bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1117: Acquired resistance to ATP-competitive and irreversible FGFR inhibitors (FGFRi's): A library-based approach
Authors: Sootome Hiroshi , Kato Suzuko , Kato Masanori , Hirai Hiroshi
Published: July 2021

Background: The emergence of secondary FGFR kinase domain (KD) mutations with FGFRi's leads to drug resistance and disease progression. The frequency of resistance mutations with individual FGFRi's is unknown. Using an unbiased library-based approach, we examined the development of acquired resistance with the irreversible FGFRi futibatinib and the ATP-competitive FGFRi's erdafitinib (ERD) and pemigatinib (PEM).

Methods: Mutant (mt) libraries were generated with random mutagenesis in two FGFR2 KD regions: #1 (amino acids [aa] 540-569), and #2 (aa 640-669). Library were transfected as TEL-fusions into Ba/F3 cells, resulting in FGFR2-dependent cell growth. Transfected cells were treated with FGFRi's at concentrations suppressing parental cell line growth. Surviving drug-resistant clones were counted, isolated, and sequenced to identify the KD mutation. The inhibitory activity of FGFRi's against resistant clones was assessed using phospho-FGFR2 ELISA.

Results: Treatment of the library-transfected Ba/F3-TEL-FGFR2 KD cells with PEM, ERD, and futibatinib resulted in an average (n=2) of 93, 27, and 13 resistant clones in region #1 and 28, 21 and 1 resistant in region #2, respectively. Sequencing (table) identified, among others, the following aa mutations: N549, E565, and K641 (regulatory triad), V564 (gatekeeper region) and K659 (activation loop), also seen in patients who developed resistance to Debio1347, infigratinib, and PEM. Among FGFR mts shown (table), futibatinib inhibited 8/10, and ERD, 5/10 mts at IC50 values comparable to wild type (wt) FGFR; however, PEM activity against all FGFR mts was attenuated by ≥5-fold vs wt.

Conclusions: Resistance mutations identified with this unbiased library-based in vitro approach were consistent with those observed in the clinic. Acquired drug resistance mutations were less frequent with futibatinib than with ATP-competitive FGFRi's. Futibatinib demonstrated the most robust inhibition of drug resistant mts.

FGFR mutationKD regionFutibatinib IC50 (nM)Erdafitinib IC50 (nM)Pemigatinib IC50 (nM)Wild-type (parent)-2.12.51.5N549DRegulatory triad4.225.5153.2N549KRegulatory triad16.732.0246.2V562L-6.32.97.7V564IGatekeeper8.32.762.4V564LGatekeeper91.956.5502.3E565ARegulatory triad6.82.911.8E565GRegulatory triad3.93.09.0K641IRegulatory triad3.855.829.6K641RRegulatory triad4.73.210.3K659MActivation loop9.546.633.8

Citation Format: Hiroshi Sootome, Suzuko Kato, Masanori Kato, Hiroshi Hirai. Acquired resistance to ATP-competitive and irreversible FGFR inhibitors (FGFRi's): A library-based approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1117.
Abstract 1229: Novel Arf1-targeting ã-dipeptides counteract triple negative breast cancer by inducing autophagic death
Authors: He Leilei , Chavanieu Alain , Vo-Hoang Yen , Teng Yong
Published: July 2021

ADP-ribosylation factor 1 (Arf1) is a crucial regulator in vesicle-mediated membrane trafficking and involved in the activation of signaling molecules. However, the molecular mechanisms underlying Arf1-dependent cancer development and progression are far from understood. We report here that Arf1 is the most amplified gene in Arf gene family in breast cancer, and knocking it down leads to an induction of autophagy via the AMPK pathway. To effectively target Arf1, we rationally designed and synthesized constrained ATC-based (4-amino-(methyl)-1,3-thiazole-5-carboxylic acid) γ-dipeptides, which can block Arf1 activation more efficiently than other commercially available Arf1 inhibitors through targeting the Arf1-GDP/ARNO complex at the Golgi. The anticancer effects of these γ-dipeptides were evaluated in triple native breast cancer MDA-MB-231 cells and their bone-seeking and lung-seeking sublines, showing reduced cell viability along with significant increased autophagy following treatment in both 2D and 3D cell cultures. Among these new inhibitors, the γ-dipeptide 10b displayed strongest cytotoxicity. The orthotopic NSC mouse model for breast cancer further showed the γ-dipeptides 10b exhibited a superior anticancer effects by inducing autophagic cell death in breast tumor, with no significant systemic toxicity. These findings reveal that Arf1-targeting γ-dipeptides developed by our collaborative team may represent a promising targeted therapeutic to improve treatment of breast cancer.

Citation Format: Leilei He, Alain Chavanieu, Yen Vo-Hoang, Yong Teng. Novel Arf1-targeting ã-dipeptides counteract triple negative breast cancer by inducing autophagic death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1229.
Abstract 126: HUB Organoids: bringing the 'patient in the lab' for preclinical and clinical development
Authors: Mullenders Jasper , Buijs Annemarie , Fielmich Lars-Eric , Sluimer Jasper , Sun Jane , Vries Robert G. , Boj Sylvia F.
Published: July 2021

Preclinical disease models are essential for the development of novel therapies and help better understand the molecular processes. In recent years, the groundbreaking technology to generate patient specific in vitro cultures based on adult stem-cell (ASC) organoids, has gained widespread interest for the development of new therapies and as a predictive diagnostic tool. ASC-derived organoids can be generated at high efficiency from both healthy and diseased tissue (resections as well as biopsies) including most carcinomas. These patient-derived cultures can be expanded for prolonged periods of time while maintaining many properties of the tissue they were derived from.

