Systematic shRNA screens for the analysis of critical signaling pathways in KRAS-mutant multiple myeloma

KRAS突变是多发性骨髓瘤（MM）的驱动突变，但没有靶向编码蛋白（KRAS）的小分子。突变型KRAS激活多种信号转导途径，这些途径以未知和潜在的高度细胞特异性组合起作用，以驱动MM细胞的生长。在上一个资助期间，我们已经建立了系统性的功能丧失（shRNA）筛选，作为慢病毒池筛选，并结合下一代测序，以达到10，000个shRNA的复杂性。我们发现，这些筛选可以鉴定突变KRAS的下游效应子途径之间的治疗相关的合作。申请项目旨在使用不同的MM细胞系牢固地建立和验证这一概念。与基因组序列数据的比较将确定特定的生物标志物（例如突变）是否可靠地指示关键信号通路之间的协同性。同时，我们建议设计和克隆一个shRNA文库，该文库以有限的复杂性靶向关键效应子通路，以便分析小细胞群体中的KRAS依赖性信号通路（例如，在体内肿瘤发生模型中）。

Mutations in KRAS are driver mutations in multiple myeloma (MM) yet no small moleculesthat target the encoded protein (KRAS) are available. Mutant KRAS activates multiple signal transduction pathways that act in unknown and potentially highly cell-specific combinations to drive growth of MM cells. In the previous funding period, we have established systematic loss-of-function (shRNA) screens as lentiviral pool screens coupled with next generation sequencing to a complexity of 10,000 shRNAs. We found that these screens can identify therapeutically relevant co-operations between downstream effector pathways of mutant KRAS. The applied-for project aims to firmly establish and validate this concept using different MM cell lines. Comparison with genome sequence data will establish whether specific biomarkers (for example mutations) reliably indicate co-operativity between critical signaling pathways. In parallel, we propose to design and clone a shRNA library that targets critical effector pathways with limited complexity in order to allow the analysis of KRASdependent signaling pathways in small cell populations (for example, in in vivo models of tumorigenesis).