bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3093: Prognostic value of novel immune basal subtypes in muscle invasive bladder cancer
Authors: Choi Woonyoung , Sundi Debasish , Lee I-ling , Siefker-Radtke Arlene , Dinney Colin , Czerniak Bogdan , Kim Seungchan , McConkey David
Published: August 2019

Using gene expression profiling we previously reported the existence of 3 molecular subtypes (basal, p53-like and luminal) characterized by distinct gene expression patterns and clinical outcomes in muscle-invasive bladder cancer (MIBC). Among the subtypes, basal tumors were associated with advanced stage at presentation and shorter survival in the absence of neoadjuvant chemotherapy (NAC). Our recent analysis identified two novel immune subtypes within the basal tumors (basal immune signature enriched - “BIE” and basal immune signature suppressed - “BIS”) using immune infiltration markers that are known to have prognostic value in multiple solid tumors. The analysis revealed that BIE had significantly improved survival outcomes while BIS was associated with the worst survival outcomes among all subtypes in TCGA MIBC data (n=408). Survival outcomes in BIE tumors were validated in an independent cohort (GSE48075, p <0.05). The characterization of the immune landscape in our novel subtypes using TCGA’s recent pan-cancer immunogenomics data showed that the BIE subtype was significantly enriched with ‘INF -γ dominant’ immune subtype out of the 6 TCGA immune subtypes (wound healing, IFN-γ dominant, inflammatory, lymphocyte depleted, immunologically quiet and TGF-β dominant, p < 0.05). Since immune activation has been associated with response to chemotherapy in other tumor types, we explored the potential significance of the novel immune subtypes using public data consisting of patients who received NAC followed by radical cystectomy (GSE52219 and GSE69795). In these cohorts, chemotherapy was active as measured by pathological downstaging rates (≤ pT1 at cystectomy). Based on pathological downstaging, BIE was the most responsive to chemotherapy while BIS and p53-like tumors were resistant to chemotherapy (p < 0.05). We also used the recently developed CIBERSORT tool to estimate stromal cell infiltration in the NAC cohort. The results predicted that the BIE subtype was highly enriched with CD8+ T cells and NK cells (p <0.001). Taken together, our results reveal the existence of novel immune subtypes within basal tumors with distinct molecular features and survival outcomes in MIBC. The clinical implications of these subtypes will be further evaluated.

Citation Format: Woonyoung Choi, Debasish Sundi, I-ling Lee, Arlene Siefker-Radtke, Colin Dinney, Bogdan Czerniak, Seungchan Kim, David McConkey. Prognostic value of novel immune basal subtypes in muscle invasive bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3093.
Abstract 3109: Protein stability screen to identify compounds destabilizing EWS-FLI1 in Ewing sarcoma
Authors: Pedot Gloria , Niggli Felix K. , Schaefer Beat W.
Published: August 2019

Ewing sarcoma is an aggressive pediatric bone and soft tissue tumor driven by the expression of a fusion oncoprotein named EWS-FLI1, which acts as an oncogenic transcription factor. Tumor cells are strictly dependent on continuous expression of the fusion protein, since downregulation of EWS-FLI1 inhibits tumor growth. Therefore, interference with the fusion oncoprotein turnover is critical for the modulation of tumor cell proliferation and survival. We demonstrated previously that EWS-FLI1 is predominantly a proteasomal substrate with high turnover mediated by poly-ubiquitination at one specific lysine. This study provided novel insights into the crucial importance of targeting EWS-FLI1 stability as novel strategy for the treatment of Ewing sarcoma. Hence, we aimed at identifying compounds that destabilize EWS-FLI1 with subsequent reduction of tumor cell growth by performing a screen of 2’486 FDA-Approved drugs and 204 novel targeted compounds in a Ewing sarcoma reporter cell line. To this, we adopted a Global Protein Stability approach as novel read-out (Global protein stability profiling in mammalian cells, Science, 2008), which relies on a reporter construct expressing two fluorescent dyes from one mRNA to monitor changes in stability of EWS-FLI1 by high-throughput flow cytometry. This drug screen identified two main enriched classes of inhibitors that significantly destabilize EWS-FLI1. Validation of these results and characterization of their mechanism of action is currently under way. We conclude that the study of EWS-FLI1 turnover represents a novel approach to identify new effective drugs that can be used as monotherapy or in combination with other drugs as novel treatment opportunities in Ewing sarcoma.

Citation Format: Gloria Pedot, Felix K. Niggli, Beat W. Schaefer. Protein stability screen to identify compounds destabilizing EWS-FLI1 in Ewing sarcoma [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 3109.
Abstract 312: Hypoxia induces EGFR inhibitor resistance in lung cancer cells via fibroblast growth factor receptor 1 (FGFR1) by promoting epithelial-mesenchymal transition (EMT)
Authors: Lu Yuhong , Zhang Gary J. , Glazer Peter M.
Published: August 2019

