Treatment of patients with relapsed or refractory chronic lymphocytic leukemia (CLL) has significantly improved more recently with the approval of several new agents, including ibrutinib, idelalisib, and venetoclax. Despite the outstanding efficacies observed with these agents, these treatments are sometimes discontinued due to toxicity, unresponsiveness, transformation of the disease and/or resistance. Constitutive NF-[kappa]B activation that protects CLL cells from apoptotic stimuli represents one of molecular mechanisms that underlie the emergence of drug resistance. As prostaglandin E (EP)4 receptor agonists have been shown to successfully inhibit the NF-[kappa]B pathway in B-cell lymphoma cells, we investigated the potential of the highly specific EP4 receptor agonist L-902688 for the potential treatment of patients with CLL. We show here that low micromolar concentrations of L-902688 can indeed induce selective cytotoxicity towards several B-cell malignancies, including CLL. Moreover, L-902688-mediated activation of the EP4 receptor in patient derived CLL cells resulted in inhibition of the NF-[kappa]B pathway, cell proliferation, and induction of apoptosis. Most importantly, we show for the first time that in combination with ibrutinib, idelalisib, or venetoclax, L-902688 induces synergistic cytotoxic activity against patient derived CLL cells. To conclude, the modulation of NF-[kappa]B activity by EP4 receptor agonists represents an innovative approach to improve the...
COBISS.SI-ID: 43415299
Reliable disease and healthy control models predictive of human responses are of vital need in preclinical evaluation of efficacy and safety of novel drugs. Chronic lymphocytic leukemia (CLL) is a malignancy of mature B cells which are highly dependent on interactions with the tissue microenvironment. In search of novel drugs for treatment of CLL, establishment of reliable in vitro models is of high importance. Availability of primary B cells obtained from patients is a good approach. However, despite the long survival of malignant B cells in vivo, they do not proliferate and die in vitro. The aim of this study was to establish CLL endogenous growth conditions as an in vitro disease model. Hence, we stimulated primary CLL cells with a) synthetic oligonucleotide DSP30 containing a CpG motif that activates immune cells by augmenting the expression of interleukin 2 (IL-2) receptors in combination with IL-2; b) Ionomycin, a calcium ionophore, which increases intracellular calcium and in combination with PMA activates protein kinase C, mimicking B cell receptor activation; and c) LPS, which activates TLR4. Malignant B cells obtained after informed consent from patients diagnosed with CLL were incubated with IL-2/DSP30, ionomycin/PMA and LPS for 24 h and 48 h. Cell viability was measured by resazurin-based metabolic activity PrestoBlue assay. Stimulation of CLL cells (N = 19) by IL-2/DSP30 increased cell viability by 2-fold (206%) after 24 h and 4-fold (389%) after 48 h. When CLL cells (N = 16) were stimulated by ionomycin/PMA, cell viability increased to 171% after 24 h and 360% after 48 h. Incubation of CLL cells (N = 16) by LPS resulted in 129% and 136% increased cell viability after 24 h and 48 h, respectively. The same trend for increased cell viability was observed in all tested CLL samples treated with IL2/DSP30 or ionomycin/PMA, however, inter-individual differences were observed, which is in agreement with the fact that CLL is very heterogeneous disease. Stimulation of CLL cells with IL-2/DSP30 and ionomycin/PMA was thus identified as a valuable in vitro model. Next, we evaluated prostaglandin EP4 receptor as a target in CLL. A selective EP4 receptor agonist, PgE1-OH, previously shown to induce apoptosis in malignant B cells (1, 2) was chosen as a model drug in the present study. PgE1-OH was cytotoxic in a dose-dependent manner in CLL cells