Accelerated Tumor Progression Research Articles

Abstract IA002: Targeting senescence heterogeneity against cancer therapy-resistance and metastases

Abstract Therapy resistance of metastatic tumor cells poses a major limitation to the cure of cancer. Aside from cell-intrinsic processes, the tumor microenvironment is a contributor to this problem. Senescent cells can originate from healthy cells that accumulate irreparable damage. Senescent cells cease to divide, but chronically secrete a wide range of factors that permanently alter their environment. As such, they are thought to impair tissue function and, when part of a tumor microenvironment, they are thought to accelerate tumor progression, metastazation and therapy resistance. Today, I will discuss how we identified that therapy-surviving cancer cells can develop a stem-like state, which is, at least partially, driven by exogenous signals from senescent cells. In addition, I will show how therapy-surviving cancer cells can develop a senescence-like state of their own, including an associated pro-inflammatory secretory phenotype. Given the importance of cancer stemness in therapy resistance, both senescent stromal cells and senescence-like cancer cells pose exciting candidates for therapeutic removal. When designing therapies for elimination of, we ran into a problem, which is that there is no such thing as (just) senescence. Instead, there are distinct subtypes, which are not merely caused by noise. I will show how we investigate these subtypes of senescence and how this is important for their targeted elimination. More specifically, I will highlight interaction between the damage-associated proteins FOXO4 and p53 as a pivot in the viability of a damaged type of senescence, which we call now call “scarred” senescence. We had already shown in the past (Baar .. de Keizer, Cell, 2017) that inhibition of FOXO4, or interference with its interaction with p53 using cell penetrating peptides could selectively eliminate senescent cells and target signs of aging in vivo. Today, I will show how we can use FOXO4-TP53 inhibitors against at least some therapy-surviving cancer cells. Last, I will show how we invested in optimization of these compounds to develop them towards clinical translation and how we can now very effectively eliminate certain types of therapy-surviving cancer cells. Altogether, this adds to a model where a senescent TME, as well as senescence-like cancer cells, can also promote cancer progression, migration, and therapy resistance by locally enforcing a state we called (cancer) “stem-lock”. And specific TP53-FOXO4 inhibitors may overcome this problem, thereby adding to a yin-yang treatment of damaging chemo-radiotherapy, followed by FOXO4-TP53-based anti-senescence treatment. Citation Format: Diana A. Putavet, Johannes Lehmann, Beatriz Subtil, Damon Hofman, Marjolein P. Baar, Peter L.J. de Keizer. Targeting senescence heterogeneity against cancer therapy-resistance and metastases [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr IA002.

Differentiated glioblastoma cells accelerate tumor progression by shaping the tumor microenvironment via CCN1-mediated macrophage infiltration

Glioblastoma (GBM) is the most lethal primary brain tumor characterized by significant cellular heterogeneity, namely tumor cells, including GBM stem-like cells (GSCs) and differentiated GBM cells (DGCs), and non-tumor cells such as endothelial cells, vascular pericytes, macrophages, and other types of immune cells. GSCs are essential to drive tumor progression, whereas the biological roles of DGCs are largely unknown. In this study, we focused on the roles of DGCs in the tumor microenvironment. To this end, we extracted DGC-specific signature genes from transcriptomic profiles of matched pairs of in vitro GSC and DGC models. By evaluating the DGC signature using single cell data, we confirmed the presence of cell subpopulations emulated by in vitro culture models within a primary tumor. The DGC signature was correlated with the mesenchymal subtype and a poor prognosis in large GBM cohorts such as The Cancer Genome Atlas and Ivy Glioblastoma Atlas Project. In silico signaling pathway analysis suggested a role of DGCs in macrophage infiltration. Consistent with in silico findings, in vitro DGC models promoted macrophage migration. In vivo, coimplantation of DGCs and GSCs reduced the survival of tumor xenograft-bearing mice and increased macrophage infiltration into tumor tissue compared with transplantation of GSCs alone. DGCs exhibited a significant increase in YAP/TAZ/TEAD activity compared with GSCs. CCN1, a transcriptional target of YAP/TAZ, was selected from the DGC signature as a candidate secreted protein involved in macrophage recruitment. In fact, CCN1 was secreted abundantly from DGCs, but not GSCs. DGCs promoted macrophage migration in vitro and macrophage infiltration into tumor tissue in vivo through secretion of CCN1. Collectively, these results demonstrate that DGCs contribute to GSC-dependent tumor progression by shaping a mesenchymal microenvironment via CCN1-mediated macrophage infiltration. This study provides new insight into the complex GBM microenvironment consisting of heterogeneous cells.

