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. 2013 Mar 26;6(268):ra21.
doi: 10.1126/scisignal.2003848.

Dysregulated RasGRP1 responds to cytokine receptor input in T cell leukemogenesis

Catherine Hartzell  1 , Olga Ksionda, Ed Lemmens, Kristen Coakley, Ming Yang, Monique Dail, Richard C Harvey, Christopher Govern, Jeroen Bakker, Tineke L Lenstra, Kristin Ammon, Anne Boeter, Stuart S Winter, Mignon Loh, Kevin Shannon, Arup K Chakraborty, Matthias Wabl, Jeroen P Roose
Affiliations

Dysregulated RasGRP1 responds to cytokine receptor input in T cell leukemogenesis

Catherine Hartzell et al. Sci Signal. .

Abstract

Enhanced signaling by the small guanosine triphosphatase Ras is common in T cell acute lymphoblastic leukemia/lymphoma (T-ALL), but the underlying mechanisms are unclear. We identified the guanine nucleotide exchange factor RasGRP1 (Rasgrp1 in mice) as a Ras activator that contributes to leukemogenesis. We found increased RasGRP1 expression in many pediatric T-ALL patients, which is not observed in rare early T cell precursor T-ALL patients with KRAS and NRAS mutations, such as K-Ras(G12D). Leukemia screens in wild-type mice, but not in mice expressing the mutant K-Ras(G12D) that encodes a constitutively active Ras, yielded frequent retroviral insertions that led to increased Rasgrp1 expression. Rasgrp1 and oncogenic K-Ras(G12D) promoted T-ALL through distinct mechanisms. In K-Ras(G12D) T-ALLs, enhanced Ras activation had to be uncoupled from cell cycle arrest to promote cell proliferation. In mouse T-ALL cells with increased Rasgrp1 expression, we found that Rasgrp1 contributed to a previously uncharacterized cytokine receptor-activated Ras pathway that stimulated the proliferation of T-ALL cells in vivo, which was accompanied by dynamic patterns of activation of effector kinases downstream of Ras in individual T-ALLs. Reduction of Rasgrp1 abundance reduced cytokine-stimulated Ras signaling and decreased the proliferation of T-ALL in vivo. The position of RasGRP1 downstream of cytokine receptors as well as the different clinical outcomes that we observed as a function of RasGRP1 abundance make RasGRP1 an attractive future stratification marker for T-ALL.

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Conflict of interest statement

Competing interests: The authors declare that they do not have any competing interests.