stimulated by IL2/DSP30, ionomcyin/PMA as well as LPS. Moreover, ionomycin/PMA activation resulted in increased levels of TNF?, which is important growth factor for CLL cells. The pre-treatment of CLL cells (N =10) with PgE1-OH decreased the production of TNF?, indicating beneficial immunomodulatory properties of PgE1-OH in CLL. This is in agreement with our previous study, in which we evaluated immunomodulatory properties of PgE1-OH in human lymphoblastoid cell lines, a predictive in vitro model for immunotoxicity screening of compounds (3). In conclusion, primary CLL cells stimulated by ionomcyin/PMA were identified as a valuable in vitro disease model
COBISS.SI-ID: 4615793
Prostaglandin EP4 receptor signalling was shown to prevent B cell receptor (BCR)-mediated proliferation, and represents a novel strategy toward improving the therapy of B cell malignancies, known to be depend on BCR signals for survival. Chronic lymphocytic leukemia (CLL) is the most common haematological malignancy diagnosed in adults and is currently an incurable disease. In our previous research we have shown that selective EP4 receptor agonist, inhibited NF-?B signalling pathway, resulting in an increased caspase-mediated apoptosis of malignant B cells. The aim of this study was to evaluate the EP4 receptor as a potential target for the treatment of CLL (1, 2). Malignant B cells were isolated from whole blood obtained after informed consent from CLL patients. The expression of EP4 receptor was higher on CLL cells compared to lymphoblastoid cell lines (LCLs) obtained from healthy donors. EP4 receptor agonist PgE1-OH induced a concentration and time dependent cytotoxicity in all 151 CLL cells tested. The anti-cancer effects were mediated via EP4 receptor as evident from stronger cytotoxic effects of selective EP4 receptor agonist compared to endogenous ligand PgE2 and the fact that EP4 receptor antagonist prevented PgE1-OH induced apoptosis. We evaluated the selectivity of PgE1-OH towards CLL cells using LCLs and peripheral blood mononuclear cells (PBMCs) obtained from healthy individuals. The average EC50 values for PgE1-OH after 24 h were 13.53 µM on CLL cells (N=151), and 55.43 µM on LCLs (N=24) and 46.36 µM on PBMC (N=21), indicating that PgE1-OH was significantly more cytotoxic to malignant B cells compared to immune cells isolated from healthy individuals. PgE1-OH exerted cytotoxic effects in all CLL cells with EC50 values ranging from 2 to 55 µM indicating inter-individual variability in response to PgE1-OH, which is in agreement with the fact that CLL is very heterogeneous disease. The analysis of the results revealed sex-dependent sensitivity of CLL cells to PgE1-OH, which was more cytotoxic to the cells of male compared to female donors. PgE1-OH was also more cytotoxic toward the cells of the carriers of the variant A allele of EP4 receptor expression-modulating polymorphism rs4495224. Moreover, male patients had higher expression levels of Ptger4, coding for EP4 receptor, compared to female patients as did the donors with the rs4495224 AA genotype compared to those with rs4495224 AC/CC genotype. A weak, but significant correlation between an increased expression of the Ptger4 gene and lower EC50 values was also shown. Furthermore, PgE1-OH was cytotoxic in CLL cells obtained from patients in Binet stage A, B as well as C and induced apoptosis in CLL cells isolated from patients with p53 deletion, known to be resistant to the standard cytotoxic therapy. PgE1-OH acted synergistically with fludarabin in therapeutic concentrations, ranging from 3 to 5 µM. Moreover, a very strong synergism was detected when the cells were treated with PgE1-OH and idelalisib (combination index ( 0.1) well as with ibrutinib, which might provide novel therapeutic options for the treatment of CLL. In conclusion, EP4 receptor was identified as a promising drug target for the treatment of CLL.
