bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract LB-173: Epitope mapping of mTERT for vaccine development
Authors: Song Yurong , Marshall Jason , Das Sudipto , Andresson Thorkell , Corbel Amanda , Sei Shizuko , Shoemaker Robert H. , Pinto Ligia
Published: August 2018

Telomerase reverse transcriptase (TERT) is one of two essential components of telomerase, an enzyme complex that generates and maintains telomeres. Telomerase is expressed mainly in embryonic and adult stem cells. Telomere biology has recently been implicated in the pathogenesis of a variety of diseases, and mutations in telomerase components result in a predisposition to solid malignancies. More recent findings show that TERT is not only expressed in late stage primary tumor cells and metastatic cells, but also expressed in incipient cancer stem cells and/or tumor-initiating cells, indicating that TERT has essential roles at every stage of tumorigenesis. However, the clinical benefit of a TERT (hTERT)-targeting vaccine, given as a single agent or in combination with chemotherapeutic agents, has been limited in patients with advanced cancer, possibly due to insufficient epitope coverage as well as tumor-induced immune suppression. Thus, targeting TERT in tumor-initiating cells in early stage lesions may be more effective in preventing cancer development and progression. To develop cancer preventive TERT vaccines, novel immunogenic epitopes must be first identified and evaluated in a relevant preclinical model of tumorigenesis. The goal of this project is to identify the epitopes of mouse TERT (mTERT) with high immunogenicity via epitope mapping using two approaches. The first one consists in washing off the MHC I-bound mTERT peptides (Mild Acid Elution; MAE) from the cell surface of mTERT-overexpressing cells (SPON-10-mTERT cells generated from spontaneous lung tumors developed in an A/J mouse), followed by mass spectrometry analysis. The initial mass spec analysis showed one 14-mer peptide from MAE with 22 PSMs, with a good XCorr score compared to peptide from PBS elution control with only 1 PSMs, with lower XCorr score. The second approach is to inject overlapping mTERT peptides covering the whole protein sequence in mice, and then splenocytes from these mice will be stimulated in vitro using mTERT peptide library to determine immunogenic peptides. Both approaches are in progress to identify potential mTERT epitopes.

Funded by NCI Contract No. HHSN261200800001E

Citation Format: Yurong Song, Jason Marshall, Sudipto Das, Thorkell Andresson, Amanda Corbel, Shizuko Sei, Robert H. Shoemaker, Ligia Pinto. Epitope mapping of mTERT for vaccine development [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 LB-173.
Abstract LB-175: The malignancy associated change hypothesis tested through 3D cellular imaging
Authors: Meyer Michael , Kelbauskas Laimonas , Katdare Rahul , Presley Chris , Hamilton Celeste , Sussman Daniel , Bell Tim , Nelson Alan
Published: August 2018

Early detection remains by far the most reliable and potent strategy in curing lung cancer. Many current approaches, however, are limited by poor sensitivity or specificity that increase health care costs and potentially risk patient health through unneeded and invasive procedures.

The association between cell morphology and cancer has been established in the cytology literature. However, the tumor field effect potentially introduces subtle changes into non-tumor cells that reside in the tumor microenvironment. This phenomenon has given rise to the malignancy associated change (MAC) hypothesis.

The Cell-CT™ platform and LuCED® test represent a promising new method for detecting lung cancer with high (92%) sensitivity and (95%) specificity that is based on alterations in cellular morphology.

We present a study of 3D morphological alterations in non-tumor cells obtained from sputum of healthy subjects and biopsy confirmed lung cancer patients. Three major cell types were analyzed from 235 patients: bronchial epithelial columnar, squamous intermediate, and mature macrophages (dust cells). We used the Cell-CT platform to measure over 700 different structural biosignatures for each cell. A classifier to test the degree to which the features discriminate between cells from normal or cancer patients was developed and its performance characterized by the area under the ROC curve (aROC) (Table 1). Hierarchical clustering analysis revealed biosignatures of nuclear chromatin organization to be the most differentiating between macrophages from healthy subjects and patients with cancer.

Table 1. Classifier development and performance characteristics

Cell TypeNumber of cells from cancer patientsNumber of cells from normal patientsSupervised Learning – aROCSquamous Intermediate4,6849530.70Macrophages7,2191,3660.75Columnar cells11,7742,9110.80

Our results confirm the MAC hypothesis based on 3D cellular imaging and suggest that cancer presence in the lung can be detected in patient's sputum based on the morphologic alterations in non-tumor cells. This is important as cancer cells are rare in early stages of cancer, posing challenges in terms of detection sensitivity and specificity.

Further work will emphasize boosting of the performance figures above based on the combination of results from the differing methods presented.

