Bruton tyrosine kinase (BTK) inhibitor therapy induces peripheral blood lymphocytosis in chronic lymphocytic leukemia (CLL), which lasts for several months. It remains unclear whether nongenetic adaptation mechanisms exist, allowing CLL cells' survival during BTK inhibitor-induced lymphocytosis and/or playing a role in therapy resistance. We show that in approximately 70% of CLL cases, ibrutinib treatment in vivo increases Akt activity above pretherapy levels within several weeks, leading to compensatory CLL cell survival and a more prominent lymphocytosis on therapy. Ibrutinib-induced Akt phosphorylation (pAktS473) is caused by the upregulation of Forkhead box protein O1 (FoxO1) transcription factor, which induces expression of Rictor, an assembly protein for the mTORC2 protein complex that directly phosphorylates Akt at serine 473 (S473). Knockout or inhibition of FoxO1 or Rictor led to a dramatic decrease in Akt phosphorylation and growth disadvantage for malignant B cells in the presence of ibrutinib (or PI3K inhibitor idelalisib) in vitro and in vivo. The FoxO1/Rictor/pAktS473 axis represents an early nongenetic adaptation to B cell receptor (BCR) inhibitor therapy not requiring PI3Kδ or BTK kinase activity. We further demonstrate that FoxO1 can be targeted therapeutically and its inhibition induces CLL cells' apoptosis alone or in combination with BTK inhibitors (ibrutinib, acalabrutinib, pirtobrutinib) and blocks their proliferation triggered by T cell factors (CD40L, IL-4, and IL-21).

• MeSH
• adenin * analogy a deriváty   farmakologie   MeSH
• chronická lymfatická leukemie * farmakoterapie   metabolismus   genetika   patologie   MeSH
• forkhead box protein O1 * metabolismus   genetika   MeSH
• fosforylace MeSH
• lidé MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nádorové proteiny metabolismus   genetika   MeSH
• piperidiny * farmakologie   MeSH
• protein RICTOR * genetika   metabolismus   MeSH
• proteinkinasa BTK metabolismus   genetika   antagonisté a inhibitory   MeSH
• protoonkogenní proteiny c-akt * metabolismus   genetika   MeSH
• pyrazoly * farmakologie   MeSH
• pyrimidiny * farmakologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Approval of ibrutinib and idelalisib has changed the therapy of chronic lymphocytic leukemia (CLL), however, there is a clear need to identify efficient combinatorial therapy with these “BCR inhibitors”. We analyses transcriptome of CLL cells from patients treated with “BCR inhibitors” and identified 2 novel mechanisms that malignant B cells utilize in vivo to adapt and survive BTK inhibition (and potentially PI3K inhibition). We will further study these molecular pathways and pre-clinically test the use of 2 novel inhibitors targeting these mechanisms in combination with “BCR inhibitors” (aim 1a/b). Additionally, we analyzed CLL cell-surface molecules that could be potentially targeted by monoclonal antibodies approved or tested in other diseases. We observed that ibrutinib and idelalisib are inducing CXCR4 expression, and that a novel therapeutic anti-CXCR4 antibody potently synergizes with ibrutinib and idelalisib (aim 2). We plan to study the adaptation mechanisms to ibrutinib versus idelalisib therapy, and pre-clinically test the 3 novel therapeutic approaches.

