Harold Varmus

Wild-Type U2AF1 Antagonizes the Splicing Program Characteristic of U2AF1-Mutant Tumors and Is Required for Cell Survival

We have asked how the common S34F mutation in the splicing factor U2AF1 regulates alternative splicing in lung cancer, and why wild-type U2AF1 is retained in cancers with this mutation. A human lung epithelial cell line was genetically modified so that U2AF1S34F is expressed from one of the two endogenous U2AF1 loci. By altering levels of mutant or wild-type U2AF1 in this cell line and by analyzing published data on human lung adenocarcinomas, we show that S34F-associated changes in alternative splicing are proportional to the ratio of S34F:wild-type gene products and not to absolute levels of either the mutant or wild-type factor. Preferential recognition of specific 3′ splice sites in S34F-expressing cells is largely explained by differential in vitro RNA-binding affinities of mutant versus wild-type U2AF1 for those same 3′ splice sites. Finally, we show that lung adenocarcinoma cell lines bearing U2AF1 mutations do not require the mutant protein for growth in vitro or in vivo. In contrast, wild-type U2AF1 is required for survival, regardless of whether cells carry the U2AF1S34F allele. Our results provide mechanistic explanations of the magnitude of splicing changes observed in U2AF1-mutant cells and why tumors harboring U2AF1 mutations always retain an expressed copy of the wild-type allele.

Insurance for broad genomic tests in oncology

Insurance coverage should precede rather than follow clinical validation of broad genomic testing in oncology Tests based on DNA sequencing methods are redefining diagnostic categories in oncology and providing a rational basis for the development and use of new cancer therapies, especially the many drugs targeted against mutant proteins that drive malignant growth (1). The medical and economic value of identifying specific genetic abnormalities in cancers has been established by the evidence-based use of targeted drugs and immunotherapies in cancer patients (see the figure for an example). For some cancer therapies, the U.S. Food and Drug Administration (FDA)–approved label calls for the use of a companion diagnostic test for the presence or absence of certain genetic changes before initiating treatment. Most recently, the FDA approved the use of the immunotherapeutic pembrolizumab for treatment of tumors that show a defect in DNA repair called microsatellite instability, irrespective of the tissue of origin; although an FDA-approved test for microsatellite instability is not yet available, many clinical laboratories are using assays for the relevant biomarkers (2).

Wild-type Splicing Factor U2AF1 inhibits splicing associated with a recurrent U2AF1 mutant in human lung cancers and is required for cell survival

