Article | Published:

Chronic lymphocytic leukemia

Bone marrow hematopoietic dysfunction in untreated chronic lymphocytic leukemia patients

Leukemia (2018) | Download Citation

Abstract

The consequences of immune dysfunction in B-chronic lymphocytic leukemia (CLL) likely relate to the incidence of serious recurrent infections and second malignancies that plague CLL patients. The well-described immune abnormalities are not able to consistently explain these complications. Here, we report bone marrow (BM) hematopoietic dysfunction in early and late stage untreated CLL patients. Numbers of CD34+ BM hematopoietic progenitors responsive in standard colony-forming unit (CFU) assays, including CFU-GM/GEMM and CFU-E, were significantly reduced. Flow cytometry revealed corresponding reductions in frequencies of all hematopoietic stem and progenitor cell (HSPC) subsets assessed in CLL patient marrow. Consistent with the reduction in HSPCs, BM resident monocytes and natural killer cells were reduced, a deficiency recapitulated in blood. Finally, we report increases in protein levels of the transcriptional regulators HIF-1α, GATA-1, PU.1, and GATA-2 in CLL patient BM, providing molecular insight into the basis of HSPC dysfunction. Importantly, PU.1 and GATA-2 were rapidly increased when healthy HSPCs were exposed in vitro to TNFα, a cytokine constitutively produced by CLL B cells. Together, these findings reveal BM hematopoietic dysfunction in untreated CLL patients that provides new insight into the etiology of the complex immunodeficiency state in CLL.

Key points

Cell-intrinsic defects in BM hematopoietic stem and progenitor cells (HSPCs) in untreated CLL patients.

Altered levels of specific nuclear factors regulating HSPC differentiation and function in untreated CLL patients.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    Forconi F, Moss P. Perturbation of the normal immune system in patients with CLL. Blood. 2015;126:573–81.

  2. 2.

    Sala R, Mauro FR, Bellucci R, De Propris MS, Cordone I, Lisci A, et al. Evaluation of marrow and blood haemopoietic progenitors in chronic lymphocytic leukaemia before and after chemotherapy. Eur J Haematol. 1998;61:14–20.

  3. 3.

    Tsopra OA, Ziros PG, Lagadinou ED, Symeonidis A, Kouraklis-Symeonidis A, Thanopoulou E, et al. Disease-related anemia in chronic lymphocytic leukemia is not due to intrinsic defects of erythroid precursors: a possible pathogenetic role for tumor necrosis factor alpha. Acta Haematol. 2009;121:187–95.

  4. 4.

    Eliasson P, Rehn M, Hammar P, Larsson P, Sirenko O, Flippin LA. et al. Hypoxia mediates low cell-cycle activity and increases the proportion of long-term reconstituting hematopoietic stem cells during in vitro culture. Exp Hematol. 2010;38:301–310.e302.

  5. 5.

    Takubo K, Goda N, Yamada W, Iriuchishima H, Ikeda E, Kubota Y, et al. Regulation of the HIF-1alpha level is essential for hematopoietic stem cells. Cell Stem Cell. 2010;7:391–402.

  6. 6.

    Zhang F-L, Shen G-M, Liu X-L, Wang F, Zhao Y-Z, Zhang J-W. Hypoxia-inducible factor 1–mediated human GATA1 induction promotes erythroid differentiation under hypoxic conditions. J Cell Mol Med. 2012;16:1889–99.

  7. 7.

    Yoon D, Ponka P, Prchal JT. Hypoxia. 5. Hypoxia and hematopoiesis. Am J Physiol Cell Physiol. 2011;300:C1215.

  8. 8.

    Schito L, Semenza GL. Hypoxia-inducible factors: master regulators of cancer progression. Trends Cancer. 2016;2:758–70.

  9. 9.