Citation Format: Michael Meyer, Laimonas Kelbauskas, Rahul Katdare, Chris Presley, Celeste Hamilton, Daniel Sussman, Tim Bell, Alan Nelson. The malignancy associated change hypothesis tested through 3D cellular imaging [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 LB-175.
Abstract LB-186: Evaluation of EGFR degradation as a mechanism of tumor evasion in EGFR-TKI resistant non-small cell lung cancer (NSCLC) cells compared to EGFR-TKI sensitive cells
Authors: Norma Hernandez-Pedro Y. , Giovanny Soca-Chafre , Mario Orozco-Morales , Pedro Barrios-Bernal , Oscar Arrieta
Published: August 2018

The resistance mechanisms to EGFR-TKIs in Non-Small Cell Lung Cancer (NSCLC) are not well elucidated. Here we evaluate EGFR degradation as a mechanism of tumor evasion in EGFR-TKI resistant NSCLC cells (H1975) compared to EGFR-TKI sensitive cells (HCC827). After erlotinib treatment, EGFR protein half-life was quantified by cycloheximide chase. EGFR expression on cell membrane was measured by flow cytometry and transcription levels were measured by Real-Time PCR. EGFR half-life was shorter in H1975 compared to HCC827 cells after cycloheximide inhibition of protein synthesis. EGFR phosphorylation levels were not affected by erlotinib in H1975 while in HCC827 they were significantly reduced (p < 0.001). EGFR expression on cell membrane decreased similarly in both H1975 and HCC827 cell lines. Also the transcription levels of EGFR remained unaffected by erlotinib treatment in both cell lines. However, there was more apoptosis in HCC827 compared to H1975 with 64% vs. 22%. Our results suggest that H1975 cells present more EGFR degradation however, they show less cell death by apoptosis and this could represent a resistance mechanism to EGFR-TKIs.

Citation Format: Hernandez-Pedro Y. Norma, Soca-Chafre Giovanny, Orozco-Morales Mario, Barrios-Bernal Pedro, Arrieta Oscar. Evaluation of EGFR degradation as a mechanism of tumor evasion in EGFR-TKI resistant non-small cell lung cancer (NSCLC) cells compared to EGFR-TKI sensitive cells [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 LB-186.
Abstract LB-188: POH1 knockdown induces cancer cell apoptosis via p53 and Bim
Authors: Chen Shi-Lu
Published: August 2018

The ubiquitin-proteasome system is implicated in cell apoptosis that is frequently dysregulated in human cancers. POH1/rpn11/PSMD14, as a part of the 19S proteasomal subunit, contributes to the progression of malignancy, but its role in apoptosis remains unclear. Here, we showed that POH1 expression was increased and associated with poor outcomes in three independent cohorts of patients with hepatocellular carcinoma (HCC), esophageal cancer (EC), and colorectal cancer (CRC). The knockdown of POH1 significantly inhibited tumor cell proliferation and induced apoptosis mediated by the mitochondrial pathway in vitro. Intratumoral injection of POH1 small interfering RNA (siRNA) significantly reduced the progression of tumor growth and induced apoptosis in vivo. Furthermore, p53 or Bim siRNA markedly attenuated the apoptosis induced by POH1 depletion. POH1 depletion resulted in cell apoptosis by increasing the stability of p53 and Bim and inhibiting their ubiquitination. Overall, POH1 knockdown induced cell apoptosis through increased expression of p53 and Bim via enhanced protein stability and attenuated degradation. Thus, POH1 may serve as a potential prognostic marker and therapeutic target in human cancers.

Citation Format: Shi-Lu Chen. POH1 knockdown induces cancer cell apoptosis via p53 and Bim [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 LB-188.
Abstract LB-193: TRAIL promotes entosis through Caspase-8
Authors: Bozkurt Emir , Dussmann Heiko , Prehn Jochen H.
Published: August 2018

Despite its apoptosis-inducing activity, tumor necrosis factor-related apoptosis inducing ligand (TRAIL) also activates survival signaling pathways. Survival signaling not only interferes with apoptosis and causes resistance, but can also lead to gain of several tumor-promoting functions such as proliferation, invasion and metastasis. Here we demonstrate that colorectal cancer cells surviving from TRAIL treatment can initiate a cell-in-cell invasion process known as entosis. Entosis refers to a unique cell-in-cell phenomenon, in which one or more viable cells are actively internalized into another cell of the same type. After formation of cell-in-cell structures (CICs), internalized cells can divide inside the host cell or they can escape, however, the vast majority of cells undergo cell death inside their hosts, through a lysosome-dependent mechanism. Although the clinical relevance of entosis is highly context-dependent, the frequency of entosis has been shown associated with tumor grade and poor outcome in several malignancies. While monitoring single-cell response upon TRAIL stimulation, we noted that some cells internalized into their neighboring cells and died inside a vacuole. We confirmed CICs by fluorescence staining of cytoplasm, nucleus, lysosome and cell membrane, in addition to TEM. Further quantification revealed that TRAIL indeed increased the number of CICs in colorectal cancer cell lines (HCT116 and LS180) and spheroids. Moreover, both pre-treatment with ROCK inhibitor (Y-27632) and knockout of Caspase-8 resulted in a significant reduction in the number of TRAIL-induced CICs. After forming cell-in-cell structures, the majority of internalized cells appeared positive for lysotracker staining and underwent cell death inside their hosts. These cells showed an increase in IETD FRET probe cleavage, additionally, we observed increased levels of cleaved caspase-8, cleaved caspase-9 and cleaved caspase-3 in these cells in presence of TRAIL. Pre-treatment with z-VAD-fmk reduced the number of internalized cells undergoing cell death and Bax/Bak double knockout cells escaped from their hosts without undergoing cell death in response to TRAIL. Our findings demonstrate that entosis can be induced by TRAIL in a caspase-8-dependent manner, and may give new insights in understanding the death, survival and escape mechanisms in internalized cells.

