An inducible Cd79b mutation confers ibrutinib sensitivity in mouse models of Myd88-driven diffuse large B-cell lymphoma.

ABSTRACT: Diffuse large B-cell lymphoma (DLBCL) is the most common aggressive lymphoma and constitutes a highly heterogenous disease. Recent comprehensive genomic profiling revealed the identity of numerous molecularly defined DLBCL subtypes, including a cluster which is characterized by recurrent aberrations in MYD88, CD79B, and BCL2, as well as various lesions promoting a block in plasma cell differentiation, including PRDM1, TBL1XR1, and SPIB. Here, we generated a series of autochthonous mouse models to mimic this DLBCL cluster and specifically focused on the impact of Cd79b mutations in this setting. We show that canonical Cd79b immunoreceptor tyrosine-based activation motif (ITAM) mutations do not accelerate Myd88- and BCL2-driven lymphomagenesis. Cd79b-mutant murine DLBCL were enriched for IgM surface expression, reminiscent of their human counterparts. Moreover, Cd79b-mutant lymphomas displayed a robust formation of cytoplasmic signaling complexes involving MYD88, CD79B, MALT1, and BTK. These complexes were disrupted upon pharmacological BTK inhibition. The BTK inhibitor-mediated disruption of these signaling complexes translated into a selective ibrutinib sensitivity of lymphomas harboring combined Cd79b and Myd88 mutations. Altogether, this in-depth cross-species comparison provides a framework for the development of molecularly targeted therapeutic intervention strategies in DLBCL.

Multiphoton characterization and live cell imaging using fluorescent adenine analogue 2CNqA.

Fluorescent nucleobase analogues (FBAs) are established tools for studying oligonucleotide structure, dynamics and interactions, and have recently also emerged as an attractive option for labeling RNA-based therapeutics. A recognized drawback of FBAs, however, is that they typically require excitation in the UV region, which for imaging in biological samples may have disadvantages related to phototoxicity, tissue penetration, and out-of-focus photobleaching. Multiphoton excitation has the potential to alleviate these issues and therefore, in this work, we characterize the multiphoton absorption properties and detectability of the highly fluorescent quadracyclic adenine analogue 2CNqA as a ribonucleotide monomer as well as incorporated, at one or two positions, into a 16mer antisense oligonucleotide (ASO). We found that 2CNqA has a two-photon absorption cross section that, among FBAs, is exceptionally high, with values of σ2PA(700 nm) = 5.8 GM, 6.8 GM, and 13 GM for the monomer, single-, and double-labelled oligonucleotide, respectively. Using fluorescence correlation spectroscopy, we show that the 2CNqA has a high 2P brightness as the monomer and when incorporated into the ASO, comparing favorably to other FBAs. We furthermore demonstrate the usefulness of the 2P imaging mode for improving detectability of 2CNqA-labelled ASOs in live cells.

An Autochthonous Mouse Model of Myd88- and BCL2-Driven Diffuse Large B-cell Lymphoma Reveals Actionable Molecular Vulnerabilities.

Based on gene expression profiles, diffuse large B cell lymphoma (DLBCL) is sub-divided into germinal center B cell-like (GCB) and activated B cell-like (ABC) DLBCL. Two of the most common genomic aberrations in ABC-DLBCL are mutations in MYD88, as well as BCL2 copy number gains. Here, we employ immune phenotyping, RNA-Seq and whole exome sequencing to characterize a Myd88 and Bcl2-driven mouse model of ABC-DLBCL. We show that this model resembles features of human ABC-DLBCL. We further demonstrate an actionable dependence of our murine ABC-DLBCL model on BCL2. This BCL2 dependence was also detectable in human ABC-DLBCL cell lines. Moreover, human ABC-DLBCLs displayed increased PD-L1 expression, compared to GCB-DLBCL. In vivo experiments in our ABC-DLBCL model showed that combined venetoclax and RMP1-14 significantly increased the overall survival of lymphoma bearing animals, indicating that this combination may be a viable option for selected human ABC-DLBCL cases harboring MYD88 and BCL2 aberrations.

Getting DNA and RNA out of the dark with 2CNqA: a bright adenine analogue and interbase FRET donor.

With the central role of nucleic acids there is a need for development of fluorophores that facilitate the visualization of processes involving nucleic acids without perturbing their natural properties and behaviour. Here, we incorporate a new analogue of adenine, 2CNqA, into both DNA and RNA, and evaluate its nucleobase-mimicking and internal fluorophore capacities. We find that 2CNqA displays excellent photophysical properties in both nucleic acids, is highly specific for thymine/uracil, and maintains and slightly stabilises the canonical conformations of DNA and RNA duplexes. Moreover, the 2CNqA fluorophore has a quantum yield in single-stranded and duplex DNA ranging from 10% to 44% and 22% to 32%, respectively, and a slightly lower one (average 12%) inside duplex RNA. In combination with a comparatively strong molar absorptivity for this class of compounds, the resulting brightness of 2CNqA inside double-stranded DNA is the highest reported for a fluorescent base analogue. The high, relatively sequence-independent quantum yield in duplexes makes 2CNqA promising as a nucleic acid label and as an interbase Förster resonance energy transfer (FRET) donor. Finally, we report its excellent spectral overlap with the interbase FRET acceptors qAnitro and tCnitro, and demonstrate that these FRET pairs enable conformation studies of DNA and RNA.

Interbase FRET in RNA: from A to Z.

Interbase FRET can reveal highly detailed information about distance, orientation and dynamics in nucleic acids, complementing the existing structure and dynamics techniques. We here report the first RNA base analogue FRET pair, consisting of the donor tCO and the non-emissive acceptor tCnitro. The acceptor ribonucleoside is here synthesised and incorporated into RNA for the first time. This FRET pair accurately reports the average structure of A-form RNA, and its utility for probing RNA structural changes is demonstrated by monitoring the transition from A- to Z-form RNA. Finally, the measured FRET data were compared with theoretical FRET patterns obtained from two previously reported Z-RNA PDB structures, to shed new light on this elusive RNA conformation.