Suppression of Lung Adenocarcinoma Progression by Nkx2-1

Despite the high prevalence and poor outcome of patients with metastatic lung cancer the mechanisms of tumour progression and metastasis remain largely uncharacterized. Here we modelled human lung adenocarcinoma, which frequently harbours activating point mutations in KRAS and inactivation of the p53 pathway, using conditional alleles in mice. Lentiviral-mediated somatic activation of oncogenic Kras and deletion of p53 in the lung epithelial cells of Kras[superscript LSL-G12D/+];p53[superscript flox/flox] mice initiates lung adenocarcinoma development4. Although tumours are initiated synchronously by defined genetic alterations, only a subset becomes malignant, indicating that disease progression requires additional alterations. Identification of the lentiviral integration sites allowed us to distinguish metastatic from non-metastatic tumours and determine the gene expression alterations that distinguish these tumour types. Cross-species analysis identified the NK2-related homeobox transcription factor Nkx2-1 (also called Ttf-1 or Titf1) as a candidate suppressor of malignant progression. In this mouse model, Nkx2-1 negativity is pathognomonic of high-grade poorly differentiated tumours. Gain- and loss-of-function experiments in cells derived from metastatic and non-metastatic tumours demonstrated that Nkx2-1 controls tumour differentiation and limitsmetastatic potential in vivo. Interrogation of Nkx2-1-regulated genes, analysis of tumours at defined developmental stages, and functional complementation experiments indicate that Nkx2-1 constrains tumours in part by repressing the embryonically restricted chromatin regulator Hmga2. Whereas focal amplification of NKX2-1 in a fraction of human lung adenocarcinomas has focused attention on its oncogenic function, our data specifically link Nkx2-1 downregulation to loss of differentiation, enhanced tumour seeding ability and increased metastatic proclivity. Thus, the oncogenic and suppressive functions ofNkx2-1 in the sametumourNational Institutes of Health (U.S.) (grant U01-CA84306 )National Institutes of Health (U.S.) (grant K99-CA151968)Howard Hughes Medical InstituteLudwig Center for Molecular OncologyNational Cancer Institute (U.S.) (Cancer Center Support (core) grant P30-CA14051

Genetically engineered mouse models of cancer reveal new insights about the antitumor immune response

Cancer is a complex disease that can originate in virtually all the tissues of the body, and tumors progress through many different stages during their development. While genetic mutations in the emerging cancer cells drive this disease, it has become increasingly clear that cancer development is strongly influenced by the surrounding microenvironment. Cells of the immune system are critical components of this extrinsic network of cancer regulators, contributing significantly to the microenvironment of most cancers and either promoting or inhibiting the initiation and progression of this disease. Genetically engineered mouse (GEM) mouse models of spontaneous cancer are starting to shape our understanding of how antitumor T cells may act to prevent or inhibit cancer progression in some settings and not others. Lessons learned from investigating spontaneous mouse cancer models have important implications for directing clinical efforts that attempt to direct a cancer patient's immune system to eradicate their disease.National Institutes of Health (U.S.) (Grant 1 U54 CA126515-01)National Cancer Institute (U.S.) (Cancer Center Support (core) Grant P30-CA14051)John D. Proctor Foundation (Margaret A. Cunningham Immune Mechanisms in Cancer Research Fellowship Award)Howard Hughes Medical Institute (Investigator)Virginia and Daniel K. Ludwig Graduate Fellowshi

MHC-II neoantigens shape tumour immunity and response to immunotherapy

The ability of the immune system to eliminate and shape the immunogenicity of tumours defines the process of cancer immunoediting1. Immunotherapies such as those that target immune checkpoint molecules can be used to augment immune-mediated elimination of tumours and have resulted in durable responses in patients with cancer that did not respond to previous treatments. However, only a subset of patients benefit from immunotherapy and more knowledge about what is required for successful treatment is needed2–4. Although the role of tumour neoantigen-specific CD8+ T cells in tumour rejection is well established5–9, the roles of other subsets of T cells have received less attention. Here we show that spontaneous and immunotherapy-induced anti-tumour responses require the activity of both tumour-antigen-specific CD8+ and CD4+ T cells, even in tumours that do not express major histocompatibility complex (MHC) class II molecules. In addition, the expression of MHC class II-restricted antigens by tumour cells is required at the site of successful rejection, indicating that activation of CD4+ T cells must also occur in the tumour microenvironment. These findings suggest that MHC class II-restricted neoantigens have a key function in the anti-tumour response that is nonoverlapping with that of MHC class I-restricted neoantigens and therefore needs to be considered when identifying patients who will most benefit from immunotherapy.National Institutes of Health Cancer Center Support Grant (P30CA14051

