Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Chronic myeloproliferative neoplasms

Downregulating Notch counteracts KrasG12D-induced ERK activation and oxidative phosphorylation in myeloproliferative neoplasm

Abstract

The Notch signaling pathway contributes to the pathogenesis of a wide spectrum of human cancers, including hematopoietic malignancies. Its functions are highly dependent on the specific cellular context. Gain-of-function NOTCH1 mutations are prevalent in human T-cell leukemia, while loss of Notch signaling is reported in myeloid leukemias. Here, we report a novel oncogenic function of Notch signaling in oncogenic Kras-induced myeloproliferative neoplasm (MPN). We find that downregulation of Notch signaling in hematopoietic cells via DNMAML expression or Pofut1 deletion significantly blocks MPN development in KrasG12D mice in a cell-autonomous manner. Further mechanistic studies indicate that inhibition of Notch signaling upregulates Dusp1, a dual phosphatase that inactivates p-ERK, and downregulates cytokine-evoked ERK activation in KrasG12D cells. Moreover, mitochondrial metabolism is greatly enhanced in KrasG12D cells but significantly reprogrammed by DNMAML close to that in control cells. Consequently, cell proliferation and expanded myeloid compartment in KrasG12D mice are significantly reduced. Consistent with these findings, combined inhibition of the MEK/ERK pathway and mitochondrial oxidative phosphorylation effectively inhibited the growth of human and mouse leukemia cells in vitro. Our study provides a strong rational to target both ERK signaling and aberrant metabolism in oncogenic Ras-driven myeloid leukemia.

This is a preview of subscription content

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

References

  1. Wharton KA, Johansen KM, Xu T, Artavanis-Tsakonas S. Nucleotide sequence from the neurogenic locus notch implies a gene product that shares homology with proteins containing EGF-like repeats. Cell. 1985;43:567–81.

    CAS  PubMed  Google Scholar 

  2. Kidd S, Kelley MR, Young MW. Sequence of the notch locus of Drosophila melanogaster: relationship of the encoded protein to mammalian clotting and growth factors. Mol Cell Biol. 1986;6:3094–108.

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Ntziachristos P, Lim JS, Sage J, Aifantis I. From fly wings to targeted cancer therapies: a centennial for notch signaling. Cancer Cell. 2014;25:318–34.

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Moloney DJ, Panin VM, Johnston SH, Chen J, Shao L, Wilson R, et al. Fringe is a glycosyltransferase that modifies Notch. Nature. 2000;406:369–75.

    CAS  PubMed  Google Scholar 

  5. Wang Y, Shao L, Shi S, Harris RJ, Spellman MW, Stanley P, et al. Modification of epidermal growth factor-like repeats with O-fucose. Molecular cloning and expression of a novel GDP-fucose protein O-fucosyltransferase. J Biol Chem. 2001;276:40338–45.

    CAS  PubMed  Google Scholar 

  6. Luo Y, Haltiwanger RS. O-fucosylation of notch occurs in the endoplasmic reticulum. J Biol Chem. 2005;280:11289–94.

    CAS  PubMed  Google Scholar 

  7. Okajima T, Xu A, Lei L, Irvine KD. Chaperone activity of protein O-fucosyltransferase 1 promotes notch receptor folding. Science. 2005;307:1599–603.

    CAS  PubMed  Google Scholar 

  8. Okajima T, Irvine KD. Regulation of notch signaling by o-linked fucose. Cell. 2002;111:893–904.

    CAS  PubMed  Google Scholar 

  9. Maillard I, Koch U, Dumortier A, Shestova O, Xu L, Sai H, et al. Canonical notch signaling is dispensable for the maintenance of adult hematopoietic stem cells. Cell Stem Cell. 2008;2:356–66.

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Artavanis-Tsakonas S, Rand MD, Lake RJ. Notch signaling: cell fate control and signal integration in development. Science. 1999;284:770–6.

    CAS  PubMed  Google Scholar 

  11. Weng AP, Ferrando AA, Lee W, Morris JPt, Silverman LB, Sanchez-Irizarry C, et al. Activating mutations of NOTCH1 in human T cell acute lymphoblastic leukemia. Science. 2004;306:269–71.

