Adhesion to activated endothelium and VCAM-1 is inhibited by idelalisib.

<p>A linear gradient of shear flow increasing from 0-45 dynes/cm² is applied over the adhered cells and the number of adherent cells is determined by the percentage of cells remaining every 50 seconds. (A) CLL cells were either treated or not treated with idelalisib and allowed to adhere to HUVEC stimulated or not stimulated (control) with TNFα (n=5). The data displayed the mean relative adhesion (±SEM) at different shear forces compared with the baseline adhesion for each condition. (B) The bar diagrams display the percentage of cells adherent to HUVEC at two representative shear forces (15 and 30 dynes/cm²). Displayed are the means (±SEM) from 5 different patients. idelalisib significantly inhibits the adhesion of CLL cells to HUVEC TNFα stimulated at low and high shear forces (*p<0.05; **p<0.01). (C) Ramos cells were treated or not treated with idelalisib and allowed to adhere to slides coated with VCAM-1 or ovalbumin (control). Idelalisib is able to interfere with VCAM-1-mediated adhesion at different shear forces (n=3, *p<0.05). The data display the mean relative adhesion (±SEM) at different shear forces compared with the baseline adhesion for each condition. </p

Idelalisib inhibits CLL cell adhesion to TNFα-stimulated EC and BMSC.

<p>CLL cells were allowed to adhere to EC (HUVEC or HMEC-1) or BMSC (9-15c or CLL-MSC). EC and BMSC were either unstimulated (control) or stimulated with TNFα (10ng/ml). The bar diagrams represent the mean relative adhesion (±SEM; n=8) of CLL cells in the presence or absence of idelalisib to EC and BMSC compared with the control. TNFα treatment increases the adhesion of CLL cells to EC and BMSC and in presence of idelalisib is significantly inhibited (*p<0.05; **p<0.01). (B) Representative phase contrast photomicrographs demonstrating CLL cell adhesion to HMEC-1 either treated or untreated (control) with TNFα. Treatment with TNFα increased the number of adherent CLL cells to HMEC-1 in comparison to the control (on the bottom left); in presence of idelalisib the number is reduced both in presence of TNFα treatment.</p

Idelalisib down-regulates integrin-induced AKT phosphorylation.

<p>(A) Overlay histograms depict the mean AKT phosphorylation (pAKT) fluorescence intensity of CD19+ CLL cells in a representative case. The solid grey histogram depicts pAKT staining of CLL cells in co-culture with HMEC-1 without idelalisib and the black line histogram depicts of CLL cells in co-culture with HMEC-1 in presence of 5 μM idelalisib. AKT phosphorylation was analyzed in CLL cells after 1 hour and 24 hours of co-culture with HMEC-1 either in presence or absence of idelalisib. pAKT was evaluated by flow cytometry in CD19⁺ CLL cells gated from 4 different patients. (B) CLL cells were cultured in medium alone and in co-culture with HMEC-1, either in presence or absence of idelalisib for 1 hour or 24 hours. The bar diagrams represent the mean ± SEM of pAKT positive cells (%) (n=4, p<0.05). (C) Displayed are immunoblots from 3 representative CLL cell samples cultured alone or in co-cultured with HMEC-1 in presence or absence of idelalisib for 24 hours. Lysates were probed with antibodies to pAKT (S473) and actin. (D) CLL cells were stimulated or no stimulated with anti-VLA4 mAbs (clone 19H8) in the presence or absence of idelalisib. Displayed are the means ± SEM from 5 different patients (*p<0.05). The immunoblots on the right hand side depict AKT activation in 3 representative CLL samples stimulated with anti-VLA4 in presence or absence of idelalisib.</p

Idelalisib affects adhesion mediated by VLA-4 and VCAM-1 interaction.

<p>(A) On the left, displayed are fluorescence histograms depicting the relative fluorescence intensity of HUVEC and HMEC-1 stained with anti-VCAM-1 mAb either treated or not treated (control) with TNFα (10ng/ml) for 24 hours. Mean fluorescence intensity ratio was calculated by dividing the mean fluorescence intensity for VCAM-1 by the mean fluorescence of the isotype control in both conditions. On the right, representative fluorescence histograms of CLL cells stained with anti-VLA-4 (grey histograms) or the corresponding isotype control (white histograms). THE MFIR is displayed below the histograms. (B) A positive correlation is displayed between VLA-4 expression (%) and the relative adhesion of CLL cells to HUVEC stimulated with TNFα (n=10). (C) The box plot displays a comparison of CLL cell adhesion to HUVEC TNFα-stimulated in presence or absence of idelalisib in VLA-4 high (n=5) and VLA-4 low (n=5) expression groups. Bar diagram represents the percentage of inhibition in CLL adhesion to HUVEC stimulated with TNFα induced by idelalisib in VLA-4 high and VLA-4 low CLL samples relative to adhesion observed without idelalisib. Data are shown as mean ±SEM (*p<0.05).</p

Idelalisib overcomes endothelial cells and bone marrow stromal cells mediated CLL cell protection.