Enhanced adaptive immune responses in lung adenocarcinoma through natural killer cell stimulation

Natural killer (NK) cells inhibit tumor development in mouse models and their presence in tumors correlates with patient survival. However, tumor-associated NK cells become dysfunctional; thus, stimulation of NK cells in cancer is emerging as an attractive immunotherapeutic strategy. In a mouse model of lung adenocarcinoma, NK cells localized to tumor stroma with immature phenotypes and low functional capacity. To test their responsiveness within established disease,we engineered a system for inducible expression of activating ligands in tumors. After stimulation, NK cells localized inside tumors, with increased cytokine production capacity. Strikingly, T cells were also recruited to tumors in an NK cell-dependent manner, and exhibited higher functionality. In neoantigen-expressing tumors, NK cell stimulation enhanced the number and function of tumor-specific T cells and, in long-term settings, reduced tumor growth. Thus, even in established disease NK cells can be activated to contribute to antitumor immunity, supporting their potential as an important target in cancer immunotherapy.National Institutes of Health (Grant U54 CA126515-01)National Institutes of Health (Grant R01-CA185020-01

Endogenous T Cell Responses to Antigens Expressed in Lung Adenocarcinomas Delay Malignant Tumor Progression

Neoantigens derived from somatic mutations in tumors may provide a critical link between the adaptive immune system and cancer. Here, we describe a system to introduce exogenous antigens into genetically engineered mouse lung cancers to mimic tumor neoantigens. We show that endogenous T cells respond to and infiltrate tumors, significantly delaying malignant progression. Despite continued antigen expression, T cell infiltration does not persist and tumors ultimately escape immune attack. Transplantation of cell lines derived from these lung tumors or prophylactic vaccination against the autochthonous tumors, however, results in rapid tumor eradication or selection of tumors that lose antigen expression. These results provide insight into the dynamic nature of the immune response to naturally arising tumors.National Institutes of Health (U.S.) (Grant 1 U54 CA126515-01)National Cancer Institute (U.S.) (Cancer Center Support (Core) Grant P30-CA14051)John D. Proctor Foundation (Margaret A. Cunningham Immune Mechanisms in Cancer Research Fellowship Award)Damon Runyon Cancer Research Foundation (Fellowship)Genentech, Inc. (Postdoctoral Fellowship)Virginia and D.K. Ludwig Fund for Cancer ResearchHoward Hughes Medical Institut

Regulatory T Cells in Tumor-Associated Tertiary Lymphoid Structures Suppress Anti-tumor T Cell Responses

Infiltration of regulatory T (Treg) cells into many tumor types correlates with poor patient prognoses. However, mechanisms of intratumoral Treg cell function remain to be elucidated. We investigated Treg cell function in a genetically-engineered mouse lung adenocarcinoma model and found Treg cells suppress anti-tumor responses in tumor-associated tertiary lymphoid structures (TA-TLS). TA-TLS have been described in human lung cancers, but their function remains to be determined. TLS in this model were spatially associated with >90% of tumors and facilitated interactions between T cells and tumor-antigen presenting dendritic cells (DCs). Costimulatory ligand expression by DCs and T cell proliferation rates increased in TA-TLS upon Treg cell depletion, leading to tumor destruction. Thus, we propose Treg cells in TA-TLS can inhibit endogenous immune responses against tumors, and targeting these cells may provide therapeutic benefit for cancer patients.National Cancer Institute (U.S.) (Cancer Center Support Grant P30-CA14051)Howard Hughes Medical InstituteNational Institutes of Health (U.S.) (Grants 1 U54 CA126515-01, R01-CA185020-01 and T32 GM007753)Damon Runyon Cancer Research FoundationJohn D. Proctor Foundation (Margaret A. Cunningham Immune Mechanisms in Cancer Research Fellowship Award)Lung Cancer Research FoundationHoward Hughes Medical Institute (Investigator)Daniel K. Ludwig Schola

Suppression of lung adenocarcinoma progression by Nkx2-1.

