bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5911: A bicistronic adenoviral vector carrying cytosine deaminase and GM-CSF genes significantly improves antitumor immunity and overall survival in colon cancer
Authors: Akbulut Hakan , Coleri Arzu , Sahin Gunce , Tang Yucheng , Deisseroth Albert , Icli Fikri
Published: August 2018

Background: Combination of cytotoxic treatments with immunotherapeutic agents seem more efficacious than using either strategy alone. Oncolytic viral vectors carrying immunostimulatory genes like GM-CSF have emerged as treatment options in various tumors. Previously, we have shown that combination of suicide gene therapy either with GM-CSF or vector-induced dendritic cells has significant anti-tumor efficacy. In the current study, we aimed to combine the cytotoxic effect of the suicide gene cytosine deaminase and the immunostimulatory effect of granulocyte macrophage colony stimulating factor in a single vector construct. Methods: We have constructed a bicistronic adenoviral vector carrying cytosine deaminase (CD) and granulocyte macrophage-colony stimulating factor (GM-CSF) genes combined with an IRES element and driven by CMV promoter (Ad-CMV-CDiresGMCSF). We tested the in vitro efficacy of the vector in various tumor cell lines and in a mouse model of colon cancer. Results: Our bicistronic vector Ad-CMV-CDiresGMCSF showed significant in-vitro cytotoxic effects on tumor cell lines similar to the vector carrying CD gene alone when 5-FC added. Likewise, the GM-CSF producing efficacy of the bicistronic vector was comparable to the vector carrying GM-CSF gene alone. In the syngeneic colon cancer model, the bicistronic vector carrying CD and GM-CSF genes yielded significant tumor shrinkage and overall survival when compared to the control suicide vector carrying CD gene alone. Accordingly, anti-tumor immune response parameters including CTL assay and immune cell infiltration of tumor tissue were significantly improved in the Ad-CMV-CDiresGMCSF vector treated group (p<0.01). Conclusions: The Ad-CMV-CDiresGMCSF vector construct suggests a potential for the treatment of established tumors by inducing significant tumor killing and tumor-specific immune response.

Citation Format: Hakan Akbulut, Arzu Coleri, Gunce Sahin, Yucheng Tang, Albert Deisseroth, Fikri Icli. A bicistronic adenoviral vector carrying cytosine deaminase and GM-CSF genes significantly improves antitumor immunity and overall survival in colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5911.
Abstract 5931: PDGFRα+ pericryptal stromal cells are the critical source of Wnts and RSPO3 for murine intestinal stem cells in vivo
Authors: Greicius Gediminas , Kabiri Zahra , Sigmundsson Kristmundur , Liang Chao , Bunte Ralph , Singh Manvendra K. , Virshup David M.
Published: August 2018

Wnts and R-spondins (RSPOs) support intestinal homeostasis by regulating crypt cell proliferation and differentiation. Ex vivo, Wnts secreted by Paneth cells in organoids can regulate the proliferation and differentiation of Lgr5-expressing intestinal stem cells. However, in vivo, Paneth cell and indeed, all epithelial Wnt production is completely dispensable, and the cellular source of Wnts and RSPOs that maintain the intestinal stem cell niche is not known. Here we investigated both the source and the functional role of stromal Wnts and RSPO3 in regulation of intestinal homeostasis. RSPO3 is highly expressed in pericryptal myofibroblasts in the lamina propria and is several orders of magnitude more potent than RSPO1 in stimulating both Wnt/β-catenin signaling and organoid growth. Stromal Rspo3 ablation ex vivo resulted in markedly decreased organoid growth that was rescued by exogenous RSPO3 protein. PdgfRα is known to be expressed in pericryptal myofibroblasts. We therefore evaluated if PdgfRα identified the key stromal niche cells. In vivo, Porcn excision in PdgfRα+ cells blocked intestinal crypt formation, demonstrating for the first time that Wnt production in the stroma is both necessary and sufficient to support the intestinal stem cell niche. Mice with Rspo3 excision in the PdgfRα+ cells had decreased intestinal crypt Wnt/β-catenin signaling and Paneth cell differentiation, and were hypersensitive when stressed with dextran sodium sulfate. The data support a model of the intestinal stem cell niche regulated by both Wnts and RSPO3 supplied predominantly by stromal pericryptal myofibroblasts marked by PdgfRα.

[G.G. and Z.K. contributed equally to this work.]

Citation Format: Gediminas Greicius, Zahra Kabiri, Kristmundur Sigmundsson, Chao Liang, Ralph Bunte, Manvendra K. Singh, David M. Virshup. PDGFRα+ pericryptal stromal cells are the critical source of Wnts and RSPO3 for murine intestinal stem cells in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5931.
Abstract 609: Neutrophils play a critical role in mediating antitumor activity of a combination therapy of radiation and immunotherapy
Authors: Tao Mi-Hua , Wu Chia-Jen , Tsai Yi-Ting , Wu Ping-Yi
Published: August 2018

