Chronic lymphocytic leukemia (CLL) is the most common form of leukemia in the Western world. It is a disease in which malignant, non-functional B cells accumulate in the peripheral blood. CLL is initially a slowly progressive disease with a heterogeneous clinical course. Many patients have no clinical problems at the time of diagnosis and need not to be treated for more than a decade. For some, however, the disease progresses rapidly and needs immediate treatment. Today, new small molecules such as idelalisib, ibrutinib and venetoclax are available for treatment of CLL and replacing chemoimmunotherapy consisting of fludarabine, cyclophosphamide, and rituximab (FCR regimen). Today, new small molecules such as idelalisib, ibrutinib and venetoclax are available for treatment of CLL. Despite the fact that these compounds are very effective and have been approved for the treatment of patients with unresponsive disease or recurrence of the disease, many patients still do not respond to treatment or have relapses of the disease. CLL thus still remains an incurable disease for which we need new medicines and therapeutic approaches. In the doctoral thesis, we therefore focused on finding new modulators of programmed cell death for the treatment of CLL. Prostaglandin E2 receptor, subtype 4 (EP4) and the nucleotide-binding oligomerization domain (NOD) receptor 2 were chosen as therapeutic targets. The EP4 receptor is a G protein-coupled receptor and one of the four E-type prostaglandin receptors. Its natural ligand is prostaglandin E2 (PGE2), which is an important regulator of B lymphocyte function. Binding of PGE2 to the EP4 receptor modulates cyclic adenosine monophosphate (cAMP)-dependent and cAMP-independent signaling pathways, among which the NF-kB signaling pathway is the most important. The EP4 receptor has been identified as a target for treatment of B cell malignancies. The EP4 receptor agonist, PGE1-OH, inhibited NF-kB signaling pathway, leading to increased susceptibility of malignant B cells to bortezomib and doxorubicin. PGE1-OH also induced cell death in leukemia and lymphoma models as well as in primary CLL cells. New EP4 receptor agonists have been made available recently. Specifically, compound L-902688 is a highly potent agonist of EP4 receptor, which is 4000-fold more selective for the EP4 receptor than for other EP1-3 receptors. L-902688 also showed good bioavailability and a long half-life in rats. Most research with the compound L-902688 has been conducted in smooth muscle cells, but it is not yet known how the compound acts in B malignant cells. vi The NOD2 receptor is a cytosolic receptor involved in the recognition of intracellular pathogens within the innate immune system. It detects muramyl dipeptide (MDP), a small structural motif located in the cell wall of bacteria. Activation of NOD2 leads to antimicrobial and proinflammatory activity, and also participates in the process of apoptosis. NOD2 receptor agonists are immunostimulatory compounds with potential antiviral, antibacterial and antitumor activity. Several generations of NOD2 receptor agonists have been synthesized at the University of Ljubljana, Faculty of Pharmacy. They have been shown to be potent immunostimulatory compounds in vitro and SG8 was identified as the most promising. In terms of structure, it comprises trans-feruloyl-glycine mimetic of N-acetylmuraminic acid, linked to a dipeptide composed of L-valine (L-Val) and D-glutamic acid (D-Glu). SG8 showed potent immunostimulatory activity in vitro, which was not retained in vivo. To improve the pharmacokinetic properties of SG8, a new generation of NOD2 agonists was synthesized. However, it is not yet known whether compounds of this generation possess the capacity to enhance the antitumor activity of immune cells. In the first part of the doctoral dissertation, we evaluated the ability of the EP4 receptor agonist, L-902688, to induce cell death in leukemia and lymphoma cell lines, primay CLLs and peripheral blood mononuclear cells (PBMCs). Blood samples from patients with CLL were collected at the University Medical Center Ljubljana, Clinical Department of Haematology (Clinical study 93/12/10 approved by the Republic of Slovenia National Medical Ethics Committee). Compound L-902688 decreased metabolic activity in all malignant cells, and demonstrated the lowest IC50 values for those of B-cell origin. IC50 values were also determined in 38 samples of CLL patients who were at different stages of the disease (stage Binet A, B, C) and had different cytogenetic aberrations (del(13q), +12, del(17p)). L-902688 was similarly cytotoxic to all 3 stages of the disease and all 3 groups of cytogenetic aberrations. Conversely, L-902688 reduced the metabolic activity of the PBMCs only at the highest concentrations tested. L-902688 had a 