Retention of somatic mutations in cancers by changes in pH sensing

描述（由申请人提供）：癌症中的体细胞基因突变随机发生，但一个重要的未解决的问题是如何保留选择性突变。保留部分取决于适应性特征，例如克服增殖障碍或免疫监视，从而适应肿瘤微环境。我们的提案测试了一个关于健康特征和适应性的新想法，有助于保留癌症中的基因突变。这一新想法是基于大多数癌症的保守特征，其具有比正常细胞（pHi 7.1-7.2）更高的细胞内pH（pHi 7.5-7.6）。我们预测，选择性体细胞突变赋予pH传感的获得或丧失，这为癌症的较高pHi提供了适应性优势。为了限制测试这一新想法的范围和风险，我们将专注于癌症中的Arg>His突变，并预测这些突变编码pH传感的增益。我们的具体假设是，癌症中pHi的增加赋予了保留选择性精氨酸至组氨酸突变的适应性优势。我们的初步分析揭示了Arg>His突变的发生率高于密码子偏好的预期，包括CpG突变效应。我们对通过Arg>His突变获得pH传感的预测是基于组氨酸在溶液中具有接近中性的pKa，因此可以在正常细胞的pH下质子化，并且在癌细胞的较高pH下不带电荷。相比之下，因为Arg具有约12的pKa，所以它可能在正常细胞和癌细胞的pHi下保持带电。在目标1中，我们使用生物信息学方法测试保留组氨酸突变的进化力量。我们将开发新的生物信息学技术来对Arg>His体细胞突变进行排序，并表征在这些位置操作的历史和同期进化力量，以量化突变偏倚，并确定pKa改变His突变在癌症中保留的程度，相对于在进化时间尺度上预期的程度。在目标2中，我们实验性地测试了重复出现的Arg>His候选突变以获得pH传感。我们将确定野生型和突变蛋白在体外和正常和转化克隆细胞中的pH依赖性功能。我们还将计算预测突变的组氨酸和组氨酸的pKa值，并使用分子动力学模拟测试蛋白质结构中pH依赖的构象变化。据我们所知，在癌症中赋予pH传感的突变，如p53-R273 H，我们将测试的最常见的突变之一，尚未报道，也没有相对于更高的pH传感的体细胞突变的保留。 癌症的pHi。如果是正确的，我们的假设将产生一个实质性的新观点，为什么癌症中的一些体细胞突变被保留。此外，成功证实我们的假设对于未来的研究将是有说服力的，以测试His>Arg突变的pH敏感性丧失，以及涉及酪氨酸、丝氨酸和苏氨酸残基的突变，这些残基在磷酸化时具有接近中性的pKa。因此，我们的发现可以广泛应用，并对靶向驱动癌症的突变的pH依赖性的治疗策略产生重大影响。

DESCRIPTION (provided by applicant): Somatic gene mutations in cancers occur randomly but a significant unresolved question is how selective mutations are retained. Retention is in part determined by fitness traits such as overcoming barriers to proliferation or immune surveillance that confer an adaptation to the tumor microenvironment. Our proposal tests a new idea on fitness traits and adaptation contributing to retention of gene mutations in cancers. This new idea is based on the conserved trait of most cancers having a higher intracellular pH (pHi 7.5-7.6) than normal cells (pHi 7.1-7.2). We predict that selective somatic mutations confer gain or loss of pH sensing that provides an adaptive advantage to the higher pHi of cancers. To limit the scope and risk of testing this new idea we will focus on Arg>His mutations in cancers with the prediction that these mutations encode a gain of pH sensing. Our specific hypothesis is that increased pHi in cancers confers an adaptive advantage for retention of selective arginine to histidine mutations. Our preliminary analysis reveals a higher incidence of Arg>His mutations than expected from codon bias, including CpG mutational effects. Our prediction on gain of pH sensing by Arg>His mutations is based on histidine having a pKa near neutral in solution and hence can be protonated at the pHi of normal cells and uncharged at the higher pHi of cancer cells. In contrast, because Arg has a pKa ~12 it likely remains charged at the pHi of normal and cancer cells. In Aim 1 we test evolutionary forces for retention of histidine mutations by using bioinformatics approaches. We will develop novel bioinformatics techniques for ranking Arg>His somatic mutations, and characterize the historical and contemporaneous evolutionary forces operating at these positions to quantify mutational biases and determine the extent to which pKa altering His mutations are retained in cancers relative to what would be expected across evolutionary timescales. In Aim 2 we experimentally test recurring Arg>His candidate mutations for gain of pH sensing. We will determine pH-dependent functions of wild type and mutant proteins in vitro and in normal and transformed clonal cells. We also will computationally predict pKa's of mutated histidines and arginines and test pH-dependent conformational changes in protein structure using molecular dynamics simulations. To our knowledge mutations in cancers conferring pH sensing, such as p53-R273H, one of the most commonly occurring mutations we will test, has not been reported, nor has the retention of somatic mutations relative to the higher pHi of cancers. If correct, our hypothesis would generate a substantial new view on why some somatic mutations in cancers are retained. Additionally, successfully confirming our hypothesis would be compelling for future studies to test His>Arg mutations for loss of pH-sensing, and mutations involving tyrosine, serine and threonine residues that have pKa's near neutral when phosphorylated. Hence, our findings could be applied broadly and have substantial impact on therapeutic strategies to target pH dependence of mutations driving cancers.