The development of small molecule tyrosine kinase inhibitors (TKIs) specific for epidermal growth factor receptors (EGFRs) with activating mutations has led to a new paradigm in the treatment of non-small cell lung cancer (NSCLC) patients. However, most patients eventually develop resistance. Hypoxia is a key micro-environmental stress in solid tumors that is associated with poor prognosis due in part to acquired resistance to conventional therapy. In this study, we show that long-term, moderate hypoxia promotes resistance to the EGFR TKI, gefitinib, in the NSCLC cell line, HCC827, which harbors an activating EGFR mutation. We also found that hypoxia induces resistance to the third generation EGFR TKI, osimernitib, in the NSCLC cell line, H1975, which developed resistance to first and second EGFR TKI resistance by harboring a second EGFR mutation, T790M. Following growth in hypoxia, HCC827 and H1975 cells show increased N-cadherin expression with downregulation of E-cadherin, characteristics of an epithelial-mesenchymal transition (EMT), which prior studies have linked to EGFR TKI resistance. Mechanistically, we show that hypoxia increases fibroblast growth factor receptor 1 (FGFR1) expression in both HCC827 and H1975 cells, accompanied by down regulated BIM expression. ZEB1, an EMT activator, was also increased by hypoxia in HCC827 H1975 cells. Knockdown of the FGFR1 by shRNA can attenuate gefitinib resistance in HCC827 cells and osimerinitib resistance in H1975 cells, respectively. Inhibition of FGFR1 function by the small molecular inhibitor, ponatinib, resensitizes hypoxia-induced resistant HCC827 clones to gefitinib treatment, and also attenuates osimerinitib resistance in hypoxic H1975 cells. These results suggest that hypoxia is a driving force for acquired resistance to EGFR TKIs through increased FGFR1 expression and coordination of EMT in NSCLC. The combination of EGFR TKIs and FGFR1 inhibitors may offer an attractive therapeutic strategy for NSCLCs.

Citation Format: Yuhong Lu, Gary J. Zhang, Peter M. Glazer. Hypoxia induces EGFR inhibitor resistance in lung cancer cells via fibroblast growth factor receptor 1 (FGFR1) by promoting epithelial-mesenchymal transition (EMT) [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 312.
Abstract 3147A: The use of the sTRA glycan for predicting responses to neoadjuvant therapy in patients with pancreatic ductal adenocarcinoma
Authors: Staal Ben , Liu Ying , Hsueh Peter , Aldakkak Mohammed , Zeh Herbert , Evans Douglas , Brand Randall E. , Tsai Susan , Haab Brian B.
Published: August 2019

The sTRA glycan has been validated as a serological and cell-surface biomarker of pancreatic ductal adenocarcinoma (PDAC). In previous work, a biomarker panel using sTRA and CA19-9 performed significantly better than CA19-9 for distinguishing PDAC from benign diseases of the pancreas; here we explored whether the subgroup identified by sTRA has a different prognosis than other subgroups. We examined the plasma levels of sTRA and CA19-9 in two cohorts of patients with resectable or borderline-resectable PDAC. In each cohort, two samples per patient were collected: one prior to any therapy, and another after neoadjuvant therapy but before surgery. In the first cohort (n = 54), the ratio of post-therapy to pre-therapy plasma sTRA was statistically higher (p < 0.05, student’s t-test using logged ratios) in patients with poor response, as assessed by surgical pathology. CA19-9 had higher ratios in a subset of the poor-response subjects but without statistical significance. In the second cohort (n = 75), the pre-treatment and post-treatment levels of sTRA were significantly higher (p < 0.01, Wilcoxon Rank-Sum test) in patients with shorter survival (<2 years). In contrast, CA19-9 did not show statistical significance in the pre-treatment or post-treatment levels, but the post:pre ratios were substantially higher in a subset of the short-survival group. In both cohorts, sTRA identified a subset of poor responders that was non-identical to the one identified by CA19-9. As a result, a biomarker panel combining the markers provided better accuracy for identifying poor responders than either marker alone. In an analysis of 24 cell line models of PDAC, the cell lines that secreted sTRA had a statistically higher resistance to chemotherapy than those not secreting sTRA, whereas the cell lines secreting CA19-9 showed no such association. Therefore, pancreatic cancers that produce sTRA may be a distinct subtype with higher resistance to chemotherapy, and sTRA in combination with CA19-9 could be a valuable serological predictor of responsiveness to chemotherapy.

Citation Format: Ben Staal, Ying Liu, Peter Hsueh, Mohammed Aldakkak, Herbert Zeh, Douglas Evans, Randall E. Brand, Susan Tsai, Brian B. Haab. The use of the sTRA glycan for predicting responses to neoadjuvant therapy in patients with pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3147A.
Abstract 3176: Biomarker identification using xenograft mouse model based clinical trial simulation and artificial intelligence data analytics
Authors: Afshar M , Bichat Francis , Duchamp Olivier , Etcheto A , France Damien , Kindermans M , Mignard Caroline , Parmentier F , Ratsima Hery
Published: August 2019

