bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2354: Targeted IRAK-M degradation as a novel and efficacious cancer-immunotherapy overcoming innate-driven immunosuppression
Authors: Gamo Kanae , Kitamoto Naomi , Tomata Yoshihide , Tominari Yusuke
Published: August 2019

IRAK-M has an important role in tightly controlling innate immune responsiveness to preserve homeostasis, mediating immune tolerance, and acts as a negative feedback regulator of TLR/IL-1R signaling pathway. Targeting IRAK-M, which expression is restricted to myeloid cells, would be potentially limiting adverse events against non-target tissues. From supporting evidence for the role of IRAK-M in innate immunosuppressive capacity of tumor-associated macrophages (TAMs) or dendritic cells (DCs), we have developed compounds targeting IRAK-M as an effective cancer-immunotherapy strategy. Since IRAK-M is a pseudokinase which is characterized by the lack of conserved motifs involved in catalytic kinase activity, drug design for inhibition of IRAK-M has been challenging because the conventional small molecule could not modulate its function in cells. Therefore, we conceived and optimized heterobifunctional for degradation comprising IRAK-M-binding moiety linked to the our proprietary XIAP binders to eliminate the IRAK-M protein via ubiquitin-proteasome system. We demonstrated a concentration-dependent degradation of IRAK-M protein in THP1 human monocytic leukemia cells with degraders, and this degradation activity was confirmed to be proteasome-dependent by rescue of depletion following pre-incubation with the proteasome inhibitor epoxomicin. In addition, to estimate the cellular degradation selectivity of our degrader to IRAK-M in an unbiased manner, we carried out whole proteome mass spectrometry in THP1 cells and exhibited good selectivity against identified quantifiable proteins including IRAK-4. As functional studies, we performed MDSC suppression assays in a co-culture model with T cells and found IRAK-M degrader could release the suppressive function of MDSC. Lead optimization steps for IRAK-M degraders have conducted by applying RaPPIDSTM which is a proprietary divergent degrader synthetic platform and already identified multiple drug candidates within a year. Finally, in LLC Lewis lung carcinoma syngeneic model, treatment with candidate resulted in significant tumor growth inhibition. Pre-clinical toxicological studies have been entered and backup compounds are also in development. Taken together, these results highlight the promise of combined IRAK-M silent binder and targeted protein degradation technology as a novel therapeutic strategy for the cancer-immunotherapy that shifts the balance between tolerance and immunity by releasing immunosuppressive activity and in turn by unleashing tumor-specific T-lymphocytes.

Citation Format: Kanae Gamo, Naomi Kitamoto, Yoshihide Tomata, Yusuke Tominari. Targeted IRAK-M degradation as a novel and efficacious cancer-immunotherapy overcoming innate-driven immunosuppression [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 2354.
Abstract 2355: IKKa serves as a molecular switch linking miRNA biogenesis and inflammation in skin tumorigenesis
Authors: Zhu Feng , Zhang Jian , Willette-Brown Jami , Su Ling , Wu Xiaolin , Hu Yinling
Published: August 2019

Our previous studies have shown that the absence of tumor suppressor IKKα leads to development of spontaneous mouse squamous cell carcinomas (SCCs) in the skin, lungs, and esophagus, which is partially mediated by aberrant epigenetic alterations and inflammation. However, it remains unknown whether there is a crosstalk between the above-mentioned mechanisms. As a part of the epigenetic machinery, miRNAs regulate many genes post-transcriptionally. Here we show that IKKα deletion in the mouse epidermis results in impaired miRNA processing with markedly reduced expression of ribonuclease Dicer, an enzyme required for miRNA maturation. We found that the lack of IKKα causes Dicer gene silencing through DNA methylation. As a result, the maturation of miR-203-3p, which is the most abundant miRNA in keratinocytes, is blocked in Ikkα-null keratinocytes. Next, we identified Myd88, an important component of innate immunity and inflammation, as one of miR-203-3p targets. The level of Myd88 expression is robustly increased in Ikkα-null keratinocytes due to the lack of miR-203-3p. We also observed increased activity of caspase 1 cleavage, an indicator of inflammasome activation, in Ikkα-null keratinocytes. Interestingly, Myd88 deletion or 5-aza-2’-deoxycytidine (DNA methyltransferase inhibitor) treatment rescued the survival of Ikkαf/f;K5Cre mice, which usually die within three weeks after birth. Ikkαf/f;Myd88f/f;K5Cre mice also develop much fewer spontaneous skin tumors. By analyzing a cohort of human head and neck SCCs from TCGA database, we found that Dicer expression is concurrently downregulated among 32% of patients with low IKKα expression. Taken together, our finding illustrated how IKKα loss mediates Dicer gene silencing, which turns off miRNA processing, including miR-203-3p maturation, and eventually triggers Myd88-dependent inflammation. Thus, our study revealed a novel IKKα/Dicer/miR-203-3p/Myd88 signaling loop, in which IKKα serves as a molecular switch linking microRNA biogenesis and inflammation in skin tumorigenesis.

