Apoptosis Induction in Cutaneous T-Cell Lymphoma Cells after Treatment with Lenalidomide

Introduction: Cutaneous T-cell lymphomas (CTCL) represent a heterogeneous group of extranodal non-Hodgkin lymphomas, derived from skin-homing mature T-cells. The two most common types of CTCL are Mycosis fungoides (MF) (50%-72%), which is generally indolent in behavior, and Sézary syndrome (SS) (1%-3%), an aggressive leukemic form of the disease. Currently, there is no effective treatment for CTCL patients. Research on new therapies for CTCL is largely centered on defining novel therapeutic targets, but in most cases the response is short and the survival rate remains the same. Therefore, the CTCL's resistance to chemotherapy and the lack of full understanding of their pathogenesis request further investigation.

Aims: With the view of a more targeted therapy, we evaluated in vitro the effectiveness of lenalidomide, an immunomodulatory agent (IMID) with clinical efficacy, among others, in plasma cell dysplasias and lymphoproliferative disorders.

Methods: Four CTCL cell lines were used: MJ and MyLa (derived from MF), SeAx and Hut-78 (both derived from peripheral blood of patients with Sézary syndrome). SeAx and Hut-78 cells were cultured in RPMI 1640, supplemented with 10% FBS and 2 mM L-glutamine. MJ and MyLa cells were cultured in RPMI 1640 and IMDM, respectively, supplemented with 20% FBS. All cell lines were maintained at 37°C in a humid atmosphere of 5% CO2. Cells were treated with various concentrations of lenalidomide (1μΜ, 10μΜ and 100μΜ) for 24, 48 and 72h. Apoptosis was determined by flow cytometry using the Annexin V/PI method. The proliferative capacity of untreated and lenalidomide- treated cells was measured using the BrdU assay.

One-way Anova and LSD/ Bonferroni methods were applied for the statistical analysis of the results.

Results: Our data indicate that all cell lines responded with enhanced apoptosis at various lenalidomide treatment conditions. Between the two MF lines tested, MyLa cells were affected the most. Specifically, MyLa cells exhibited a statistically significant augmentation on their apoptosis compared to untreated cells after treatment with 10μΜ and 100μΜ lenalidomide for 24h (9.7 and 8.66 vs 4.83, p=0.000 and p<0,001, respectively) and 48h (6.1 and 4.46 vs 3,36, p=0.000 and p<0,007, respectively), as well as after treatment with 10μΜ Lenalidomide for 72h (5.3 vs 4.7, p=0.000). Similarly, MJ cells also responded with enhanced apoptosis after 24h and 48h of treatment with 100μΜ lenalidomide (8.8 vs 7.43 and 11.13 vs 8.66, p=0.000, respectively) and 72h of treatment with 1 μM lenalidomide (10.63 vs 7.96, p=0.000). Regarding the response of the two SS cell lines to lenalidomide treatment, SeAx cells were significantly affected, exhibiting high apoptotic rates compared to untreated cells after treatment with 1μΜ lenalidomide for 48h (13.76 vs 5.1, p=0.000) and 1μΜ, 10μΜ and 100 μΜ lenalidomide for 72h (1.5, 2.4 and 7.03 vs 0.13, p=0.000, respectively). Lenalidomide had a rather moderate effect on the apoptosis of Hut-78 cells, which presented with statistically significant enhanced apoptosis only after treatment with 1μΜ lenalidomide for 72h (1.33 vs 0.8, p<0,009). The proliferation capacity of the CTCL cell lines tested presented no statistically significant changes under any of the treatment conditions.

Conclusions: Our observations demonstrate that lenalidomide doesn't appear to have any significant effect on the proliferation of the CTCL cell lines. On the contrary, it seems to lead both MF and SS cell lines to significantly enhanced apoptosis. Interestingly, the MF cell lines seem to be more sensitive to lenalidomide treatment in terms of apoptosis induction compared to SS cell lines. Moreover, in all CTCL cell lines tested, high concentrations of lenalidomide contributed to enhanced apoptosis after a shorter period of treatment, compared to lower concentrations, which seemed to be more effective after a longer period, with the exception of SeAx cells. Although these initial results need to be further confirmed both in vitro and in vivo, they appear very encouraging for the integration of lenalidomide treatment, alone or in combination, in CTCL therapy.