bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4495: Elucidating BCL11A’s function in triple negative breast cancer
Authors: Stevers Nicholas O. , Tinsley Samantha L. , Moody Rebecca , Lin Chang-Ching , Meagher Jennifer , Stuckey Jeanne A. , Lo Miao-Chia , Sun Duxin
Published: July 2017

It has been recently shown that the B-cell CLL/lymphoma 11A (BCL11A) transcription factor is highly expressed in triple-negative breast cancer (TNBC) and has critical functions in promoting tumor growth and mammary stem cell maintenance. Studies in our group have found that the first twelve amino acids (aa 1-12) of BCL11A are a conserved sequence that allows binding to retinoblastoma binding protein 4 (RBBP4). RBBP4, a histone binding protein, is involved in many epigenetic complexes, including the Polycomb repressive complex 2 (PRC2), the nucleosome remodeling and deacetylase (NuRD) complex, and SIN3A complex. Through these interactions BCL11A may exert transcriptional and epigenetic regulation.

Herein we investigate the importance of BCL11A’s interaction with RBBP4 and its function in TNBC. BCL11A-complex interactions were observed through pull-down experiments of TNBC cell lysate utilizing aa 2-16 of BCL11A and a scramble control sequence. Our results demonstrate that this 16-amino acid sequence is sufficient to bind to RBBP4 and pull down the PRC2, NuRD, and SIN3A complexes. Furthermore, the NuRD and Sin3a complexes pulled down by BCL11A aa 2-16 retained their functionality as observed through post pull-down histone deacetylase (HDAC) activity assays. These results indicate significantly higher HDAC activity in the wildtype sequence as compared to the scrambled sequence. To further investigate this interaction, protein crystallization was conducted, the results of which validate the binding between BCL11A and RBBP4. Additionally, a BCL11A peptide (aa 2-12) and a scramble control were transfected into the TNBC cell line SUM149. Analysis via flow cytometry shows a reduction of the ALDH+ cell population in the BCL11A peptide transfected cells versus scramble control. We are currently investigating the necessity of this 12-amino acid sequence in interactions with RBBP4, PRC2, NuRD and SIN3A via co-Immunoprecipitation experiments using an N-terminal truncated BCL11A protein (del 2-12). The effects of full-length BCL11A and del 2-12 on the ALDH+ cell population are also being examined. These experiments will further delineate this sequence’s importance in BCL11A-RBBP4 interactions.

Citation Format: Nicholas O. Stevers, Samantha L. Tinsley, Rebecca Moody, Chang-Ching Lin, Jennifer Meagher, Jeanne A. Stuckey, Miao-Chia Lo, Duxin Sun. Elucidating BCL11A’s function in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4495. doi:10.1158/1538-7445.AM2017-4495
Abstract 4496: Dissecting cellular heterogeneity using single cell RNA sequencing in human lung tumors
Authors: Courtois Elise T. , Li Huipeng , Tan Yuliana , Tan Daniel , Prabhakar Shyam , Robson Paul
Published: July 2017

Intra-tumor heterogeneity is a key determinant of tumor biology, treatment response and patient survival. It is therefore essential to comprehensively characterize the phenotypes and interactions of the diverse cells within the tumor ecosystem. However, traditional molecular profiling studies have largely relied on bulk-tissue analysis, which obscures the signatures of distinct cell populations.

We present here an unbiased analysis of functional heterogeneity in NSCLC (Non Small Cell Lung Cancer) cancers (Lung Adenocarcinoma and Squamous Cell Carcinoma) and their microenvironment using RNA-seq profiling of unsorted single cells from 20 primary tumors and matched normal lung tissue (NL). Using a new clustering algorithm RCA (Reference Component Analysis), we identified the major cell types composing the normal lung tissue as well as the tumor ecosystem such as epithelial, fibroblast, endothelial and immune cells. Notably, we were able to identify resident alveolar macrophages, in tumor and normal tissue and their single-cell differential expression analysis revealed substantial differences in their gene expression. Similarly, we identified blood-vessel endothelial cells in matched normal and tumor tissue, and identified the differentially expressed genes in both cell types. Our unbiased approach allowed us to study the diversity of the epithelial malignant cells present in different tumors, highlighting the tumor and patient specific variability. Together, the unbiased study of the NSCLC at the single cell level by sc-RNAseq offers a powerful tool to provide insights into the transcriptomic variability within the key cellular players of the tumorigenesis.

Citation Format: Elise T. Courtois, Huipeng Li, Yuliana Tan, Daniel Tan, Shyam Prabhakar, Paul Robson. Dissecting cellular heterogeneity using single cell RNA sequencing in human lung tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4496. doi:10.1158/1538-7445.AM2017-4496
Abstract 4503: Role of ER-associated degradation in rhabdomyosarcoma
Authors: Sannino Sara , Guerriero Christopher J. , Brodsky Jeffrey L.
Published: July 2017

