Immunoregulatory effects of Lurbinectedin in chronic lymphocytic leukemia

Abstract

Despite significant therapeutic improvements chronic lymphocytic leukemia (CLL) remains an incurable disease and there is a persistent pursuit of new treatment alternatives. Lurbinectedin, a selective inhibitor of active transcription of protein-coding genes, is currently in phase II/III clinical trials for solid tumors such as small-cell lung cancer (SCLC). In this study, we aimed to evaluate the activity of Lurbinectedin on circulating mononuclear cells from CLL patients and to determine whether Lurbinectedin could affect the cross-talk between B-CLL cells and the tumor microenvironment. We found that Lurbinectedin induced a dose- and time-dependent death in all cell types evaluated, with B cells, monocytes and monocytic myeloid derived suppressor cells (Mo-MDSC) being the most susceptible populations. At sub-apoptotic doses, Lurbinectedin decreased the expression of CCR7 in B-CLL cells and impaired their migration towards CCL19 and CCL21. Furthermore, low concentrations of Lurbinectedin stimulated the synthesis of pro-IL1β in monocytes and nurse-like cells, without inducing the inflammasome activation. Altogether, these results indicate that Lurbinectedin might have antitumor activity in CLL due to its direct action on leukemic cells in combination with its effects on the tumor microenvironment. Our findings encourage further investigation of Lurbinectedin as a potential therapy for CLL.

Keywords: CCR7; Chronic lymphocytic leukemia (CLL); IL1β; Lurbinectedin; Tumor microenvironment.

Fig. 1

B cells are sensitive to Lurbinectedin. a CLL PBMC were incubated with Lurbinectedin 1, 3, 10, 30 nM or vehicle for 24, 48 and 72 h. Cells were stained with anti-CD19-PECy5 mAb and Annexin V-FITC. Shown are the individual values and the mean ± SEM from 10 samples. b The experiment on (a) was repeated using HD PBMC. Shown are the individual values and the mean ± SEM from 13 samples after 24 h of treatment. Statistical analysis was performed using Friedman test followed by the Dunn’s multiple comparison post-test. c, d B-CLL cells were treated with Lurbinectedin 10 nM for 24 h. c Then, γ-H2AX and PARP fragmentation were evaluated by western blot. d Bands on the immunoblots were quantified. Results are shown as the mean ± SEM of the ratio γ-H2AX/tubulin and PARP/tubulin. Statistical analysis was performed using Wilcoxon’s matched-pairs signed-rank test. Lur Lurbinectedin; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig. 2

Effects of Lurbinectedin on tumor-microenvironment cells. a Monocytes were treated with different concentrations of Lurbinectedin for 24 h. Cell death was determined by Annexin V-FITC staining and flow cytometry analysis. The individual values and the mean ± SEM are shown, n = 6. b The sensitivity of Mo-MDSC to Lurbinectedin was determined by incubating PBMC with or without Lurbinectedin 1 nM for 24 h. Cells were stained with anti-CD3 PECy5, anti-CD19 PECy5, anti-CD56 PeCy5, anti-CD14-PE and anti-HLA-DR-FITC antibodies. Mo-MDSC population was selected as Lin⁻ (CD3⁻, CD19⁻, CD56⁻), CD14⁺ and HLA-DR^low. c NLC were incubated with vehicle, Lurbinectedin 10 or 30 nM for 24 h. Then, cells were labeled with Annexin V-FITC and analyzed by flow cytometry. The individual values and the mean ± SEM are shown, n = 6. d CLL PBMC were plated over immobilized anti-CD3 antibody (anti-CD3i) or isotype control. After 48 h of incubation, Lurbinectedin or vehicle were added for another 72 h of culture. Finally, cells were labeled with anti-CD4-PECy5 and anti-CD8-PE antibodies and analyzed by flow cytometry. The percentage of total non-viable T lymphocytes after 5 days of culture is shown (mean ± SEM), n = 11. Statistical significance was determined by the Friedman test followed by Dunn’s post-test of multiple comparisons (a, c and d) or Wilcoxon signed-rank test (b). Lur Lurbinectedin, N.S no statistically significant difference, *p < 0.05; **p < 0.01; ****p < 0.0001