Citation Format: Emir Bozkurt, Heiko Dussmann, Jochen H. Prehn. TRAIL promotes entosis through Caspase-8 [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 LB-193.
Abstract LB-200: A pan-cancer analysis of stage-dependent gene expression program
Authors: Kim Man-Sun , Kim Dongsan , Kim Jeong-Rae , Lim Yong Pyo
Published: August 2018

Cancer cells undergo diverse gene expression alterations according to their development and progression due to the accumulation of mutations. Understanding these phenomena not only provide us an insight into the underlying mechanisms of cancer development, but also offer useful information on the prediction of patient's prognosis. For some cancer types, previous studies attempted to identify key genes that change with the development of cancer. However, identification of cancer stage-dependent gene expression dysregulations based on pan-cancer dataset have not been addressed. Here, based on gene expression profiles and clinical data from 20 major cancer types and ~6,000 individual patients, we identified both gene expression programs that specifically change from each cancer-type and across all cancer types. Based on the pan-cancer analysis, we found that cell cycle and p53 dependent pathways are the most commonly dysregulated pathways across all cancer types according to cancer development. Next, we investigated whether the identified the stage-dependent gene expression programs from the pan-cancer analysis also hold from each cancer-type analysis. Interestingly, we identified that only about half cancer types also showed consistent gene expression alterations. These results suggest that although most cancer share common dysregulation mechanisms as cancer progress, also show that some cancer type have quite unique cancer development programs depending on the cancer type.

Keywords: stage-dependent gene expression, systems biology, PANCANCER

Citation Format: Man-Sun Kim, Dongsan Kim, Jeong-Rae Kim, Yong Pyo Lim. A pan-cancer analysis of stage-dependent gene expression program [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 LB-200.
Abstract LB-202: Role of the FOXA transcription factors in shaping distinct transcriptional programs in human pancreatic cancers
Authors: Milan Marta , Natoli Gioacchino
Published: August 2018

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest types of cancer and is characterized by a high intra-tumor heterogeneity, with the coexistence of well- and poorly-differentiated cells in virtually all the tumor cases. FOXA1/2 play a fundamental role in pancreas development and are believed to act as pioneer factors, namely to create accessible chromatin by displacing nucleosomes early in development and making genomic regulatory elements available to other transcriptional regulators. Therefore, we asked whether FOXA1/2 also control differentiation in human PDAC. Whereas FOXA1 showed a remarkable propensity to be expressed selectively by well-differentiated tumor areas and cell lines, FOXA2 showed a broad profile of expression throughout PDAC stages. Through the combination of genomic and functional assays we found that FOXA2 contributed to activate distinct transcriptional programs in tumor cells of different grades. Such complexity in FOXA2 activity specifically depended on its interaction with distinct transcription factors selectively expressed in high- or low-grade tumor cells. While in low-grade (well-differentiated) PDAC cells FOXA2 binding specificity relied on the interaction with the epithelial transcription factor HNF1beta, in high-grade cells it depended on the cooperation with HOXB8 and HOXB9. Knocking out these transcription factors was sufficient to abolish differential recruitment of FOXA2 to grade specific genomic sites. In summary, we found that FOXA2 is broadly express throughout PDAC stages and, through the interaction with different transcription factors, shapes the transcriptional program of the different tumor grades in pancreatic cancer. These findings help to shed light on the complexity of PDAC heterogeneity and on the circuitries that maintain PDAC differentiation and grading.

Citation Format: Marta Milan, Gioacchino Natoli. Role of the FOXA transcription factors in shaping distinct transcriptional programs in human pancreatic cancers [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 LB-202.
Abstract LB-207: A novel role for polycomb group protein BMI1 in prostate cancer
Authors: Zhu Sen , Zhang Dongyu , Yan Lin , Jiang Weihua , Liu Qipeng , Wang Rui , Xia Bo , Zhao Jonathan C. , Wang Rong-Fu , Shi Xiaobing , Lam Hung-ming , Dong Xuesen , Yu Jindan , Chen Kaifu , Cao Qi
Published: August 2018

BMI1, a Polycomb Group (PcG) protein, plays a critical role in epigenetic regulation of cancer stem cell self-renewal, cell differentiation and proliferation. BMI1 is upregulated in multiple types of cancer, including prostate cancer. As a key component of Polycomb Repressive Complex 1 (PRC1), BMI1 can exert its oncogenic functions by enhancing the enzymatic activities of RING1B to ubiquitinate histone H2A at lysine 119 and repress gene transcription. Here we report a PRC1-independent role of BMI1 that is critical for castrate-resistant prostate cancer (CRPC) progression. BMI1 binds the androgen receptor (AR) and prevents MDM2-mediated AR protein degradation, resulting in sustained AR signaling in prostate cancer cells. More importantly, we demonstrate that targeting BMI1 effectively inhibits tumor growth of xenografts that have developed resistance to surgical castration and enzalutamide treatment. These results suggest that blocking BMI1 alone or in combination with anti-AR therapy can be more efficient to suppress prostate tumor growth.

Citation Format: Sen Zhu, Dongyu Zhang, Lin Yan, Weihua Jiang, Qipeng Liu, Rui Wang, Bo Xia, Jonathan C. Zhao, Rong-Fu Wang, Xiaobing Shi, Hung-ming Lam, Xuesen Dong, Jindan Yu, Kaifu Chen, Qi Cao. A novel role for polycomb group protein BMI1 in prostate 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 LB-207.
Abstract LB-218: Pathologist reproducibility of combined positive score (CPS) for the evaluation of PD-L1 in gastric or gastroesophageal junction adenocarcinoma tumors
Authors: Guerrero Lindsay , Kulangara Karina , Truong Yen , Schwefel Sue , Hanks Debra
Published: August 2018

Background: The clinically relevant and highly reproducible combined positive score (CPS) algorithm is used in conjunction with PD-L1 IHC 22C3 pharmDx for evaluation of programmed death ligand 1 (PD-L1) expression in gastric or gastroesophageal junction (GEJ) adenocarcinoma specimens. PD-L1 IHC 22C3 pharmDx and the CPS algorithm were approved by the United States Food and Drug Administration (FDA) on September 22, 2017 to aid in identifying patients likely to respond to pembrolizumab.

