Leaders in Pharmaceutical Business Intelligence (LPBI) Group

Using RNA-seq and targeted nucleases to identify mechanisms of drug resistance in acute myeloid leukemia

Reporter: Aviva Lev-Ari, PhD, RN

The evolution from microarrays to transcriptome deep-sequencing (RNA-seq) and from RNA interference to gene knockouts using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and Transcription Activator-Like Effector Nucleases (TALENs) has provided a new experimental partnership for identifying and quantifying the effects of gene changes on drug resistance. Here we describe the results from deep-sequencing of RNA derived from two cytarabine (Ara-C) resistance acute myeloid leukemia (AML) cell lines, and present CRISPR and TALEN based methods for accomplishing complete gene knockout (KO) in AML cells. We found protein modifying loss-of-function mutations in deoxycytidine kinase (dck) in both Ara-C resistant cell lines. The first kinase in the dNTP salvage pathway is  DCK, the rate limiting enzyme in the metabolic activation of Ara-C. Numerous studies have shown DCK expression is frequently downregulated in cells that are unresponsive to Ara-C2, 3, 4, 5, 6, 7. CRISPR and TALEN-based KO of Dckdramatically increased the IC50 of Ara-C and introduction of a DCK overexpression vector into Dck KO clones resulted in a significant increase in Ara-C sensitivity. This effort demonstrates the power of using transcriptome analysis and CRISPR/TALEN-based KOs to identify and verify genes associated with drug resistance.

Source: www.nature.com

See on Scoop.it – Cardiovascular Disease: PHARMACO-THERAPY