| Popis: |
Kronična limfocitna levkemija (KLL) je najbolj pogosta podvrsta levkemije v zahodnem svetu, pri kateri pride do kopičenja malignih, nefunkcionalnih limfocitov B v periferni krvi. KLL je sprva počasi napredujoča bolezen, ki ima zelo heterogen klinični potek. Veliko bolnikov ob postavitvi diagnoze nima kliničnih težav in so lahko tudi več kot desetletje brez terapije. Pri nekaterih pa bolezen napreduje hitro in potrebujejo takojšnje zdravljenje. Danes so za zdravljenje KLL na voljo nova tarčna zdravila, kot so idelalisib, ibrutinib in venetoklaks, ki vse bolj nadomeščajo kemoimunoterapijo sestavljeno iz fludarabina, ciklofosfamida in rituksimaba (shema FCR). Kljub temu, da so nova tarčna zdravila zelo učinkovita in odobrena za zdravljenje bolnikov z neodzivno boleznijo ali pri ponovitvi bolezni, bolniki razvijajo odpornost nanje in imajo ponovitve bolezni. KLL tako še vedno ostaja neozdravljiva bolezen, za katero potrebujemo nove učinkovine in terapevtske pristope. V doktorski nalogi smo se zato usmerili v iskanje novih modulatorjev programirane celične smrti za zdravljenje KLL, kjer smo kot terapevtski tarči predpostavili receptor za prostaglandin E2, podtip 4 (EP4) in receptor nukleotid-vezoče oligomerizacijske domene 2 (NOD2). Receptor EP4 je s proteinom G sklopljen receptor in eden izmed štirih prostaglandinskih receptorjev tipa E. Njegov naravni ligand je prostaglandin E2 (PGE2), ki je pomemben regulator delovanja limfocitov B. Vezava PGE2 na receptor EP4 modulira od ciklični adenozin-monofosfat (cAMP)-odvisne in od cAMP-neodvisne signalne poti, med katerimi je najpomembnejša signalna pot NF-kB. Receptor EP4 je bil prepoznan kot tarča za zdravljenje B celičnih levkemij in limfomov. Agonist EP4, PGE1-OH je inhibiral signalno pot jedrnega dejavnika kappa B (NF-kB), kar je vodilo v povečano občutljivost malignih celic B na učinkovini bortezomib in doksorubicin. Prav tako je PGE1-OH sprožil celično smrt na modelih levkemij in limfomov kot tudi na primarnih celicah KLL. Danes so na voljo zelo specifični agonisti EP4, kot je na primer spojina L-902688, ki je 4000-krat bolj selektivna za receptor EP4 kot za ostale receptorje EP1-3. L-902688 je v raziskavah na podganah pokazala dobro biološko razpoložljivost ter dolgo razpolovno dobo. Večina raziskav s to spojino je bilo narejenih na gladkih mišičnih celicah, ni pa znano kako spojina deluje na malignih celicah B. Receptor NOD2 je citosolni receptor, udeležen v prepoznavanju znotrajceličnih patogenov v okviru prirojenega imunskega sistema. Njegov naravni ligand je muramil dipeptid (MDP), majhen strukturni motiv, ki se nahaja v celični steni bakterij. Aktivacija receptorja ii NOD2 vodi do protimikrobnega in vnetnega delovanja, prav tako pa sodeluje tudi pri procesu apoptoze. Agonisti NOD2 so dobre imunostimulatorne spojine in imajo s tem posredno tudi potencialno protivirusno, protibakterijsko in protitumorno delovanje. Sintetiziranih je bilo že več generacij agonistov receptorjev NOD2, ki so se v testih in vitro izkazale kot močne imunostimulatorne spojine. Kot najbolj perspektivna spojina se je izkazala spojina SG8 strukturno gledano gre za spojino, ki vsebuje trans-feruloil-glicinski mimetik N-acetilmuraminske kisline, ki je povezan z dipeptidom, sestavljenim iz L-valina (L-Val) in D-glutaminske kisline (D-Glu). Spojina SG8 je v testih in vitro izkazala zelo dobro imunostimulatorno delovanje, ki pa se žal ni ohranilo in vivo. Da bi se izboljšale njene farmakokinetične lastnosti, so sintetizirali novo generacijo agonistov NOD2. Ker lahko nekateri agonisti NOD2 povečajo protitumorno delovanje imunskih celic, smo v doktorski nalogi preverili ali lahko le-to povečajo tudi izbrani predstavniki nove generacijo agonistov NOD2. V prvem delu doktorske naloge smo ovrednotili sposobnost agonista receptorja EP4, L-902688, da povzroči celično smrt na celičnih linijah levkemij in limfomov, primarnih celicah KLL in mononuklearnih celicah periferne krvi (PBMC). Zbiranje krvnih vzorcev bolnikov s KLL je potekalo na Univerzitetnem kliničnem centru Ljubljana, Kliničnem oddelku za hematologijo Interne klinike (klinični študiji 93/12/10 in 0120-136/2019/4, odobreni s strani Komisije Republike Slovenije za medicinsko etiko). Ugotovili smo, da je spojina L-902688 zmanjšala metabolno aktivnost pri vseh malignih celicah, najnižja vrednost IC50 pa je bila določena na celičnih linijah B-celičnega izvora. Vrednosti IC50 smo določili tudi na 38 vzorcih bolnikov s KLL, ki so bili v različnem stadiju bolezni (stadij Binet A, B, C) in imeli različne citogenetske spremembe (del(13q), +12, del(17p)). L-902688 je deloval podobno citotoksično na celice iz vseh 3 skupin stadijev bolezni in citogenetskih sprememb. Spojina L-902688 je izkazovala dobro selektivno toksičnost, saj je