Course Of Tumor Development Research Articles

Survey for Activating Oncogenic Mutation Variants in Metazoan Germline Genes.

Most cancers present with mutations or amplifications in distinctive tumor promoter genes that activate principal cell-signaling cascades promoting cell proliferation, dedifferentiation, cell survival, and replicative immortality. Somatic mutations found in this these driver proto-oncogenes invariably result in constitutive activation of the encoded protein. A salient feature of the activating mutations observed throughout many thousands of clinical tumor specimens reveals these driver missense mutations are recurrent and restricted to just one or very few codons of the entire gene, suggesting they have been positively selected during the course of tumor development. The purpose of this study is to investigate whether these characteristic oncogenic driver mutations are observed in the germline genes of any metazoan species. Six well-known tumor promoter genes were chosen for this survey including BRAF, KRAS, JAK2, PIK3CA, EGFR, and IDH1/2. The sites of all driver mutations were found to occur in highly conserved regions of each gene comparing protein sequences throughout diverse phyla of metazoan species. None of the oncogenic missense mutations were found in germlines of any species of current genome and protein databases. Despite many tumors readily selecting these somatic mutations, the conclusion drawn from this study is that these variants are negatively rejected if encountered as a germline mutation. While cancer expansion ensues from dysregulated growth elicited by these mutations, this effect is likely detrimental to embryonic development and/or survival of multicellular organisms. Although all oncogenic mutations considered here are gain-of-function where five of the six increase activity of the encoded proteins, clonal advancement promotes tumor growth by these genomic changes without conferring selection advantages benefiting the organism or species.

Abstract 135: Resolving the tumorigenesis continuum of DICER1 syndrome with novel lineage-trackable genetically engineered mouse model

Abstract Background: DICER1 syndrome is a rare cancer predisposition syndrome associated with germline DICER1 mutations, which develops pulmonary or extra-pulmonary manifestations mostly in pediatric patients. Unlike classic tumor suppressors, the 2nd hit in DICER1 gene is a missense mutation that renders the RNase IIIb domain defective; therefore, this mutant form of DICER1 is the only protein expressed in these cancer cells, leading to a biased 5p-miRNA production deficiency. Many DICER1 cancers are sarcomas; we therefore postulate the cell of origin is mesenchymal. We recently developed a RNase IIIb-mutant mouse strain and embryonic activation of this mutant along with silencing of the other Dicer1 allele in Mullerian mesenchymal progenitor cells led to the development of tumors resembling sarcomas in human patients. To expand the model and enable lineage tracking, we developed a tamoxifen inducible, tdTomato-trackable, Hypermethylated in Cancer 1 (HIC1)-creERT2 driven transgenic mouse strain. Hic1 marks mesenchymal progenitors. With this strain, we created a model that histologically recapitulates the 3 renal tumors in DICER1 syndrome: cystic nephromas, Wilms tumors, and anaplastic sarcomas. Objective: to identify oncogenic events underlying Dicer1 mutation-driven murine kidney tumor development with single cell RNA-sequencing (scRNA-seq). Method: To build the tumorigenesis continuum, we harvested kidneys from Dicer1+/fl-D1693N and Dicer1fl/fl-D1693N mice at 1, 3, 6, months post tamoxifen injection, sorted out tdTomato+/CD45-/Epcam- cells, and performed scRNA-seq. Five endpoint tumors were included for analyses. Single cell transcriptomic profiles were integrated and clustered using Seurat to identify cell populations that emerge and expand along the tumor development continuum. Differential gene expression, pathway analysis were conducted to identify genes/oncogenic pathways that are activated along the course of tumor development. Cell differentiation trajectory analysis will be performed using Monocle. Results: Integration of 5 tumors revealed diverse cell populations: epithelial, mesenchymal, endothelial cells, lymphocytes, and macrophages. We further clustered the mesenchymal population and identified sub-populations such as highly proliferative cells, muscle satellite cells, and terminally differentiated muscle cells. These mesenchymal cells show high expression of blastema markers (Ncam1, Sox11) - a histological component in Wilms tumors. Temporal trajectory of tumorigenesis with time point samples is currently being analyzed. Conclusion: With scRNA-seq, we begin to unravel heterogeneity of these murine tumors and will identify critical oncogenic events driving the development of DICER1 syndrome-associated cancer, enabling us to utilize this model to develop therapeutic approaches to improve patient management. Citation Format: Joyce Zhang, Shary Chen, Yana Moscovitz, Branden Lynch, Maxwell Douglas, Janine Senz, Wilder Scott, Michael Underhill, Yemin Wang, David Huntsman. Resolving the tumorigenesis continuum of DICER1 syndrome with novel lineage-trackable genetically engineered mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 135.