At Hubrecht Organoid Technology (HUB), we have created comprehensive ‘organoid living biobanks' from tumors of different origin like colon, lung, breast, pancreas and ovary. These biobanks have been characterized at the genetic and transcriptional level. These analyses have shown that our ‘organoid living biobanks' capture disease heterogeneity at the genetic and molecular level.

Because HUB Organoids can be expanded long term, drug screening is feasible in these models. HUB has developed procedures for (high-throughput) compound screening in organoids including small molecules and biologics. In this setting, HUB Organoids have been used to identify novel targets, identify lead compounds and stratify potential responders based on their genetic or molecular profile.

In addition, HUB Organoids can serve as a patient avatar in clinical development or diagnostics. In this setting, HUB Organoids could add a functional test for therapy response in a patient centered setting providing a unique tool for personalized medicine.

Citation Format: Jasper Mullenders, Annemarie Buijs, Lars-Eric Fielmich, Jasper Sluimer, Jane Sun, Robert G. Vries, Sylvia F. Boj. HUB Organoids: bringing the 'patient in the lab' for preclinical and clinical development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 126.
Abstract 1275: Potent ENPP1 inhibitors activating STING pathway in tumor microenvironment
Authors: Peng Zhiyuan , Guo Kun , Li Ying-Ying , Kong Deming , Yang Jinfu , (Shelley) Qin Xiaoli , Hao Xiaolin
Published: July 2021

Cyclic GMP-AMP synthase (cGAS) recognizes and binds double-stranded DNA (dsDNA) in the cytosol to generate 2′3′-cGAMP, a cyclic dinucleotide and an activator of STING-dependent innate immunity. Emerging studies revealed that tumor derived 2′3′-cGAMP could act as an immune transmitter and directly trigger anti-tumor immunity. Up to date, Ecto-nucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1/NPP1), a membrane-bound nucleotide hydrolase, was discovered as the predominant enzyme hydrolyzing 2'3'cGAMP. The breakdown of 2'3' cGAMP by ENPP1 may dampen STING-dependent anti-tumor innate immune response. Employing genomic approach, we found that ENPP1 expression varies dramatically among different tumor tissues. Interestingly, ENPP1 expression in breast cancer tissues is overall double the level in normal breast tissues. These results made ENPP1 an attractive cancer immunotherapy target, to enhance STING-dependent innate immunity in tumor microenvironment.We have discovered a novel class of potent ENPP1 inhibitors with pico-molar IC50 in rhENPP1 enzymatic assay. In a cell-based enzymatic assay, compound ZXP-8202 showed an EC50 of 20 nM. To further assess its efficacy on STING-dependent interferon production, 2'3'-cGAMP was added to the culture medium of a high ENPP1-expressing cell line MDA-MB-231 in the absence and presence of ZXP-8202. IFNB1 production in THP1 monocyte was induced when THP-1 cells were incubated with the conditioned medium from MDA-MB-231 cells. In this IFNB1 assay, ZXP-8202 had an EC50 of 10nM. To confirm the ZXP-8202 potency in this culture system reflecting cellular ENPP1 activity, MDA-MB-231 was also treated with 2'3'-cGAMP(PS)2(Rp/Sp), a potent activator of STING but resistant to ENPP1 hydrolysis. The presence of ZXP-8202 showed no effect on 2'3'-cGAMP(PS)2(Rp/Sp) stimulated IFNB1 production in THP-1 cells when cultured in conditioned medium. These results demonstrated ZXP-8202 as a potent ENPP1 inhibitor, blocking 2'3-cGAMP hydrolysis by ENPP1, subsequently activating STING pathway. ZXP-8202 was dosed in CT26 syngeneic mouse model for 14 days and achieved about 70% tumor growth inhibition. No body weight loss was observed at this efficacious dose during treatment. The studies on further characterization of ENPP1 inhibitors and their mechanism of actions are ongoing and the results will be reported in due course.

Citation Format: Zhiyuan Peng, Kun Guo, Ying-Ying Li, Deming Kong, Jinfu Yang, Xiaoli (Shelley) Qin, Xiaolin Hao. Potent ENPP1 inhibitors activating STING pathway in tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1275.
Abstract 1632: Antibody mediated crosslinking of CD38 triggers costimulatory signaling and promotes T cell effector function
Authors: Hwang Mun Kyung , Wang Anlai , Song Zhili , Dai Shujia , Zhang Bailin , Theilhaber Joachim , Pao Lily , Wiederschain Dmitri , Zhu Chen
Published: July 2021

CD38 is a multi-functional ecto-enzyme that regulates NAD+ metabolism. Monoclonal antibodies against CD38, including daratumumab and isatuximab have been successfully developed to treat patients with multiple myeloma, a cancer known to express high levels of CD38. However, CD38 is also expressed by normal immune cells, including B, NK, myeloid and T cell subsets. Using monoclonal antibodies against CD38, we show that CD38 crosslinking can augment T cell antigen receptor signaling, leading to enhanced T cell proliferation and cytokine production. Gene profiling analysis supports the contention that anti-CD38 ligation promotes T cell activation. Studies of CD38 mutants demonstrate that the co-stimulatory effect of anti-CD38 is independent of the ecto-enzymatic activity of CD38. Further analysis of activated CD4 T cells by mass spectrometry shows that CD38 is in close proximity with multiple adhesion and co-stimulatory molecules, including LFA-1, ICOS and OX40. These data support a role for CD38 as a co-stimulatory molecule in T cells, independent of its enzymatic activity.