Citation Format: Feng Zhu, Jian Zhang, Jami Willette-Brown, Ling Su, Xiaolin Wu, Yinling Hu. IKKa serves as a molecular switch linking miRNA biogenesis and inflammation in skin tumorigenesis [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 2355.
Abstract 2357: Identification of microRNAs associated with melanoma immunity and immunotherapy outcome
Authors: Sloane Robert A. Szczepaniak , Andrews Miles C. , Han Guangchun , Haydu Lauren , Burton Elizabeth , Jeffrey Gershenwald E. , Davies Michael A. , Wang Linghua , Woodman Scott , Wargo Jennifer
Published: August 2019

Introduction:Resistance to immunotherapy is a significant clinical challenge, preventing or limiting response to PD1 checkpoint blockade in approximately 60% of melanoma patients. We sought to identify microRNAs (miRs) that modulate the tumour immune microenvironment and potentially influence outcome to immunotherapy.Methods:To identify miRs associated with immune infiltration and exclusion in melanoma, we first compared miR expression in the human skin cutaneous melanoma (SKCM) dataset from The Cancer Genome Atlas (TCGA) with a selection of immune correlates including histologic assessment of tumour infiltrating lymphocytes (TIL), gene expression-based cytolytic score using Granzyme A and Perforin 1, and transcriptomic estimation of immune cell populations using CIBERSORT. In parallel, we performed miR sequencing of 22 pre-PD-1 inhibitor treatment melanoma tumors to identify differentially-expressed miRs according to PD-1 inhibitor response. We then tested the immune effects of candidate immunomodulatory miRs using microRNA mimics in a panel of human melanoma cell lines, evaluating immunomodulatory cytokine secretion using a bead-based immunoassay (LEGENDplex) and cell surface expression of HLA-A and PD-L1 by flow cytometry.Results:We identified 14 microRNAs associated with immune infiltration or exclusion in the TCGA melanoma dataset. In vitro characterisation of these miRs showed that they regulate secretion of IL-6 and VEGF, and expression of HLA-A in human melanoma cell lines. Furthermore, we identified 25 downregulated and 8 upregulated miRs in responder vs non-responder melanoma lesions (log2 fold change >=1, padj<0.1), including several miRs with high-confidence targets involved in oncogenesis, cancer germline antigens and modification of the extracellular matrix that may impact upon susceptibility to immune attack.Conclusions:This study identified several immunomodulatory miRs associated with immune infiltration in melanoma tumors of TCGA. Several of these candidate immunomodulatory miRs show evidence of activity relevant to the efficacy of PD-1 checkpoint blockade, whilst additional response/resistance-associated miRs were identified. These miRs represent strong candidates for ongoing in vitro and in vivo studies in combination with immunotherapy.

Citation Format: Robert A. Szczepaniak Sloane, Miles C. Andrews, Guangchun Han, Lauren Haydu, Elizabeth Burton, Gershenwald E. Jeffrey, Michael A. Davies, Linghua Wang, Scott Woodman, Jennifer Wargo. Identification of microRNAs associated with melanoma immunity and immunotherapy outcome [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 2357.
Abstract 2359: The MCPyV st-ag and IL33/ST2 axis: critical role in merkel cell carcinoma
Authors: Rasheed Kashif , Fismen Silje , Grimstad Øystein , Sveinbjørnsson Baldur , Moens Ugo Lionel
Published: August 2019

Merkel Cell polyomavirus (MCPyV) is a causal factor in Merkel Cell Carcinoma (MCC). The oncogenic potential is mediated through its viral oncoproteins large T-antigen (LT-ag) and small t-antigen (st-ag). Many cancers arise from sites of infection, chronic irritation and inflammation, and inflammatory signaling pathways are often activated by oncogenic mutations. Chemokines are a family of cytokines that regulate leukocyte trafficking in immunity and inflammation. The aberrant expression of chemokines and chemokine receptors in tumors may regulate the trafficking of leukocytes into the tumor microenvironment. IL33, a member of IL-1 family cytokines, is involved in the response to viral infection and plays important roles in type-2 innate immunity, tumorigenesis and tumor immune evasion.

We assessed IL33/ST2 expression in MCPyV-positive (MKL2) and –negative (MCC13) MCC cell lines in order to examine a possible role of IL33/ST2 pathway in MCC.

Using the human RT2 Profiler PCR Inflammatory Cytokines and Receptors Array, we did a PCR array analysis of 84 different human cytokines and receptor genes. Expression of IL-33 and ST2 was determined by qRT-PCR, immunohistochemistry and western-blot analysis. Luciferase analysis was done to monitor the effect of MCPyV early proteins on IL33 promoter activity and the effect of IL33 on MCPyV early and late promoter activity. Furthermore, mitogen-activated protein kinase (MAPK) and the NF-κB pathways were also assessed after stimulating MCC13 cells with recombinant human (rh) IL33 protein. Finally, proliferation activity after rhIL33 stimulation was assessed by MTT-assay.

PCR array analysis showed higher transcript levels of IL33 in MCC13 cells transfected with st-ag compared to control cells. Western blot analysis confirmed increased IL33 expression by st-ag. Luciferase analysis showed higher IL33 promoter activity in st-ag transfected MCC13 cells. Interestingly, IL33 also increased MCPyV early and late promoter activity. Furthermore, rhIL33 activated both MAPK and the NF-κB pathways and blocking ST2 receptor using polyclonal goat ST2 antibody abolished IL33 activity. Dose-dependent effect of rhIL33 showed higher proliferation activity of MCC13 cells. Immunohistochemical analysis of IL33 and ST2 showed a significant stronger expression in MCC tissues compared to normal skin.