Abstract 2694: The role of innate lymphoid cells and innate like T cells in hepatocellular carcinoma

Abstract The major focus of cancer immunotherapy has been on conventional T cells due to their unique recognition of specific peptide antigens. Despite advances in the cancer immunotherapy field, including the use of atezolizumab and bevacizumab as first-line therapy for unresectable hepatocellular carcinoma (HCC), the overall mortality rate of HCC continues to rise. Innate lymphoid cells (ILC) and innate-like T cells (ILTC) are broadly categorized within the innate lymphoid population as playing an important role in inflammation, tissue repair and immune tolerance. Their ability to react rapidly to stimulants in their local tissue environment as well as to recruit and activate a variety of other immune cells makes these cells attractive targets for anti-tumor therapy. However, previous studies looking at ILCs and ILTCs in cancer have revealed both pro- and anti-tumor functions. Furthermore, the complex interplay between these cell populations in the tumor microenvironment is not well understood, particularly over the natural course of tumor development. In this study, we aim to better elucidate the role of ILC and ILTC populations in HCC progression in terms of both their kinetics and role as potential therapeutic targets. We performed hydrodynamic tail vein injections in mice with two different plasmid combinations to alter expression of commonly mutated oncogenes and tumor suppressors in human HCC (MYC;TP53 and MYC;CTNNB1). Histologic analysis of the livers was performed to determine appropriate time points to assess ILC and ILTC populations. We then designed a comprehensive 29-plex spectral flow panel to assess broad kinetic changes in the frequency of ILC, ILTC, and conventional T cell populations. We show that in early time points (4-7 days post-injection) when neoplastic transformation of hepatocytes has already occurred, mucosal-associated invariant T (MAIT) cells, group 1 ILCs, ILC2s, and ILC3s expand in frequency for both tumor models. Cytokine analysis shows a generalized decreased trend across timepoints in effector cytokine production in MAITs and group 1 ILCs, suggesting gradual dysfunction over time. Taken together, these preliminary findings show that ILC and ILTC populations expand early during tumor progression but gradually decrease in functionality with tumor progression. Citation Format: Patrick Huang, Benjamin Ruf, Rajiv Trehan, Dana Soika, Chi Ma, Tim F. Greten, Firouzeh Korangy. The role of innate lymphoid cells and innate like T cells in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2694.

Transcriptional background effects on a tumor driver gene in different pigment cell types of medaka.

The Xiphophorus melanoma receptor kinase gene, xmrk, is a bona fide oncogene driving melanocyte tumorigenesis of Xiphophorus fish. When ectopically expressed in medaka, it not only induces development of several pigment cell tumor types in different strains of medaka but also induces different tumor types within the same animal, suggesting its oncogenic activity has a transcriptomic background effect. Although the central pathways that xmrk utilizes to lead to melanomagenesis are well documented, genes and genetic pathways that modulate the oncogenic effect and alter the course of disease have not been studied so far. To understand how the genetic networks between different histocytes of xmrk-driven tumors are composed, we isolated two types of tumors, melanoma and xanthoerythrophoroma, from the same xmrk transgenic medaka individuals, established the transcriptional profiles of both xmrk-driven tumors, and compared (1) genes that are co-expressed with xmrk in both tumor types, and (2) differentially expressed genes and their associated molecular functions, between the two tumor types. Transcriptomic comparisons between the two tumor types show melanoma and xanthoerythrophoroma are characterized by transcriptional features representing varied functions, indicating distinct molecular interactions between the driving oncogene and the cell-type-specific transcriptomes. Melanoma tumors exhibit gene signatures that are relevant to proliferation and invasion, while xanthoerythrophoroma tumors are characterized by expression profiles related to metabolism and DNA repair. We conclude the transcriptomic backgrounds, exemplified by cell-type-specific genes that are downstream of xmrk effected signaling pathways, contribute the potential to change the course of tumor development and may affect overall tumor outcomes.