Figures

Fig. 1
Fig. 1
Increased RasGRP1 abundance is a common feature of human T-ALL. (A and B) In silico prediction of the effect of changing (A) the catalytic rates and (B) the concentrations of RasGAP and RasGRP1 on Ras activation. The SOS concentration was set at a basal amount (5 simulation concentration units) here. Each data point reported represents the average of 10,000 simulation trajectories at 200 simulation time units. The error bars indicate standard deviations, which measure cell-to-cell variability. Qualitatively similar results were observed when the SOS concentration was increased from 5 to 15 (see figs. S1, B and C). (C) Western blotting analysis of RasGRP1 protein abundance in a panel of established human T-ALL lines with abundance in normal T cells arbitrarily set at 1. Data are a representative example of three independent experiments. (D) Real-time RT-PCR analysis of RasGRP1 expression in bone marrow samples of pediatric T-ALL patients (T, 18 years or younger) or healthy donors (N). RasGRP1 mRNA abundances were normalized to those of GAPDH, and RasGRP1 expression was arbitrarily set to 1.0 in the sample from healthy donor N-1. (E and F) Standard Affymetrix analyses were used to generate and normalize signal intensities of RasGRP1 mRNA abundances in samples from 107 pediatric patients enrolled in Children's Oncology Group studies 9900/9404 and AALL03B1/AALL0434. Analyses of RMA-normalized data were plotted and indicated a statistically significant 128-fold range in the abundance of RasGRP1 mRNA, but not of any other RasGEF mRNA. (G)RasGRP1 mRNA abundance plotted as a function of clinical outcome. Low RasGRP1 mRNA abundance correlated with IF. Patients with greater RasGRP1 mRNA abundance in general went into remission followed by relapse (RE) or displayed complete continuous remission (CCR).
Fig. 2
Fig. 2
Retroviral insertions in the Rasgrp1 locus are frequent in mouse T-ALL and result in enhanced Rasgrp1 expression, whereas CIS in the genes encoding other RasGEFs or RasGAPS are rare. (A) Overview of the four most frequent insertion sites as determined by our in vivo BalbC mouse screen. Fathers of mice injected with SL3-3 were mutagenized by ENU. (B) Frequency of SL3-3 CIS in the other seven RasGEF-encoding genes. (C) Numbers of SL3-3 insertions in seventeen RasGAP-encoding genes. (D) Genomic locations of SL3-3 insertions in Rasgrp1. See figs. S2 and S3A for a detailed map of the insertions in Rasgrp1. (E) Increased Rasgrp1 mRNA abundance in clonal SL3-3 lymphoma cell lines that were generated from the primary tumor 3397S, which had an SL3-3 insertion in the Rasgrp1 promoter, as well as from tumors 1713S and 1156S, which had insertions in intron 1. The abundances of Rasgrp1 mRNA in these lines were compared to that in the control T-ALL 98 cell line in which Rasgrp1 mRNA abundance, as determined by real-time RT-PCR, was arbitrarily set at 1.0. Data are means and standard errors from three independent experiments.
Fig. 3
Fig. 3
Rasgrp1 and K-RasG12D oncogenes induce Ras activation to different extents and drive proliferation in distinct manners. (A) Analysis of basal RasGTP and Rasgrp1 abundances in the indicated Rasgrp1 and K-RasG12D T-ALL lines. Only 20% of the RasGTP pull-down material was loaded for all K-RasG12D lines to remain in the dynamic range for quantification by Western blotting analysis, which was later corrected by a factor of five. The abundance of RasGTP in 1156S-O cells was arbitrarily set to 1.0 by normalizing to the abundance of α-tubulin protein. (B) Analysis of the p53-P21CIP cell cycle arrest pathway in the indicated T-ALL lines. Phosphorylated p53 (pp53) was measured 6 hours after seeding the cells in culture medium, whereas p21 abundance was determined after 24 hours. Protein loading controls and positive controls for cell cycle arrest triggered by doxorubicin are presented in fig. S4. (C) In vitro proliferation assays were performed with cells incubated over a 100-hour time period with complete medium (condition 1, brown), medium with 20% of normal serum content (condition 2, orange), or complete medium with doxorubicin (condition 3, blue). Cells were seeded at 106/ml, and live cells were counted over the time period. A combination of IL-2, IL-7, and IL-9 was added to the culture medium to ensure sufficient growth factor presence. For additional growth curves and measurement of proliferation in the absence of cytokines, see figs. S4 and S5. Data in (A) to (C) are representative of three or more experiments. (D) Analysis of the in vivo proliferative potential of K-RasG12D and Rasgrp1 T-ALL cells. 1 × 105 T-ALL cells were injected subcutaneously into nude mice and proliferation was determined by measuring tumor volume (in cm3) over time. For additional images and growth curves see fig. S6. Each curve is derived from a single mouse.
