Discovery and Validation of Tumor Immunoevasion Mechanisms

描述（由申请人提供）：目前还没有全面或令人满意的解释肿瘤如何逃避免疫反应。我们对肿瘤免疫逃避的了解大多局限于单一药物治疗靶向的个体机制。然而，肿瘤进化出多种冗余策略来规避免疫监视。这种可塑性有助于仅靶向一种/几种途径的癌症免疫疗法的适度功效。我们面临的挑战是：全面破译肿瘤逃避免疫监视的机制。具体来说，我们如何确定哪些可能是多余的，哪些可能是理想的药物靶点（例如，非冗余机制）？同样，哪些对肿瘤免疫逃避至关重要的途径可能会协同作用，并需要几种药物才能实现有效的免疫治疗？为了应对这一挑战，我们将建立检测方法，有意选择允许肿瘤逃避免疫反应的突变。利用免疫选择压力的力量，我们将确定单独和联合工作的机制，使肿瘤逃避免疫。我们的中心假设是，肿瘤进化免疫抑制基因表达模式，其微环境特异性，以逃避免疫攻击。我们进一步假设，这些基因表达的变化可以通过功能基因组学方法快速识别。我们以前曾使用转座子为基础的诱变，以确定启动肿瘤的突变。转座子是移动的遗传元件，其可以被设计成引起可快速识别的突变。在此，我们将定制基于转座子的诱变，以鉴定允许肿瘤在存在大量杀肿瘤T细胞的情况下茁壮成长的基因。我们的具体目标是识别和验证促进神经胶质瘤（一种侵袭性脑癌）逃避免疫监视的基因。为了实现这一点，诱变转座子将在培养的神经胶质瘤细胞中动员。将突变的神经胶质瘤细胞植入具有预先建立的免疫记忆的小鼠中，以选择仅在实现免疫逃避时生长的肿瘤细胞。与此同时，我们将在通过输注肿瘤特异性T细胞治疗的自发性胶质瘤小鼠中进行类似的遗传筛选。转座子插入将被克隆，测序和统计分析，以确定在许多逃避免疫的肿瘤中重复突变的基因。候选基因将在组织培养和动物模型实验中进行功能验证，以建立其表达和免疫逃避之间的因果关系。影响：确定肿瘤需要逃避免疫反应才能茁壮成长的综合途径，这将最终导致开发提高癌症免疫治疗疗效的药物。

DESCRIPTION (provided by applicant): There are currently no comprehensive or satisfactory explanations for how tumors evade the immune response. Much of our knowledge about tumor immunoevasion is limited to individual mechanisms that have been therapeutically targeted with single drugs. However, tumors evolve multiple, redundant strategies to circumvent immune surveillance. This plasticity has contributed to the modest efficacy of cancer immunotherapies that only target one/several pathway(s). The challenge we face is: to comprehensively decipher the mechanisms tumors use to evade immune surveillance. Specifically, how can we determine which of these may be redundant and which may be ideal drug targets (e.g., non-redundant mechanisms)? Similarly, which pathways crucial for tumor immunoevasion might synergize and require several drugs to achieve effective immunotherapy? In order to address this challenge we will establish assays that intentionally select for mutations that allow a tumor to escape an immune response. Using the power of immunologic selective pressure, we will indentify mechanisms that work alone and in combination to allow a tumor to escape immunity. Our central hypothesis is that tumors evolve immunosuppressive gene expression patterns specific to their microenvironment to evade immune attack. We further hypothesize that those changes in gene expression can be rapidly indentified by functional genomics approaches. We have previously used transposon-based mutagenesis to indentify mutations that initiate tumors. Transposons are mobile genetic elements that can be designed to cause mutations that are rapidly identifiable. Herein we will customize transposon-based mutagenesis to identify genes that allow a tumor to thrive despite the presence of high numbers of tumoricidal T cells. Our specific aim is to identify and validate genes that promote escape from immune surveillance in gliomas, an aggressive form of brain cancer. To accomplish this, mutagenic transposons will be mobilized in cultured glioma cells. Mutagenized glioma cells will be implanted into mice with pre-established immunological memory to select for tumor cells that only grow when immune evasion is achieved. In parallel, we will carry out a similar genetic screen in mice with spontaneously arising gliomas that are treated by infusion of tumor-specific T cells. Transposon insertions will be cloned, sequenced and statistically analyzed to identify genes that are repetitively mutated in many tumors that escape immunity. Candidate genes will be functionally validated in tissue culture and animal model experiments to establish cause and affect relationships between their expression and immunoevasion. IMPACT: The identification of integrated pathways that tumors require to escape immune responses in order to thrive, which will ultimately lead to the development of drugs that increase the efficacy of cancer immunotherapy.