Due to their higher proliferation rate, most cancers experience impaired ATP generation, hypoxia, hypoglycemia, and increased genetic mutations that may perturb cellular and protein homeostasis. Persistent stress conditions induce the unfolded protein response (UPR) as well as increased expression of molecular chaperones, such as Hsp70, to modulate cell fate. Our recent studies demonstrate how perturbing cytosolic Hsp70 activity with a small molecule modulator, MAL3-101, induces rhabdomyosarcoma cell (RMS13) death, patient-derived cells of one of the most common soft-tissue cancers in children. MAL3-101 addition activates PERK- and CHOP-dependent RMS13 cell death in a dose dependent manner (Sabnis et al., PNAS, 2016). To identify the mechanism that links UPR-triggered cell death and cytosolic Hsp70 inhibition, an isogenic MAL3-101-resistant cell line was generated (MAR4C). Even though both RMS13 and MAR4C cells were equally sensitive to a diverse panel of therapeutic compounds, we now find that they have different efficiencies of ER-associated degradation (ERAD), a quality control pathway that degrades misfolded proteins in the secretory pathway and that lessens proteotioxic stress. Specifically, MAR4C cells remove both soluble and membrane associated ERAD substrates faster than RMS13 cells and possess increased levels of ubiquitinated proteins upon proteasomal inhibition. These data suggest that higher ERAD efficiency is required for MAR4C cell survival upon Hsp70 inhibition and highlight an unexpected link between cytosolic Hsp70 inhibition and the ERAD pathway in cancer cells. Moreover, RNAseq analysis suggests that select redox-related ER quality control chaperones are differentially expressed between MAR4C and RMS13 cells, and lower levels of the BiP and PDI chaperones and Peroxiredoxin 4 are found in MAR4C cells. Because new therapeutic agents as well as reliable biomarkers are critically needed in rhabdomyosarcoma and other cancers, this study sheds light on one mechanism of cancer pathogenesis and suggests that different cancer subtypes modulate ER quality protein quality uniquely.

Citation Format: Sara Sannino, Christopher J. Guerriero, Jeffrey L. Brodsky. Role of ER-associated degradation in rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4503. doi:10.1158/1538-7445.AM2017-4503
Abstract 4508: Penfluridol-induced endoplasmic reticulum stress leads to autophagy-mediated pancreatic tumor growth suppression
Authors: Ranjan Alok , Ramachandran Sharavan , Gupta Nehal , Srivastava Sanjay
Published: July 2017

Pancreatic cancer is the fourth leading cause of cancer-related deaths in the United States. Experimental and clinical evidences suggested that high basal state autophagy in pancreatic tumors could induce resistance to chemotherapies. Recently, we have demonstrated that penfluridol suppresses pancreatic tumor growth by autophagy-mediated apoptosis both in vitro and in vivo. (Ranjan and Srivastava, Scientific Reports: 2016;6:26165; PMID: 27189859), however the mechanism of autophagy induction by penfluridol was not clear. Several studies have established that endoplasmic reticulum (ER) stress could lead to autophagy and inhibit tumor progression. In the current study, we demonstrated that penfluridol induced ER stress in BxPC-3, AsPC-1 and Panc-1, pancreatic cancer cell lines as indicated by up regulation of ER stress markers such as BIP, CHOP and IRE1α after treatment with penfluridol in a concentration-dependent manner. Inhibiting ER stress by pre-treatment with pharmacological inhibitors such as sodium phenylbutyrate and mithramycin or by silencing CHOP using CHOPsiRNA, blocked penfluridol-induced autophagy. These results clearly indicated that penfluridol induced ER stress lead to autophagy in our model. Western blot analysis of subcutaneously implanted AsPC-1 and BxPC-3 tumors as well as orthotopically implanted Panc-1 tumors demonstrated upregulation of BIP, CHOP and IRE1α expression in the tumors lysates from penfluridol treated mice as compared to tumors from control mice. Altogether, our study established that penfluridol induced ER stress mediated autophagy in pancreatic tumor. Our study opened a new therapeutic target for advanced chemotherapies against pancreatic cancer. (Supported in part by RO1 grant CA129038, awarded by National Cancer Institute, NIH).

Citation Format: Alok Ranjan, Sharavan Ramachandran, Nehal Gupta, Sanjay Srivastava. Penfluridol-induced endoplasmic reticulum stress leads to autophagy-mediated pancreatic tumor growth suppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4508. doi:10.1158/1538-7445.AM2017-4508
Abstract 4512: The landscape of hypoxia-driven alternative splicing in breast cancer
Authors: Choudhry Hani , Oikonomopoulos Spyridon , Yu Peng , Ivan Cristina , Ivan Mircea , Harris Adrian L. , Ragoussis Jiannis
Published: July 2017

Tumor hypoxia is generally associated with poor patient outcome and resistance to therapy. Hypoxia has an impact on multiple pathways inside the cell and a widespread effect on phenotype. Here we determined the changes in transcript architecture that arise as result of alternative splicing in hypoxic cells. A panel of breast cancer cell lines that include the Luminal A and B, HER2+, Triple-Negative subcategories grown in hypoxic and normoxic conditions were subjected for deep RNA-sequencing. Depending on the cell lines we found between 23- 927 splicing events with 3-45% of them also present in the differentially expressed fraction of genes. This points to an extensive isoform switching control independently of the transcriptional control of the genes themselves. The splicing events appear on genes responsible for mRNA processing (splicing, nuclear export and catabolism) as well as initiation of translation. Members of the SR family of proteins involved in RNA splicing, appear to be potential regulators of the biogenesis of the hypoxia specific splicing events. The differentially spliced exons are frequently part of both the coding and noncoding isoforms of a given gene potentially indicating a switch between these two types under hypoxia. Ten of the hypoxia specific splicing events were also found to be associated with the hypoxia status of primary breast cancer samples taken from The Cancer Genome Atlas (TCGA) database. Altogether, these data demonstrate the important role of hypoxia in driving alternative splicing events in breast cancer.