Our study unveiled a novel st-ag dependent IL33 function in MCC. Therefore, neutralizing the IL-33/ST2 axis may thereby represent a therapeutic target in MCPyV-positive MCC patients.

Citation Format: Kashif Rasheed, Silje Fismen, Øystein Grimstad, Baldur Sveinbjørnsson, Ugo Lionel Moens. The MCPyV st-ag and IL33/ST2 axis: critical role in merkel cell carcinoma [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 2359.
Abstract 2374: Non-canonical TGF-beta signaling modulates pancreatic ductal adenocarcinoma progression
Authors: Hussain S. Mazher , Miranda-Carboni Gustavo , Alvarez Marcus A. , Elahi Abul , Hedrick Leah E. , Shibata David , Glazer Evan S.
Published: August 2019

Introduction: While the genomic and proteomic characterization of pancreatic ductal adenocarcinoma (PDAC) is extraordinarily complex, the transforming growth factor-beta (TGF-β) pathway is critically important. TGF-β has a context dependent role- it is tumor suppressive in early PDAC and tumor promotive in metastatic PDAC. Our hypothesis is that non-canonical TGF-β signaling induces metastatic PDAC. Non-canonical signaling may help explain the paradoxical nature of TGF-β in PDAC.

Methods: Two cell lines were utilized- Panc-1, a cell line derived from primary PDAC, was compared to Capan-1, a cell line derived from metastatic PDAC- in order to model an early PDAC (Panc-1) and a more aggressive PDAC (Capan-1). We investigated the gene and protein expression of markers of epithelial to mesenchymal transition (Snail and Twist), Akt, β-catenin, and PD-L1 upon treatment with TGF-β and the Akt inhibitor MK-2206.

Results: While TGF-β increased Snail gene and protein expression in Panc-1 cells, it had nil effect on Capan-1 cells where TWIST1 protein expression increased with TGF-β. TGF-β increased pAkt expression in both cell lines. TGF-β increased active and total β-catenin expression in Capan-1 but not Panc-1 cell lines. We then found that PD-L1 expression was generally diminished by Akt inhibition in both cell lines but this was reversed with TGF-β receptor inhibition and gemcitabine treatment in the Capan-1 cells.

Conclusions: Our data suggests that modulation of the non-canonical TGF-β pathway may lead to increased epithelial to mesenchymal transition through multiple mechanisms. PD-L1 expression is a response to chemotherapy in metastatic PDAC when multiple proteins in that pathway are inhibited. Our data suggests that (1) personalized therapy based on the degree of non-canonical TGF-β pathway activation will be critical, (2) patients with uncontrolled non-canonical TGF-β pathway activation may have lower survival, and (3) there is an opportunity to utilize anti-PD-L1 based therapy in combination with other therapy in selected patients based on their response to chemotherapy.

Citation Format: S. Mazher Hussain, Gustavo Miranda-Carboni, Marcus A. Alvarez, Abul Elahi, Leah E. Hedrick, David Shibata, Evan S. Glazer. Non-canonical TGF-beta signaling modulates pancreatic ductal adenocarcinoma progression [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 2374.
Abstract 2379: In vivo drug screening platform accelerated 4-1BB agonistic antibody development
Authors: Yang Benny(Yi) , Ni Jian , Li Yanan , Shen Yuelei , Lin Qingcong
Published: August 2019

4-1BB (TNFRSF9/CD137) belongs to the TNFR superfamily. 4-1BB is expressed by activated CD4 helper T cells, B cells, natural killer cells, natural killer T cells, dendritic cells, and activated endothelium. A majority of regulatory T cells (Tregs) also express 4-1BB, but it remains unclear whether agonistic antibody treatment exerts pro- or anti-suppressive effect on these cells. 4-1BB enhances tumor rejection because it is upregulated on T cells following activation and its engagement increases T cell proliferation and pro-inflammatory cytokine production. The clinical development of 4-1BB targeting therapy was slow due to the toxicity associated with overt immune activation. Fortunately, new therapeutic modalities using 4-1BB targeting aptamers as well as therapeutic combinations with other immuno-modulatory and traditional anti-cancer treatments have revived excitement for the use of 4-1BB agonists in the clinic.

We focused on discovering novel 4-1BB therapeutic antibodies for cancer patients. Candidate molecules obtained through the classical hybridoma technology were screened by high-throughput flow cytometry. Then, we took the advantages of Biocytogen’s humanized mice and screened the purified antibodies in B-h4-1BB mice bearing MC-38 tumor cells. We found some antibodies that worked as equivalent as BMS’s Urelumab. To determine which isotype should be adopted, we engrafted the antigen binding domain onto various human Fc backbones. Interestingly, we found that the human IgG1 isotype appeared to be more effective against tumors than other isotypes in B-4-1BB mice bearing MC-38 tumors. This study provides a reliable basis for our follow-up mechanism research and clinical evaluation for this target.