Citation Format: Mun Kyung Hwang, Anlai Wang, Zhili Song, Shujia Dai, Bailin Zhang, Joachim Theilhaber, Lily Pao, Dmitri Wiederschain, Chen Zhu. Antibody mediated crosslinking of CD38 triggers costimulatory signaling and promotes T cell effector function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1632.
Abstract 1673: Uveal melanoma study patients with low CD163:CD3 ratio in tumor biopsy and low serum IL-6 showed enhanced tumor shrinkage (TS) and overall survival (OS) on tebentafusp
Authors: Hassel Jessica C. , Benlahrech Adel , Stanhope Sarah , Naidoo Revashnee , Leach Emma , Collins Laura , Abdullah Shaad , Ranade Koustubh , Sacco Joseph
Published: July 2021

Background: Tebentafusp, an investigational TCR–anti-CD3 bispecific fusion protein (ImmTAC) that targets gp100 and activates T cells, has shown monotherapy activity in metastatic melanoma [1-3]. In a phase I/II trial enrolling metastatic uveal melanoma patients we explored biomarkers of clinical outcomes.

Methods: The effect of CD163+ macrophages on ImmTAC-mediated T cell proliferation and melanoma cell killing was assessed in vitro. Advanced uveal melanoma HLA-A*02:01+ patients (N = 127) were treated with weekly 68mcg tebentafusp after intra-patient dose escalation (NCT02570308). Serum samples (up to N=123) were profiled for 11 cytokines and chemokines. Digital image analysis of IHC enumerated immune cells per mm2 in tumor biopsies (up to N=112). Median cutoff was used for biomarker correlations with outcomes.

Results: In vitro, increasing numbers of CD163+ macrophages resulted in greater inhibition of ImmTAC-mediated T cell proliferation and killing of MEL624 melanoma cells. The numbers of tumor infiltrating T cells (CD3, CD4, CD8) and CD163+ macrophages at baseline were not associated with TS. However, below median CD163:CD3 ratio was associated with greater TS (odds ratio OR=2.9, p=0.014) and OS (hazard ratio HR=0.4, p=0.001), attributable to CD163:CD8 (OR=3.6, p=0.004) but not CD163:CD4 (OR=1.9, p=0.2). Low serum IL-6 levels at baseline trended with TS (OR=2.1, p=0.09) and OS (HR=0.3, p<0.001). Subset with below median CD163:CD3 in tumor biopsies and serum IL-6 had the best outcomes (Table).

Conclusions: Uveal melanoma patients with low levels of pre-treatment CD163:CD3 in tumor biopsy and serum IL-6 may derive the most benefit from tebentafusp. This hypothesis will be tested in samples from a randomized trial of tebentafusp (NCT03070392). [1] NCT01211262; ASCO 2019 [2] Clinical Cancer Research; 2020[3] #195; ESMO IO; 2020

Subset% with tumor shrinkageMean change in tumor SLD ± S.E.1-year OS %All biomarker evaluable43 (N=95)5.4 ± 3 (N=95)58 (N=105)Low IL-6/Low CD163:CD370 (N=27)-9.4 ± 6.1 (N=27)93 (N=30)Low IL-6/High CD163:CD330 (N=20)10.2 ± 5.0 (N=20)64 (N=22)High IL-6/Low CD163:CD340 (N=20)14.2 ± 7.7 (N=20)41 (N=22)High IL-6/High CD163:CD329 (N=28)9.9 ± 4.0 (N=28)32 (N=31)

Citation Format: Jessica C. Hassel, Adel Benlahrech, Sarah Stanhope, Revashnee Naidoo, Emma Leach, Laura Collins, Shaad Abdullah, Koustubh Ranade, Joseph Sacco. Uveal melanoma study patients with low CD163:CD3 ratio in tumor biopsy and low serum IL-6 showed enhanced tumor shrinkage (TS) and overall survival (OS) on tebentafusp [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1673.
Abstract 1695: Activation of B cells by CD73 blocking antibodies
Authors: Hair James , Garcon Fabien , Hsueh Michelle , Dallaway Laura , Bibikova Elena , Torchia Maria Letizia Giardino , Moody Gordon , Schuller Alwin , Dovedi Simon J. , Cooper Zachary A. , Sachsenmeier Kris , Kumar Rakesh , Eyles Jim , Wilkinson Robert W.
Published: July 2021

The catabolism of ATP into immunosuppressive adenosine contributes to the dysfunction of tumor infiltrating leukocytes (TIL). CD73 is an ectonucleotidase which catabolizes conversion of AMP to adenosine and is expressed on a range of immune cells including B cells. Oleclumab (MEDI9447) is a monoclonal antibody specific for human CD73 and is currently in clinical development for the treatment of cancer. B cells constitute a significant proportion of human TIL; however, their importance to Immuno-Oncology (IO) treatments remains unclear. Recent publications demonstrate that B cells sustain inflammation and predict response to immune checkpoint blockade in human melanoma, their presence within the tumor microenvironment is correlated with improved prognosis in several human tumor types, and CD73 expression has been shown to vary on different human B cell subtypes. Further investigations into the role of CD73 in control of human B cell function are therefore warranted. We show that blockade of CD73 by oleclumab and other monoclonal antibodies on peripheral blood B cells from healthy human donors upregulates expression of CD69, CD83 and CD86, and induces secretion of IL-6, MIP-1α and MIP-1β. Activation is mediated by antibodies that block and internalize CD73 and is reduced by pharmacological inhibition of BTK, thus highlighting involvement of the canonical B-cell receptor signaling pathway. Furthermore, our analyses highlight a differential effect of CD73 blockade on individual B-cell populations, with the most robust increases in activation marker expression being observed on naïve subsets that retain expression of IgD. Our research has identified that oleclumab activates human peripheral blood B cells. Given the renewed interest in B-cell biology in IO, this is an area we believe warrants further clinical investigation.