COBISS.SI-ID: 4572017
Chronic lymphocytic leukemia (CLL) is the most common type of leukemia in adults in Western countries affecting B cells and is characterized by great heterogeneity in the clinical course. Some patients remain free of symptoms and do not require treatment for decades, whereas others rapidly develop an aggressive form requiring immediate therapeutic intervention. Treatment can include chemotherapy, chemoimmunotherapy, or drugs which target the signaling pathways that promote the survival and/or growth of CLL cells.1 However, the increasing rise of resistance to these medicines presents a severe problem and promotes the search of innovative and effective targets for the treatment of CLL patients. The EP4 receptor is one of the four prostaglandin E (EP) receptors (EP1, EP2, EP3 and EP4) that recognize prostaglandin E2 (PGE2) as its natural binding ligand and was identified as a principal molecule conveying the growth-suppressive effect of PGE2 in immature and mature malignant B cells.2–4 Our research group demonstrated that EP4 receptor agonist 1-hydroxyprostaglandin E1 induced caspase-mediated apoptosis and a decreased NF-?B activity in Burkitt's lymphoma cells resulting in an elevated sensitivity of cells towards bortezomib- and doxorubicin-induced chemotherapeutic effects.5 Due to the promising results obtained in previous studies, selected EP4 agonists have been evaluated for their potential to induce apoptosis of CLL cell lines (MEC-1 and MEC-2) as well as primary CLL cells in comparison to non-malignant control cells. Among them, selective EP4 receptor agonist L-902688 demonstrates cytotoxic activity in both CLL derived cell lines and primary CLL cells in the low micromolar range and displays selectivity towards peripheral blood mononuclear cells after 24 h of treatment. Moreover, L-902688 also causes the inhibition of proliferation determined by CFSE staining and induces apoptosis confirmed by Annexin V/Sytox Blue staining and detection of increased sub-G1 population in cell cycle distribution analysis. Activation of the EP4 receptor modulates several signaling pathways. Examining the phosphorylation levels of several downstream effectors such as Akt, MEK, ERK, CREB, p105 and I?B?, we have found that L-902688 decreases NF-?B activity. The observed effect is beneficial since this pathway is aberrantly activated in CLL cells. Moreover, L-902688 has also been tested in combination with drugs, which are already used in treatment of CLL (ibrutinib, idelalisib) since the utilization of drug combinations endowed with synergistic effects enables the reduction of therapeutically efficient doses, thereby decreasing the toxicity against normal cells. Our results show that L-902688-ibrutinib/idelalisib combinations exhibit promising synergistic anticancer activity towards CLL cells. To conclude, the EP4 receptor agonists with their ability to modulate NF-?B activity represent an interesting alternative for development of new therapies for CLL. In particular, EP4 receptor agonists could be an excellent adjuncts to already existing therapies for CLL patients on account of their promising synergistic activity.
COBISS.SI-ID: 4742001
Introduction: Chronic lymphocytic leukemia (CLL) is a hematologic malignancy, characterized by progressive accumulation of mature appearing monoclonal CD5+, CD19+, CD20+ and CD23+ B lymphocytes in the blood, bone marrow, and secondary lymphoid organs. Multiple therapies for CLL are available, such as chemoimmunotherapy, consisting of fludarabine, cyclophosphamide and anti-CD20 monoclonal antibody rituximab, and novel targeted therapies, including ibrutinib, idelalisib and venetoclax.1 However, proteasome inhibitors (PIs), albeit effective treatment for multiple myeloma, haven't been applied to CLL.2 We hypothesized that as an alternative to current therapies or as part of combination strategy, PIs could be used in the manegment of relapsed and refractory CLL. Results: We evaluated the potency of FDA-approved PIs carfilzomib (CFZ), bortezomib (BTZ), and ixazomib (IXA) on fludarabin resistant cell line MEC-1.2,3 CFZ exhibited the most potent cytotoxic effects and it induced caspase-mediated cell death confirmed with pan-caspase inhibitor Q-VD-OPh and by flow cytometry. In addition, CFZ synergized with ibrutinib, idelalisib and venetoclax. Since CFZ resistance is likely to occur with prolonged exposure to malignant cells, we developed CFZ resistant cell line. Cells were grow in the presence of gradually increasing concentrations of CFZ from 5 nM until exhibiting persistent growth in the presence of 50 nM CFZ, and termed MEC-1 CFZR. We tested the potency of CFZ, BTZ, and IXA on MEC-1 CFZR and compared to MEC-1 EC50 values were 7-, 1-2-, and 1-2- higher, respectively. Next, we investigated the mechanism of CFZ resistance. MEC-1 CFZR was treated with PIs in presence/absence of selective p-gp inhibitor elacridar for 24 hours, followed by viability determination with MTS assay. Furthermore, p-gp activity assay was performed by flow cytometry, with rhodamine 123 used as a p-gp substrate. Results show resistance of MEC-1 CFZR to CFZ is due to p-gp upregulation, since cytotoxicity of CFZ significantly increased and rhodamin 123 efflux significantly decreased when elacridar was added. Conclusions: We have demonstrated PIs are cytotoxic to fludarabine resistant CLL. Furthermore, we've developed CFZ resistant in vitro model of CLL and identified p-gp upregulation as a mechanism driving CFZ resistance. Since p-gp inhibition restored cytotoxicity of CFZ, we propose addition of elacridar should be used in the case of CFZ failure. In addition, MEC-1 CFZR exhibited minimal cross-resistance to other PIs, indicating BTZ and IXA could be used as an alternative for CFZ resistant CLL.
COBISS.SI-ID: 4742513