Použití ibrutinibu a idelalisibu změnilo terapii chronické lymfatické leukémie (CLL), ale je třeba identifikovat efektivní terapeutické kombinace s těmito “BCR inhibitory”. Analyzovali jsme transkriptom CLL buněk získaných od pacientů během podávání “BCR inhibitorů” a identifkovali 2 nové mechanismy adaptace, které maligní B lymfocyty využívají in vivo pro přežití při inhibici BTK (a potenciálně PI3K inhibici). Budeme dále studovat tyto molekulární dráhy a pre-klinicky testovat použití 2 nových inhibitorů cílících na tyto mechanismy v kombinaci s „BCR inhibitory” (cíl 1a/b). Dále jsme analyzovali expresi molekul na povrchu CLL buněk, které by potenciálně mohly být cíleny monoklonálními protilátkami schválenými či testovanými u jiných chorob. Zjistili jsme, že ibrutinib i idelalisib indukují expresi CXCR4 a pozorovali potentní synergický účinek s novou terapeutickou anti-CXCR4 protilátkou v kombinaci s ibrutinibem a idelalisibem (cíl 2). Plánujeme dále studovat adaptační mechanismy u terapie ibrutinibem versus idelalisibem a pre-klinicky testovat 3 zmíněné nové terapeutické přístupy.

• Klíčová slova
• rezistence, resistance, chronic lymphocytic leukemia, therapy, Chronická lymfocytární leukémie, adaptace, adaptation, mTOR, mTOR, BTK, BTK, CXCR4, CXCR4, ibrutinib, ibrutinib, BCR signalling, BCR signalizace, therapy, idelalisib, BCR inhibitor, idelalisib, BCR inhibitor, PI3K, PI3K,

• NLK Publikační typ
• závěrečné zprávy o řešení grantu AZV MZ ČR

Peptides that form transmembrane barrel-stave pores are potential alternative therapeutics for bacterial infections and cancer. However, their optimization for clinical translation is hampered by a lack of sequence-function understanding. Recently, we have de novo designed the first synthetic barrel-stave pore-forming antimicrobial peptide with an identified function of all residues. Here, we systematically mutate the peptide to improve pore-forming ability in anticipation of enhanced activity. Using computer simulations, supported by liposome leakage and atomic force microscopy experiments, we find that pore-forming ability, while critical, is not the limiting factor for improving activity in the submicromolar range. Affinity for bacterial and cancer cell membranes needs to be optimized simultaneously. Optimized peptides more effectively killed antibiotic-resistant ESKAPEE bacteria at submicromolar concentrations, showing low cytotoxicity to human cells and skin model. Peptides showed systemic anti-infective activity in a preclinical mouse model of Acinetobacter baumannii infection. We also demonstrate peptide optimization for pH-dependent antimicrobial and anticancer activity.

• MeSH
• Acinetobacter baumannii účinky léků   MeSH
• antibakteriální látky farmakologie   chemie   chemická syntéza   MeSH
• antimikrobiální peptidy chemie   farmakologie   chemická syntéza   MeSH
• antitumorózní látky * farmakologie   chemie   chemická syntéza   MeSH
• kationické antimikrobiální peptidy farmakologie   chemie   chemická syntéza   MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• racionální návrh léčiv * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Venetoclax (VEN), a B-cell lymphoma 2 (BCL2) inhibitor, has a promising single-agent activity in mantle cell lymphoma (MCL), acute lymphoblastic leukemia (ALL), and large BCLs, but remissions were generally short, which call for rational drug combinations. Using a panel of 21 lymphoma and leukemia cell lines and 28 primary samples, we demonstrated strong synergy between VEN and A1155463, a BCL-XL inhibitor. Immunoprecipitation experiments and studies on clones with knockout of expression or transgenic expression of BCL-XL confirmed its key role in mediating inherent and acquired VEN resistance. Of note, the VEN and A1155463 combination was synthetically lethal even in the cell lines with lack of expression of the proapoptotic BCL2L11/BIM and in the derived clones with genetic knockout of BCL2L11/BIM. This is clinically important because BCL2L11/BIM deletion, downregulation, or sequestration results in VEN resistance. Immunoprecipitation experiments further suggested that the proapoptotic effector BAX belongs to principal mediators of the VEN and A1155463 mode of action in the BIM-deficient cells. Lastly, the efficacy of the new proapoptotic combination was confirmed in vivo on a panel of 9 patient-derived lymphoma xenografts models including MCL (n = 3), B-ALL (n = 2), T-ALL (n = 1), and diffuse large BCL (n = 3). Because continuous inhibition of BCL-XL causes thrombocytopenia, we proposed and tested an interrupted 4 days on/3 days off treatment regimen, which retained the desired antitumor synergy with manageable platelet toxicity. The proposed VEN and A1155463 combination represents an innovative chemotherapy-free regimen with significant preclinical activity across diverse BCL2+ hematologic malignancies irrespective of the BCL2L11/BIM status.

