Mutations of the RNA Binding Protein hnRNP U Modulate MYC Expression in MYC-Driven Lymphomas

Introduction Deregulated expression of the MYC oncogene is a common feature of B-cell lymphomas. Sustained MYC expression can promote cell activation and proliferation while simultaneously sensitizing cells to apoptosis or can directly trigger apoptosis via proteotoxic stress. Identifying genetic drivers of lymphomagenesis that facilitate tolerance of MYC overexpression are thus important for understanding its pathobiology. Burkitt lymphoma (BL) is an aggressive B-cell cancer, characterized by translocations that juxtapose potent enhancers, most commonly belonging to the immunoglobulin heavy or light chain loci, with the MYC oncogene. MYC translocations are also found in 10% of tumors with diffuse large B-cell lymphoma morphology. When a BCL2 translocation is also present, these high-grade B-cell lymphomas (HGBCL-BCL2) have an aggressive phenotype and respond poorly to treatment. To discover novel treatment options, it is important to fully understand the molecular mechanisms of these diseases.

Methods Patient samples used in this study were sequenced as part of the ongoing Burkitt Lymphoma Genome Sequencing project and the Lymphoma/Leukemia Molecular Profiling Project. Simple somatic mutations were identified from whole genome or exome sequencing using an ensemble of variant callers (Strelka2, LoFreq, Mutect2, SAGE). MYC rearrangements were identified using GRIDSS and Manta. HNRNPU shRNA-seq and eCLIP in HepG2 and K562 cells were obtained from the ENCODE project consortium. Differential expression analysis was performed with DESeq2. Peak-calling and individual crosslink sites were detected with PureCLIP. All functional studies were performed in the BL cell line Raji using an HNRNPU expression vector or siRNA targeting endogenous HNRNPU. HNRNPU and MYC expression was quantified through qPCR and western blot. Cell viability and proliferation were assayed with resazurin reduction.

Results We identified RNA binding proteins as commonly mutated in BL and HGBCL-BCL2. In particular, mutations affecting HNRNPU were identified in 10.6% of HGBCL-BCL2 and 5.2% of BL tumors. hnRNP U is an RNA- and DNA-binding protein that plays a central role in gene expression regulation, notably in transcription and regulating mRNA stability. Most HNRNPU mutations are predicted to introduce a premature stop codon near its N-terminus.

To identify a functional consequence of decreased HNRNPU expression, we identified differentially expressed transcripts upon HNRNPU knockdown in K562 and HepG2 cells. MYC transcriptional targets were significantly differentially expressed in these experiments. The modulation of MYC was further supported in experiments with the Raji cell line. Specifically, HNRNPU knockdown lead to a decreased expression of MYC at both the mRNA and protein level, while overexpression of hnRNP U lead to increased MYC expression. Taken together, our data support the notion that hnRNP U is a modulator of MYC.

Overexpressing hnRNP U in Raji cells results in cellular stress and a decrease in cell proliferation. We propose a model where HNRNPU mutations moderate MYC expression, thus buffering MYC-induced apoptosis and proteotoxic stress. This parallels the effect of loss of function mutations in the RNA helicase DDX3X commonly observed in MYC-driven lymphomas. DDX3X and HNRNPU mutations rarely co-occur in patients, supporting their similar roles.

Preliminary findings from crosslinking immunoprecipitation suggest that hnRNP U binds directly to the MYC transcript in poly(G) tracts in intron 1. This region is predicted to fold into G-quadruplexes, a secondary structure that can be bound by hnRNP U. Most HNRNPU mutations are mutually exclusive with cases that harbor MYC intron-1 breaks. Taken together, these findings suggest that the region 5' of intron 1 is necessary for HNRNPU-mediated modulation of MYC.

ConclusionHNRNPU mutations are novel recurrent driver mutations specifically within MYC translocated B-cell lymphomas. HNRNPU acts as a modulator of MYC expression, and the most common mutations are predicted to negatively impact this role. Further experiments to explore the role of HNRNPU binding on MYC expression and the potential role in buffering the proteotoxic stress associated with MYC translocations are ongoing. Insights into the mechanisms contributing to disease will support ongoing work to establish in vitro and in vivo models for future therapeutic investigations.

Coyle:Allakos, Inc.: Consultancy; Dren Bio, Inc.: Consultancy. Scott:NanoString: Patents & Royalties; Roche: Research Funding; Janssen: Consultancy, Research Funding; Abbvie: Consultancy; Incyte: Consultancy; AstraZeneca: Consultancy, Honoraria.