We have asked how the common S34F mutation in the splicing factor U2AF1 regulates alternative splicing in lung cancer, and why wild-type U2AF1 is retained in cancers with this mutation. A human lung epithelial cell line was genetically modified so that U2AF1S34F is expressed from one of the two endogenous U2AF1 loci. By altering levels of mutant or wild-type U2AF1 in this cell line and by analyzing published data on human lung adenocarcinomas, we show that S34F-associated changes in alternative splicing are proportional to the ratio of S34F:wild-type gene products and not to absolute levels of either the mutant or wild-type factor. Preferential recognition of specific 3′ splice sites in S34F-expressing cells is largely explained by differential in vitro RNA-binding affinities of mutant versus wild-type U2AF1 for those same 3′ splice sites. Finally, we show that lung adenocarcinoma cell lines bearing U2AF1 mutations do not require the mutant protein for growth in vitro or in vivo. In contrast, wild-type U2AF1 is required for survival, regardless of whether cells carry the U2AF1S34F allele. Our results provide mechanistic explanations of the magnitude of splicing changes observed in U2AF1-mutant cells and why tumors harboring U2AF1 mutations always retain an expressed copy of the wild-type allele. Author Summary Large-scale genomics studies have identified recurrent mutations in many genes that fall outside the conventional domain of proto-oncogenes. They include genes encoding factors that mediate RNA splicing; mutations affecting four of these genes are present in up to half of proliferative myeloid disorders and in a significant number of solid tumors, including lung adenocarcinoma. Here we have characterized several properties of a common mutant version of the U2AF1 splicing factor, a component of the U2 auxiliary factor complex, in lung cells. We have found that mutant-associated changes in splice site selection are primarily influenced by the ratio of mutant and wild-type U2AF1 gene products; thus increasing wild-type U2AF1 levels represses the mutant-induced splicing program. We show that the altered splice site preferences of mutant U2AF1 can be attributed to changes in its binding to relevant 3′ splice sites. We also show that mutant U2AF1 is different from some oncogenes: the growth properties of lung cancer cell lines carrying the mutant allele are unaffected by loss of the mutant gene, while the wild-type allele is absolutely required for survival. These results advance our understanding of the molecular determinants of the mutant-associated splicing program, and they highlight previously unappreciated roles of wild-type U2AF1 in the presence of the recurrent U2AF1S34F mutation.The splicing factor gene, U2AF1, is recurrently mutated in a variety of human cancers, including lung adenocarcinomas. The most frequent U2AF1 mutant, U2AF1 p.Ser34Phe (S34F), induces specific changes in splicing that we collectively refer to as S34F-associated splicing, but it is unclear how these splicing changes are regulated. Moreover, while a wild-type U2AF1 allele is retained in all cancers expressing a U2AF1 mutation, the functional significance of the retained wild-type allele is unknown. Our analysis of published data on human lung adenocarcinomas indicates that lung adenocarcinomas carrying a U2AF1 S34F allele exhibit a wide range of mutant to wild-type U2AF1 (S34F:WT) mRNA ratios, which can be partially attributed to copy number variation at the U2AF1 locus. S34F:WT mRNA ratios, rather than absolute levels of U2AF1 S34F or total U2AF1 mRNA, correlate positively with the magnitude of S34F-associated splicing in lung adenocarcinoma transcriptomes. To examine the effect of S34F:WT ratios on S34F-associated splicing directly, we modified a human bronchial epithelial cell line so that U2AF1 S34F is expressed at one of the two endogenous U2AF1 loci and the S34F:WT mRNA ratio approximates one. By altering the levels of mutant or wild-type U2AF1 in this engineered cell line, we show that the degree of S34F-associated splicing is proportional to the ratio of S34F:WT gene products and not to absolute levels of either the mutant or wild-type factor. Further, we show that in nearly all cases, S34F-associated splicing alterations are largely explained by the different RNA binding affinities of recombinant protein complexes containing wild-type or mutant U2AF1. Together, these observations suggest that wild-type U2AF1 is a negative regulator of S34F-associated splicing, at least in part through differential binding to 3 splice sites. Finally, we show that the U2AF1 S34F allele does not behave like some oncogenes: the mutated gene does not induce cell transformation, and lung adenocarcinoma cell lines are not dependent on it for growth in vitro or in vivo. Wild-type U2AF1, however, is absolutely required for cell survival, regardless of whether the cells carry the U2AF1 S34F allele. We conclude that wild-type U2AF1 has two important functions in U2AF1 S34F-expressing lung cancers: it controls the magnitude of S34F-associated splicing, and it is essential for cell survival.

How Cancer Genomics Drives Cancer Biology: Does Synthetic Lethality Explain Mutually Exclusive Oncogenic Mutations?

Large-scale analyses of cancer genomes are revealing patterns of mutations that suggest biologically significant ideas about many aspects of cancer, including carcinogenesis, classification, and preventive and therapeutic strategies. Among those patterns is mutual exclusivity, a phenomenon observed when two or more mutations that are commonly observed in samples of a type of cancer are not found combined in individual tumors. We have been studying a striking example of mutual exclusivity: the absence of coexisting mutations in the KRAS and EGFR proto-oncogenes in human lung adenocarcinomas, despite the high individual frequencies of such mutations in this common type of cancer. Multiple lines of evidence suggest that toxic effects of the joint expression of KRAS and EGFR mutant oncogenes, rather than loss of any selective advantages conferred by a second oncogene that operates through the same signaling pathway, are responsible for the observed mutational pattern. We discuss the potential for understanding the physiological basis of such toxicity, for exploiting it therapeutically, and for extending the studies to other examples of mutual exclusivity.

Generation of pulmonary neuro-endocrine cells and tumors resembling small cell lung cancers from human embryonic stem cells

SUMMARY By blocking an important signaling pathway (called NOTCH) and interfering with expression of two tumor suppressor genes in cells derived from human embryonic stem cells, the authors have developed a model for studying highly lethal small cell lung cancers. ABSTRACT Cell culture models based on directed differentiation of human embryonic stem cells (hESCs) may reveal why certain constellations of genetic changes drive carcinogenesis in specialized human cell lineages. Here we demonstrate that up to 10 percent of lung progenitor cells derived from hESCs can be induced to form pulmonary neuroendocrine cells (PNECs), the putative normal precursors to small cell lung cancers (SCLCs), by inhibition of NOTCH signaling. By using small inhibitory RNAs in these cultures to reduce levels of retinoblastoma (RB) protein, the product of a gene commonly mutated in SCLCs, we can significantly expand the number of PNECs. Similarly reducing levels of TP53 protein, the product of another tumor suppressor gene commonly mutated in SCLCs, or expressing mutant KRAS or EGFR genes, did not induce or expand PNECs, consistent with lineage-specific sensitivity to loss of RB function. Tumors resembling early stage SCLC grew in immunodeficient mice after subcutaneous injection of PNEC-containing cultures in which expression of both RB and TP53 was blocked. Single-cell RNA profiles of PNECs are heterogeneous; when RB levels are reduced, the profiles show similarities to RNA profiles from early stage SCLC; when both RB and TP53 levels are reduced, the transcriptome is enriched with cell cycle-specific RNAs. Taken together, these findings suggest that genetic manipulation of hESC-derived pulmonary cells will enable studies of the initiation, progression, and treatment of this recalcitrant cancer.