Citation Format: James Hair, Fabien Garcon, Michelle Hsueh, Laura Dallaway, Elena Bibikova, Maria Letizia Giardino Torchia, Gordon Moody, Alwin Schuller, Simon J. Dovedi, Zachary A. Cooper, Kris Sachsenmeier, Rakesh Kumar, Jim Eyles, Robert W. Wilkinson. Activation of B cells by CD73 blocking antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1695.
Abstract 1751: Decoding the immune system response to leptomeningeal metastasis
Authors: Remsik Jan , Tong Xinran , Sener Ugur , Li Min Jun , Wilcox Jessica , Isakov Danielle , Derderian Camille , Chabot Kiana , Schietinger Andrea , Boire Adrienne
Published: July 2021

For decades, the central nervous system was considered to be an immune privileged organ with limited access to systemic immunity. However, the leptomeninges, the cerebrospinal fluid (CSF)-filled anatomical structure that protects the brain and spinal cord, represent a relatively immune-rich environment. Despite the presence of immune cells, complications in the CSF, such as infectious meningitis and a neurological development of cancer known as leptomeningeal metastasis, are difficult to treat and are frequently fatal. We show that immune cells entering the CSF are held in an ‘idle' state that limits their cytotoxic arsenal and antigen presentation machinery. To understand this underappreciated neuroanatomic niche, we used unique mouse models and rare patient samples to characterize its cellular composition and critical signaling events in health and disease at a single-cell resolution. Revealing the mediators of CSF immune response will allow us to re-evaluate current therapeutic protocols and employ rational combinations with immunotherapies, therefore turning the patient's own immune system into an active weapon against pathogens and cancer.

Citation Format: Jan Remsik, Xinran Tong, Ugur Sener, Min Jun Li, Jessica Wilcox, Danielle Isakov, Camille Derderian, Kiana Chabot, Andrea Schietinger, Adrienne Boire. Decoding the immune system response to leptomeningeal metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1751.
Abstract 1775: Metformin-mediated natural killer cell cytotoxicity in head and neck squamous cell carcinoma
Authors: Crist McKenzie , Lehn Maria , Wise-Draper Trisha
Published: July 2021

Head and Neck Squamous Cell Carcinoma (HNSCC) is the 6th most common cancer worldwide. Even with intensive treatment, up to 50% of patients will relapse. Checkpoint inhibitors, including antibodies targeting programmed death 1 (PD-1), have dramatically improved survival outcomes but only elicit a response in 20% of patients. PD-1 is expressed on both T-cells and Natural Killer (NK) cells, and depletion of NK cells has been linked to poor prognosis in several solid tumors. NK cells are essential to the innate immune response, do not require MHC-1 expression for recognition, and can stimulate an anti-cancer response through release of cytotoxic granules and direct killing of cancer cells. Upregulation of several interleukins including IL-6, which induces the JAK/STAT3 pathway and subsequently inhibits cancer cell apoptosis, is correlated with a poor prognosis in HNSCC patients. Importantly, inhibition of phosphorylated STAT3 (pSTAT3) increases NK-specific cancer cytotoxicity, NK cell cytokine release, and anti-tumor cell chemokines. Therefore, we hypothesize that impaired NK cell cytotoxicity results in a poor response to immunotherapy. We have shown that a common anti-diabetes drug with anti-cancer properties, metformin, downregulates NK pSTAT3 with subsequent increase in cytotoxicity of peripheral NK cells in HNSCC patients. Metformin also decreases pSTAT3 pathway related cytokine release from HNSCC cell lines. However, it remains unclear whether metformin-mediated enhanced NK cytotoxicity was through a direct effect on NK cells or indirect through inhibition of pSTAT3 in tumor cells and subsequent decrease in inhibitory cytokine release. In addition, inhibiting pSTAT3 could lead to further activation of innate immunity through activation of NK cell cytotoxicity complementing current PD-1 inhibitor therapy and improved treatment response. Here we report the contribution of metformin-mediated direct and indirect effects on NK cell cytotoxic functions and the combined effect of metformin and pembrolizumab using patient samples from a clinical trial and ex vivo models. Understanding metformin-mediated regulation of pSTAT3 and downstream pathways will provide necessary insight to potentially overcome resistance to PD-1 inhibitors and identifying novel treatment combinations for this disease.

Citation Format: McKenzie Crist, Maria Lehn, Trisha Wise-Draper. Metformin-mediated natural killer cell cytotoxicity in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1775.
Abstract 1917: Immunomodulatory effects of a novel, enhanced potency gibbon ape leukaemia virus (GALV) fusogenic membrane glycoprotein-expressing herpes simplex virus platform with increased efficacy combined with anti PD-1 therapy
Authors: Roulstone Victoria , Kyula Joan , Thomas Suzanne , Kuncheria Linta , Bommareddy Praveen K. , Smith Henry , Whittock Harriet , Coffin Robert S. , Harrington Kevin
Published: July 2021