Fig. 4
Fig. 4
Potential role of cytokine signaling in Rasgrp1 T-ALL. (A and B) We used realtime RT-PCR analysis to measure the abundances of IL2, IL7, and IL9 mRNAs in the indicated T-ALL cell lines that were maintained in an exponential log phase in tissue culture in complete medium. In (B), CD4+ T cells that were activated with PMA and ionomycin were used as a positive control for IL9 expression. Relative mRNA abundances are plotted as the Log2 of the inverse function of the ΔCT (difference in cycles), normalized to the abundance of GAPDH mRNA. Data are means and standard errors from three independent experiments. (C) Real-time RT-PCR analysis of cytokine mRNAs from T-ALL that proliferated subcutaneously in nude mice. Means (horizontal bars) and abundances in individual tumors (symbols) are plotted. (D) Quantification of the frequency of GFP-containing (GFP+) T-ALL cells in individual mouse organs as determined by flow cytometric analysis. Five mice of each group were injected with 4,000 T-ALL cells and were analyzed 14 days later. See fig. S7 for results with the 3397 T-ALL cell line. Data are representative of two independent experiments, and each dot represents an individual mouse. Note the statistically significantly reduction in GFP+ cells in the bone marrow and liver when Rasgrp1 was knocked down with shRNA (P < 0.05. (E and F) Histological analysis of T-ALL. (E) H&E staining of blood smears at 17 days after subcutaneous injection for GFP-labeled 1156S-O-GFP T-ALL demonstrated a high frequency of T-ALL cells circulating in the blood (blue cells). Injection of 20,000 cells for 1156S-O-shRNA Rasgrp1 cells resulted in fewer T-ALL in the blood, monitored at 22 days after injection. (F) Analysis of bone marrow and liver sections from the mice in (D) with H&E and Rasgrp1-staining. The morphology of the bone marrow and liver as well as the presence of T-ALL blasts and Rasgrp1 abundance were analyzed by sections and H&E and Rasgrp1 staining. Representative images are shown from three mice for each condition tested.
Fig. 5
Fig. 5
DAG has the potential to activate Rasgrp1 and stimulate increased RasGTP production in T-ALL. (A to C) PMA induced rapid and enhanced Ras activation in a Rasgrp1-dependent manner. RasGTP pull-down assays and analysis of Rasgrp1 abundance in the indicated T-ALL cell lines that were serum-starved and then stimulated with PMA (25 ng/ml) for the indicated times. The amount of RasGTP in resting 1156S-O T-ALL was arbitrarily set at 1.0. For downstream ERK activation patterns, see fig. S8. Data in (A) to (C) are representative of three or more experiments. (D and E) The deterministic steady-state amount of RasGTP modeled as a function of DAG and Rasgrp concentrations at different low amounts of Sos. The amounts of Sos were arbitrarily set at 5 and 15 activation units.
Fig. 6
Fig. 6
The abundance of Rasgrp1, but not Sos1, is important for cytokine-induced Ras activation in T-ALL. (A and B) RasGTP production as a function of Rasgrp concentration and receptor input at (A) t = 5 simulation time units and (B) t = 15 simulation time units. Each data point represents the average of 10,000 simulation trajectories. (C to E) Cytokine stimulation induces strong, but transient, Ras activation in the Rasgrp1 1156S-O and C6 T-ALL cell lines and enhanced production of RasGTP in the K-RasG12D T-ALL 3 cell line. Lentivirally expressed shRNA led to Rasgrp1 knockdown in 1156S-O and T-ALL C6 by 55 and 38%, respectively, which resulted in decreases in cytokine-induced RasGTP abundance. Note that Sos1 abundance was unchanged. (F) Computational predictions of receptor-induced Ras activation as described in (A) and (B) but in which all input from SOS has been removed. (G) In contrast to the substantial decrease in cytokine-induced Ras activation in T-ALL C6 after a 38% reduction in Rasgrp1 abundance, a reduction in Sos1 abundance by 42% in the same C6 T-ALL cell line had no substantial effect on RasGTP generation. As a specificity control for the shRNA for murine Sos1, C6 T-ALL cells were transduced with lentivirus expressing shRNA specific for human SOS1. Data in (C) to (E) and in (G) are representative of three independent experiments. (H) Patterns of phosphorylation of ERK, Akt, and IκBα in unstimulated human T-ALL lines. Data are representative of two experiments. (I) Pathway analysis of individual subcutaneous T-ALL tumors (from experiments in Fig. 3D and fig. S6). The patterns of phosphorylation of ERK, Akt, and IκB demonstrate complex heterogeneity in K-RasG12D T-ALL as well as plasticity in Rasgrp1 T-ALL. Data are representative of three experiments.
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