Bayesian Inference of Tissue Heterogeneity for Individualized Prediction of Glioma Growth.

Reliably predicting the future spread of brain tumors using imaging data and on a subject-specific basis requires quantifying uncertainties in data, biophysical models of tumor growth, and spatial heterogeneity of tumor and host tissue. This work introduces a Bayesian framework to calibrate the two-/three-dimensional spatial distribution of the parameters within a tumor growth model to quantitative magnetic resonance imaging (MRI) data and demonstrates its implementation in a pre-clinical model of glioma. The framework leverages an atlas-based brain segmentation of grey and white matter to establish subject-specific priors and tunable spatial dependencies of the model parameters in each region. Using this framework, the tumor-specific parameters are calibrated from quantitative MRI measurements early in the course of tumor development in four rats and used to predict the spatial development of the tumor at later times. The results suggest that the tumor model, calibrated by animal-specific imaging data at one time point, can accurately predict tumor shapes with a Dice coefficient 0.89. However, the reliability of the predicted volume and shape of tumors strongly relies on the number of earlier imaging time points used for calibrating the model. This study demonstrates, for the first time, the ability to determine the uncertainty in the inferred tissue heterogeneity and the model-predicted tumor shape.

Regional mutational signature activities in cancer genomes.

Cancer genomes harbor a catalog of somatic mutations. The type and genomic context of these mutations depend on their causes and allow their attribution to particular mutational signatures. Previous work has shown that mutational signature activities change over the course of tumor development, but investigations of genomic region variability in mutational signatures have been limited. Here, we expand upon this work by constructing regional profiles of mutational signature activities over 2,203 whole genomes across 25 tumor types, using data aggregated by the Pan-Cancer Analysis of Whole Genomes (PCAWG) consortium. We present GenomeTrackSig as an extension to the TrackSig R package to construct regional signature profiles using optimal segmentation and the expectation-maximization (EM) algorithm. We find that 426 genomes from 20 tumor types display at least one change in mutational signature activities (changepoint), and 306 genomes contain at least one of 54 recurrent changepoints shared by seven or more genomes of the same tumor type. Five recurrent changepoint locations are shared by multiple tumor types. Within these regions, the particular signature changes are often consistent across samples of the same type and some, but not all, are characterized by signatures associated with subclonal expansion. The changepoints we found cannot strictly be explained by gene density, mutation density, or cell-of-origin chromatin state. We hypothesize that they reflect a confluence of factors including evolutionary timing of mutational processes, regional differences in somatic mutation rate, large-scale changes in chromatin state that may be tissue type-specific, and changes in chromatin accessibility during subclonal expansion. These results provide insight into the regional effects of DNA damage and repair processes, and may help us localize genomic and epigenomic changes that occur during cancer development.

Leveraging Nanodrug Delivery System for Simultaneously Targeting Tumor Cells and M2 Tumor-Associated Macrophages for Efficient Colon Cancer Therapy.

Tumor-associated macrophages (TAMs) widely exist in the solid tumors, which participate in the entire course of tumor development and execute momentous impacts. Therefore, manipulating TAMs has been identified as an expecting strategy with immense potential for cancer therapy. Herein, a nanodrug delivery system was leveraged for simultaneously targeting tumor cells and M2-type TAMs for efficient colon cancer therapy. The broad-spectrum anticancer chemotherapeutic drug doxorubicin (DOX) was hitchhiked in a mannose-modified bovine serum albumin (MAN-BSA) carrier. The DOX@MAN-BSA nanodrug delivery system was verified to possess feasible physical performances for unhindered systemic circulation and active targeting on colon tumors. DOX@MAN-BSA nanoparticles could be preferentially swallowed by colon tumor cells and M2 TAMs through mannose receptor-mediated endocytosis. Further in vivo antitumor therapy in CT26 colon tumor-bearing mice has achieved remarkable suppression efficacy with satisfactory biosafety. Leveraging the nanodrug delivery system for simultaneously targeting tumor cells and M2 TAMs has contributed a feasible strategy to collaboratively repress the malignant tumor cells and the collusive M2 TAMs for efficient cancer therapy.

