Alexander J. Ritter

IGF2BP3 is an oncofetal RNA-binding protein that promotes leukemogenesis, but the mechanisms by which it remodels post-transcriptional gene regulation remain incompletely understood. Because IGF2BP3 binds extensively within 3′ untranslated regions and has been implicated in microRNA (miRNA)-mediated regulation, we asked whether IGF2BP3 controls access of the RNA-induced silencing complex (RISC) to endogenous messenger RNA (mRNA) targets in leukemia cells. Using AGO2 miR–eCLIP in control and IGF2BP3-deficient MLL–AF4 B-lymphoblastic leukemia cells, we found that loss of IGF2BP3 broadly redistributes AGO2 occupancy toward 3′ UTRs of IGF2BP3-bound transcripts. AGO2 gains were enriched near miRNA target sites and on transcripts associated with proliferative and oncogenic pathways. Chimeric AGO2-miRNA reads revealed transcript- and miRNA-specific remodeling of RISC interactions, with enhanced miR-181 occupancy on oncogenic transcripts following IGF2BP3 loss. Functionally, miR-181a overexpression impaired leukemic cell growth, partially phenocopying IGF2BP3 deletion. Motif analyses indicated that IGF2BP3 and AGO2 converge on related 3′ UTR sequence environments, while biochemical competition assays demonstrated that purified IGF2BP3 can displace AGO2-containing RISC complexes from a shared RNA substrate. Together, these findings support a model in which IGF2BP3 restricts RISC access to select 3′ UTR regulatory elements, thereby reshaping miRNA–target interactions and sustaining leukemic gene expression programs. Rather than acting solely as an mRNA stabilizing factor, IGF2BP3 functions as a transcript-selective regulator of RISC accessibility in leukemia.

The Nr4a nuclear hormone receptors are transcriptionally upregulated in response to antigen recognition by the T cell receptor (TCR) in the thymus and are implicated in clonal deletion, but the mechanisms by which they operate are not clear. Moreover, their role in central tolerance is obscured by redundancy among the Nr4a family members and by their reported functions in Treg generation and maintenance. Here we take advantage of competitive bone marrow chimeras and the OT-II/RIPmOVA model to show that Nr4a1 and Nr4a3 are essential for the upregulation of Bcl2l11/BIM and thymic clonal deletion by self-antigen. Importantly, thymocytes lacking Nr4a1/3 acquire an anergy-like signature after escaping clonal deletion and Treg lineage diversion. We further show that the Nr4a family helps mediate a broad transcriptional program in self-reactive thymocytes that resembles anergy and may operate at the margins of canonical thymic tolerance mechanisms to restrain self-reactive T cells after thymic egress.

Accurate detection and quantification of mRNA isoforms from nanopore long-read sequencing remains challenged by technical noise, particularly in single cells. To address this, we introduce Isosceles, a computational toolkit that outperforms other methods in isoform detection sensitivity and quantification accuracy across single-cell, pseudo-bulk and bulk resolution levels, as demonstrated using synthetic and biologically-derived datasets. Here we show Isosceles improves the fidelity of single-cell transcriptome quantification at the isoform-level, and enables flexible downstream analysis. As a case study, we apply Isosceles, uncovering coordinated splicing within and between neuronal differentiation lineages. Isosceles is suitable to be applied in diverse biological systems, facilitating studies of cellular heterogeneity across biomedical research applications.

Alternative splicing (AS) is emerging as an important regulatory process for complex biological processes. Transcriptomic studies therefore commonly involve the identification and quantification of alternative processing events, but the need for predicting the functional consequences of changes to the relative inclusion of alternative events remains largely unaddressed. Many tools exist for the former task, albeit each constrained to its own event type definitions. Few tools exist for the latter task; each with significant limitations. To address these issues we developed junctionCounts, which captures both simple and complex pairwise AS events and quantifies them with straightforward exon-exon and exon-intron junction reads in RNA-seq data, performing competitively among similar tools in terms of sensitivity, false discovery rate and quantification accuracy. Its partner utility, cdsInsertion, identifies transcript coding sequence (CDS) information via in silico translation from annotated start codons, including the presence of premature termination codons. Finally, findSwitchEvents connects AS events with CDS information to predict the impact of individual events to the isoform-level CDS. We used junctionCounts to characterize splicing dynamics and NMD regulation during neuronal differentiation across four primates, demonstrating junctionCounts’ capacity to robustly characterize AS in a variety of organisms and to predict its effect on mRNA isoform fate.

RNA-binding proteins (RBPs) are emerging as a novel class of therapeutic targets in cancer, including in leukemia, given their important role in posttranscriptional gene regulation, and have the unexplored potential to be combined with existing therapies. The RBP insulin-like growth factor 2 messenger RNA–binding protein 3 (IGF2BP3) has been found to be a critical regulator of MLL-AF4 leukemogenesis and represents a promising therapeutic target. Here, we study the combined effects of targeting IGF2BP3 and menin-MLL interaction in MLL-AF4–driven leukemia in vitro and in vivo, using genetic inhibition with CRISPR-Cas9–mediated deletion of Igf2bp3 and pharmacologic inhibition of the menin-MLL interaction with multiple commercially available inhibitors. Depletion of Igf2bp3 sensitized MLL-AF4 leukemia to the effects of menin-MLL inhibition on cell growth and leukemic initiating cells in vitro. Mechanistically, we found that both Igf2bp3 depletion and menin-MLL inhibition led to increased differentiation in vitro and in vivo, seen in functional readouts and by gene expression analyses. IGF2BP3 knockdown had a greater effect on increasing survival and attenuating disease than pharmacologic menin-MLL inhibition with small molecule MI-503 alone and showed enhanced antileukemic effects in combination. Our work shows that IGF2BP3 is an oncogenic amplifier of MLL-AF4–mediated leukemogenesis and a potent therapeutic target, providing a paradigm for targeting leukemia at both the transcriptional and posttranscriptional level.

Disclosures. Rao:AbbVie, Inc: Consultancy, Speakers Bureau.