Citation Format: Hani Choudhry, Spyridon Oikonomopoulos, Peng Yu, Cristina Ivan, Mircea Ivan, Adrian L. Harris, Jiannis Ragoussis. The landscape of hypoxia-driven alternative splicing in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4512. doi:10.1158/1538-7445.AM2017-4512
Abstract 4527: Estimating tumor cell dissemination rates through lymph and blood via computational modeling
Authors: Barrette Anne Marie , Lenoir Benedicte , Skobe Mihaela , Birtwistle Marc R.
Published: July 2017

Metastasis is the primary cause of death from most cancer types, but the relative rates of tumor cell migration through lymphatic versus blood routes remains an open question. Here we combine data from a xenograft model of metastatic breast cancer with a computational model of tumor cell growth and dissemination to help answer this question. Orthotopic mouse xenografts using the triple negative breast cancer cell line MDA-MB-231 allowed for monitoring of metastatic cells within the primary tumor, bloodstream, lymph nodes, and lungs. Experiments resembling a pulse-chase method helped inform and parameterize the model to the rate of dissemination to the lymph nodes and lungs. Our ordinary differential equation model describes primary tumor growth as the balance between proliferation and death, with the proliferative fraction decreasing with tumor size, and the apoptotic or necrotic fraction increasing with tumor size. Intravasation to both lymph and blood rapidly increases early in tumor growth, followed by a decay to a low rate of tumor cell release well before the primary tumor grows to its saturating size. The mouse data guided our parameter estimations based on the assumptions that disseminated tumor cell death rate is greatest in the harsh environment of the blood and lowest in the more accommodating lymph node, and that metastatic cells seeding in the lymph nodes and lungs follow similar growth dynamics as the primary tumor only with far fewer total cells. We are currently performing sensitivity analysis to explore the range of relative rates of tumor cell dissemination that fit the in vivo data, and thus gain insight into tumor cell dynamics within the different compartments. These analyses will allow us to estimate what the relative contributions of blood versus lymphatic tumor cell dissemination are to overall metastatic burden, as well as suggest experiments that further constrain these estimates. Such combined experimental and modeling analysis lend insight into where one might detect pre-metastatic cells early in disease progression or suggest new targets for anti-metastasis drugs based on the dominant mode of dissemination.

Citation Format: Anne Marie Barrette, Benedicte Lenoir, Mihaela Skobe, Marc R. Birtwistle. Estimating tumor cell dissemination rates through lymph and blood via computational modeling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4527. doi:10.1158/1538-7445.AM2017-4527
Abstract 4532: Adrenal metastases in lung cancer: Clinical implications of a mathematical model
Authors: Mason Jeremy M. , Ciccarella AnneMarie , Marx-Rubiner Lori , Bazhenova Lyudmila , Newton Paul K. , Bethel Kelly , Nieva Jorge J. , Kuhn Peter
Published: July 2017

Adrenal gland metastases are common in lung cancer. It is well recognized that aggressive treatment of solitary adrenal metastases leads to improved outcomes but the exact nature of adrenal deposits is not well understood. Controversy exists as to the routing of cancer cells to the adrenal gland with some believing that this transmission is lymphatic, in contrast to the more generally accepted theory of hematogenous spread. Using an autopsy dataset of 3827 untreated cancer patients, we use the metastatic distribution of common primary cancer types to create Markov models of progression. The anatomical sites of spread in the body represent states in the model, while the transition probability between states represents the probability of metastatic spread from one site to another. We then use the Markov models to run Monte Carlo simulations of random walkers representing circulating tumor cells traveling within the body to simulate metastatic spread. We calculate mean first passage times (MFPT) to each site as a representative of time to metastasis formation. Analysis of 6 common cancer types (bladder [n=120 autopsies; 289 metastases], breast [n=432; 2235], colorectal [n=161; 420], lung [n=560; 859], ovarian [n=418; 806], and prostate [n=62; 212]) from the dataset showed distinct metastatic distributions across the populations. MFPT calculations to anatomical sites in the 6 analyzed primary cancer types indicated model progression times to the adrenal gland similar to the regional and distal lymph nodes in only lung cancer. The times associated with adrenal gland in the other primary cancers were similar to other metastatic sites of hematogenous spread. The Markov models created strongly support the lymphatic theory for metastatic spread to the adrenal glands. After performing a literature review to look for the biological plausibility of the simulated results, we believe evidence supports this theory and validates the models. This could explain improved survival for patients in whom solitary adrenal metastases are managed aggressively with surgical or radiation modalities. In order to further validate this theory, we are calling for clinical trials prospectively testing this hypothesis.