The growing number of anti-cancer drugs available at different stages of clinical development combined with the broadening potential use of combination therapy further complexifies the early identification of companion markers, markers of synergy as well as novel indications for existing and new drug combinations. Well characterized patient derived xenograft mouse models (PDX), combined with Artificial Intelligence tools that can integrate and analyze the broad range of generated data can help address this challenge. PDX experiments can providing an opportunity to simulate a clinical assessment using multiple mice models.In this study, we developed a PDX platform combined with the KEM® Artificial Intelligence data analytics, that is based on Formal Concept Analysis, to simulate a clinical trial and identify biomarkers of response. The platform was tested on colon cancer patient derived PDX. Respectively mRECIST response and survival of respectively 21 and 26 PDXs against Oxalipaltin combined with 5-Fluorouracil and folinic acid (Folfox) was experimentally assessed against a placebo, simulating a clinical trial-like setting with 2 arms. Biomarkers of response (mRECIST) and survival were identified using KEM®, combined with statistical modelling (Cox survival-modelling). 24 candidate biomarker genes were identified including PGAP3, ERBB2, NOTCH2, WDR70, and ZNF227. Alone or combined, these biomarkers are significantly linked to an increase or decrease of the survival PDX (p ranging from 2.2e-10 to 0.048, odd-ratio ranging from 0.12 to 10.00), with the potential to be used as inclusion or exclusion biomarkers. This work demonstrates the ability of a combined PDX / Artificial Intelligence platform to simulate clinical trials and identify biomarkers of drug efficacy and synergy, thus fostering the design of precision medicine clinical trials.

Citation Format: M Afshar, Francis Bichat, Olivier Duchamp, A Etcheto, Damien France, M Kindermans, Caroline Mignard, F Parmentier, Hery Ratsima. Biomarker identification using xenograft mouse model based clinical trial simulation and artificial intelligence data analytics [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 3176.
Abstract 3178: Comparative study of methods and platforms for T-cell receptor repertoire
Authors: Dutta Mukta , Smit Mariya , Collins Heather , Malge Anjali , Anderson Steve , Braastad Corey
Published: August 2019

High-throughput sequencing technologies facilitate in-depth sequencing of T-cell receptor (TCR) repertoires to gain insights into human adaptive immunity. These studies can help decipher the diversity of T-cell receptors, relevant to physiological and disease conditions, and potential therapy responses. A variety of TCR kits and programs have been developed for the identification of V, D and J gene segments, complementarity-determining region 3 (CDR3) sequence extraction and clonality analysis. However, there is still a deficit of systematic comparative studies to assist in the selection of an optimal platform. Here, we present a detailed comparison of 4 state-of-the-art TCR platform on samples with different complexities (ranging from cancers to immune disorders) by taking into account aspects such as clonotype detection [unique V(D)J combination] and CDR3 identification. Our results provide new insights into the effect of method selection on analysis results, and will consequently inform users in the selection of an appropriate analysis method for the desired application(s).

Citation Format: Mukta Dutta, Mariya Smit, Heather Collins, Anjali Malge, Steve Anderson, Corey Braastad. Comparative study of methods and platforms for T-cell receptor repertoire [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 3178.
Abstract 3181: A novel, bioluminescent assay for the selective detection of target cell killing in mixed cultures
Authors: Binkowski Brock , Paguio Aileen , Eggers Christopher , Butler Braeden , Beck Michael , Fan Frank
Published: August 2019

Efforts to develop and commercialize T cell immunotherapies would benefit from sensitive and easy-to-use assays to monitor target cell killing. We have developed an approach to selectively measure the death of target cells mediated by CAR/TCR-T cells or other types of effector cells. The method relies on the release of a HiBiT-tagged protein from target cells following cell lysis. Once released, HiBiT, an 11 a.a. peptide tag, binds to cell-impermeable Large BiT (LgBiT), a 17.6 kDa protein, to reconstitute NanoBiT Luciferase. In the presence of furimazine substrate, the luminescent signal is proportional to the amount of target cell killing, and cell lysis can be quantified at a single time point using Maximum Release and Spontaneous Release controls. The assay is compatible with a wide range of target cells per well (e.g. 100-500,000 cells/well), and low levels of Spontaneous Release allow the sensitive detection of low levels of target cell lysis (e.g. 1-5%). Using a modified format, target cell killing can be measured continuously in a bench-top luminometer for 24 hours or more. We demonstrate these approaches using commercially available CAR-T cells and target cells stably expressing a HiBiT-tagged protein via random integration of plasmid DNA or viral transduction. We also demonstrate the use of a thaw-and-use format to facilitate MOA-based lot release assays. The approach provides simple, non-radioactive assay formats for selectively monitoring target cell killing in mixed culture experiments.

Citation Format: Brock Binkowski, Aileen Paguio, Christopher Eggers, Braeden Butler, Michael Beck, Frank Fan. A novel, bioluminescent assay for the selective detection of target cell killing in mixed cultures [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 3181.
Abstract 3182: Directed screening TCRs against Ras mutations in mCRC patients
Authors: Yuan Jijun
Published: August 2019

Ras gene is involved in many signaling pathway to regulate cell proliferation, metabolism and motility, for example MAPK and PI3K pathway. Ras mutation is constitutively active to drive uncontrolled cell proliferation that results in malignant transformation. Ras mutation frequently occurs in human malignancy, for example pancreatic cancer (98%), colorectal cancer (45%) and lung cancer (31%). Drug development targeting Ras mutation was not succeed despites decades of effort in small molecule fields.

T cell receptor (TCRs) can recognize processed and presented neoantigens on human leukocytes antigen (HLA). Ras G12D mutation was report as neoantigen and could be recognized by tumor infiltrating lymphocytes restricted by HLA-C0802 in mCRC patients. We assume that human TCR repertoire encompassing specific TCRs against Ras mutation restricted by other HLAs in Chinese patients.

