Murine Retrovirally‐Transduced Bone Marrow Engraftment Models of MLL‐Fusion‐Driven Acute Myelogenous Leukemias (AML)

Matthew C. Stubbs1, Andrei V. Krivtsov2

1 Incyte Corporation, Wilmington, Delaware, 2 Department of Pediatric Oncology, Dana‐Farber Cancer Institute, Boston, Massachusetts
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 14.42
DOI:  10.1002/cpph.28
Online Posting Date:  September, 2017
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

MLL‐rearranged leukemia represents approximately 5% to 10% of adult acute myelogenous leukemia (AML) and nearly half of all infant/pediatric acute leukemia cases. These leukemias have a poor prognosis, and there are no approved therapeutic options. The rearrangement in the MLL gene leads to aberrant expression of MLL‐fusion proteins. These are transforming in murine bone marrow and, in particular, on stem cells and myeloid progenitors derived from bone marrow or fetal liver. The commonality of the MLL fusions is the in‐frame fusion of 8 to 11 N‐terminal exons of MLL1 (KMT2a) with the C‐terminus of a partner fusion gene. Currently, over 80 different fusion partners are known. The protocols detailed in this unit focus on bone marrow–derived models only, using one particular MLL fusion, MLL‐AF9. These models have proven effective for drug screening to predict clinical response. © 2017 by John Wiley & Sons, Inc.

Keywords: murine leukemia model; AML; bone marrow transplant; retroviral transduction techniques; cancer stem cells; myeloid progenitor cells; leukemia therapeutics

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Leukemic Transformation of Hematopoietic Stem and Progenitor Cells From Whole Bone Marrow
  • Basic Protocol 2: Implantation of MLL‐AF‐9 Expressing Mouse Cells Into Recipient Mice
  • Basic Protocol 3: In Vivo Analysis of the MLL‐Fusion‐Driven Mouse Leukemia Model for Disease Progression and Pharmacological Studies
  • Basic Protocol 4: In Vivo Analysis of Therapeutic Agents in Mice Bearing MLL‐Rearranged AML
  • Basic Protocol 5: Final Readout: Leukemic Burden
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Leukemic Transformation of Hematopoietic Stem and Progenitor Cells From Whole Bone Marrow

  Materials
  • HEK 293T cells that have not grown to confluence (ATCC CRL‐3216)
  • IMDM medium (Gibco/ThermoFisher Scientific)
  • Fetal bovine serum (FBS; Gibco/ThermoFisher Scientific)
  • OptiMEM medium (Gibco/ThermoFisher Scientific)
  • FuGENE 9 (Promega)
  • Maxiprep‐purified Ψ‐Eco (Phoenix‐ECO) plasmid (10 to 100 µg) containing gag, pol, and packaging envelope; available from Gary Nolan, Stanford University; https://web.stanford.edu/group/nolan/_OldWebsite/MTAs/mtas.html)
  • Maxiprep purified pMIG‐MLL‐AF9 plasmid (10 to 100 µg; AddGene, cat. no. 71443)
  • Murine IL‐3, murine IL‐6, murine SCF (Peprotech)
  • Polybrene (Sigma/Millipore)
  • 8 mg/ml 5‐fluorouracil (5‐FU; e.g., Sigma‐Aldrich) in DPBS (without Ca or Mg)
  • Dulbecco's phosphate‐buffered saline (DPBS) without Ca or Mg (PBS; Corning/Cellgro, cat. no. 21‐031‐CV)
  • C57BL/6 mice, female, 6‐8 weeks old (Charles River, Jackson Labs, Taconic)
  • 5‐fluorouracil (Sigma, cat. no. F6627)
  • 70% ethanol (ThermoFisher Scientific)
  • RPMI cell culture medium containing GlutaMAX (Gibco/ThermoFisher Scientific)
  • Red blood cell lysis buffer (Qiagen)
  • Cytokines: mIL‐3 (Peprotech, cat. no. 213‐13), mIL‐6 (Peprotech, cat. no. 216‐16), and mSCF (Peprotech, cat. no. 250‐03)
  • Methylcellulose with cytokines (Stem Cell Technologies, cat. no. M3234)
  • 10‐cm tissue culture treated plates (Corning)
  • 15‐ and 50‐ml conical tubes (e.g., Corning Falcon)
  • Fluorescence microscope
  • 1‐, 3‐, 5‐, and 10‐ml syringes (BD Biosciences)
  • 0.22‐μm filter units with luer lock (ThermoFisher Scientific)
  • Cryovials (Corning)
  • Flat‐bottom tissue culture treated 96‐well plates (Corning Costar)
  • 5‐ml FACS tubes
  • Refrigerated centrifuge
  • Surgical scissors and forceps
  • 27‐ and 26½‐G needles
  • 70‐µm cell strainers for 50‐ml conical tube (Corning Falcon, cat. no. 352350)
  • 6‐well culture dishes (Corning)
  • 2‐ml microcentrifuge tubes
  • 16‐G blunt‐end needles (Stem Cell Technologies)
  • Inverted light microscope with a 4× objective
  • 6‐well cell culture dishes (Corning)
  • 35‐mm plates (Falcon)
  • Additional reagents and equipment for cell culture including cell counting (Phelan & May, ), fluorescence activated cell sorting (FACS; Robinson et al., ), injection of mice (Donovan & Brown, ), and euthanasia of mice (Donovan & Brown, )