Overexpression of EZH2/NSD2 Histone Methyltransferase Axis Predicts Poor Prognosis and Accelerates Tumor Progression in Triple-Negative Breast Cancer.

Two histone methyltransferases, enhancer of zeste homolog 2 (EZH2) and nuclear SET domain-containing 2 (NSD2), are aberrantly expressed in several types of human cancers. However, the regulatory relationship between EZH2 and NSD2 and their prognostic values in breast cancer (BC) have not been fully elucidated. In this study, we demonstrated that EZH2 and NSD2 were overexpressed in BC compared with benign lesions and normal tissues using tissue microarray, immunohistochemistry, and bioinformatic databases. Both EZH2 and NSD2 expression were associated with pathological grade of tumor and lymph node metastasis. A comprehensive survival analysis using Kaplan-Meier Plotter database indicated that EZH2 expression was negatively correlated with relapse-free survival (RFS), overall survival (OS), distant metastasis-free survival (DMFS), and postprogression survival (PPS) in 3951 BC patients, and NSD2 expression was negatively correlated with RFS and DMFS. Notably, EZH2 and NSD2 expression were coordinately higher in triple-negative breast cancer (TNBC) than that in other subtypes. Stable knockdown of EZH2 using lentiviral shRNA vector significantly reduced the proliferation, migration and invasion abilities of TNBC cell line MDA-MB-231 and MDA-MB-468, and downregulated NSD2 expression as well as the levels of H3K27me3 and H3K36me2, two histone methylation markers catalyzed by EZH2 and NSD2, respectively. By contrast, overexpression of EZH2 using adenovirus vector displayed an inverse phenotype. Furthermore, knockdown of NSD2 in EZH2-overexpressing cells could dramatically attenuate EZH2-mediated oncogenic effects. Bioinformatic analysis further revealed the function and pathway enrichments of co-expressed genes and interactive genes of EZH2/NSD2 axis, suggesting that EZH2/NSD2 axis was associated with cell division, mitotic nuclear division and transition of mitotic cell cycle in TNBC. Taken together, EZH2/NSD2 axis may act as a predictive marker for poor prognosis and accelerate the progression of TNBC.

Antioxidants Promote Intestinal Tumor Progression in Mice.

Dietary antioxidants and supplements are widely used to protect against cancer, even though it is now clear that antioxidants can promote tumor progression by helping cancer cells to overcome barriers of oxidative stress. Although recent studies have, in great detail, explored the role of antioxidants in lung and skin tumors driven by RAS and RAF mutations, little is known about the impact of antioxidant supplementation on other cancers, including Wnt-driven tumors originating from the gut. Here, we show that supplementation with the antioxidants N-acetylcysteine (NAC) and vitamin E promotes intestinal tumor progression in the ApcMin mouse model for familial adenomatous polyposis, a hereditary form of colorectal cancer, driven by Wnt signaling. Both antioxidants increased tumor size in early neoplasias and tumor grades in more advanced lesions without any impact on tumor initiation. Importantly, NAC treatment accelerated tumor progression at plasma concentrations comparable to those obtained in human subjects after prescription doses of the drug. These results demonstrate that antioxidants play an important role in the progression of intestinal tumors, which may have implications for patients with or predisposed to colorectal cancer.

Regulation of tumor immune suppression and cancer cell survival by CXCL1/2 elevation in glioblastoma multiforme.

The invasiveness and high immune suppression of glioblastoma multiforme (GBM) produce poor survival of afflicted patients. Unfortunately, in the past decades, no therapeutic approach has remarkably improved the survival time of patients with GBM. Our analysis of the TCGA database and brain tumor tissue arrays indicated that CXCL1 and CXCL2 overexpression is closely associated with GBM's aggressiveness. Our results showed that elevation of CXCL1 or CXCL2 facilitated myeloid cell migration and simultaneously disrupted CD8+ T cell accumulation at tumor sites, causing accelerated tumor progression. Yet, blockade of CXCL1/2 significantly prevented myeloid-derived suppressor cell migration and thereby increased CD8+ T cell accumulation in vitro and in vivo. CXCL1/2 also promoted the paracrine factor S100A9 and further activated Erk1/2 and p70S60k, whereas blocking CXCL1/2 down-regulated these prosurvival factors. The combination of targeting CXCL1/2 and standard temozolomide chemotherapy improved upon the antitumor efficacy of chemotherapy alone, extending the overall survival time in GBM.

