bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4683: Distinct subsets of dysfunctional CD8+ T cells underlie response to checkpoint blockade
Authors: Miller Brian C. , Sen Debattama R. , Al-Abosy Rose , Bi Kevin , Yates Kathleen B. , Gjini Evisa , Felt Kristen , Manguso Robert T. , Rodig Scott J. , Sharpe Arlene H. , Haining Nicholas
Published: August 2018

T-cell exhaustion is a heterogeneous state, with recent work in chronic viral infections revealing at least two subtypes with different functional properties: "stem-like" and "terminal" exhausted cells. Whether these two populations exist in tumors and have different roles to control tumor growth remains unknown. We performed single-cell RNA-seq on exhausted CD8+ T cells from chronic virally infected mice, generating unique transcriptional signatures of both exhausted populations. By transcriptional enrichment and flow cytometry, we discovered both the stem-like and terminal exhausted populations in the B16 mouse melanoma model and confirmed their presence in human melanoma samples with multiplex immunofluorescence. ATAC-seq revealed that these two subpopulations are defined by state-specific changes in chromatin accessibility. These states are shared between exhausted CD8+ T cells from both viral and tumor models, demonstrating a common epigenetic program of T-cell dysfunction. The two populations have different functional properties, with the stem-like cells having a more polyfunctional cytokine response whereas terminal cells are more cytotoxic. When transferred into new tumor-bearing mice, stem-like cells were better able to control tumor growth, suggesting superior long-term functionality. Checkpoint blockade with anti-PD-1 is known to increase CD8+ T cell numbers in the tumor microenvironment, but it is not known which subpopulation responds to therapy. Treatment with anti-PD-1 antibody resulted in proliferation of the stem-like population, which differentiate into the more cytotoxic terminal exhausted cells. We have shown that two subpopulations of exhausted CD8+ T cells exist in mouse and human tumors, and anti-PD-1 therapy activates the stem-like CD8+ T cells to promote tumor control.

Citation Format: Brian C. Miller, Debattama R. Sen, Rose Al-Abosy, Kevin Bi, Kathleen B. Yates, Evisa Gjini, Kristen Felt, Robert T. Manguso, Scott J. Rodig, Arlene H. Sharpe, Nicholas Haining. Distinct subsets of dysfunctional CD8+ T cells underlie response to checkpoint blockade [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 4683.
Abstract 4684: NSD1 inactivation defines an immune cold, DNA hypomethylated subtype in squamous cell carcinoma
Authors: Brennan Kevin , Gevaert Olivier , Sunwoo John B. , Shin June Ho
Published: August 2018

Normal 0 false false false EN-US JA X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:Cambria; mso-ascii-font-family:Cambria; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Cambria; mso-hansi-theme-font:minor-latin;} Chromatin modifying enzymes are frequently mutated in cancer, resulting in widespread epigenetic deregulation. Recent reports indicate that inactivating mutations in the histone methyltransferase NSD1 define an intrinsic subtype of head and neck squamous cell carcinoma (HNSC) that features pronounced DNA hypomethylation. We have identified a similar hypomethylated subtype of lung squamous cell carcinoma (LUSC) that is enriched for both inactivating mutations and deletions in NSD1. We identified cancer DNA methylation subtypes by first applying the MethylMix algorithm to whole genome DNA methylation and gene expression data from The Cancer Genome Atlas to identify DNA methylation deregulated genes, followed by consensus clustering to patients based on their profiles of these deregulated genes. The ‘NSD1 subtypes' of HNSC and LUSC are highly correlated at the DNA methylation and gene expression levels, featuring ectopic expression of developmental transcription factors and genes that are also hypomethylated in Sotos syndrome, a congenital disorder caused by germline NSD1 mutations. Further, the NSD1 subtype of HNSC displays an ‘immune cold' phenotype characterized by low infiltration of tumor-associated leukocytes, particularly pro-inflammatory M1 macrophages and CD8+ T cells, as well as low expression of genes encoding the immunotherapy target PD-1 immune checkpoint receptor and its ligands. Using an in vivo model, we demonstrate that NSD1 inactivation results in reduced T cell infiltration into the tumor microenvironment, implicating NSD1 as a tumor cell-intrinsic driver of an immune cold phenotype. NSD1 inactivation therefore drives widespread oncogene expression across cancer sites, and has implications for immunotherapy.

Citation Format: Kevin Brennan, Olivier Gevaert, John B. Sunwoo, June Ho Shin. NSD1 inactivation defines an immune cold, DNA hypomethylated subtype in squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4684.
Abstract 4688: Single-cell discrimination of altered human T cell states in the bladder tumor microenvironment
Authors: Oh David Yoonsuk , Kwek Serena S. , Mangul Serghei , Raju Siddharth S. , Targ Sasha , Burra Arun , Chow Eric , Aran Dvir , Porten Sima , Meng Maxwell V. , Friedlander Terence W. , Ye Chun Jimmie , Fong Lawrence
Published: August 2018