The goal of cancer immunotherapy is to elicit robust tumor-specific immune responses that can generate durable tumor regression and/or eradication. Recent clinical trials using immune checkpoint blockades specific to programmed cell death-1/ligand-1 (PD-1/PD-L1) have shown durable clinical responses in various cancer types but benefit only a small portion of patients. We previously demonstrated that combination of radiation and immunotherapy of interleukin 12 (IL-12) and granulocyte macrophage colony-stimulator factor (GM-CSF) greatly improved the therapeutic efficacy than either single therapy alone. In analysis of the tumor-infiltrating leukocytes post combination therapy, we found that the number of CD11b+F4/80-/Ly6G+ neutrophils were greatly increased. In this study, we characterized these tumor-associated neutrophils (TANs) before and after treatment and investigated their role in mediating the antitumor activity of the combination therapy. We isolated TANs from the regressing tumors and demonstrated that these TANs were impaired in their immunosuppressive activities. Flowcytometric analysis revealed that several co-stimulatory molecules, including class II major histocompatibility complex, CD40 and CD86, were increased on these TANs. Accordingly, these TANs gained the ability to stimulate T cell proliferation. Antibody-depletion experiments showed that TANs were required for tumor regression in RT/12GM combination therapy. Together, our data demonstrate that combination of radiotherapy and IL-12/GM-CSF improved antitumor immunity through enhancing antigen presentation activity and reduced immunosuppressive functions of TANs.

Citation Format: Mi-Hua Tao, Chia-Jen Wu, Yi-Ting Tsai, Ping-Yi Wu. Neutrophils play a critical role in mediating antitumor activity of a combination therapy of radiation and immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 609.
Abstract 610: Dynamic immune landscape of the peripheral blood mononuclear cells from recurrent hepatocellular carcinoma patients undergoing radiofrequency ablation treatment
Authors: Chen Dexi , Zhao Yang , Ouyang Yabo , Liu Kai , Rao Wei , Wei Feili , Liu Xiaoni , Shi Ying , Wang Shanshan , Pang Lijun , Qiao Luxin , Zang Yunjin , Yin Xiaoming
Published: August 2018

Hepatocellular carcinoma (HCC) is one of the most common malignancies and the third most lethal malignancy worldwide. Radiofrequency ablation (RFA) is the most commonly used curative treatment for the patients not suitable for surgical resection or liver transplantation. It can cause regional and global anti-tumor immune response, but is insufficient to prevent HCC recurrence in many patients. Peripheral blood mononuclear cells (PBMC) are considered to be the best target for the study of the global immune status after RFA. Previous studies on PBMC were mainly based on traditional flow cytometry, which could not differentiate the complicated immune cell subtypes. In this study, we used mass cytometry to comprehensively characterize the phenotypic and functional alterations of the PBMC in recurrent HCC patients who had received RFA treatment. Fifty-four (54) blood samples from 13 patients were analyzed. Manual gating, SPADE analysis and viSNE analysis were used to interpret the mass cytometry data. The percentage of monocytes showed a fast increase after RFA, and reduced to the normal level before the HCC recurrence (P=0.026). A long-lasting reduction of B cells count were also found after the first RFA (P=0.021), and the B cell count remained at a low level before and after the secondary RFA. The percentage of CD4+ T cells in CD45+ cells was significantly reduced after the first RFA, then restored to the pretreatment level at the time of recurrence, and reduced again after the second RFA(P=0.024). CD8+ T cells showed the same alteration trend as CD4+ cells (P=0.03). We also examined the expression of functional markers in T cells and Tregs. Alterations of CD28 expression were most frequently found in T cells. The Treg population showed a large heterogeneity among the patients with TIM3, LAG3 or CTLA4 positive Tregs being the largest populations. These results suggest a more specific treating modality should be used in combination with the RFA treatment.

Citation Format: Dexi Chen, Yang Zhao, Yabo Ouyang, Kai Liu, Wei Rao, Feili Wei, Xiaoni Liu, Ying Shi, Shanshan Wang, Lijun Pang, Luxin Qiao, Yunjin Zang, Xiaoming Yin. Dynamic immune landscape of the peripheral blood mononuclear cells from recurrent hepatocellular carcinoma patients undergoing radiofrequency ablation treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 610.
Abstract 62: Effect of cancer cell-derived exosome on energy metabolism associated with macrophage activation
Authors: Kam Yoonseok , Dranka Brian P.
Published: August 2018

Macrophage is a critical microenvironmental component which can control tumor progression, and dynamic changes in cellular metabolism accompany the polarization. As well as other immune cells, the activation of macrophages can be modulated by cancer cells. Cancer-derived exosome is a newly emerging microenvironmental factor enables cancer cells to communicate with macrophages. In this study, examined were the activation-associated bioenergetic changes and the effect of cancer cell-derived exosome on the changes by a) monitoring the early metabolic kinetics and b) profiling bioenergetic phenotype resulted. In vitro activation of RAW264.7 macrophage cell line by injecting lipopolysaccharide (LPS) and interferon γ (IFNγ) in a Seahorse XF analyzer induced an immediate early increase in glycolytic rate. It also showed a delayed down-regulation of mitochondrial respiration, which resulted in a secondary elevation of glycolytic rate. This secondary glycolytic response was only obtained by LPS-IFNγ co-stimulation and mediated by induced nitric oxide synthase (iNOS) signaling. In contrast to the dynamic changes in metabolic phenotype, the total ATP production rate appeared to be stably maintained according to the XF data normalized by cell number. However, macrophage activation significantly increased glycolysis-dependent portion of ATP production. ATPs were generated almost solely by glycolysis after the delayed mitochondrial down-regulation by iNOS. Co-injection of cancer cell-derived exosomes with LPS slightly but significantly accelerated the immediate early glycolytic response as well as the delayed mitochondrial down-regulation. Furthermore, exosomes induced the immediate early glycolytic response even in the absence of LPS. These results imply that presence of cancer cell-derived exosomes may promote macrophage activation and accelerate pro-inflammatory immune response in a tumor microenvironment.

