A genetic screen to identify novel regulators of Ikaros-mediated cell cycle arrest

Abstract

Failure to regulate cellular proliferation is one of the hallmarks of cancer. The development of pre-B cells is demarcated by alternating stages of quiescence, in which immunoglobulin receptors are sequentially rearranged, and clonal expansion, in which signals from the assembled pre-B cell receptor lead to proliferation. Ikaros (encoded by Ikzf1 in mice) is a transcription factor that regulates gene expression in cycling B cell progenitors to enforce proliferative arrest. In humans IKZF1 mutations are prevalent in subsets of haematological malignancies and result in inappropriate proliferation, therefore the regulation of the cell cycle by Ikaros may be fundamental to its tumour suppressor function. The study of Ikaros-mediated cell cycle arrest in pre-B cells is complicated by the role of Ikaros in the regulation of genes involved in pre-B cell differentiation. I used 3T3 fibroblasts as a reductionist model to study the regulation of the cell cycle by Ikaros independently of pre-B cell receptor signalling. Using this model I performed an RNAi screen to discover novel regulators that cooperate with Ikaros to arrest the cell cycle. Amongst a number of candidates I identified the scaffolding protein SSeCKS (Akap12) as necessary for Ikaros-mediated proliferative arrest. Overexpression of Ikaros and SSeCKS synergistically arrested the cell cycle and silenced the expression of the proto-oncogene Myc. Utilising fibroblasts in which Ikaros and SSeCKS could be inducibly expressed I performed RNAseq to profile the global gene expression of cells that had undergone Ikaros and SSeCKS-mediated cell cycle arrest. Ikaros and SSeCKS together regulated the expression of hundreds of genes to coordinate proliferative arrest. The insights gained from these analyses may be applied to pre-B cells to deepen our understanding of the role of Ikaros in the regulation of proliferation in normal development and in the leukaemic state.