Basic Protocol 2: Implantation of MLL‐AF‐9 Expressing Mouse Cells Into Recipient Mice

  Materials
  • C57BL/6 mice, female, 6‐ to 8‐weeks‐old (Charles River, Jackson Labs, Taconic)
  • Ethanol wipes
  • Working vivarium (with approved protocols in place for the work to be performed)
  • Irradiator capable of holding rodents
  • Irradiator safe housing, such as a mouse pie cage (Braintree Scientific)
  • Heat lamp (Braintree Scientific)
  • 1‐ml syringes (BD Biosciences)
  • 27‐G needles (BD Biosciences)
  • Additional reagents and equipment for injection of mice (Donovan & Brown. )

Basic Protocol 3: In Vivo Analysis of the MLL‐Fusion‐Driven Mouse Leukemia Model for Disease Progression and Pharmacological Studies

  Materials
  • Engrafted mice ( protocol 2)
  • Red blood cell lysis buffer (Qiagen)
  • Dulbecco's phosphate‐buffered saline (DPBS) without Ca or Mg (Corning/Cellgro, cat. no. 21‐031‐CV)
  • Fetal bovine serum (FBS; Gibco/ThermoFisher Scientific)
  • Fluorescein‐tagged CD45.1 and CD45.2 antibodies (Affymetrix/eBiosciences/ThermoFisher Scientific)
  • Luciferin (in vivo grade; Promega)
  • Working vivarium (with approved protocols in place for the work)
  • Capillary tubes (ThermoFisher Scientific)
  • Vacutainer tubes for blood collection containing heparin or EDTA (BD Biosciences)
  • 15‐ml conical tubes (Corning)
  • Gauze pads
  • 5‐ml FACS tubes
  • Centrifuge
  • Flow cytometer with appropriate fluorescence excitation/detection capabilities (Robinson et al., )
  • 1‐ml syringes (BD Biosciences)
  • 27‐G needles (BD Biosciences)
  • In vivo imager (e.g., Xenogen IVIS; Perkin Elmer IVIS Lumina or Spectrum) and accompanying software
  • Additional reagents and equipment for anesthesia of mice (Donovan & Brown, ), flow cytometry (Robinson et al., ), and injection of mice (Donovan & Brown, )

Basic Protocol 4: In Vivo Analysis of Therapeutic Agents in Mice Bearing MLL‐Rearranged AML

  Materials
  • Treated mice (see protocols above)
  • Test compounds (examples of standards of care for leukemia include 5‐azacytidine, cytarabine, or decitabine; one example of a directed agent is the DOT1L inhibitor EPZ‐5676 for MLL‐rearranged leukemias; Stein & Tallman, )
  • Needles for intraperitoneal injection (27‐G is suitable; BD Biosciences)
  • Needles for oral gavage (Braintree Scientific)
  • 1‐ml syringes (BD Biosciences)
  • 50‐ml conical tubes (Falcon)
  • Additional reagents and equipment for euthanasia of mice (Donovan & Brown, ) and mouse necropsy (Treuting & Snyder, )