    Ghosh AK, Secreto CR, Knox TR, Ding W, Mukhopadhyay D, Kay NE. Circulating microvesicles in B-cell chronic lymphocytic leukemia can stimulate marrow stromal cells: implications for disease progression. Blood. 2010;115:1755–64.

  10. 10.

    Koczula KM, Ludwig C, Hayden R, Cronin L, Pratt G, Parry H. et al. Metabolic plasticity in CLL: adaptation to the hypoxic niche. Leukemia. 2016;30:65–73.

  11. 11.

    Valsecchi R, Coltella N, Belloni D, Ponente M, ten Hacken E, Scielzo C, et al. HIF-1α regulates the interaction of chronic lymphocytic leukemia cells with the tumor microenvironment. Blood. 2016;127:1987–97.

  12. 12.

    Whyatt D, Karis A, Harkes I, Verkerk A, Gillemans N, Elefanty A, et al. The level of the tissue-specific factor GATA-1 affects the cell-cycle machinery. Genes Funct. 1997;1:11–24.

  13. 13.

    Whyatt D, Lindeboom F, Karis A, Ferreira R, Milot E, Hendriks R, et al. An intrinsic but cell-nonautonomous defect in GATA-1-overexpressing mouse erythroid cells. Nature. 2000;406:519–24.

  14. 14.

    Foa R, Massaia M, Cardona S, Tos AG, Bianchi A, Attisano C, et al. Production of tumor necrosis factor alpha by B-cell chronic lymphocytic leukemia cells: a possible regulatory role of TNF in the progression of the disease. Blood. 1990;76:393–400.

  15. 15.

    Michalevicz R, Porat R, Vechoropoulos M, Baron S, Yanoov M, Cycowitz Z, et al. Restoration of in vitro hematopoiesis in B-chronic lymphocytic leukemia by antibodies to tumor necrosis factor. Leuk Res. 1991;15:111–20.

  16. 16.

    Perfetto SP, Ambrozak D, Nguyen R, Chattopadhyay P, Roederer M. Quality assurance for polychromatic flow cytometry. Nat Protoc. 2006;1:1522–30.

  17. 17.

    Perfetto SP, Ambrozak D, Nguyen R, Chattopadhyay PK, Roederer M. Quality assurance for polychromatic flow cytometry using a suite of calibration beads. Nat Protoc. 2012;7:2067–79.

  18. 18.

    Grigorakaki C, Morceau F, Chateauvieux S, Dicato M, Diederich M. Tumor necrosis factor alpha-mediated inhibition of erythropoiesis involves GATA-1/GATA-2 balance impairment and PU.1 overexpression. Biochem Pharmacol. 2011;82:156–66.

  19. 19.

    Xiao W, Koizumi K, Nishio M, Endo T, Osawa M, Fujimoto K, et al. Tumor necrosis factor alpha inhibits generation of glycophorin A+ cells by CD34+ cells. Exp Hematol. 2002;30:1238–47.

  20. 20.

    Cvejic A. Mechanisms of fate decision and lineage commitment during haematopoiesis. Immunol Cell Biol. 2016;94:230–5.

  21. 21.

    Lunger I, Fawaz M, Rieger MA. Single-cell analyses to reveal hematopoietic stem-cell fate decisions. FEBS Lett. 2017;591:2195–212.

  22. 22.

    Nakajima H. Role of transcription factors in differentiation and reprogramming of hematopoietic cells. Keio J Med. 2011;60:47–55.

  23. 23.

    Nombela-Arrieta C, Pivarnik G, Winkel B, Canty KJ, Harley B, Mahoney JE, et al. Quantitative imaging of haematopoietic stem and progenitor cell localization and hypoxic status in the bone marrow microenvironment. Nat Cell Biol. 2013;15:533–43.

  24. 24.

    Arinobu Y, Mizuno S-i, Chong Y, Shigematsu H, Iino T, Iwasaki H. et al. Reciprocal activation of GATA-1 and PU.1 marks initial specification of hematopoietic stem cells into myeloerythroid and myelolymphoid lineages. Cell Stem Cell. 2007;1:416–27.

