Increased baseline RASGRP1 signals enhance stem cell fitness during native hematopoiesis

Abstract

Oncogenic mutations in RAS genes, like KRASG12D or NRASG12D, trap Ras in the active state and cause myeloproliferative disorder and T cell leukemia (T-ALL) when induced in the bone marrow via Mx1CRE. The RAS exchange factor RASGRP1 is frequently overexpressed in T-ALL patients. In T-ALL cell lines overexpression of RASGRP1 increases flux through the RASGTP/RasGDP cycle. Here we expanded RASGRP1 expression surveys in pediatric T-ALL and generated a RoLoRiG mouse model crossed to Mx1CRE to determine the consequences of induced RASGRP1 overexpression in primary hematopoietic cells. RASGRP1-overexpressing, GFP-positive cells outcompeted wild type cells and dominated the peripheral blood compartment over time. RASGRP1 overexpression bestows gain-of-function colony formation properties to bone marrow progenitors in medium containing limited growth factors. RASGRP1 overexpression enhances baseline mTOR-S6 signaling in the bone marrow, but not in vitro cytokine-induced signals. In agreement with these mechanistic findings, hRASGRP1-ires-EGFP enhances fitness of stem- and progenitor- cells, but only in the context of native hematopoiesis. RASGRP1 overexpression is distinct from KRASG12D or NRASG12D, does not cause acute leukemia on its own, and leukemia virus insertion frequencies predict that RASGRP1 overexpression can effectively cooperate with lesions in many other genes to cause acute T-ALL.

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Fig. 1: T-ALL patient analysis and mouse models.
Fig. 2: In vivo accumulation of hRASGRP1-overexpressing cells in RoLoRiG mouse model.
Fig. 3: hRASGRP1 overexpression in a bone marrow transfer model.
Fig. 4: Cytokine-induced bone marrow colony formation and signaling.
Fig. 5: RasGRP1 overexpression drives spontaneous bone marrow colony formation and S6 signaling.
Fig. 6: Increased fitness in Native hematopoiesis in RoLoRiG+/+ mice.
Fig. 7: Co-insertion table of SL3-3 leukemia virus insertions.

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Acknowledgements

This work was supported by an Alex’ Lemonade Stand Foundation Innovator Award, the NIH/NCI (R01 – CA187318), and the NIH/NHLBI (R01 – HL120724) (all to JPR). Further support came from a Leukemia & Lymphoma Society grant (to MM) and the Rothschild Fellowship for postdoctoral fellows in the Natural, Exact or Life Sciences and Engineering (to LK), and PD is a Mark Foundation Momentum Fellow supported by a fellowship from the Mark Foundation for Cancer Research; and by NCI grants CA021765 (St Jude Comprehensive Cancer Center Support Grant), an NCI R35 Outstanding Investigator Award (R35 CA197695) and a St. Baldrick’s Foundation Robert J. Arceci Innovation award. We thank the members of the Roose lab and the Heme-Onc community at UCSF for useful suggestions and comments. We thank UCSF flow cytometry facility and DRC Center Grant NIH P30 DK063720. We thank Emmanuelle Passague and her lab for kindly providing us Mx1-CRE mice.

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LK, DR-M, OK, and MM performed experiments and analyzed results. PD made the mouse construct. ZG and CGM generated and analyzed human T-ALL genomic data. LK and DR-M. made the figures; JR designed the research and secured the majority of the funding. LK, DR-M, and JR wrote the paper.

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Correspondence to Jeroen P. Roose.

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Jeroen Roose is a co-founder and scientific advisor of Seal Biosciences, Inc. and on the scientific advisory committee for the Mark Foundation for Cancer Research. C.G.M. receives research funding from Loxo Oncology, Abbvie, and Pfizer, and speaking fees from Amgen.

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Karra, L., Romero-Moya, D., Ksionda, O. et al. Increased baseline RASGRP1 signals enhance stem cell fitness during native hematopoiesis. Oncogene 39, 6920–6934 (2020). https://doi.org/10.1038/s41388-020-01469-8

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