2-10-fold higher IC50 value in PBMCs (39.54 [micro]M) than in malignant cells (3,26-18,42 [micro]M). Next, we also demonstrated that EP4-selective receptor antagonist (CJ-042794) suppressed the L-902688-mediated inhibitory effects on metabolic activity, thus corroborating that EP4 activation is indeed responsible for the observed effects in CLL cells. We also found that treatment of CLL cells with L-902688 resulted in inhibition of NF-kB signaling pathway, inhibition of cell proliferation, and induction of caspase-mediated apoptosis. Next, we investigated the effects of L-902688 on the cytotoxic effects of the recently approved small molecules for the treatment of CLL (ibrutinib, idelalisib, venetoclax) and CD20-directed monoclonal antibodies (rituximab, ofatumumab and obinutuzumab). Ibrutinib and idelalisib decreased the metabolic activity of MEC-1 cells at all concentrations tested, whereas they had no effect on viability assuming cytostatic activity of both compounds against these cells. Treatment of cells with a combination of ibrutinib or idelalisib and L-902688 induced the cytotoxic activities throughout the concentration ranges, as shown by the decreased metabolic activities and viabilities of MEC-1 cells. Venetoclax induced a dose-dependent decrease of the metabolic activity and viability of MEC-1 cells. Treatment of cells with a combination of venetoclax and L-902688 increased the cytotoxic activity of venetoclax, as seen by the decreased metabolic activities and viabilities of these MEC-1. In addition, L-902688 also potentiated cytotoxic activity of ibrutinib or idelalisib in primary CLL cells. The cytotoxic effects of ibrutinib, idelalisib, or venetoclax in the combination with L-902688 were evaluated in silico using the CompuSyn software. The results demonstrated that the tested combinations act synergistically. Compound L-902688 was also tested in a combination with CD20-directed monoclonal antibodies. It was found to potentiate the cytotoxic activity of rituximab, ofatumumab and obinutuzumab, which manifested as decreased metabolic activity of CLL cells when the cells were incubated with a combination of L-902688 and monoclonal antibody. The cytotoxic effects of the tested combinations were further evaluated in the CompuSyn program and it was found that L-902688 acts synergistically with obinutuzumab and rituximab, while it acts additively with ofatumumab. The finding that L-902688 enhances the cytotoxic effect of new targeted drugs, small molecules as well as monoclonal antibodies opens up new possibilities for the development of therapeutic modalities for treatment of patients with CLL. In the second part of the doctoral dissertation, we evaluated the ability of new NOD2 agonists to enhance the antitumor activity of immune cells against CLL cells. The compounds were synthesized at the Faculty of Pharmacy and have improved pharmacokinetic properties compared to previous generations. First, NOD2 receptor agonists were tested in macrophages obtained by defferentiation of THP-1 monocyte cell line using phorbol-12-myristate-13-acetate (PMA). Differentiated THP-1 cells (PMA, 3 days) increased the expression of cell markers CD11b and CD68, demonstrating the viii success of differentiation and generation of macrophages in culture. Macrophages were then exposed to NOD2 agonists and MEC-1 cells for 24 or 48 h. MDP was used as a positive control, as it is known that several parts of bacterial cell walls such as MDP or lipopolysaccharide (LPS) can stimulate the antitumor activity of macrophages. Neither MDP, nor any of the tested compounds (10 %M) increased the cytotoxic potential of macrophages against MEC-1 cells. To better recapitulate conditions in vivo, PBMCs were used instead of macrophages differentiated from THP-1 cells. They were exposed to selected NOD2 agonists and MEC-1 cells for 24 h. Among all the tested compounds, SG101 markedly increased the cytotoxicity of PBMC cells against MEC-1 cells. The finding that SG101 may potentiate the antitumor activity of immune cells presents a promising opportunity for the development of potential (co)therapy for the treatment of CLL. The main purpose of the doctoral thesis is to contribute to the improvement of the therapy of patients with CLL. Although effective drugs are available today to treat CLL, many patients still do not respond to treatment or have relapses of the disease. CLL thus still remains an incurable disease for which we need new compounds and therapeutic approaches. The finding that L-902688 may potentiate the cytotoxic activity of selected compounds and that NOD2 agonists may enhance the antitumor activity of immune cells, opens up new possibilities for the development of therapeutic options for the treatment of patients with CLL.
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