Abstract 2468: Genetic metastatic colorectal cancer model with in vivo imaging capabilities

Abstract Introduction: Colorectal cancer (CRC) is the second leading cancer related cause of death worldwide, responsible for nearly 2 million deaths annually, most commonly due to complications from metastasis. A mouse model that reproduces human CRC is critically important to support pre-clinical research in the field. Recently a novel inducible genetically engineered mouse model in an immune competent B6 background was developed that metastasizes to the lung, liver, and peritoneum recapitulating what is seen in CRC patients. This model uses the Lgr5 (L) promoter to drive tamoxifen inducible Cre recombinase with the following conditional alleles: Apc (A), mutant Kras G12D (K), Tgfbr2 (T), and p53 (P) (LAKTP). After establishing the model in our center, we found mice developing tumors in the absence of tamoxifen induction. Because the original model lacked the ability to monitor the tumor development and progression in vivo in real time we created a LAKTP based mouse model that would allow for easy monitoring of tumor growth over time, allowing us to determine the frequency and time course of tumor development in the absence of tamoxifen induction. Methods: C57BL/6J mice, with each individual mutation (L, A, K, T, and P), were purchased from Jackson Laboratory and used for breeding in-house. Following breeding, congenic C57BL/6J LAKTP mice were characterized prior to breeding in conditional Luciferase (Rosa26 LSL Luciferase) which was also purchased from Jackson Laboratory. Luciferin was provided by intraperitoneal injection prior to capturing the bioluminescence signal using an imager from Spectral Imaging Systems. Tissue samples were fixed in formalin and embedded in paraffin prior sectioning for histology and immunohistochemistry. Results: Approximately 30% of our LAKTP mice died or required sacrifice due to meeting humane endpoints around 12 weeks of life in the absence of tamoxifen induction. Upon dissection, these mice exhibited large tumors located in the small intestine that led to non-survival bowel obstructions. Ten percent of these mice had liver metastases as well. Breeding in the conditional Luciferase allele allowed for detection of developing, non-induced tumors as early as 6 weeks of life. In addition to being useful for monitoring tumor growth in vivo, immunostaining of tumor tissue with anti-luciferase antibodies allows for detection of cells derived from Lgr5 positive intestinal stem cells. Conclusion: The LAKTP mouse model is an important tool for studying CRC, but exhibits tumor development due to leaky Cre activity in the absence of tamoxifen in approximately 30% of mice by 12 weeks. Our strategy of breeding in conditional luciferase allows for in vivo imaging such that mice can be partitioned based on bioluminescence for trials that require equivalent tumor burden at the beginning of interventions. In addition, bioluminescence imaging also allows for tumor size to be monitored over time. Citation Format: Tyler Leiva, James Griffith, Shaoxuan Guo, Megan Lerner, Lacey McNally, William Berry, Katherine Morris. Genetic metastatic colorectal cancer model with in vivo imaging capabilities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2468.

The fate and function of NK cells in the suppressive tumor microenvironment

Abstract Natural killer (NK) cells are an important cell subset of the innate immune system. Accumulating evidence indicates that NK cells are dysfunctional in the tumor microenvironment during cancer development. However, the dynamic changes in phenotypes and functional interactions within the tumor microenvironment during tumor development and progression are unknown. Here, we used both the mouse E0771 breast cancer and B16F0 melanoma tumor models to mimic different clinical stages of human cancers and characterized the dynamic distributions and qualities of NK cells in different organs and tumors associated with tumor progression. We found that NK cells are dynamically involved in the immune responses to cancer with different distributions and phenotypic profiles in tumor sites and other peripheral organs during the course of tumor development and progression. In the early stages of tumor development, NK cells exhibit effector properties. In the later cancer stages, NK cells have impaired cytotoxic capacities and dysfunctional states. Importantly, we found that melanoma and breast cancer cells promote NK cells to become senescent NK cells in vitro and in vivo in tumor microenvironments. These studies provide a better understanding of the dynamic and functional role of NK cells in anti-tumor immunity, which may facilitate the development of novel immunotherapies targeting NK cells for cancer treatment. the Melanoma Research Alliance, American Cancer Society, and the National Institutes of Health.