Systemic PPARγ Antagonism Reduces Metastatic Tumor Progression in Adipocyte-Rich Bone in Excess Weight Male Rodents.

Primary tumors are widely associated with an excess in body fat. The role of adipose tissue on tumor cell homing to bone is yet poorly defined. In this study, we aimed to assess whether bone colonization by tumor cells is favored by an adipocyte-rich bone marrow. We delineated the accompanying alterations of the bone microenvironment and established a treatment approach that interferes with high fat diet (HFD)-induced bone metastasis formation. We were able to show that adipocytes affect skeletal tumor growth in a metastatic model of breast cancer in male rats and melanoma in male mice as well as in human breast cancer bone biopsies. Indeed, HFD-induced bone marrow adiposity was accompanied by accelerated tumor progression and increased osteolytic lesions. In human bone metastases, bone marrow adiposity correlated with tumor cell proliferation. By antagonization of the adipocyte differentiation and storage pathway linked to the peroxisome proliferator-activated receptor gamma (PPARγ) with bisphenol-A-diglycidylether (BADGE), we were able to decelerate tumor progression and subsequent osteolytic damage in the bones of two distinct metastatic animal models exposed to HFD. Overall these data show that adipose tissue is a critical factor in bone metastases and cancer-induced bone loss. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

SMURF1, a promoter of tumor cell progression?

Overexpression of HECT-type E3 ubiquitin ligase SMURF1 is correlated with poor prognosis in patients with various cancers, such as glioblastoma, colon cancer, and clear cell renal cell carcinoma. SMURF1 acts as a tumor promoter by ubiquitination modification and/or degradation of tumor-suppressing proteins. Combined treatment of Smurf1 knockdown with rapamycin showed collaborative antitumor effects in mice. This review described the role of HECT, WW, and C2 domains in regulating SMURF1 substrate selection. We summarized up to date SMURF1 substrates regulating different type cell signaling, thus, accelerating tumor progression, invasion, and metastasis. Furthermore, the downregulation of SMURF1 expression, inhibition of its E3 activity and regulation of its specificity to substrates prevent tumor progression. The potential application of SMURF1 regulators, specifically, wisely choose certain drugs by blocking SMURF1 selectivity in tumor suppressors, to develop novel anticancer treatments.

Abstract PO-063: Regulatory T cells regulate fibroblast differentiation during pancreatic carcinogenesis

Abstract Pancreatic ductal adenocarcinoma (PDA) is characterized by an extensive fibrotic stroma, which includes vascular elements, infiltrating immune cells, extracellular matrix and a large number of fibroblasts. The tumor microenvironment as a whole has been believed to be immunosuppressive. However, how different cell populations within the stroma contribute to the establishment of the immunosuppression is not fully understood. We sought to investigate the crosstalk between cancer cells, fibroblasts and immune cells within pancreatic cancer with the goal to identify novel targets for PDA treatment. Targeting regulatory T cell (Treg), which acts to suppress immune responses, is considered as one potential approach to relieve the immunosuppression of pancreatic cancer. However, we previously showed that Treg depletion in a genetically engineered mouse model of pancreatic cancer (KC;Foxp3DTR) failed to relieve immunosuppression and led to accelerated tumor progression. We discovered that Treg depletion reprogramed the fibroblast population, with loss of tumor-restraining, smooth muscle actin-expressing fibroblasts. Conversely, Treg depletion resulted in differentiation of inflammatory fibroblast subsets with an increase in chemokines Ccl3, Ccl6, and Ccl8. To fully understand the contribution of Tregs to pancreatic carcinogenesis, we performed single-cell RNA-sequencing (scRNA-seq) with Pancreatic intraepithelial neoplasia (PanIN) from KC mouse and Treg depleted PanIN lesions from KC;Foxp3DTR mouse. scRNA-seq allows us to address cell type identification and heterogeneity of cell responses by unsupervised clustering, which uncovered dramatic differences in several stromal cell populations including fibroblast, CD4 T cell and macrophage subsets between the KC and KC;Foxp3DTR pancreata. We have expanded list of genes that are regulated in tumor associated fibroblasts upon Treg depletion through scRNA-seq. For example, Il33 was one of down-regulated genes in fibroblasts upon Treg depletion. We also obtained potential ligands/receptors interactions between tumor cells, fibroblasts and immune cells through interactome analysis. Our data describe an unexpected crosstalk between Tregs and fibroblasts and point to new mechanisms regulating fibroblast differentiation in pancreatic cancer. Citation Format: Yaqing Zhang, Katelyn L. Donahue, Wei Yan, Zeribe C. Nwosu, Kristee L. Brown, Sion Yang, Howard C. Crawford, Costas A. Lyssiotis, Timothy L. Frankel, Filip Bednar, Marina Pasca di Magliano. Regulatory T cells regulate fibroblast differentiation during pancreatic carcinogenesis [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-063.