Objective: Assessment of pathologist reproducibility in the gastric and GEJ adenocarcinoma Early Validation Program (EVP).

Methods: Immunohistochemical (IHC) staining for PD-L1 was performed on thirty gastric or GEJ adenocarcinoma surgical resection specimens with PD-L1 IHC 22C3 pharmDx. All specimens were evaluated by in-house pathologists and assigned a reference CPS. Inter-pathologist and intra-pathologist reproducibility was assessed in ten pathologists from five clinical labs. Acceptance criteria was based on a minimum of 85% agreement with the reference CPS.

Results: Nine pathologists met acceptance criteria for inter-observer reproducibility (87-100% agreement) and intra-observer reproducibility (87-100% agreement). One pathologist had 90% inter-pathologist agreement and 80% intra-pathologist agreement. This pathologist received additional training and achieved 87% intra-pathologist agreement upon re-testing. Upon completion of EVP, all ten participating pathologists were certified to score gastric or GEJ adenocarcinoma specimens stained with PD-L1 IHC 22C3 pharmDx. Inter-pathologist (87.6% overall agreement) and intra-pathologist (93% overall agreement) studies performed in analytical validation demonstrate similar reproducibility.

Conclusion: The CPS algorithm is highly reproducible within and among pathologists for gastric or GEJ adenocarcinoma specimens stained with PD-L1 IHC 22C3 pharmDx, as demonstrated in analytical validation.

Citation Format: Lindsay Guerrero, Karina Kulangara, Yen Truong, Sue Schwefel, Debra Hanks. Pathologist reproducibility of combined positive score (CPS) for the evaluation of PD-L1 in gastric or gastroesophageal junction adenocarcinoma tumors [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 LB-218.
Abstract LB-254: Combined inhibition of MEK and autophagy promotes regression of pancreatic cancer
Authors: Kinsey Conan , Guillen Katrin , Camolotto Soledad , Boespflug Amelie , Shea Jill , Seipp Michael , Scaife Courtney , McMahon Martin
Published: August 2018

Pancreatic ductal adenocarcinoma (PDAC) is a recalcitrant disease responsible for ~43,000 deaths in the USA in 2017. Despite an advanced understanding of the genetics, biochemistry and biology of pancreatic cancer, there is no effective pathway-targeted therapy for PDAC such that the standard of care treatment for most patients remains conventional cytotoxic chemotherapy. Although the clinical picture remains grim, progress has been made in understanding how alterations in tumor suppressors and proto-oncogenes contribute to PDAC initiation and progression. Whereas initiating mutations in KRAS promote the growth of premalignant pancreatic intra-epithelial neoplasia, progression to cancer malignancy requires cooperating alterations of tumor suppressors such as TP53, CDKN2A, and/or SMAD4. Downstream of KRAS oncoproteins, the RAF→MEK→ERK MAP kinase signaling pathway plays a central role in the genesis and maintenance of PDAC. However, to date, pharmacological inhibition of this pathway has demonstrated little clinical benefit in PDAC patients. Here we show that inhibition of KRAS→RAF→MEK→ERK signaling in PDAC cell lines elicits autophagy, a process of cellular self-consumption, which protects pancreatic cancer cells from the potentially cytotoxic effects of pathway inhibition. Mechanistically, inhibition of MEK1/2 leads to activation of ULK1 through the LKB1-AMPK-ULK1 axis. Combined inhibition of MEK1/2 and autophagy (with chloroquine) displays synergistic anti-proliferative effects against PDAC cell lines in vitro. Most strikingly, whereas single agent therapy had modest effects, combined treatment of xenografted patient-derived PDAC tumors with trametinib plus chloroquine/hydroxychloroquine elicited striking tumor regression. Finally, the observed anti-tumor effects of combination trametinib plus chloroquine were observed against a NRAS mutated melanoma and a BRAF mutated colorectal patient-derived xenografts. These data may warrant testing this combination therapy in patients with KRAS mutated pancreatic cancers, a disease for which new pathway-targeted therapies are urgently required.

Citation Format: Conan Kinsey, Katrin Guillen, Soledad Camolotto, Amelie Boespflug, Jill Shea, Michael Seipp, Courtney Scaife, Martin McMahon. Combined inhibition of MEK and autophagy promotes regression of pancreatic 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 LB-254.
Abstract LB-276: Application of a CRISPR/Cas9 screen to identify novel therapeutic strategies with CDK4/6 inhibitors in pancreatic cancer
Authors: Goodwin Craig , Javaid Sehrish , Cox Adrienne D. , Wood Kris , Der Channing
Published: August 2018

