Application of CRISPR/Cas9 to Autophagy Research.

The ability to efficiently modulate autophagy activity is paramount in the study of the field. Conventional broad-range autophagy inhibitors and genetic manipulation using RNA interference (RNAi), although widely used in autophagy research, are often limited in specificity or efficacy. In this chapter, we address the problems of conventional autophagy-modulating tools by exploring the use of three different CRISPR/Cas9 systems to abrogate autophagy in numerous human and mouse cell lines. The first system generates cell lines constitutively deleted of ATG5 or ATG7 whereas the second and third systems express a Tet-On inducible-Cas9 that enables regulated deletion of ATG5 or ATG7. We observed the efficiency of autophagy inhibition using the CRISPR/Cas9 strategy to surpass that of RNAi, and successfully generated cells with complete and sustained autophagy disruption through the CRISPR/Cas9 technology.

The tyrosine kinase Syk regulates the survival of chronic lymphocytic leukemia B cells through PKCdelta and proteasome-dependent regulation of Mcl-1 expression.

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by accumulation of mature monoclonal CD5+ B cells. The disease results mainly from a failure of cells to undergo apoptosis, a process largely influenced by the existence of constitutively activated components of B-cell receptor signaling and the deregulated expression of anti-apoptotic molecules. Recent evidence pointing to a critical role of spleen tyrosine kinase (Syk) in ligand-independent BCR signaling prompted us to examine its role in primary B-CLL cell survival. We demonstrate that pharmacological inhibition of constitutive Syk activity and silencing by siRNA led to a dramatic decrease of cell viability in CLL samples (n=44), regardless of clinical and biological status and induced typical apoptotic cell death with mitochondrial failure followed by caspase 3-dependent cell death. We also provide functional and biochemical evidence that Syk regulated B-CLL cell survival through a novel pathway involving PKCdelta and a proteasome-dependent regulation of the anti-apoptotic protein Mcl-1. Together, our observations are consistent with a model wherein PKCdelta downstream of Syk stabilizes Mcl-1 through inhibitory phosphorylation of GSK3 by Akt. We conclude that Syk constitutes a key regulator of B-CLL cell survival, emphasizing the clinical utility of Syk inhibition in hematopoietic malignancies.