Bladder cancer can be responsive to immunotherapies such as anti-PD-1 and anti-PD-L1 checkpoint inhibitors, but overall response rates are low. Tumor-resident T cells demonstrate considerable heterogeneity as far as antigenic repertoire and phenotype. Whether the bladder tumor environment is enriched for specific types of T cells with a particular functional profile and antigenic specificity, and whether these tumor-specific populations are associated with treatment or response to immunotherapy, remains unclear. We performed single-cell RNA sequencing of CD4+ and CD8+ T cells from localized human bladder tumors and paired adjacent non-malignant bladder. We also assessed resected bladder tumors treated with neoadjuvant chemotherapy or atezolizumab (anti-PD-L1) as part of an ongoing phase II trial. We find both known and unexpected CD4+ T cell functional populations that are specific to the tumor microenvironment, including regulatory T cells and a novel population of cytotoxic CD4+ T cells. Furthermore, by combining whole-transcriptome data with paired T cell receptor identification on single cells, we find that tumor-specific CD4+ populations are clonally expanded and utilize an antigenic repertoire which is distinct from uninvolved bladder. These findings provide evidence for specialization of both phenotype and antigenic specificity of CD4+ T cells in the bladder tumor environment, and indicate that a limited number of tumor-specific antigens may drive in situ expansion and differentiation of specialized CD4+ subsets whose function may be critical to tumor control.

Citation Format: David Yoonsuk Oh, Serena S. Kwek, Serghei Mangul, Siddharth S. Raju, Sasha Targ, Arun Burra, Eric Chow, Dvir Aran, Sima Porten, Maxwell V. Meng, Terence W. Friedlander, Chun Jimmie Ye, Lawrence Fong. Single-cell discrimination of altered human T cell states in the bladder tumor microenvironment [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 4688.
Abstract 4690: FoxP3+T cells program/re-program the prostatic tumor microenvironment
Authors: Kumar Sanjay , III James Stoke , Malik Shalie , Singh Udai P. , Ponnazhagan Selvarangan , Manne Upender , Mishra Manoj K.
Published: August 2018

Foxp3+ T cells play a critical role in maintaining immunological tolerance by regulating functions of other immune cells, thus preventing autoimmunity. Previous studies have demonstrated the importance of Foxp3+ T cells in the tumor, however, the role increasingly recruited FoxP3+ T cells in the programming of prostatic tumor microenvironment is not well illustrated. Therefore, in this study, we have investigated the role of Foxp3+ T cells in the prostatic tumor microenvironment with the notion that increased Foxp3+ T cells facilitate tumor growth. To accomplish this, we carried out tumor induction studies in C57/BL-6 mice using transgenic adenocarcinoma of mouse prostate (TRAMP cell lines: -C1, -C2 and -C3) as tumor models. Mice were administered with TRAMP (C1-3) cells using the Matrigel system. TRAMP-C3 cells are non-tumorigenic while TRAMP-C1 and TRAMP-C2 cells form tumors. Mice were sacrificed by cervical dislocation when tumor sizes reached ~18-20mm in diameter. To prepare single cell suspension, spleen and tumor were dissected out and cells were stained with various cell surface as well as intracellular immune markers to determine the number of Foxp3+ T cells and other immune cells in the prostatic tumor microenvironment. Our findings suggest that the high number of Foxp3+ T cells was found in the spleen and tumor of TRAMP-C1 and TRAMP-C2 mice than the spleen and tiny tumor of TRAMP-C3 mice. Furthermore, the number of cytotoxic T lymphocytes and NK cells were also observed to be low in the tumorigenic TRAMP-C1 and -C2 mice as compared to non-tumorigenic mice (TRAMP-C3). Therefore, our findings suggest that the number of Foxp3+ T cells may play a critical role in shaping the prostatic tumor microenvironment.

Citation Format: Sanjay Kumar, James Stoke III, Shalie Malik, Udai P. Singh, Selvarangan Ponnazhagan, Upender Manne, Manoj K. Mishra. FoxP3+T cells program/re-program the prostatic tumor microenvironment [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 4690.
Abstract 4699: Identification of novel potential biomarkers of response to sunitinib in glioblastoma
Authors: Noman Abu Shadat Mohammod , Abdulkarim Bassam , Sabri Siham
Published: August 2018

Introduction: Glioblastoma multiforme (GBM) is the most advanced and aggressive form of primary malignant brain tumors in adults. O6-Methylguanine methyltransferase (MGMT) is a DNA repair protein well known for its role in resistance to temozolomide used in standard treatment of patients newly diagnosed with GBM. Sunitinib is an oral multitargeting receptor tyrosine kinase (RTK) inhibitor with antiangiogenic and antiproliferative activities targeting several RTKs. Our group has previously shown the role of MGMT as a negative regulator of angiogenesis and invasion and the differential antiproliferative effect of sunitinib based on expression of MGMT in GBM. Recent gene expression profiling (GEP) studies showed that besides its effects on RTKs, sunitinib affects the expression level of other genes at the transcriptional level in different cancer types. We hypothesized that: (i) the mechanism of action of sunitinib may not be limited to direct inhibition of its known targets (RTKs) in GBM and (ii) expression of MGMT may affect response of GBM tumor cells to sunitinib. We performed GEP analysis to identify genes that could be potentially modulated in response to sunitinib in GBM cell lines isogenic for MGMT. Empty vector (EV)-transfected T98/EV cells with constitutive expression of MGMT and its knockdown counterpart T98/MGMT-shRNA were treated with sunitinib or vehicle control. Treatment of T98/EV and T98/MGMT-shRNA cell lines with sunitinib significantly changed the expression of over 2,000 gene transcripts (analysis performed at Genome Quebec Innovation center, McGill University). Lists of significant genes were fitted into previously known biochemical pathways, available among the canonical pathways from various databases provided by ConsensusPathDB (CPDB). Strikingly, immune system was among the top significantly enriched pathways (p-value<10-3), with some immune pathways not previously reported for response to sunitinib.

