Mutated mTOR regulator RRAGC proteins decrease interactions with FLCN

Follicular lymphoma is a B-cell lymphoma that remains incurable with conventional therapies. Ying et al. (2016) present a new study exploring the biological and genetic features of follicular lymphoma and identifying potential new therapeutic targets. The authors identify recurrent mutations in the mTOR regulator RRAGC, a small G-protein, in approximately 10% of follicular lymphoma cases. Mutations in RRAGC localized to one protein surface area surrounding the GTP/GDP–binding sites. In stable retrovirally transfected HEK293T cells, multiple RRAGC mutations showed higher mTOR activation. A similar phenotype was observed in lymphoma cell lines and in yeast. Mutated RRAGC proteins showed increased binding to RPTOR, a binding protein for RRAG heterodimers, and decreased interactions with FLCN , a known tumour suppressor that plays a role in mTOR signalling and the causative gene of BHD syndrome.

By Sanger sequencing of RRAGC in 128 follicular lymphoma cases, the authors identified novel RRAGC heterozygous mutations in 9.4% of cases, including multiple mutation hotspots.   All the follicular lymphoma RRAGC mutations were located on the surface of the nucleotide-binding domain (G-domain) of RRAGC, a region implicated in protein–protein interaction.  This suggests a potential mechanism of action (Gong et al., 2011) that the mutant residues could potentially contribute to GTP/GDP binding.

Transiently transfected HEK293T or HELA cells showed that the co-transfection of RRAGC with RRAGB stabilized both proteins and also stabilized RPTOR protein levels independently of RRAGC-mutation status. To identify the effects of mutant RRAGC on mTOR activity the authors stably expressed HA-tagged RRAGC, both wild-type and mutants, in HEK293T cells in complete medium or in medium lacking leucine. RRAGC Asn90 mutants displayed elevated mTOR activity in the presence of leucine. Modestly elevated mTOR activity levels were also detected in all other RRAGC mutants, suggesting a mild intrinsic activation phenotype of follicular lymphoma RRAGC mutants.

A similar mTOR activation phenotype was observed in multiple lymphoma cell lines expressing HA-tagged RRAGC-mutant proteins generated through lentiviral infection and cell sorting.  In yeast, the authors saw similar results for the RRAGC homologue Gtr2, by monitoring TOR activity, supporting the hypothesis that specific mutations at Gtr2/RRAGC affect the activity of TOR/mTOR.

To address the mechanism underlying the mTOR activation by RRAGC mutations, the authors checked for interactions with RPTOR and FLCN. In transient co-transfection experiments in HEK293T cells, FLCN co-immunoprecipitated with HA-RPTOR. HA-RPTOR co-immunoprecipitated RRAGB and RRAGC WT as expected. Higher amounts of the RRAGC mutants were co-immunoprecipitated, particularly Asn90. Leucine starvation did not influence the RRAGC–RPTOR interaction. HA-RPTOR co-immunoprecipitated FLCN in the absence of co-transfected RRAGB/C, but the amount of FLCN was higher in the presence of the RRAGB/C heterodimer. In the absence of leucine, FLCN showed reduced phosphorylation, as previously described (Baba et al., 2006).

In stably transduced HEK293T cells or lymphoma cell lines, the authors observed increased binding of RPTOR and, more strikingly, a substantially impaired interaction of mutant RRAGC proteins with FLCN, suggesting that this impairment is also involved in mTOR regulation and underlies the pathogenesis of follicular lymphoma RRAGC mutations. This hypothesis is consistent with the known tumour features observed in BHD mouse models and in patients (Schmidt et al., 2015; Hasumi et al., 2009).. Since the measured mTOR activation for some RRAGC mutations was relatively mild, additional RRAGC mutant-regulated pathways might also be involved.

Adding to this study, very recently, Okosun et al. (2016) reported a 17% incidence of RRAGC mutations in follicular lymphoma, with a comparatively higher incidence of mutations targeting residues surrounding amino acid 90. The authors also observed mTOR activation and higher RPTOR binding.

Together, these studies identify a previously unknown link between the amino acid signalling pathway to mTOR and the pathogenesis of follicular lymphoma, and they suggest mTOR activation and inhibition as a potential novel therapy strategy in follicular lymphoma. In addition, follow-up studies may help unravel the role of FLCN in mTOR signalling.

  • Ying ZX, Jin M, Peterson LF, Bernard D, Saiya-Cork K, Yildiz M, Wang S, Kaminski MS, Chang AE, Klionsky DJ, & Malek SN (2016). Recurrent Mutations in the MTOR Regulator RRAGC in Follicular Lymphoma. Clinical cancer research : an official journal of the American Association for Cancer Research, 22 (21), 5383-5393 PMID: 27267853

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