Basic Protocol 5: Final Readout: Leukemic Burden

  Materials
  • Spleen obtained by mouse necropsy (Treuting & Snyder, )
  • Red blood cell lysis buffer (Qiagen)
  • RPMI cell culture medium (Gibco/ThermoFisher Scientific)
  • Fetal bovine serum (FBS; Gibco/ThermoFisher Scientific)
  • Dulbecco's phosphate‐buffered saline (DPBS) (Corning/Cellgro, cat. no. 21‐031‐CV)
  • 50‐ml conical tubes (Falcon)
  • 10‐cm culture dish (Falcon)
  • 10‐ml syringes (BD Biosciences)
  • 18‐G needles (BD Biosciences)
  • Cell strainers (BD Biosciences)
  • Centrifuge (ThermoFisher Scientific)
  • Cytospin (ThermoFisher Scientific)
  • Glass slides (ThermoFisher Scientific)
  • Wright‐Giemsa stain (Jorgensen Labs, cat. no. J‐322)
NOTE: This procedure may be performed on a lab bench or in a tissue culture hood, depending on the level of sterility needed.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
  Balgobind, B. V., Raimondi, S. C., Harbott, J., Zimmermann, M., Alonzo, T. A., Auvrignon, A., … van den Heuvel‐Eibrink, M. M. (2009). Novel prognostic subgroups in childhood 11q23/MLL‐rearranged acute myeloid leukemia: Results of an international retrospective study. Blood, 114(12), 2489–2496. doi: 10.1182/blood‐2009‐04‐215152.
  Berson, A. E., Knobel, K. M., Rood, D., Chen, K., Lamons, D., McNally, M. A., … Lebkowski, J. S. (1996). Selection of murine lymphoid and hematopoietic cells using polystyrene tissue culture devices containing covalently immobilized antibody. Biotechniques, 20(6), 1098–1103.
  Cozzio, A., Passegue, E., Ayton, P. M., Karsunky, H., Cleary, M. L., & Weissman, I. L. (2003). Similar MLL‐associated leukemias arising from self‐renewing stem cells and short‐lived myeloid progenitors. Genes & Development, 17(24), 3029–3035. doi: 10.1101/gad.1143403.
  Deshpande, A. J., Chen, L., Fazio, M., Sinha, A. U., Bernt, K. M., Banka, D., … Armstrong, S. A. (2013). Leukemic transformation by the MLL‐AF6 fusion oncogene requires the H3K79 methyltransferase Dot1l. Blood, 121(13), 2533–2541. doi: 10.1182/blood‐2012‐11‐465120.
  DiMartino, J. F., Ayton, P. M., Chen, E. H., Naftzger, C. C., Young, B. D., & Cleary, M. L. (2002). The AF10 leucine zipper is required for leukemic transformation of myeloid progenitors by MLL‐AF10. Blood, 99(10), 3780–3785. doi: 10.1182/blood.V99.10.3780.
  DiMartino, J. F., Miller, T., Ayton, P. M., Landewe, T., Hess, J. L., Cleary, M. L., & Shilatifard, A. (2000). A carboxy‐terminal domain of ELL is required and sufficient for immortalization of myeloid progenitors by MLL‐ELL. Blood, 96(12), 3887–3893.
  Donovan, J. and Brown, P. (1998). Anesthesia. Current Protocols in Immunology, 27, 1.4.1–1.4.5. doi: 10.1002/0471142735.im0104s27.
  Donovan, J., & Brown, P. (2006a). Euthanasia. Current Protocols in Immunology, 73, 1.8.1–1.8.4. doi: 10.1002/0471142735.im0108s73.
  Donovan, J., & Brown, P. (2006b). Parenteral injections. Current Protocols in Immunology, 73, 1.6.1–1.6.10. doi: 10.1002/0471142735.im0106s73.
  Duran‐Struuck, R., & Dysko, R. C. (2009). Principles of bone marrow transplantation (BMT): Providing optimal veterinary and husbandry care to irradiated mice in BMT studies. Journal of the American Association for Laboratory Animal Science, 48(1), 11–22.