Conclusion: Our study opens new avenues to further understand the mechanisms of action of sunitinib, investigate its immunomodulatory effects, and identify biomarkers for selection of patients who might benefit from the combination of antiangiogenic and immunebased strategies in GBM.

Funding Acknowledgment: Cancer Research Society Operating Grant Competition #22716, and Higher Education Quality Enhancement Project (HEQEP)- Window -4 (Grant Id. CP-4023), University Grant Commission (UGC), Bangladesh.

Citation Format: Abu Shadat Mohammod Noman, Bassam Abdulkarim, Siham Sabri. Identification of novel potential biomarkers of response to sunitinib in glioblastoma [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 4699.
Abstract 47: Brain metastatic versus non-metastatic breast cancer exosomes influence astrocyte response
Authors: Sayyad Megan , Puchalapalli Madhavi , Hu Bin , Koblinski Jennifer
Published: August 2018

Brain metastasis is a devastating, late-stage event affecting 10-30% of breast cancer patients, but it is unclear how breast cancer cells can cross the blood-brain barrier (BBB) and colonize the unique brain environment. Exosomes, endosomal-derived extracellular vesicles, have been reported to support premetastatic niche formation through recruitment of tumor growth-supporting cells, creating sites conducive to cancer development. Still, it is not known how exosomes might prime the distinctive brain environment for metastasis despite the ability of circulating exosomes to readily cross the BBB. Glial cells, namely astrocytes and microglia, are important contributors to brain metastasis, and astrocytes in particular secrete exosomes that promote tumor outgrowth in the brain. We therefore investigated the effects of breast cancer exosomes on astrocytes in promoting brain premetastatic niche formation. Using ultracentrifugation, we isolated exosomes from 4T1 metastatic and 67NR nonmetastatic mouse mammary carcinoma and MDA-MB-231 human triple-negative breast cancer cell-conditioned media. Exosomes were characterized using Western immunoblot, dynamic light scatter analysis, and transmission electron microscopy, and labeled with a fluorescent lipid dye. Mouse and human astrocytes were then treated with either 4T1 or MDA-MB-231 exosomes, respectively, and vesicle internalization was assessed using confocal microscopy. Astrocytes were indeed found to internalize cancer exosomes in vitro. Using qPCR analysis, we explored gene expression changes in mouse astrocytes treated with 4T1 exosomes compared to those treated with 67NR exosome nonmetastatic controls. 4T1 exosomes were found to significantly upregulate expression of tenascin C, an astrocyte-derived brain extracellular matrix protein, suggesting the brain matrix could be remodeled prior to cancer cell arrival. Further, astrocytes treated with 4T1 exosomes exhibited significant upregulation of inflammation-associated genes, namely COX-2, IL-6, CCL4 and PDGF-alpha, genes also associated with cancer and metastasis, specifically to the brain. Alternatively, 67NR exosome treated-astrocytes exhibited significant downregulation in these genes of interest, suggesting that there is a unique response profile associated with nonmetastatic versus metastatic breast cancer-derived exosomes. Taken together, these findings suggest that breast cancer exosomes could drive transformation of the brain environment towards a prometastatic site.

Citation Format: Megan Sayyad, Madhavi Puchalapalli, Bin Hu, Jennifer Koblinski. Brain metastatic versus non-metastatic breast cancer exosomes influence astrocyte 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 47.
Abstract 4703: The inhibitor of apoptosis protein (IAP) antagonist Debio 1143 enhances the immune response to anti-PD1/L1 inhibitors in vitro and in vivo
Authors: Attinger Antoine , Gavillet Bruno , Chessex Anne Vaslin , Wiedemann Norbert , Vuagniaux Gregoire
Published: August 2018

Background: Debio 1143 is an oral antagonist of IAPs, currently in clinical development, which sensitizes tumor cells to radiation- or chemotherapy-induced apoptosis. IAPs inhibitors modulate NF-κB signaling and drive the expression of genes relevant for inflammation and immunity. Here, we hypothesized that Debio 1143 could improve antitumor immunity by directly enhancing T-lymphocyte activation and by improving the effects of immune checkpoint inhibitors in vitro and in vivo.

Methods: Ex vivo human PBMC anti-CD3/CD28 stimulation and modified mixed-lymphocyte reaction assays were performed to evaluate the immunostimulatory potential of Debio 1143 alone or in combination with the anti-PD-1 antibody nivolumab (N= 5 donors). T cells' proliferation and activation were measured by flow cytometry and cytokine release was measured by ELISA. The antitumor activity of an anti-PD-L1 antibody (5 mg/kg BIW IP) was tested either alone or in combination with Debio 1143 (100 mg/kg QD1-5 PO) in MBT-2 immunocompetent mouse model of bladder cancer over 3 weeks (n=8 /group).

Results: Debio 1143 significantly enhanced CD4+ and CD8+ intracellular IFNγ expression in a concentration-dependent manner following anti-CD3/CD28 stimulation. This result was confirmed using a mixed-lymphocyte reaction assay, where Debio 1143 at concentrations achieved in clinical studies significantly increased IFNγ expression by activated CD4+ cells, and this effect was even further increased in presence of nivolumab. In MBT-2 tumor-bearing mice, the combination of Debio 1143 and anti-PD-L1 antibody significantly decreased tumor growth (P=0.001 using two-sided t-test) and increased survival, whereas monotherapies only displayed moderate activities (median survival time of 42 days vs. 33 or 28 days for Debio 1143 or anti-PD-L1 alone, respectively).