    CAS  PubMed  Google Scholar 

  12. Kong G, Du J, Liu Y, Meline B, Chang YI, Ranheim EA, et al. Notch1 gene mutations target KRAS G12D-expressing CD8+ cells and contribute to their leukemogenic transformation. J Biol Chem. 2013;288:18219–27.

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Ashworth TD, Pear WS, Chiang MY, Blacklow SC, Mastio J, Xu L, et al. Deletion-based mechanisms of Notch1 activation in T-ALL: key roles for RAG recombinase and a conserved internal translational start site in Notch1. Blood. 2010;116:5455–64.

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Chiang MY, Xu L, Shestova O, Histen G, L’Heureux S, Romany C, et al. Leukemia-associated NOTCH1 alleles are weak tumor initiators but accelerate K-ras-initiated leukemia. J Clin Invest. 2008;118:3181–94.

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Kannan S, Sutphin RM, Hall MG, Golfman LS, Fang W, Nolo RM, et al. Notch activation inhibits AML growth and survival: a potential therapeutic approach. J Exp Med. 2013;210:321–37.

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Lobry C, Ntziachristos P, Ndiaye-Lobry D, Oh P, Cimmino L, Zhu N, et al. Notch pathway activation targets AML-initiating cell homeostasis and differentiation. J Exp Med. 2013;210:301–19.

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Klinakis A, Lobry C, Abdel-Wahab O, Oh P, Haeno H, Buonamici S, et al. A novel tumour-suppressor function for the Notch pathway in myeloid leukaemia. Nature. 2011;473:230–3.

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Yao D, Huang Y, Huang X, Wang W, Yan Q, Wei L, et al. Protein O-fucosyltransferase 1 (Pofut1) regulates lymphoid and myeloid homeostasis through modulation of Notch receptor ligand interactions. Blood. 2011;117:5652–62.

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Zhang J, Wang J, Liu Y, Sidik H, Young KH, Lodish HF, et al. Oncogenic Kras-induced leukemogeneis: hematopoietic stem cells as the initial target and lineage-specific progenitors as the potential targets for final leukemic transformation. Blood. 2009;113:1304–14.

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Shi S, Stanley P. Protein O-fucosyltransferase 1 is an essential component of Notch signaling pathways. Proc Natl Acad Sci USA. 2003;100:5234–9.

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Du J, Liu Y, Meline B, Kong G, Tan LX, Lo JC, et al. Loss of CD44 attenuates aberrant GM-CSF signaling in Kras G12D hematopoietic progenitor/precursor cells and prolongs the survival of diseased animals. Leukemia. 2013;27:754–7.

    CAS  PubMed  Google Scholar 

  22. Chang YI, You X, Kong G, Ranheim EA, Wang J, Du J, et al. Loss of Dnmt3a and endogenous Kras cooperate to regulate hematopoietic stem and progenitor cell functions in leukemogenesis. Leukemia. 2015;29:1847–56.

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Kong G, Chang Y-I, Damnernsawad A, You X, Du J, Ranheim RA, et al. Loss of wild-type Kras promotes activation of all Ras isoforms in oncogenic Kras-induced leukemogenesis. Leukemia. 2016;30:1542–51.

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Clausen BE, Burkhardt C, Reith W, Renkawitz R, Forster I. Conditional gene targeting in macrophages and granulocytes using LysMcre mice. Transgenic Res. 1999;8:265–77.

    CAS  PubMed  Google Scholar 

  25. Ye M, Iwasaki H, Laiosa CV, Stadtfeld M, Xie H, Heck S, et al. Hematopoietic stem cells expressing the myeloid lysozyme gene retain long-term, multilineage repopulation potential. Immunity. 2003;19:689–99.

    CAS  PubMed  Google Scholar 

  26. Oh P, Lobry C, Gao J, Tikhonova A, Loizou E, Manent J, et al. In vivo mapping of notch pathway activity in normal and stress hematopoiesis. Cell Stem Cell. 2013;13:190–204.

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Van Meter ME, Diaz-Flores E, Archard JA, Passegue E, Irish JM, Kotecha N, et al. K-RasG12D expression induces hyperproliferation and aberrant signaling in primary hematopoietic stem/progenitor cells. Blood. 2007;109:3945–52.

