Targeting metabolism and survival in chronic lymphocytic leukemia and Richter syndrome cells by a novel NF-κB inhibitor

Abstract

IT-901 is a novel and selective NF-κB inhibitor with promising activity in pre-clinical models. Here we show that treatment of chronic lymphocytic leukemia cells (CLL) with IT-901 effectively interrupts NF-κB transcriptional activity. CLL cells exposed to the drug display elevated mitochondrial reactive oxygen species, which damage mitochondria, limit oxidative phosphorylation and ATP production, and activate intrinsic apoptosis. Inhibition of NF-κB signaling in stromal and myeloid cells, both tumor-supportive elements, fails to induce apoptosis, but impairs NF-κB-driven expression of molecules involved in cell-cell contacts and immune responses, essential elements in creating a pro-leukemic niche. The consequence is that accessory cells do not protect CLL cells from IT-901-induced apoptosis. In this context, IT-901 shows synergistic activity with ibrutinib, arguing in favor of combination strategies. IT-901 is also effective in primary cells from patients with Richter syndrome (RS). Its anti-tumor properties are confirmed in xenograft models of CLL and in RS patient-derived xenografts, with documented NF-κB inhibition and significant reduction of tumor burden. Together, these results provide pre-clinical proof of principle for IT-901 as a potential new drug in CLL and RS.

Figure 1.

IT-901 blocks nuclear factor-kappa B (NF-κB) activity in primary chronic lymphocytic leukemia (CLL) cells and derived cell line. (A) DNA binding activity of the p65 and p50 subunits in primary CLL cells (n=13) was analyzed using an ELISA kit, applying the same amount of nuclear extracts. Leukemic cells were co-cultured on a stromal layer (HS-5) to maximize the activation of the pathway, in the presence of increasing doses of drug or vehicle (NT) for 6 hours (h). (B) Cumulative DNA binding activity of NF-κB (p65 and p50) in 2 different cell line models of CLL, Mec-1 (n=5), and OSU-CLL (n=3). (C, D, G and H) Cytoplasmic (C) and nuclear (N) fractions obtained from primary CLL cells (C) or cell lines (D) cultured as indicated above were resolved by SDS-PAGE and blotted with specific antibodies to detect the expression of NF-κB1 subunits p105/p50 (arrow head) and RelA (p65). Lamin A/C and β-tubulin were used as nuclear and cytoplasmic markers, respectively. Nuclear p65 and p50 band intensities in primary CLL samples and CLL cell lines are reported in (G) and (H), respectively. (E and F) Total lysates from primary CLL cells (E) or CLL cell lines (F) cultured alone and exposed to different doses of IT-901 or vehicle (NT) for 6 h were resolved by SDS-PAGE and expression of the NF-κB complex analyzed using specific antibodies. Actin was used as a loading control. OD: optical density.

Figure 2.

IT-901 induces mitochondrial damage and compromises mitochondrial respiration. (A) Representative plots and cumulative data of mitochondrial reactive oxygen species (mROS) concentration in chronic lymphocytic leukemia (CLL) cells. Data are represented as fold change (FC) over the vehicle (n=10). (B) Box plot reporting the catalase (CAT) mRNA expression levels in vehicle (NT)- or IT-901-treated primary cells (n=10). (C) Representative plots and cumulative data of inner mitochondrial membrane potential (ΔΦm) in primary CLL cells (n=10) exposed to vehicle (NT) or increasing doses of IT-901 for 6 hours (h). CCCP was used as positive control. (D) Dynamic mitochondrial metabolic profile (OCR; pmoles/min) of a representative CLL patient treated with vehicle (red line) or IT-901 10 μM (blue line) for 6 h. Maximal respiration (calculated as: OCR after FCCP injection-late OCR measurement after RT/AA addition) and ATP production (calculated as last rate measurement of OCR before Oligo injection-minimum rate measurement after Oligo injection) in primary CLL patients (n=7). (E) Box plots reporting ATP-synthase (ATP5A1) and Cytochrome C Oxidase Assembly Protein (SCO2) mRNA expression levels in vehicle- or IT-901-treated primary cells (n=7). CCCP: carbonyl cyanide m-chlorophenyl hydrazone; Oligo: oligomycin; FCCP: carbonyl cyanide p-trifluoromethoxyphenylhydrazone; RT/AA: rotenone + antimycin A; OCR: oxygen consumption rate.

Figure 3.

IT-901 rapidly induces apoptosis selectively in primary chronic lymphocytic leukemia (CLL) cells. (A) Representative plots and cumulative data of apoptotic staining with Annexin-V and propidium iodide (PI) of CLL patient cells (n=25) after 24–48 hours (h) of exposure to increasing doses of IT-901 or vehicle (NT). (B) Apoptotic data were plotted according to the mutational status of IgHV genes [mutated (M) vs. unmutated (UM)] and to the presence of specific genetic abnormalities (deletion 13/normal vs. deletion 17/mutation in TP53). (C) Total lysates from primary CLL cells cultured alone and exposed to different doses of IT-901 or vehicle (NT) for 6 h were resolved by SDS-PAGE and expression of pro- and anti-apoptotic proteins and activation of the caspase pathway analyzed using specific antibodies. Actin was used as a loading control. (D) Cumulative data of apoptosis obtained from Mec-1 (n=12) and OSU-CLL cell lines (n=9) at 24 h and both cell lines (n=21) at 48 h, exposed to increasing doses of IT-901. (E) Total lysates of CLL cell lines, cultured alone and exposed to different doses of IT-901 or vehicle (NT) for 6 h were resolved by SDS-PAGE and expression of pro- and anti-apoptotic proteins and activation of the caspase pathway analyzed using specific antibodies. Actin was used as a loading control. (F) Cumulative data of mROS and apoptosis of CLL cell lines (n=6) measured in the presence of N-acetyl-cysteine (NAC) and/or IT-901. (G) Cumulative data of apoptosis in normal B and T lymphocytes purified from 6 different healthy donors and exposed to increasing doses of IT-901 for the indicated time points. FL: full-length; CL: cleaved.