Circulating miR-146a expression as a non-invasive predictive biomarker for acute lymphoblastic leukemia

Dysregulation of non-coding microRNAs during the course of tumor development, invasion and/or progression to the distant organs, makes them a promising candidate marker for the diagnosis of cancer and associated malignancies. This exploratory study aims at evaluating the usefulness of plasma concentration of circulating mir-146a as a non-invasive biomarker for acute lymphoblastic leukemia (ALL). Total RNA including miRNA was isolated from 110 plasma samples of patients (n = 66), healthy controls (n = 24) and follow up (n = 20) cases and reverse transcribed. Relative concentrations were assessed using real-time quantitative PCR and fold-change was calculated by 2−ΔΔCt method. Finally, relative concentrations were correlated to clinicopathological factors. Patients (n = 66) were analyzed to determine fold expression of miR-146a in plasma samples of ALL. Before chemotherapy, pediatric (n = 42) and adult (n = 24) showed overexpression of miR-146a compared with healthy controls (P < 0.0001). There was no effect of age and gender on mir-146a expression in plasma. mirR-146a expression was independent of clinical and hematological features. Moreover, miR-146a levels in plasma of paired samples (n = 20) after treatment showed significant decrease in expression (P < 0.001). Expression of plasma miR-146a may be utilized as non-invasive marker to diagnose and predict prognosis in pediatric and adult patients with ALL. Moreover predicted targets may be utilized for ALL therapy in future.

Abstract 2667: RAS-induced transformation of mammary epithelial cells relies upon a ZEB1-dependent cellular reprogramming through a paracrine process

Abstract Despite an enhanced interest, the origin of cancer stem cells (CSCs) remains equivocal. A strong body of evidence suggests that, in some adult carcinomas, CSC may originate from normal mature differentiated cells through a dedifferentiation process and the acquisition of stem-like properties. At the crux of this concept is the epithelial-mesenchymal transition (EMT), an embryonic transdifferentiation process that can be abnormally reactivated over the course of tumor development as a result of oncogenic or microenvironmental cues. High expression of EMT genes and stemness features are hallmarks of claudin-low (CL) breast cancers, a rare subtype of breast malignancies, generally considered as the most primitive breast cancers. We have recently reported that this molecular subtype of breast cancers exhibits a significant diversity, comprising three main subgroups (CL1, CL2 and CL3), that emerge from unique evolutionary processes. Genetic and epigenetic analyses support the hypothesis that CL1 tumors generate from the transformation of a normal mammary stem cells, whereas CL2 and CL3 arise from the reactivation of an EMT process over the course of tumor progression. Interestingly, CL tumors share the frequent activation of the RAS-MAPK pathway, suggesting a potent role for the RAS pathway in the development of CL tumors. Based on these findings, we wondered whether RAS pathway activation could induce the reactivation of EMT-TFs allowing the emergence of CSCs displaying CL2 and CL3 characteristics. By expressing an oncogenic form of RAS in differentiated epithelial cells (HME), we observed a phenotypic switch towards a CD24-/low/CD44+ phenotype, associated with CSC properties, and suggesting an acquisition of cellular plasticity after RAS activation. By using CRISPR/Cas9 knock-down experiments we have shown i) the emergence of CD24-/low/CD44+ cells, ii) the acquisition of transformation and stemness features, and iii) that the cellular plasticity acquired after RAS activation in differentiated HME strongly rely on ZEB1. Single-cell RNA sequencing analyses after RAS activation in HME-RAS cells suggested two different outcomes: senescence or EMT. Treatment with a senolytic drug resulted in reduction of RAS-induced senescent cells associated with a decrease in the emergence of CD24-/low/CD44+ cells and ZEB1 expression. Finally, a co-culture reporter system and a depletive approach with cytokine-neutralizing antibodies highlighted a paracrine effect of IL-6 and IL-1α, produced by senescent RAS-expressing cells on their neighboring differentiated cells, leading to the acquisition of ZEB1-dependent cellular plasticity. Hence, we unveiled a new paracrine process, in which cytokines secreted by cells undergoing oncogene-induced senescence drive ZEB1 expression, thus promoting cellular plasticity correlated with the emergence of CSCs. Citation Format: ALAIN PUISIEUX, Hadrien De Blander, Laurie Tonon, Frédérique Fauvet, Roxane Pommier, Christelle Lamblot, Rahma Benhassoun, Benjamin Gibert, Maria Ouzounova, Anne-Pierre Morel. RAS-induced transformation of mammary epithelial cells relies upon a ZEB1-dependent cellular reprogramming through a paracrine process [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 2667.