Inhibitory effect of ginsenoside Rg3 on cancer stemness and mesenchymal transition in breast cancer via regulation of myeloid-derived suppressor cells.

Ginsenoside Rg3 (Rg3) has been studied in several cancer models and is suggested to act through various pharmacological effects. We investigated the anticancer properties of Rg3 through myeloid-derived suppressor cell (MDSC) modulation in FM3A mouse mammary carcinoma cells. The effects of Rg3 on MDSCs and consequent changes in cancer stem-like cells (CSCs) and epithelial-mesenchymal transition (EMT) were evaluated by diverse methods. MDSCs promoted cancer by enhancing breast cancer stemness and promoting EMT. Rg3 at a dose without obvious cytotoxicity downregulated MDSCs and repressed MDSC-induced cancer stemness and EMT. Mechanistic investigations suggested that these inhibitory effects of Rg3 on MDSCs and corresponding cancer progression depend upon suppression of the STAT3-dependent pathway, tumor-derived cytokines, and the NOTCH signaling pathway. In a mouse model, MDSCs accelerated tumor progression, and Rg3 delayed tumor growth, which is consistent with the results of in vitro experiments. These results indicated that Rg3 could effectively inhibit the progression of breast cancer. The anticancer effect of Rg3 might be partially due to its downregulation of MDSCs and consequent repression of cancer stemness and EMT in breast cancer. Hence, we suggest the regulation of MDSCs through Rg3 treatment as an effective therapeutic strategy for breast cancer patients.

Long Non-Coding RNA SNHG14 Regulates SPIN1 Expression to Accelerate Tumor Progression in Non-Small Cell Lung Cancer by Sponging miR-382-5p.

BackgroundNon-small cell lung cancer (NSCLC) is the most common type of lung carcinoma. Long non-coding RNA (lncRNA) small nucleolar RNA host gene 14 (SNHG14) was identified to participate in tumor progression. However, the mechanism and functions of SNHG14 were rarely reported in NSCLC progression.MethodsThe relative gene expression was tested by qRT-PCR. Cell viability, apoptosis, migration and invasion were measured by MTT assay, flow cytometry, and transwell migration and invasion assays, respectively. The interactions between miR-382-5p and SNHG14 or SPIN1 were predicted by starBase and confirmed by the dual-luciferase reporter assay and RNA pull-down assay. The protein level of SPIN1 was evaluated by Western blot assay.ResultsThe levels of SNHG14 and SPIN1 were significantly increased, while the level of miR-382-5p was apparently reduced in NSCLC tissues and cells. SNHG14 was verified to sponge miR-382-5p and SPIN1 was identified as a direct target of miR-382-5p. SNHG14 depletion repressed cell viability, migration and invasion, but induced the apoptotic rate by targeting miR-382-5p. miR-382-5p overexpression blocked cell viability, metastasis and promoted cell apoptosis by regulating SPIN1. SNHG14 silencing down-regulated SPIN1 expression by sponging miR-382-5p.ConclusionSNHG14 facilitated NSCLC progression by regulating SPIN1 expression via targeting miR-382-5p.

Abstract 6680: Inclusion of inhibitory neoantigens can abolish efficacy of otherwise protective therapeutic anti-tumor vaccines