We design a novel synthetic presentation mRNA construct which harboring all pathogenic Ras mutations. The construct significantly enhanced Ras mutation peptides presentation on HLAs (>60%) compared with conventional used construct. We developed a directed screening method to identify T cell clone against Ras mutation restricted by Chinese pop HLAs in tumor infiltrating lymphocytes and sequenced TCRs by using 10xGenomics. We identified several TCRs that showed effector function against Ras mutation in China mCRC patients for further developing TCR-T cell therapy.

Citation Format: Jijun Yuan. Directed screening TCRs against Ras mutations in mCRC patients [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 3182.
Abstract 3184: Targeting multiple solid tumor types with anti-CD70 allogeneic CAR-T cells
Authors: Dequeant Mary Lee , Karnik Sushant , Padalia Zinkal , Tham Minh Thu , Ho Tony , Carson Julie , Terrett Jonathan A.
Published: August 2019

CD70 (CD27 ligand) is highly expressed in multiple hematologic malignancies, such as non-Hodgkin lymphoma, multiple myeloma, and chronic lymphocytic leukemia, and is uniquely highly expressed in the solid tumor type clear cell renal cell carcinoma (ccRCC). Malignancies showing high CD70 expression have been the subject of multiple clinical trials evaluating anti-CD70 agents, such as antibodies with enhanced antibody-dependent cell-mediated cytotoxicity and antibody drug conjugates (ADCs). The focus on high expression indications seems to be a requirement, particularly for ADCs. We have shown previously that CTX130, a CRISPR/Cas9 gene-edited allogeneic anti-CD70 CAR-T, is potently cytotoxic against even the lowest expressing RCC cells (e.g. ACHN). With this in mind, we evaluated CD70 expression in other solid tumor indications, including pancreatic, lung, and ovarian cancers, and assessed the cytotoxic activity of CTX130 against cell lines derived from these tumor types. These studies show that high expression is not a requirement for potent activity for CAR-T cells as it appears to be for CD70-targeted ADCs. Further, these data highlight a translational medicine route for CTX130 in many solid tumor indications.

Citation Format: Mary Lee Dequeant, Sushant Karnik, Zinkal Padalia, Minh Thu Tham, Tony Ho, Julie Carson, Jonathan A. Terrett. Targeting multiple solid tumor types with anti-CD70 allogeneic CAR-T 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 3184.
Abstract 3185: CAR-T cells can eradicate human uveal melanoma and immune-therapy resistant malignant melanoma in IL-2 transgenic NOD/SCID IL2 receptor gamma knockout mice
Authors: Forsberg Elin , Lindberg Mattias , Jespersen Henrik , Alsén Samuel , Bagge Roger Olofsson , Donia Marco , Svane Inge Marie , Nilsson Ola , Ny Lars , Nilsson Lisa , Nilsson Jonas
Published: August 2019

Immune therapies including checkpoint blockade and adoptive T cell transfer show great promise for the treatment of melanoma, with long-term effects in some patients. However, not all patients respond to current immune therapies and are in need of other treatment strategies. For example, around half of the patients with metastatic malignant melanoma will not be cured with available therapies today. For metastatic uveal melanoma (a rare melanoma of the eye) available immune therapies have less effect, and there is currently no approved therapy for these patients. Our objective is to find novel immune therapies with the potential to treat immune-therapy resistant melanoma, with the use of chimeric antigen receptor T cells (CAR-T). So far, no CAR-T therapy is approved for use in solid tumors. We have previously developed a humanized mouse model, where tumor cells and T cells from the same patient are grafted in IL-2 transgenic NOD/SCID IL2 receptor gamma knockout (NOG) mice, and found that responses in the mouse model correlated to responses in the corresponding patients in a clinical trial of adoptive T cell transfer (Jespersen et al, Nature Communications, 2017). In order to test the potential for CAR-T therapy in immune-therapy resistant melanoma in this mouse model, we used TCGA to determine the expression in melanoma biopsies of targets for commercially available CAR-T cells. Interestingly, we were able to use this model to treat immune-therapy resistant malignant melanoma and uveal melanoma patient-derived xenografts with human CAR-T cells and obtain curative responses, but only in IL-2 transgenic mice and not in regular NOG mice. These findings prove potential for the use of CAR-T cell therapy in immune-therapy resistant melanoma, and indicate the utility of this mouse model in the translation of CAR-T therapies. We are currently working on the development of CAR-T cells derived from patient-specific T cells.

Citation Format: Elin Forsberg, Mattias Lindberg, Henrik Jespersen, Samuel Alsén, Roger Olofsson Bagge, Marco Donia, Inge Marie Svane, Ola Nilsson, Lars Ny, Lisa Nilsson, Jonas Nilsson. CAR-T cells can eradicate human uveal melanoma and immune-therapy resistant malignant melanoma in IL-2 transgenic NOD/SCID IL2 receptor gamma knockout mice [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 3185.
Abstract 3193: Combining immunotherapies: A strategy for improving CD8 T cell-mediated antitumor responses
Authors: Fan Aaron E. , Sultan Hussein , Kumai Takumi , Celis Esteban
Published: August 2019

Adoptive cell therapy (ACT) of retrovirally transduced (RV) CD8 T cells is a powerful technique that has shown promise in tumor eradication in cancer patients. However, some major barriers to current methods are that ACT is expensive, time consuming, and requires harmful and toxic adjunct procedures. Our laboratory has demonstrated the use of TriVax, a potent peptide vaccination strategy that dramatically expands ACT cell populations and bypasses the necessity for adjunct procedures. The purpose of this project was to enhance current methods of ACT+TriVax by testing an antigen-specific antitumor response of RV CD8 T cells and if it could be improved with constitutively active STAT5 (CA-STAT5) expression, a protein activated downstream several cytokine pathways that have been shown to play a role in increasing CD8 T cell persistence and resistance to apoptosis. We tested the hypothesis that CA-STAT5 in CD8 T cells enhances an antitumor effect by increasing T cell persistence and efficacy.