    PubMed  PubMed Central  Google Scholar 

  28. Maraver A, Fernandez-Marcos PJ, Herranz D, Canamero M, Munoz-Martin M, Gomez-Lopez G, et al. Therapeutic effect of gamma-secretase inhibition in KrasG12V-driven non-small cell lung carcinoma by derepression of DUSP1 and inhibition of ERK. Cancer Cell. 2012;22:222–34.

    CAS  PubMed  Google Scholar 

  29. Zhang J, Kong G, Rajagopalan A, Lu L, Song J, Hussaini M, et al. p53-/- synergizes with enhanced NrasG12D signaling to transform megakaryocyte-erythroid progenitors in acute myeloid leukemia. Blood. 2017;129:358–70.

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Palomero T, Lim WK, Odom DT, Sulis ML, Real PJ, Margolin A, et al. NOTCH1 directly regulates c-MYC and activates a feed-forward-loop transcriptional network promoting leukemic cell growth. Proc Natl Acad Sci USA. 2006;103:18261–6.

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Grieselhuber NR, Klco JM, Verdoni AM, Lamprecht T, Sarkaria SM, Wartman LD, et al. Notch signaling in acute promyelocytic leukemia. Leukemia. 2013;27:1548–57.

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Wang H, Zou J, Zhao B, Johannsen E, Ashworth T, Wong H, et al. Genome-wide analysis reveals conserved and divergent features of Notch1/RBPJ binding in human and murine T-lymphoblastic leukemia cells. Proc Natl Acad Sci USA. 2011;108:14908–13.

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Herranz D, Ambesi-Impiombato A, Sudderth J, Sanchez-Martin M, Belver L, Tosello V, et al. Metabolic reprogramming induces resistance to anti-NOTCH1 therapies in T cell acute lymphoblastic leukemia. Nat Med. 2015;21:1182–9.

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Yeh TC, Marsh V, Bernat BA, Ballard J, Colwell H, Evans RJ, et al. Biological characterization of ARRY-142886 (AZD6244), a potent, highly selective mitogen-activated protein kinase kinase 1/2 inhibitor. Clin Cancer Res. 2007;13:1576–83.

    CAS  PubMed  Google Scholar 

  35. Kong G, Wunderlich M, Yang D, Ranheim EA, Young KH, Wang J, et al. Combined MEK and JAK inhibition abrogates murine myeloproliferative neoplasm. J Clin Invest. 2014;124:2762–73.

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Hitosugi T, Fan J, Chung TW, Lythgoe K, Wang X, Xie J, et al. Tyrosine phosphorylation of mitochondrial pyruvate dehydrogenase kinase 1 is important for cancer metabolism. Mol Cell. 2011;44:864–77.

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 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.

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Chang YI, Damnernsawad A, Allen LK, Yang D, Ranheim EA, Young KH, et al. Evaluation of allelic strength of human TET2 mutations and cooperation between Tet2 knockdown and oncogenic Nras mutation. Br J Haematol. 2014;166:461–5.

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Merlevede J, Droin N, Qin T, Meldi K, Yoshida K, Morabito M, et al. Mutation allele burden remains unchanged in chronic myelomonocytic leukaemia responding to hypomethylating agents. Nat Commun. 2016;7:10767.

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Ambrogio C, Gomez-Lopez G, Falcone M, Vidal A, Nadal E, Crosetto N, et al. Combined inhibition of DDR1 and Notch signaling is a therapeutic strategy for KRAS-driven lung adenocarcinoma. Nat Med. 2016;22:270–7.

    CAS  PubMed  Google Scholar 

  41. Xu J, Chi F, Guo T, Punj V, Lee WN, French SW, et al. NOTCH reprograms mitochondrial metabolism for proinflammatory macrophage activation. J Clin Invest. 2015;125:1579–90.

    PubMed  PubMed Central  Google Scholar 

  42. Kishton RJ, Barnes CE, Nichols AG, Cohen S, Gerriets VA, Siska PJ, et al. AMPK is essential to balance glycolysis and mitochondrial metabolism to control T-ALL cell stress and survival. Cell Metab. 2016;23:649–62.