Citation Format: Benny(Yi) Yang, Jian Ni, Yanan Li, Yuelei Shen, Qingcong Lin. In vivo drug screening platform accelerated 4-1BB agonistic antibody development [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 2379.
Abstract 2383: The molecular binding mechanism of tislelizumab, an investigational anti-PD-1 antibody, is differentiated from pembrolizumab and nivolumab
Authors: Feng Yingcai , Hong Yuan , Sun Hanzi , Zhang Bo , Wu Hongfu , Li Kang , Liu Xuesong (Mike) , Liu Ye
Published: August 2019

Programmed cell death protein 1 (PD-1) is an immune checkpoint receptor expressed by activated T, B, and NK cells, which interacts with its ligand PD-L1/L2 to inhibit T-cell proliferation and effector functions such as tumor cell killing and cytokine production. Two anti-PD-1 antibodies approved by the FDA, pembrolizumab and nivolumab, have shown efficacy in many cancer types, nevertheless there are some indications where limited efficacy is observed. Tislelizumab (BGB-A317), an investigational anti-PD-1 antibody, has demonstrated significant clinical activity (85.7% ORR, including 61.4% CR) in relapsed/refractory classical Hodgkin’s lymphoma (R/R cHL). Additionally, tislelizumab is being studied in global pivotal trials in a number of malignancies, including non-small cell lung cancer, hepatocellular carcinoma, and esophageal squamous cell carcinoma. However, how tislelizumab binds to PD-1 has yet to be shown, particularly in comparison to pembrolizumab and nivolumab. Here we report the co-crystal structure of PD-1 extracellular domain and the Fab of tislelizumab. Tislelizumab interacts with IgV-like domain of PD-1 with an interface area of 1112 Å2. Structure-guided mutagenesis of PD-1 and surface plasmon resonance were performed to compare the binding of tislelizumab, pembrolizumab and nivolumab to mutant and wild type PD-1. The dissociation rate (kd) of tislelizumab from wild type PD-1 is about 100-fold and 50-fold slower than that of pembrolizumab and nivolumab, respectively. Gln75, Thr76, Asp77 and Arg86 on PD-1 are critical epitopes for tislelizumab, but mutation of them showed little effect on binding of PD-1 to pembrolizumab and nivolumab. Both the co-crystal structure and mutagenesis study identified the unique epitopes of tislelizumab that correlate to the extremely slow-off property of tislelizumab after binding to PD-1. In conclusion, we observed that tislelizumab is differentiated from pembrolizumab and nivolumab by its unique binding epitopes as well as binding kinetics.

Citation Format: Yingcai Feng, Yuan Hong, Hanzi Sun, Bo Zhang, Hongfu Wu, Kang Li, Xuesong (Mike) Liu, Ye Liu. The molecular binding mechanism of tislelizumab, an investigational anti-PD-1 antibody, is differentiated from pembrolizumab and nivolumab [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 2383.
Abstract 2385: Discovery of highly specific claudin 6 monoclonal antibodies
Authors: Stafford L Joe
Published: August 2019

Claudin 6 (CLDN6) is an oncofetal tight junction protein involved in the cell-to-cell adhesion of epithelial and endothelial cell sheets. Although silenced in healthy adult human tissues, CLDN6 expression has been found in ovarian, gastric, pediatric, and other cancer tissues and can lead to a poor prognosis. Monoclonal antibody (MAb) discovery against CLDN6 has been encumbered by the high homology of endogenously expressed claudin 9 (CLDN9), which varies from CLDN6 by only 3 amino acids in the extracellular domain. Our MPS Antibody Discovery platform utilizes proprietary technologies to overcome key barriers to MAb discovery, enabling the isolation of MAbs against multipass membrane proteins such as CLDN6. Using the MPS platform, we have successfully isolated and characterized panels of highly specific MAbs to CLDN6. Nine chickens were immunized with the target antigen, and titer was achieved in all but one animal. Following 2 rounds of phage panning, we screened 2,000 clones for CLDN6 specificity, identifying 788 hits and 68 unique VH CDR3 families. From these clones, 72 antibodies were selected for binding to CLDN6 and CLDN6/9. Two lead antibodies, IM-171 and IM-136, showed high specificity and high affinity to CLDN6 but not to claudins 3, 4, or 9. Using high-resolution Shotgun Mutagenesis Epitope Mapping, we confirmed that our lead candidate MAbs bind distinct residues on CLDN6 compared to previously described MAbs. Specificity analysis with our Membrane Proteome Array revealed no significant binding of either of our lead MAbs to any other membrane protein in the human proteome, including any of the other 20+ claudin proteins. Both of our lead MAbs bound endogenously expressed CLDN6 on ovarian carcinoma (PA-1) cells. Our preclinical CLDN6 antibodies are now being developed for use in various formats, including bispecifics, antibody-drug conjugates, and CAR-T applications.

Citation Format: L Joe Stafford. Discovery of highly specific claudin 6 monoclonal antibodies [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 2385.
Abstract 2387: NKG2A-antagonizing monoclonal antibodies identified through phage display
Authors: Gao Jing , Hu Lili , Sui Ruirui , Guo Cuicui , Wang Lina , Yang Teddy , Han Jack , Duan Qing , Liu Louis
Published: August 2019

The inhibitory receptor NKG2A-CD94 is mainly expressed on NK cells and some T cell and mediates an inhibitory signal through association with a non-classical MHC class I molecule HLA-E, which is commonly over-expressed in human cancers. Targeting NKG2A with monoclonal antibody holds potentials in immunotherapy of cancer. However, proteins with homologous sequences such as NKG2C/CD94 or NKG2E/CD94 complexes generally deliver an activating signal upon ligand binding. NKG2A-specific antagonizing antibodies would be needed to further explore therapeutic potentials of targeting NKG2A for immunotherapy of cancer.

