The T-box transcription factors TBX2 and TBX3 are overexpressed in several cancers and are able to bypass senescence by repressing ARF and p21 WAF1/CIP1/SDII . Although these studies suggest that they may both contribute to the oncogenic process by repressing common targets, whether they have redundant or distinct roles in cancers where they are both overexpressed remains to be elucidated. Importantly, when Tbx2 function is inhibited in melanoma cells lacking Tbx3, the cells senesce, but whether this is possible in melanoma cells overexpressing both proteins is not known. An understanding of this issue may have important implications for the design of an effective pro-senescence therapy. In this study, the authors used a sh-RNA approach to knock down TBX2 and TBX3 individually in 2 human melanoma cell lines that overexpress both these factors and then examined their specific involvement in the oncogenic process. They demonstrate, using in vitro and in vivo cell proliferation, as well as colony-and tumor-forming ability and cell motility assays, that TBX2 and TBX3 have distinct roles in melanoma progression. In the tested lines, although TBX2 could promote proliferation and transformation and was required by primary melanoma cells for immortality, TBX3 was required for tumor formation and cell migration. These findings were reproducible in a human breast cancer cell line, which confirms that TBX2 and TBX3, although highly homologous, do not have redundant roles in the transformation process of cancers where they are both overexpressed. These results have important implications for the development of new cancer treatments and in particular for melanoma, which is a highly aggressive and intractable cancer.

Treatment of individuals with cystic fibrosis (CF) has been transformed by small molecule therapies that target select pathogenic variants in the CF transmembrane conductance regulator (CFTR). To expand treatment eligibility, we stably expressed 43 rare missense CFTR variants associated with moderate CF from a single site in the genome of human CF bronchial epithelial (CFBE41o-) cells. The magnitude of drug response was highly correlated with residual CFTR function for the potentiator ivacaftor, the corrector lumacaftor, and ivacaftor-lumacaftor combination therapy. Response of a second set of 16 variants expressed stably in Fischer rat thyroid (FRT) cells showed nearly identical correlations. Subsets of variants were identified that demonstrated statistically significantly higher responses to specific treatments. Furthermore, nearly all variants studied in CFBE cells (40 of 43) and FRT cells (13 of 16) demonstrated greater response to ivacaftor-lumacaftor combination therapy than either modulator alone. Together, these variants represent 87% of individuals in the CFTR2 database with at least 1 missense variant. Thus, our results indicate that most individuals with CF carrying missense variants are (a) likely to respond modestly to currently available modulator therapy, while a small fraction will have pronounced responses, and (b) likely to derive the greatest benefit from combination therapy.

No abstract

Missense DNA variants have variable effects upon protein function. Consequently, interpreting their pathogenicity is challenging, especially when they are associated with disease variability. To determine the degree to which functional assays inform interpretation, we analyzed 48 CFTR missense variants associated with variable expressivity of cystic fibrosis (CF). We assessed function in a native isogenic context by evaluating CFTR mutants that were stably expressed in the genome of a human airway cell line devoid of endogenous CFTR expression. 21 of 29 variants associated with full expressivity of the CF phenotype generated <10% wild-type CFTR (WT-CFTR) function, a conservative threshold for the development of life-limiting CF lung disease, and five variants had moderately decreased function (10% to ∼25% WT-CFTR). The remaining three variants in this group unexpectedly had >25% WT-CFTR function; two were higher than 75% WT-CFTR. As expected, 14 of 19 variants associated with partial expressivity of CF had >25% WT-CFTR function; however, four had minimal to no effect on CFTR function (>75% WT-CFTR). Thus, 6 of 48 (13%) missense variants believed to be disease causing did not alter CFTR function. Functional studies substantially refined pathogenicity assignment with expert annotation and criteria from the American College of Medical Genetics and Genomics and Association for Molecular Pathology. However, four algorithms (CADD, REVEL, SIFT, and PolyPhen-2) could not differentiate between variants that caused severe, moderate, or minimal reduction in function. In the setting of variable expressivity, these results indicate that functional assays are essential for accurate interpretation of missense variants and that current prediction tools should be used with caution.