Fig 2: Hypoxia and proteasomal inhibition independently cause mTORC1 suppression in MM.A A relative correlation between mRNA expression level of TSC2 and HIF1β in CD138 + bone marrow plasma cells from healthy subjects (n = 22) and newly diagnosed MM patients (n = 559). Axis units are arbitrary. Error bars represent S.E.M. B, C Shown are Kaplan–Meier plots for the overall survival (OS) and progression-free survival (PFS) of MM patients with differential expression of TSC2 (B) and HIF1β (C) from GMMG HD4 and MM5 trials. D Subcutaneous tumor growth of wt, NPRL2 KO, and TSC2 KO MM.1S cells into NCG mice (n = 4). Shown is the average relative tumor weight ± S.E.M. and the asterisk represents p < 0.05 of unpaired two-tailed student’s t-test between wt and KO. ns, not significant. E Cells were cultured in fresh media for 4 h in either normoxia or hypoxia. mTORC1 response was assessed by immunoblotting of its downstream phosphorylated and total effectors, S6K1, S6, and 4E-BP1. p97 was used to confirm equal protein loading in each lane. Shown are technical duplicates in each condition of a typical experiment of three repetitions. F, G Cells were cultured in fresh media for 16 h, then treated with IXZ [32 nM]. Following the treatment, cells were collected for immunoblotting in increasing time points as indicated. Shown are immunoblots for downstream effectors of mTORC1. Ubiquitin immunoblot was used to assess proteasomal inhibition. Shown are a typical immunoblots out of three independent experiments. nor, normoxia. hyp, hypoxia, IXZ, ixazomib.

Fig 3: WDR24-R329K mutation inhibits amino-acid-induced mTORC1 activation(A) IB analysis of WCLs derived from WDR24WT or WDR24R329K knockin Huh7 cells. Cells were starved of aa for 2 h or starved for 2 h and then restimulated with aa for 10 min before harvesting. Similar results were obtained in three independent experiments.(B) IF analysis of mTOR lysosomal localization in WDR24WT or WDR24R329K knockin Huh7 cells. LAMP2 was a lysosomal marker. Cells were starved of aa for 2 h or starved for 2 h and then restimulated with aa for 10 min. Scale bar, 10 μm. Similar results were obtained in three independent experiments.(C) Quantification of lysosome enrichment of mTOR in (B). Data are represented as mean ± SD. **p < 0.01, two-tailed Student’s t test.(D) IB analysis of WCL and lysosomal enriched fraction. Huh7 cells stably expressing HA-LAMP1 were transfected with Myc-WDR24-WT or Myc-WDR24-R329K mutant. Cells were starved of aa for 2 h or starved for 2 h and then restimulated with aa for 10 min before harvesting. Similar results were obtained in three independent experiments.(E) IB analysis of WCLs derived from WDR24WT or WDR24R329K knockin Huh7 cells stably expressing Tet-on-FLAG-PRMT1. Cells were treated with doxycycline for 48 h to induce PRMT1 expression. Cells were then starved of aa for 2 h or starved for 2 h and then restimulated with aa for 10 min before harvesting. Similar results were obtained in three independent experiments.(F) IB analysis of WCLs derived from WDR24WT or WDR24R329K knockin Huh7 cells stably expressing FLAG-RagBQ99L. Cells were starved of aa for 2 h or starved for 2 h and then restimulated with aa for 10 min before harvesting. Similar results were obtained in three independent experiments.(G) IB analysis of WCLs derived from WDR24WT or WDR24R329K knockin Huh7 cells depleted of NPRL2. Cells were starved of aa for 2 h or starved for 2 h and then restimulated with aa for 10 min before harvesting. Similar results were obtained in three independent experiments.See also Figure S6 and Table S1.

Fig 4: PRMT1 regulates mTORC1 activation upstream of Rag GTPases(A) The key complexes of the mTORC1 pathway in amino-acid-sensing branch.(B) IB analysis of WCLs derived from HEK293T cells stably expressing FLAG-Rap2a or FLAG-RagBQ99L with or without PRMT1 depletion. Cells were starved of aa for 50 min or starved for 50 min and then restimulated with aa for 10 min before harvesting. Similar results were obtained in three independent experiments.(C) IB analysis of WCL derived from HEK293T cells depleted of PRMT1 or PRMT1 and NPRL2. Cells were starved of aa for 50 min or starved for 50 min and then restimulated with aa for 10 min before harvesting. Similar results were obtained in three independent experiments.(D) IB analysis of WCL derived from HEK293T cells depleted of PRMT1, KPTN, or both. Cells were starved of aa for 50 min or starved for 50 min and then restimulated with aa for 10 min before harvesting. Similar results were obtained in three independent experiments.(E and F) IB analysis of WCLs derived from cells stably expressing Tet-on-FLAG-PRMT1 and depleted of WDR24 (E) or WDR59 (F). 1 μg/mL doxycycline was added for 48 hours to induce PRMT1 expression. Cells were starved of aa for 50 min or starved for 50 min and then restimulated with aa for 10 min before harvesting. Similar results were obtained in three independent experiments.See also Figure S4 and Table S1.

Fig 5: Disrupting the electrostatic interaction abrogates amino acid signaling in HEK-293T cells.A, generation of stable cell lines expressing wild-type Depdc5 or Depdc5(R1407D). Endogenous Depdc5 was first knocked out in HEK-293T cells using CRISPR-Cas9, and HA-tagged wild-type Depdc5 or Depdc5(R1407D) was then stably expressed in the knockout line. Co-immunoprecipitation experiment shows comparable GATOR1 complex formation between wild-type Depdc5 or Depdc5(R1407D) with Nprl2 and Nprl3 (lanes 3 and 4). B, amino acid starvation experiment to monitor mTORC1 signaling when cells are deprived of amino acids. C, Depdc5(R1407D) fails to restore cellular response to amino acids deprivation in Depdc5-knockout cell line. Asterisk denotes hyperphosphorylated 4EBP. Cross denotes unphosphorylated 4EBP, which only appears when mTORC1 activity is turned off (lanes 2 and 6). D, cell size analysis of HEK-293T cells expressing wild-type Depdc5 under starvation (black) or fed (red) conditions. The average size of cells under starvation condition is 17.1 μm, while that under fed condition is 18.5 μm. E, cell size analysis of HEK-293T cells expressing Depdc5(R1407D) under starvation (black) or fed (red) conditions. The average size of cells under starvation condition is 18.0 μm, while that under fed condition is 18.3 μm. F, amino acid starvation experiment in HEK-293T cells expressing charge-neutralizing RagC mutants. Cellular response to amino acid deprivation was blunted. G, amino acid starvation experiment in HEK-293T cells expressing charge-reversal RagC mutants. Cellular response to amino acid deprivation was abrogated. H, long-term starvation assay to monitor autophagy mediated by wild-type GATOR1 or GATOR1[Depdc5(R1407D)]. Depdc5(R1407D) severely delays response to amino acid deprivation. I, quantification of S6K1 phosphorylation in response to amino acid deprivation. J, quantification of ULK1 abundance in response to amino acid deprivation.