Citation Format: Yoonseok Kam, Brian P. Dranka. Effect of cancer cell-derived exosome on energy metabolism associated with macrophage activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 62.
Abstract 628: Immunometabolic signature of papillary thyroid carcinoma
Authors: Weiss Vivian L. , Baregamian Naira
Published: August 2018

Introduction: Papillary thyroid carcinoma (PTC) is the most common subtype of thyroid cancer with rapidly rising incidence. The interface between PTC tumor metabolic reprogramming and metabolic immunomodulation remains poorly understood.

Experimental Procedures: We sought to investigate the immunometabolic interaction patterns of PTC tumors and to determine whether a correlative relationship exists between tumor-infiltrating immune cell phenotypes and the expression of tumor metabolic enzymes. We performed a computational immunogenomic analysis to calculate leukocyte fractions within 496 PTC tumors from The Cancer Genome Atlas (TCGA). Immunogenomic analysis was performed using CIBERSORT (Newman AM et al., Nature Methods2015), a deconvolution method to calculate leukocyte fractions using RNA gene expression signatures. The PTC immune cell fractions were then compared to the RNA expression of tumor metabolic enzymes within the TCGA data using regression analysis. Matching targeted metabolomic analysis (Metabolon, Inc.) of 20 human PTC tumors was performed to determine tumor metabolite profiles associated with perturbed tumor cellular metabolic pathways based on the TCGA data analysis.

Results: The immunogenomic and PTC metabolic pathway enzyme expression analysis demonstrated significant correlation between immune cell types and major metabolic pathway enzymes. Specifically, tumor glycolytic enzymes upstream of the TCA cycle correlated with CD8 (hexokinase-1, p<0.0001) and T regulatory (enolase, p<0.0001) cell infiltrates. Enhanced pyruvate cycling into the TCA cycle correlated with the presence of several immune cell types (malic enzyme 2 vs. follicular helper T cells, p<0.0001; pyruvate carboxylase vs CD8 T cells, p<0.0001). The expression of TCA cycle enzymes correlated with the fraction of macrophages (aconitase, p<0.0001; IDH1, p<0.05). Significant differences in PTC tumor and normal thyroid tissue metabolite profiles were observed in glycolysis and TCA cycle, further supporting our findings of immunometabolic plasticity it PTC. Together, these findings highlight the central role TCA cycle activity plays in PTC tumor immunometabolism.

Conclusion: We demonstrate, for the first time, a distinct immunometabolic tumor signature supported by tumor metabolic reprogramming in PTC tumors. This novel characterization approach provides an important foundation for future functional metabolic studies within the tumor and immunologic microenvironment. A mechanistic understanding of the complex integration of immune networks and cellular metabolism can be harnessed for future therapeutic immunometabolic modulation in PTC.

Citation Format: Vivian L. Weiss, Naira Baregamian. Immunometabolic signature of papillary thyroid carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 628.
Abstract 63: Microenvironment-derived mitochondria prime glioma chemoresistance by augmenting NAD+metabolism and PARP-dependent DNA repair
Authors: Liu Yang , Gilbert Mark , Yang Chunzhang
Published: August 2018

Background: Mitochondria, the major organelles of cellular metabolism, can be released into the microenvironment and affect adjacent cells, supporting recipient cell metabolism and protecting them from unfavorable conditions. While our knowledge of intercellular mitochondria transfer under normal physiological conditions is expanding, a key question remains unanswered: do microenvironment-derived mitochondria influence the survival and malignant transformation of cancer cells?

Methods: In this study, we have established in vitro and in vivo models to aid in the understanding of mitochondrial transfer between astrocytes and glioma cells.

Results: We demonstrate that mitochondria are released from normal astrocytes through extracellular vesicles, and are adopted by neighboring glioma cells. Mitochondrial transfer not only improves oxidative metabolism in recipient glioma cells, but also supports the detoxification of chemotherapeutic agents by fueling PARP-dependent DNA repair with ATP and NAD+. Targeting astrocyte mitochondria-releasing pathways increased the cytotoxic effects of chemotherapy, reduced xenograft progression, and prolonged overall survival. Our findings suggest that microenvironment-derived mitochondria confer both metabolic capability and chemoresistance to cancer cells.

Conclusion: Targeting microenvironment-derived mitochondria may be important in reducing the capacity for cancer cells to recover from cancer treatment, thereby enhancing treatment efficacy.

Citation Format: Yang Liu, Mark Gilbert, Chunzhang Yang. Microenvironment-derived mitochondria prime glioma chemoresistance by augmenting NAD+metabolism and PARP-dependent DNA repair [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 63.
Abstract 641: Cell free DNA in the supernatant of pleural effusion can detect driver and resistance mutations and can guide TKI treatment decisions
Authors: Hummelink Karlijn , Muller Mirte , Linders Dorothe , Noort Vincent van der , Nederlof Petra , Burgers Sjaak , Meijer Gerrit , Heuvel Michel van den , Broek Daan van den , Monkhorst Kim
Published: August 2018

Introduction:

Molecular profiling of tumors has become the mainstay of diagnostics for metastasized solid malignancies and guides personalized treatment, especially in non-small cell lung cancer (NSCLC). In current practice it is often challenging to obtain sufficient tumor material for reliable molecular analysis. Cell free (cf) DNA in blood or other bio-sources could present an alternative approach to obtain genetic information from the tumor. In a retrospective cohort we analyzed the added value of cfDNA analysis in pleural effusions for molecular profiling.