Despite the high prevalence and poor outcome of patients with metastatic lung cancer the mechanisms of tumour progression and metastasis remain largely uncharacterized. Here we modelled human lung adenocarcinoma, which frequently harbours activating point mutations in KRAS and inactivation of the p53 pathway, using conditional alleles in mice. Lentiviral-mediated somatic activation of oncogenic Kras and deletion of p53 in the lung epithelial cells of Kras(LSL-G12D/+);p53(flox/flox) mice initiates lung adenocarcinoma development. Although tumours are initiated synchronously by defined genetic alterations, only a subset becomes malignant, indicating that disease progression requires additional alterations. Identification of the lentiviral integration sites allowed us to distinguish metastatic from non-metastatic tumours and determine the gene expression alterations that distinguish these tumour types. Cross-species analysis identified the NK2-related homeobox transcription factor Nkx2-1 (also called Ttf-1 or Titf1) as a candidate suppressor of malignant progression. In this mouse model, Nkx2-1 negativity is pathognomonic of high-grade poorly differentiated tumours. Gain- and loss-of-function experiments in cells derived from metastatic and non-metastatic tumours demonstrated that Nkx2-1 controls tumour differentiation and limits metastatic potential in vivo. Interrogation of Nkx2-1-regulated genes, analysis of tumours at defined developmental stages, and functional complementation experiments indicate that Nkx2-1 constrains tumours in part by repressing the embryonically restricted chromatin regulator Hmga2. Whereas focal amplification of NKX2-1 in a fraction of human lung adenocarcinomas has focused attention on its oncogenic function, our data specifically link Nkx2-1 downregulation to loss of differentiation, enhanced tumour seeding ability and increased metastatic proclivity. Thus, the oncogenic and suppressive functions of Nkx2-1 in the same tumour type substantiate its role as a dual function lineage factor

Conditional mouse lung cancer models using adenoviral or lentiviral delivery of Cre recombinase

The development of animal models of lung cancer is critical to our understanding and treatment of the human disease. Conditional mouse models provide new opportunities for testing novel chemopreventatives, therapeutics and screening methods that are not possible with cultured cell lines or xenograft models. This protocol describes how to initiate tumors in two conditional genetic models of human non-small cell lung cancer (NSCLC) using the activation of oncogenic K-ras alone or in combination with the loss of function of p53. We discuss methods for sporadic expression of Cre in the lungs through engineered adenovirus or lentivirus, and provide a detailed protocol for the administration of the virus by intranasal inhalation or intratracheal intubation. The protocol requires 1–5 min per mouse with an additional 30–45 min to set-up and allow for the recovery of mice from anesthesia. Mice may be analyzed for tumor formation and progression starting 2–3 weeks after infection.Howard Hughes Medical InstituteNational Cancer Institute (U.S.) (Cancer Center Support Grant)Massachusetts Institute of Technology. Ludwig Center for Molecular Oncolog

Control of PI(3) kinase in Treg cells maintains homeostasis and lineage stability.

Foxp3(+) regulatory T cells (Treg cells) are required for immunological homeostasis. One notable distinction between conventional T cells (Tconv cells) and Treg cells is differences in the activity of phosphatidylinositol-3-OH kinase (PI(3)K); only Tconv cells downregulate PTEN, the main negative regulator of PI(3)K, upon activation. Here we found that control of PI(3)K in Treg cells was essential for lineage homeostasis and stability. Mice lacking Pten in Treg cells developed an autoimmune-lymphoproliferative disease characterized by excessive T helper type 1 (TH1) responses and B cell activation. Diminished control of PI(3)K activity in Treg cells led to reduced expression of the interleukin-2 (IL-2) receptor α subunit CD25, accumulation of Foxp3(+)CD25(-) cells and, ultimately, loss of expression of the transcription factor Foxp3 in these cells. Collectively, our data demonstrate that control of PI(3)K signaling by PTEN in Treg cells is critical for maintaining their homeostasis, function and stability