Citation Format: Jeremy M. Mason, AnneMarie Ciccarella, Lori Marx-Rubiner, Lyudmila Bazhenova, Paul K. Newton, Kelly Bethel, Jorge J. Nieva, Peter Kuhn. Adrenal metastases in lung cancer: Clinical implications of a mathematical model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4532. doi:10.1158/1538-7445.AM2017-4532
Abstract 4535: Tumor cell division rate heterogeneity explains in vitro clonal dominance
Authors: Palm Margriet M. , Elemans Marjet , Beltman Joost B.
Published: July 2017

The cells that make up a tumor exhibit large phenotypic and genotypic variation, which strongly influences the effectiveness of cancer therapy. A typical manifestation of intratumor heterogeneity is the presence of a few clones that represent most of the tumor population, i.e. clonal dominance. Such heterogeneity could potentially be caused by the occurrence of different ratios of cancer stem cells and differentiated cells per clone, or, alternatively, clonal dominance may be caused by heterogeneity of division rates between clones. Previously published work, applying lineage tracing with genetic barcodes to in vitro iterated growth and passage experiments demonstrated that clonal dominance was accompanied by clone loss and partial overlap in dominant clones between biological replicates [1]. To identify the source of these observations, we build a computational model that simulates the iterated growth and passage experiments. By varying the 'division rules' in the model, we can quantitatively predict the evolution of the clone distribution for the aforementioned hypotheses. In this manner, we show that clonal dominance does not appear in simulations where all cells divide at a uniform rate, even when the population is subdivided in cancer stem cells and differentiated cells. However, when the division rates vary between clones and are inherited from parent cells, dominant clones appear. These simulations also closely match the in vitro evolution of clone loss and clonal overlap between biological replicates. Altogether, our findings suggest that tumor cells exhibit a heritable variability in the division rates of individual cells.

References

[1] Porter, S. et al. (2014). Lentiviral and targeted cellular barcoding reveals ongoing clonal dynamics of cell lines in vitro and in vivo. Genome Biology, 15(5), R75.

Citation Format: Margriet M. Palm, Marjet Elemans, Joost B. Beltman. Tumor cell division rate heterogeneity explains in vitro clonal dominance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4535. doi:10.1158/1538-7445.AM2017-4535
Abstract 4556: MEDI5395: An armed oncolytic Newcastle disease virus
Authors: Carroll Danielle , Harper James , Jon Travers , Burke Shannon , Franks Ruth , Navarro Christel , Cheng Xing , Wilkinson Robert , Jin Hong
Published: July 2017

Oncolytic viruses are live, replication-competent viruses that infect and/or replicate selectively in tumour cells leading to the destruction of the infected cell. Cell lysis occurs as a natural consequence of the viral life cycle and released virions can infect and kill neighbouring tumour cells leading to an amplified therapeutic effect. Oncolysis has the added benefit of releasing multiple tumour antigens that may further induce an immune-mediated therapeutic response. Newcastle Disease Virus (NDV) is an avian paramyxovirus, which has proven safety and demonstrated efficacy against a variety of preclinical cancer models and in PhI clinical studies as an oncolytic agent, oncolysate or whole cell vaccine. Using reverse genetics, we have generated a recombinant strain of NDV that overcomes environmental and regulatory concerns uncoupling oncolytic potency and avian pathogenicity. Furthermore we have enhanced the immune modulatory properties of NDV by engineering the virus to express granulocyte/macrophage colony-stimulating factor (GM-CSF). We have evaluated the biological characteristics of recNDVGM-CSF (MEDI5395) in vivo and in vitro. MEDI5395 selectively replicates in and kills a wide variety of human and mouse tumour cell lines. Additionally infection of cancer cells with MEDI5395 results in the increased production and secretion of pro-inflammatory cytokines and chemokines which are able to recruit mediators of both the innate and adaptive immune responses. MEDI5395 is a potent activator of the type I interferon response. In vivo, using a range of syngeneic and xenograft models we have demonstrated that NDV treatment has robust anti-tumour activity. In a HT1080 fibrosarcoma xenograft model a single administration (intra-tumoural or systemic) was able to cure 80% of tumour bearing mice. In syngeneic mouse tumour models, which support minimal viral replication MEDI5395 treatment causes significant changes in the local immune suppressive microenvironment and results in long-lasting anti-tumour immune responses. These responses are further enhanced in models that permit greater replication and also when combined with immune checkpoint blockade. The inherent properties of NDV (self-propagation, tumour-selective replication, and immunostimulatory properties) coupled with the ability to genetically engineer NDV to express therapeutic transgenes may provide a multi-modal attack on the tumour, delivering greater benefit to patients.

Citation Format: Danielle Carroll, James Harper, Travers Jon, Shannon Burke, Ruth Franks, Christel Navarro, Xing Cheng, Robert Wilkinson, Hong Jin. MEDI5395: An armed oncolytic Newcastle disease virus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4556. doi:10.1158/1538-7445.AM2017-4556
Abstract 456: Functional analysis of DEC1 and DEC2 in cancer metabolism
Authors: Sato Fuyuki , Muragaki Yasueru
Published: July 2017