Methods:

We retrospectively analyzed both the supernatant and the cell pellet of 44 pleural effusions sampled from 39 patients with KRAS (23) or EGFR (16) positive tumors for the original driver gene mutation as well as for EGFR T790M resistance mutations. Patients were diagnosed with either NSCLC (32), colon carcinoma (4), appendiceal carcinoma (2) or adenocarcinoma of unknown primary (1). Samples collected in the context of routine clinical care were stored in the NKI-AVL biobank. We used Bio-Rad QX200 droplet digital PCR for analysis.

Results:

The original driver gene mutation could be detected in 37 of the 44 pleural effusions by analysis of both supernatant (35/44 positive) and cell pellet (29/44 positive). In 7 out of 20 pleural effusions from patients with EGFR mutation positive tumors, a T790M mutation was detected. All 7 supernatants were positive as were 5 of the 7 cell pellets. The EGFR T790M mutation was confirmed in all supernatants (4/4) and in 3 of the 4 cell pellets sampled from patients with T790M positive tumors (4).

Conclusions:

Cell free DNA in pleural effusion proved to be a valuable bio-source and can be used to detect driver gene mutations as well as resistance mechanisms like EGFR T790M in pleural effusion.

Citation Format: Karlijn Hummelink, Mirte Muller, Dorothe Linders, Vincent van der Noort, Petra Nederlof, Sjaak Burgers, Gerrit Meijer, Michel van den Heuvel, Daan van den Broek, Kim Monkhorst. Cell free DNA in the supernatant of pleural effusion can detect driver and resistance mutations and can guide TKI treatment decisions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 641.
Abstract 652: Mutational profile of papillary thyroid microcarcinoma with extensive lymph node metastasis: Comparison of primary thyroid cancer and metastatic lymph nodes
Authors: Jeon Min Ji , Kim Won Gu , Chun Sung Min , Song Dong Eun , Kim Tae Yong , Shong Young Kee , Kim Won Bae
Published: August 2018

Introduction: Papillary thyroid microcarcinoma (PTMC) has excellent clinical outcomes, but distant metastasis of PTMC can be fatal. We previously reported that change to an aggressive pathologic subtype of metastatic lymph nodes (LNs) were associated with distant metastasis of PTMC. This study aimed to evaluate and compare the mutational profile of primary tumor and metastatic LNs of PTMC. Method: We included 16 PTMC patients with lateral cervical LN metastases. One of them has distant metastases. DNAs from formalin-fixed, paraffin-embedded archival samples of 16 sets of the normal thyroid tissue, primary PTMC, and the largest metastatic LN were used for targeted next-generation sequencing by Oncopanel AMC version 2.0. Results: A total of 7 somatic variants were confirmed in primary PTMC compared to normal thyroid tissue. BRAF V600E mutation was the most common and seen in 12 primary PTMCs (75%) and 11 metastatic LNs (69%). Missense mutation of KMT2A, RAF1, and ROS1 was detected only in one primary PTMC (6.3%), respectively. Frame-shift mutation of JAK2, inframe deletion of ACVR2A and nonsense mutation of AR was detected one primary PTMC (6.3%) with its metastatic LN, respectively. When we compare the mutational profile of primary tumor and metastatic LNs, there were no newly developed somatic mutations in metastatic LNs. We also found ALK/STRN rearrangement in one PTMC with its metastatic LN (6.3%). CCDC6/RET rearrangement and RET intergenic rearrangement was detected only in 2 metastatic LNs (12.5%) and one metastatic LN (6.3%), respectively. BARD1 deletion was detected in one primary PTMC with its metastatic LNS (6.3%) and FLT4 deletion was detected only in one primary PTMC (6.3%). We also found JAK1, AURKC, and TP53 deletions, which were detected only in one metastatic LN, respectively (6.3%). Conclusion: The mutation frequency of PTMC was really low even in patients with extensive LN metastasis. The mutational status of primary tumor and their regional metastatic LNs were not significantly different and this suggests the minor role of genetic mutations in the process of LN metastases.