• MeSH
• antitumorózní látky farmakologie   terapeutické užití   MeSH
• apoptóza účinky léků   MeSH
• benzothiazoly MeSH
• bicyklické sloučeniny heterocyklické * farmakologie   terapeutické užití   MeSH
• chemorezistence * MeSH
• isochinoliny MeSH
• lidé MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• protein bcl-X * metabolismus   antagonisté a inhibitory   MeSH
• protein BCL2L11 * metabolismus   genetika   MeSH
• protoonkogenní proteiny c-bcl-2 metabolismus   antagonisté a inhibitory   MeSH
• sulfonamidy * farmakologie   terapeutické užití   MeSH
• synergismus léků MeSH
• xenogenní modely - testy antitumorózní aktivity MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Several in vitro models have been developed to mimic chronic lymphocytic leukemia (CLL) proliferation in immune niches; however, they typically do not induce robust proliferation. We prepared a novel model based on mimicking T-cell signals in vitro and in patient-derived xenografts (PDXs). Six supportive cell lines were prepared by engineering HS5 stromal cells with stable expression of human CD40L, IL4, IL21, and their combinations. Co-culture with HS5 expressing CD40L and IL4 in combination led to mild CLL cell proliferation (median 7% at day 7), while the HS5 expressing CD40L, IL4, and IL21 led to unprecedented proliferation rate (median 44%). The co-cultures mimicked the gene expression fingerprint of lymph node CLL cells (MYC, NFκB, and E2F signatures) and revealed novel vulnerabilities in CLL-T-cell-induced proliferation. Drug testing in co-cultures revealed for the first time that pan-RAF inhibitors fully block CLL proliferation. The co-culture model can be downscaled to five microliter volume for large drug screening purposes or upscaled to CLL PDXs by HS5-CD40L-IL4 ± IL21 co-transplantation. Co-transplanting NSG mice with purified CLL cells and HS5-CD40L-IL4 or HS5-CD40L-IL4-IL21 cells on collagen-based scaffold led to 47% or 82% engraftment efficacy, respectively, with ~20% of PDXs being clonally related to CLL, potentially overcoming the need to co-transplant autologous T-cells in PDXs.

• MeSH
• buňky stromatu * metabolismus   patologie   MeSH
• chronická lymfatická leukemie * patologie   genetika   farmakoterapie   MeSH
• inhibitory proteinkinas farmakologie   MeSH
• interleukiny genetika   metabolismus   MeSH
• kokultivační techniky * MeSH
• lidé MeSH
• ligand CD40 * metabolismus   genetika   MeSH
• myši MeSH
• proliferace buněk * MeSH
• T-lymfocyty imunologie   metabolismus   MeSH
• xenogenní modely - testy antitumorózní aktivity MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Východiska: Folikulární lymfom (FL) je nejčastějším indolentním non-Hodgkinským lymfomem v západním světě. U většiny pacientů se jedná o indolentní onemocnění, ale u cca 20 % případů dochází po úvodní léčbě k časnému relapsu, což je spojeno s kratším celkovým přežitím. Další prognosticky závažnou událostí je histologická transformace FL do agresivního lymfomu, nejčastěji do difuzního velkobuněčného B-lymfomu. Díky genomickým studiím a myším modelům se nám lépe daří chápat molekulární podstatu vzniku FL a evoluci „agresivních“ subklonů buněk. Zároveň se také v posledních letech podařilo popsat deregulace molekulárních drah přispívajících k histologické transformaci FL. Cíl: V tomto přehledovém článku shrnujeme komplexní mechanizmy, které jsou na molekulární úrovni zodpovědné za vznik, progresi a agresivitu FL a jeho transformaci. Domníváme se, že tato pozorování u FL mají obecnější přesah pro pochopení mechanizmů, které vedou k evoluci „agresivity“ nádorového onemocnění jako je divergentní evoluce, intraklonální variabilita a nádorová plasticita.