  25. 25.

    Fukuchi Y, Ito M, Shibata F, Kitamura T, Nakajima H. Activation of CCAAT/enhancer-binding protein α or PU.1 in hematopoietic stem cells leads to their reduced self-renewal and proliferation. Stem Cells. 2008;26:3172–81.

  26. 26.

    Burda P, Laslo P, Stopka T. The role of PU.1 and GATA-1 transcription factors during normal and leukemogenic hematopoiesis. Leukemia. 2010;24:1249–57.

  27. 27.

    van Lochem EG, van der Velden VHJ, Wind HK, te Marvelde JG, Westerdaal NAC, van Dongen JJM. Immunophenotypic differentiation patterns of normal hematopoiesis in human bone marrow: reference patterns for age-related changes and disease-induced shifts. Cytom Part B Clin Cytom. 2004;60B:1–13.

  28. 28.

    Walsh JC, DeKoter RP, Lee H-J, Smith ED, Lancki DW, Gurish MF, et al. Cooperative and Antagonistic Interplay between PU.1 and GATA-2 in the specification of myeloid cell fates. Immunity. 2002;17:665–76. 2002/11/01/

  29. 29.

    Kikushige Y, Ishikawa F, Miyamoto T, Shima T, Urata S, Yoshimoto G, et al. Self-renewing hematopoietic stem cell is the primary target in pathogenesis of human chronic lymphocytic leukemia. Cancer Cell. 2011;20:246–59.

  30. 30.

    Bresnick EH, Katsumura KR, Lee HY, Johnson KD, Perkins AS. Master regulatory GATA transcription factors: mechanistic principles and emerging links to hematologic malignancies. Nucleic Acids Res. 2012;40:5819–31.

  31. 31.

    Moriguchi T, Yamamoto M. A regulatory network governing Gata1 and Gata2 gene transcription orchestrates erythroid-lineage differentiation. Int J Hematol. 2014;100:417–24.

  32. 32.

    Mizrahi K, Askenasy N. Physiological functions of TNF family receptor/ligand interactions in hematopoiesis and transplantation. Blood. 2014;124:176.

  33. 33.

    Rusten LS, Jacobsen SE. Tumor necrosis factor (TNF)-alpha directly inhibits human erythropoiesis in vitro: role of p55 and p75 TNF receptors. Blood. 1995;85:989.

  34. 34.

    Bojarska-Junak A, Hus I, Szczepanek EW, Dmoszyńska A, Roliński J. Peripheral blood and bone marrow TNF and TNF receptors in early and advanced stages of B-CLL in correlation with ZAP-70 protein and CD38 antigen. Leuk Res. 2008;32:225–33.

  35. 35.

    Vicente C, Conchillo A, Garcia-Sanchez MA, Odero MD. The role of the GATA2 transcription factor in normal and malignant hematopoiesis. Crit Rev Oncol Hematol. 2012;82:1–17.

  36. 36.

    Tipping AJ, Pina C, Castor A, Hong D, Rodrigues NP, Lazzari L, et al. High GATA-2 expression inhibits human hematopoietic stem and progenitor cell function by effects on cell cycle. Blood. 2009;113:2661–72.

  37. 37.

    Ferrajoli A, Keating MJ, Manshouri T, Giles FJ, Dey A, Estrov Z, et al. The clinical significance of tumor necrosis factor alpha plasma level in patients having chronic lymphocytic leukemia. Blood. 2002;100:1215–9.

  38. 38.

    Hartmann EM, Rudelius M, Burger JA, Rosenwald A. CCL3 chemokine expression by chronic lymphocytic leukemia cells orchestrates the composition of the microenvironment in lymph node infiltrates. Leuk Lymphoma. 2016;57:563–71.

  39. 39.