Citation Format: Min Ji Jeon, Won Gu Kim, Sung Min Chun, Dong Eun Song, Tae Yong Kim, Young Kee Shong, Won Bae Kim. Mutational profile of papillary thyroid microcarcinoma with extensive lymph node metastasis: Comparison of primary thyroid cancer and metastatic lymph nodes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 652.
Abstract 657: ESR1 mutations abundance in local recurrence of endocrine-treated breast cancer
Authors: Zundelevich Adi , Dadiani Maya , Kahana-Edwin Smadar , Gadot Moran , Sela Tal , Marin Irina , Necula Daniela , Pavlovsky Anya , Barshack Iris , Kaufman Bella , Gal-Yam Einav N.
Published: August 2018

Emerging mutations in the ESR1 gene that encodes for estrogen receptor (ER) have been recently associated with resistance to endocrine therapy in ER positive metastatic breast cancer patients. These mutations promote the active conformation of the receptor, conferring resistance to endocrine therapy, and were associated with shorter progression free survival. ESR1 mutations were found to rarely exist in primary tumors (~1%) but are relatively common in metastatic, endocrine therapy-resistant lesions, with an estimated prevalence of ~10-50%. Nevertheless, not much is known about the incidence of these mutations in local recurrence (breast and adjacent lymph nodes) and its clinical significance. The objective of this study is to determine the prevalence of ESR1 mutations in local recurrence of endocrine-treated breast cancer patients. To this end, we collected a cohort of breast cancer patients that had at least one local or loco-regional recurrence during or after adjuvant endocrine treatment for primary breast cancer. We analyzed the 5 most common ESR1 hotspot mutations (D538G, L536R, Y537S/N/C) using droplet digital PCR technology in 44 samples from 34 patients (some of the patients had more than one recurrence). ESR1 mutations were identified in 10/34 (29%) patients. Specifically, mutations were distributed between D538G (5/34), Y537S+ D538G (4/34) and L536R (1/34). ESR1 Mutations developed after or on Tamoxifen only treatment in 50% of the positive patients (5/10 patients) and after or on Tamoxifen + Aromatase Inhibitor (AI) treatment in the other 50% (5/10 patients). Notably, one patient developed ESR1 mutation while on neoadjuvant endocrine therapy. ESR1 mutations persisted in local recurrence samples of mutation positive patients that had more than one local recurrence and the chance of detecting a mutation in patients that had more than one recurrence was higher than in those with a single recurrence. This study demonstrates that ESR1 mutations are common in local recurrence of hormone receptor positive breast cancer. Further studies to establish the clinical significance of these mutations and their impact on survival are underway. Since local recurrences are amenable to curative therapy, the identified unexpected high prevalence of ESR1 mutations at this stage of the disease may have important consequences for choosing the optimal adjuvant treatment option for these patients.

Citation Format: Adi Zundelevich, Maya Dadiani, Smadar Kahana-Edwin, Moran Gadot, Tal Sela, Irina Marin, Daniela Necula, Anya Pavlovsky, Iris Barshack, Bella Kaufman, Einav N. Gal-Yam. ESR1 mutations abundance in local recurrence of endocrine-treated breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 657.
Abstract 700: Characterization of new AML PDX models: Engraftment kinetics and mutational profile
Authors: Kaur Pali , Sachs Victoria , Christie Amanda L. , Weinstock David M. , Keck James G.
Published: August 2018

Acute myeloid leukemia (AML) is a genetically heterogeneous cancer of progenitor cells in myeloid hematopoiesis. Due to heterogeneity, most of AMLs are not responsive to targeted therapy. In addition, treatment outcome varies greatly depending upon patient age, disease etiology and mutational status. Previously, a large repository of well-characterized patient-derived- xenografts (PDXs) of hematologic malignancies was established (Townsend et al., 2016). This repository was generated by injecting primary bone marrow and blood samples from patients with leukemia into NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice. An average success rate of 23.2% engraftment was observed in first passage (P0). In this study, we have characterized ten AML PDX models in NSG-SGM3 mice by serial transplantation. NSG-SGM3 (NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ) mice have been found to have increased proliferation and survival of engrafted AML cells along with decreased disease latency (Wunderlich et al., 2010). Leukemic engraftment in these models was confirmed by flow analysis for hCD45+ hCD33+ in the peripheral blood, bone marrow and spleen of engrafted mice. Kinetics of engraftment varied from one model to another; and ranged from 10% - 80% of hCD33+ in peripheral blood 8 week post AML cell injection (1X106 cells/mouse). Four models displayed positive response to Cytarabine (Ara-C) treatment with reduction in levels of human CD33+ cells following a 5-day treatment cycle. Data will be presented on model characterization to show distinct engraftment kinetics, correlation with disease mutations and response to AML standard of care drug Cytarabine. These models have varying mutational profiles and treatment histories. Mutations include FLT3, FLT3-ITD, NPM1, TP53, DNMT3A and CUX1 alone or in combination. Treatment phases at the time of collection varied from untreated to relapse post-chemotherapy. These AML PDX models are useful tools for testing new experimental therapeutics. Diversity of these AML PDX models will facilitate focus on specific AML targets, thus providing an invaluable tool set to support drug discovery.

Citation Format: Pali Kaur, Victoria Sachs, Amanda L. Christie, David M. Weinstock, James G. Keck. Characterization of new AML PDX models: Engraftment kinetics and mutational profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 700.
Abstract 710: CD73 inhibitors (CD73i) reverse the AMP/adenosine-mediated impairment of immune effector cell activation by immune checkpoint inhibitors (ICI)
Authors: Becker Annette , Narasappa Nell , Yin Fangfang , Zhang Kristen , DiRenzo Daniel , Park Timothy , Kalisiak Jaroslaw , Lawson Ken , Jeffrey Jenna , Powers Jay P. , Schindler Ulrike , Walters Matthew J. , Tan Joanne B.
Published: August 2018

INTRODUCTION: CD73 catalyzes the extracellular generation of adenosine (ADO) from adenosine monophosphate (AMP). ADO suppresses immune responses, including those of T cells, NK cells and dendritic cells through activation of A2aR and A2bR receptors. Exhausted T cells and NK cells express high levels of several immune checkpoint proteins, including PD-1 and TIGIT. We present here preclinical data on the ability of CD73i to reverse effector cell suppression from exposure to ADO even in the presence of ICI.