Background: Follicular lymphoma (FL) is the most common indolent non-Hodgkin‘s lymphoma in the Western world. It is an indolent disease in most patients, but about 20% of patients experience an early relapse after initial treatment, which is associated with shorter overall survival. A histological transformation into an aggressive lymphoma, most frequently diffuse large-cell B-lymphoma, represents another prognostically unfavorable event in the course of the disease. Thanks to recent genomic studies and mouse models, we are able to better understand the molecular nature of the FL onset and evolution of “aggressive” subclones of cells. Recently, deregulation of several molecular pathways associated with the histological transformation has also been described. Purpose: This review summarizes the complex molecular mechanisms responsible for FL onset, progression, aggressiveness, and transformation. We believe that the observations in FL have some general implications for understanding the mechanisms leading to the evolution of cancer “aggressiveness,” such as divergent evolution, intraclonal variability and tumor plasticity.

• Klíčová slova
• histologická transformace, transformovaný folikulární lymfom,
• MeSH
• difúzní velkobuněčný B-lymfom genetika   patologie   MeSH
• folikulární lymfom * genetika   patologie   MeSH
• lidé MeSH
• nádorová transformace buněk * genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• přehledy MeSH

The involvement of microRNAs (miRNAs) in orchestrating self-renewal and differentiation of stem cells has been revealed in a number of recent studies. And while in human pluripotent stem cells, miRNAs have been directly linked to the core pluripotency network, including the cell cycle regulation and the maintenance of the self-renewing capacity, their role in the onset of differentiation in other contexts, such as determination of neural cell fate, remains poorly described. To bridge this gap, we used three model cell types to study miRNA expression patterns: human embryonic stem cells (hESCs), hESCs-derived self-renewing neural stem cells (NSCs), and differentiating NSCs. The comprehensive miRNA profiling presented here reveals novel sets of miRNAs differentially expressed during human neural cell fate determination in vitro. Furthermore, we report a miRNA expression profile of self-renewing human NSCs, which has been lacking to this date. Our data also indicates that miRNA clusters enriched in NSCs share the target-determining seed sequence with cell cycle regulatory miRNAs expressed in pluripotent hESCs. Lastly, our mechanistic experiments confirmed that cluster miR-17-92, one of the NSCs-enriched clusters, is directly transcriptionally regulated by transcription factor c-MYC.

• MeSH
• buněčná diferenciace genetika   MeSH
• embryonální kmenové buňky MeSH
• lidé MeSH
• mikro RNA * genetika   metabolismus   MeSH
• nervové kmenové buňky * metabolismus   MeSH
• stanovení celkové genové exprese MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• 5' nepřekládaná oblast genetika   MeSH
• alternativní sestřih * MeSH
• proteosyntéza MeSH
• sestřih RNA * genetika   MeSH
• Publikační typ
• komentáře MeSH
• úvodníky MeSH