    Sivina M, Hartmann E, Kipps TJ, Rassenti L, Krupnik D, Lerner S, et al. CCL3 (MIP-1alpha) plasma levels and the risk for disease progression in chronic lymphocytic leukemia. Blood. 2011;117:1662–9.

  40. 40.

    Wang Y, Gao A, Zhao H, Lu P, Cheng H, Dong F, et al. Leukemia cell infiltration causes defective erythropoiesis partially through MIP-1[alpha]/CCL3. Leukemia. 2016 ;30:1897–908. 09//print

  41. 41.

    Lotz M, Ranheim E, Kipps TJ. Transforming growth factor beta as endogenous growth inhibitor of chronic lymphocytic leukemia B cells. J Exp Med. 1994;179:999.

  42. 42.

    Blank U, Karlsson S. TGF-beta signaling in the control of hematopoietic stem cells. Blood. 2015;125:3542–50.

  43. 43.

    Fan X, Valdimarsdottir G, Larsson J, Brun A, Magnusson M, Jacobsen SE, et al. Transient disruption of autocrine TGF-beta signaling leads to enhanced survival and proliferation potential in single primitive human hemopoietic progenitor cells. J Immunol. 2002;168:755–62.

  44. 44.

    Gerber HP, Malik AK, Solar GP, Sherman D, Liang XH, Meng G, et al. VEGF regulates haematopoietic stem-cell survival by an internal autocrine loop mechanism. Nature. 2002;417:954–8.

  45. 45.

    Xue Y, Chen F, Zhang D, Lim S, Cao Y. Tumor-derived VEGF modulates hematopoiesis. J Angiogenes Res. 2009;1:9.

  46. 46.

    Fortunel NO, Hatzfeld A, Hatzfeld JA. Transforming growth factor beta: pleiotropic role in the regulation of hematopoiesis. Blood. 2000;96:2022–36.

  47. 47.

    Zermati Y, Fichelson S, Valensi F, Freyssinier JM, Rouyer-Fessard P, Cramer E, et al. Transforming growth factor inhibits erythropoiesis by blocking proliferation and accelerating differentiation of erythroid progenitors. Exp Hematol. 2000;28:885–94.

  48. 48.

    Zermati Y, Varet B, Hermine O. TGF-β1 drives and accelerates erythroid differentiation in the Epo-dependent UT-7 cell line even in the absence of erythropoietin. Exp Hematol. 2000;28:256–66.

  49. 49.

    Dong XM, Yin RH, Yang Y, Feng ZW, Ning HM, Dong L, et al. GATA-2 inhibits transforming growth factor-beta signaling pathway through interaction with Smad4. Cell Signal. 2014;26:1089–97.

  50. 50.

    Paggetti J, Haderk F, Seiffert M, Janji B, Distler U, Ammerlaan W, et al. Exosomes released by chronic lymphocytic leukemia cells induce the transition of stromal cells into cancer-associated fibroblasts. Blood. 2015;126:1106–17.

  51. 51.

    Hoshino A, Costa-Silva B, Shen TL, Rodrigues G, Hashimoto A, Tesic Mark M, et al. Tumour exosome integrins determine organotropic metastasis. Nature. 2015;527:329–35.

  52. 52.

    Lin LY, Du LM, Cao K, Huang Y, Yu PF, Zhang LY, et al. Tumour cell-derived exosomes endow mesenchymal stromal cells with tumour-promotion capabilities. Oncogene. 2016;35:6038–42.

  53. 53.

    Kumar B, Garcia M, Weng L, Jung X, Murakami JL, Hu X, et al. Acute myeloid leukemia transforms the bone marrow niche into a leukemia-permissive microenvironment through exosome secretion. Leukemia. 2018;32:575–87.

  54. 54.

    Pando A, Reagan JL, Quesenberry P, Fast LD. Extracellular vesicles in leukemia. Leuk Res. 2018;64:52–60.

  55. 55.

