Association with TFIIIC limits MYCN localisation in hubs of active promoters and chromatin accumulation of non-phosphorylated RNA polymerase II.

MYC family oncoproteins regulate the expression of a large number of genes and broadly stimulate elongation by RNA polymerase II (RNAPII). While the factors that control the chromatin association of MYC proteins are well understood, much less is known about how interacting proteins mediate MYC's effects on transcription. Here, we show that TFIIIC, an architectural protein complex that controls the three-dimensional chromatin organisation at its target sites, binds directly to the amino-terminal transcriptional regulatory domain of MYCN. Surprisingly, TFIIIC has no discernible role in MYCN-dependent gene expression and transcription elongation. Instead, MYCN and TFIIIC preferentially bind to promoters with paused RNAPII and globally limit the accumulation of non-phosphorylated RNAPII at promoters. Consistent with its ubiquitous role in transcription, MYCN broadly participates in hubs of active promoters. Depletion of TFIIIC further increases MYCN localisation to these hubs. This increase correlates with a failure of the nuclear exosome and BRCA1, both of which are involved in nascent RNA degradation, to localise to active promoters. Our data suggest that MYCN and TFIIIC exert an censoring function in early transcription that limits promoter accumulation of inactive RNAPII and facilitates promoter-proximal degradation of nascent RNA.

HER3 overexpression: a predictive marker for poor prognosis in advanced ALK-positive non-small cell lung cancer treated with ALK inhibitors.

Background: Anaplastic lymphoma kinase (ALK)-targeted tyrosine kinase inhibitors (TKIs) improve patient survival; however, some patients develop ALK-TKI resistance with unidentified mechanisms. We investigated ErbB family and c-MET expression in patients with ALK-positive non-small cell lung cancer (NSCLC) to understand their roles in the ALK-TKI response. Methods: We studied 72 patients with advanced ALK-positive NSCLC with EML4-ALK fusion variant subtyping and immunostaining for c-MET, EGFR, HER2, and HER3 on tissue specimens both pre- (primary) and post-treatment (secondary) with ALK-TKI. We investigated the association of their expression with survival outcomes and assessed the effectiveness of combining ALK and EGFR inhibitors in ALK-positive NSCLC cell lines stimulated with the HER3-specific ligand HRG1. Results: High expression of c-MET, EGFR, HER2, and HER3 was observed in 4.9%, 18.0%, 1.6%, and 25.8% of primary tumors, respectively, and 18.5%, 37.0%, 10.7%, and 35.7% of secondary tumors, respectively. HER3 overexpression in primary tumors showed inferior survival (P=0.132). In the subgroup with EML4-ALK variant 1/2 (V1/V2), HER3 overexpression was significantly associated with inferior survival in both primary and secondary tumors (P=0.022 and P=0.004, respectively). Combination treatment with lorlatinib and erlotinib significantly reduced HRG1-induced activation of RTK signaling in ALK-positive NSCLC cells. Conclusions: HER3 overexpression has potential as a prognostic marker in ALK-positive NSCLCs, including ALK-TKI naïve and treated cases, especially those with EML4-ALK V1/V2. Assessing HER3 expression may be crucial for treatment planning and outcome prediction in these patients.

A nanobody inhibitor of Fascin-1 actin-bundling activity and filopodia formation.

Fascin-1-mediated actin-bundling activity is central to the generation of plasma membrane protrusions required for cell migration. Dysregulated formation of cellular protrusions is observed in metastatic cancers, where they are required for increased invasiveness, and is often correlated with increased Fascin-1 abundance. Therefore, there is interest in generating therapeutic Fascin-1 inhibitors. We present the identification of Nb 3E11, a nanobody inhibitor of Fascin-1 actin-bundling activity and filopodia formation. The crystal structure of the Fascin-1/Nb 3E11 complex reveals the structural mechanism of inhibition. Nb 3E11 occludes an actin-binding site on the third ß-trefoil domain of Fascin-1 that is currently not targeted by chemical inhibitors. Binding of Nb 3E11 to Fascin-1 induces a conformational change in the adjacent domains to stabilize Fascin-1 in an inhibitory state similar to that adopted in the presence of small-molecule inhibitors. Nb 3E11 could be used as a tool inhibitor molecule to aid in the development of Fascin-1 targeted therapeutics.