Oncolytic viral immunotherapies are attractive because they are self-amplifying, can kill tumors through multiple, immunologically visible mechanisms and have the ability to promote anti-tumor immune responses. RP1 is a novel, enhanced potency oncolytic HSV that has been genetically engineered to express human GM-CSF and the gibbon ape leukemia virus fusogenic membrane glycoprotein with the R sequence deleted (GALV-GP R-), providing constitutive fusion activity. GALV-GP R- binds pit-1 receptors on human and rat, but not mouse, cells to mediate cell-cell fusion. RP1 potently kills human tumor-derived cells, including a panel of melanoma and head and neck cell lines, as demonstrated here. In vivo, using a human melanoma model in nude mice, higher titres (2-3 log) of virus were retrieved from tumors treated with the virus expressing GALV-GP R- (i.e. RP1) versus an equivalent non-GALV-GP R- expressing virus. No virus was retrieved from uninjected tumors. In addition to enhanced direct effects on injected tumors, contralateral uninjected tumors were significantly smaller in groups treated with RP1 versus the GALV-GP R- non-expressing virus despite restriction of virus replication to the injected tumor. This effect was seen in nude mice, i.e. without an adaptive immune system, suggesting contribution of innate (e.g. NK cell-mediated) immunity to the contralateral effects observed. In the immunocompetent mouse 4434 melanoma model, RP1 also reduced tumor burden in both injected and uninjected tumors, again with absolute restriction of virus replication to injected tumors. Animals treated with RP1 had splenomegaly and a population of tumor-infiltrating PD-L1+ cells within injected tumors at one week post-treatment, albeit without a contribution of fusion mediated by GALV-GP R- in this model. FACS-based analysis identified these PD-L1+ cells as predominantly CD45+/CD11b(med/low)/LY6G+ neutrophils. Co-treatment with RP1 and an anti-mouse PD-1 antibody increased anti-tumor effects and the levels of CD3+ cells in uninjected tumors. Data from detailed RNAseq transcriptional analyses from injected and uninjected tumors will also be presented.

Citation Format: Victoria Roulstone, Joan Kyula, Suzanne Thomas, Linta Kuncheria, Praveen K. Bommareddy, Henry Smith, Harriet Whittock, Robert S. Coffin, Kevin Harrington. Immunomodulatory effects of a novel, enhanced potency gibbon ape leukaemia virus (GALV) fusogenic membrane glycoprotein-expressing herpes simplex virus platform with increased efficacy combined with anti PD-1 therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1917.
Abstract 1985: Activation of EGFR/IGF1R - beta catenin - CD44 pathway in periampullary cancer
Authors: Maji Suvendu , Bankura Biswabandhu , Panda Chinmay Kumar , Alam Neyaz
Published: July 2021

Background: Periampullary cancer (PC) is a global medical burden, although infrequent but less than 5% of patients experience an overall survival of five years or more. The study aims to analyze the importance of Epidermal Growth Factor Receptor (EGFR)/Insulin-Like Growth Factor 1 Receptor (IGF1R) - Beta-catenin (β-catenin)- CD44 pathway in the development of periampullary cancer of Indian patients. Materials and Methods: Expression profile of EGFR, IGF1R, β-catenin, and CD44 was verified by immunohistochemical analysis in primary tumor samples (N=14) and respective adjacent normal tissues. Results: In periampullary carcinoma patients, a high level of EGFR expression was seen in 64% of the samples along with co-expression of IGF1-R in 77.8% samples. Furthermore, the high expression of EGFR and IGF1R significantly correlated with the increased expression of β-catenin along with CD44 expression of the tumors. Conclusion: The EGFR/IGF1R - β-catenin- CD44 pathway seems to be important in the development of PC with clinical importance.

Key-words: Periampullary cancer; EGFR; IGF1R; β-catenin; CD44

Key Messages: Interplay of EGFR, IGF1R, β-Catenin and CD44 expressions is important phenomena in the development of PC.

Citation Format: Suvendu Maji, Biswabandhu Bankura, Chinmay Kumar Panda, Neyaz Alam. Activation of EGFR/IGF1R - beta catenin - CD44 pathway in periampullary cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1985.
Abstract 2006: The contributions of iron regulatory proteins 1 and 2 to ferroptosis activation and ferroptotic cell death
Authors: Hermann Evan , Oliveira Thais , Montgomery McKale
Published: July 2021

The essentiality of iron for cell growth and proliferation, coupled with its capacity to promote damaging free radical production, has made it a desirable target for cancer treatment and prevention. One such approach may be through the activation of ferroptosis, a form of iron-mediated programmed cell death. However, we must first understand how cancer cells manipulate the homeostatic regulators of iron metabolism to promote malignancy before we can fully harness iron's therapeutic potential. The iron regulatory proteins 1 and 2 (IRP1 and IRP2) are the master regulators of intracellular iron homeostasis because they coordinate the expression of proteins involved in iron storage, uptake, and utilization. Yet, the roles and regulation of IRPs during cellular ferroptosis remain unknown. The primary objective of this work was to examine the reciprocal relationship between ferroptosis activation and IRP mRNA binding activity. Utilizing tetracycline-inducible plasmids, we found that overexpression of IRP1 or IRP2 significantly increases sensitivity to ferroptotic cell death in HEK293T cells. Intriguingly however, ferroptosis induction by erastin treatment differentially influences IRP1 and IRP2 mRNA binding activity in a cell-type dependent manner, with those cell types expressing higher levels IRP2 exhibiting increased sensitivity to ferroptosis activation. Yet, IRP2 knockout cells are still subject to ferroptotic cell death, as are cells that lack IRP1 expression. Collectively, our findings suggest that while increased expression of IRP1 and IRP2 can promote ferroptotic cell death, expression of IRP1 or IRP2 is sufficient to convey sensitivity to ferroptosis activation. To identify mechanism driving increased IRP mRNA binding activity during cellular ferroptosis, we are currently assessing both iron-dependent and -independent regulators of IRP1 and IRP2 function and stability. As iron is an essential, yet potentially toxic nutrient, such findings will be important because they are expected to provide novel insights for exploiting the toxic nature of iron without compromising the essential homeostatic control mechanisms.