We have developed and characterized panels of monoclonal antibodies (mAbs) that specifically recognize NKG2A. Balb/c or SJL mice had been immunized with recombinant NKG2A/CD94 protein, recombinant plasmids encoding hNKG2A or stable cell line with ectopic expression of hNKG2A. Splenocytes from such immunized animals were collected for construction of phage-displayed antibody library in a single chain variable fragment (scFv) format. The NKG2A-specific antibodies, that had no binding to NKG2C, NKG2E or CD94, were isolated through multiple rounds of phage panning and ELISA-based screening. Such antibodies also block HLA-E ligand binding and enhanced cytotoxicity of NK cell lines and primary NK cells on cancer cells.

In summary, isolation and characterization of a panel of NKG2A-specific antagonizing antibodies enable us to further determine the feasibility of NKG2A-targeted immunotherapy of cancer.

Citation Format: Jing Gao, Lili Hu, Ruirui Sui, Cuicui Guo, Lina Wang, Teddy Yang, Jack Han, Qing Duan, Louis Liu. NKG2A-antagonizing monoclonal antibodies identified through phage display [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 2387.
Abstract 2388: In vitro assessment of tumor associated I.O. markers in cancer cell lines following Vicinium treatment
Authors: Chooniedass Shilpa , Dillon Rachelle L. , Premsukh Arjune , MacDonald Glen C. , Cizeau Jeannick , Adams Gregory P.
Published: August 2019

Vicinium is a fusion protein that comprises a scFv fragment specific for the Epithelial Cell Adhesion Molecule (EpCAM) genetically fused to a truncated form of Pseudomonas exotoxin A, ETA, via a flexible linker. Vicinium is used for the treatment of loco-regionally accessible tumors and is currently in a phase III clinical trial (VISTA) for the treatment of high-grade non-muscle invasive bladder cancer and a phase I study in combination with durvalumab for the same indication. In a different phase I study in late stage squamous cell carcinoma of the head and neck, direct injection of Vicinium led to shrinkage of the principal injected tumor as well as non-targeted tumors in some patients suggesting the activation of a T cell-mediated anti-tumor response through cross-priming. Supporting this hypothesis, in vitro studies have demonstrated that Vicinium mediated tumor cell killing elicits biological features of an immunogenic cell death (ICD). Tumor cells’ ability to evade the innate and adaptive immune response plays a major role in cancer development and progression. Altered expression of immune modulators is a key mechanism responsible for tumor escape. Recent studies have shown that exposure to chemotherapeutic drugs results in the selection of tumor cells expressing immunosuppressive markers such as PD-L1, CD47 and CD39. Following in vitro treatment with Vicinium, the enrichment of tumor cell population expressing I.O. markers was examined and compared to chemotherapeutic agents. Unlike small molecule chemotherapies, Vicinium treatment did not lead to an enrichment of CD47 or CD39 positive tumor cell populations. Taken together, the data suggests that Vicinium does not induce an immunosuppressive environment.

Citation Format: Shilpa Chooniedass, Rachelle L. Dillon, Arjune Premsukh, Glen C. MacDonald, Jeannick Cizeau, Gregory P. Adams. In vitro assessment of tumor associated I.O. markers in cancer cell lines following Vicinium treatment [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 2388.
Abstract 2389: Role of Midkine in molecular targeted therapy to TIP1 for lung cancer treatment
Authors: Kapoor Vaishali , Singh Abhay K. , Thotala Dinesh , Hallahan Dennis
Published: August 2019

Non-small cell lung cancer (NSCLC) ranks among the highest cancer-related mortalities world-wide. Targeted therapy has been a growing topic of investigation to improve therapeutic efficacy for NSCLC. A few targeted therapies that exploit aberrant protein expression profiles have been approved for NSCLC. However, marginal improvement in efficacy observed with these therapeutic approaches highlights the need for discovery of additional therapeutic targets. We identified Tax interacting protein 1 (TIP1) which is overexpressed in various cancers as a molecular targets. We focused on antibody-based targeting of the chaperone protein TIP1. The functional domain of TIP1 (PDZ domain) caps the C-terminus of many cellular proteins that regulate important cellular functions. Knocking down TIP1 revealed that it plays an important role in cell signaling, cancer development, and progression. Comparing antibodies targeting different epitopes of TIP1, we found that antibodies against the PDZ domain of TIP1 were most effective in inducing cytotoxicity of lung cancer cells but not normal cells. Anti-PDZ/TIP1 antibodies injected into mice bearing lung cancer bind specifically to cancer and substantially enhance tumor control. A mass spectrometry-based approach identified Midkine (MDK) as a putative protein that modulates this cytotoxicity by anti-TIP1 antibodies. Additional studies suggested that the β-catenin/Wnt signaling may be involved in induction of MDK after blocking of TIP1. These studies led to the hypothesis that MDK is upregulated by the anti-PDZ/TIP1 antibody via the β-catenin/Wnt signaling pathway, which subsequently modulates downstream signaling mechanisms. This project will systematically characterize the mechanisms of cytotoxicity by the anti-PDZ/TIP1 antibodies. To achieve this, a number of novel methodologies including genome-wide CRISPR knockout, proteomics and mass cytometry to characterize the link between TIP1 and MDK will be applied. The project will guide development of combination therapies to optimize efficacy of NSCLC treatment. One strategy proposed, is dual-targeting of TIP1 and MDK for enhanced efficacy. This project will lead to identification of novel molecular targets for NSCLC treatment that have never been considered before.