Impaired hematopoiesis and leukemia development in mice with a "knock-in" allele of U2af1(S34F)

Mutations affecting the spliceosomal protein U2AF1 are commonly found in myelodysplastic syndromes (MDS) and secondary acute myeloid leukemia (sAML). We have generated mice that carry Cre-dependent “knock-in” alleles of U2af1(S34F), the murine version of the most common mutant allele of U2AF1 encountered in human cancers. Cre-mediated recombination in murine hematopoietic lineages caused changes in RNA splicing, as well as multilineage cytopenia, macrocytic anemia, decreased hematopoietic stem and progenitor cells, low-grade dysplasias, and impaired transplantability, but without lifespan shortening or leukemia development. In an attempt to identify U2af1(S34F)-cooperating changes that promote leukemogenesis, we combined U2af1(S34F) with Runx1 deficiency in mice and further treated the mice with a mutagen, N-Ethyl-N-Nitrosourea (ENU). Overall, three of sixteen ENU-treated compound transgenic mice developed AML. However, AML did not arise in mice with other genotypes or without ENU treatment. Sequencing DNA from the three AMLs revealed somatic mutations homologous to those considered to be drivers of human AML, including predicted loss-or gain-of-function mutations in Tet2, Gata2, Idh1, and Ikzfl. However, the engineered U2af1(S34F) missense mutation reverted to wild type (WT) in two of the three AML cases, implying that U2af1(S34F) is dispensable, or even selected against, once leukemia is established. SIGNIFICANCE STATEMENT Somatic mutations in four splicing factor genes (U2AF1, SRSF2, SF3B1, and ZRSR2) are found in MDS and MDS-related AML, blood cancers with few effective treatment options. However, the pathophysiological effects of these mutations remain poorly characterized, in part due to the paucity of disease-relevant models. Here, we report the establishment of mouse models to study the most common U2AF1 mutation, U2af1(S34F). Production of the mutant protein specifically in the murine hematopoietic compartment disrupts hematopoiesis in ways resembling human MDS. We further identified deletion of the Runx1 gene and other known oncogenic mutations as changes that might collaborate with U2af1(S34F) to give rise to frank AML in mice.We have generated mice that carry Cre-dependent knock-in alleles of U2af1(S34F), the murine version of a mutant allele commonly encountered in human myelodysplastic syndromes and acute myeloid leukemia (AML). Cre-mediated recombination in murine hematopoietic lineages caused changes in RNA splicing, as well as multilineage cytopenia, macrocytic anemia, decreased hematopoietic stem and progenitor cells, low-grade dysplasias, and impaired transplantability, but without lifespan shortening or leukemia development. Mice with Cre-dependent U2af1(S34F) and homozygous Runx1 knockout alleles were mutagenized with low-dose N-Ethyl-N-Nitrosourea, and three of fourteen mice developed AML. However, AML did not arise in mice with other genotypes in our relatively small cohort. Sequencing DNA from the three AMLs revealed somatic mutations considered to be drivers of human AML, including mutations in Tet2, Gata2, Idh1, and Ikzf1. However, the U2af1 missense mutation reverted to WT in two of the three AML cases, implying that U2af1(S34F) is dispensable for maintaining leukemia.

Improving support for young biomedical scientists

Expand grant programs to encourage innovative research Over the past several years, we and others in the biomedical research community have become increasingly concerned that younger scientists are not being adequately supported as independent academic investigators and that, of equal importance, these newly launched investigators are being strongly discouraged from tackling novel scientific problems (1–6). Both issues can prevent talented trainees from aspiring to careers in biomedical research, despite the extraordinary opportunities offered by new technologies and recent discoveries. We view this situation as an existential threat to our profession, demanding that we urgently confront the underlying problems. It is widely recognized that career pathways for young scientists have changed dramatically and that over 80% of those who receive biomedical Ph.D.s today will be employed in positions other than academic faculty (1, 5). The U.S. National Academies of Sciences, Engineering, and Medicine recently released a report that addresses many important aspects of these cultural changes (7). Here we focus on the problems faced by those who will renew the ranks of academic research faculty, with proposals that complement the recommendations in that report. Drawing on lessons from Europe and the United States, we propose three steps that could be taken by funding agencies, specifically the U.S. National Institutes of Health (NIH) but also others across the world, to support young investigators in more constructive and effective ways.

