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Increased baseline RASGRP1 signals enhance stem cell fitness during native hematopoiesis

Oncogene volume 39, pages 6920–6934 (2020)Cite this article

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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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References

  1. Terwilliger T, Abdul-Hay M. Acute lymphoblastic leukemia: a comprehensive review and 2017 update. Blood Cancer J. 2017;7:e577. https://doi.org/10.1038/bcj.2017.53.

    Article  CAS  Google Scholar 

  2. von Lintig FC, Huvar I, Law P, Diccianni MB, Yu AL, Boss GR. Ras activation in normal white blood cells and childhood acute lymphoblastic leukemia. Clin Cancer Res. 2000;6:1804–10.

    Google Scholar 

  3. Hartzell C, Ksionda O, Lemmens E, Coakley K, Yang M, Dail M, et al. Dysregulated RasGRP1 responds to cytokine receptor input in T cell leukemogenesis. Sci Signal. 2013;6:ra21. https://doi.org/10.1126/scisignal.2003848.

    Article  CAS  Google Scholar 

  4. Ksionda O, Melton AA, Bache J, Tenhagen M, Bakker J, Harvey R, et al. RasGRP1 overexpression in T-ALL increases basal nucleotide exchange on Ras rendering the Ras/PI3K/Akt pathway responsive to protumorigenic cytokines. Oncogene. 2016;35:3658–68. https://doi.org/10.1038/onc.2015.431.

    Article  CAS  Google Scholar 

  5. Jackson EL, Willis N, Mercer K, Bronson RT, Crowley D, Montoya R, et al. Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras. Genes Dev. 2001;15:3243–8. https://doi.org/10.1101/gad.943001.

    Article  CAS  Google Scholar 

  6. Schubbert S, Shannon K, Bollag G. Hyperactive Ras in developmental disorders and cancer. Nat Rev Cancer. 2007;7:295–308. https://doi.org/10.1038/nrc2109.

    Article  CAS  Google Scholar 

  7. Bowen DT, Frew ME, Hills R, Gale RE, Wheatley K, Groves MJ, et al. RAS mutation in acute myeloid leukemia is associated with distinct cytogenetic subgroups but does not influence outcome in patients younger than 60 years. Blood. 2005;106:2113–9. https://doi.org/10.1182/blood-2005-03-0867.

    Article  CAS  Google Scholar 

  8. Liu Y, Easton J, Shao Y, Maciaszek J, Wang Z, Wilkinson MR, et al. The genomic landscape of pediatric and young adult T-lineage acute lymphoblastic leukemia. Nat Genet. 2017;49:1211–8. https://doi.org/10.1038/ng.3909.

    Article  CAS  Google Scholar 

  9. Kuhn R, Schwenk F, Aguet M, Rajewsky K. Inducible gene targeting in mice. Science. 1995;269:1427–9.

    Article  CAS  Google Scholar 

  10. Chan IT, Kutok JL, Williams IR, Cohen S, Kelly L, Shigematsu H, et al. Conditional expression of oncogenic K-ras from its endogenous promoter induces a myeloproliferative disease. J Clin Invest. 2004;113:528–38. https://doi.org/10.1172/JCI20476.

    Article  CAS  Google Scholar 

  11. Braun BS, Tuveson DA, Kong N, Le DT, Kogan SC, Rozmus J, et al. Somatic activation of oncogenic Kras in hematopoietic cells initiates a rapidly fatal myeloproliferative disorder. Proc Natl Acad Sci USA. 2004;101:597–602.

    Article  CAS  Google Scholar 

  12. Kindler T, Cornejo MG, Scholl C, Liu J, Leeman DS, Haydu JE, et al. K-RasG12D-induced T-cell lymphoblastic lymphoma/leukemias harbor Notch1 mutations and are sensitive to gamma-secretase inhibitors. Blood. 2008;112:3373–82.

    Article  CAS  Google Scholar 

  13. Sabnis AJ, Cheung LS, Dail M, Kang HC, Santaguida M, Hermiston ML, et al. Oncogenic Kras initiates leukemia in hematopoietic stem cells. PLoS Biol. 2009;7:e59.

    Article  Google Scholar 

  14. Wang J, Liu Y, Li Z, Du J, Ryu MJ, Taylor PR, et al. Endogenous oncogenic Nras mutation promotes aberrant GM-CSF signaling in granulocytic/monocytic precursors in a murine model of chronic myelomonocytic leukemia. Blood. 2010;116:5991–6002. https://doi.org/10.1182/blood-2010-04-281527.

    Article  CAS  Google Scholar 

  15. Li Q, Haigis KM, McDaniel A, Harding-Theobald E, Kogan SC, Akagi K, et al. Hematopoiesis and leukemogenesis in mice expressing oncogenic NrasG12D from the endogenous locus. Blood. 2011;117:2022–32. https://doi.org/10.1182/blood-2010-04-280750.