    Quesenberry PJ, Goldberg L, Aliotta J, Dooner M. Marrow hematopoietic stem cells revisited: they exist in a continuum and are not defined by standard purification approaches; then there are the microvesicles. Front Oncol. 2014;4:56.

  56. 56.

    Asada N, Takeishi S, Frenette PS. Complexity of bone marrow hematopoietic stem-cell niche. Int J Hematol. 2017;106:45–54.

  57. 57.

    Janel A, Dubois-Galopin F, Bourgne C, Berger J, Tarte K, Boiret-Dupre N, et al. The chronic lymphocytic leukemia clone disrupts the bone marrow microenvironment. Stem Cells Dev. 2014;23:2972–82.

  58. 58.

    Boissard F, Fournie JJ, Quillet-Mary A, Ysebaert L, Poupot M. Nurse-like cells mediate ibrutinib resistance in chronic lymphocytic leukemia patients. Blood Cancer J. 2015;5:e355.

  59. 59.

    Lagneaux L, Delforge A, Dorval C, Bron D, Stryckmans P. Excessive production of transforming growth factor beta by bone marrow stromal cells in B-cell chronic lymphocytic leukemia inhibits growth of hematopoietic precursors and interleukin-6 production. Blood. 1993;82:2379.

  60. 60.

    Damm F, Mylonas E, Cosson A, Yoshida K, Della Valle V, Mouly E, et al. Acquired initiating mutations in early hematopoietic cells of CLL patients. Cancer Discov. 2014;4:1088.

  61. 61.

    Marsilio S, Khiabanian H, Fabbri G, Vergani S, Scuoppo C, Montserrat E, et al. Somatic CLL mutations occur at multiple distinct hematopoietic maturation stages: documentation and cautionary note regarding cell fraction purity. Leukemia. 2017;32:1040.

Download references

Acknowledgments

This work was supported by funding provided by the Mayo Clinic Center for Biomedical Discovery to K.L.M., W.D., and N.E.K. B.A.M. is supported by a NIH T32 Training Grant in Basic Immunology (NIH AI07425).

Author contributions

B.A.M., H.Z., N.E.K., and K.L.M. designed experiments; B.A.M., H.Z., M.M., K.A.G., and C.R.S. performed and analyzed experiments; B.A.M., N.E.K., and K.L.M. wrote the manuscript.

Author information

Affiliations

  1. Department of Immunology, Mayo Clinic, Rochester, MN, 55905, USA

    • Molly G. Mikkelson
    • , Kimberly A. Gwin
    •  & Kay L. Medina
  2. Division of Hematology, Mayo Clinic, Rochester, MN, 55905, USA

    • Henan Zhang
    • , Charla R. Secreto
    • , Wei Ding
    • , Sameer A. Parikh
    •  & Neil E. Kay
  3. Mayo Clinic Graduate School of Biomedical Sciences, Mayo Clinic, Rochester, MN, 55905, USA

    • Bryce A. Manso

Authors

  1. Search for Bryce A. Manso in:

  2. Search for Henan Zhang in:

  3. Search for Molly G. Mikkelson in:

  4. Search for Kimberly A. Gwin in:

  5. Search for Charla R. Secreto in:

  6. Search for Wei Ding in:

  7. Search for Sameer A. Parikh in:

  8. Search for Neil E. Kay in:

  9. Search for Kay L. Medina in:

Conflict of interest

Research funding has been provided to the institution from Pharmacyclics, Morphosys, and AbbVie for clinical studies in which Dr. Sameer Parikh is a principal investigator. Dr. Sameer Parikh has also participated in Advisory Board meetings of Pharmacyclics, AstraZeneca, and AbbVie (he was not personally compensated for his participation).

Corresponding author

Correspondence to Kay L. Medina.

Electronic supplementary material

About this article

Publication history

Received

Revised

Accepted

Published

DOI

https://doi.org/10.1038/s41375-018-0280-0