While mutational activation of KRAS occurs in nearly all pancreatic ductal adenocarcinomas (PDAC), clinically relevant agents directly targeting KRAS remain elusive. The second most frequent aberration in PDAC is loss of the tumor suppressor CDKN2A, which encodes INK4A, an endogenous inhibitor of the CDK4 and CDK6 cell cycle regulatory proteins that promote G1 cell cycle progression. KRAS and INK4A mutations cooperate to accelerate PDAC metastatic development, in part, by converging to block RB tumor suppressor activity. While no anti-KRAS targeted therapies have reached the clinic, pharmacologic restoration of CDKN2A function by inhibition of CDK4/6 may be an effective anti-KRAS therapeutic strategy. We found that CDK4/6 inhibitors (e.g., palbociclib) elicited single-agent activity in a subset of tested PDAC cell lines. Furthermore, concurrent treatment with the ERK inhibitor SCH772984 caused a potent synergistic reduction in anchorage-dependent and -independent cell growth, induced apoptosis, and increased senescence and G1 arrest. We next sought to identify novel genes that regulate sensitivity to palbociclib. Using a CRISPR/Cas9 loss-of-function screen, we individually silenced expression of 2500 genes from the “druggable genome” in combination with palbociclib. We identified genes that caused synergistic growth suppression in combination, suggesting small molecule combinations to overcome de novo or acquired CDK4/6 inhibitor resistance in the clinic. We also identified genes whose loss imparts a survival advantage, suggesting possible resistance mechanisms to single-agent CDK4/6 inhibition. In total, these data suggest that CDK4/6 inhibitors alone, or in combination, may benefit patients clinically.

Citation Format: Craig Goodwin, Sehrish Javaid, Adrienne D. Cox, Kris Wood, Channing Der. Application of a CRISPR/Cas9 screen to identify novel therapeutic strategies with CDK4/6 inhibitors in pancreatic 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 LB-276.
Abstract LB-278: In vivo evaluation model of human T cell activation by immune checkpoint inhibitors using human PBMC-transferred NOG mouse
Authors: Nishime Chiyoko , Katano Ikumi , Nishinaka Eiko , Kawai Kenji , Ito Ryoji , Hata Jun-ichi , Yamamoto Taichi
Published: August 2018

Within the past decade, immune checkpoint inhibitors, such as anti-programmed death (PD)-1 antibodies (e.g. pembrolizumab; Keytruda and Nivolumab: OPDIVO), treatment has become one of the most advancing therapeutic options in oncology. The PD-1/PD-L1 pathway plays a crucial role in mediating self tolerance and controlling self damage to suppress effector/self-reactive T cells and protects against autoimmune reactions. Therapeutic treatment of anti-PD-1 antibodies clearly suppresses tumor growth and induces T cell activation in cancer patients by blockade of the PD-1/PD-L1 pathway, but over activation of T cells exhibits immune-related adverse effects with exacerbation of autoimmune reactions. A human peripheral blood mononuclear cells (hPBMC) transferred humanized NOG mouse has been used to evaluate the efficacy and safety of therapeutic antibodies due to the efficient engraftment of human T cells in the mice. The human T cells were spontaneously activated in the mice because of xenogenic-reaction and the mice frequently died of graft-versus-host disease (GVHD) (Ito R et al., Transplantation, 2009). In the present study, we evaluate the potential adverse effects of 2 therapeutic antibodies, Keytruda and OPDIVO by using conventional NOG mice transferred with hPBMC. These mice showed a weight loss, wasting, rough fur, and skin thickening suggesting of GVHD symptoms. These changes exacerbated in either antibody treated hPBMC-NOG mice with increase of activation marker for T cells, and they died earlier than saline-treated group. By immune-cytoloical analysis, human T cells was significantly decreased in number in hPBMC-NOG mice even though the antibodies

were treated. These results demonstrated the hPBMC-NOG mice might become powerful preclinical tools to analyze human T cells activated by therapeutic immune checkpoint inhibitors.

Citation Format: Chiyoko Nishime, Ikumi Katano, Eiko Nishinaka, Kenji Kawai, Ryoji Ito, Jun-ichi Hata, Taichi Yamamoto. In vivo evaluation model of human T cell activation by immune checkpoint inhibitors using human PBMC-transferred NOG mouse [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 LB-278.
Abstract LB-279: Efficacy of fractionated injections of CLR 131 in a OPM-2 SCID nude mouse model
Authors: Longcor Jarrod , Banach Maria , Friend John
Published: August 2018

Background: Multiple myeloma remains an incurable disease. Treatment with immunomodulators and proteasome inhibitors have provided significant benefits to patients but they still relapse. The introduction of new treatments like daratumumab have provided further benefit. However, patients continue to experience issues and relapse. CLR 131 is a novel radioiodinated therapeutic that exploits the selective uptake and retention of phospholipid ethers (PLEs) by malignant cells to provide targeted delivery of iodine-131 directly to tumor cells. This study evaluates the effect of fractionated injections of CLR 131 in OPM-2 tumor bearing mice in comparison to equivalent doses of CLR 131 and the standard dosing of bortezomid.

Methods: The OPM-2 cell line (human multiple myeloma) was purchased from American Type Culture Collection (ATCC, Rockville, MD) and maintained in McCoy's 5a media supplemented with 10% fetal bovine serum. Female CB17 SCID mice approximately 5-7 weeks of age were injected subcutaneously with 1x107 viable cells (in ~100 µL Dulbecco's PBS) into the right flank. The study was initiated when tumor size had reached a pre-determined size (approximately 150-200 mm3). The mice were given potassium iodide at a concentration of 0.1% in their drinking water to block possible free iodide in the drug formulation three days prior to injection and continuing through two weeks post-injection.