Citation Format: Vaishali Kapoor, Abhay K. Singh, Dinesh Thotala, Dennis Hallahan. Role of Midkine in molecular targeted therapy to TIP1 for lung cancer treatment [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 2389.
Abstract 2404: Mechanistic insight into anti-tumor activity of DKN-01, a therapeutic antibody with immune modulatory activity
Authors: Haas Michael , Heath Heidi , Schurpf-Huber Franziska , Wu Yinyuan , Zhang Xinjun , He Xi , Yang Min , Newman Walter , Kagey Michael
Published: August 2019

Wnt signaling is a fundamental pathway that is dysregulated in oncology. The Wnt signaling modulator DKK1 is expressed in a variety of tumor types and elevated levels frequently correlate with poor survival. DKK1 promotes tumor growth by stimulating proliferation, metastasis, and angiogenesis, and has been implicated in contributing to an immune suppressive tumor microenvironment. DKN-01 is a humanized monoclonal therapeutic antibody that binds DKK1 with high affinity and selectivity. It is currently being evaluated clinically as a monotherapy and in combination with chemotherapies and other immunotherapies in a variety of solid tumors. Here we demonstrate that efficacy from a murine version of DKN-01 (mDKN-01) in a syngeneic tumor model requires NK cells. mDKN-01 also reduces lung metastasis, further supporting a model where DKK1 neutralization stimulates a NK mediated anti-cancer response. In addition, DKN-01 has efficacy in a human prostate xenograft model that expresses high levels of DKK1 and retains functioning NK cells. Taken together, these data support an innate immunotherapeutic mechanism of action of DKN-01 and suggest that tumor expression of DKK1 may predict response. As such, evaluation of peripheral blood and tumor biopsies from patients undergoing treatment with DKN-01 in combination with chemotherapy or other immunotherapies are underway to demonstrate innate immune mediated anti-tumor activity and validate DKK1 expression as a biomarker.

Citation Format: Michael Haas, Heidi Heath, Franziska Schurpf-Huber, Yinyuan Wu, Xinjun Zhang, Xi He, Min Yang, Walter Newman, Michael Kagey. Mechanistic insight into anti-tumor activity of DKN-01, a therapeutic antibody with immune modulatory activity [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 2404.
Abstract 2410: Development of a custom genotyping platform and genetic prediction of prostate cancer risks in sub-Saharan Africa
Authors: Lachance Joseph , Harlemon Maxine , Kachambwa Paidamoyo , Ajayi Olabode , Kim Michelle , Adams Marcia , Pugh Elizabeth , Peterson Lindsay , Rebbeck Timothy
Published: August 2019

Prostate cancer is a highly heritable disease that disproportionally affects African and African-American men. With this in mind, the MADCaP (Men of African Descent Carcinoma of the Prostate) Network has developed a custom genotyping platform. This array is optimized for detection of novel genetic associations in sub-Saharan African populations. The MADCaP Array contains approximately 1.6 million markers, many of which overlap the H3Africa Consortium Array and the OncoArray. It includes an imputation backbone that successfully tags 94% of common (MAF > 0.05) genetic variants in African populations (r2 threshold = 0.8). To aid in fine-mapping, the MADCaP Array has a high density of markers in genomic regions surrounding known cancer associations, including 8q24. Markers on the MADCaP Array also include over 27,000 prostate eQTLs. Using the MADCaP Array, we conducted a pilot study of 800 individuals with individual-level phenotype information. These samples include equal numbers of prostate cancer cases and controls, and they were collected from study sites in Ghana, Nigeria, Senegal, and South Africa. Here, we assess the extent to which polygenic risk scores are able to predict prostate cancer risks in African populations. We also identify non-African GWAS signals that replicate well in African populations. Additional analyses in this study include testing whether specific genetic ancestries are over-represented in cases, and quantifying the extent to which runs of homozygosity are found in the genomes of cases and controls.

Citation Format: Joseph Lachance, Maxine Harlemon, Paidamoyo Kachambwa, Olabode Ajayi, Michelle Kim, Marcia Adams, Elizabeth Pugh, Lindsay Peterson, Timothy Rebbeck. Development of a custom genotyping platform and genetic prediction of prostate cancer risks in sub-Saharan Africa [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 2410.
Abstract 2414: Prognosis prediction of colorectal cancer using gene expression profiles
Authors: Pan Feixia , Chen Tianhui , Sun Xiaohui , Li Kuanrong , Jiang Xiyi , Försti Asta , Zhu Yimin , Lai Maode
Published: August 2019

Background: Investigation on prognostic markers for colorectal cancer (CRC) deserves efforts, but data from China are scarce. This study aimed to build a prognostic algorithm using differentially expressed gene (DEG) profiles and to compare it with the TNM staging system in their predictive accuracy for Chinese CRC prognosis.