NK and NKT cells have distinct properties and functions in cancer.

Natural killer (NK) and natural killer T (NKT) cells are two important cell subsets of the innate immune system. NK and NKT cells share many phenotypes and functions for anti-tumor immunity; however, the dynamic changes in phenotypes and functional interactions within the tumor microenvironment during tumor development and progression are unknown. Here we report that NK and NKT cells have distinct properties, metabolic profiles, and functions during tumor development. Using the mouse E0771 breast cancer and B16 melanoma models, we found that both NK and NKT cells are dynamically involved in the immune responses to cancer but have distinct distributions and phenotypic profiles in tumor sites and other peripheral organs during the course of tumor development and progression. In the early stages of tumor development, both NK and NKT cells exhibit effector properties. In the later cancer stages, NK and NKT cells have impaired cytotoxic capacities and dysfunctional states. NK cells become senescent cells, while NKT cells, other than invariant NKT (iNKT) cells, are exhausted in the advanced cancers. In contrast, iNKT cells develop increases in activation and effector function within the breast tumor microenvironment. In addition, senescent NK cells have heightened glucose and lipid metabolism, but exhausted NKT cells display unbalanced metabolism in tumor microenvironments of both breast cancer and melanoma tumor models. These studies provide a better understanding of the dynamic and distinct functional roles of NK and NKT cells in anti-tumor immunity, which may facilitate the development of novel immunotherapies targeting NK and NKT cells for cancer treatment.

An Unusual Presentation of Dermatofibrosarcoma Protuberans: a Small Inflamed Erosion with Spontaneous Hemorrhage on the Alae of the Nose

A case of dermatofibrosarcoma protuberans (DFSP), which has been diagnosed as inflammatory granuloma at the first time and treated with laser ablation, because it has an atypical clinical history, including a short course of tumor development, spontaneous hemorrhage from lesion, and ulceration in surface. The lesion recurred rapidly and underwent a surgical resection of the tumor with margins being negative. Histopathological examinations revealed the final diagnosis of dermatofibrosarcoma protuberans. No recurrence has been observed with 1-year follow-up. This case indicates that dermatofibrosarcoma protuberans may have non-typical morphology; early biopsy in rapidly growing nodule is necessary.

A31 A Reservoir of Tumor-Specific CD8 T Cells in Lung Cancer Resides in the Draining Lymph Node

Recent work has described the population of CD8 T cells that respond to anti-PD-1 therapy (marked by TCF1 and PD-1), but it remains unclear how these T cells are maintained within the immunosuppressive tumor microenvironment (TME) of lung cancer. To understand this, we developed a genetically engineered model in which Kras-G12D expressing p53 deficient lung adenocarcinomas express a known neoantigen called the iNversion INduced Joined neoAntigen (NINJA). NINJA allows us to follow neoantigen-specific CD8 T cells over the course of tumor development. We find that ∼20% of tumor-specific T cells in early 8-wk tumors are TCF1+, but by 17-20 wks, this TCF1+ has significantly shrunk, and there has been a concomitant increase in the expression of markers of T-cell terminal differentiation (Tim3). This is consistent with the idea that T cells receive signals in the TME that drive terminal differentiation and restrict responses to immunotherapy. We reasoned that if the signals driving terminal differentiation were provided in the TME, neoantigen-specific T cells in the tumor-draining lymph node (dLN) may remain less differentiated over the course of tumor development. Analysis of tumor-specific T cells in the dLNs of 8-wk and 17-wk tumors showed that they were mostly TCF1+. Moreover, single-cell transcriptional analyses suggested that these cells were less differentiated than their counterparts in tumors. T-cell receptor (TCR) signals are a major driver of terminal differentiation, and we observed that tumor-specific T cells in the dLN were not receiving TCR signals, while all T cells in the TME were positive for TCR signals. This suggested at least two models for how antitumor T cells function: 1) tumor-reactive T cells in dLNs and TME could function independently of one another, or 2) tumor-reactive T cells might have a role in sustaining the antitumor T-cell response over the course of lung-tumor development through migration. Consistent with the latter model, TCR sequencing of dLN and TME neoantigen-specific T cells showed a close clonal relationship: 13 of the top 15 clones in the TME were present in the dLN, and the hierarchy of clonal dominance was maintained. This was also true in 17-wk tumor-bearing mice, suggesting that the population of tumor-specific CD8 T cells in the dLN serves as a reservoir of less differentiated cells that can continuously replenish the T cells in the TME, helping to sustain the antitumor T-cell response over the course of tumor development. Critically, it is unclear whether current immunotherapeutic strategies leverage this reservoir of T cells, raising the possibility that this population of dLN tumor-specific T cells could be targeted to provide significant additional benefit for patients receiving immunotherapy.

