title: Leflunomide induces apoptosis in fludarabine-resistant and clinically refractory CLL cells.
abstract: OBJECTIVE
  Environmental conditions in lymph node proliferation centers protect chronic lymphocytic leukemia (CLL) cells from apoptotic triggers. This situation can be mimicked by in vitro stimulation with CD40 ligand (CD40L) and interleukin 4 (IL-4). Our study investigates the impact of the drug leflunomide to overcome apoptosis resistance of CLL cells.
  
  
  METHODS
  CLL cells were stimulated with CD40L and IL-4 and treated with fludarabine and the leflunomide metabolite A771726.
  
  
  RESULTS
  Resistance to fludarabine-mediated apoptosis was induced by CD40 activation alone stimulating high levels of BCL-XL and MCL1 protein expression. Apoptosis resistance was further enhanced by a complementary Janus-activated kinase (JAK)/STAT signal induced by IL-4. In contrast, CLL proliferation required both a CD40 and a JAK/STAT signal and could be completely blocked by pan-JAK inhibition. Leflunomide (A771726) antagonized CD40L/IL-4-induced proliferation at very low concentrations (3 μg/mL) reported to inhibit dihydroorotate dehydrogenase. At a concentration of 10 μg/mL, A771726 additionally attenuated STAT3/6 phosphorylation, whereas apoptosis of CD40L/IL-4-activated ("resistant") CLL cells was achieved with higher concentrations (IC(50): 80 μg/mL). Apoptosis was also effectively induced by A771726 in clinically refractory CLL cells with and without a defective p53 pathway. Induction of apoptosis involved inhibition of NF-κB activity and loss of BCL-XL and MCL1 expression. In combination with fludarabine, A771726 synergistically induced apoptosis (IC(50): 56 μg/mL).
  
  
  CONCLUSIONS
  We thus show that A771726 overcomes CD40L/IL-4-mediated resistance to fludarabine in CLL cells of untreated as well as clinically refractory CLL cells. We present a possible novel therapeutic principle for attacking chemoresistant CLL cells.
authors.affiliation: Department of Medicine V, Im Neuenheimer Feld 410, 69120 Heidelberg, Germany.
authors.forename: Sascha
authors.initials: S
authors.lastname: Dietrich
authors.forename: Oliver H
authors.initials: OH
authors.lastname: Krämer
authors.forename: Esther
authors.initials: E
authors.lastname: Hahn
authors.forename: Claudia
authors.initials: C
authors.lastname: Schäfer
authors.forename: Thomas
authors.initials: T
authors.lastname: Giese
authors.forename: Michael
authors.initials: M
authors.lastname: Hess
authors.forename: Theresa
authors.initials: T
authors.lastname: Tretter
authors.forename: Michael
authors.initials: M
authors.lastname: Rieger
authors.forename: Jennifer
authors.initials: J
authors.lastname: Hüllein
authors.forename: Thorsten
authors.initials: T
authors.lastname: Zenz
authors.forename: Anthony D
authors.initials: AD
authors.lastname: Ho
authors.forename: Peter
authors.initials: P
authors.lastname: Dreger
authors.forename: Thomas
authors.initials: T
authors.lastname: Luft
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D000814:Aniline Compounds / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D023201:CD40 Ligand / Q000502:physiology; D049109:Cell Proliferation / Q000187:drug effects; D003437:Crotonates; D019008:Drug Resistance, Neoplasm; D006801:Humans; D006885:Hydroxybutyrates / Q000494:pharmacology*; D015847:Interleukin-4 / Q000502:physiology; D007555:Isoxazoles / Q000494:pharmacology; D053612:Janus Kinases / Q000378:metabolism; D000077339:Leflunomide; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D008875:Middle Aged; D009570:Nitriles; D050791:STAT Transcription Factors / Q000378:metabolism; D015398:Signal Transduction; D014052:Toluidines; D014407:Tumor Cells, Cultured / Q000187:drug effects; D014740:Vidarabine / Q000031:analogs & derivatives* / Q000494:pharmacology
chemical_list: D000814:Aniline Compounds; D000970:Antineoplastic Agents; D003437:Crotonates; D006885:Hydroxybutyrates; C508598:IL4 protein, human; D007555:Isoxazoles; D009570:Nitriles; D050791:STAT Transcription Factors; D014052:Toluidines; D023201:CD40 Ligand; C527525:teriflunomide; D015847:Interleukin-4; D053612:Janus Kinases; D014740:Vidarabine; D000077339:Leflunomide; C024352:fludarabine