Citation Format: Evan Hermann, Thais Oliveira, McKale Montgomery. The contributions of iron regulatory proteins 1 and 2 to ferroptosis activation and ferroptotic cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2006.
Abstract 2179: Identification of ROS1 alterations in ctDNA from colorectal cancer patients in China
Authors: Wang Mojin , Zhu Lili , Huang Depei , Shen Xudong , Zhang Hushan , She Xueke
Published: July 2021

Background: ROS proto-oncogene 1 (ROS1) is an oncogenic driver involved in various cancer types for which has FDA approved therapies. ROS1 has shown to undergo genetic rearrangements in a variety of human cancers including glioblastoma, non-small cell lung cancer (NSCLC), ovarian cancer, gastric adenocarcinoma and colorectal cancer. The rearrangements create fusion proteins in which the kinase domain of ROS1 becomes constitutively active and drives cellular proliferation. Although previous data have reported ROS1 fusions occur in 0.2% to 2.4% of colorectal cancers, the frequency and type of ROS1 alterations in Chinese CRC patients have not been fully reported.

Methods: This study queried data from 1,083 colorectal cancer samples which underwent a targeted next-generation sequencing assay performed by 3DMed Clinical Laboratory Inc., a College of American Pathologists (CAP) certified and Clinical Laboratory Improvement Amendments (CLIA) certified laboratory of 3D Medicines Inc. between January, 2017 and June, 2020 in China, to identify the incidence of ROS1 alterations detected in ctDNA in colorectal cancer patients. The detected genetic alteration types included single-nucleotide variants (SNVs), copy number variations (CNVs) and gene rearrangements/fusions.

Results: ROS1 alterations were found in 114 patients among the 1,083 (10.5%) CRC patients' cohort, including 72 males and 42 females, median age was 58 years old. The ROS1 alterations included 110 (96.5%) SNVs, 3 (2.6%) CNVs and 1 (0.9%) GOPC-ROS1 fusion. Among the 110 single nucleotide variants, we identified 91 (82.7%) of ROS1 SNVs were nonsynonymous mutations. The top 5 co-occurring mutations in samples with ROS1 alterations were TP53 (71, 62.2%), CYP2C19 (48, 42.1%), APC (48, 42.1%), KRAS (46, 40.3%) and DPYD (42, 36.8%).

Conclusion: Our study indicated that ctDNA can be used as a nonvasive methodology to detect cancer-specific genetic aberrations in plasma for CRC. ROS1 alterations occurred modestly in Chinese colorectal cancer patients. Co-occurring mutations may provide different biological subsets of patients with ROS1-mutant colorectal cancer to reveal patient outcomes and response to therapy. Further investigation could be focused on the effect of ROS1 nonsynonymous mutations on the ROS1 proteins in vitro to identify whether these are more actionable than currently recognized.

Citation Format: Mojin Wang, Lili Zhu, Depei Huang, Xudong Shen, Hushan Zhang, Xueke She. Identification of ROS1 alterations in ctDNA from colorectal cancer patients in China [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2179.
Abstract 2189: Subtype-specific epigenomic landscape and 3D genome structure in bladder cancer
Authors: Tejaswi Tejaswi , Zhang Baozhen , Wang Qixuan , Hou Ye , Jin Qiushi , Xu Jie , Yang Hongbo , Liu Tingting , DeGraff David J. , Yue Feng
Published: July 2021

Urinary bladder cancers (BLCA) are commonly diagnosed urologic malignancy in the United States. Muscle invasive BLCA is a morbid and expensive disease to treat, thus increased molecular understanding is necessary. BLCA has recently been characterized by their distinct expression of luminal and basal genes, which could be used to predict key clinical features such as disease progression and overall survival. Transcriptionally, FOXA1, GATA3, and PPARg have been shown to be essential for luminal subtype-specific gene regulation and subtype switching, while TP63, STAT3, and TFAP2 family members are critical for regulation of basal subtype specific genes. Despite these advances, the underlying epigenetic mechanisms and 3D chromatin architecture responsible for subtype-specific regulation in bladder cancer remains unknown. Here, we determined the genome-wide transcriptome, enhancer landscape, and transcription factor binding profiles (FOXA1 and GATA3) in luminal and basal subtypes of bladder cancers. Furthermore, we report the first-ever mapping of genome-wide chromatin interactions by Hi-C in both bladder cancer cell lines and primary patient tumors. We show that subtype-specific transcription is accompanied by specific open chromatin and epigenomic marks, at least partially driven by distinct TF binding at distal-enhancers of luminal and basal bladder cancers. Finally, we identified a novel clinically relevant transcriptional factor, Neuronal PAS Domain Protein 2 (NPAS2), in luminal bladder cancers that regulates other subtype-specific genes and influences cancer cell proliferation and migration. In summary, our work identifies a subtype-specific epigenomic and 3D genome structure in urinary bladder cancers and suggests a novel link between the circadian TF NPAS2 and a clinical bladder cancer subtype.