Conclusion: These data show a key mechanistic role for future combination therapy of the IAP antagonist Debio 1143 and immune-checkpoint agents in cancer patients. This synergy will be further explored in a phase-Ib dose-finding clinical study combining Debio 1143 and Avelumab (anti-PD-L1) in patients with advanced solid malignancies and non-small cell lung cancer (CT# 03270176).

Citation Format: Antoine Attinger, Bruno Gavillet, Anne Vaslin Chessex, Norbert Wiedemann, Gregoire Vuagniaux. The inhibitor of apoptosis protein (IAP) antagonist Debio 1143 enhances the immune response to anti-PD1/L1 inhibitors in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4703.
Abstract 4711: Modulation of NFκ-B signaling to optimize antitumor characteristics in macrophages
Authors: Liu Esther , Hoover Alyssa A. , Harris Whitney R. , Barham Whitney , Tikhomirov Oleg , Khabele Dineo , Mirafzahli Zahra , Giorgio Todd D. , Wilson Andrew J. , Yull Fiona E.
Published: August 2018

While it is known that nuclear factor κ-B (NF-κB) signaling within macrophages regulates expression of downstream targets that can mediate either pro- or antitumor functions, the mechanisms that define predominant characteristics are not well understood. As macrophages are present in the tumor microenvironment of diverse tumor types, their characteristics have the potential to either support or hinder tumor growth and metastatic spread. Therefore, a method to shift the balance of tumor associated macrophages from pro- to antitumor functions could potentially target tumor cells directly and also recruit and activate other immune cells, resulting in significant therapeutic benefit. Our studies are designed to gain an understanding of how to use modulation of NF-κB signaling as the “pinch point” to generate antitumor phenotypes and determine how to achieve this modulation in a patient. We are using immortalized bone marrow-derived macrophages or ex vivo tumor-associated macrophages in cell culture approaches and inducible transgenic mice in in vivo approaches to investigate the mechanisms by which NF-κB defines macrophage characteristics. Interactions with tumor cells are also being investigated using co-culture and murine models. Our data suggests that high levels of NFκ-B activity in macrophages induce both direct tumor cell killing and immune stimulating responses. We are testing two different translational approaches. The first is liposomal muramyl tripeptide ethanolamine that acts as an immune stimulant by increasing NF-κB activity in macrophages. The second is preferential delivery of siRNA against the inhibitor of NFκ-B (IκB-alpha) to tumor-associated macrophages using polymeric nanoparticles. We hope to better understand the mechanisms by which NFκ-B regulates macrophage functions to inform development of a novel macrophage-based immunotherapy that could be effective across a wide spectrum of solid tumors and metastatic disease.

Citation Format: Esther Liu, Alyssa A. Hoover, Whitney R. Harris, Whitney Barham, Oleg Tikhomirov, Dineo Khabele, Zahra Mirafzahli, Todd D. Giorgio, Andrew J. Wilson, Fiona E. Yull. Modulation of NFκ-B signaling to optimize antitumor characteristics in macrophages [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 4711.
Abstract 4715: Mifepristone extends both length and quality of life in a patient with advanced non-small cell lung cancer that progressed despite chemotherapy and check-point inhibitors
Authors: Check Jerome H. , Check Diane , Poretta Trina
Published: August 2018

The US FDA approved an investigator initiated phase II 40 patient study to treat patients with stage IIIB or IV non-small cell lung cancer who have progressed despite a minimum of 2 rounds of chemo or immunotherapy with the progesterone receptor modulator mifepristone taken orally at 300mg/day (www.clinicaltrials.gov). The first patient in the study reported at the 2017 AACR meeting with stage IV lung cancer with brain metastasis has had non-progressive disease with excellent quality of life (ECOG-zero) after 24 months on mifepristone therapy. His lung cancer was PD-L1 negative, and thus he did not receive check-point inhibitors. We report, herein, the clinical outcome of the second patient with stage IV disease who not only progressed despite 3 chemotherapy regimens but also nivolumab (she was positive for the PD-L1 marker). The second patient in the study began 300mg/day oral mifepristone at age 68. Her stage IV lung cancer had progressed despite 4 cycles of carboplatin, permetrexed, and bevacizumab, 6 cycles of carboplatin and docetaxel, and 6 cycles of erlotinib. Finally she progressed and treatment was stopped after 11 cycles of nivolumab (PD-L1 marker present). After 1 year of oral mifepristone there have been no new metastatic lesions. She is ECOG 1 (related to chronic obstructive lung disease). Her cancer is stable with slight reduction in some metastatic lesions. More importantly she states she feels better than at any time since her initial diagnosis. The mechanism of action of mifepristone is hypothesized to be related to suppressing a key immunosuppressive protein that seems to be required for rapidly growing cells (e.g., possibly cancer stem cells), to proliferate known as the progesterone induced blocking factor (PIBF). This protein stablizes perforin granules in natural killer cells, thus obviating their mechanism for cytotoxicity. Mifepristone has been shown to suppress the intracytoplasmic immunosuppressive isoform in cancer cell line studies. Mifepristone can also diminish T-cell cytotoxicity. Progesterone receptor modulators may provide a new treatment option for advanced non-small cell lung cancer, even those positive for the PD-L1 marker that progresses despite treatment with chemotherapy and check-point inhibitors. Because intracytoplasmic PIBF is present in only mesenchymal, embryonic, trophoblast, or cancer cells suppressing this protein does not engender any health risks - oral mifepristone is well tolerated.