Abstract Identification of neoantigens that can elicit strong anti-tumor responses has become critical to vaccine design for cancer immunotherapy. Conventional methods for in silico neoantigen identification have yielded poor predictive value, highlighting the need for methods that identify bona fide neoantigen targets. The Genocea ATLAS™ platform uses autologous antigen presenting cells and T cells to identify pre-existing CD4+ and/or CD8+ T cell responses to patient-specific mutations and therefore selects confirmed neoantigens that can be used for vaccines and cell therapies. Screened neoantigens are characterized as stimulatory or inhibitory based on up- or downregulation of inflammatory cytokine secretion compared to baseline controls. In the mouse B16F10 melanoma model, therapeutic immunization with stimulatory neoantigen peptides arrested tumor growth whereas in contrast, inhibitory antigen immunization (henceforth referred to as inhibigens) resulted in accelerated tumor progression. The presence of an inhibigen in an otherwise protective vaccine formulation completely abolished protection. Global IFNγ responses to neoantigens were abrogated in these mice as measured by ELISpot suggesting that inhibigen pro-tumor responses can be immunodominant. Analysis of tumor-infiltrating lymphocytes (TILs) from mice immunized with ATLAS-identified neoantigen ± inhibigen peptide vaccines revealed significant alterations in the tumor microenvironment. Inclusion of inhibigens resulted in low T cell infiltration into tumors and increased expression of TIL inhibitory surface markers (e.g. PD-1, LAG-3). Immunological mechanisms of the inhibitory phenomenon are currently being explored. These data promote rational methods for neoantigen identification and highlight the potential advantages of excluding deleterious inhibigens from cancer vaccines and immunotherapies. GEN-009, a personalized cancer vaccine filtered for inclusion of only ATLAS-identified neoantigens (excluding inhibigens) is currently being evaluated in a Phase 1/2a clinical trial (NCT03633110). Citation Format: Victoria L. DeVault, Hanna Starobinets, Sanmit Adhikari, Simran Singh, Stephanie Rinaldi, Brendan Classon, Jessica B. Flechtner, Hubert Lam. Inclusion of inhibitory neoantigens can abolish efficacy of otherwise protective therapeutic anti-tumor vaccines [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6680.

Abstract 124: Recycling endosomes in mature epithelia restrain tumorigenic signaling

Abstract The effects of polarized membrane trafficking in mature epithelial tissue on cell growth and cancer progression have not been fully explored in vivo. A majority of colorectal cancers have reduced and mislocalized Rab11, a small GTPase dedicated to trafficking of recycling endosomes. Patients with low Rab11 protein expression have poor survival rates. Using genetic models across species, we show that intact recycling endosome function restrains aberrant epithelial growth elicited by APC or RAS mutations. Loss of Rab11 protein led to epithelial dysplasia in early animal development and synergized with oncogenic pathways to accelerate tumor progression initiated by carcinogen, genetic mutation, or aging. Transcriptomic analysis uncovered an immediate expansion of the intestinal stem cell pool along with cell-autonomous Yki/Yap activation following disruption of Rab11a-mediated recycling endosomes. Intestinal tumors lacking Rab11a traffic exhibited marked elevation of nuclear Yap, upd3/IL6-Stat3, and amphiregulin-MAPK signaling, while suppression of Yki/Yap or upd3/IL6 reduced gut epithelial dysplasia and hyperplasia. Examination of Rab11a function in enteroids or cultured cell lines suggested that this endosome unit is required for suppression of the Yap pathway by Hippo kinases. Thus, recycling endosomes in mature epithelia constitute key tumor suppressors, loss of which accelerates carcinogenesis. Statement of significance: Recycling endosome traffic in mature epithelia constitutes a novel tumor suppressing mechanism. Citation Format: Sayantani Goswami. Recycling endosomes in mature epithelia restrain tumorigenic signaling [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 124.

LncRNA DLX6-AS1 aggravates the development of ovarian cancer via modulating FHL2 by sponging miR-195-5p

BackgroundOvarian cancer (OC) is a huge burden on women’s lives. Recently, the implication of long non-coding RNAs (lncRNAs) in cancers, including OC, has aroused much attention. The objective of this study was to explore the role and functional mechanism of lncRNA distal-less homeobox 6 antisense 1 (DLX6-AS1) in OC.MethodsThe expression of DLX6-AS1, miR-195-5p, and four and a half LIM domains protein 2 (FHL2) was measured by quantitative real-time polymerase chain reaction (qRT-PCR). The cell proliferation, apoptosis, migration, and invasion were assessed by cell count kit 8 (CCK-8), flow cytometry and transwell assays, respectively. The protein levels of proliferating cell nuclear antigen (PCNA), cleaved-caspase-3 (C-caspase 3), N-cadherin, Vimentin, E-cadherin and FHL2 were quantified by western blot. The relationship between miR-195-5p and DLX6-AS1 or FHL2 was predicted by bioinformatics tool starBase and verified by luciferase reporter assay and RNA immunoprecipitation (RIP) assay. Xenograft tumor model was established to observe the role of DLX6-AS1 in vivo.ResultsDLX6-AS1 and FHL2 were up-regulated in OC tissues and cells, while miR-195-5p was down-regulated. DLX6-AS1 knockdown inhibited proliferation, migration, and invasion but induced apoptosis of OC cells. However, miR-195-5p inhibition reversed these effects. Overexpression of miR-195-5p also depleted proliferation, migration, and invasion but promoted apoptosis of OC cells, while FHL2 overexpression overturned these influences. DLX6-AS1 knockdown blocked tumor growth in vivo.ConclusionDLX6-AS1, as an oncogene in OC, accelerated tumor progression by up-regulating FHL2 via mediating miR-195-5p, suggesting that DLX6-AS1 was a hopeful target for the lncRNA-targeted therapy in OC.