Citation Format: Tejaswi Tejaswi, Baozhen Zhang, Qixuan Wang, Ye Hou, Qiushi Jin, Jie Xu, Hongbo Yang, Tingting Liu, David J. DeGraff, Feng Yue. Subtype-specific epigenomic landscape and 3D genome structure in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2189.
Abstract 2251: Gene expression analysis by RNA-sequencing highlights differential regulated pathways involved in cell cycle and inflammation in orbitofacial neurofibromas
Authors: Imada Eddie L. , Edward Deepak P. , Arnold Antje , Al-Hussain Hailah , Strianese Diego , Marchionni Luigi , Rodriguez Fausto J.
Published: July 2021

Although most commonly benign, neurofibromas (NFs) can have devastating functional and cosmetic effects in addition to the possibility of malignant transformation. Orbitofacial NFs in particular may cause progressive, disfiguring tumors of the lid, brow, temple, face and orbit, and anecdotal evidence suggest that they may have increased local aggressiveness compared to NFs developing at other sites. The purpose of this study was to identify biological differences between orbitofacial NFs and those occurring at other anatomic sites. We performed global RNA-sequencing in orbitofacial (n=10) and non-orbitofacial (n=9) NFs. A total of 880 mRNA transcripts were differentially expressed between the two groups (adjusted p>0.05), The top 10 genes relatively overexpressed in orbitofacial NF included NEFL, TREM2, CST1, GAP43, ADORA3, MIA, SYT6, FCGR3A, SPP1, and FCGR1A. The top 10 genes relatively underexpressed in orbitofacial NF included XG, WISP2, MMP27, CXCL14, MFAP5, APLNR, MYOC, SLITRK6, STMN2, and TDRD1. Gene enrichment analyses demonstrated a variety of gene sets differentially affected including pathways involved in cell proliferation, interferon and immune related pathways. Comparisons with publicly available databases of various Schwann cell tumors and models using CAT plots demonstrated the highest overlap with differentially expressed genes in plexiform NF vs MPNST (>10%). In summary, we identified gene expression differences between orbitofacial NF and NFs occurring at other anatomic locations. Further investigation may be warranted, given that orbitofacial NF are notoriously difficult to treat and associated with disproportionate morbidity.

Citation Format: Eddie L. Imada, Deepak P. Edward, Antje Arnold, Hailah Al-Hussain, Diego Strianese, Luigi Marchionni, Fausto J. Rodriguez. Gene expression analysis by RNA-sequencing highlights differential regulated pathways involved in cell cycle and inflammation in orbitofacial neurofibromas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2251.
Abstract 2358: miRNA-483-3p overexpression unleashes invasiveness of metastatic colorectal cancer by NDRG1 targeting and upregulation of the HER3-AKT axis
Authors: Candiello Ermes , Reato Gigliola , Verginelli Federica , D'Ambrosio Antonio , Gambardella Gennaro , Albano Raffaella , Luraghi Paolo , Comoglio Paolo M. , Bertotti Andrea , Trusolino Livio , Boccaccio Carla
Published: July 2021

In colorectal cancer, the genetic mechanisms underlying the metastatic switch are still poorly understood. Here we show that overexpression of miRNA-483-3p, encoded by a frequently amplified gene locus encompassing also insulin-like growth factor 2, confers an aggressive phenotype to stem-like cells derived from colorectal cancer metastases (m-colospheres). As result of miRNA-483-3p ectopic overexpression, m-colospheres displayed (i) increased proliferative response to exogenous EGFR family ligands EGF and NRG1; (ii) increased spontaneous and growth factor-induced in vitro invasiveness and epithelial-mesenchymal transition (EMT); (iii) enhanced stem-cell frequency and resistance to differentiation. By transcriptomic analyses and functional validation, we found that miRNA-483-3p targets NDRG1, a known ‘metastasis suppressor', which is responsible for degradation of EGFR family members, in particular HER3. As result, ectopic or native miRNA-483-3p overexpression was associated with hyper-activation of the signaling pathway triggered by HER3, including AKT and GSK3β, responsible for activation of EMT transcription factors. Consistently, treatment of miRNA-483-3p overexpressing m-colospheres with HER3 specific antibodies counteracted their proliferative and invasive phenotype. The pro-invasive role of miRNA-483-3p in patients was further confirmed by (i) analysis of colorectal tumors, where miRNA-483-3p expression levels directly correlated with expression of EMT transcription factors and poor-prognosis, and (ii) downregulation of naturally occurring miRNA-483-3p overexpression, which prevented invasion of tumors formed by m-colosphere transplantation. These results indicate that miRNA-483-3p can support colorectal cancer invasion through a signaling pathway amenable to targeting in human patients.

Citation Format: Ermes Candiello, Gigliola Reato, Federica Verginelli, Antonio D'Ambrosio, Gennaro Gambardella, Raffaella Albano, Paolo Luraghi, Paolo M. Comoglio, Andrea Bertotti, Livio Trusolino, Carla Boccaccio. miRNA-483-3p overexpression unleashes invasiveness of metastatic colorectal cancer by NDRG1 targeting and upregulation of the HER3-AKT axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2358.
Abstract 2486: Pleiotropic anti-cancer effects of STING in estrogen receptor positive breast cancer cells
Authors: Liu Chun-Yu , Chen Ji-Lin , Huang Chun-Teng , Chu Pei-Yi , Tseng Mei-Fang , Lee Yu-Hsuan , Wang Wan-Lun , Tseng Ling-Ming
Published: July 2021

Purpose: Stimulator of interferon genes (STING) is an upstream regulator of interferon and NF-κB, the key mediators of immune and inflammatory responses associated with cancer, triggered by cytosolic DNA. Downregulation of STING in breast tumor tissues has been reported. STING elicits caspase-8-dependent cell apoptosis via NF-κB in breast cancer cells. STING agonists are considered as potent anti-cancer agents. The current study aims to assess the biological functions of STING in breast cancer progression.

Experimental design: Data of mRNA expression levels and recurrence-free survival in patients with breast cancer from The Cancer Genome Atlas and Kaplan Meier-plotter databases were analyzed. Transwell assays were used to evaluate cell migrated and invaded abilities. Sphere formation was performed in stem cell-selective medium and ultra-low attachment plates. Two STING agonists, ADU-S100 and diABZI STING agonist-1 trihydrochloride, were used for in vitro studies. The molecular events were examined by Western blot analysis.