Methods: DEGs in six-paired tumor and corresponding normal tissues were determined using RNA-Sequencing. Subsequently, matched tumor and normal tissues from 127 Chinese patients were assayed for further validation. Univariate and multivariate Cox regressions were used to identify informative DEGs. A predictive index (PI) was derived as a linear combination of the products of the DEGs and their Cox regression coefficients. The predictive performance of the PI was evaluated with receiver operating characteristics (ROC) curves and the area under curves (AUCs). A logistic regression model was built, including both the DEGs-based PI and tumors’ TNM stages.

Results: We identified significant associations of 13 DEGs (out of 75 candidate DEGs) with CRC survival. A PI based on these 13 DEGs (PI-13) predicted CRC survival probability more accurately than the TNM staging system [AUCs for 3-year survival probability 0.76 (95% confidence interval: 0.67, 0.83) vs. 0.63 (0.53, 0.71)] but comparable to a simplified PI (PI-5) using five DEGs (LOC646627, SCARA5, CDKN2A, ATP6V1A, and DNMT3B). The predictive accuracy was improved further by combining PI-5 and the TNM staging system [AUC for 3-year survival probability: 0.81 (0.73, 0.88)].

Conclusion: Prognosis prediction based on informative DEGs might yield a higher accuracy than the TNM staging system.

Citation Format: Feixia Pan, Tianhui Chen, Xiaohui Sun, Kuanrong Li, Xiyi Jiang, Asta Försti, Yimin Zhu, Maode Lai. Prognosis prediction of colorectal cancer using gene expression profiles [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 2414.
Abstract 2441: Association between NRF1 regulatory gene networks and cancer and aging
Authors: Bhawe Kaumudi , Felty Quentin , Yoo Changwon , Roy Deodutta
Published: August 2019

There is a growing convergence between molecular aspects of aging and cancer. Cellular senescence acts as a barrier through preventing the proliferation of cells under oncogenic stress. Therefore, we decided to examine the role of nuclear respiratory factor 1 (NRF1) transcription factor in human brain cancer and brain aging. In contrast to higher expression of NRF1 in human glioma tumors, lower NRF1 levels are observed in Alzheimer’s disease (AD) brain tissue. Using the glioma and AD transcriptomic data of NRF1 target genes, we conducted Transcription Factor Target Enrichment Analysis that detects transcription factor (TF) activity based on quantification of the collective transcriptional activation of their target genes. NRF1 TF activity was significantly lower in AD brain tissues from four independent cohorts compared to non-AD brain tissues. In contrast, we observed higher NRF1 transcription factor activity in glioma. NRF1 activity as well as expression of several cell cycle pathway genes including CDK1,2,6,9 and NRF1 target genes such as NR2E1, APOE and MAPT are significantly altered in these two population cohorts. We also discovered that expression of NRF1, RB1 and E2F influenced survival in glioma. Specifically, the mean overall survival for patients with a high NRF1, E2F1,2,6,7,8 and RB1 gene signature was 20.91 months (95% CI = 9.37-32.46), compared to 101.99 months (95% CI = 74.28-129.70), in high NRF1 expressing patients that lacked the specific gene signature. NRF1 based gene signatures combined with IDH1 mutation status are necessary and sufficient for explaining the observed differences in survival curves. We examined the association of NRF1, the E2Fs and RB1 through Bayesian graphical modeling and observed gender differences in gene networks between NRF1 and E2F family members in the glioma patient sub-populations as compared to normal aging human brain samples. Furthermore, the differentially expressed NRF1 regulated genes are part of the KEGG pathways associated with spliceosome, ribosome and lysosome function as well as a diverse array of other functions are enriched, indicating ways in which NRF1 may be involved in glioma biology. In summary these findings support our postulate that NRF1 is an important transcription factor in the human brain, where it plays an active role in both aging and cancer.

Citation Format: Kaumudi Bhawe, Quentin Felty, Changwon Yoo, Deodutta Roy. Association between NRF1 regulatory gene networks and cancer and aging [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 2441.
Abstract 2457: Prediction of selective genetic vulnerabilities from large-scale functional screens in cancer cells
Authors: Rauscher Benedikt
Published: August 2019

Cancer genomes often harbor hundreds of molecular aberrations. Such genetic variants can be drivers or passengers of tumorigenesis and create new vulnerabilities for potential therapeutic exploitation. To identify genotype-dependent vulnerabilities, a large number of forward genetic screens in different genetic backgrounds have been conducted. To aggregate and mine functional data from published screens in a standardized format, we recently developed GenomeCRISPR, a curated database for high-throughput CRISPR-Cas9 experiments. We then devised MINGLE, a computational framework that integrates genetic screens in large panels of cancer cell lines to predict genetic interactions between variant and knockout alleles. We showed that our method identifies specific known and previously unknown genetic relationships. For example, we identified a synthetic lethal dependency between RNF43 and members of the Glucosidase II complex. Additional experiments confirmed that the Glucosidase II complex acts as a regulator of Wnt/beta-catenin signalling. In our first study we analyzed a dataset consisting of screens performed in sixty cell lines. Since then the amount of data available for this analysis has increased several fold. We are continuously integrating the new data that become available as we work towards the next iteration of a genetic interaction map in cancer cells. We are specifically interested in using the increased resolution of this new map to understand how genetic networks differ between individual cancer types.