Basic helix-loop-helix (bHLH) transcription factor DEC1 (bHLHE40/Stra13/Sharp2) and DEC2 (bHLHE41/Sharp1) are clock genes that show a circadian rhythm in various tissues. However, the function of DEC1 and DEC2 in metabolism has not been poorly understood. AMP-activated protein kinase (AMPK) activity plays important roles in the metabolic process and circadian rhythm. However, how circadian rhythm of phosphorylation of AMPK (pAMPK) is regulated has been yet understood. The aim of this study is to investigate whether there is a direct correlation between DEC1 and DEC2 expression and AMPK activity in normal and cancer cells. There was an inverse relationship between DEC1 expression and AMPK activity. DEC1 protein and pAMPK showed a circadian rhythm in the mouse liver with different peak levels. Knocking down DEC1 expression increased pAMPK, whereas overexpression of DEC1 decreased it. DEC1 bound to the E-box of the LKB1 promoter, decreased LKB1 activity and total protein levels. In addition, pAMPK immunoreactivity was strongly detected in the liver and lung of DEC2 knockout mice compared with that of wild-type mice. Interestingly, medium change induced the circadian rhythm of DEC2, but not DEC1 in cancer cells. We conclude that DEC1 and DEC2 negatively regulates AMPK activity via LKB1, and play important roles in cancer cell metabolism.

Citation Format: Fuyuki Sato, Yasueru Muragaki. Functional analysis of DEC1 and DEC2 in cancer metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 456. doi:10.1158/1538-7445.AM2017-456
Abstract 4570: Intratumoral IFN-α gene transfer reduces trafficking of Tregs into tumor by inhibition of CCL17 expression
Authors: Aoki Kazunori , Shibasaki Chihiro , Hashimoto Hisayoshi , Narumi Kenta , Kudo Chie
Published: July 2017

Type I interferon (IFN) is a pleiotropic cytokine regulating the cancer cell death and immune response. IFN-α can effectively induce an antitumor immunity by the activation of tumor-specific T cells and maturation of dendritic cells in various animal models. It is widely known that an induction of tolerance by the tumor is one of the critical mechanisms involved in tumor progression and resistance for immune therapy. Extensive studies have shown that CD4+Foxp3+ regulatory T cells (Tregs) are critical in controlling antitumor immune responses in the tumor microenvironment. Since the effect of type I IFN on an immunotolerant microenvironment is unknown, we investigated the immunological effects of intratumoral IFN-α gene transfer on tumor-infiltrating Tregs in a mouse model.

Here, to examine whether the expression of IFN-α affects the immunotolerant microenvironment in tumors, CT26 murine colon cancer tumors were directly injected once with 1 x 108 PFU of IFN-α-expressing adenovirus (Ad-mIFN). The injection of Ad-mIFN significantly suppressed the tumor growth. We found that intratumoral IFN-α gene transfer significantly decreased the frequency of Tregs per CD4+ T cells in tumors compared with control vector injection.

We hypothesized that Treg-homing cytokines such as CCL17 and CCL22 cytokines are involved in the reduction of tumor-infiltrating Tregs by the Ad-mIFN injection. The IFN-α expression significantly suppressed of CCL17 and CCL22 expression of tumors, and the expression of CCR4 and CCR5 expression on Tregs was not changed. IFN-α protein significantly suppressed CCL17 of CT26 cells in vitro in a dose-dependent manner. Then, we constructed CCL17 shRNA-transduced CT26 cells (CT26shCCL17) to examine the immunological role of CCL17 in vivo. The frequency of Tregs per CD4+ T cells was significantly reduced in subcutaneous tumors of CT26shCCL17 cells (25.9 %), and tumor growth was inhibited compared with that of control shRNA-transduced CT26 cells (39.4%), suggesting CCL17 has an important role to migrate Tregs into tumors. The study demonstrated that IFN-α gene delivery creates an environment strongly supporting the enhancement of antitumor immunity through the suppression of Tregs.

Citation Format: Kazunori Aoki, Chihiro Shibasaki, Hisayoshi Hashimoto, Kenta Narumi, Chie Kudo. Intratumoral IFN-α gene transfer reduces trafficking of Tregs into tumor by inhibition of CCL17 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4570. doi:10.1158/1538-7445.AM2017-4570
Abstract 4577: Appropriately delivered curcumin causes recruitment of natural killer cells into glioblastoma brain, stabilizing M1 polarization of tumor-associated microglia
Authors: Mukherjee Sumit , Fried Angela , Hussaini Rahman , White Richard , Chatterjee Aheli , Banerjee Probal
Published: July 2017