Results: Data from public database showed that lower transcripts levels of STING in Her2 and luminal B breast cancer tissues than normal breast tissues. Luminal-type breast cancer cell lines harbored low STING transcripts compared with other subtypes. Importantly, breast cancer patients with low expression levels of STING was associated with worse recurrence-free survival. Results of immunoblotting indicated that breast cancer cell lines have lower STING protein expressions than normal MCF10A cells. Knockdown of STING led to aggressive phenotypes of MCF10A and BT474 cells. In contrast, overexpression of STING suppressed the abilities of migration, invasion and sphere formation of MCF7 and BT474 cells. The markers of epithelial were increased while the markers of mesenchymal and stemness were decreased by STING overexpression. STING agonist slightly suppressed cell viability of breast cancer cells but not MCF10A cells. Treatment of STING agonists reduced cell migrated ability with upregulation of E-cadherin and γ-catenin.

Conclusion: These findings suggest that STING serves tumor-suppressive effects in breast cancer.

Citation Format: Chun-Yu Liu, Ji-Lin Chen, Chun-Teng Huang, Pei-Yi Chu, Mei-Fang Tseng, Yu-Hsuan Lee, Wan-Lun Wang, Ling-Ming Tseng. Pleiotropic anti-cancer effects of STING in estrogen receptor positive breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2486.
Abstract 259: Comparison of Illumina NovaSeq 6000 and MGISEQ-2000 in profiling xenograft models
Authors: Qian Wubin , Chen Xiaobo , Li Henry Q. , Guo Sheng
Published: July 2021

BACKGROUND: Comprehensive genomic profiling of tumor models, including patient-derived xenografts, cell lines and organoids, is an integral prerequisite for productive in vivo studies. There are now two leading sequencing platforms from Illumina and MGI Tech. In this study, we compared the performance of the two platforms in sequencing xenograft tumors, which contain both human tumor cells and mouse stromal cells.

METHODS: We performed genomic profiling on several xenograft tumors by both sequencers, including RNA-seq and whole-exome sequencing (WES) for 3 samples, and whole-genome sequencing (WGS) for 1 sample. We then performed a comprehensive assessment of sequencer performance using multiple criteria.

RESULTS: The mouse ratios from the two sequencers were very close in all RNA-seq/WES/WGS datasets, indicating no or similar species bias for both platforms. The RNA-seq datasets showed high gene expression concordance, with Pearson correlation coefficients of 0.993, 0.992 and 0.993. The WES datasets showed high mutation concordance, with the mutation overlapping rates of 98.9%, 99.3% and 99.1%, and the mutation ratio correlation coefficients of 0.971, 0.976 and 0.977. The WGS data showed overall overlapping rate of 95.5%, and mutation ratio correlation coefficient of 0.955, while for SNPs and INDELs, the overlapping rate was 97.1% and 86.1%.

CONCLUSIONS: Our study shows that the MGISEQ and Illumina sequencers have comparable performance in sequencing xenograft tumors.

Citation Format: Wubin Qian, Xiaobo Chen, Henry Q. Li, Sheng Guo. Comparison of Illumina NovaSeq 6000 and MGISEQ-2000 in profiling xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 259.
Abstract 2823: Exploring the role of neutrophil myeloperoxidase in lung cancer
Authors: Valadez-Cosmes Paulina , Cosic Nejra , Kienzl Melanie , Maitz Kathrin , Raftopoulou Sofia , Houghton A. McGarry , Heinemann Akos , Davies Michael J. , Kargl Julia
Published: July 2021

The aim of this study is to investigate whether myeloperoxidase, a neutrophil-derived molecule in the tumor microenvironment, can influence tumor growth in lung cancer. Lung cancer is the leading cause of cancer related deaths worldwide. Chronic inflammation plays a key role in lung cancer and some studies point out the role of neutrophils as critical mediators of lung carcinogenesis. In this context, some of the neutrophil granule components have been proposed to contribute to tumor development. One of these molecules is myeloperoxidase. Myeloperoxidase is a heme peroxidase enzyme that generates reactive oxygen/nitrogen species and is most abundantly expressed in neutrophils. Upon neutrophil activation, during acute inflammation or in the tumor microenvironment, MPO is secreted into the extracellular milieu during degranulation and utilizes H2O2 to hypochlorous acid that reacts with surrounding proteins and cell surface molecules and thereby can alter signaling pathways and cell functions. So far, we have found some evidence on myeloperoxidase acting in favor of tumor development. In vivo, LSL-K-ras/MPO-/- mice revealed prolonged survival and reduced tumor burden when compared to LSL-K-ras/MPO+/+ mice. Consistently, in a heterotopic syngeneic tumor mouse model, MPO knockout mice (C57/B6-J/MPO-/-) displayed smaller tumors in comparison to the WT littermates. In vitro, myeloperoxidase is able to bind, internalize and preserve its enzymatic activity in human lung adenocarcinoma A549 cells. Furthermore, A549 cells showed increased proliferation and reduced apoptosis after MPO treatment as well as an enhanced phosphorylation of Akt and Erk proteins. According to our current results, myeloperoxidase may play a role in the development of lung cancer by regulating cancer cell function. Moreover, this project sheds light on the role of neutrophil-derived molecules as key players in the context of tumor growth.

Citation Format: Paulina Valadez-Cosmes, Nejra Cosic, Melanie Kienzl, Kathrin Maitz, Sofia Raftopoulou, A. McGarry Houghton, Akos Heinemann, Michael J. Davies, Julia Kargl. Exploring the role of neutrophil myeloperoxidase in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2823.