<p>(A) CLL cells were co-cultured with HUVEC, UV-2, NKtert and KUSA-H1 in presence or absence of 5μM idelalisib. The gates in the contour plots exemplify viable CLL cells in one representative sample. (B) The bar diagrams represent the mean relative viabilities of CLL cells co-cultured in normal conditions (control) or in presence of 5μM idelalisib with EC and BMSC. Viabilities in idelalisib-treated samples were normalized to the corresponding viabilities of control samples at the respective time-point (100%) to account for differences in spontaneous apoptosis. Displayed is mean±SEM from 7 different samples (*p<0.05; **p<0.01). </p

ET-1 induces CLL activation and proliferation through ET_A receptor.

<p>Purified CLL cells, pretreated or not with 0.1 µM BQ-123, were stimulated with 100 nM ET-1 for 4 hours (n = 7) in panel A, or with an agonist specific for toll-like receptor 9 (TLR9) (CpG oligonucleotides) and IL-2 with/without ET-1 addition for 5 days (n = 15) in panel B, or with endothelial cell contact (HC) for 4 days (n = 11) in panel C. Histograms depict the formazan release by metabolically active CLL cells as fold change compared to unstimulated control (untreated CLL cells cultured alone). Data are presented as mean±SEM of 6 independent experiments. Of note, the CLL activation is significantly increased upon ET-1 stimulation and inhibited by blocking ET_AR signaling in all conditions. (D) After 1 h of serum-starvation, CLL cells (n = 8) were stimulated or not with ET-1 overnight. PI analysis was performed to define cell cycle phases. Histograms represent the percentage of CLL cells in S-phase after treatment compared to unstimulated control. (E) CFSE-labeled CLL cells were cultured for 4 days alone in complete medium (control) or on endothelial layers. Where indicated, CLL cells were incubated for 20 min with 0.1 µM BQ-123 before co-culture. The proliferative measure was inspected for 4 days, gating the CD19+ live CLL cells. The histograms represent cumulative data at 96 hours of 5 independent experiments by using 9 CLL patients. Data are shown as mean values ± SEM of the percentage of dividing CLL cells. In panel F, the percentages of divided CLL cells at 48 h, 72 h and 96 h measured by CFSE dilution in a representative CLL sample are shown. HUVEC cells stimulate CLL cells to divide. The addition of BQ-123 counteracts the EC-mediated proliferative stimuli. (*p<0.05, **p<0.01, ***p<0.001).</p

ET-1 inhibits the pro-apoptotic effect of idelalisib, MEK inhibitor and fludarabine.

<p>CLL cells (n = 8) were treated with idelalisib at 0.5 µM (panel A) or PD98059, a MEK inhibitor, at 50 µM (panel B). When indicated, CLL cells were also incubated with BQ-123 at 0.1 µM before ET-1 stimulation. CLL cell viability was assessed by flow cytometry by using Annexin V and PI staining. CLL viable cells were defined as Annexin V-/PI-. Histograms represent cell viability as mean±SEM at 48 hours of 8 CLL patients evaluated in 4 independent experiments. Control is defined as CLL cells cultured without any treatment. Flow cytometric contour plots of one representative case are shown in panel C. The gates in the plots exemplify viable CLL cells (Annexin V-/PI-, circled). ET-1 signaling reduces CLL sensitivity to both idelalisib and PD98059 (**p<0.001). BQ-123 restores the drug effect on CLL cells (*p<0.01 for idelalisib, **p<0.001 for PD98059). In addition, CLL cells (n = 8) were cultured (panel D) alone in complete medium or (panel E) in contact with HUVEC layer (HC). Fludarabine was added at 1 µM. Cells were also treated with 100 nM ET-1 and, as indicated, pretreated with 0.1 µM BQ-123 (20 min). Histograms represent mean±SEM of the percentage of live cells at 48 h of 8 CLL patients evaluated in 4 independent experiments. Control is defined as CLL cells cultured without any treatment alone in panel D and in co-culture in panel E. Flow cytometric contour plots of one representative case are shown in panel F. The gates in the plots exemplify viable CLL cells (Annexin V-/PI-, circled). Of note, inhibition of fludarabine-induced apoptosis is evident in presence of ET-1 both in CLL cultured alone and in CLL in co-culture. Again, treatment with BQ-123 improves CLL sensitivity to fludarabine-mediated apoptosis. (*p<0.05, **p<0.01, ***p<0.001).</p

ET-1 mediates survival signal on CLL cells.