Radiotherapy Increases 12-LOX and CCL5 Levels in Esophageal Cancer Cells and Promotes Cancer Metastasis via THP-1-Derived Macrophages.

BackgroundDioxygenase 12-lipoxygenase (12-LOX) plays an important role in tumorigenesis and promotes angiogenesis and proliferation in several tumors, including prostate and breast tumors. Radiotherapy enhances the expression of 12-LOX in esophageal squamous cell carcinoma (ESCC). Two types of macrophages can be found in the tumor microenvironment. The M2 subtype accelerates tumor progression; however, the relationship between 12-LOX and macrophages is not well established. Here, we explore this interaction and its effect on ESCC to induce tumor progression.Methods and ResultsRT-qPCR and Western blot analyses were used to evaluate the mRNA and protein expression levels of 12-LOX and chemokine (C-C motif) ligand 5 (CCL5) in ESCC after radiotherapy. CCL5 expression was increased by 12-LOX upregulation but was suppressed by the well-established 12-LOX inhibitor, baicalein. Furthermore, CCL5 attracted and repolarized human myeloid leukemia mononuclear cells (THP-1)-derived macrophages. Finally, ESCC co-culture with THP-1-derived macrophages led to a strong cancer migratory capacity.ConclusionRadiation-induced 12-LOX overexpression in ESCC upregulates CCL5 expression, thereby attracting THP-1-derived macrophages and promoting their polarization to the M2 subtype, which enhances cellular metastasis.

RELL1, a novel oncogene, accelerates tumor progression and regulates immune infiltrates in glioma

BackgroundDiffuse gliomas are the most prevalent primary brain tumors. The Chinese Glioma Genome Atlas (CGGA) and The Cancer Genome Atlas (TCGA) databases provide evidence of the relationships between gliomas and altered molecular pathways. In glioma pathogenesis, the microenvironment has emerged as a potential indicator of tumor progression. MethodsWe investigated and confirmed the role of RELT-like 1 (RELL1) oncogene in glioblastoma and low-grade glioma by data mining large cohorts of TCGA and the CGGA. Correlations between immune cells and RELL1 were verified using the CIBERSORT algorithm. ResultsOur analysis revealed that RELL1 expression was much higher in grade-dependent glioma patients with poor prognosis. Comparable prognostic values for isocitrate dehydrogenase 1 (IDH1) and RELL1 were observed during in-depth analyses of the integrated correlations. Moreover, RELL1 was found to be associated with numerous tumor-infiltrating immune cells in glioma-affected patients. ConclusionWe conclude that RELL1 might serve as a potential therapeutic target or prognostic marker in glioblastoma and diffuse glioma cases.

Thrombin is a therapeutic target for non-small-cell lung cancer to inhibit vasculogenic mimicry formation

Tumor cells transform into endothelial cells by epithelial-to-mesenchymal transition, which is characterized by vasculogenic mimicry (VM). VM not only accelerates tumor progression but also increases drug-induced resistance. However, very little is currently known about the molecular determinants that enable VM. Targeting VM might bring a new breakthrough in cancer treatment. Thrombin is the key enzyme of the blood coagulation system and could contribute to tumor progression. Nevertheless, the association between thrombin and VM formation remains largely unknown. We found that VM was associated with the overall survival of non-small-cell lung cancer (NSCLC) patients, and that thrombin expression was closely related to VM formation. This research revealed that thrombin induced VM formation via PAR-1-mediated NF-κB signaling cascades. The novel thrombin inhibitors r-hirudin and DTIP inhibited VM formation and spontaneous metastases in subcutaneous tumors. Clinical pathological analysis confirmed that NSCLC patients with thrombin-positive/PAR-1-high expression had the poorest prognosis and were the most likely to form VM. The promotional activity of thrombin in VM formation and tumor metastasis was abolished in PAR-1-deficient NSCLC cells. The EGFR inhibitor gefitinib had no effect on VM and increased VEGF expression in tumors. The combination therapy of DTIP and gefitinib achieved a better therapeutic effect than either agent alone. This study is the first to illustrate that thrombin substantially contributes, together with PAR-1, to VM formation and to illustrate that VM might be a target of r-hirudin and DTIP to suppress tumor progression. The anticoagulants r-hirudin and DTIP could be employed for antitumor therapy. Combination therapy with DTIP with an EGFR inhibitor might achieve superior therapeutic effects.