Results: All treatment groups showed a reduction in tumor growth with the fractionated dose of CLR 131 producing the greatest inhibition. The control group showed exponential growth throughout the study, increasing in volume by an average 11 fold from baseline. This compared with a 3.5 fold increase from baseline in the fractionated dose group and 7.5 fold for the bortezomid group.

Conclusions: The results of the study indicated that a fractioned injection of CLR 131 (2 injections of ~50 µCi) on human multiple myeloma (OPM-2) tumor bearing model showed a significant inhibition of tumor growth as well as a survival benefit. While a single injection of CLR 131 at either ~50 uCi and ~100 uCi were effective, there was an important improvement with by using fractionated dosing. The implication is that in this clinically predictive in vivo model CLR 131, which is currently in a Phase 2 clinical trial for multiple myeloma, might show improved clinical efficacy in patients if a fractionated dose is utilized.

Citation Format: Jarrod Longcor, Maria Banach, John Friend. Efficacy of fractionated injections of CLR 131 in a OPM-2 SCID nude mouse model [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 LB-279.
Abstract LB-282: Dynamic regulation of CD73 expression in cancer associated fibroblasts enforces immunosuppression via a feedforward adenosinergic pathway
Authors: Yu Miao , Guo Gang , Huang Lei , Deng Libin , Bollag Roni , Mellor Andrew , Shi Huidong , Cui Yan
Published: August 2018

CD73 is the major extracellular adenosine-generating ecto-5'-nucleotidase (Nt5e) that metabolizes extracellular (e) AMP and generates immunosuppressive adenosine (eADO). Elevated CD73 expression is often exploited by tumors and other cellular constituents of the tumor microenvironment (TME) as a mechanism of immune evasion. Clinically, a high level of CD73 expression in the tumor microenvironment (TME) is linked to poor prognosis. Experimental evidence also demonstrates that host CD73 activity in both hematopoietic and non-hematopoietic compartments augments the suppressive milieu of the TME. Nevertheless, the identities of CD73-expressing non-hematopoietic cells and factors that regulate CD73 activity of these cells have yet to be clearly defined. Our recent results unravel that cancer associated fibroblasts (CAFs) constitute a crucial non-hematopoietic CD73hi compartment in the TME of human colorectal cancer (CRC) and murine MC38 CRC and EG7 lymphoma ectopic models. Strikingly, the CD73 expression profile among cohorts of published human CRC and breast cancer patient data sets confirmed its significant positive association with that of CAF-related markers, both of which are negatively correlated with poor survival. Cellular and transcriptomic analyses of CAFs in the EG7 TME reveal that CD73 expression in CAFs increases progressively during tumor progression, associated with elevated tissue hypoxia. Mechanistically, we demonstrate that the progressive elevation of CD73 expression in CAFs during tumor progression is caused by elevated eADO in the TME, which is resulted by the hypoxia/necrosis-associated eATP release. Furthermore, this eADO/adenosingeric pathway-induced CD73 upregulation in CAFs is mostly mediated by activation of A2BR and downstream MAPK signaling, which exacerbates eADO accumulation and serves as a feedforward loop of re-enforcing the immunosuppressive TME. Our study demonstrated that clinically and experimentally, high level of CD73 expression in CAFs represents another crucial immune checkpoint in the TME. Targeted inactivation or blockade of A2BR signaling may serve as a new strategy to prevent or alleviate the initiation of the eADO-CD73-associated amplification of immunosuppression in the TME, thereby improving the outcome of immunotherapy.

Citation Format: Miao Yu, Gang Guo, Lei Huang, Libin Deng, Roni Bollag, Andrew Mellor, Huidong Shi, Yan Cui. Dynamic regulation of CD73 expression in cancer associated fibroblasts enforces immunosuppression via a feedforward adenosinergic pathway [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 LB-282.
Abstract LB-284: Engineering a hypermutant mouse model of NSCLC to modulate immune response
Authors: Smith Olivia C. , Westcott Peter M. , Canner David A. , Rideout William M. , Bhutkar Arjun , Beytagh Mary C. , Tammela Tuomas , Jacks Tyler E.
Published: August 2018

Immune checkpoint inhibitors are an exciting new class of cancer therapy with unprecedented response rates, and have shed light on the importance of understanding the immune context of tumors. Despite achieving durable clinical responses for a subset of patients, immune checkpoint therapies produce variable response rates, even within tumor type, suggesting a need for improved understanding of the tumor-immune cell axis. Recent clinical trials using checkpoint blockade against CTLA-4 and PD-1 have underscored the relevance of tumor mutational landscapes in predicting these response rates. For example, non-small cell lung cancer (NSCLC) patients with high mutational burden and PD-L1 expression respond better to anti-PD-1 treatment than those with high PD-L1 expression alone. While genetically engineered mouse models (GEMMs) of cancer are powerful platforms for testing new therapies, existing models fail to recapitulate the mutational burden and signatures of human cancers, limiting their ability to model relevant immune responses. We have therefore used CRISPR-Cas9 to knockout the DNA mismatch repair (MMR) gene Msh2 in the widely-used Kras and P53 mutant (KP) GEMM of NSCLC. Specifically, lentivirus expressing a short guide RNA (sgRNA) targeting Msh2, and Cre-recombinase, is delivered intra-tracheally into KP mice carrying a Cas9 allele. Whole-exome sequencing reveals elevated mutational burdens in Msh2KO tumors, as compared to non-targeted controls, and mutational signatures characteristic of MMR deficient human tumors. Histological characterization indicates elevated T cell infiltration of Msh2KO tumors at early time points, while tumors at later stages show minimal infiltration and appear like immune “cold” human tumors. Our future efforts will test checkpoint blockade therapies combined with immunogenic chemotherapy to facilitate neo-antigen presentation in late stage tumors. Development and characterization of this “hypermutant” model should prove valuable for future preclinical development of immunotherapies in NSCLC.