Figure 4.

Silencing of the p65 subunit confirms results obtained with IT-901. (A) chronic lymphocytic leukemia (CLL) cell lines were infected with lentiviral particles containing plasmids coding for an shRNA scramble (Scr) or shRNA p65 (A and B). Expression of the NF-κB complex was checked by western blot and p50 and p65 expression quantified over actin, showing a significant reduction for p65, while for p50 statistical significance was not reached. Silencing of p65 diminished also the expression of the anti-apoptotic protein XIAP and of the full-length (FL) Caspase3 protein. Actin was used as a loading control. (B) Apoptosis analysis by flow cytometry of cell lines infected with shRNA lentiviral particles (n=5).

Figure 5.

IT-901 inhibits the nurturing properties of nurse-like cells (NLC). (A) Confocal microscopy analysis of nuclear factor-kappa B (NF-κB) activation in NLC alone or co-cultured with autologous CLL cells in the presence of vehicle (+CLL) or IT-901 (+CLL+IT-901). Cumulative results of the nuclear p65 fluorescence intensity, in NLC, are reported in the graph. (B) Giemsa staining of vehicle- or IT-901-treated NLC. (C and D) mRNA expression level (C; n=8) and phenotypic analysis by flow cytometry (D) of molecules involved in cell-cell interactions (ITGA4, ICAM1 and VCAM1) and immune-modulatory pathway (CD274 and CD86) in NLC cultured as indicated above. (E) Cumulative data of apoptosis of primary leukemic cells co-cultured over HS-5 cells in the presence of increasing doses of IT-901 for 24h. a.u.: arbitrary units.

Figure 6.

IT-901 co-operates with ibrutinib in inducing apoptosis of chronic lymphocytic leukemia (CLL) cells. Cumulative results of apoptosis of primary CLL cells (n=16) cultured alone (A) or over autologous nurse-like cells (NLC) (B) for 48 hours (h) in the presence of ibrutinib (2.5 μM) and IT-901 (5 and 10 μM) alone or in combination. Statistical significance of the combined effect of IT-901 and ibrutinib compared to single drugs alone was calculated according to the effect-based strategy, using the Highest Single Agent approach, as described by Foucquier and Guedj. *Over single box plot indicates statistical significance compared to untreated (NT).

Figure 7.

IT-901 limits in vivo growth and spread of chronic lymphocytic leukemia (CLL) cells. (A) Representative scheme of the in vivo model. Mec-1 cells were intravenously injected in tail vein of NSG mice, left to engraft for ten days before starting the treatment with IT-901 or vehicle. (B) Images of kidneys obtained from vehicle- or IT-901-treated mice. (C) Mec-1 engraftment in different organs evaluated by flow cytometry after labeling of leukemic cells with anti-human-CD45 and -CD19 antibodies. Cumulative data of engraftment in kidneys, liver, spleen and bone marrow (BM) (n=8 different mice/group). (D) Immunohistochemical analyses and quantification of CD20 and nuclear p65 staining (reported as percentage of positive cells) in kidneys of vehicle- or IT-901-treated mice. (E) Viability of Mec-1 cells, purified from kidneys and liver of vehicle- or IT-901-treated mice, analyzed by flow cytometry (69% vs. 85% of viable cells in kidneys and 70% vs. 82% of viable cells in liver, respectively). (F) Kaplan-Meier curves showing survival of mice treated with IT-901 (n=8; red line; 43 days) compared to vehicle (n=8; blue line; 31 days). Engr: engraftment; Tx: treatment; Stop: drug holidays; Sac: euthanasia.

Figure 8.

IT-901 is active on primary Richter syndrome (RS) cells and in patient-derived xenograft (PDX). (A) Cumulative data of apoptosis of primary or PDX-tumor-derived RS cells. (B) Western blot analysis of the expression of the nuclear factor-kappa B (NF-κB) complex, pro- and anti-apoptotic proteins and caspase-3 in RS cells exposed to the indicated doses of IT-901 for 6 hours (h). Actin was used as loading control. (C) Tumor masses from vehicle- or IT-901-treated RS-PDX mice compared for tumor volume (cm³) and weight (g) (6 mice/group; double-flank injected). (D) Immunohistochemistry analysis of p65 expression within the tumor mass. Staining was reported as percentage of positive cells. (E) Cytoplasmic (C) and nuclear (N) fractions obtained from RS cells purified from the tumor mass were resolved by SDS-PAGE and expression of the NF-κB complex analyzed. β-tubulin and Lamin A/C were used as cytoplasmic and nuclear controls, respectively. FL: full-length; CL: cleaved.