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Lagadinou ED, Sach A, Callahan K, Rossi RM, Neering SJ, Minhajuddin M, et al. BCL-2 inhibition targets oxidative phosphorylation and selectively eradicates quiescent human leukemia stem cells. Cell Stem Cell. 2013;12:329–41.

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Jacque N, Ronchetti AM, Larrue C, Meunier G, Birsen R, Willems L, et al. Targeting glutaminolysis has antileukemic activity in acute myeloid leukemia and synergizes with BCL-2 inhibition. Blood. 2015;126:1346–56.

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Liu Y, Marks K, Cowley GS, Carretero J, Liu Q, Nieland TJ, et al. Metabolic and functional genomic studies identify deoxythymidylate kinase as a target in LKB1-mutant lung cancer. Cancer Discov. 2013;3:870–9.

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Kerr EM, Gaude E, Turrell FK, Frezza C, Martins CP. Mutant Kras copy number defines metabolic reprogramming and therapeutic susceptibilities. Nature. 2016;531:110–3.

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Castro-Malaspina H, Schaison G, Passe S, Pasquier A, Berger R, Bayle-Weisgerber C, et al. Subacute and chronic myelomonocytic leukemia in children (juvenile CML). Clinical and hematologic observations, and identification of prognostic factors. Cancer. 1984;54:675–86.

    CAS  PubMed  Google Scholar 

  48. Passmore SJ, Hann IM, Stiller CA, Ramani P, Swansbury GJ, Gibbons B, et al. Pediatric myelodysplasia: a study of 68 children and a new prognostic scoring system. Blood. 1995;85:1742–50.

    CAS  PubMed  Google Scholar 

  49. Passmore SJ, Chessells JM, Kempski H, Hann IM, Brownbill PA, Stiller CA. Paediatric myelodysplastic syndromes and juvenile myelomonocytic leukaemia in the UK: a population-based study of incidence and survival. Br J Haematol. 2003;121:758–67.

    PubMed  Google Scholar 

  50. Chou TC. Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol Rev. 2006;58:621–81.

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We are grateful to Patrick Nyman and Dr. Paul Lambert for providing the Rosa26LSL DNMAML-GFP/+ mice and to Dr. Pamela Stanley for sharing the Pofut1fl/fl mice. We appreciate the critical comments from Drs. Emery Bresnick and Paul Lambert on the manuscript. We would like to thank the University of Wisconsin Carbone Comprehensive Cancer Center (UWCCC) for use of its Shared Services (Flow Cytometry Laboratory and Experimental Pathology Laboratory) to complete this research. This work was supported by the National Natural Science Foundation of China (NO.81600100) to G.K., Alexander von Humboldt Foundation (Alfred Toepfer Faculty Fellow) and NIH-MIRA grant R35GM124806 to X.Z., grants from American Cancer Society LIB-125064 and NIH HL103827 to L.Z., and R01 grants CA152108 and HL113066, and a Scholar Award from the Leukemia & Lymphoma Society to J.Z.. This work was also supported in part by NIH/NCI P30 CA014520--UW Comprehensive Cancer Center Support.

Author contributions

Conception and design: G. Kong, X. You, and J. Zhang. Acquisition of data: G. Kong, X. You, Z. Wen, Y.-I Chang, C. Letson, J. F. Zhang, Y. Zhou, Y. Liu, A. Rajagopalan. Analysis and interpretation of data: G. Kong, J. F. Zhang, X. Zhang, E. A. Ranheim, A. Rajagopalan, E. Padron, W. S. Pear, L. Zhou, and J. Zhang. Writing, review, and/or revision of the manuscript: G. Kong, E. A. Ranheim, E. Stieglitz, M. Loh, W. S. Pear, L. Zhou, and J. Zhang. Technical or material support: S. Qian, I. Hofmann-Zhang, D. Yang, E. Stieglitz, M. Loh, X. Zhong, W. S. Pear and L. Zhou. Study supervision: J. Zhang

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Guangyao Kong or Jing Zhang.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Electronic supplementary material

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Kong, G., You, X., Wen, Z. et al. Downregulating Notch counteracts KrasG12D-induced ERK activation and oxidative phosphorylation in myeloproliferative neoplasm. Leukemia 33, 671–685 (2019). https://doi.org/10.1038/s41375-018-0248-0

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41375-018-0248-0

Search

Quick links