Impaired hematopoiesis and leukemia development in mice with a conditional knock-in allele of a mutant splicing factor gene U2af1

Significance Somatic mutations in some splicing factor genes are frequently found in myelodysplastic syndromes (MDS) and MDS-related acute myeloid leukemia (AML), blood cancers with few effective treatment options. However, the pathophysiological effects of these mutations remain poorly characterized. Here, we report the establishment of mouse models to study a common splicing factor mutation, U2AF1(S34F). Production of the mutant protein in the murine hematopoietic compartment disrupts hematopoiesis in ways resembling human MDS. We further identified deletion of the Runx1 gene and other known oncogenic mutations as changes that might collaborate with U2af1(S34F) to give rise to frank AML in mice. However, the U2af1(S34F) mutation was absent in two of the three AML cases, raising the possibility that this mutant protein plays a dispensable role in tumor maintenance. Mutations affecting the spliceosomal protein U2AF1 are commonly found in myelodysplastic syndromes (MDS) and secondary acute myeloid leukemia (sAML). We have generated mice that carry Cre-dependent knock-in alleles of U2af1(S34F), the murine version of the most common mutant allele of U2AF1 encountered in human cancers. Cre-mediated recombination in murine hematopoietic lineages caused changes in RNA splicing, as well as multilineage cytopenia, macrocytic anemia, decreased hematopoietic stem and progenitor cells, low-grade dysplasias, and impaired transplantability, but without lifespan shortening or leukemia development. In an attempt to identify U2af1(S34F)-cooperating changes that promote leukemogenesis, we combined U2af1(S34F) with Runx1 deficiency in mice and further treated the mice with a mutagen, N-ethyl-N-nitrosourea (ENU). Overall, 3 of 16 ENU-treated compound transgenic mice developed AML. However, AML did not arise in mice with other genotypes or without ENU treatment. Sequencing DNA from the three AMLs revealed somatic mutations homologous to those considered to be drivers of human AML, including predicted loss- or gain-of-function mutations in Tet2, Gata2, Idh1, and Ikzf1. However, the engineered U2af1(S34F) missense mutation reverted to WT in two of the three AML cases, implying that U2af1(S34F) is dispensable, or even selected against, once leukemia is established.

Hyperactivation of extracellular signal-regulated kinase (ERK) by RAS-mediated signaling or inhibition of dual specificity phosphatase 6 (DUSP6) is associated with toxicity in lung adenocarcinoma cells with mutations in KRAS or EGFR

We recently described the synthetic lethality that results when mutant KRAS and mutant EGFR are coexpressed in human lung adenocarcinoma (LUAD) cells, revealing the biological basis for the mutual exclusivity of KRAS and EGFR mutations in lung cancers. We have now further defined the biochemical events responsible for the toxic effects of signaling through the RAS pathway. By combining pharmacological and genetic approaches, we have developed multiple lines of evidence that signaling through extracellular signal-regulated kinases (ERK1/2) mediates the toxicity. These findings imply that tumors with mutant oncogenes that drive signaling through the RAS pathway must restrain the activity of ERK1/2 to avoid cell toxicities and enable tumor growth. In particular, a dual specificity phosphatase, DUSP6, regulates phosphorylated (P)-ERK levels in lung adenocarcinoma cells, providing negative feedback to the RAS signaling pathway. Accordingly, inhibition of DUSP6 is cytotoxic in LUAD cells driven by either mutant KRAS or mutant EGFR, phenocopying the effects of co-expression of mutant KRAS and EGFR. Together, these data suggest that targeting DUSP6 or other feedback regulators of the EGFR-KRAS-ERK pathway may offer a strategy for treating certain cancers by exceeding an upper threshold of RAS-mediated signaling.

A Prize for Cancer Prevention

This years Lasker-DeBakey Prize for Clinical Research to Douglas Lowy and John Schiller celebrates the science behind one of the greatest advances in the history of cancer research: the development of vaccines that prevent infection and thus prevent tumor induction by pathogenic strains of human papilloma virus (HPV).