Oncogenic RUNX3: A Link between p53 Deficiency and MYC Dysregulation.

The RUNX transcription factors serve as master regulators of development and are frequently dysregulated in human cancers. Among the three family members, RUNX3 is the least studied, and has long been considered to be a tumor-suppressor gene in human cancers. This idea is mainly based on the observation that RUNX3 is inactivated by genetic/epigenetic alterations or protein mislocalization during the initiation of tumorigenesis. Recently, this paradigm has been challenged, as several lines of evidence have shown that RUNX3 is upregulated over the course of tumor development. Resolving this paradox and understanding how a single gene can exhibit both oncogenic and tumor-suppressive properties is essential for successful drug targeting of RUNX. We propose a simple explanation for the duality of RUNX3: p53 status. In this model, p53 deficiency causes RUNX3 to become an oncogene, resulting in aberrant upregulation of MYC.

Development of a Mouse Glioblastoma Orthotopic Model Using the GLi-261 Cell Line

Glioblastoma is the most common and most aggressive type of brain tumor, with an almost 100 % mortality rate over 5 years. The search for new effective approaches to the treatment of this disease requires the development of adequate experimental models.Objective: to develop and put into practice an orthotopic model of mouse glioblastoma.Materials and methods: GLi-261 mouse glioma cells were orthotopically inoculated into the putamen of C57Bl/6 mice brain. Tumor dynamics was investigated by Preclinical MRI System 7.0T/17cm (Flexiscan) highfield magnetic resonance imager (MR Solutions, UK). Temcital® (temozolomide) was used as a positive control in the treatment of experimental glioblastoma. The neurological status of animals in the course of tumour development was assessed by specific tests.Results: a GLi-261 cell-based mouse glioblastoma orthotopic model was developed using stereotactic equipment for accurate inoculation of tumour cells, magnetic resonance imaging for non-invasive determination of tumour volume and dynamics, and special tests for determination of the neurological status of the biological test systems. This model was used to demonstrate the effectiveness of temozolomide (the «gold standard» for glioblastoma treatment).Conclusions: this model has been introduced into practice at the IBC Generium, LLC, and can be used as an in vivo test system for preclinical evaluation of efficacy of new antitumour drugs being developed, as well as brain cancer treatment regimens using combination therapy.

Longitudinal immune characterization of syngeneic tumor models to enable model selection for immune oncology drug discovery

BackgroundThe ability to modulate immune-inhibitory pathways using checkpoint blockade antibodies such as αPD-1, αPD-L1, and αCTLA-4 represents a significant breakthrough in cancer therapy in recent years. This has driven interest in identifying small-molecule-immunotherapy combinations to increase the proportion of responses. Murine syngeneic models, which have a functional immune system, represent an essential tool for pre-clinical evaluation of new immunotherapies. However, immune response varies widely between models and the translational relevance of each model is not fully understood, making selection of an appropriate pre-clinical model for drug target validation challenging.MethodsUsing flow cytometry, O-link protein analysis, RT-PCR, and RNAseq we have characterized kinetic changes in immune-cell populations over the course of tumor development in commonly used syngeneic models.ResultsThis longitudinal profiling of syngeneic models enables pharmacodynamic time point selection within each model, dependent on the immune population of interest. Additionally, we have characterized the changes in immune populations in each of these models after treatment with the combination of α-PD-L1 and α-CTLA-4 antibodies, enabling benchmarking to known immune modulating treatments within each model.ConclusionsTaken together, this dataset will provide a framework for characterization and enable the selection of the optimal models for immunotherapy combinations and generate potential biomarkers for clinical evaluation in identifying responders and non-responders to immunotherapy combinations.