Citation Format: Jerome H. Check, Diane Check, Trina Poretta. Mifepristone extends both length and quality of life in a patient with advanced non-small cell lung cancer that progressed despite chemotherapy and check-point inhibitors [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 4715.
Abstract 4717: MAP kinase inhibition induces metabolic reprogramming in T-cells leading to induction of stem cell memory CD8 cells that enhance potency of adoptive cell therapy and anti-OX40 antibody
Authors: Verma Vivek , Ahmad Shamim , Zeng Peng , Mkrtichyan Mikayel , Barry Simon T. , Smith Paul D. , Rodriguez Paulo C. , Janik John E. , Gupta Seema , Khleif Samir N.
Published: August 2018

Adoptive T-cell therapy has demonstrated remarkable activity in refractory malignancies; however, the short survival of these cells in some patients results in treatment failure. CD8+ T-cells with self-renewable stem cell memory (TSCM) phenotype persist longer than the effector cells. Hence pharmacologic agents that maintain lymphocytes in TSCM phenotype may benefit the adoptive T cell-transfer strategies as well as combination immunotherapies. Small-molecule inhibitors of mitogen-activated protein kinase (MEKi) have been used for treatment of solid tumors. Here we report that inhibition of MEK enhances immune-driven antitumor response. We show that MEKi prevents the terminal differentiation and exhaustion of CD8+ T-cells in the tumor microenvironment. Interestingly, we further observed that MEKi leads to a significant increase in the CD62LhiCD44loSca1+ cells with low mitochondrial potential that are defined as TSCM CD8+ cells. Furthermore, ex vivo MEKi-treated CD8+ cells demonstrated a significantly higher antitumor activity in tumor-bearing mice. These MEKi treated CD8+ T-cells have higher mitochondrial respiration, which is fueled by fatty acid oxidation and exhibit significantly enhanced spare respiratory capacity (SRC). In addition, MEKi-treated cells have higher metabolic fitness, making them resistant to terminal exhaustion upon cell activation. Since OX40 co-stimulatory signals potentiate the self-renewal and homeostatic capacity of memory T-cells, we treated the tumor-bearing mice with MEKi in combination with OX40 agonistic antibody. Combining MEKi with OX40 agonist antibody significantly enhanced anti-OX40 antitumor effect. These data highlight the ability of pharmacologic MEK inhibition in inducing TSCM, which can be used ex vivo to enhance the potency of T-cell therapies and in vivo in combination immune therapy.

Citation Format: Vivek Verma, Shamim Ahmad, Peng Zeng, Mikayel Mkrtichyan, Simon T. Barry, Paul D. Smith, Paulo C. Rodriguez, John E. Janik, Seema Gupta, Samir N. Khleif. MAP kinase inhibition induces metabolic reprogramming in T-cells leading to induction of stem cell memory CD8 cells that enhance potency of adoptive cell therapy and anti-OX40 antibody [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 4717.
Abstract 4727: MicroRNA-155 as a regulator of NK cell mediated solid tumor rejection
Authors: Prasad Aman , Chan Wing Keung
Published: August 2018

Natural Killer (NK) cells are granular innate lymphocytes that orchestrate immune response by releasing interferon gamma (IFN-γ): a critical cytokine heavily involved in intercellular immune and inflammatory processes. Prior work from our lab has linked microRNA-155 (miR-155) to significant cell proliferation and NK cell mediated IFN-γ release. Previously, our lab has also shown that miR-155 can increase hematological malignancy rejection and NK cell cytotoxicity in T-cell lymphomas. However, it is unclear how miR-155 can affect NK cell cytotoxicity in solid tumor models. IFN-γ has been reported in the literature to directly induce apoptosis in glioblastoma solid tumors. We therefore hypothesize that miR-155 induction can increase NK cell mediated cytotoxicity and IFN-γ mediated apoptosis for solid tumor rejection. To test this, 10x106 GL261 cells were injected subcutaneously into the flanks of both wild type (WT) littermates and miR-155 transgenic (miR-155Tg) mice. CD3(-)NK1.1(+) effector splenic NK cells were isolated from WT and miR-155Tg mice for total RNA extraction and subsequent gene expression profiling on NK cell activation pathways. We found that tumor growth in WT mice was at least 2-fold greater than that of miR-155Tg mice (average 2cmx2cm vs 0.7cmx0.7cm, n=6). Tumor weights from WT and miR-155 mice were 1052 mg vs 340 mg, respectively, with an average of 2 mice per group. Furthermore, we have successfully created an IFN-γ receptor 1 (IFN-γR1) shRNA knock-down (KD) lentiviral construct in a stable glioblastoma cell line. IFN-γR1 knockdown was measured to be 90% as determined by real-time PCR analysis. Future experiments will be performed wherein IFN-γR1 KD glioblastoma cells will be injected in miR-155Tg and WT mice to evaluate tumor rejection. In summary, we demonstrate a model system to investigate the effect of miR-155 on solid tumor glioblastoma multiforme. Further characterization of gene expression profiles of purified NK cells from both WT and miR-155 mice will provide important insights on the mechanisms of NK cell medicated tumor rejection.

