Translational Research Career Development: Overcoming Resistance to Radiotherapy

头颈癌（HNC）是世界上第六大常见恶性肿瘤，诊断两次 在退伍军人中也是如此。放射治疗（RT）是癌症的重要组成部分 治疗;然而，其疗效受到放射抗性的限制，癌症有时 返回到治疗区域内。对RT的抗性至少部分是由适应性免疫系统介导的。 治疗诱导的信号传导事件。然而，我们还不了解使用的途径， 长期目标是更好地理解， 并随后靶向癌细胞用来逃避癌症的耐药机制。 辐射引起的死亡。组合适应性反应疗法（CART）代表了一种新的 该平台允许快速和系统地识别治疗组合， 克服治疗抗性并导致合成致死性。CART利用 反相蛋白质微阵列分析（RPPA），提供高通量，灵敏， 定量方法来分析差异蛋白质表达，以确定目标， 组合疗法CART方法对RT的适用性以前没有被 研究了这个职业发展应用的中心目标是发展自己 作为一个独立的翻译科学家，在HNC的专业知识，并成为领导者， 转化肿瘤学领域，采用CART方法提高疗效 在3D培养和异种移植模型中的RT。拟议研究的理由是合理的 结合新确定的全身治疗，如转氨酶抑制剂CB-839， RT将导致最大的疗效，同时最大限度地减少潜在的毒性。以强为导 初步数据表明转氨酶在RT适应性耐药中发挥作用，我们将 追求三个具体目标：第一（SA 1），我们将确定RT与 转氨酶抑制剂（CB-839）导致有氧呼吸减少和细胞死亡增加 在2D和3D文化中。为了追求这一点，上调有氧呼吸途径，包括那些 由转氨酶催化的，将被选择性地单独靶向或与RT组合在3D中， 增殖和凋亡标记物的分析。海马技术将用于评估 有氧呼吸其次（SA 2），我们将通过以下方法验证谷氨酰胺酶抑制的功效： 在临床前异位细胞系和患者中测试其单独和与RT组合， 衍生的异种移植动物模型。最后（SA 3），我们将鉴定其他新的HNC信号传导 通过使用RPPA的RT显著改变的途径。为了达到这个目的， 口腔肿瘤来源的细胞系将在3D培养物中生长并进行非致死性RT 通过RPPA评估的蛋白质水平的剂量，以鉴定候选靶蛋白， 这种创新的方法使用了一种尖端的，高通量的， 敏感和定量的方法（RPPA），以确定全新的治疗靶点， 在蛋白质水平上与RT联合使用。这项研究意义重大，因为 它正在测试一种合理选择的治疗方法（CB-839），以提高RT的疗效。 实验将为未来的临床试验奠定基础，并有望确定 其他未知但有效的治疗组合。总而言之，该提案概述了 识别和测试新治疗靶点的复杂、合理和快速的方法 这对与头颈癌作斗争的退伍军人来说是不成比例的好处。

Head and neck cancer (HNC) is the sixth most common malignancy worldwide, diagnosed twice as frequently in veterans. Radiation therapy (RT) is an important component of cancer treatment; however, its efficacy is limited by radioresistance, with the cancer sometimes returning within the treated area. Resistance to RT is, at least in part, mediated by adaptive signaling events induced by treatment. However, we do not yet understand the pathways used by cells to evade the cellular damage caused by RT. The long-term goal is to better understand, and subsequently target, the mechanisms of resistance that cancer cells use to evade a radiation-induced death. Combinatorial adaptive response therapy (CART) represents a novel platform that allows for the rapid and systematic identification of treatment combinations that overcome therapeutic resistance and result in synthetic lethality. CART takes advantage of Reverse Phase Protein Microarray Analysis (RPPA), providing a high throughput, sensitive, and quantitative approach to analyze differential protein expression to identify targets for combinatorial therapy. The applicability of the CART approach to RT has not previously been investigated. The central objective of this career development application is to develop myself as an independent translational scientist with expertise in HNC, and to become a leader in the field of translational oncology, with implementation of a CART approach to increase the efficacy of RT in 3D culture and xenograft models. The rationale for the proposed research is rationally combining newly identified systemic treatments, such as the glutaminase inhibitor, CB-839, with RT will result in maximal efficacy while minimizing potential toxicities. Guided by strong preliminary data implicating glutaminase as playing a role in adaptive resistance to RT, we will pursue three specific aims: First (SA1), we will determine whether the combination of RT with a glutaminase inhibitor (CB-839) results in decreased aerobic respiration and increased cell death in 2D and 3D culture. To pursue this, upregulated aerobic respiration pathways, including those catalyzed by glutaminase, will be selectively targeted alone or in combination with RT in 3D with analysis of proliferation and apoptosis markers. Seahorse technology will be used to assess aerobic respiration. Second (SA2), we will validate the efficacy of glutaminase inhibition by testing it both alone and in combination with RT in preclinical heterotopic cell line and patient- derived xenograft animal models. Finally (SA3), we will identify other novel HNC signaling pathways that are significantly altered by RT using RPPA. For this aim, spheroids grown from oral cavity tumor derived cell lines will be grown in 3D culture and subjected to non-lethal RT doses with protein levels assessed by RPPA to identify candidate target proteins that are differentially expressed with RT. This innovative approach uses a cutting-edge, high-throughput, sensitive, and quantitative method (RPPA) to identify entirely novel therapeutic targets to be used in combination with RT, at the protein level. The proposed research is significant because it is testing a rationally selected treatment (CB-839) to increase the efficacy of RT. These experiments will lay the groundwork for future clinical trials and are expected to identify additional unknown, yet effective treatment combinations. In sum, this proposal outlines a sophisticated, rational, and rapid approach to identifying and testing novel therapeutic targets which would be of disproportionate benefit to veterans battling head and neck cancer.