Leflunomide Overcomes CD40L/IL-4-Mediated Resistance to Fludarabine-Induced Apoptosis and Effectively Blocks Proliferation of CLL Cells

Chronic lymphocytic leukemia (CLL) has a poor prognosis if resistance to purine analogues, such as fludarabine, occurs. Therefore strategies to overcome fludarabine resistance in poor-risk CLL are highly warranted. Proliferation and resistance to fludarabine-induced apoptosis of CLL cells can be induced in vitro with CD40L and IL-4. This experimental setting may mimic the anti-apoptotic environment protecting CLL cells in lymph nodes in vivo. CD40 signalling involves NF-kB activation, whereas IL-4 acts through the JAK/STAT pathway. Leflunomide is an oral drug approved for treatment of rheumatoid and psoriasis arthritis. Leflunomide exerts its immunosuppressive effects by directly inhibiting dihydroorotate dehydrogenase (DHODH), an enzyme involved in pyrimidine synthesis, but has also been reported to interfere with NF-kB. Serum concentrations of more than 100 μg/ml of the active metabolite (A771726) can be achieved in patients with a 20 mg tablet per day.

We established a long-term culture system in order to study proliferation and apoptosis resistance of human CLL cells in vitro. This system involves stimulation of CLL cells with a CD40L-transfected baby hamster kidney cell line (BHK) and IL-4 (50 U/ml). Thymidine incorporation assays and intracellular KI-67 FACS analyses were used to measure proliferation. Apoptosis was quantified using AnnexinV/7-AAD FACS analyses. Intracellular nuclear FACS analyses for phospho-p65, phospho-STAT1, phospho-STAT3, survivin and bcl-xL as well as quantitative Western Blot analyses were used to examine the mechanism of apoptosis resistance in CLL cells. Fludarabine (10μg/ml), the pan-JAK inhibitor Pyridone 6 (0.15 μg/ml) and/or Leflunomide (A771726: 0–100 μg/ml) were investigated in the context of a CD40L/IL-4 signal.

Proliferation of CLL cells required a complementary CD40 and JAK/STAT signal and could be blocked by the JAK inhibitor. In contrast, resistance to fludarabine-mediated apoptosis could be induced by CD40 activation alone. This coincided with persistently high levels of intracellular phospho-p65 (NF-kB), survivin and bcl-xL. Apoptosis resistance was further enhanced by a complementary JAK/STAT signal but could not be blocked by the JAK inhibitor. Leflunomide inhibited proliferation at very low concentrations (IC50: 4 μg/ml), but induced apoptosis of CD40L+IL-4-activated (“resistant”) CLL cells only at higher concentrations (IC50: 72 μg/ml). Furthermore leflunomide in combination with fludarabine showed synergistic effects on induction of apoptosis of CLL cells (IC50: 50 μg/ml). The anti-proliferative effect of low Leflunomide concentrations was likely due to inhibition of DHODH, as inhibition of STAT1/3 phosphorylation was observed at higher concentrations only (50-100 μg/ml). Similarly, in concentrations which induced apoptosis of CD40L/IL4-activated CLL cells, leflunomide efficiently inhibited phosphorylation of RelA resulting in low expression levels of the anti-apoptotic proteins bcl-xL and survivin. In contrast, fludarabine alone was inefficient to block bcl-xL and survivin expression in CD40L/IL-4 stimulated CLL cells.

Leflunomide can overcome CD40L/IL4-mediated fludarabine resistance at clinically achievable concentrations by inhibiting phosphorylation of STAT1/3 and NF-kB-induced expression of survivin and bcl-xL. Furthermore leflunomide has a strong, JAK/STAT-independent, anti-proliferative effect on CD40L/IL4-activated CLL cells already at very low concentrations. Therefore leflunomide might be a promising candidate drug to attack CLL cells in chemo-resistant niches.

Luft:Celgene: Research Funding.