Citation Format: Aman Prasad, Wing Keung Chan. MicroRNA-155 as a regulator of NK cell mediated solid tumor rejection [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 4727.
Abstract 4732: Myeloid-derived suppressor cells potentiate colorectal carcinogenesis: Identification of a novel RIPK3-PGE2 circuit in tumor microenvironment
Authors: Li Yongsheng , Yan Guifang , Zhao Huakan , Zhang Qi
Published: August 2018

The role of receptor-interacting protein kinase 3 (RIPK3), a central factor mediating necroptosis, in tumor immunity is largely unknown. Here we show the significantly decreased RIPK3 in the tumor microenvironment of colorectal cancer (CRC) correlated with the accumulation of myeloid-derived suppressor cells (MDSCs). Deficiency of RIPK3 boosted the infiltration and immunosuppressive activity of MDSCs in vitro and in vivo. Mechanistically, the reduction of RIPK3 in MDSCs and colorectal cancer cells elicited NF-κB expression in the nuclei and the transcription of COX-2 which catalyses the synthesis of prostaglandin E2 (PGE2). PGE2 exacerbated the immunosuppressive activity of MDSCs, i.e. increased arginase 1 (Arg-1), blunted CD8+ T cell activation and accelerated tumor growth. Moreover, PGE2 inhibited RIPK3 expression while enhanced the expression of NF-κB and COX-2 in MDSCs and CRC cells. Inhibition of COX-2 or PGE2 receptors significantly reversed the immunosuppressive activity of MDSCs and dampened tumorigenesis. The patient databases also delineated the correlation of RIPK3 and COX-2 with CRC survival. Our findings demonstrate a novel signaling circuit by which RIPK3 and PGE2 regulate tumor immunity, and provide potential targets for the immunotherapy of CRC.

Citation Format: Yongsheng Li, Guifang Yan, Huakan Zhao, Qi Zhang. Myeloid-derived suppressor cells potentiate colorectal carcinogenesis: Identification of a novel RIPK3-PGE2 circuit in tumor microenvironment [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 4732.
Abstract 4743: Inhibition of the Hif1a-mediated checkpoint refuels NK activation in cancer
Authors: Ni Jing , Bühler Lea , Stojanovic Ana , Arnold Annette , Sexl Veronika , Cerwenka Adelheid
Published: August 2018

Solid tumors remain a major challenge for immunotherapy of cancer due to their immunosuppressive microenvironment. Hypoxia is a common feature of solid tumors and might affect immune cell activation within the tumor tissue. Upon hypoxia the transcription factor Hypoxia inducible factor 1a (HIF-1α) that affects many cellular processes including cell proliferation, survival and effector function becomes induced. Its impact on innate immune cells such as Natural Killer (NK) cells, however, is still incompletely understood. Natural Killer (NK) cells are considered as highly potent effector cells against tumors. Within the tumor tissue, however, NK effector function is often impaired. Our microarray analyses of tumor-infiltrated NK cells revealed upregulated levels of Hif1a. To determine the role of the HIF-1α in NK cell function, we generated the mouse strain Hif1aΔ/ΔNcr1-iCreTg in which HIF-1α is deficient in the NKp46+ lineage. Hif1a deficient NK cells showed greatly enhanced anti-tumor activity compared to wild-type NK cells, leading to a pronounced reduction of tumor growth. Tumor-infiltrated Hif1a deficient NK cells expressed lower levels of checkpoint molecules and show enhanced responses to tumor cells indicating a less exhausted phenotype. Hif1a deficient NK cells preferentially utilize oxidative phosphorylation (OXPHOS) than glycolysis to fuel their energy resulting in enhanced IFN-γ production in the hypoxic environment. In addition, using HIF-1α inhibitors we were able to greatly enhance human NK cell anti-tumor activity. Accordingly, our data reveal HIF-1α as a negative metabolic checkpoint for NK cells. Moreover, inhibition of HIF-1α holds high promise to refuel NK cell activation in cancer with a high potential for improving current strategies of NK cell-based cancer immunotherapy.

Citation Format: Jing Ni, Lea Bühler, Ana Stojanovic, Annette Arnold, Veronika Sexl, Adelheid Cerwenka. Inhibition of the Hif1a-mediated checkpoint refuels NK activation in 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 4743.
Abstract 475: Characterization of miR-21 expression in budding colon cancer cells using digital images of multiplex fluorescence stained slides obtained by confocal scanning microscopy
Authors: Nielsen Boye Schnack , Knudsen Kirsten Nguyen , Lindebjerg Jan , Kalmár Alexandra , Molnár Béla , Sorensen Flemming , Hansen Torben
Published: August 2018

This study was undertaken to analyze the presence and prevalence of microRNA-21 (miR-21) positive budding cancer cells in colon cancer using confocal scanning microscopy. Budding cancer cells are locally invasive cancer cells with increased metastatic properties and characteristics of epithelial to mesenchymal transition (EMT). Expression of miR-21 in stromal fibroblastic cells in colorectal cancers is well documented (1,2), whereas miR-21 expression in budding cells is poorly described. In order to characterize miR-21 positive budding cells, we first developed a multiplex fluorescence staining method by combining miR-21 in situ hybridization with immunohistochemical staining for cytokeratin and budding cell marker laminin-γ2, and stained 16 colon cancer cases (stage II, n=5, stage III, n=11). We then employed a confocal scanning microscope to obtain 15-55 GB digital images covering areas of 10-40 mm2 of the invasive front. The high resolution of the confocal digital images allowed detailed examination of the 4-fluorophore-stained slides using extended focus and z-stack images, e.g. in the discrimination of epithelial cells from adjacent stromal cells. All cytokeratin-positive budding cells were evaluated for the presence of miR-21 and laminin-γ2. Five out of the 16 cases had more than 10% miR-21 positive budding cells, a few of which were also laminin-γ2 positive. All five cases were stage III cancers. The presence of miR-21 in the budding cancer cells was not associated with the level of tumor budding. These observations suggest that the miR-21 expression in tumor budding cells increases with the progression of the cancer and is independent of laminin-γ2. References: 1. Nielsen BS et al, Clin Exp Mets 2011, 28:27-38. 2. Kjaer-Frifeldt S et al, Br J Cancer 2012, 107:1169-1174.

