Inactivating NF1 Mutations Are Enriched in Advanced Breast Cancer and Contribute to Endocrine Therapy Resistance.

PURPOSE: Advanced breast cancer (ABC) has not been subjected to the same degree of molecular scrutiny as early primary cancer. Breast cancer evolves with time and under the selective pressure of treatment, with the potential to acquire mutations with resistance to treatment and disease progression. To identify potentially targetable mutations in advanced breast cancer, we performed prospective molecular characterization of a cohort of patients with ABC. EXPERIMENTAL DESIGN: Biopsies from patients with advanced breast cancer were sequenced with a 41 genes targeted panel in the ABC Biopsy (ABC-Bio) study. Blood samples were collected at disease progression for circulating tumor DNA (ctDNA) analysis, along with matched primary tumor to assess for acquisition in ABC in a subset of patients. RESULTS: We sequenced 210 ABC samples, demonstrating enrichment compared with primary disease for potentially targetable mutations in HER2 (in 6.19% of samples), AKT1 (7.14%), and NF1 (8.10%). Of these enriched mutations, we show that NF1 mutations were frequently acquired in ABC, not present in the original primary disease. In ER-positive cancer cell line models, loss of NF1 resulted in endocrine therapy resistance, through both ER-dependent and -independent mechanisms. NF1 loss promoted ER-independent cyclin D1 expression, which could be therapeutically targeted with CDK4/6 inhibitors in vitro. Patients with NF1 mutations detected in baseline circulating tumor DNA had a good outcome on the CDK4/6 inhibitor palbociclib and fulvestrant. CONCLUSIONS: Our research identifies multiple therapeutic opportunities for advanced breast cancer and identifies the previously underappreciated acquisition of NF1 mutations.

Specialized transcription factories.

We have previously suggested a model for the eukaryotic genome based on the structure of the bacterial nucleoid where active RNA polymerases cluster to loop the intervening DNA. This organization of polymerases into clusters--which we call transcription 'factories'--has important consequences. For example, in the nucleus of a HeLa cell the concentration of soluble RNA polymerase II is approximately 1 mM, but the local concentration in a factory is 1000-fold higher. Because a promoter can diffuse approximately 100 nm in 15 s, one lying near a factory is likely to initiate; moreover, when released at termination, it will still lie near a factory, and the movement and modifications (e.g. acetylation) accompanying elongation will leave it in an 'open' conformation. Another promoter out in a long loop is less likely to initiate, because the promoter concentration falls off with the cube of the distance from the factory. Moreover, a long tether will buffer it from transcription-induced movement, making it prone to deacetylation, deposition of HP1 (heterochromatin protein 1), and incorporation into heterochromatin. The context around a promoter will then be self-sustaining: productive collisions of an active promoter with the factory will attract factors increasing the frequency of initiation, and the longer an inactive promoter remains inactive, the more it becomes embedded in heterochromatin. We review here the evidence that different factories may specialize in the transcription of different groups of genes.

Paired-type homeodomain transcription factors are imported into the nucleus by karyopherin 13.

We report that the paired homeodomain transcription factor Pax6 is imported into the nucleus by the Karyopherin beta family member Karyopherin 13 (Kap13). Pax6 was identified as a potential cargo for Kap13 by a yeast two-hybrid screen. Direct binding of Pax6 to Kap13 was subsequently confirmed by in vitro assays with recombinant proteins, and binding in vivo was shown by coimmunoprecipitation. Ran-dependent import of Pax6 by Kap13 was shown to occur by using a digitonin-permeabilized cells assay. Kap13 binds to Pax6 via a nuclear localization sequence (NLS), which is located within a segment of 80 amino acid residues that includes the homeodomain. Kap13 showed reduced binding to Pax6 when either region located at each end of the homeodomain (208 to 214 and 261 to 267) was deleted. The paired-type homeodomain transcription factor family includes more than 20 members. All members contain a region similar to the NLS found in Pax6 and are therefore likely to be imported by Kap13. We confirmed this hypothesis for Pax3 and Crx, which bind to and are imported by Kap13.

Karyopherin beta 2B participates in mRNA export from the nucleus.

Transport of macromolecules between the cell nucleus and cytoplasm occurs through the nuclear pores and is mediated by soluble carriers known as karyopherins (Kaps), transportins, importins, or exportins. We report that Kap beta2B (transportin-2) forms complexes with the mRNA export factor TAP in the presence of RanGTP, as shown by coimmunoprecipitation from HeLa cells. The interaction strictly depends on the presence of RanGTP. In digitonin-permeabilized cells, Kap beta2B mediates TAP-GFP export from the nuclei in the presence of RanGTP. A TAP mutant that does not coimmunoprecipitate with Kap beta2B is also not exported by Kap beta2B. In the permeabilized cells assay, TAP is also exported independently of Kap beta2B by direct interaction with nucleoporins, in agreement with previous reports. The export rate is, however, significantly lower than the Kap beta2B-mediated pathway. Both Kap beta2B and TAP are present and enriched in the poly(A)(+) RNA complexes isolated from HeLa cell nuclear lysates. Poly(A)(+) RNA strongly accumulates in the nuclei of HeLa cells treated with Kap beta2B short interfering RNA, indicating that Kap beta2B is involved in the export of at least a large proportion of the mRNA species. The export of beta-actin and GAPDH mRNA is also inhibited, whereas 28S RNA is not affected. The data support the conclusion that Kap beta2B participates directly in the export of a large proportion of cellular mRNAs, and TAP connects Kap beta2B to the mRNAs to be exported.