Chronic lymphocytic leukemia (CLL) cells have variably low surface IgM (sIgM) levels/signaling capacity, influenced by chronic antigen engagement at tissue sites. Within these low levels, CLL with relatively high sIgM (CLLhigh) progresses more rapidly than CLL with low sIgM (CLLlow). During ibrutinib therapy, surviving CLL cells redistribute into the peripheral blood and can recover sIgM expression. Return of CLL cells to tissue may eventually recur, where cells with high sIgM could promote tumor growth. We analyzed time to new treatment (TTNT) following ibrutinib in 70 patients with CLL (median follow-up of 66 months) and correlated it with pretreatment sIgM levels and signaling characteristics. Pretreatment sIgM levels correlated with signaling capacity, as measured by intracellular Ca2+ mobilization (iCa2+), in vitro (r = 0.70; P < .0001). High sIgM levels/signaling strongly correlated with short TTNT (P < .05), and 36% of patients with CLLhigh vs 8% of patients with CLLlow progressed to require a new treatment. In vitro, capacity of ibrutinib to inhibit sIgM-mediated signaling inversely correlated with pretherapy sIgM levels (r = -0.68; P = .01) or iCa2+ (r = -0.71; P = .009). In patients, sIgM-mediated iCa2+ and ERK phosphorylation levels were reduced by ibrutinib therapy but not abolished. The residual signaling capacity downstream of BTK was associated with high expression of sIgM, whereas it was minimal when sIgM expression was low (P < .05). These results suggested that high sIgM levels facilitated CLL cell resistance to ibrutinib in patients. The CLL cells, surviving in the periphery with high sIgM expression, include a dangerous fraction that is able to migrate to tissue and receive proliferative stimuli, which may require targeting by combined approaches.

• MeSH
• adenin analogy a deriváty   MeSH
• chronická lymfatická leukemie * metabolismus   MeSH
• imunoglobulin M MeSH
• lidé MeSH
• piperidiny MeSH
• vápník MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

We aim to study follicular lymphomas (FL) that experience transformation (tFL) or early progression, and the molec. pathways defining the aggressiveness of intra-clonal subpopulation of chronic lymphocytic leukemia (CLL) cells. Our data suggest that the mechanisms for “aggressiveness” in FL and CLL are partially analogical, and involve (de)regulation of miRNAs. We plan to i) perform the first global miRNA expression profiling (Illumina) in paired samples of FL-tFL, and define their functional role. We observed that miR-150 is down-modulated in tFL, which leads to over-expression of FOXP1 transcription factor, and adverse outcome in FL. We will also test if tFL-associated miRNAs can be used as predictors of relapse, survival or transformation in FL. ii) in CLL we will study miRNAs and their targets in the proliferative intra-clonal subpopulation of cells that has interacted in the microenvironment, has active PI3K/BCR/MAPK signalling, and differential expression of multiple miRNAs (including miR-150). Additionally, we will test miRNAs as predictors of response to FCR therapy in CLL.

Plánujeme studovat folikulární lymfomy (FL), u nichž dojde k časnému relapsu nebo transformaci (tFL) a také molek. dráhy, které řídí chování „agresivní“ intra-klonální populace buněk chronické lymfatické leukémie (CLL). Naše předběžné výsledky naznačují, že mechanismy řídící “agresivitu” FL a CLL jsou částečně analogické a zahrnují (de)regulaci miRNA. Plánujeme: i) provést první komplexní expresní profilování miRNA u párových vzorků FL a tFL (Illumina) a definovat zde jejich biologickou funkci. Předběžná data ukazují, že miR-150 je sníženě exprimována u všech tFL. To vede k zvýšení exprese transkripčního faktoru FOXP1 a asociuje s agresivitou FL. Budeme také testovat využití tFL-asociovaných miRNA jako prediktorů časného relapsu, přežití či transformace FL ii) u CLL budeme studovat miRNA a jejich cíle v intra-klonální subpopulaci buněk, které recentně interagovaly v mikroprostředí a mají aktivní PI3K/BCR/MAPK signalizaci. Tyto buňky mají rozdílnou expresi množství miRNA (včetně miR-150). Navíc otestujeme využití vybraných miRNA jako prediktorů odpovědi na FCR léčbu u CLL.

• Klíčová slova
• microRNA, microRNA, prognóza, prognosis, predikce, prediction, chronic lymphocytic leukemia, folikulární lymfom, chronická lymfatická leukémie, folicullar lymphoma,

• NLK Publikační typ
• závěrečné zprávy o řešení grantu AZV MZ ČR