Citation Format: Boye Schnack Nielsen, Kirsten Nguyen Knudsen, Jan Lindebjerg, Alexandra Kalmár, Béla Molnár, Flemming Sorensen, Torben Hansen. Characterization of miR-21 expression in budding colon cancer cells using digital images of multiplex fluorescence stained slides obtained by confocal scanning microscopy [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 475.
Abstract 4776: Antitumor activity of pan-HER inhibitors in HER2-positive gastric cancer
Authors: Yoshioka Takahiro , Shien Kazuhiko , Takahashi Yuta , Kurihara Eisuke , Namba Kei , Ogoshi Yusuke , Torigoe Hidejiro , Sato Hiroki , Yamamoto Hiromasa , Soh Junichi , Fujiwara Toshiyoshi , Toyooka Shinichi
Published: August 2018

Purpose: Molecularly targeted therapy has enabled outstanding advances in cancer treatment. Whereas various anti-HER2 drugs have been developed, trastuzumab is still the only anti-HER2 drug presently available for gastric cancer. Among HER2 targeting drug, afatinib and neratinib inhibit the activation of all HER family protein, and are called pan-HER inhibitors. In this study, we examined the effect of these pan-HER inhibitors to gastric cancer cells. Materials and Methods: We determined the molecular profiles of 12 gastric cancer cell lines. Protein level of HER2 and down-signal pathway molecules were analyzed by Western blotting, and copy number assay or gene expression assay were performed using qPCR. To detect HER2 mutation, we also performed direct-sequence of these cell lines. And next, we examined the antitumor effect of the pan-HER inhibitors afatinib and neratinib in those cell lines, in vitro and in vivo. In addition, we analyzed HER2 alteration in 123 primary gastric cancers resected from Japanese patients to clarify possible candidates with the potential to respond to these drugs. Results: HER2 was amplified in 5 out of 12 gastric cancer cell lines. Gene expression or protein level of HER2 were generally correlated with the copy number of HER2. HER2 mutation was found in one cell line, ECC10, at kinase domain (L755S). In the drug sensitivity analysis, both afatinib and neratinib showed an anti-tumor effect in all the HER2 amplified cell lines both in vitro and in vivo except MKN7 cell line. When the molecular profiles of the cells were compared based on the drug sensitivities, we found that cancer cells with lower mRNA expression levels of IGFBP7, a tumor suppressor gene by inhibiting the activation of IGF-1R, were less sensitive to pan-HER inhibitors. A combination therapy consisting of pan-HER inhibitors and an IGF-1R inhibitor, picropodophyllin, demonstrated a notable synergistic effect. Regarding HER2 alteration in 123 clinical samples, we found 19 cases of HER2 amplification and 3 cases of oncogenic mutations. Conclusion: Afatinib and neratinib are promising therapeutic options for the treatment of HER2-amplified gastric cancer. In addition to HER2 amplification, IGFBP7 might be a biomarker of sensitivity to these drugs, and IGF-1R-targeting therapy can overcome drug insensitiveness in HER2-amplified gastric cancer.

Citation Format: Takahiro Yoshioka, Kazuhiko Shien, Yuta Takahashi, Eisuke Kurihara, Kei Namba, Yusuke Ogoshi, Hidejiro Torigoe, Hiroki Sato, Hiromasa Yamamoto, Junichi Soh, Toshiyoshi Fujiwara, Shinichi Toyooka. Antitumor activity of pan-HER inhibitors in HER2-positive gastric 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 4776.
Abstract 4777: Antitumor effect of neratinib in lung cancers harboring HER2 oncogene alterations
Authors: Ogoshi Yusuke , Toyooka shinichi , Soh Jyunichi , Yamamoto hiromasa , Shien Kazuhiko , torigoe Hidejiro , sato Hiroki , Yoshioka Takahiro , Namba Kei , Takahashi Yuta , Kurihara Eisuke
Published: August 2018

Background The HER2-targeted therapy for non-small cell lung cancer (NSCLC) harboring HER2 oncogene alterations is one of promising strategy to improve the clinical outcome of treatment for NSCLC. In this study, we investigated the antitumor effect of neratinib, an irreversible pan-HER tyrosine kinase inhibitor (TKI) in NSCLC cells harboring HER2 alterations including mutation and amplification.

Materials and Methods We examined the sensitivity of neratinib against normal bronchial epithelial cells BEAS-2B which ectopically overexpressing wild-type or mutant HER2. Furthermore, we examined the antitumor activity of neratinib in addition to afatinib in several NSCLC cell lines harboring HER2 or EGFR alterations in in vitro and in vivo experiments, and investigated the association between their genetic alterations and sensitivity to neratinib treatment.

