Clinical and molecular characterization of newly detected mutations in receptor tyrosine kinases, adhesion molecules and their downstream effectors

最近，一项针对原发性MM的深度测序方法在BRAF、组蛋白修饰酶、RNA加工机制基因和NFκB通路基因中发现了突变。然而，根据迄今为止已知的MM分子改变，这些突变的发生频率都很低。在第一个资助期的Z3项目中，我们在GAIIx （Illumina）上建立了外显子组测序，并对5个初代MM、相应的种系DNA和6个MM细胞系进行了测序，以进一步分析MM的遗传景观。许多突变聚集在粘附和生长因子受体信号网络中，包括几种受体酪氨酸激酶（rtk）（例如TGFBR， EGFR, IGF1R, ERBB3, NTRK1/2, EPHB2）。在这个网络中，几乎100%的原发性MM （n=43）受到至少一个突变的影响，约50%受到一个以上突变的影响。这一新的观察结果与最近的另一项发现（Akt/PI3K和MEK的平行抑制导致75%的MM患者样本（TP1）凋亡）表明RTK-粘附-和RTK/RAS/PI3K-网络在MM的发病机制中起重要作用，并表明某些RTK的突变对这些网络的失调至关重要。因此，在申请的项目中，我们将调查来自DSMM （Deutsche Studiengruppe multiple Myelom）试验的MM样本和具有可用akt激活状态的样本中选定基因（例如rtk）突变的频率和临床影响。此外，我们将研究EGFR的影响，EGFR在我们的数据集中发生突变，并在其他癌症实体中作为有希望的药物靶点对MM细胞生长和存活的影响。具体而言，计划进行以下实验：i)我们将使用GS Junior （Roche）对86例MM患者的RTK-和EGFR配体结合域的外显子区域以及频繁突变的基因KRAS和DIS3的所有编码区域进行深度测序（约200倍）。通过经典细胞遗传学或SNP 6.0阵列检测，突变将与细胞遗传学改变相关，并与生存等临床特征相关（与Z2合作）。ii)我们将收集新的MM病例，从药理学上确定akt激活状态（TP1），并在约20个akt依赖性MM和约20个akt非依赖性MM中对EGFR-、IGF1R-、ERBB3-、NTRK1/2-和EPHB2编码区进行突变筛选。iii)我们将研究EGFR功能在可转染MM细胞系中的作用。具体来说，我们将对EGFR单独进行siRNA敲低，并与KRAS联合进行，在具有野生型ras的MM细胞系中敲入突变的EGFR，并在处理和未处理的细胞系中研究EGFR（与Z4N合作）和KRAS的蛋白质水平和激活状态。此外，我们将研究egfr抑制剂（如吉非替尼）在egfr敲低或敲入前后对突变或未突变MM细胞系细胞存活的影响。iv)最后，我们将通过确定它们的相互作用状态（与Z4N合作）和降低单个候选基因（如IGF1R， EPHB2）或两个候选基因（如EGFR + EPHA2， IGF1R + ITGB1）的基因表达，以及研究对肿瘤细胞存活和RAS/PI3K信号传导的影响（与TP1合作），来分析不同rtk（见上文）和膜站粘附分子（如β1-整合素，NCAM2）的功能。

A recent deep sequencing approach in primary MM identified mutations in BRAF, in histone modifying enzymes, in genes of the RNA processing machinery and in genes of the NFκB pathway. However, in accordance with the hitherto known molecular alterations in MM, each of these mutations occurred with low frequency. Within the Z3 project of the first funding period we established exome sequencing on a GAIIx (Illumina) and sequenced five primary MM, corresponding germline DNA and six MM cell lines to further analyze the genetic landscape of MM. A bioinformatics strategy that integrated published filtered mutational data of 38 primary MM was developed. Many mutations clustered in an adhesion and growthfactor receptor signaling network, including several receptor tyrosine kinases (RTKs) (for example, TGFBR, EGFR, IGF1R, ERBB3, NTRK1/2, EPHB2). Almost 100% of primary MM (n=43) were affected by at least one mutation and ~50% by more than one mutation within this network. This new observation together with another recent finding that parallel inhibition of Akt/PI3K and MEK led to apoptosis in 75% of MM patient samples (TP1) indicates an important role for the RTK-adhesion- and RTK/RAS/PI3K-networks in the pathogenesis of MM and suggests that mutations of certain RTKs critically contribute to the deregulation of these networks. In the applied-for project we will therefore investigate the frequencies and clinical impact of mutations in selected genes (e.g. RTKs) in MM samples from the DSMM (Deutsche Studiengruppe Multiples Myelom) trials and samples with available Akt-activation status. In addition, we will investigate the influence of EGFR, which was mutated in our dataset and serves as a promising drug target in other cancer entities on MM cell growth and survival. Specifically, the following experiments are planned: i) we will deep sequence (~200- fold) the exonic regions of the RTK- and ligand binding domain of EGFR as well as all coding regions of the frequently mutated genes KRAS and DIS3 in 86 MM patients using the GS Junior (Roche). Mutations will be correlated with cytogenetic alterations, as detected by classical cytogenetics or by SNP 6.0-arrays, and with clinical features such as survival (in cooperation with Z2). ii) We will collect new MM cases, pharmacologically determine the Aktactivation status (TP1) and perform mutational screens of the EGFR-, IGF1R-, ERBB3-, NTRK1/2- and EPHB2 coding regions in ~20 Akt-dependent and ~20 Akt-independent MM. iii) We will investigate the role of EGFR function in a broad panel of transfectable MM cell lines. Specifically, we will perform siRNA knockdown of EGFR alone and in combination with KRAS, knock-in of mutated EGFR in MM cell lines with wildtype-RAS and investigate the protein level and activation status of EGFR (in co-operation with Z4N) and KRAS in the treated and untreated cell lines. Moreover, we will investigate the influence of EGFRinhibitors (e.g. gefitinib) on the survival of cells of mutated or unmutated MM cell lines before and after EGFR-knock-down or knock-in. iv) Finally, we will analyze the function of different RTKs (see above) and membrane standing adhesion molecules (e.g. β1-integrins, NCAM2) by determining their interaction status (co-operation with Z4N) and knocking down the gene expression of single candidates (e.g. IGF1R, EPHB2) or two candidates in parallel (e.g. EGFR + EPHA2, IGF1R + ITGB1) and by studying the effect on tumor cell survival and RAS/PI3K signaling (collaboration with TP1).