    Article  CAS  Google Scholar 

  16. Oki-Idouchi CE, Lorenzo PS. Transgenic overexpression of RasGRP1 in mouse epidermis results in spontaneous tumors of the skin. Cancer Res. 2007;67:276–80.

    Article  CAS  Google Scholar 

  17. Klinger MB, Guilbault B, Goulding RE, Kay RJ. Deregulated expression of RasGRP1 initiates thymic lymphomagenesis independently of T-cell receptors. Oncogene. 2005;24:2695–704.

    Article  CAS  Google Scholar 

  18. Oki T, Kitaura J, Watanabe-Okochi N, Nishimura K, Maehara A, Uchida T, et al. Aberrant expression of RasGRP1 cooperates with gain-of-function NOTCH1 mutations in T-cell leukemogenesis. Leukemia. 2012;26:1038–45. https://doi.org/10.1038/leu.2011.328.

    Article  CAS  Google Scholar 

  19. Norment AM, Bogatzki LY, Klinger M, Ojala EW, Bevan MJ, Kay RJ. Transgenic expression of RasGRP1 induces the maturation of double-negative thymocytes and enhances the production of CD8 single-positive thymocytes. J Immunol. 2003;170:1141–9.

    Article  CAS  Google Scholar 

  20. Jun JE, Rubio I, Roose JP. Regulation of Ras exchange factors and cellular localization of Ras activation by lipid messengers in T cells. Front Immunol. 2013;4:239. https://doi.org/10.3389/fimmu.2013.00239.

    Article  CAS  Google Scholar 

  21. Ksionda O, Limnander A, Roose JP. RasGRP Ras guanine nucleotide exchange factors in cancer. Front Biol (Beijing). 2013;8:508–32. https://doi.org/10.1007/s11515-013-1276-9.

    Article  CAS  Google Scholar 

  22. Winter SS, Dunsmore KP, Devidas M, Wood BL, Esiashvili N, Chen Z, et al. Improved survival for children and young adults with t-lineage acute lymphoblastic leukemia: results from the children’s oncology group AALL0434 methotrexate randomization. J Clin Oncol. 2018;36:2926–34. https://doi.org/10.1200/JCO.2018.77.7250.

    Article  CAS  Google Scholar 

  23. Ksionda, O, Melton, AA, Bache, J, Tenhagen, M, Bakker, J, Harvey, R et al. RasGRP1 overexpression in T-ALL increases basal nucleotide exchange on Ras rendering the Ras/PI3K/Akt pathway responsive to protumorigenic cytokines. Oncogene, https://doi.org/10.1038/onc.2015.431 (2015).

  24. Anders S, Pyl PT, Huber W. HTSeq–a Python framework to work with high-throughput sequencing data. Bioinforma. 2015;31:166–9. https://doi.org/10.1093/bioinformatics/btu638.

    Article  CAS  Google Scholar 

  25. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15:550. https://doi.org/10.1186/s13059-014-0550-8.

    Article  CAS  Google Scholar 

  26. Kortum RL, Sommers CL, Pinski JM, Alexander CP, Merrill RK, Li W, et al. Deconstructing Ras signaling in the thymus. Mol Cell Biol. 2012;32:2748–59. https://doi.org/10.1128/MCB.00317-12.

    Article  CAS  Google Scholar 

  27. Priatel JJ, Teh SJ, Dower NA, Stone JC, Teh HS. RasGRP1 transduces low-grade TCR signals which are critical for T cell development, homeostasis, and differentiation. Immunity. 2002;17:617–27.

    Article  CAS  Google Scholar 

  28. Morrison SJ, Scadden DT. The bone marrow niche for haematopoietic stem cells. Nature. 2014;505:327–34. https://doi.org/10.1038/nature12984.

    Article  CAS  Google Scholar 

  29. Zhang CC, Lodish HF. Cytokines regulating hematopoietic stem cell function. Curr Opin Hematol. 2008;15:307–11. https://doi.org/10.1097/MOH.0b013e3283007db5.

    Article  CAS  Google Scholar 

  30. Vigil D, Cherfils J, Rossman KL, Der CJ. Ras superfamily GEFs and GAPs: validated and tractable targets for cancer therapy?. Nat Rev Cancer. 2010;10:842–57. https://doi.org/10.1038/nrc2960.

    Article  CAS  Google Scholar 

  31. Myers DR, Wheeler B, Roose JP. mTOR and other effector kinase signals that impact T cell function and activity. Immunol Rev. 2019;291:134–53. https://doi.org/10.1111/imr.12796.

