Analytical Validation and Performance of a 7-Gene Next-Generation Sequencing Panel in Uveal Melanoma.

INTRODUCTION: Gene expression profiling (GEP) is widely used for prognostication in patients with uveal melanoma (UM). Because biopsy tissue is limited, it is critical to obtain as much genomic information as possible from each sample. Combined application of both GEP and next-generation sequencing (NGS) allows for analysis of RNA and DNA from a single biopsy sample, offers additional prognostic information, and can potentially inform therapy selection. This study evaluated the analytical performance of a targeted custom NGS panel for mutational profiling of 7 genes commonly mutated in UM. METHODS: One hundred five primary UM tumors were analyzed, including 37 formalin-fixed paraffin-embedded (FFPE) and 68 fine-needle aspiration biopsy specimens. Sequencing was performed on the Ion GeneStudio S5 platform to an average read depth of >500X per region of interest. RESULTS: The 7-gene panel achieved a positive percent agreement of 100% for detection of both single-nucleotide variants and insertions/deletions, with a technical positive predictive value of 98.8% and 100%, respectively. Intra-assay and inter-assay concordance studies confirmed the assay's reproducibility and repeatability. DISCUSSION/CONCLUSION: The 7-gene panel is a robust, highly accurate NGS test that can be successfully performed, along with GEP, from a single small-gauge needle biopsy sample or FFPE specimen.

The Conserved ATM Kinase RAG2-S365 Phosphorylation Site Limits Cleavage Events in Individual Cells Independent of Any Repair Defect.

Many DNA lesions associated with lymphoid malignancies are linked to off-target cleavage by the RAG1/2 recombinase. However, off-target cleavage has mostly been analyzed in the context of DNA repair defects, confounding any mechanistic understanding of cleavage deregulation. We identified a conserved SQ phosphorylation site on RAG2 365 to 366 that is involved in feedback control of RAG cleavage. Mutation of serine 365 to a non-phosphorylatable alanine permits bi-allelic and bi-locus RAG-mediated breaks in the same cell, leading to reciprocal translocations. This phenomenon is analogous to the phenotype we described for ATM kinase inactivation. Here, we establish deregulated cleavage itself as a driver of chromosomal instability without the associated repair defect. Intriguingly, a RAG2-S365E phosphomimetic rescues the deregulated cleavage of ATM inactivation, reducing the incidence of reciprocal translocations. These data support a model in which feedback control of cleavage and maintenance of genome stability involves ATM-mediated phosphorylation of RAG2.

RAG2's acidic hinge restricts repair-pathway choice and promotes genomic stability.

V(D)J recombination-associated DNA double-strand breaks (DSBs) are normally repaired by the high-fidelity classical nonhomologous end-joining (cNHEJ) machinery. Previous studies implicated the recombination-activating gene (RAG)/DNA postcleavage complex (PCC) in regulating pathway choice by preventing access to inappropriate repair mechanisms such as homologous recombination (HR) and alternative NHEJ (aNHEJ). Here, we report that RAG2's "acidic hinge," previously of unknown function, is critical for several key steps. Mutations that reduce the hinge's negative charge destabilize the PCC, disrupt pathway choice, permit repair of RAG-mediated DSBs by the translocation-prone aNHEJ machinery, and reduce genomic stability in developing lymphocytes. Structural predictions and experimental results support our hypothesis that reduced flexibility of the hinge underlies these outcomes. Furthermore, sequence variants present in the human population reduce the hinge's negative charge, permit aNHEJ, and diminish genomic integrity.

MOZ increases p53 acetylation and premature senescence through its complex formation with PML.

Monocytic leukemia zinc finger (MOZ)/KAT6A is a MOZ, Ybf2/Sas3, Sas2, Tip60 (MYST)-type histone acetyltransferase that functions as a coactivator for acute myeloid leukemia 1 protein (AML1)- and Ets family transcription factor PU.1-dependent transcription. We previously reported that MOZ directly interacts with p53 and is essential for p53-dependent selective regulation of p21 expression. We show here that MOZ is an acetyltransferase of p53 at K120 and K382 and colocalizes with p53 in promyelocytic leukemia (PML) nuclear bodies following cellular stress. The MOZ-PML-p53 interaction enhances MOZ-mediated acetylation of p53, and this ternary complex enhances p53-dependent p21 expression. Moreover, we identified an Akt/protein kinase B recognition sequence in the PML-binding domain of MOZ protein. Akt-mediated phosphorylation of MOZ at T369 has a negative effect on complex formation between PML and MOZ. As a result of PML-mediated suppression of Akt, the increased PML-MOZ interaction enhances p21 expression and induces p53-dependent premature senescence upon forced PML expression. Our research demonstrates that MOZ controls p53 acetylation and transcriptional activity via association with PML.

Non-consensus heptamer sequences destabilize the RAG post-cleavage complex, making ends available to alternative DNA repair pathways.

V(D)J recombination entails double-stranded DNA cleavage at the antigen receptor loci by the RAG1/2 proteins, which recognize conserved recombination signal sequences (RSSs) adjoining variable (V), diversity (D) and joining (J) gene segments. After cleavage, RAG1/2 remain associated with the coding and signal ends (SE) in a post-cleavage complex (PCC), which is critical for their proper joining by classical non-homologous end joining (NHEJ). Certain mutations in RAG1/2 destabilize the PCC, allowing DNA ends to access inappropriate repair pathways such as alternative NHEJ, an error-prone pathway implicated in chromosomal translocations. The PCC is thus thought to discourage aberrant rearrangements by controlling repair pathway choice. Since interactions between RAG1/2 and the RSS heptamer element are especially important in forming the RAG-SE complex, we hypothesized that non-consensus heptamer sequences might affect PCC stability. We find that certain non-consensus heptamers, including a cryptic heptamer implicated in oncogenic chromosomal rearrangements, destabilize the PCC, allowing coding and SEs to be repaired by non-standard pathways, including alternative NHEJ. These data suggest that some non-consensus RSS, frequently present at chromosomal translocations in lymphoid neoplasms, may promote genomic instability by a novel mechanism, disabling the PCC's ability to restrict repair pathway choice.

Excised V(D)J recombination byproducts threaten genomic integrity.

Signal joints were long considered to be inert byproducts of V(D)J recombination that protect the genome from illegitimate rearrangements. However, increasing evidence suggests that signal joints are not inert and could pose a threat to genomic stability. A recent study from Nadel and colleagues shows that episomal signal joints readily undergo trans recombination, resulting in their insertion into chromosomal DNA.