Myeloid Cell–Derived TGFβ Signaling Regulates ECM Deposition in Mammary Carcinoma via Adenosine-Dependent Mechanisms

TGFβ plays a crucial role in the tumor microenvironment by regulating cell-cell and cell-stroma interactions. We previously demonstrated that TGFβ signaling on myeloid cells regulates expression of CD73, a key enzyme for production of adenosine, a protumorigenic metabolite implicated in regulation of tumor cell behaviors, immune response, and angiogenesis. Here, using an MMTV-PyMT mouse mammary tumor model, we discovered that deletion of TGFβ signaling on myeloid cells (PyMT/TGFβRIILysM) affects extracellular matrix (ECM) formation in tumor tissue, specifically increasing collagen and decreasing fibronectin deposition. These changes were associated with mitigated tumor growth and reduced metastases. Reduced TGFβ signaling on fibroblasts was associated with their proximity to CD73+ myeloid cells in tumor tissue. Consistent with these findings, adenosine significantly downregulated TGFβ signaling on fibroblasts, an effect regulated by A2A and A2B adenosine receptors. METABRIC dataset analysis revealed that patients with triple-negative breast cancer and basal type harbored a similar signature of adenosine and ECM profiles; high expression of A2B adenosine receptors correlated with decreased expression of Col1 and was associated with poor outcome. Taken together, our studies reveal a new role for TGFβ signaling on myeloid cells in tumorigenesis. This discovered cross-talk between TGFβ/CD73 on myeloid cells and TGFβ signaling on fibroblasts can contribute to ECM remodeling and protumorigenic actions of cancer-associated fibroblasts. SIGNIFICANCE: TGFβ signaling on fibroblasts is decreased in breast cancer, correlates with poor prognosis, and appears to be driven by adenosine that accelerates tumor progression and metastasis via ECM remodeling.

Abstract PR13: Polymerase-mediated ultramutagenesis: A new approach for modeling the high mutational load of human cancer

Abstract Cancer is characterized by increased mutation rate, and recent work has led to a deeper understanding of mutator phenotypes and underlying molecular mechanisms. Differences in the mutational landscape of individual cancers underlie key aspects of clinical behavior. For example, overall base substitution rate is the best predictor of immune therapy response. Most genetically engineered mouse models of cancer (GEMMs) reiterate a few driver events but fail to recapitulate the high mutational loads that are the ultimate cause of most human cancer, making GEMMs inadequate for many aspects of tumor behavior, such as immune responses. The highest base substitution rates in cancers (≥100/Mb) result from specific heterozygous single amino acid substitutions (usually P286R) in the proofreading domain of DNA polymerase epsilon (POLE), rendering POLE highly error prone. POLE mutations are most common in endometrial cancer but occur with lower incidence in a wide range of cancers. We hypothesized that GEMMs could be generated by recapitulating PoleP286R via conditional knockin. Previously, we showed that constitutive expression of PoleP286R throughout the body triggered malignancies of diverse lineages. Mice harboring LSL-PoleP286R and the endometrial BAC-Sprr2f-Cre developed endometrial cancers starting at 1 year. Endometrial cancers exhibited histologic features previously reported in human POLE tumors, and metastasized in all mice (p<0.00001, n=36 vs n=28 controls). Of note, 100% penetrance was achieved through a single (monoallelic) amino acid change, with experimental animals efficiently generated in a single generation. Whole-genome and targeted cancer gene panel sequencing of primary tumors (n=12) and cell lines (n=6) revealed distinctive mutational signatures and the highest nucleotide substitution rates described in murine cancers, with some tumors exceeding 100 base substitutions/Mb. Since most DNA replication errors are repaired by mismatch repair (MMR), and some human tumors harbor POLE mutations and defective MMR (dMMR), we introduced dMMR via conditional Msh2 knockout. This further accelerated tumor progression and mortality (p<0.00001, n=36 Pole alone vs. n=28 Pole+Msh2), revealing clear synergism. In both models, there were robust antitumor immune responses, as evidenced by increased T-cell infiltrates, accelerated tumor growth following T-cell depletion by anti-CD4 antibody, and clinical regression following anti-CTLA4/PDL1 checkpoint therapy. In summary, this is a robust approach to model the high mutational loads intrinsic to human cancer. Expression of PoleP286R in a specific lineage is sufficient to result in cancers with greater ease than previous strategies such as dMMR alone, and with mutational loads in the range of human cancers. The approach may be useful for diverse models and investigations. This abstract is also being presented as Poster A34. Citation Format: Hao-Dong Li, Changzheng Lu, He Zhang, Ileana C. Cuevas, Subhransu S. Sahoo, Yang-Xin Fu, Diego H. Castrillon. Polymerase-mediated ultramutagenesis: A new approach for modeling the high mutational load of human cancer [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr PR13.