Our results show that TriVax administration selectively expanded frequencies of the ACT cell population expressing gp100-TCR in both blood and spleen. When co-transduced with CA-STAT5, an even higher fold expansion of antigen-specific cells was observed. +CA-STAT5 T cells were able to expand more robustly than -CA-STAT5 T cells upon repeated antigen stimulation (vaccine boost), demonstrating nearly 4000-fold increases in antigen-specific CD8 T cells. +CA-STAT5 T cells also seemed to persist longer in vivo over time, and they expressed lower levels of surface PD-1. Using B16F10 melanoma, ACT+TriVax of these cell populations into tumor-bearing mice demonstrated a powerful antitumor effect, leading to tumor regression in treated groups. CA-STAT5 seemed to recapitulate similar antitumor effects our laboratory observed previously with combinatorial anti-PD-L1 treatment or IL2/anti-IL2 mAb complexes (IL2Cx), suggesting a potential role for STAT5 in resisting the PD-1/PD-L1 inhibitory pathway. When compared to tumor-bearing mice treated with ACT+TriVax with anti-PD-L1 antibody, our approach using +CA-STAT5 T cells generated a comparable antitumor response. Altogether, these results demonstrate that RV CD8 T cells expressing gp100-TCR and CA-STAT5 are capable of antigen-dependent expansion in response to TriVax. CA-STAT5 plays a role in increasing T cell proliferation and persistence, as well as increasing efficacy through mechanisms that seem to indicate resistance to PD-1/PD-L1 inhibition.

Citation Format: Aaron E. Fan, Hussein Sultan, Takumi Kumai, Esteban Celis. Combining immunotherapies: A strategy for improving CD8 T cell-mediated antitumor responses [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 3193.
Abstract 3211: MTL-CEBPA combined with radiofrequency ablation and immunotherapy enhances immunological anti-tumour response in an HCC mouse model
Authors: Sodergren Mikael H. , Huang Kai-Wen , Reebye Vikash , Chee Cheng-Ean , Zacharoulis Dimitris , Habib Robert , Blakey David , Rossi John , Habib Nagy
Published: August 2019

The transcription factor CEBPA (CCAAT/enhancer-binding protein alpha) is recognised for its antiproliferative effects. MTL-CEBPA is a small activating RNA drug which upregulates gene expression of CEBPA and amongst other effects causes and immunomodulatory effect on peripheral granulocytes. Radiofrequency ablation (RFA) is standard treatment for some tumour types such as liver cancer and induces modulation of both innate and adaptive immune systems. To investigate any synergistic effect of MTL-CEBPA with RFA and immune checkpoint inhibition we initiated a reverse translation experiment, where syngeneic BNL hepatocellular carcinoma tumour cells were injected in the two opposite flanks of immunocompetent BALB/c mice (n=8 in each group). Treatments for hepatoma bearing mice included: 1) RFA on one flank (day 0), 2) Anti-PD-1 inhibition immunotherapy (RMP1-14 antibody, BioXCell, West Lebanon, NH, USA at 200 μg IV/mouse/dose on days 0, 2 & 5) and 3) MTL-CEBPA (3mg/kg IV/mouse/dose on days 0, 2 & 5) as well as combinations of all 3 interventions. We found that tumour control on the opposite flank was augmented by addition of RFA and most significant in the triple combination group (RFA + anti-PD1 + MTL-CEBPA) in which 2/8 animals showed a complete response and 5/8 a partial response. This was also the only group that showed a statistically significant increase in CD8+ (Cytotoxic) as well as CD49b+/CD45+ (Natural Killer) tumour infiltrating lymphocytes. These data suggest a clinical role for combination treatment with checkpoint blockade, RFA and MTL-CEBPA through synergistic priming of the immune tumour response, enabling RFA to have a pronounced anti-tumour abscopal effect.

Citation Format: Mikael H. Sodergren, Kai-Wen Huang, Vikash Reebye, Cheng-Ean Chee, Dimitris Zacharoulis, Robert Habib, David Blakey, John Rossi, Nagy Habib. MTL-CEBPA combined with radiofrequency ablation and immunotherapy enhances immunological anti-tumour response in an HCC mouse model [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 3211.
Abstract 3216: CKD-516, a novel vascular disrupting agent, enhances anticancer activity of anti-PD-1 antibody in SMAD4-deficient colon cancer model
Authors: Kim Soo Jin , Kim Hark Kyun , Ryu Keun Ho , Hong Chung Il
Published: August 2019