Glioblastoma (GBM) is the most insidious form of primary adult brain tumor with a mean life expectancy of 12-24 months. Previously, we have used the non-invasive strategies of (1) intranasal delivery of a glioblastoma-directed curcumin (CC) adduct (CC-CD68Ab) and (2) the intraperitoneal (i.p.) infusion of a lipid-encapsulated formulation of CC-phytosome to rescue orthotopically GL261-implanted GBM mice and study the effect of CC on the phenotype of the tumor-associated microglial cells (TAMs). Both treatment regimens not only caused tumor remission in 50-60% of GL261-implanted GBM mice, but also induced a dramatic change in the tumor-associated Iba1+ TAMs, suppressing the tumor-promoting Arginase1high, IL10high, iNOSlow, IL12low M2-type TAMs, while simultaneously inducing the Arginase1low, IL10low, iNOShigh, IL12high M1-type TAMs. Concurrently, we observed a marked induction and phosphorylation-mediated activation of microglial p65 NF-kB and STAT1, with concomitant suppression and inactivation of STAT3. p-STAT1high, p-STAT3low M1 microglia are known to cause IL12-dependent recruitment/activation of tumoricidal NKp46high natural killer (NK) cells. In determining the kinetics of CC-induced M1 repolarization of M2-TAMs, we observed that five days of CCP treatment (i.p.) was sufficient to induce a dramatic change in the tumor-associated Iba1+ TAMs, inhibiting the tumor-promoting M2-type TAM population, while activating M1-type microglia. As expected, we observed a concomitant recruitment of NKp46high NK cells into the GBM tumor. Cognizant of the fact that NK cells activate and stabilize the M1-microglia, we eliminated the NK cells in these GBM mice using the NK cell-targeted antibody NK1.1 Ab to observe a significant reduction in CC-evoked repolarization of TAMs. Our results demonstrate a unique oncoimmunotherapeutic function of CC, which when appropriately delivered, not only eliminates GBM directly, but also indirectly elicits recruitment and activation of tumoricidal NK cells and M1-TAMs. Furthermore, maintenance of this CC-evoked M1 phenotype is contingent upon CC-induced recruitment and activation of NK cells. Thus, when delivered appropriately, CC functions on GBM both directly as well as by stimulating and recruiting an army of immune cells that eliminate both GBM and GBM-initiating cells.

Citation Format: Sumit Mukherjee, Angela Fried, Rahman Hussaini, Richard White, Aheli Chatterjee, Probal Banerjee. Appropriately delivered curcumin causes recruitment of natural killer cells into glioblastoma brain, stabilizing M1 polarization of tumor-associated microglia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4577. doi:10.1158/1538-7445.AM2017-4577
Abstract 4583: Heterogeneity in immune biomarker expression: Detailed analysis of a case with SCLC transformation after EGFR-TKI treatment
Authors: Suda Kenichi , Murakami Isao , Yu Hui , Kim Jihye , Ellison Kim , Rivard Christopher J. , Mitsudomi Tetsuya , Hirsch Fred R.
Published: July 2017

Introduction: The expression status of immune markers are of current research interest due to their potential roles as predictive biomarkers for immunotherapy in cancers. Currently there is little information on how the microenvironment of tumors and/or therapy-inducible histological transformation may affect the expression of these immune markers. Methods: A 76-year-old never-smoking female with epidermal growth factor receptor (EGFR) mutated lung adenocarcinoma (AC) acquired resistance to gefitinib. After her death, autopsy revealed small-cell lung cancer (SCLC) transformation and EGFR T790M secondary mutation as mutually exclusive resistance mechanisms. Two liver metastases (SCLC vs. AC with T790M) and two lymph node metastases (SCLC vs. AC with T790M) were analyzed to compare the expression status of immune markers by immunohistochemistry (IHC) and an immune-oncology gene expression panel (HTG EdgeSeq Immuno-Oncology Assay). Results: IHC analysis revealed that PD-L1 was expressed in 5% of tumor cells with AC histology (T790M) but not in tumor cells with SCLC transformation (Table 1). The liver metastasis with SCLC transformation showed negative stromal PD-L1 expression and scant tumor infiltrating lymphocytes, while the other lesions demonstrated stromal PD-L1 staining and infiltration of CD8-positive T cells. PD-1 was positive in lymphocytes from lymph node metastases, but not in those from liver metastases. Data generated using an IO panel indicated higher expression of galectin 9 and higher level of T cell stimulatory checkpoints in lesions with SCLC transformation. Bioinformatic analysis also demonstrated lower expression of type I interferon regulated genes in lesions with SCLC transformation. Conclusion: These data highlight the heterogeneity of expression of immune markers depending on the metastatic sites and histological transformation, and indicate that a biopsy from one lesion may not be representative of immune marker status for all lesions.

Summary of immune marker IHC staining in all specimensSpecimensPD-L1 (tumor)PD-L1 (stroma)TILsCD8 (T cells)PD-1 (T cells)Liver metastasis (AC / T790M)5%5%+++negativeLiver metastasis (SCLC)negativenegativescantn.a.n.a.Lymph node metastasis (AC / T790M)5%5%++++Lymph node metastasis (SCLC)negative10%++++

Citation Format: Kenichi Suda, Isao Murakami, Hui Yu, Jihye Kim, Kim Ellison, Christopher J. Rivard, Tetsuya Mitsudomi, Fred R. Hirsch. Heterogeneity in immune biomarker expression: Detailed analysis of a case with SCLC transformation after EGFR-TKI treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4583. doi:10.1158/1538-7445.AM2017-4583
Abstract 4596: Antibody-drug conjugates bearing pyrrolobenzodiazepine or tubulysin payloads alter the tumor immune microenvironment and synergize with multiple immunotherapies
Authors: Rios-Doria Jonathan , Harper Jay , Rothstein Raymond , Wetzel Leslie , Chesebrough Jon , Marrero Allison , Chen Cui , Strout Patrick , Mulgrew Kathy , McGlinchey Kelly , Fleming Ryan , Bezabeh Binyam , Meekin John , Stewart David , Kennedy Maureen , Martin Philip , Buchanan Andrew , Dimasi Nazzareno , Michelotti Emil , Hollingsworth Robert
Published: July 2017