GEN-009, a neoantigen vaccine containing ATLAS selected neoantigens, to generate broad sustained immunity against immunogenic tumor mutations and avoid inhibitory peptides.

3107 Background: Tumor-specific neoantigens provide personalized targets for immunotherapy. Vaccines against epitopes predicted by in silico approaches very rarely induce CD4+ and CD8+ ex vivo T cell responses regardless of formulation. ATLAS selects neoantigens for vaccine inclusion using ex vivo screening of all patient-specific mutations to identify pre-existing CD4+ or CD8+ T cell responses and to exclude Inhibigens, which are inhibitory peptides that suppress immunity and accelerate tumor progression. The Inhibigen burden correlates with patient outcomes in observational studies and rapid tumor progression in mouse models. Methods: GEN-009-101 is a phase 1/2a study testing safety, immunogenicity and clinical activity in immune responsive tumors. After next-generation tumor sequencing and ATLAS testing of autologous leukocytes, up to 20 stimulatory synthetic long peptides adjuvanted with poly-ICLC comprise each personalized vaccine. Eight vaccinated patients have been followed for sustained immunological responses and clinical outcomes. Results: The 40 doses given across patients have induced only mild local discomfort and no DLT. Vaccination has generated immune responses against 99% of administered peptides, with both CD8+ and CD4+ responses in ex vivo fluorospot assays. To date, no patients have developed recurrent disease. Broad immunity develops as early as Day 29 and is sustained for over 12 months. Immune response against individual peptides is correlated with peptide concentration (OR = 1.26, p≤0.0001) but not with other classifiers such as GRAVY index (Grand Average of Hydropathy), tumor type, injection site or sex. The Inhibigen burden prior to treatment again correlates with disease progression. Conclusions: GEN-009 identifies tumor specific immune targets from the individual patient’s tumor mutagens. Initial clinical data show that ATLAS antigen selection may be critical to the induction of broad, rapid and sustained immunity against tumor specific neoantigens. Clinical vaccination with PD-1 blockade is in process. Clinical trial information: NCT03633110 . [Table: see text]

Exploratory analysis of factors associated with hyperprogression in advanced non-small cell lung cancer (NSCLC) treated with PD1/PDL1 inhibitors.

e21634 Background: Hyperprogressive disease(HPD) is a recently recognized type of accelerated tumor progression with immune checkpoint inhibitor(ICI) therapy. A recent meta-analysis identified serum lactate dehydrogenase(LDH) above upper limit of normal(ULN), liver metastases and > 2 metastatic sites to be associated with increased likelihood of HPD. Methods: We performed a single-center retrospective analysis of NSCLC patients treated with ICI from 2/2017-1/2020. We defined HPD using combined criteria of failure to complete > 4 cycles of ICI and radiological progression with development of new lesions or marked clinical progression where restaging was not achievable because of rapid decline in performance status. Treatment discontinuation/interruption because of toxicity alone, was not designated as HPD. Results: HPD was seen in 9/41(22%) patients treated with at least 1 cycle of ICI. In 6/9 there was marked radiological progression (new lesions/significant progression of existing lesions); in the remaining 3 restaging was not possible because of rapid decline which mandated treatment cessation. Patients with HPD were more likely to have a raised LDH, liver metastases or > 2 metastatic sites but these differences were not statistically significant. 9/9 versus 5/32 patients in the HPD/non-HPD cohorts respectively progressed within 10 weeks of treatment (p < 0.001) and 7/9 versus 12/32 respectively did not survive beyond 6months (p = 0.057). Conclusions: This small study has shown a non-statistically significant trend for association between HPD, raised LDH, liver metastases and presence of > 2 metastatic sites. A better understanding of risk/predictive factors for HPD is essential for improved patient selection for treatment with ICI. [Table: see text]