Tankyrase Inhibition Blocks Wnt/ß-Catenin Pathway and Reverts Resistance to PI3K and AKT Inhibitors in the Treatment of Colorectal Cancer.

PURPOSE: Oncogenic mutations in the KRAS/PI3K/AKT pathway are one of the most frequent alterations in cancer. Although PI3K or AKT inhibitors show promising results in clinical trials, drug resistance frequently emerges. We previously revealed Wnt/ß-catenin signaling hyperactivation as responsible for such resistance in colorectal cancer. Here we investigate Wnt-mediated resistance in patients treated with PI3K or AKT inhibitors in clinical trials and evaluate the efficacy of a new Wnt/tankyrase inhibitor, NVP-TNKS656, to overcome such resistance. EXPERIMENTAL DESIGN: Colorectal cancer patient-derived sphere cultures and mouse tumor xenografts were treated with NVP-TNKS656, in combination with PI3K or AKT inhibitors.We analyzed progression-free survival of patients treated with different PI3K/AKT/mTOR inhibitors in correlation with Wnt/ß-catenin pathway activation, oncogenic mutations, clinicopathological traits, and gene expression patterns in 40 colorectal cancer baseline tumors. RESULTS: Combination with NVP-TNKS656 promoted apoptosis in PI3K or AKT inhibitor-resistant cells with high nuclear ß-catenin content. High FOXO3A activity conferred sensitivity to NVP-TNKS656 treatment. Thirteen of 40 patients presented high nuclear ß-catenin content and progressed earlier upon PI3K/AKT/mTOR inhibition. Nuclear ß-catenin levels predicted drug response, whereas clinicopathologic traits, gene expression profiles, or frequent mutations (KRAS, TP53, or PIK3CA) did not. CONCLUSIONS: High nuclear ß-catenin content independently predicts resistance to PI3K and AKT inhibitors. Combined treatment with a Wnt/tankyrase inhibitor reduces nuclear ß-catenin, reverts such resistance, and represses tumor growth. FOXO3A content and activity predicts response to Wnt/ß-catenin inhibition and together with ß-catenin may be predictive biomarkers of drug response providing a rationale to stratify colorectal cancer patients to be treated with PI3K/AKT/mTOR and Wnt/ß-catenin inhibitors.

Clinical response to a lapatinib-based therapy for a Li-Fraumeni syndrome patient with a novel HER2V659E mutation.

UNLABELLED: Genomic characterization of recurrent breast and lung tumors developed over the course of 10 years in a 29-year-old patient with a germline TP53 mutation (Li-Fraumeni Syndrome) identified oncogenic alterations in the HER2 and EGFR genes across all tumors, including HER2 amplifications, an EGFR-exon 20 insertion, and the first-in-humans HER2V659E mutation showing a phenotypic convergent evolution toward HER2 and EGFR alterations. Following the identification of HER2-activating events in the most recent lung carcinoma and in circulating tumor cells, we treated the reminiscent metastatic lesions with a lapatinib-based therapy. A symptomatic and radiologic clinical response was achieved. HER2V659E sensitivity to lapatinib was confirmed in the laboratory. SIGNIFICANCE: The precise knowledge of the genomic alterations present in tumors is critical to selecting the optimal treatment for each patient. Here, we report the molecular characterization and clinical response to a lapatinib-based therapy for the tumors of a Li-Fraumeni patient showing prevalence of HER2 and EGFR genomic alterations.

In vivo role of different domains and of phosphorylation in the transcription factor Nkx2-1.

BACKGROUND: The transcription factor Nkx2-1 (also known as TTF-1, Titf1 or T/EBP) contains two apparently redundant activation domains and is post-translationally modified by phosphorylation. We have generated mouse mutant strains to assess the roles of the two activation domains and of phosphorylation in mouse development and differentiation. RESULTS: Mouse strains expressing variants of the transcription factor Nkx2-1 deleted of either activation domain have been constructed. Phenotypic analysis shows for each mutant a distinct set of defects demonstrating that distinct portions of the protein endow diverse developmental functions of Nkx2-1. Furthermore, a mouse strain expressing a Nkx2-1 protein mutated in the phosphorylation sites shows a thyroid gland with deranged follicular organization and gene expression profile demonstrating the functional role of phosphorylation in Nkx2-1. CONCLUSIONS: The pleiotropic functions of Nkx2-1 are not all due to the protein as a whole since some of them can be assigned to separate domains of the protein or to specific post-translational modifications. These results have implication for the evolutionary role of mutations in transcription factors.

Conditional inactivation of the E-cadherin gene in thyroid follicular cells affects gland development but does not impair junction formation.

We have conditionally inactivated the E-cadherin gene in the thyroid follicular cells of mouse embryo to unravel its role in thyroid development. We used the Cre-loxP system in which the Cre-recombinase was expressed under the control of the tissue-specific thyroglobulin promoter that becomes active at embryonic d 15. At postnatal d 7, thyroid follicle lumens in the knockout mice were about 30% smaller with respect to control mice and had an irregular shape. E-cadherin was almost completely absent in thyrocytes, beta-catenin was significantly reduced, whereas no change in gamma-catenin was detected. alpha-Catenin was also reduced on the cell plasma membrane. Despite the dramatic loss of E-cadherin and beta-catenin, cell-cell junctions were not affected, the distribution of tight junction proteins was unaltered, and no increase of thyroglobulin circulating in the blood was observed. In addition, we found that other members of the cadherin family, the R-cadherin and the Ksp-cadherin, were expressed in thyrocytes and that their membrane distribution was not altered in the E-cadherin conditional knockout mouse. Our results indicate that E-cadherin has a role in the development of the thyroid gland and in the expression of beta-catenin, but it is not essential for the maintenance of follicular cell adhesion.

TTF-1 phosphorylation is required for peripheral lung morphogenesis, perinatal survival, and tissue-specific gene expression.

Thyroid transcription factor-1 (TTF-1) is a 43-kDa, phosphorylated member of the Nkx2 family of homeodomain-containing proteins expressed selectively in lung, thyroid, and the central nervous system. To assess the role of TTF-1 and its phosphorylation during lung morphogenesis, mice bearing a mutant allele, in which seven serine phosphorylation sites were mutated, Titf1PM/PM, were generated by homologous recombination. Although heterozygous Titf1PM/+ mice were unaffected, homozygous Titf1PM/PM mice died immediately following birth. In contrast to Titf1 null mutant mice, which lack peripheral lung tissues, bronchiolar and peripheral acinar components of the lung were present in the Titf1PM/PM mice. Although lobulation and early branching morphogenesis were maintained in the mutant mice, abnormalities in acinar tubules and pulmonary hypoplasia indicated defects in lung morphogenesis later in development. Although TTF-1PM protein was readily detected within the nuclei of pulmonary epithelial cells at sites and abundance consistent with that of endogenous TTF-1, expression of a number of known TTF-1 target genes, including surfactant proteins and secretoglobulin 1A, was variably decreased in the mutant mice. Vascular endothelial growth factor mRNA was decreased in association with decreased formation of peripheral pulmonary blood vessels. Genes mediating surfactant homeostasis, vasculogenesis, host defense, fluid homeostasis, and inflammation were highly represented among those regulated by TTF-1. Thus, in contrast to the null Titf1 mutation, the Titf1PM/PM mutant substantially restored lung morphogenesis. Direct and indirect transcriptional targets of TTF-1 were identified that are likely to play important roles in lung formation and function.