    Article  CAS  Google Scholar 

  32. Mues M, Roose JP. Distinct oncogenic Ras signals characterized by profound differences in flux through the RasGDP/RasGTP cycle. Small GTPases. 2017;8:20–25. https://doi.org/10.1080/21541248.2016.1187323.

    Article  CAS  Google Scholar 

  33. Wang J, Kong G, Liu Y, Du J, Chang YI, Tey SR, et al. Nras(G12D/+) promotes leukemogenesis by aberrantly regulating hematopoietic stem cell functions. Blood. 2013;121:5203–7. https://doi.org/10.1182/blood-2012-12-475863.

    Article  CAS  Google Scholar 

  34. Soriano P. Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet. 1999;21:70–71.

    Article  CAS  Google Scholar 

  35. Okabe M, Ikawa M, Kominami K, Nakanishi T, Nishimune Y. ‘Green mice’ as a source of ubiquitous green cells. FEBS Lett. 1997;407:313–9.

    Article  CAS  Google Scholar 

  36. Sun J, Ramos A, Chapman B, Johnnidis JB, Le L, Ho YJ, et al. Clonal dynamics of native haematopoiesis. Nature. 2014;514:322–7. https://doi.org/10.1038/nature13824.

    Article  CAS  Google Scholar 

  37. Li Q, Bohin N, Wen T, Ng V, Magee J, Chen SC, et al. Oncogenic Nras has bimodal effects on stem cells that sustainably increase competitiveness. Nature. 2013;504:143–7. https://doi.org/10.1038/nature12830.

    Article  CAS  Google Scholar 

  38. Ksionda O, Mues M, Wandler AM, Donker L, Tenhagen M, Jun J, et al. Comprehensive analysis of T cell leukemia signals reveals heterogeneity in the PI3 kinase-Akt pathway and limitations of PI3 kinase inhibitors as monotherapy. PLoS ONE. 2018;13:e0193849. https://doi.org/10.1371/journal.pone.0193849.

    Article  CAS  Google Scholar 

  39. Romero-Moya D, Bueno C, Montes R, Navarro-Montero O, Iborra FJ, Lopez LC, et al. Cord blood-derived CD34+ hematopoietic cells with low mitochondrial mass are enriched in hematopoietic repopulating stem cell function. Haematologica. 2013;98:1022–9. https://doi.org/10.3324/haematol.2012.079244.

    Article  CAS  Google Scholar 

  40. Downward J. Cancer biology: signatures guide drug choice. Nature. 2006;439:274–5.

    Article  CAS  Google Scholar 

  41. Schubbert S, Bollag G, Shannon K. Deregulated Ras signaling in developmental disorders: new tricks for an old dog. Curr Opin Genet Dev. 2007;17:15–22. https://doi.org/10.1016/j.gde.2006.12.004.

    Article  CAS  Google Scholar 

  42. Stine RR, Matunis EL. Stem cell competition: finding balance in the niche. Trends Cell Biol. 2013;23:357–64. https://doi.org/10.1016/j.tcb.2013.03.001.

    Article  CAS  Google Scholar 

  43. Myers DR, Norlin E, Vercoulen Y, Roose JP. Active Tonic mTORC1 Signals Shape Baseline Translation in Naive T Cells. Cell Rep.2019;27:1858–74 e1856. https://doi.org/10.1016/j.celrep.2019.04.037.

    Article  CAS  Google Scholar 

  44. Seita J, Weissman IL. Hematopoietic stem cell: self-renewal versus differentiation. Wiley Interdiscip Rev Syst Biol Med. 2010;2:640–53. https://doi.org/10.1002/wsbm.86.

    Article  CAS  Google Scholar 

  45. Wilson A, Trumpp A. Bone-marrow haematopoietic-stem-cell niches. Nat Rev Immunol. 2006;6:93–106. https://doi.org/10.1038/nri1779.

    Article  CAS  Google Scholar 

  46. Zhang Y, Gao S, Xia J, Liu F. Hematopoietic hierarchy - an updated roadmap. Trends Cell Biol. 2018;28:976–86. https://doi.org/10.1016/j.tcb.2018.06.001.

    Article  Google Scholar 

  47. Knudson AG Jr. Mutation and cancer: statistical study of retinoblastoma. Proc Natl Acad Sci USA. 1971;68:820–3.

    Article  Google Scholar 

  48. Gilliland DG, Griffin JD. The roles of FLT3 in hematopoiesis and leukemia. Blood. 2002;100:1532–42.

    Article  CAS  Google Scholar 

  49. Montes R, Ayllon V, Prieto C, Bursen A, Prelle C, Romero-Moya D. et al. Ligand-independent FLT3 activation does not cooperate with MLL-AF4 to immortalize/transform cord blood CD34+ cells. Leukemia. 2014;28:666–74. https://doi.org/10.1038/leu.2013.346.

