Analysis of 54 Follicular Lymphomas By Whole Exome Sequencing Identifies Multiple Novel Recurrently Mutated Pathways

Introduction: Follicular lymphoma (FL) constitutes the second most common non-Hodgkin’s lymphoma in the Western world. FL carries multiple recurrently mutated genes that are under active investigation. However, due to the relatively small number of published sequenced cases, knowledge regarding the coding genome in FL is still evolving.

Methods: To further our understanding of the genetic basis of FL, we used solution exon capture of sheared and processed genomic DNA isolated from highly purified light chain restricted B-cells and paired CD3+ T-cells from 54 FL cases for paired-end massively parallel sequencing (WES). Data were subsequently analyzed using bioinformatics pipelines including the variant callers MuTect v.1.1.4, Strelka v.1.0.13, and VarScan2 v.2.3.7. Candidate somatically acquired gene mutations with variant allele frequencies (VAFs) >0.15 were confirmed using Sanger sequencing.  Selected mutations were validated in an expansion cohort of 120 FL.

Results:  We identified heterozygous missense mutations in the mTOR regulator RRAGC in 10% of FL. The RRAGC mutations targeted multiple hotspot residues (amino acid 115, 118 and 119).  RRAGC forms heterodimers with either RRAGA or RRAGB that under conditions of amino acid sufficiency facilitate recruitment of mTOR through the raptor subunit to lysosomal membranes. At the lysosomal surface, multiple protein complexes, each containing various proteins regulate mTOR activation through RHEB.

To gain insights into the functional consequences of RRAGC mutations, we performed 3-dimensional modeling of FL-associated RRAGC mutations and located the mutations into relatively close proximity to the RRAGC GTP/GDP binding site. Energy calculations did not identify strong effects of mutated amino acid residues on the binding of GTP/GDP to RRAGC. 

We performed studies of the effects of RRAGC mutants on mTOR activity as measured through S6-kinase phosphorylation. In transient transfection systems (293T and HELA) achieving expression slightly above endogenous RRAGC levels, performed under conditions of leucine starvation or sufficiency, we did not identify differences in baseline mTOR activation. In stably transfected 293T cell lines (expressing RRAGB and RRAGC proteins above endogenous levels), that were starved for leucine for 1 hour, we detected modestly elevated p-S6K levels in RRAGC mutant versus wild type transfectants, suggesting a mild intrinsic activation phenotype of RRAGC mutations. Experiments in lentivirally-transfected lymphoma cell lines, including RRAGC binding studies to raptor and folliculin (a RRAGC regulator) are in progress and will be updated at the meeting.  Curiously, we did not identify mutations in the other three small GTP binding proteins that are part of the same amino acid sensing pathway (RRAGA, RRAGB or RRAGD), potentially pointing to a unique advantage conferred by RRAGC mutants on FL B cells.

We identified additional mutations (combined ~15%) in other mTOR components linked to lysosomal amino acid sensing, including recurrent mutations in the v-ATPase subunit ATP6V1B2 and the accessory subunit ATP6VAP1. The mutations in RRAGC and v-ATPase together highlight a previously unidentified role of the amino acid sensing pathway that regulates mTOR in FL pathogenesis. 

We have discovered a high frequency of mutations (40%) in the surrogate light chain gene IGLL5 in FL, a critical component of the pre-B-cell receptor.  Mutations sharply cluster in the N-terminal 70 amino acid of IGLL5, a region known as the non-Ig domain of IGLL5. The non-Ig domain of IGLL5 has been implicated in influencing pre-B-cell receptor signaling and receptor surface expression as well as interaction with extracellular ligands. The mutational data suggest an unexpected role of IGLL5 in the pathogenesis of FL and work is in progress studying IGLL5 expression in primary FL samples.

Conclusion: This large WES study of 54 FL identifies novel recurrently mutated genes and pathways in FL, including frequent mutations in genes involved in amino acid signaling to mTOR (RRAGC and v-ATPase) as well as pre-B-cell receptor signaling (the surrogate light chain gene IGLL5) and multiple other novel recurrently mutated genes that will be updated at the meeting. These data substantially broaden our understanding of the genetic basis of FL and provide clues to therapeutically targeting specific pathways in FL.