METHODS: CD73i effects in a monotherapeutic setting were assessed by CD3/CD28/CD2 T cell stimulation and cytolytic assays. Combinatorial settings were assessed using mixed lymphocyte reactions (MLRs). In vivo effects of CD73i + ICI were determined using syngeneic tumor models.

RESULTS: CD73 is expressed across a wide range of tumor types, including those with limited response to anti-PD-1 therapy. CD73i completely rescued AMP-mediated inhibition of T cell proliferation and effector function as well as NK cell cytolytic function. AMP abrogated the enhanced allogeneic CD4+ T cell activation and IFN-γ production mediated by blocking PD-1/PD-L1 and TIGIT, an effect that was reversed by CD73i. Mechanistically, addition of AMP in MLRs repressed expression of activation markers and immune checkpoint proteins. Thus, activation of the adenosinergic pathway may limit the efficacy of ICI. TCGA data from anti-PD-1-treated melanoma patients identified CD73 expression as a negative prognostic factor. Finally, co-administration of a CD73i with an anti-PD-1 mAb resulted in significant reduction of tumor volume associated with increases in immune cell infiltration.

CONCLUSIONS: CD73 inhibition, alone or in combination with anti-PD-1 and anti-TIGIT antibodies, translates into potent enhancement of immune cell activation in a variety of studies. These data provide a rationale for CD73i + ICI combinations.

Citation Format: Annette Becker, Nell Narasappa, Fangfang Yin, Kristen Zhang, Daniel DiRenzo, Timothy Park, Jaroslaw Kalisiak, Ken Lawson, Jenna Jeffrey, Jay P. Powers, Ulrike Schindler, Matthew J. Walters, Joanne B. Tan. CD73 inhibitors (CD73i) reverse the AMP/adenosine-mediated impairment of immune effector cell activation by immune checkpoint inhibitors (ICI) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 710.
Abstract 719: A tumor mitochondria vaccine protects against experimental renal cell carcinoma
Authors: Pierini Stefano , Facciabene Andrea , Uribe-Herranz Mireia , Rafail Stavros , Fang Chongyun , Tanyi Janos
Published: August 2018

Mitochondria provide energy for cells via oxidative phosphorylation. Reactive oxygen species, a byproduct of this mitochondrial respiration, can damage mitochondrial DNA (mtDNA), and somatic mtDNA mutations have been found in all colorectal, ovarian, breast, urinary bladder, kidney, lung, and pancreatic tumors studied. The resulting altered mitochondrial proteins or tumor-associated mitochondrial Ags (TAMAs) are potentially immunogenic, suggesting that they may be targetable Ags for cancer immunotherapy. In this article, we show that the RENCA tumor cell line harbors TAMAs that can drive an antitumor immune response. We generated a cellular tumor vaccine by pulsing dendritic cells with enriched mitochondrial proteins from RENCA cells. Our dendritic cell-based RENCA mitochondrial lysate vaccine elicited a cytotoxic T cell response in vivo and conferred durable protection against challenge with RENCA cells when used in a prophylactic or therapeutic setting. By sequencing mtDNA from RENCA cells, we identified two mutated molecules: COX1 and ND5. Peptide vaccines generated from mitochondrial-encoded COX1 but not from ND5 had therapeutic properties similar to RENCA mitochondrial protein preparation. Thus, TAMAs can elicit effective antitumor immune responses, potentially providing a new immunotherapeutic strategy to treat cancer.

Citation Format: Stefano Pierini, Andrea Facciabene, Mireia Uribe-Herranz, Stavros Rafail, Chongyun Fang, Janos Tanyi. A tumor mitochondria vaccine protects against experimental renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 719.
Abstract 743: ABT-806-derived antibody-drug conjugates (ADCs) inhibit growth of malignant mesothelioma in vivo
Authors: Chia Puey Ling , Cao Diana , Rigopoulos Angela , Gan Hui , Reilly Ed , Phillips Andrew , John Thomas , Scott Andrew M.
Published: August 2018

Introduction: Malignant mesothelioma (MM) is an aggressive malignancy of the pleura with limited therapeutic options, and is associated with a poor prognosis. EGFR is known to be highly over-expressed in mesothelioma with reported EGFR overexpression between 44 to 97%. We have developed an anti-EGFR antibody (ABT-806), which is tumour specific and robustly inhibits EGFR-expressing tumours. We aimed to establish the validity and feasibility of targeting tumour expressed EGFR in MM using ABT-806 novel ADCs (ABT-414 and ABBV-221).

Methods: We evaluated EGFR and mAb 806 immunohistochemistry in 4 MM cell lines (MSTO-211H, NCI-H2052, NCI-H28, NCI-H2452) and performed in-vitro cell proliferation assays (CPA) to evaluate the antineoplastic potential of mAb806-related ADCs. In-vivo therapeutic studies using the biphasic mesothelioma cell line (MSTO-211H) were conducted with treatment groups involving ABT-414, ABBV-221, ADC control, cisplatin chemotherapy. We also performed quantitative biodistribution and imaging of mAb806 ADC uptake (89Zr mAb806 ADC) to allow correlation of mAb806 ADC concentration in tumours.

