Enrichment of kinase fusions in ESR1 wild-type, metastatic breast cancer revealed by a systematic analysis of 4854 patients.

BACKGROUND: Kinase fusions are rare and poorly characterized in breast cancer (BC). We aimed to characterize kinase fusions within a large cohort of advanced BC. PATIENTS AND METHODS: A total of 4854 patients with BC were analyzed by Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) targeted DNAseq and MSK-Fusion targeted RNAseq during the study time period. RESULTS: Twenty-seven of 4854 (0.6%) patients harbored fusions: 11 FGFR (five FGFR2, three FGFR3, three FGFR1), five BRAF, four NTRK1, two RET, two ROS1, one ALK, one ERBB2, and one MET. A history of endocrine therapy was present in 15 (56%) of fusion-positive BC; eight of the 15 cases had available pre-treatment samples, of which six were fusion-negative. None of the fusion-positive BC samples harbored ESR1 hotspot mutations. Two patients with acquired LMNA-NTRK1 fusions and metastatic disease received larotrectinib and demonstrated clinical benefit. CONCLUSION: Kinase fusions in BC are extremely rare, and appear to be enriched in hormone-resistant, metastatic carcinomas and mutually exclusive with ESR1 mutations. The present study expands the spectrum of genetic alterations activating mitogen-activated protein kinase (MAPK) signaling that can substitute for ESR1 mutations in this setting. Molecular testing at progression after endocrine therapy should include fusion testing, particularly in the absence of ESR1 hotspot alterations, in an effort to identify additional therapeutic options which may provide substantial clinical benefit.

Constitutively activated Stat3 protects fibroblasts from serum withdrawal and UV-induced apoptosis and antagonizes the proapoptotic effects of activated Stat1.

Stats1 and 3 (signal transducers and activators of transcription) can be activated simultaneously, although not necessarily to the same degree or duration, by the interaction of cells with the same polypeptide ligand (EGF, PDGF, or high concentrations of IL-6, for example). However, these two Stat proteins can mediate opposing effects on cell growth and survival. Stat1 activation slows growth and promotes apoptosis. In contrast, activated Stat3 can protect cells from apoptosis. Furthermore, a constitutively active form of Stat3, Stat3-C (bridged by S-S linkages between cysteines instead of phosphotyrosines) can induce cellular transformation of fibroblasts. We have determined that fibroblasts transformed by Stat3-C are more resistant to proapoptotic stimuli than nontransformed cells. Also, to examine the potential opposing roles in apoptosis of Stat1 and Stat3, we studied the cervical carcinoma-derived cell line, Me180, which undergoes Stat1-dependent, IFN gamma-induced apoptosis. Me180 cells that express Stat3-C are protected against IFN gamma-mediated apoptosis.

Activation of STAT proteins and growth control.

This review will discuss how STAT (Signal Transducers and Activators of Transcription) proteins, a group of transcription factors that transmit signals from the extracellular surface of cells to the nucleus, are involved in growth control. I will discuss the anatomy of a STAT protein, how it works as a transcription factor, the molecules that regulate its "activity", the phenotypes of mice that lack individual STAT proteins and their involvement in growth, differentiation, apoptosis, and transformation. Finally, a number of examples will be presented of how dysregulated STAT signaling may be involved in the pathogenesis of cancer.

Stat3 as an oncogene.

STATs are latent transcription factors that mediate cytokine- and growth factor-directed transcription. In many human cancers and transformed cell lines, Stat3 is persistently activated, and in cell culture, active Stat3 is either required for transformation, enhances transformation, or blocks apoptosis. We report that substitution of two cysteine residues within the C-terminal loop of the SH2 domain of Stat3 produces a molecule that dimerizes spontaneously, binds to DNA, and activates transcription. The Stat3-C molecule in immortalized fibroblasts causes cellular transformation scored by colony formation in soft agar and tumor formation in nude mice. Thus, the activated Stat3 molecule by itself can mediate cellular transformation and the experiments focus attention on the importance of constitutive Stat3 activation in human tumors.

A single amino acid substitution in the v-Eyk intracellular domain results in activation of Stat3 and enhances cellular transformation.

