Transcriptional effects of CRISPR-induced MAF repression in t(14;16) multiple myeloma
Multiple myeloma (MM) is a cancer of terminally differentiated B-lymphocytes (plasma cells) that accounts for 20% of hematological cancers and 2% of all cancer cases in the United States.
Genetic factors play a significant role in the development and prognosis of MM. Translocations involving the immunoglobulin heavy chain (IgH) locus on chromosome 14 and one of four recurrent partners (MYC, CCND, WHSC1, and MAF) result in spiked expression of the target gene, and are likely primary oncogenic events in a subset of MM. In patients with a t(14;16) MAF translocation, overexpression of Maf, a transcription factor, results in a distinct expression signature linked to poor outcome in patients. Despite the advent of novel therapies, t(14;16)-positive MM patients continue to show similarly poor prognosis. This study leveraged a two-vector dCAS9-KRAB and sgRNA CRISPR system to investigate the transcriptional effects resulting from MAF repression in the t(14;16) positive MM cell line JJN3. The dCAS9-KRAB system is a distinct version of CRISPR/cas9 that is used to silence target gene expression in mammalian cells. In this study, a 20-base pair MAF guide RNA was designed, constructed using overlapping PCR, and cloned into the pLX-sgRNA vector. In tandem, a stable puromycin selected dCAS9-KRAB-JJN3 culture was generated. After MAF-sgRNA lentiviral production in HEK293T cells, dCAS9-KRAB-JJN3 will be treated with the lentivirus and qRT-PCR will be used to confirm MAF knockdown and potential dysregulation of MAF transcriptional target genes such as cyclin D2 (CCND2), and integrin beta 7 (ITGB7). Understanding transcriptional effects of MAF repression may lead to a better understanding of resistance to emerging therapies in this patient subset of MM.