

In summary, we have established reporter-based in vitro assays and in vivo efficacy validation platforms, which can be used for STING agonists efficacy evaluation for cancer therapy and combination IO target therapy.

Citation Format: Xiaohe Shi, Feifei Fan, Ruicheng Wei, Xiangyang Zuo, Xiutao Lu, Quanhong Sun. A comprehensive in vitro and in vivo system to evaluate STING agonist efficacy in cancer therapeutics [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6644.
Abstract 6680: Inclusion of inhibitory neoantigens can abolish efficacy of otherwise protective therapeutic anti-tumor vaccines
Authors: DeVault Victoria L. , Starobinets Hanna , Adhikari Sanmit , Singh Simran , Rinaldi Stephanie , Classon Brendan , Flechtner Jessica B. , Lam Hubert
Published: September 2020

Identification of neoantigens that can elicit strong anti-tumor responses has become critical to vaccine design for cancer immunotherapy. Conventional methods for in silico neoantigen identification have yielded poor predictive value, highlighting the need for methods that identify bona fide neoantigen targets. The Genocea ATLAS™ platform uses autologous antigen presenting cells and T cells to identify pre-existing CD4+ and/or CD8+ T cell responses to patient-specific mutations and therefore selects confirmed neoantigens that can be used for vaccines and cell therapies. Screened neoantigens are characterized as stimulatory or inhibitory based on up- or downregulation of inflammatory cytokine secretion compared to baseline controls. In the mouse B16F10 melanoma model, therapeutic immunization with stimulatory neoantigen peptides arrested tumor growth whereas in contrast, inhibitory antigen immunization (henceforth referred to as inhibigens) resulted in accelerated tumor progression. The presence of an inhibigen in an otherwise protective vaccine formulation completely abolished protection. Global IFNγ responses to neoantigens were abrogated in these mice as measured by ELISpot suggesting that inhibigen pro-tumor responses can be immunodominant. Analysis of tumor-infiltrating lymphocytes (TILs) from mice immunized with ATLAS-identified neoantigen ± inhibigen peptide vaccines revealed significant alterations in the tumor microenvironment. Inclusion of inhibigens resulted in low T cell infiltration into tumors and increased expression of TIL inhibitory surface markers (e.g. PD-1, LAG-3). Immunological mechanisms of the inhibitory phenomenon are currently being explored. These data promote rational methods for neoantigen identification and highlight the potential advantages of excluding deleterious inhibigens from cancer vaccines and immunotherapies. GEN-009, a personalized cancer vaccine filtered for inclusion of only ATLAS-identified neoantigens (excluding inhibigens) is currently being evaluated in a Phase 1/2a clinical trial (NCT03633110).

Citation Format: Victoria L. DeVault, Hanna Starobinets, Sanmit Adhikari, Simran Singh, Stephanie Rinaldi, Brendan Classon, Jessica B. Flechtner, Hubert Lam. Inclusion of inhibitory neoantigens can abolish efficacy of otherwise protective therapeutic anti-tumor vaccines [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6680.