CKD-516 is a potent vascular disrupting agent (VDA), selectively acting on tumor vessels. A clinical study of CKD-516 combined with irinotecan is undergoing in colorectal cancer. There have been exponential gains in immune-oncology (I-O) in recent times through the development of immune checkpoint inhibitors (ICIs). ICIs demonstrated durable response and some patients achieved disease control for several years. However, there are still critical unmet medical needs for the combination therapies due to limited response rate of ICIs. The first step of the cancer-immunity cycle is dendritic cells (DCs) maturation which is necessary for the anticancer immunity. It is well known that mature DCs play critical roles in priming immune responses in cancer patients. We found that CKD-516 is able to induce DC maturation through Rho signaling pathway in DCs. A tumor suppressor gene, SMAD4 is associated with several cancers including stomach cancer, colorectal cancer, and pancreas cancer. The loss of SMAD4 is a poor prognostic biomarker in these cancer types and its mutation is related to poor clinical outcome on immunotherapies. A SMAD4-deficient mouse colorectal cancer cell line (3349LM) was primary cultured from a spontaneous intestinal adenocarcinoma formed in a Villin-Cre;Smad4(F/F);Trp53(F/F) mouse. CKD-516 showed a synergistic effect with anti-PD-1 antibody in 3349LM syngeneic model. In addition, 3349LM is also a MSS type cancer. Thus, CKD-516 displayed a therapeutic potential for several cancer patients who do not respond to immunotherapy. In summary, CKD-516 is a novel VDA with immune boosting effect. In SMAD4 deficient cancer model, CKD-516 has shown synergistic effects in combination with PD-1 antibody. Therefore, these data suggest that CKD-516 potentiates the anticancer activity of immunotherapy.

Citation Format: Soo Jin Kim, Hark Kyun Kim, Keun Ho Ryu, Chung Il Hong. CKD-516, a novel vascular disrupting agent, enhances anticancer activity of anti-PD-1 antibody in SMAD4-deficient colon cancer model [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 3216.
Abstract 322: Discovery of synergistic PI3K inhibitor combination therapies using high throughput approaches in HNSCC
Authors: Michmerhuizen Nicole Lynn , Leonard Elizabeth , Heenan Caitlin , Nair Vivek , Matovina Chloe , Harris Micah , Herbst Gabrielle , Kim Daniel , Bachand Amanda , Zhai Jingyi , Foltin Susan K. , Kulkarni Aditi , Carey Thomas E. , Bradford Carol R. , Jiang Hui , Brenner John C.
Published: August 2019

Recent head and neck squamous cell carcinoma (HNSCC) sequencing studies have identified the phosphatidylinositol 3-kinase (PI3K) pathway as the most frequently mutated, oncogenic pathway in this cancer type. Despite frequent activating mutation or amplification in PIK3CA (the gene encoding the catalytic subunit of PI3K), targeted PI3K inhibitors have shown limited clinical efficacy as monotherapies. To identify factors that might predict sensitivity and resistance to PI3K inhibitors, we tested a panel of more than 40 patient-derived HNSCC cell lines and observed varying sensitivity to PI3K inhibitors. While all cell lines were much more sensitive than fibroblasts to these drugs, responses varied widely; for pan-PI3K inhibitor BKM120, IC50 values ranged from 0.2 to 4.2 μM across the OSCC cell line panel. Responses to BKM120 do not correlate with PIK3CA mutation status copy number, RNA expression, p110α protein levels, and AKT phosphorylation at baseline and after inhibitor treatment, among other genetic or phenotypic features.

In order to characterize potential mechanisms of PI3K inhibitor resistance in HNSCC, we have also developed and optimized an unbiased, high-throughput screening approach. We have used this assay to test ~1400 inhibitors as monotherapies and in combination with PI3K inhibitors HS-173 and BKM120 in ten HNSCC models. Our initial screening data suggested that combinations of PI3K inhibitors and ALK inhibitors are among the most synergistic in a subset of cell lines. We have evaluated the effects of these drug combinations on apoptosis, cell cycle, and downstream signaling in several in vitro HNSCC models, observing synergy in response to FDA-approved agents PI3K inhibitor pictilisib and ALK inhibitor brigatinib in various systems using a range of techniques. We next plan to expand these analyses to organoid and xenograft models to evaluate their effects in more clinically relevant settings.

Future studies will also examine additional factors that may predict PI3K inhibitor responses and will seek to better understand promising combination treatments and advance them for effective use in HNSCC patients.

Citation Format: Nicole Lynn Michmerhuizen, Elizabeth Leonard, Caitlin Heenan, Vivek Nair, Chloe Matovina, Micah Harris, Gabrielle Herbst, Daniel Kim, Amanda Bachand, Jingyi Zhai, Susan K. Foltin, Aditi Kulkarni, Thomas E. Carey, Carol R. Bradford, Hui Jiang, John C. Brenner. Discovery of synergistic PI3K inhibitor combination therapies using high throughput approaches in HNSCC [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 322.
Abstract 3223: Overcome LKB1 mutated cancer resistance to anti-PD1 treatment
Authors: Deng Jiehui , Thennavan Aatish , Pan Yuanwang , Dolgalev Igor , Chen Ting , Silver Heather , Harris Matthew , Pyon Val , Li Fei , Lee Chelsea , Tsirigos Aristotelis , Rothenberg Eli , Perou Charles M. , Wong Kwok-Kin
Published: August 2019

