Fig 2: Characterization of the xenograft model of the NB4 cell line in NSG mice. Female 12-week-old NSG mice were injected subcutaneously into the left thigh with 1 × 106 non-T cell activation linker-knockdown (NTAL-KD) cells, and received an equal number of control (CT) cells in the right thigh subcutaneously. (A,B) Relationship between bioluminescence and size of the tumor mass. The bioluminescent signal intensity was measured 2, 5, 7, 9, 11, and 13 days after injection of CT tumor cells (right flank) and NTAL-KD cells (left flank) expressing GFP-LUC. Images were acquired with IVIS Lumina representing the location of the cells and the tumor size. (C) After 2 weeks, the animals were euthanized and the tumors excised, weighed and processed. A graph of tumor masses derived from NB4 (CT and NTAL-KD) after two weeks: NB4: CT (1.286 g ± 0.3137) and NTAL-KD (0.8540 g ± 0.3402) (n = 5, P = 0.0143). (D) Immunohistochemical detection quantitative analysis of Ki67 proliferation marker. Data are reported as means and standard deviations. (E) Western blot analysis of proteins NTAL, total Ras, p-p44/42 MAPK (ERK1/2-Thr202/204), total p44/42 MAPK (ERK1/2) and caspase-3 tumors originating from NB4 (CT and NTAL-KD) (animals A1 = 1, A2 = 2 and A3 = 3). Bar graphs show the NTAL-KD to CT ratio from the three animals analyzed. Values are show as the mean ± SEM.

Fig 3: CRLF2-signaling induces direct interaction between activated PTPN11 and RAS, and PTPN11-activity is required for ALL cell growth.a Direct interaction between RAS and phosphorylated PTPN11 was monitored via PLA using high-content microscopy. Serum-starved MUTZ-5 cells were induced (or not) with 20 ng/mL TSLP for 10 min. Cells were fixed and permeabilized in a 96 well plate. Antibodies against phosphorylated PTPN11 and pan-RAS were used in conjunction with PLA-probes to allow the amplification and staining of interaction-specific PLA-spots. The negative control (NC) are two cytosolic proteins not expected to interact. Fluorescent-microscopy images show examples of PLA-spots (scale bars = 20 µm). At least 250 cells per well were analyzed using Operetta-CLS automated high-content microscopy platform. The bar-graph shows the averages of three independent experiments (each performed in triplicates). Error bars are SD and P values were determined in one-way ANOVA and post-hoc Bonferroni multiple comparison. b MUTZ-5 cells were pre-incubated with DMSO or 25 µM II-B08 (PTPN11 inhibitor) for 2 h and then stimulated or not with 20 ng/mL TSLP for 10 min before cell lysis. Each cell lysate was split up for RAS-GTP pull-down assay and for WB (whole-cell lysates). c MUTZ-5 cells were treated as in b before fixation. A PLA described in a was performed. d MUTZ-5 cells were seeded at 1.6 × 105/mL density and cultured for 7 days with either 0.5% DMSO (vehicle control), 50 µM Salirasib (indirect pan-RAS inhibitor), 25 µM II-B08, 50 µM Vemurafenib (pan-Raf inhibitor), 1 µM PD0325901 (MEK1/2-inhibitor), or 5 µM Ruxolitinib (JAK-inhibitor), in presence of 20 ng/mL TSLP. Percentage of viable cells was determined in an NC-250 automated cell counter.

Fig 4: Stable expression of oncogenic H-RAS 12V inhibits growth and decreases proliferation in RAS wild type eRMS cells. (A) Under log phase growth conditions, the RAS WT eRMS cell line, RMS-YM, displayed lower levels of RAS-GTP than the N-RAS mutant RD cell line. Under serum stimulation conditions, RAS-GTP levels were still lower in RMS-YM cells than RD cells. Cells were collected either in log phase, after 48 h of serum starvation, or 48 h of serum starvation followed by 1 h of growth in serum media. RD cells collected in log phase were included as a positive control. (B) Immunoblot confirmation of ectopic expression of H-RAS 12V, H-RAS WT, or H-RAS 17N in RMS-YM cells. (C) Expression of oncogenic H-RAS caused growth suppression in RMS-YM cells. Wild type and dominant negative H-RAS also impaired growth in RMS-YM cells, though not to the same degree as oncogenic H-RAS, and did not meet statistical significance. (D) RMS-YM cell proliferation decreased in response to expression of oncogenic H-RAS, as measured by BrdU assay. (E) Whereas H-RAS 12V and H-RAS WT drove upregulation of p16 in Rh28 and RD cells with modest increase in p21 in Rh28, RMS-YM cells showed upregulation of p21 with no change to p16. Expression of dominant negative H-RAS led to a two-fold increase of p53. (F) Expression of H-RAS 12V and H-RAS WT constructs increased AKT and ERK activation as assessed by levels of pAKT and pERK. Actin used as a loading control. (*p < 0.05; **p < 0.01; ****p < 0.0001).

Fig 5: EFR3A regulates plasma membrane lipid content to promote RAS oncogenesis.a, c Representative EM micrographs of plasma membrane (PM) sheets from human HEK-HT cells stably infected with a lentivirus(es) encoding Cas9 with no sgRNA (vector) or the indicated sgRNAs and transiently transfected with (a) GFP-P4M-SidM (a marker of PM PI(4)P) or (c) GFP-LactC2 (a marker of PM phosphatidylserine). Gold-conjugated GFP-antibodies seen as black dots are bound to the probe of interest. Scale bar: 100 nm. b, d The corresponding mean ± SEM of the number of gold particles per 1 µm2 of plasma membrane sheets representative of amount of (b) PI(4)P and (d) PS on the plasma membrane. e, f Mean ± SEM of (e) gold particles per 1 µm2 of plasma membrane sheets representative of the amount of KRAS on the plasma membrane and the (f) the extent of nanoclustering of KRAS on the plasma membrane as measured by Lmax from human HEK-HT cells transiently expressing GFP-KRASG12V and stably infected with a lentiviruses encoding Cas9 and sgEFR3A/B in the absence and presence of exogenous phosphatidylserine (PS). g Mean ± SEM of gold particles per 1 µm2 of the plasma membrane sheets from human HEK-HT cells transiently transfected with GFP-LactC2 and stably infected with a lentiviruses encoding Cas9 and sgEFR3A/B in the absence and presence of exogenous phosphatidylserine (PS). a–g EM analysis is based on 30 images. Significance values were calculated by the two-sided bootstrap test for (b, d–g): *p < 0.05, **p < 0.01, and ***p < 0.001. Specific p values for (b) are 0.00062274 (sgEFR3A), 0.00111426 (sgEFR3B), 0.00167654 (sgEFR3A/B), d are 0.000436671 (sgEFR3A), 0.056486805 (sgEFR3B), 0.000536135 (sgEFR3A/B), e is 0.00680191 (sgEFR3A/B + PS), f is 0.001 (sgEFR3A/B + PS), and (g) is 0.00051866 (sgEFR3A/B + PS).