Recurrent Mutations in the MTOR Regulator RRAGC in Follicular Lymphoma

Abstract

Purpose This study was performed to further our understanding of the biological and genetic basis of follicular lymphoma (FL) and to identify potential novel therapy targets. Experimental Design We analyzed previously generated whole exome sequencing data of 23 FL cases and one transformed FL case and expanded findings to a combined total of 125 FL/ 3 t-FL. We modeled the 3D-location of RRAGC-associated hotspot mutations. We performed functional studies on novel RRAGC mutants in stable retrovirally transduced HEK293T cells, stable lentivirally transduced lymphoma cell lines and in Saccharomyces cerevisiae. Results We report recurrent mutations, including multiple amino acid hotspots, in the small G-protein RRAGC, which is part of a protein complex that signals intracellular amino acid concentrations to MTOR, in 9.4% of FL cases. Mutations in RRAGC distinctly clustered on one protein surface area surrounding the GTP/GDP binding sites. Mutated RRAGC proteins demonstrated increased binding to RPTOR (raptor) and substantially decreased interactions with the product of the tumor suppressor gene FLCN (folliculin). In stable retrovirally transfected 293T cells, cultured in the presence or absence of leucine, multiple RRAGC mutations demonstrated elevated MTOR activation as evidenced by increased RPS6KB/S6-kinase phosphorylation. Similar activation phenotypes were uncovered in yeast engineered to express mutations in the RRAGC homolog Gtr2 and in multiple lymphoma cell lines expressing HA-tagged RRAGC mutant proteins. Conclusions Our discovery of activating mutations in RRAGC in ~10% of FL provides the mechanistic rationale to study mutational MTOR activation and MTOR inhibition as a potential novel actionable therapeutic target in FL.

Control of B lymphocyte development and functions by the mTOR signaling pathways

“…Loss of function mutations in tumor suppressors such as PTEN (Figure 2), or activating mutations in upstream kinases such as protein kinase C delta (PKCδ) or Syk, are the most common causes of mTOR hyperactivity, although various mutations in mTOR itself have also been associated with increased cancer risk [121]. For example, recent studies indicate that up to 17% of follicular lymphoma patients have activating mutations in the mTORC1 regulator RRAGC, enabling constitutive mTORC1 activation independent of amino acid levels [122,123]. Conversely, a polymorphism in the mTOR promoter that leads to decreased mTOR expression has been negatively associated with several different cancer types including acute lymphoblastic leukemia (ALL) [124].…”

Follicular lymphoma–associated mutations in vacuolar ATPase ATP6V1B2 activate autophagic flux and mTOR

“…In addition, we detected the mutations p.D367E>D/E, p.R400R>R/W, and p.R471R>R/S ( Figure 1A). We found that clonal mutations in RRAGC and ATP6V1B2 in FL did not occur together, suggesting that the corresponding proteins have overlapping functions in a shared pathway (see below) (24,25).…”

“…The spectrum of ATP6V1B2 mutations in 144 FL and 14 transformed FL cells. Recent reports of relatively frequent mutations in the v-ATPase subunit ATP6V1B2 in FL, and mTOR-activating mutations in RRAGC, along with findings of involvement of both proteins in a lysosomal amino acid-sensing pathway to mTOR, prompted us to study the underlying biology of these mutations (18,24,25,27,30). We first determined the frequency and nature of ATP6V1B2 mutations in 144 FL and 14 transformed FL (t-FL) cells using direct Sanger sequencing.…”

“…The identification of mTOR-activating mutations in the small G protein RRAGC, a component of the amino acid signaling pathway to mTOR, has highlighted a potential opportunity to target this pathway in FL (24)(25)(26). Upstream of RRAGC in this pathway lies the multisubunit vacuolar-type H + -translocating ATPase (v-ATPase) (27)(28)(29), which has also been identified as a commonly mutated target in FL (18,24,30).…”

Atypical tuberous sclerosis complex presenting as familial renal cell carcinoma with leiomyomatous stroma

“…Regarding melanocytic neoplasms as they were observed in the proband and his mother, there have been so far only exceptional reports hinting toward an association between these and TSC [23], which could correlate with the relatively significant 10% prevalence of mTOR pathway alterations in melanoma [24]. Similarly, follicular lymphoma has not been reported to be associated with TSC yet, although 10% of follicular lymphomas were reported to display aberrant mTOR pathway activation in one recent study [25,26]. Further probing for mTOR pathway dysregulation and possible TSC gene alterations in both melanocytic lesions and follicular lymphoma may be interesting in that regard.…”