Results: mAb806 elicited strong binding to three of four MM cell lines (MSTO-211H, NCI-H2052 and NCI-H28). CPAs also demonstrated ABT-414 and ABBV- 221 had significant cell growth inhibition demonstrated in the range between 1 to 10ug/ml for MM cell lines. In MSTO-211H xenograft model significant anti-tumour response to both ABT-414 and ABBV-221 (p<0.01), was demonstrated. High, specific targeting of 89Zr-ch806 to MM tumour in-vivo was also shown.

Conclusion: ABT-806 ADCs show potent anti-tumour activity in MM model, and warrant further exploration as a potential therapy for MM.

Citation Format: Puey Ling Chia, Diana Cao, Angela Rigopoulos, Hui Gan, Ed Reilly, Andrew Phillips, Thomas John, Andrew M. Scott. ABT-806-derived antibody-drug conjugates (ADCs) inhibit growth of malignant mesothelioma in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 743.
Abstract 752: CovisoLinK: New bacterial transglutaminase Q-Tag substrate for the development of site specific antibody-drug conjugates
Authors: Sivado Eva , Thomas Vincent , Alaoui Meddy El , Jallas Anne-Catherine , Dyson Mike R. , McCafferty John , Alaoui Said El , Valsesia-Wittmann Sandrine
Published: August 2018

CovIsoLink™ (Covalently Isopeptide crosslinking) relates to methods for enzymatic covalently coupling drugs and other compounds through transglutaminase site specific generated in the targeted proteins including, polypeptides, proteins and immunoglobulins (patent pending1). Transglutaminases (TGases) catalyze covalent cross- linking of specific glutamine residues to the primary amine of peptide-bound lysine residues or primary amines of other compounds such as polyamines2. Using an in house peptide library and the transglutaminase colorometric assay3, we identified several amino acid sequences that were recognized as glutamine donor substrates. The optimum peptide sequences were selected and we further confirmed that these sequences have improved affinity compared with the conventional small peptides Z-QG. In different experiments we engineered Fc-containing polypeptide at the C-terminal domain and showed that mTG incorporates with high efficiency several types of amine donors to the engineered antibodies. CovIsoLink™ is now used to develop new antibody drug conjugates (ADCs) since the major advantage of this method is to obtain a homogenous immunoconjugate with uniform stoichiometry. We developed and characterized different recombinant anti Her2 IgG1 Mab carrying optimized enzymatic substrates (tag) by genetic insertion in the coding sequence of MAb. We then evaluated the best linkers and conformation to incorporate different compounds through bacterial Transglutaminase (mTG) enzymatic reaction. We set up experimental conditions, production, purification, HPLC/MS analysis and control of the immunoreactivity of CovIsoLink™ Her2-ADC. Using mTG, we obtained site specific conjugation of different modified drugs with optimized linker on the anti Her2 IgG1 antibody. By HIC analysis, we validated a specific and reproducible DAR reaching DAR2 depending on drugs and experimental conditions. In vitro and In vivo characterization and dose response studies of CovIsolink-ADC specificity and reactivity are currently performed in Her2 positive models demonstrating an improved efficacy by comparison with Kadcyla (T-DM1).1) PCT/EP2014/0792278, 2) El Alaoui S et al, Int J Cancer, 1991; 3) Perez AM et al, Anal Biochem, 2009.

Citation Format: Eva Sivado, Vincent Thomas, Meddy El Alaoui, Anne-Catherine Jallas, Mike R. Dyson, John McCafferty, Said El Alaoui, Sandrine Valsesia-Wittmann. CovisoLinK: New bacterial transglutaminase Q-Tag substrate for the development of site specific antibody-drug conjugates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 752.
Abstract 758: Head-to-head comparison of ADC and SMDC products, alone and in combination with antibody-IL2 fusion proteins
Authors: Cazzamalli Samuele , Corso Alberto Dal , Widmayer Fontaine , Neri Dario
Published: August 2018

In an attempt to improve the therapeutic index of cancer chemotherapy, monoclonal antibodies and small organic ligands have been proposed as delivery vehicles, allowing the construction of antibody-drug conjugates (ADCs) and of small molecule-drug conjugates (SMDCs). Four ADC products have gained marketing authorization for cancer therapy, while SMDCs are still being investigated in clinical trials. SMDCs could in principle offer certain advantages compared to ADCs, such as a more rapid and uniform diffusion into the tumor mass, lower cost-of-goods and lack of immunogenicity, but a direct head-to-head comparison has not yet been reported.

Here we describe a novel small molecule-drug conjugate, based on a high-affinity ligand specific to carbonic anhydrase IX. The product features a peptide linker, suitable for cleavage in the tumor extracellular environment, and monomethyl auristatin E as cytotoxic payload. We compared the therapeutic efficacy of the novel SMDC and of an anti-CAIX ADC, which were injected at equimolar doses in nude mice bearing SKRC-52 renal cell carcinoma xenografts. Both products showed a potent anti-cancer activity, although no complete tumor eradication was observed. Instead, when the SMDC was administered together with L19-IL2 (a clinical-stage fusion protein capable of delivering interleukin-2 to the tumor neo-vasculature), all treated mice in the combination group could be rendered tumor-free, in a process which favored the influx of natural killer cells into the tumor mass. The combination of L19-IL2 and the new small molecule-drug conjugate also eradicated cancer in 100% of immunocompetent mice, bearing subcutaneously-grafted CT26 colorectal cancer cells, which stably expressed carbonic anhydrase IX. These findings may be of clinical significance, since carbonic anhydrase IX is over-expressed in the majority of clear-cell renal cell carcinomas and in approximately 30% of colorectal cancers. The targeted delivery of interleukin-2 helps potentiate the action of targeted cytotoxics leading to cancer eradication in models that cannot be cured by conventional chemotherapy.