Citation Format: Olivia C. Smith, Peter M. Westcott, David A. Canner, William M. Rideout, Arjun Bhutkar, Mary C. Beytagh, Tuomas Tammela, Tyler E. Jacks. Engineering a hypermutant mouse model of NSCLC to modulate immune response [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 LB-284.
Abstract LB-293: IL-2 diphtheria toxin fusion protein with anti-PD1: in mouse B16-F10 melanoma model
Authors: Fu Juan , Pardoll Drew , Cheung Laurene
Published: August 2018

In tumor immunotherapy, regulatory T lymphocytes (Tregs) play an important role in the suppression of beneficial anti-tumor responses. In the non-immunogenic B16-F10 mouse melanoma model, tumors are found to be highly infiltrated by Tregs. DAB389IL-2 (Ontak, denileukin diftitox) is an IL-2 diphtheria toxin-related fusion protein that has been showed to deplete Tregs in both humans and mice. We treated mice with established B16F10 tumors with s-DAB389IL-2 (produced as a secreted protein from C. diphtheria) and observed a significant decrease in tumor growth rate in C57BL/6 mice. Also, s-DAB389IL-2 treatment increased the frequency of CD8+IFNγ+ T cells (TILs) in tumors and spleens. S-DAB389IL-2 treatment followed by PD-1 blockade resulted in greater tumor growth inhibition than either monotherapy alone. These data suggest that s-DAB389IL-2 plus anti-PD1 treatment can improve anti-tumor responses in non-immunogenic B16F10 melanoma and inhibit tumor growth. Additionally, we mutated a vascular leak inducing motif to construct s-DAB389IL-2 (V6A), which cause less vascular leak in vitro. S-DAB389IL-2 (V6A) inhibited tumor growth with similar efficacy to the original fusion toxin. As vascular leak is the major adverse effect caused by Ontak, DAB389IL-2 (V6A) shows promise as a better tolerated drug for further studies as a cancer immunotherapy.

Citation Format: Juan Fu, Drew Pardoll, Laurene Cheung. IL-2 diphtheria toxin fusion protein with anti-PD1: in mouse B16-F10 melanoma model [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 LB-293.
Abstract LB-302: Oncolytic poliovirus-mediated inflammation and immunity in breast cancer
Authors: Landa Karenia , Holl Eda , Brown Michael , Herndon James , Grimm Lars , Force Jeremy , Bigner Darell , Hwang Shelley , Gromeier Matthias , Nair Smita
Published: August 2018

Breast cancer is the most common malignancy among women in the United States and the second leading cause of cancer deaths in women. Disease prognosis, local recurrence rates and response to therapy are all related to receptor status of the individual cancer. Triple negative breast cancer (TNBC; ER-, PR-, and HER2-negative) is a subset of breast cancer with poor prognosis and high rate of local recurrence and metastasis with limited therapeutic options.

The oncolytic poliovirus, PVSRIPO, is a recombinant polio:rhinovirus chimera that has been engineered to eliminate neuropathogenicity and has demonstrated promising responses in patients with recurrent glioblastoma. PVSRIPO is oncolytic in tumor cells and enters cells via its receptor CD155, which is overexpressed in solid malignancies, including TNBC. Treatment of primary TNBC tissue with PVSRIPO induces IFN-I and pro-inflammatory cytokines. In a murine immunotherapy model, intratumoral injection of PVSRIPO induces IFN-I, IL12 and IFN-γ and rapid recruitment of neutrophils into the tumor followed by infiltration of DCs, T cells and B cells. Treatment of orthotopic breast tumors with PVSRIPO results in delayed tumor growth and increased survival in an immunocompetent murine immunotherapy model.

We have designed a pilot study to examine the effects of intratumoral injection of PVSRIPO into six women with TNBC with at least 1 cm of residual tumor after chemotherapy. The primary exploratory objective is to describe the changes in the amount of tumor infiltrating immune cells in tumor tissue pre- and post-injection of PVSRIPO. We will also collect blood at different time points during PVSRIPO treatment to evaluate inflammatory and immune signatures. We hypothesize that intratumoral administration of PVSRIPO will cause inflammation thus stimulating innate and adaptive immune activation.

This study is funded by the Department of Defense Breast Cancer Research Program award W81XWH-16-1-0354 (PI, Smita Nair).

Citation Format: Karenia Landa, Eda Holl, Michael Brown, James Herndon, Lars Grimm, Jeremy Force, Darell Bigner, Shelley Hwang, Matthias Gromeier, Smita Nair. Oncolytic poliovirus-mediated inflammation and immunity in 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 LB-302.
Abstract LB-362: A TGF-beta linked ECM-dysregulation programme in CAFs drives immune evasion and immunotherapy resistance
Authors: Chakravarthy Ankur Ravinarayana , Khan Lubaba , Bensler Nathan P. , Bose Pinaki , Carvalho Daniel Diniz De
Published: August 2018

Background

One of the key hallmarks of cancer is metastasis, which involves extensive extracellular matrix remodelling to permit extravasation from the primary tumour site before colonisation of distant sites. In this study, we defined the landscape of ECM-associated transcriptional dysregulation and performed in-silico associative analyses in terms of multiple -omics platforms, cellular composition and immunological parameters.