KRAS/STK11 (LKB1) mutant lung cancers are a subtype of cancer that respond poorly in the clinic to immunotherapies with shorter progression-free survival and overall survival comparing with other KRAS mutant lung cancer patients. Interestingly, this group of patients have high tumor mutational burden (TMB) comparable with other KRAS mutant lung cancers, which usually is an indicator for better response to anti-PD1 treatment. By using genetically engineered mouse model (GEMMs), we found that LKB1 loss of function can cause high level of nonsynonymous mutations, as a consequence of both replication dependent and independent DNA repair machinery dysfunctions. This LKB1 mutant dependent increased mutational load might further synergize with smoking mutagen exposures. When LKB1 function is defective, the neoantigens are not properly presented to T cells, which is required for proper T cell activation and effector T cells expansion. This will lead to insensitivity to anti-PD1 antibodies in high TMB tumors and tolerance to neoantigens produced by cancer cells. We further demonstrated that using a small molecule drug that impacts on the LKB1 downstream effector pathway, we can reverse this process through activating innate immunity in tumors, which will restore the antigen presentation functions. Combinational treatment of this small molecule with anti-PD1 can reverse the tumor progression in vivo, through releasing the suppressive tumor infiltrating lymphocytes (TILs) in LKB1 mutant lung cancers. Our study not only helps to further our understanding of the mechanism that contributes to immune evasion in high TMB tumors, but also provides a potential solution for LKB1 mutant cancers to overcome resistance to anti-PD1 treatment in the clinic.

Citation Format: Jiehui Deng, Aatish Thennavan, Yuanwang Pan, Igor Dolgalev, Ting Chen, Heather Silver, Matthew Harris, Val Pyon, Fei Li, Chelsea Lee, Aristotelis Tsirigos, Eli Rothenberg, Charles M. Perou, Kwok-Kin Wong. Overcome LKB1 mutated cancer resistance to anti-PD1 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 3223.
Abstract 3236: T-cell intrinsic mechanisms of resistance to PD-1 checkpoint blockade
Authors: Moogk Duane , Wang Lin , Li Kaitao , Yuan Zhou , Weber Jeffrey , Osman Iman , Zhu Cheng , Krogsgaard Michelle
Published: August 2019

Although much clinical progress has been made in harnessing the immune system to recognize and target cancer, there is still a significant lack of an understanding of how tumors evade immune recognition and the mechanisms that drive tumor resistance to both T cell and checkpoint blockade immunotherapy. Our objective is to understand how tumor-mediated signaling through inhibitory receptors, including PD-1, combine to affect the process of T cell recognition of tumor antigen and signal activation in melanoma patients. The broader goal of the project is to understand the basis of resistance to PD-1 blockade and potentially identify new molecular targets to enable T cells to overcome dysfunction mediated by multiple inhibitory receptors (IR). Our biophysical measurements have shown that that the activities of TCR-proximal signaling components affect T cell antigen recognition and sensitivity at the earliest stages of T cell stimulation. We have shown that T cell antigen recognition is influenced by PD-1 and other inhibitory receptors via Shp-1/2 by targeting CD28 and Lckby directly suppressing TCR-pMHC-CD8 binding. Phospho-proteomics and flow cytometry-based analysis of melanoma patient-derived T cells from PD-1 blockade responders and non-responders identified additional mediators, signaling components and pathways associated with blockade resistance. CRISPR/Cas9 mediated genome editing was utilized to determine if resistance is mediated by the continued signaling of multiple IRs by perturbing IR signaling in mouse models of PD-1 blockade.Targeting these interactions and understanding the basis of resistance to PD-1 blockade would potentially allow identification of novel biomarkers of resistance or new molecular targets to enable T cells to overcome dysfunction during PD-1 checkpoint blockade.

Citation Format: Duane Moogk, Lin Wang, Kaitao Li, Zhou Yuan, Jeffrey Weber, Iman Osman, Cheng Zhu, Michelle Krogsgaard. T-cell intrinsic mechanisms of resistance to PD-1 checkpoint blockade [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 3236.
Abstract 3251: Fibroblast activation protein (FAP)-selective delivery of CD40 agonistic DARPin®molecule for tumor-localized immune activation
Authors: Rigamonti Nicolo , Schlegel Anja , Barsin Sophie , Schwestermann Jonas , Mangold Susanne , Kaufmann Yvonne , Zitt Christof , Veitonmäki Niina , Levitsky Victor , Metz Clara
Published: August 2019