The receptor tyrosine kinase Eyk, a member of the Axl/Tyro3 subfamily, activates the STAT pathway and transforms cells when constitutively activated. Here, we compared the potentials of the intracellular domains of Eyk molecules derived from c-Eyk and v-Eyk to transform rat 3Y1 fibroblasts. The v-Eyk molecule induced higher numbers of transformants in soft agar and stronger activation of Stat3; levels of Stat1 activation by the two Eyk molecules were similar. A mutation in the sequence Y933VPL, present in c-Eyk, to the v-Eyk sequence Y933VPQ led to increased activation of Stat3 and increased transformation efficiency. However, altering another sequence, Y862VNT, present in both Eyk molecules to F862VNT markedly decreased transformation without impairing Stat3 activation. These results indicate that activation of Stat3 enhances transformation efficiency and cooperates with another pathway to induce transformation.

Epidermal growth factor-induced growth inhibition requires Stat1 activation.

Epidermal growth factor (EGF) is a mitogen for most epithelial cells. Paradoxically, the growth of some cultured cell lines, containing high numbers of EGF receptors, are inhibited by EGF. Here we demonstrate that growth inhibition by EGF in several cell lines correlates with the activation of the signal transducer and activator of transcription (Stat) 1. In contrast, in normal fibroblasts and several cell lines that are growth stimulated by EGF, we observed no or very transient activation of Stat1. A causal association between Stat1 activation by EGF and growth inhibition was suggested by the expression of a dominant-negative Stat1 in A431 cells, resulting in the loss of Stat1 DNA binding and concomitant resistance to growth inhibition by EGF. We conclude that, in the cells examined, EGF-induced arrest of growth requires activated Stat1.

Stat3 activation is required for cellular transformation by v-src.

Stat3 activation has been associated with cytokine-induced proliferation, anti-apoptosis, and transformation. Constitutively activated Stat3 has been found in many human tumors as well as v-abl- and v-src-transformed cell lines. Because of these correlations, we examined directly the relationship of activated Stat3 to cellular transformation and found that wild-type Stat3 enhances the transforming potential of v-src while three dominant negative Stat3 mutants inhibit v-src transformation. Stat3 wild-type or mutant proteins did not affect v-ras transformation. We conclude that Stat3 has a necessary role in v-src transformation.

Transcriptionally active Stat1 is required for the antiproliferative effects of both interferon alpha and interferon gamma.

Type I (alpha, beta) and type II (gamma) interferons (IFNs) can restrict the growth of many cell types. INF-stimulated gene transcription, a key early event in IFN response, acts through the Janus kinase-signal transducers and activators of transcription pathway, in which both IFN-alpha and IFN-gamma activate the transcription factor Stat1. A cell line lacking Stat1 (U3A) was not growth-arrested by IFN-alpha or IFN-gamma, and experiments were carried out with U3A cells permanently expressing normal or various mutant forms of Stat1 protein. Only cells in which complete Stat1 activity was available (Stat1alpha) were growth-inhibited by IFN-gamma. A mutant that supports 20-30% normal transcription did not cause growth restraint. In contrast, IFN-alpha growth restraint was imposed by cells producing Stat1beta, which lacks transcriptional activation potential. This parallels earlier results showing the truncated Stat1 can function in IFN-alpha gene activation. In addition to experiments on long-term cultured cells, we also found that wild-type primary mouse embryonic fibroblasts were inhibited by IFNs, but fibroblasts from Stat1-deficient mouse embryos were not inhibited by IFNs.

Alterations in a yeast protein resembling HIV Tat-binding protein relieve requirement for an acidic activation domain in GAL4.

The acidic transcriptional activation motif functions in all eukaryotes, which suggests that it makes contact with some universal component of the transcriptional apparatus. Transcriptional activation by the yeast regulatory protein GAL4 requires an acidic region at its carboxyl terminus. Here we implement a selection scheme to determine whether GAL4 can still function when this C-terminal domain has been deleted. It can, when accompanied by a mutation in the SUG1 gene which is an essential gene in yeast. Analysis of mutant SUG1 in combination with various alleles of GAL4 indicates that SUG1 acts through a transcriptional pathway that depends on GAL4, but requires a region of GAL4 other than the C-terminal acidic activation domain. The predicted amino-acid sequence of SUG1 closely resembles that of two human proteins, TBP1 and MSS1, which modulate expression mediated by the human immunodeficiency virus tat gene.