    Article  CAS  Google Scholar 

  50. Sanjuan-Pla A, Bueno C, Prieto C, Acha P, Stam RW, Marschalek R, et al. Revisiting the biology of infant t(4;11)/MLL-AF4+ B-cell acute lymphoblastic leukemia. Blood. 2015;126:2676–85. https://doi.org/10.1182/blood-2015-09-667378.

    Article  CAS  Google Scholar 

  51. Sun C, Chang L, Zhu X. Pathogenesis of ETV6/RUNX1-positive childhood acute lymphoblastic leukemia and mechanisms underlying its relapse. Oncotarget. 2017;8:35445–59. https://doi.org/10.18632/oncotarget.16367.

    Article  Google Scholar 

  52. Beck-Engeser GB, Lum AM, Huppi K, Caplen NJ, Wang BB, Wabl M. Pvt1-encoded microRNAs in oncogenesis. Retrovirology. 2008;5:4.

    Article  Google Scholar 

  53. Xu Z, Wang M, Wang L, Wang Y, Zhao X, Rao Q, et al. Aberrant expression of TSC2 gene in the newly diagnosed acute leukemia. Leuk Res. 2009;33:891–7. https://doi.org/10.1016/j.leukres.2009.01.041.

    Article  CAS  Google Scholar 

  54. Kalaitzidis D, Sykes SM, Wang Z, Punt N, Tang Y, Ragu C, et al. mTOR complex 1 plays critical roles in hematopoiesis and Pten-loss-evoked leukemogenesis. cell stem cell. 2012;11:429–39. https://doi.org/10.1016/j.stem.2012.06.009.

    Article  CAS  Google Scholar 

  55. Kentsis A, Look AT. Distinct and dynamic requirements for mTOR signaling in hematopoiesis and leukemogenesis. cell stem cell. 2012;11:281–2. https://doi.org/10.1016/j.stem.2012.08.007.

    Article  CAS  Google Scholar 

  56. Magee JA, Ikenoue T, Nakada D, Lee JY, Guan KL, Morrison SJ. Temporal changes in PTEN and mTORC2 regulation of hematopoietic stem cell self-renewal and leukemia suppression. cell stem cell. 2012;11:415–28. https://doi.org/10.1016/j.stem.2012.05.026.

    Article  CAS  Google Scholar 

  57. Sanjuan-Pla A, Romero-Moya D, Prieto C, Bueno C, Bigas A, Menendez P. Intra-bone marrow transplantation confers superior multilineage engraftment of murine aorta-gonad mesonephros cells over intravenous transplantation. Stem Cells Dev. 2016;25:259–65. https://doi.org/10.1089/scd.2015.0309.

    Article  CAS  Google Scholar 

  58. Pietras EM, Reynaud D, Kang YA, Carlin D, Calero-Nieto FJ, Leavitt AD, et al. Functionally distinct subsets of lineage-biased multipotent progenitors control blood production in normal and regenerative conditions. Cell Stem Cell. 2015;17:35–46. https://doi.org/10.1016/j.stem.2015.05.003.

    Article  CAS  Google Scholar 

  59. Herault A, Binnewies M, Leong S, Calero-Nieto FJ, Zhang SY, Kang YA, et al. Myeloid progenitor cluster formation drives emergency and leukaemic myelopoiesis. Nature. 2017;544:53–58. https://doi.org/10.1038/nature21693.

    Article  CAS  Google Scholar 

  60. Roose JP, Diehn M, Tomlinson MG, Lin J, Alizadeh AA, Botstein D, et al. T cell receptor-independent basal signaling via Erk and Abl kinases suppresses RAG gene expression. PLoS Biol. 2003;1:E53.

    Article  Google Scholar 

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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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  1. Olga Ksionda

    Present address: Liggins Institute, The University of Auckland, Auckland, New Zealand

  2. Marsilius Mues

    Present address: Miltenyi Biotec GmbH, Friedrich-Ebert-Str. 68, 51429, Bergisch Gladbach, Germany

  3. These authors contributed equally: Laila Karra, Damia Romero-Moya, Olga Ksionda

Authors and Affiliations

  1. Department of Anatomy, University of California, San Francisco, San Francisco, CA, 94143, USA

    Laila Karra, Damia Romero-Moya, Olga Ksionda, Milana Krush, Marsilius Mues, Philippe Depeille & Jeroen P. Roose

  2. Department of Pathology and Hematological Malignancies Program, St. Jude Children’s Research Hospital, Memphis, TN, 38105, USA

    Zhaohui Gu & Charles Mullighan

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Contributions

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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