<p>(A) Serum-starved CLL cells were stimulated with 100 nM ET-1 for 1 hour, pretreating or not cells with 0.1 µM BQ-123 (20 min). Western blot analysis of CLL cells was performed with anti-phospho-Akt, anti-Akt, anti-phospho-ERK, anti-ERK and anti-β-actin antibodies. ET-1 stimulates Akt and ERK phosphorylation through ET_AR. The immunoblots depict Akt and ERK activation in a representative case. Histograms represent densitometric quantification (pAkt/total Akt ratio and pERK/total ERK ratio) of bands relative to phospho-Akt, total Akt, phospho-ERK and total ERK normalized on β-actin. Data are presented as mean±SEM of 6 CLL patients relative to unstimulated control (*p<0.05, ***p<0.001). (B) CLL cells (n = 6) were cultured with the addition of recombinant ET-1 peptide at 100 nM for 96 hours. CLL cell viability was assessed by flow cytometry using Annexin V and PI staining. A lymphocyte gate was set according to the different relative size and granularity (forward scatter and side scatter) and viable cells were defined as Annexin V-/PI-. Histograms represent mean±SEM at 96 h of 6 CLL patients evaluated in 3 independent experiments. Two representative cases are depicted on the right, showing cell viability at 48 h, 72 h and 96 h. Control is defined as viability of CLL cells cultured alone in complete medium. ET-1 stimulation improves CLL survival. The effect is inhibited by pre-treating CLL cells with BQ-123 (0.1–1 µM) (**p<0.01, ***p<0.001). (C) CLL cells (n = 11), pretreated or not with BQ-123 (0.1–1 µM), were cultured on HUVEC cell layer for 96 hours. CLL cell viability was assessed by flow cytometry, as described for panel B. Histograms represent data as mean±SEM of 11 CLL patients evaluated in 4 independent experiments. Two representative cases are depicted on the right, showing cell viability at 48 h, 72 h and 96 h. Control is defined as viability of CLL cells cultured alone in complete medium. Note that BQ-123 significantly reduces the CLL pro-survival effect mediated by HUVEC contact (***p<0.001). (D) CLL cells were stimulated with 10 µg/mL of anti-IgM for 48 hours with or without 0.1 µM BQ-123. Dot plots represent cell viability of 6 CLL patients evaluated in 3 independent experiments. The blockade of ET_AR reduces the pro-survival effect of BCR triggering in CLL cells (*p<0.05). (E) Serum-starved CLL cells were pretreated or not with 0.1 µM BQ-123 for 20 minutes before stimulation with anti-IgM for 10 minutes. The immunoblots depict ERK phosphorylation in a representative case.</p

CLL cells express ET-1 and ET_A receptor.

<p>(A) ET-1 and ET_AR expression levels were evaluated by quantitative reverse-transcription PCR on CLL cells (n = 10) and normal B lymphocytes (n = 6) purified from peripheral blood. Histograms depict mean±SEM of ET-1 and ET_AR relative expression. Results were normalized to the expression of GAPDH housekeeping gene. CLL cells show higher expression levels of both ET-1 and ET_AR mRNA compared to normal B cells (*p<0.05). (B) CLL cells or normal B lymphocytes purified from peripheral blood were allowed to adhere and then stained with anti-ET-1 antibody. A representative case of 3 independent CLL samples and 3 normal B cell samples is shown. Original magnification, 400X for left panels, and 1000X for right panels. CLL cells show more intense stainings of ET-1 peptide than normal B cells. (C) Displayed are flow cytometric histograms depicting the relative fluorescence intensity of 2 CLL samples and 2 normal B-cell samples stained with anti-CD19 and anti-ET_AR Abs. Mean fluorescence intensity ratio (MFIR) is displayed above the histograms and is calculated by dividing the mean fluorescence intensity for ET_AR by the mean fluorescence of the isotype control. Histograms on the right summarize MFIR data of B cells from 7 CLL patients and 6 normal controls. Data are presented as mean±SEM. Increased expression of ET_AR on the surface of CLL cells was measured as compared to normal B cells (*p<0.05). (D) The immunoblots depict higher ET_AR expression levels in CLL cells than in normal B lymphocytes purified from peripheral blood detected by Western blot analysis. (E) Immunohistochemical evaluation of CLL-infiltrated lymph nodes (CLL LN) (n = 4) stained with antibodies against ET-1 or ET_A receptor showing positive CLL cells. A representative staining of lymph nodes from healthy donors (n = 3) is displayed, showing a faint ET-1 expression on normal B lymphocytes identified by CD20 staining. Prostate cancer is shown as positive control. Original magnification, 200X in the above panels and 400X in the bottom panels.</p

Classification of the patients according with the results of the ELISpot assay.

<p>IFD = invasive fungal disease. IM = invasive mucormycosis; IA = invasive aspergillosis. Grey boxes indicate the patients with a defined diagnosis considered for the computation of sensitivity, specificity and receiver-operating characteristic analysis.</p