title: A chemical genetics approach for the functional assessment of novel cancer genes.
abstract: Assessing the functional significance of novel putative oncogenes remains a significant challenge given the limitations of current loss-of-function tools. Here, we describe a method that employs TALEN or CRISPR/Cas9-mediated knock-in of inducible degron tags (Degron-KI) that provides a versatile approach for the functional characterization of novel cancer genes and addresses many of the shortcomings of current tools. The Degron-KI system allows for highly specific, inducible, and allele-targeted inhibition of endogenous protein function, and the ability to titrate protein depletion with this system is able to better mimic pharmacologic inhibition compared with RNAi or genetic knockout approaches. The Degron-KI system was able to faithfully recapitulate the effects of pharmacologic EZH2 and PI3Kα inhibitors in cancer cell lines. The application of this system to the study of a poorly understood putative oncogene, SF3B1, provided the first causal link between SF3B1 hotspot mutations and splicing alterations. Surprisingly, we found that SF3B1-mutant cells are not dependent upon the mutated allele for in vitro growth, but instead depend upon the function of the remaining wild-type alleles. Collectively, these results demonstrate the broad utility of the Degron-KI system for the functional characterization of cancer genes.
authors: Oncology Disease Area, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts.
authors: Qianhe
authors: Q
authors: Zhou
authors: Oncology Translational Medicine, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts.
authors: Adnan
authors: A
authors: Derti
authors: Oncology Translational Medicine, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts.
authors: David
authors: D
authors: Ruddy
authors: Oncology Translational Medicine, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts.
authors: Daniel
authors: D
authors: Rakiec
authors: Oncology Translational Medicine, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts.
authors: Iris
authors: I
authors: Kao
authors: Oncology Disease Area, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts.
authors: Michelle
authors: M
authors: Lira
authors: Oncology Disease Area, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts.
authors: Veronica
authors: V
authors: Gibaja
authors: Oncology Disease Area, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts.
authors: HoMan
authors: H
authors: Chan
authors: Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts.
authors: Yi
authors: Y
authors: Yang
authors: Oncology Disease Area, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts.
authors: Junxia
authors: J
authors: Min
authors: Oncology Disease Area, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts.
authors: Michael R
authors: MR
authors: Schlabach
authors: Oncology Disease Area, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts. frank.stegmeier@novartis.com.
authors: Frank
authors: F
authors: Stegmeier
mesh_terms: D049109:Cell Proliferation; D052138:Genes, Neoplasm; D045325:HCT116 Cells; D006801:Humans; D009154:Mutation; D009369:Neoplasms / Q000235:genetics*; D010750:Phosphoproteins / Q000235:genetics; D055550:Protein Stability; D059748:Proteolysis; D000072260:RNA Splicing Factors; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017413:Ribonucleoprotein, U2 Small Nuclear / Q000235:genetics
chemical_list: D010750:Phosphoproteins; D000072260:RNA Splicing Factors; D011993:Recombinant Fusion Proteins; D017413:Ribonucleoprotein, U2 Small Nuclear; C112491:SF3B1 protein, human