Inhibition of Rag GTPase signaling in mice suppresses B cell responses and lymphomagenesis with minimal detrimental trade-offs

Abstract

SUMMARY B lymphocytes are exquisitely sensitive to fluctuations in nutrient signaling by the Rag GTPases, and 15% of follicular lymphomas (FLs) harbor activating mutations in RRAGC . Hence, a potential therapeutic approach against malignant B cells is to inhibit Rag GTPase signaling, but because such inhibitors are still to be developed, efficacy and safety remain unknown. We generated knockin mice expressing a hypomorphic variant of RagC (Q119L); RagC Q119L/+ mice are viable and show attenuated nutrient signaling. B lymphocyte activation is cell-intrinsically impaired in RagC Q119L/+ mice, which also show significant suppression of genetically induced lymphomagenesis and autoimmunity. Surprisingly, no overt systemic trade-offs or phenotypic alterations caused by partial suppression of nutrient signaling are seen in other organs, and RagC Q119L/+ mice show normal longevity and normal age-dependent health decline. These results support the efficacy and safety of moderate inhibition of nutrient signaling against pathological B cells.

Integrin β1 regulates marginal zone B cell differentiation and PI3K signaling

“…3 B and Data S1 ). Of note, Rap1b deletion generates a loss of MZ B cells ( Chen et al, 2008 ; Su et al, 2015 ), and various GTPases have been associated with the regulation of MZ B cells and PCs ( Guinamard et al, 2000 ; Chen et al, 2016 ; Ortega-Molina et al, 2021 ). β1-integrin–deficient MZ B cells showed an additional downregulation of genes, including Dock8 , Pax5 , and Vav2 , which was not observed in β1 KO transitional B cells ( Fig.…”

Protocol for the assessment of mTOR activity in mouse primary hepatocytes

“…This major step allows for a time-lapse assessment of nutrient-dependent activation of the mTOR signaling pathway through amino acid deprivation followed by replenishment. Note: We have previously performed nutrient signaling experiments on other primary cultures of mouse cells: Mouse Embryonic Fibroblasts ( de la Calle Arregui et al, 2021 ) ( Ortega-Molina et al., 2021 ) and naïve B lymphocytes ( Ortega-Molina et al, 2019 ), using standard RPMI without amino acids, supplemented with dFBS. However, this medium resulted suboptimal for the assessment of mTORC1 activity in primary hepatocytes from adult mice: poor acute activation of mTORC1 was observed with this medium and with several iterations aimed to more closely resemble Hepatocyte Attachment Medium ( Table 1 and Figure 6 , See troubleshooting 5 ).…”

“…This protocol can be adapted to other signaling cascades and is compatible with multiple readouts. For complete details on the use and execution of this protocol, please refer to Ortega-Molina et al. (2021) .…”

“…For complete details on the use and execution of this protocol, please refer to Ortega-Molina et al. (2021) .…”

The metabolic plasticity of B cells

“…Interestingly, a moderate, partial increase in nutrient-Rag GTPase signaling results in enhanced B-cell activation, enlarged GC and increased high-affinity antibody production (Ortega-Molina et al, 2019). Moreover, moderately activating mutations in RagC have been exclusively found in human follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) samples (Green et al, 2015;Okosun et al, 2016), while a mild inhibition of nutrient signaling to mTORC1 impairs the GC response (Ortega-Molina et al, 2021). In addition to its function controlling B-cell growth and proliferation, mTORC1 plays a key role in regulating antibody class-switch recombination, a process that is also tuned by nutrient availability and oxygen tension (Keating et al, 2013;Zhang et al, 2013;Cho et al, 2016).…”