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 PI3Ka 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.

The Augmented R-Loop Is a Unifying Mechanism for Myelodysplastic Syndromes Induced by High-Risk Splicing Factor Mutations

“…Importantly, these splicing factor mutations are not restricted to MDS; they have also been identified in various solid tumors, such as melanoma (Furney et al, 2013; Harbour et al, 2013; Martin et al, 2013) and bladder (Papaemmanuil et al, 2011), pancreatic (Biankin et al, 2012), and lung (Imielinski et al, 2012) cancer. It is also striking to note that all mutations identified to date are heterozygous and that mutations in different splicing factors are mutually exclusive in MDS patients (Dvinge et al, 2016), which is consistent with the synthetic lethality between different splicing mutants in a murine model (Lee et al, 2016a) and the requirement for a wild-type (WT) copy to support their basic cellular functions (Fei et al, 2016; Lee et al, 2016b; Zhou et al, 2015). …”

How do messenger RNA splicing alterations drive myelodysplasia?

“…allele in the context of human lung epithelial 11 and uveal melanoma 44 cells, respectively, had no effect on cell proliferation or survival in vitro. In contrast, deletion of the wild-type U2AF1 or SF3B1 allele resulted in death of cells expressing only the mutant allele.…”

“…In contrast, several recent studies have identified that ablation of the mutant RNA splicing factor allele appears to have no effect on cancer maintenance. 11,25,44 This was demonstrated in 2 studies where deletion of the mutant U2AF1 S34F or SF3B1 …”

“…1-3 Although the nature of these alterations as heterozygous missense mutations was initially assumed to suggest that they confer a gain of function, it is also now clear that the wild-type allele is absolutely required for cell survival in the setting of expression of the mutant allele 11,23,44 (Figure 2). In contrast, several recent studies have identified that ablation of the mutant RNA splicing factor allele appears to have no effect on cancer maintenance.…”

“…For example, mutations in U2AF1 tend to significantly coexist with those in ASXL1, whereas mutations in SF3B1 frequently coexist with those in DNMT3A. [41][42][43] Further efforts to understand the mechanistic and biological contributions of these coexisting mutations on splicing and hematopoiesis may greatly facilitate our understanding of how mutations in RNA splicing promote MDS development.What is the role of RNA splicing factors in MDS development vs maintenance?Mutations in SF3B1, SRSF2, and U2AF1 are consistently expressed in the heterozygous state concomitant with expression of the wild-type allele.1-3 Although the nature of these alterations as heterozygous missense mutations was initially assumed to suggest that they confer a gain of function, it is also now clear that the wild-type allele is absolutely required for cell survival in the setting of expression of the mutant allele 11,23,44 (Figure 2). In contrast, several recent studies have identified that ablation of the mutant RNA splicing factor allele appears to have no effect on cancer maintenance.…”