Peptidyl nucleoside antibiotics (PNAs) are a diverse class of natural products with promising biomedical activities. These compounds have tripartite structures composed of a core saccharide, a nucleobase, and one or more amino acids. In particular, amipurimycin and the miharamycins are novel 2-aminopurinyl PNAs with complex nine-carbon core saccharides and include the unusual amino acids (–)-cispentacin and N5-hydroxyarginine, respectively. Despite their interesting structures and properties, these PNAs have heretofore eluded biochemical scrutiny. Herein is reported the discovery and initial characterization of the miharamycin gene cluster in Streptomyces miharaensis (mhr) and the amipurimycin gene cluster (amc) in Streptomyces novoguineensis and Streptomyces sp. SN-C1. The gene clusters were identified using a comparative genomics approach, and heterologous expression of the amc cluster as well as gene interruption experiments in the mhr cluster support their role in the biosynthesis of amipurimycin and the miharamycins, respectively. The mhr and amc biosynthetic gene clusters characterized encode enzymes typical of polyketide biosynthesis instead of enzymes commonly associated with PNA biosynthesis, which along with labeled precursor feeding studies, implies that the core saccharides found in the miharamycins and amipurimycin are partially assembled as polyketides rather than derived solely from carbohydrates. Furthermore, in vitro analysis of Mhr20 and Amc18 established their roles as ATP-grasp ligases involved in the attachment of the pendant amino acids found in these PNAs, and Mhr24 was found to be an unusual hydroxylase involved in the biosynthesis of N5-hydroxyarginine. Finally, analysis of the amc cluster and feeding studies also led to the proposal of a biosynthetic pathway for (–)-cispentacin.

Herbicidins are adenosine-based nucleoside antibiotics with an unusual tricyclic undecose core decorated with a (5-hydroxy)tiglyl moiety. Feeding studies are herein reported demonstrating that the tricyclic core is derived from d-glucose and d-ribose, whereas the tiglyl moiety is derived from an intermediate of l-isoleucine catabolism. Identification of the gene cluster for herbicidin A biosynthesis in Streptomyces sp. L-9-10 as well as its verification by heterologous expression in a non-producing host are described, and the results of in vitro characterization of a carboxyl methyltransferase encoded in the cluster, Her8, are presented. Based on these observations, a biosynthetic pathway is proposed for herbicidins.

Infantile myofibromatosis (IM) is an aggressive neoplasm composed of myofibroblast-like cells in children. Although typically localized, it can also present as multifocal disease, which represents a challenge for effective treatment. IM has previously been linked to activating somatic and germline point mutations in the PDGFRβ tyrosine kinase encoded by the PDGFRB gene. Clinical panel-based targeted tumor sequencing of a tumor from a newborn with multifocal IM revealed a novel PDGFRB rearrangement, which was reported as being of unclear significance. Additional sequencing of cDNA from tumor and germline DNA confirmed a complex somatic/mosaic PDGFRB rearrangement with an apparent partial tandem duplication disrupting the juxtamembrane domain. Ectopic expression of cDNA encoding the mutant form of PDGFRB markedly enhanced cell proliferation of mouse embryo fibroblasts (MEFs) compared to wild-type PDGFRB and conferred tumor-forming capacity on nontumorigenic 10T1/2 fibroblasts. The mutated protein enhanced MAPK activation and retained sensitivity to the PDGFRβ inhibitor imatinib. Our findings reveal a new mechanism by which PDGFRB can be activated in IM, suggest that therapy with tyrosine kinase inhibitors including imatinib may be beneficial, and raise the possibility that this receptor tyrosine kinase might be altered in a similar fashion in additional cases that would similarly present annotation challenges in clinical DNA sequencing analysis pipelines.

Disruption of the CDKN2A (INK4A/ARF) and B (INK4B) genes, which encode three function-independent tumor suppressors, is one of the most common events in human cancer. Because their relative importance in tumor prevention appears to be species-and context-specific, studying their regulation can shed light on mechanisms by which they are bypassed in malignant transformation. We previously unveiled a new pathway in which TGFb selectively induces Arf at mouse Cdkn2a in eye development and cultured fibroblasts. As TGFb signaling is often derailed in cancer development or progression, we investigated its control of CDKN2A/B in human cancer. Computational analyses of sequencing and array data from nearly 11,000 patients with cancer in TCGA showed discordant expression of ARF and INK4A in most cancer subtypes, with gene copy-number loss and promoter methyl-ation involved in only a subset. Using HeLa cells as a model, we found that exogenous TGFb induced ARF mRNA and protein, and ARF knockdown limited TGFb-mediated growth suppression. TGFb-mediated ARF mRNA induction required SMAD2/3, p38MAPK, and SP1, and ARF mRNA was induced without added RNAPII recruitment. Chromatin immunoprecipitation unveiled a remote enhancer element engaged by TGFb by a mechanism that partially depended on p38MAPK. CRISPR-based editing of this enhancer limited induction of ARF and INK4B by TGFb, but not by oncogenic RAS. Implications: Our findings reveal new molecular mechanisms by which CDKN2A/B regulation is coupled to external cues, and those findings represent entry points to further explore pharmacologic strategies to restore their expression in cancer.

<div>Abstract<p>Disruption of the <i>CDKN2A</i> (<i>INK4A/ARF</i>) and <i>B</i> (<i>INK4B</i>) genes, which encode three function-independent tumor suppressors, is one of the most common events in human cancer. Because their relative importance in tumor prevention appears to be species- and context-specific, studying their regulation can shed light on mechanisms by which they are bypassed in malignant transformation. We previously unveiled a new pathway in which TGFβ selectively induces <i>Arf</i> at mouse <i>Cdkn2a</i> in eye development and cultured fibroblasts. As TGFβ signaling is often derailed in cancer development or progression, we investigated its control of <i>CDKN2A/B</i> in human cancer. Computational analyses of sequencing and array data from nearly 11,000 patients with cancer in TCGA showed discordant expression of <i>ARF</i> and <i>INK4A</i> in most cancer subtypes, with gene copy-number loss and promoter methylation involved in only a subset. Using HeLa cells as a model, we found that exogenous TGFβ induced <i>ARF</i> mRNA and protein, and <i>ARF</i> knockdown limited TGFβ-mediated growth suppression. TGFβ-mediated <i>ARF</i> mRNA induction required SMAD2/3, p38MAPK, and SP1, and <i>ARF</i> mRNA was induced without added RNAPII recruitment. Chromatin immunoprecipitation unveiled a remote enhancer element engaged by TGFβ by a mechanism that partially depended on p38MAPK. CRISPR-based editing of this enhancer limited induction of <i>ARF</i> and <i>INK4B</i> by TGFβ, but not by oncogenic RAS.</p>Implications:<p>Our findings reveal new molecular mechanisms by which <i>CDKN2A/B</i> regulation is coupled to external cues, and those findings represent entry points to further explore pharmacologic strategies to restore their expression in cancer.</p></div>