Results BEAS-2B cells ectopically overexpressing wild-type HER2 or mutants (A775insYVMA, G776VC, G776LC, P780insGSP, V659E, G660D, and S310F) showed constitutive autophosphorylation of HER2 and activation of downstream signaling by Western blotting. These BEAS-2B cells were sensitive to neratinib, but insensitive to erlotinib, a first generation EGFR-TKI. Neratinib also showed antiproliferative effects on both HER2-altered (H2170, Calu-3, and H1781) and EGFR-mutant (HCC827, PC9, and HCC4006) NSCLC cell lines. Neratinib administration showed strong antitumor effect on tumor growth in mouse xenograft model using HER2-altered lung cancer cell lines.

Conclusions Our study strongly suggests that neratinib is a promising therapeutic option for the treatment of HER2-altered NSCLC.

Citation Format: Yusuke Ogoshi, shinichi Toyooka, Jyunichi Soh, hiromasa Yamamoto, Kazuhiko Shien, Hidejiro torigoe, Hiroki sato, Takahiro Yoshioka, Kei Namba, Yuta Takahashi, Eisuke Kurihara. Antitumor effect of neratinib in lung cancers harboring HER2 oncogene alterations [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 4777.
Abstract 4778: Assessing efficacy of neratinib in HER2-driven lung cancer
Authors: Li Shuai , Liu Shengwu , Chen Ting , Deng Jiehui , Wu Min , Kuraguchi Mari , Tran Cam Anh , Kirschmeier Paul T. , Avogadri-Connors Francesca , Cutler Richard E. , Lalani Alshad S. , Wong Kwok-Kin
Published: August 2018

HER2 mutations have been classified as oncogenic drivers that are present in 2-4% of lung adenocarcinomas. However, due to limited preclinical studies and clinical trials, there is still no available standard of care for this group of lung cancer patients. To fulfill the clinical need for targeting HER2 mutations in lung cancer patients, we performed a comprehensive preclinical study to evaluate the efficacy of neratinib, an irreversible small-molecule pan-HER tyrosine kinase inhibitor that potently binds and inhibits EGFR, HER2, and HER4. We used the previously described genetically modified mouse model (GEMM) of lung adenocarcinomas driven by the major mutant form of human HER2 exon 20 insertions (A775_G776insYVMA) for testing neratinib either as a single agent or in combination with other therapies including trastuzumab in vivo. Treatment efficacy was evaluated based on both monitoring tumor volume changes and survival. Magnetic resonance imaging (MRI) was used for quantifying tumor burden. Tolerability and the overall health of animals were evaluated based on body weight change. We found that neratinib treatment alone provided a modest yet significant response in inhibiting HER2 mutant lung tumors in this model. Pharmacodynamic studies showed that neratinib effectively abolished HER2 phosphorylation and inhibited major downstream signaling targets such as pAKT and pERK. Moreover, combination therapy with neratinib and trastuzumab showed a greater treatment effect by reducing tumor burden with significantly longer progression-free survival and overall survival compared to single agents tested. In all neratinib-based regimens, body weight of treated animals remained stable. On this basis, our study provides a strong rationale to test the combination of neratinib with trastuzumab for the treatment of HER2 mutant NSCLC patients.

Citation Format: Shuai Li, Shengwu Liu, Ting Chen, Jiehui Deng, Min Wu, Mari Kuraguchi, Cam Anh Tran, Paul T. Kirschmeier, Francesca Avogadri-Connors, Richard E. Cutler, Alshad S. Lalani, Kwok-Kin Wong. Assessing efficacy of neratinib in HER2-driven lung 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 4778.
Abstract 4782: Inhibition of FGFR in breast cancer metastasis
Authors: Lin Hang , Akhand Saeed S. , Wendt Michael K.
Published: August 2018

Metastatic breast cancer is the most advanced stage of breast cancer. This type of cancer can spread other organs and cause 90% of cancer-related death. However, little is known about the molecular mechanisms which drive metastasis in breast cancer. Epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) promote tumor invasion and metastasis. Previously, it has been reported that fibroblast growth receptor 1 plays a key role during EMT: MET cycle. Therefore, optimizing FGFR inhibitors is crucial for therapeutic targeting of late-stage breast cancer. Here, we examined the efficacies of three FGFR kinase inhibitors, AZD4547, JNJ-42756493 and FIIN4, in the 4TO7 cell line both in vitro and in vivo. The 4TO7 cell line is a syngeneic model of systemic dormancy. Mammary fat pad engraftment of the 4T07 cells onto immune competent Balb/C mice results in systemic dissemination, but no macroscopic metastasis. In contrast, the 4T07 cells can escape cellular dormancy in 3D culture and form robust pulmonary tumors in mice upon tail vein inoculation. Furthermore, the 4TO7 cell line robustly responses to exogenous stimulation with FGF2. The phosphorylation of Erk1/2 downstream of FGFR was stimulated after a five-minute treatment with FGF2 and was effectively blocked with all the inhibitors tested. Furthermore, we developed a 3D culture approach that combines tumor spheroid formation in a non-adherent round bottom dish followed by placement on a bed of matrix. Our in vitro results demonstrate that both JNJ-42756493 and FIIN4 have more potent anti-proliferative activities in 3D culture and longer residence time as compared to AZD4547. In vivo, inhibition of FGFR is highly effective against 4TO7 tumors in the pulmonary microenvironment. Importantly, FIIN4 showed the least toxicity. This result suggested that covalent inhibition of FGFR is a promising cancer therapy for patients with metastatic breast cancer.

Citation Format: Hang Lin, Saeed S. Akhand, Michael K. Wendt. Inhibition of FGFR in breast cancer metastasis [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 4782.