Dysregulated RasGRP1 Responds to Cytokine Receptor Input in T Cell Leukemogenesis

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. Here, 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 we did not observe in rare early T cell precursor (ETP) T-ALL patients with KRAS and NRAS mutations, such as K-RasG12D. Leukemia screens in wild-type mice, but not in mice expressing the mutant K-RasG12D that encodes a constitutively active Ras, yielded frequent retroviral insertions that led to increased Rasgrp1 expression. Rasgrp1 and oncogenic K-RasG12D promoted T-ALL through distinct mechanisms. In K-RasG12D T-ALLs, we found that enhanced Ras activation did not lead to cell cycle arrest. 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, suggesting that patients with this cancer should be screened for increased abundance of RasGRP1 to customize treatment.

Distinct oncogenic Ras signals characterized by profound differences in flux through the RasGDP/RasGTP cycle

“…Several groups including our own observed that Ras-PI3K signaling is universally utilized in T-ALL, but with heterogeneous activation patterns. [41][42][43][44][45][46][47] By contrast, the high usage of PI3K-Akt signals is not seen in other hematologic malignancies. 48 Given that PI3K inhibitors like GDC0941 are very potent agents, 49 such inhibition may be useful as therapy for T-ALL, although the heterogeneous activation patterns observed in different T-ALL may complicate a general application.…”

“…29 Studies with different mouse models and with pediatric T-ALL patient samples pointed to a role for RasGRP1 in T-ALL leukemogenesis. 20,[30][31][32][33] Likewise, the inactivating function of RasGAPs on the Ras pathway also appears critical; combined T cell-specific deletion of NF1 and p120 RasGAPs in mice leads to development of T-ALL 34 and NF1 is mutated in both pediatric and adult T-ALL patients. [35][36][37] In our recent study by Ksionda et al 38 we describe the details of cytokine receptor-Ras pathways in T-ALL and how Ras signals in T-ALL with RasGRP1 overexpression not only differ from those in developing normal T cells (thymocytes) but also from Ras signals in Ras MUT T-ALL.…”

“…While both T-ALL and thymocytes load Ras with GTP in response to TCR stimulation, only T-ALL cells activate Ras following cytokine stimulation. Overexpression of RasGRP1 is critical for this cytokine receptor-Ras pathway 33 and thus RasGRP1 created a rewiring that is not seen in normal thymocytes. Both TCR stimulation and cytokineinduced Ras signaling via RasGRP1 yield strong and sustained PI3K-Akt signals in T-ALL.…”

“…In contrast, cells featuring wild type Ras but elevated expression of RasGRP1 do not display high basal levels of RasGTP when measured by traditional RasGTP pulldown assays. 46 However, RasGTP pulldowns only measure the net amount of RasGTP at a certain time point by fishing in the cell lysate with the portion of RAF that binds RasGTP and miss the turnover rate of nucleotide exchange on Ras. This RasGDP/GTP cycling can be directly assessed employing a digitonin nucleotide exchange assay that our collaborators in Dr. Ignacio Rubio's group optimized.…”

“…We noted in the past that not all Ras MUT T-ALL can uncouple the arrest programs to allow growth of leukemic blasts. 46 Perhaps this helps explain why Ras MUT is relatively rare in T-ALL? In contrast, overexpression of RasGRP1 in T-ALL does not lead to constitutive Ras signaling but only to a high RasGDP/GTP turnover, and should not induce cell cycle arrest.…”