Immunogenic cell death (ICD) is the process by which certain cytotoxic drugs induce apoptosis of tumor cells in a manner that stimulates the immune system. In this study, we investigated whether ADCs conjugated with pyrrolobenzodiazepine dimer (PBD) or tubulysin payloads induced ICD, modulated the immune microenvironment, and could combine with IO drugs to enhance antitumor activity. We show that these payloads on their own induced an immune response that prevented the growth of tumors following subsequent tumor cell challenge. ADCs had greater antitumor activity in immunocompetent vs. immunodeficient mice, demonstrating a contribution of the immune system to the antitumor activity of these ADCs. These ADCs also induced immunological memory. In the CT26 model, depletion of CD8+ T cells abrogated the activity of these ADCs when used alone or in combination with a PD-L1 antibody, confirming a role for T cells in the antitumor activity. Combinations of ADCs in different tumor models with IO drugs including PD-1 or PD-L1 antibodies, or OX40 ligand or GITR ligand fusion proteins produced synergistic antitumor responses. Importantly, synergy was observed in some cases with suboptimal doses of ADCs, potentially providing an approach to achieve potent antitumor responses while minimizing ADC-induced toxicity. Immunophenotyping studies in different tumor models revealed broad immunomodulation of lymphoid and myeloid cells by ADCs and ADC/IO combinations. These results suggest that it may be possible to develop novel combinatorial therapies with PBD- and tubulysin-based ADCs and IO drugs that may increase clinical responses.

Citation Format: Jonathan Rios-Doria, Jay Harper, Raymond Rothstein, Leslie Wetzel, Jon Chesebrough, Allison Marrero, Cui Chen, Patrick Strout, Kathy Mulgrew, Kelly McGlinchey, Ryan Fleming, Binyam Bezabeh, John Meekin, David Stewart, Maureen Kennedy, Philip Martin, Andrew Buchanan, Nazzareno Dimasi, Emil Michelotti, Robert Hollingsworth. Antibody-drug conjugates bearing pyrrolobenzodiazepine or tubulysin payloads alter the tumor immune microenvironment and synergize with multiple immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4596. doi:10.1158/1538-7445.AM2017-4596
Abstract 4597: A novel immuno-oncology approach: targeting cell-surface mesothelin with a fusion construct containing human granzyme B
Authors: Cheung Lawrence H. , Tijani Rasheed , Khan Nasir , Thuning-Roberson Claire , Rosenblum Michael G.
Published: July 2017

Immunological approaches to target tumors have gained considerable traction recently because of excellent clinical results. In addition, targeted treatment with antibody-drug conjugates (ADCs) can also be effective in achieving regression of solid tumors. The challenge for ADCs continues to be efficacy, stability of the construct and development of resistance. We have developed a unique fusion protein composed of 1) a novel human single-domain antibody (SD1) that uniquely targets Region III of the mesothelin glycoprotein and 2) an engineered version of the human serine protease Granzyme B (GrB). Mesothelin is highly expressed in aggressive cancers such as malignant mesothelioma, pancreatic and ovarian cancers, and NSCLC. Granzyme B generates an intense, irreversible pro-apoptotic effect through direct cleavage of caspases, release of cytochrome C from mitochrondria, and cleavage of nuclear matrix. Previous ADCs targeting mesothelin have faced the drawbacks of toxic payloads, formation of neutralizing antibodies, and competition with the serum protein MUC16/CA125. SD1-GrB represents a new class of completely human immunotoxins containing a payload with a unique mechanism of action. Additionally, because of its unique binding properties, SD1 does not compete with circulating CA125 and is not subject to cleavage. The SD1-GrB construct was designed as a homodimer (~200kDa) containing an IgG Fc domain and was expressed using transient-transfection of HEK293E cells. The soluble SD1-GrB was isolated from conditioned media and purified to homogeneity as assessed by SDS-PAGE and Western. Enzymatic activity of the GrB component was assessed by IEPD and found to be similar to authentic, native GrB. Binding to mesothelin was confirmed by surface plasmon resonance. Additionally, binding to the human lung adenocarcinoma cell lines H460 and HCC1703 was demonstrated by ELISA. Initial cytotoxicity (IC50) of SD1-GrB against H460 and HCC1703 was in the 30 nM range. Assessment against a larger panel of cell lines is ongoing followed by in vivo efficacy in a human tumor xenograft model. Our preliminary evaluation of SD1-GrB ADC suggests this construct has a unique mechanism of action and is highly cytotoxic against target cells. This molecule appears to be an excellent candidate for further pre-clinical development as a therapeutic agent. Research conducted, in part, by the Clayton Foundation for Research.