Citation Format: Benedikt Rauscher. Prediction of selective genetic vulnerabilities from large-scale functional screens in cancer cells [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 2457.
Abstract 2459: Identifying genetic drivers of uterine serous carcinoma patient prognosis with regulatory network analysis
Authors: Myers Emily , Tran Lynn K. , Tran Paul M. , Mysona David , Purohit Sharad , Wallbillich J. J. , She Jin-Xiong
Published: August 2019

Uterine serous carcinoma (USC) is a subtype of endometrial cancer (EC), which is the most common gynecological malignancy in the developed world. Though USC is a Type II EC, which are only responsible for 20% of EC cases, Type II EC causes 74% of EC deaths. Understanding the molecular mechanisms driving USC tumor biology could reveal novel therapies and improve patient outcomes. We developed and validated gene expression signatures capable of predicting USC patient’s prognosis. However, little was understood about why differential expression of these particular genes predicts patient prognosis. As such, we investigated the key transcription factors driving the phenotypic difference between the two groups by using the Passing Attributes between Networks for Data Assimilation (PANDA) algorithm. As described by Glass et al., PANDA uses message passing to integrate information from multiple data types to construct robust interaction networks between transcription factors and genes. Following regulatory network generation, we selected potential key regulators based on network analyses. First, we use linear regression analysis to select TFs involved in gene interactions with the greatest edge weight differential between the two groups. Second, we identify TFs involved in unique regulatory interactions to each group. Third, we analyze the TF-gene signature gene interaction networks for both patient populations to select hub transcription factors. TFs selected in all three analyses as potential key regulators are ranked using summed residual values to determine in which patient group the TFs were weighted more strongly. A literature review revealed that the TFs most heavily weighted in the poor survival group are implicated as suppressors of tumor growth. The TFs most heavily weighted in the good survival group are implicated in promoting tumor growth. This suggests that fast-growing tumors correlate with good survival and slow-growing tumors correlate with poor survival. We hypothesize that this effect is due to the impact of tumor growth rate on chemosensitivity, and we plan to test this hypothesis in future studies.

Citation Format: Emily Myers, Lynn K. Tran, Paul M. Tran, David Mysona, Sharad Purohit, J. J. Wallbillich, Jin-Xiong She. Identifying genetic drivers of uterine serous carcinoma patient prognosis with regulatory network analysis [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 2459.
Abstract 246: Glioblastoma cell-modified cell membrane lipid-extracted nanoparticles (CLENs) for imaging and treatment: Influence of magnetic nanoparticle on physiochemical properties of CLENs, cellular uptake, and cytotoxicity
Authors: Song Woongjae , Campbell Robert
Published: August 2019

Background: Glioblastoma multiforme (GBM), located in the cerebral hemispheres of the brain, is the most aggressive form of cancer. Surgery associated with chemotherapy with radiation treatment is the defined way to treat the disease. The delivery of chemotherapeutic agents frequently result in serious side effects. To address this issue we have taken the first steps in the development of CLENs (cell membrane lipid-extracted nanoliposomes) to enhance selective delivery of drugs to the brain.

Method: The CLENs used for the experiments were composed of various ratios of DOPC, DOTAP, cholesterol, and lipid extract (LE) material derived from the target cells (i.e., U87-MG), and DPPE-PEG5000 (PEG). Studies include the determination of drug (cisplatin) loading, magnetite (MAG-C) loading in CLENs, quantitative studies for particle size, zeta potential, SQUID (superconducting quantum interference device) measurements, and fluorescence detection to study cellular interactions.

Results: The average size for the CLENs fell within the range of 130 to 190 nm. The zeta potential reported for CLENs was negatively-charged. The inclusion of 15mol % LE resulted in the most significant cellular uptake by target cell U87-MG. Overall, the uptake by these cells was less compared to CLENS previously published for other organ tissue environments such as with breast tissue. The inclusion of cationic lipid DOTAP in CLENs did not alter the size distribution; however, the values for zeta potential increased from -8 ± 5 to 12 ± 6 mv. The additional inclusion of DOTAP in CLENs increased cellular uptake approximately 4-fold compared to CLENs without DOTAP (fluorescence intensity (A.U) from 145 ± 52 to 525 ± 49)). The inclusion of DOTAP increased the efficiency of loading Cisplatin in CLENs by approximately 30%. Studies involving the incorporation of MAG-C in U87-MG CLENs are currently underway, as well magnetization studies involving SQUID.

Conclusion: All CLENs demonstrated practical physicochemical properties similar to conventional liposomes. The cellular uptake results suggested that cellular uptake of CLENs in vitro, although relatively low overall, varied according to the total LE content. Furthermore, the inclusion of DOTAP in CLENs significantly increased uptake and the efficiency of drug loading.

Citation Format: Woongjae Song, Robert Campbell. Glioblastoma cell-modified cell membrane lipid-extracted nanoparticles (CLENs) for imaging and treatment: Influence of magnetic nanoparticle on physiochemical properties of CLENs, cellular uptake, and cytotoxicity [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 246.