Methods

We defined the landscape of ECM gene dysregulation by integrating curated Gene Ontology ECM genes with genes differentially expressed between cancer and normal tissues from TCGA. ssGSEA scores for this set were then correlated with purity (ABSOLUTE), tested for stromal association using microdissected tumours and MethylCIBERSORT, and neoepitope counts derived using Topiary. Single-cell RNAseq data was used to validate CAF-origin. Linear models were used to identify transcriptomic, proteomic and genomic correlates, and finally, RNAseq data from three cohorts of PD1-blockade treated patients and 0.632 bootstrap evaluation of logistic regression or Random Forest models based on ECM genes, Cytolytic Activity, Mutational burden or their combinations was performed.

Results

A core set of 58 ECM genes is dysregulated and significantly enriched amongst genes differentially expressed between cancer and normal tissues pan-cancer. Those upregulated in cancers were found to be a negative prognostic factor pan-cancer and through integrative analysis of microdissected samples, deconvolution and single-cell RNAseq, were found to originate in Cancer Associated Fibroblasts. Transcriptome, proteome and genomics analyses implicated TGF-beta as a driver of this ECM-dysregulation signature and most notably, this programme was associated with heavily-mutated, neoantigen-high, CD8 high cancers, M1-macrophage-skewed tumours suggesting a role as a putative adaptation permitting immune evasion. Indeed, ECM dysregulation scores were significantly better predictors of response to PD1-blockade in pretreatment biopsies compared to cytolytic activity, an interferon gene signature, and mutational-burden, while TGF-beta expression alone was not, implicating a key role for CAF-specific ECM gene dysregulation in immune evasion.

Conclusion

A TGF-beta associated, CAF-derived signature of ECM-gene dysregulation broadly in operation across cancer types constitutes an immune evasion mechanism that indicates refractoriness to PD1-blockade.

Citation Format: Ankur Ravinarayana Chakravarthy, Lubaba Khan, Nathan P. Bensler, Pinaki Bose, Daniel Diniz De Carvalho. A TGF-beta linked ECM-dysregulation programme in CAFs drives immune evasion and immunotherapy resistance [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 LB-362.
Abstract LB-367: [18F]FAZA PET imaging reveals precise pharmacodynamics in vivo of the novel chemotherapeutic IACS-010759
Authors: Gammon Seth T. , Pisaneschi Federica , Bandi Madhavi , Smith Melinda , Rao Yi , Nanda Vashisht G. Yennu , Sun Yuting , Davies Michael , Francesco Emilia Di , Marszalek Joseph , Piwnica-Worms David
Published: August 2018

Genetic deletions and mutations resulting in defects in glycolysis, force these tumors to depend on oxidative phosphorylation (OxPhos) for growth. IACS-010759 is a nanomolar inhibitor of Complex I, and in phase I clinical trials at MD Anderson for both AML and solid tumors. While ex vivo monitoring of inhibition of oxygen consumption in leukocytes was sufficient for AML, non-invasive methods of monitoring target engagement in solid tumors was desired. Tumors, particularly those that rely on oxidative phosphorylation, yield hypoxic and reducing environments. These conditions are ideal for trapping 2-nitroimidazole based imaging agents, such as F18-labeled fluoroazomycin arabinoside ([18F]FAZA). IACS-010759 inhibited oxygen consumption in vitro with IC50 values ranging from 1.3 nM to 6 nM across a variety of cell lines with a diversity of glycolysis defects. In H460 NSLC, SKMEL5 melanoma, A375R melanoma, and D423-Fluc orthotopic GBM in vivo, at the MTD 10 mg/kg, a robust, up to 6 fold, (2 way ANOVA, p<0.0001) decrease in FAZA T/B ratio was observed. A slight, 1.5-fold, but detectable increase in [18F]FDG was also observed at 10mg/kg in A375 and A375R cells (p=0.002), but smaller than the decrease [18F]FAZA retention. Thus neither cell death nor loss of perfusion could explain the reduction in [18F]FAZA retention. To further test the robustness of the mechanism of inhibition, the converse experiment was conducted. Both 2,4-dinitrophenol and pyruvate were utilized to stimulate oxygen consumption in vivo, and as predicted [18F]FAZA retention increases (p<0.05 in both cases). Finally, [18F]FAZA retention by PET yielded the same IC50 for IACS-010759 as by IHC in a dose-dependent manner, with an apparent IC50 of 1.4 mg/kg (95%CI 0.48 to 4.1 mg/kg, n=12 mice) for A375R melanoma tumors. The PET measurement was more precise than an independently scored IHC metric based upon staining for pimonidazole from the same mice. [18F]FAZA can be a powerful PD marker for the complex-I inhibitor IACS-010759 in preclinical models, and is translatable to upcoming clinical trials in patients.

Citation Format: Seth T. Gammon, Federica Pisaneschi, Madhavi Bandi, Melinda Smith, Yi Rao, Vashisht G. Yennu Nanda, Yuting Sun, Michael Davies, Emilia Di Francesco, Joseph Marszalek, David Piwnica-Worms. [18F]FAZA PET imaging reveals precise pharmacodynamics in vivo of the novel chemotherapeutic IACS-010759 [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 LB-367.