  Krivtsov, A. V., Figueroa, M. E., Sinha, A. U., Stubbs, M. C., Feng, Z., Valk, P. J., … Armstrong, S. A. (2013). Cell of origin determines clinically relevant subtypes of MLL‐rearranged AML. Leukemia, 27(4), 852–860. doi: 10.1038/leu.2012.363.
  Krivtsov, A. V., Twomey, D., Feng, Z., Stubbs, M. C., Wang, Y., Faber, J., … Armstrong, S. A. (2006). Transformation from committed progenitor to leukaemia stem cell initiated by MLL‐AF9. Nature, 442(7104), 818–822. doi: 10.1038/nature04980.
  Lavau, C., Szilvassy, S. J., Slany, R., & Cleary, M. L. (1997). Immortalization and leukemic transformation of a myelomonocytic precursor by retrovirally transduced HRX‐ENL. EMBO Journal, 16(14), 4226–4237. doi: 10.1093/emboj/16.14.4226.
  Martin, M. E., Milne, T. A., Bloyer, S., Galoian, K., Shen, W., Gibbs, D., … Hess, J. L. (2003). Dimerization of MLL fusion proteins immortalizes hematopoietic cells. Cancer Cell, 4(3), 197–207. doi: 10.1016/S1535‐6108(03)00214‐9.
  Phelan, K., & May, K. M. (2015). Basic techniques in mammalian cell tissue culture. Current Protocols in Cell Biology, 66, 1.1.1–1.1.22. doi: 10.1002/0471143030.cb0101s66.
  Robinson, J. P., Darzynkiewicz, Z., Dean, P. N., Orfao, A., Rabinovitch, P. S., Stewart, C., … Wheeless, L. L. (Eds.). (2017). Current Protocols in Cytometry, Hoboken, NJ: John Wiley & Sons.
  Schott, J. W., Hoffmann, D., & Schambach, A. (2015). Retrovirus‐based vectors for transient and permanent cell modification. Current Opinion in Pharmacology, 24, 135–146. doi: 10.1016/j.coph.2015.09.004.
  Somervaille, T. C., & Cleary, M. L. (2006). Identification and characterization of leukemia stem cells in murine MLL‐AF9 acute myeloid leukemia. Cancer Cell, 10(4), 257–268. doi: 10.1016/j.ccr.2006.08.020.
  Stein, E. M., & Tallman, M. S. (2016). Emerging therapeutic drugs for AML. Blood, 127(1), 71–78. doi: 10.1182/blood‐2015‐07‐604538.
  Stubbs, M. C., Kim, Y. M., Krivtsov, A. V., Wright, R. D., Feng, Z., Agarwal, J., … Armstrong, S. A. (2008). MLL‐AF9 and FLT3 cooperation in acute myelogenous leukemia: Development of a model for rapid therapeutic assessment. Leukemia, 22(1), 66–77. doi: 10.1038/sj.leu.2404951.
  Swift, S., Lorens, J., Achacoso, P., & Nolan, G. P. (2001). Rapid production of retroviruses for efficient gene delivery to mammalian cells using 293T cell‐based systems. Current Protocols in Immunology, 31, 10.17.14–10.17.29. doi: 10.1002/0471142735.im1017cs31.
  Szilvassy, S. J., Weller, K. P., Chen, B., Juttner, C. A., Tsukamoto, A., & Hoffman, R. (1996). Partially differentiated ex vivo expanded cells accelerate hematologic recovery in myeloablated mice transplanted with highly enriched long‐term repopulating stem cells. Blood, 88(9), 3642–3653.
  Treuting, P. M., and Snyder, J. M. (2015). Mouse necropsy. Current Protocols in Mouse Biology, 5, 223‐233. doi: 10.1002/9780470942390.mo140296.
  Zuber, J., Radtke, I., Pardee, T. S., Zhao, Z., Rappaport, A. R., Luo, W., … Lowe, S. W. (2009). Mouse models of human AML accurately predict chemotherapy response. Genes & Development, 23(7), 877–889. doi: 10.1101/gad.1771409.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library