imela 2-10 krat višjo IC50 vrednost na celicah PBMC (39,54 [mikro]M), kot na malignih celicah (3,26-18,42 [mikro]M). Z uporabo selektivnega antagonista receptorja EP4 (CJ-042794) smo potrdili, da spojina L-902688 posreduje svoje citotoksične učinke na celicah KLL preko receptorja EP4. Ugotovili smo tudi, da L-902688 na celicah KLL povzroča inhibicijo signalne poti NF-kB, inhibicijo celične proliferacije ter inducira apoptozo, ki je posredovana preko kaspaz. Nato smo na modelni celični liniji MEC-1 ovrednotili citotoksično delovanje L-902688 v kombinaciji z novimi tarčnimi zdravili za zdravljenje KLL (ibrutinib, idelalisib, iii venetoklaks) in monoklonskimi protitelesi usmerjenimi proti CD20 (rituksimab, ofatumumab in obinutuzumab). Ugotovili smo, da v testnih koncentracijah ibrutinib in idelalisib zmanjšata metabolno aktivnost celic MEC-1, medtem ko v teh koncentracijah nimata učinka na viabilnost. Sklepamo, da v teh koncentracijah spojini delujeta citostatično. Ko smo celicam dodali ibrutinib ali idelalisib v kombinaciji z L-902688, smo videli tako znižano metabolno aktivnost kot viabilnost celic MEC-1. Ko smo celice MEC-1 inkubirali z venetoklaksom, je le-ta zmanjšal njihovo metabolno aktivnost in viabilnost. Ko smo celice tretitali z venetoklaksom v kombinaciji z L-902688, se je njegova citotoksičnost povečala, kar smo izmerili kot znižano metabolno aktivnost in viabilnost celic MEC-1. Prav tako smo L-902688 v kombinaciji z ibrutinibom ali idelalisibom testirali tudi na primarnih celicah KLL. V obeh primerih je L-902688 povečal citotoksično delovanje ibrutiniba ali idelalisiba. Citotoksične učinke ibrutiniba, idelalisiba ali venetoklaksa v kombinaciji z L-902688 smo ovrednotili tudi s programom CompuSyn. Analiza podatkov je pokazala, da testirane kombinacije spojin delujejo sinergistično. Citotoksičnost spojine L-902688 smo na celicah KLL ovrednotili tudi v kombinaciji z monoklonskimi protitelesi. Ugotovili smo, da je L-902688 povečal citotoksičnost rituksimaba, ofatumumaba in obinutuzumaba, kar smo videli kot znižano metabolno aktivnost celic KLL, ko smo celice inkubirali s kombinacijo L-902688 in monoklonskega protitelesa. Citotoksične učinke testiranih kombinacij smo analizirali še v programu CompuSyn in ugotovili, da L-902688 deluje sinergistično z obinutuzumabom in rituksimabom, medtem ko z ofatumumabom deluje aditivno. Ugotovitev, da L-902688 povača citotoksičnost novih tarčnih zdravil kot tudi monoklonskih protiteles odpira nove terapevtske možnosti za zdravljenje bolnikov s KLL. V drugem delu doktorske naloge smo ovrednotili sposobnost novih agonistov receptorja NOD2, da povečajo protitumorno delovanje imunskih celic proti celicam KLL. Spojine so sintetizirali na Fakulteti za farmacijo in imajo izboljšane farmakokinetične lastnosti v primerjavi s prejšnjimi generacijami. Najprej smo njihovo sposobnost, da povečajo protitumorno delovanje imunskih celic ocenili na makrofagih, ki smo jih pridobili z diferenciacijo monocitne celične linije THP-1 s pomočjo forbol 12-miristat 13-acetata (PMA). Pri celicah THP-1, ki smo jih 3 dni gojili v prisotnosti PMA, smo videli povišanje izražanja celičnih označevalcev CD11b in CD68, kar dokazuje uspešnost diferenciacije in pridobitev makrofagov v kulturi. Makrofage smo nato izpostavili agonistom receptorja NOD2 in celicam MEC-1 za 24 ali 48 h. Kot pozitivno kontrolo smo uporabili MDP, saj je iv znano, da lahko z deli bakterijske celične stene kot sta MDP ali lipopolisaharid (LPS) stimuliramo protitumorno delovanje makrofagov. Rezultati so pokazali, da ne MDP, ne katera izmed izbranih spojin v testni koncentraciji (10 [mikro]M) ni povečala citotoksičnega potenciala makrofagov proti celicam MEC-1. Da bi bolje ponazorili pogoje in vivo, smo namesto makrofagov, diferenciranih iz celic THP-1, uporabili PBMC. Slednje smo izpostavili izbranim agonistom NOD2 in celicam MEC-1 za 24 h. Izmed vseh izbranih spojin je spojina SG101 izrazito povečala citotoksičnost PBMC proti celicam MEC-1. Ugotovitev, da lahko spojina SG101 poveča protitumorno delovanje imunskih celic, predstavlja obetavno možnost za razvoj potencialne (ko)terapije za zdravljenje KLL. Poglaviten namen doktorskega dela je bil prispevati k izboljšanju terapije bolnikov s KLL. Čeprav so danes za zdravljenje KLL na voljo učinkovita zdravila, bolniki razvijajo odpornost nanje in imajo ponovitve bolezni. KLL tako še vedno ostaja neozdravljiva bolezen, za katero potrebujemo nove učinkovine in terapevtske pristope. Ugotovitvi, da L-902688 povača citotoksičnost novih tarčnih zdravil kot tudi monoklonskih protiteles ter da lahko agonisti NOD2 stimulirajo protitumorno delovanje imunskih celic, odpirata nove terapevtske možnosti za zdravljenje bolnikov s KLL. 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. |