CD40 is a co-stimulatory molecule belonging to the tumor necrosis factor receptor superfamily which can activate both innate and adaptive immune system, making it an interesting target for tumor immunotherapy. Systemic administration of agonistic CD40 antibodies (Ab) has shown signs of activity in cancer patients, but dose-limiting toxicity impaired the clinical efficacy. New approaches are therefore needed to increase the therapeutic index of CD40-targeting molecules and achieve better clinical outcomes. Here, we report an alternative approach designed to activate CD40 specifically in the tumor microenvironment (TME), and not systemically, in order to increase efficacy and reduce systemic toxicity. This novel approach is based on a bispecific DARPin® molecule, targeting CD40 and fibroblast activation protein (FAP) alpha, intended to induce immune activation only when clustered by binding to FAP-expressing cells in the TME. The bispecific FAP x CD40 DARPin®molecule was tested in a reporter assay and in additional cell assays using primary human 1) B cells, 2) macrophages and 3) dendritic cells. These studies demonstrated CD40 activation only in the presence of FAP-positive, but not with FAP-negative cells, confirming a mechanism of action strictly dependent on FAP-mediated cross-linking. A surrogate mouse-specific FAP x CD40 DARPin® molecule (mFAP x CD40) was generated and tested in similar in vitro assays and showed FAP-dependent activation of CD40 and comparable results as the human construct. In vivo experiments, performed in tumor-free mice, showed a comparable half-life between mFAP x CD40 and an anti-mouse CD40 Ab (clone FGK45). However, mFAP x CD40, in contrast to the FGK45 Ab, did not increase the serum level of IL-6, supporting a mode of action that is dependent on FAP-mediated crosslinking of CD40 receptor. In additional studies mFAP x CD40 was active and inhibited the growth of FAP+ tumors. There were no signs of toxicity with mFAP x CD40 in contrast to the FGK45 Ab which resulted in body weight loss. In conclusion, we have generated bispecific agonist FAP x CD40 DARPin® molecules able to activate the CD40 pathway with a targeting (FAP)-dependent mechanism of action.

Citation Format: Nicolo Rigamonti, Anja Schlegel, Sophie Barsin, Jonas Schwestermann, Susanne Mangold, Yvonne Kaufmann, Christof Zitt, Niina Veitonmäki, Victor Levitsky, Clara Metz. Fibroblast activation protein (FAP)-selective delivery of CD40 agonistic DARPin®molecule for tumor-localized immune activation [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 3251.
Abstract 3254: A novel TGFβRI inhibitor suppresses tumor progression by increasing T-cell infiltration into tumor
Authors: Zusheng Xu , Lou Yangtong , Zhao Qiang , Wang Wei
Published: August 2019

TGF-beta (TGF-β) pathways, despite serving as tumor suppressor in early stage of carcinogenesis, have shown to be one of the key tumor-promoting factors in later-stage tumor progression. TGF-β receptor type 1 (TGFβRI) inhibitors have shown great potentials in their anti-tumor and anti-metastasis efficacy in preclinical studies and some of them are being tested in the clinical settings. A specific TGFβRI inhibitor YL-13027 was developed at Yingli Pharmaceutical. Our previous data have shown that YL-13027 significantly inhibited the T cell differentiation into Treg and the secretion of IL-10 in vitro, as well as tumor growth in immune-competent mice with CT26 tumors. Our further studies indicated that the anti-tumor efficacy of YL-13027 was largely mediated by T cells. More importantly, YL-13027 synergistically enhanced the anti-tumor efficacy of anti-PD-L1 antibody in CT26 model. YL-13027 displayed significant anti-tumor efficacy in other syngeneic mouse tumor models tested including orthotopic murine breast carcinoma 4T1 model in BALB/c mice and murine hepatocellular carcinoma Hepa1-6 model in C57BL/6 mice. YL-13027 is well tolerated by the animals with no treatment-related toxicity observed in these efficacy studies. Analyses of tumors treated with YL-13027 revealed that YL-13027 significantly increased the number of tumor-infiltrating lymphocytes in tumors. These data suggest that YL-13027, a novel TGFβRI inhibitor, represents a promising and safe immune modulator and shows great potential as a cancer immuno-therapeutics.

Citation Format: Xu Zusheng, Yangtong Lou, Qiang Zhao, Wei Wang. A novel TGFβRI inhibitor suppresses tumor progression by increasing T-cell infiltration into tumor [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 3254.
Abstract 3282: BMI and other factors in relation to overall survival of female breast cancer in China
Authors: Dong Yunqiu , Duijnhoven Franzel J. B. van , Wang Lu , Wu Ming , Qian Yun , Kampman Ellen
Published: August 2019

The aim of this study is to determine the relationship between body mass index(BMI) at diagnosis and various factors including the epidemiologic and clinical factors on survival of female breast cancers. We analysed the data of 653 patients in any stage from the Wuxi Breast Cancer Cohort. Cox Proportional Hazard regression models were conducted to compare the overall survival(OS) outcomes among BMI groups, and adjusted by covariates which yielded a >10% change in the hazard ratios(HRs) estimate, including age, stage, menopause status, physical activity in work. There were significant differences among BMI groups in age(P=0.000), WHR(P =0.000), physical activity in work(P =0.019),red meat intake(P =0.029), oil intake(P =0.000),tumor stage(P =0.021),menopause status(P =0.001). Overweight(OW,BMI=24 to 27.9kg/m2) and obesity(OB, BMI≥28kg/m2) showed better OS comparing with normal weight(NW, BMI=18.5 to 23.9kg/m2)(respectively, adjusted HRs=0.45, 95%CI=0.15 to 1.34, adjusted HRs=0.59,95%CI=0.21 to 1.69), meanwhile underweight(UW, BMI<18.5kg/m2) showed a poorer OS(adjusted HRs=4.65,95%CI=0.93 to 23.37). There was no significant relationship between the BMI and some other factors in OS. In conclusion, the findings of our current study indicate that BMI might play a role in the prognosis of female breast cancer in China, but the evidence was not strong enough.

Citation Format: Yunqiu Dong, Franzel J. B. van Duijnhoven, Lu Wang, Ming Wu, Yun Qian, Ellen Kampman. BMI and other factors in relation to overall survival of female breast cancer in China [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 3282.