Citation Format: Lawrence H. Cheung, Rasheed Tijani, Nasir Khan, Claire Thuning-Roberson, Michael G. Rosenblum. A novel immuno-oncology approach: targeting cell-surface mesothelin with a fusion construct containing human granzyme B [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4597. doi:10.1158/1538-7445.AM2017-4597
Abstract 4598: Triggering of OX40 on T cells by a novel monoclonal antibody elicits robust antitumor immunity
Authors: Long Hua , White Ann , Wei Jie , Jiang Brittany , Feldman Reid , Pappas Danielle , Al-Shamkhani Aymen , Lin John
Published: July 2017

T cell costimulation is an attractive strategy for cancer treatment in addition to check point inhibitors. Costimulatory molecule OX40 is a member of the TNF receptor superfamily that is transiently expressed on activated T cells. Activation of OX40 leads to enhanced T cell proliferation and cytokine secretion and in turn results in better anti-tumor efficacy as shown by multiple mouse syngeneic tumor models. PF-04518600 (PF-8600) is a fully human IgG2 agonist antibody to human OX40 with high affinity and specificity. We used in vitro and in vivo methods to evaluate the co-stimulatory functions of PF-8600. PF-8600 demonstrated better agonist activity in a luciferase reporter cell line expressing OX40 and NFκB as compared to a human IgG1 antibody. In human primary T cells, PF-8600 dose dependently increased T cell proliferation and cytokine secretion in vitro. Furthermore in a mouse line expressing human OX40, PF-8600 in a mouse IgG1 framework increased the proliferation of OT1 cells and inhibited EG7 tumor growth as compared to isotype control antibody, further confirming the mechanism of action as an OX40 agonist. As regulatory T cells are a major inhibitory population in the tumor microenvironment, their specific depletion in the tumor is highly desirable. In an in vitro assay using monocyte derived macrophages as effector cells, PF-8600 mediated the depletion of OX40 expressing cells similar to a human IgG1 antibody, and this activity is dependent on the binding to hFcγRIIA. In human FcγR knock-in mice, anti-OX40-hIgG1 showed increased Treg reduction in the tumor comparing to anti-OX40-hIgG2, but less T cell proliferation and activation in the periphery. In combination with an anti-PD-L1 antibody, both anti-OX40-hIgG1 and hIgG2 showed similar enhanced anti-tumor activity in the MC38 colon carcinoma model as comparing to anti-PD-L1 alone. Both in vitro and in vivo results demonstrated PF-8600 enhanced T cells functions and inhibited tumor growth in a mouse syngeneic tumor model. The activity of the hIgG2 antibody is similar to that of an hIgG1 in both phagocytosis assays and hFcγR KI in vivo assays. Based on the mechanism of action and robust anti-tumor efficacy in preclinical models, PF-8600 is currently in clinical development in a broad spectrum of malignancies.

Citation Format: Hua Long, Ann White, Jie Wei, Brittany Jiang, Reid Feldman, Danielle Pappas, Aymen Al-Shamkhani, John Lin. Triggering of OX40 on T cells by a novel monoclonal antibody elicits robust antitumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4598. doi:10.1158/1538-7445.AM2017-4598
Abstract 4599: Antibody targeting PDZ domain of TIP-1 induces proliferation arrest through AKT/mTOR signaling inhibition in lung cancer and glioblastoma
Authors: Kapoor Vaishali , Dadey David , Hoye Kelly , Collins Andrea , Thotala Dinesh , Hallahan Dennis
Published: July 2017

Antigens that are over-expressed in cancer in response to radiation are being used as novel targets. We showed tax interacting protein 1 (TIP-1) to be radiation-inducible that translocated to the surface of the cancer cell following irradiation. TIP-1, which consists of a single PDZ domain plays an important role in cell signaling, cancer development, and progression. TIP-1’s involvement in various survival pathways makes it an attractive target for anticancer therapeutics. We used antibodies specific to this PDZ domain to determine its role in cancer cell survival. We monitored proliferation in lung cancer (A549 and H460) and glioblastoma (D54 and U251) cells after 24, 48, 72 and 96h treatment with the anti-PDZ antibody. We observed a time-dependent proliferation arrest with anti-PDZ antibody treatment which was associated with increased apoptosis. The anti-PDZ antibody when combined with radiation (3Gy) led to reduced proliferation and colony formation. Anti-PDZ antibody had no effect on the proliferation of normal lung (MRC-5) and endothelial (HUVEC) cells. Cells treated with anti-PDZ antibody showed decreased levels of the phosphorylated forms of AKT, mTOR, and a downstream substrate of mTOR, 4EBP1. Anti-PDZ antibody treatment also led to an overall reduction in basal levels of AKT, mTOR, and 4EBP1. Further, we evaluated the effect of the anti-PDZ antibody on tumor growth in heterotopic mouse models of lung cancer (A549) and glioma (U251). We observed significant growth delay in mice treated with anti-PDZ antibody treatment when compared to mice treated with the isotype control. The combination of the anti-PDZ antibody with radiation showed an additive effect. Immunoblot analysis of tumor tissues also showed downregulation of phosphorylated and total levels of AKT, mTOR and 4EBP1 in the tumors treated with anti-PDZ antibody. Overall, our results suggest that TIP-1 is a promising therapeutic target for treatment of lung cancer and glioblastoma. Antibodies specific to the PDZ domain of TIP-1 enhance the efficacy of radiotherapy. The anti-PDZ antibodies need to be optimized further before translating it into the clinic.

Citation Format: Vaishali Kapoor, David Dadey, Kelly Hoye, Andrea Collins, Dinesh Thotala, Dennis Hallahan. Antibody targeting PDZ domain of TIP-1 induces proliferation arrest through AKT/mTOR signaling inhibition in lung cancer and glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4599. doi:10.1158/1538-7445.AM2017-4599