Genetic studies elucidate a link between testis-specific serine/threonine kinases (TSSKs) and male infertility in mammals, but the underlying mechanisms are unclear. Here, we identify a TSSK homolog in Drosophila, CG14305 (termed dTSSK), whose mutation impairs the histone-to-protamine transition during spermiogenesis and causes multiple phenotypic defects in nuclear shaping, DNA condensation, and flagellar organization in spermatids. Genetic analysis demonstrates that kinase catalytic activity of dTSSK, which is functionally conserved with human TSSKs, is essential for male fertility. Phosphoproteomics identify 828 phosphopeptides/449 proteins as potential substrates of dTSSK enriched primarily in microtubule-based processes, flagellar organization and mobility, and spermatid differentiation and development, suggesting that dTSSK phosphorylates various proteins to orchestrate postmeiotic spermiogenesis. Among them, the two substrates, protamine-like protein Mst77F/Ser9 and transition protein Mst33A/Ser237, are biochemically validated to be phosphorylated by dTSSK in vitro, and are genetically demonstrated to be involved in spermiogenesis in vivo. Collectively, our findings demonstrate that broad phosphorylation mediated by TSSKs plays an indispensable role in spermiogenesis.

The development of acquired drug resistance hampers the long-term success of B-RAF inhibitor therapy for melanoma patients. Here we show (V600E)B-RAF copy-number gain as a mechanism of acquired B-RAF inhibitor resistance in 4 out of 20 (20%) patients treated with B-RAF inhibitor. In cell lines, (V600E)B-RAF overexpression and knockdown conferred B-RAF inhibitor resistance and sensitivity, respectively. In (V600E)B-RAF amplification-driven (versus mutant N-RAS-driven) B-RAF inhibitor resistance, extracellular signal-regulated kinase reactivation is saturable, with higher doses of vemurafenib down-regulating phosho-extracellular signal-regulated kinase and re-sensitizing melanoma cells to B-RAF inhibitor. These two mechanisms of extracellular signal-regulated kinase reactivation are sensitive to the MEK1/2 inhibitor AZD6244/selumetinib or its combination with the B-RAF inhibitor vemurafenib. In contrast to mutant N-RAS-mediated (V600E)B-RAF bypass, which is sensitive to C-RAF knockdown, (V600E)B-RAF amplification-mediated resistance functions largely independently of C-RAF. Thus, alternative clinical strategies may potentially overcome distinct modes of extracellular signal-regulated kinase reactivation underlying acquired B-RAF inhibitor resistance in melanoma.

Despite the success of therapies targeting oncogenes in cancer, clinical outcomes are limited by residual disease that ultimately results in relapse. This residual disease is often characterized by non-genetic adaptive resistance, that in melanoma is characterised by altered metabolism. Here, we examine how targeted therapy reprograms metabolism in BRAF-mutant melanoma cells using a genome-wide RNA interference (RNAi) screen and global gene expression profiling. Using this systematic approach we demonstrate post-transcriptional regulation of metabolism following BRAF inhibition, involving selective mRNA transport and translation. As proof of concept we demonstrate the RNA processing kinase U2AF homology motif kinase 1 (UHMK1) associates with mRNAs encoding metabolism proteins and selectively controls their transport and translation during adaptation to BRAF-targeted therapy. UHMK1 inactivation induces cell death by disrupting therapy induced metabolic reprogramming, and importantly, delays resistance to BRAF and MEK combination therapy in multiple in vivo models. We propose selective mRNA processing and translation by UHMK1 constitutes a mechanism of non-genetic resistance to targeted therapy in melanoma by controlling metabolic plasticity induced by therapy.