Tumor dormancy as an alternative step in the development of chemoresistance and metastasis - clinical implications.

The ability of a tumor to become dormant in response to suboptimal conditions has recently been recognized as a key step in tumor progression. Tumor dormancy has been found to be implicated in several tumor types as the culprit of therapy resistance and metastasis development, the deadliest features of a cancer. Several lines of evidence indicate that the development of these traits may rely on the de-differentiation of committed tumor cells that regain stem-like properties during a dormant state. Presently, dormancy is classified into cell- and population-level, according to the preponderance of cellular mechanisms that keep tumor cells quiescent or to a balance between overall cell division and death, respectively. Cellular dormancy is characterized by autophagy, stress-tolerance signaling, microenvironmental cues and, of prime relevance, epigenetic modifications. It has been found that the epigenome alters during cellular quiescence, thus representing the driving force for short-term cancer progression. Population-level dormancy is characterized by processes that counteract proliferation, such as inappropriate blood supply and intense immune responses. The latter two mechanisms are not mutually exclusive and may affect tumor masses both simultaneously and subsequently. Overall, tumor dormancy may represent an additional step in the acquisition of cancer characteristics, and its comprehension may clarify both theoretical and practical aspects of cancer development. Clinically, only a deep understanding of dormancy may explain the course of tumor development in different patients, thus representing a process that may be targeted to prevent and/or treat advanced-stage cancers. That is especially the case for breast cancer, against which the mTOR inhibitor everolimus displays potent antitumor activity in patients with metastatic disease by impeding autophagy and tumor dormancy onset. Here we will also discuss other targeted therapies directed towards tumor dormancy onset, e.g. specific inhibitors of SFK and MEK, or aimed at keeping tumor cells dormant, e.g. prosaposin derivatives, that may shortly enter clinical assessment in breast, and possibly other cancer types.

Hiding in Plain Sight: Epigenetic Plasticity in Drug-Induced Tumor Evolution.

Cancer is a heterogeneous disease with key differences at the cellular and molecular levels. Acquisition of these differences during the course of tumor development manifests into functional and phenotypic heterogeneity leading to tumor diversity, also referred to as intra-tumor heterogeneity (ITH). Within a tumor, there are subpopulations of cells capable of tumor initiation and maintenance. These cells often exhibit resistance to standard-of-care anti-cancer drugs. However, the role of various subpopulations (clones) in drug resistance remains to be investigated. Moreover, the jury is still out about whether drug resistance is a result of clonal selection of preexisting cells, or the cells acquire resistance by dynamic re-wiring of their epigenome. Therefore, we investigated the drug-induced tumor evolution in patient-derived primary cells of head and neck squamous cell carcinoma. Our data demonstrated the role of a preexisting poised epigenetic state in drug-induced adaptive evolution of tumor cells. Importantly, the combination of chemotherapy and epigenetic inhibitors can prevent/delay drug-induced tumor evolution.

Abstract IA07: Influence of cancer initiation on tumor progression in SCLC

Abstract The vast majority of cancer patients die from metastatic disease, but the molecular and cellular mechanisms that drive metastatic progression often remain poorly understood. Small cell lung cancer (SCLC) is a neuroendocrine form of lung cancer and one of the most metastatic and lethal cancers. Most SCLC patients are usually first diagnosed when they already have metastatic disease, and SCLC tumors are thus thought to acquire metastatic ability early in the course of tumor development. However, how early events shape the evolution and metastatic progression of SCLC is largely unknown. We will present evidence, using genetically engineered mouse models of SCLC, that distinct mechanisms drive metastatic progression depending on the cell of origin. In one mouse model, we found that tumors gain metastatic ability through amplification of the transcription factor Nfib; this amplification is accompanied by a striking opening of the chromatin at many sites in the genome. In the second model, metastatic progression is not associated with Nfib-driven chromatin alterations. Gene expression profiling studies reveal distinct mechanisms of metastatic progression in the two groups, as well as markers predictive of the two metastatic paths. Our data indicate that Nfib-independent metastases arise from pulmonary neuroendocrine cells. These observations demonstrate that the identity of tumor-initiating cells can determine the molecular mechanisms of tumor evolution. Citation Format: Dian Yang, Monte M. Winslow, Julien Sage. Influence of cancer initiation on tumor progression in SCLC [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr IA07.