Citation Format: Samuele Cazzamalli, Alberto Dal Corso, Fontaine Widmayer, Dario Neri. Head-to-head comparison of ADC and SMDC products, alone and in combination with antibody-IL2 fusion proteins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 758.
Abstract 772: High MDC1 expression in cervical cancer cells can affect the chemo- and radiotherapeutic response as its depletion leads to increased cell death
Authors: Singh Neeru , Bhakuni Rashmi , Kirubakaran Sivapriya
Published: August 2018

Cervical cancer is the third most frequent cancer and common cause of death in women worldwide. The work presented identifies mediator of DNA damage checkpoint 1 (MDC1) as an important molecular target to increase sensitivity of cervical cancer cells to chemo or radiotherapy. MDC1 functions as an adaptor protein for recruitment and retention of many other DNA damage repair proteins in ataxia telangiectasia mutated (ATM) pathway for double-stranded DNA damage repair. It is reported to be highly expressed in cervical cancer cells. Also, its expression tends to increase with increase in malignancy. We have studied in detail MDC1 mRNA expression in three cervical cancer cell lines, HeLa, SiHa and CasKi, in response to various genotoxic stresses including some known inhibitors, UV exposure or gamma irradiation through quantitative PCR. The cellular response to the DNA damage resulted in increase in MDC1 expression, which declined with increase in treatment time period. Protein expression and activation by Western blotting with anti-MDC1 and anti-phosphoMDC1 antibody indicated a higher level of phosphorylated as compared to unphosphorylated MDC1. The significance of this increase in MDC1 expression was studied by generating stable cell lines knocked down for MDC1 expression. The modified cell lines were assessed for apoptosis through various assays, including flow cytometry, and showed greater cell death in response to DNA damage. In summary, high MDC1 expression can significantly affect chemo or radiotherapeutic response and its inhibition can improve sensitivity to these treatments.

Citation Format: Neeru Singh, Rashmi Bhakuni, Sivapriya Kirubakaran. High MDC1 expression in cervical cancer cells can affect the chemo- and radiotherapeutic response as its depletion leads to increased cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 772.
Abstract 779: Integrative analysis reveals therapeutic targets to the DNA methyltransferase inhibitor SGI-110 in hepatocellular carcinoma
Authors: Liu Minmin , Hinoue Toshinori , Zhou Wanding , Ohtani Hitoshi , El-Khoueiry Anthony , Daniels John , O'Connell Casey , Dorff Tanya B. , Weisenberger Daniel J. , Liang Gangning
Published: August 2018

Objective There is an urgent need for developing more effective therapies for Hepatocellular carcinoma (HCC) because of its aggressiveness. Guadecitabine (SGI-110) is a second-generation DNA methyltransferase inhibitor (DNMTi) currently in clinical trials for HCC and shows greater stability and performance over first generation DNMTi. The aim of this study is to identify potential therapeutic targets of SGI-110 for clinical trials.

Design HCC cell lines (SNU398, HepG2 and SNU475) were used to evaluate effects of SGI-110 by an integrative analysis of DNA methylation, nucleosome accessibility and gene expression profiles following the transient SGI-110 treatment and its clinic relevant by comparing HCC clinic data from TCGA.

Results These HCC cell lines represent the three DNA methylation subtypes of primary HCC tumors based on TCGA data. After SGI-110 treatment, all cell lines were sensitive to SGI-110 with prolonged anti-proliferation effects. Up-regulated genes including tumor suppressors were positively correlated with nucleosome accessibility and negatively correlated with gene promoter DNA methylation, while the down-regulated genes, such as oncogenes, were negatively correlated with nucleosome accessibility and positively correlated with gene body DNA methylation. Furthermore, SGI-110 down-regulated PRC2 complex genes by demethylating their gene bodies, resulting in re-activation of PRC2 repressed gene promoters which are independent on DNA methylation. In addition, SGI-110 up-regulated endogenous retroviruses (ERVs) to reactivate immune pathways. Finally, about 45% of frequently altered genes in primary HCC tumors can be re-shaped into a “normal like” expression status through the treatment.

Conclusion Our integrative analysis has successfully linked the anti-tumor effects of SGI-110 to detailed epigenetic alterations in HCC cells and identified potential therapeutic targets. We also provide rationale of combination treatment with immune checkpoint therapies.

Citation Format: Minmin Liu, Toshinori Hinoue, Wanding Zhou, Hitoshi Ohtani, Anthony El-Khoueiry, John Daniels, Casey O'Connell, Tanya B. Dorff, Daniel J. Weisenberger, Gangning Liang. Integrative analysis reveals therapeutic targets to the DNA methyltransferase inhibitor SGI-110 in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 779.