Fig. 3

Sensitivity of B-CLL cells to Lurbinectedin in the presence of microenvironmental stimuli. a NLC cells were differentiated as previously described. Next, Lurbinectedin (10 nM) was added to the co-culture for 24 h and the percentage of CD19⁺ cell death was determined by flow cytometry. The individual values and the mean ± SEM are shown (n = 11). b B-CLL cells were co-cultured with HS5 stromal cell line for 72 h. Then, Lurbinectedin 10 nM or vehicle was added for 24 h and CD19⁺ cell death was assessed by flow cytometry. The individual values and the mean ± SEM are shown (n = 10). c CLL PBMC were plated in wells with or without immobilized anti-CD3 antibody and after 48 h of incubation Lurbinectedin 3 nM was added. Then, cells were incubated for another 48 h before the addition of 100 nM ABT-199 for the last 24 h. Cell death was evaluated by flow cytometry. The results show the individual values and the mean ± SEM, n = 13. Statistical analysis was performed by the Friedman test followed by Dunn’s post-test of multiple comparisons. Lur Lurbinectedin, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig. 4

Lurbinectedin downregulates the expression of CCR7 on B-CLL cells and inhibits the migration towards CCL19 and CCL21. CLL PBMC were cultured for 24 h at 37 °C. Then, Lurbinectedin 1 nM, 3 nM or vehicle were added for 24 h. After that, cells were labeled with anti-CD19-PECy5 and anti-CCR7-PE (a) or anti-CXCR4-PE (b) antibodies and analyzed by flow cytometry. Figure shows the individual values and the mean ± SEM, n = 14. c, d The effect of Lurbinectedin over CLL CD19⁺ cell migration was measured by a migration assay on transwell plates (for more details on the procedure see the methods section). The results show individual values and mean ± SEM of the migration index to CCL19 (n = 7) and CCL21 (n = 6). The statistical analysis was performed using Friedman test followed by Dunn’s post-test. e CLL B lymphocytes were treated with Lurbinectedin 3 nM or vehicle for 24 h. Cells were stimulated with the chemokines and whole protein extracts were obtained. Cell extracts were then analyzed by Western blot using antibodies directed toward P-Erk 1/2. Figure shows a representative example of the bands obtained. f Quantification of the experiment described on (e) as mean ± SEM of the intensity ratio of the P-Erk vs actin, n = 8. Statistical analysis was performed by two-ways ANOVA followed by Tukey multiple comparison post-test. g CLL B lymphocytes were treated with Lurbinectedin 3 nM or vehicle for 24 h. Cells were stimulated with the chemokines and stained for P-Akt as was described in materials and methods. Results showed representative histograms and the corresponding MFI. Isotype control is depicted in grey. h Mean ± SEM of the MFI of p-Akt staining are shown (n = 9). The statistical analysis was performed by a two-ways ANOVA followed by Tukey’s post-test of multiple comparisons. Lur Lurbinectedin, MFI mean fluorescence intensity, N.S no statistically significant difference; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig. 5

Lurbinectedin increases IL-1β production by myeloid cells. a Monocytes were purified from CLL samples and treated with 3 nM Lurbinectedin or vehicle for 24 h. IL-1β secretion was quantified by ELISA. The results are shown as individual values associating, in each sample, the concentration of the control and treated condition, n = 5. b NLC were treated with Lurbinectedin 3 nM for 24 h. In the indicated cases 2 mM ATP was added for the last 90 min of culture. The concentration of IL-1β secreted was quantified by ELISA. The results are expressed as individual values associating, in each sample, the control and treated condition, n = 7. c NLCs were differentiated over sterile microscope coverslips. Then, NLC were treated for 24 h with Lurbinectedin 3 nM and labeled with anti-IL-1β antibody. Images of a representative experiment are shown (n = 3). d Purified HD monocytes were treated for 24 h with Lurbinectedin 3 nM or vehicle and then pro-IL1β mRNA level was evaluated by qRT-PCR. Results were normalized to GAPDH human gene and were represented as relative units (2^−ΔΔct). The mean ± SEM of 6 experiments is shown. e NLC were differentiated from HD monocytes co-cultured with B-CLL cells over sterile coverslips for 14 days. Leukemic cells were removed and NLC were treated with 3 nM Lurbinectedin for 24 h. For the positive control, 2 mM ATP was added for 1 h. FAM-FLICA staining was carried out according to the fabricant indications. Representative images of confocal microscopy and the quantification of the percentage of NLC positive for activated caspase-1 are shown (mean ± SEM; n = 6). a–d Statistical analysis was carried out using the Wilcoxon test or e Friedman test followed by Dunn’s post-test. Lur Lurbinectedin, N.S no statistically significant difference; *p < 0.05; **p < 0.01