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

点击翻译按钮获取中文摘要

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).