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
• Stromal Cells * metabolism   pathology   MeSH
• Leukemia, Lymphocytic, Chronic, B-Cell * pathology   genetics   drug therapy   MeSH
• Protein Kinase Inhibitors pharmacology   MeSH
• Interleukin-21 MeSH
• Interleukins genetics   metabolism   MeSH
• Coculture Techniques * MeSH
• Humans MeSH
• CD40 Ligand * metabolism   genetics   MeSH
• Mice MeSH
• Cell Proliferation * MeSH
• T-Lymphocytes immunology   metabolism   MeSH
• Xenograft Model Antitumor Assays MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Protein Kinase Inhibitors MeSH
• Interleukin-21 MeSH
• Interleukins MeSH
• CD40 Ligand * MeSH

T cells are key components in environments that support chronic lymphocytic leukemia (CLL), activating CLL-cell proliferation and survival. Here, we review in vitro and in vivo model systems that mimic CLL-T-cell interactions, since these are critical for CLL-cell division and resistance to some types of therapy (such as DNA-damaging drugs or BH3-mimetic venetoclax). We discuss approaches for direct CLL-cell co-culture with autologous T cells, models utilizing supportive cell lines engineered to express T-cell factors (such as CD40L) or stimulating CLL cells with combinations of recombinant factors (CD40L, interleukins IL4 or IL21, INFγ) and additional B-cell receptor (BCR) activation with anti-IgM antibody. We also summarize strategies for CLL co-transplantation with autologous T cells into immunodeficient mice (NOD/SCID, NSG, NOG) to generate patient-derived xenografts (PDX) and the role of T cells in transgenic CLL mouse models based on TCL1 overexpression (Eµ-TCL1). We further discuss how these in vitro and in vivo models could be used to test drugs to uncover the effects of targeted therapies (such as inhibitors of BTK, PI3K, SYK, AKT, MEK, CDKs, BCL2, and proteasome) or chemotherapy (fludarabine and bendamustine) on CLL-T-cell interactions and CLL proliferation.

• Keywords
• B cells, CD40L, Eμ-TCL1, IL-21, IL-4, T cells, chronic lymphocytic leukemia, co-culture, fludarabine, ibrutinib, interactions, interleukin, microenvironment, models, therapy resistance, venetoclax, xenograft,
• Publication type
• Journal Article MeSH
• Review MeSH

Better understanding of GBM signalling networks in-vivo would help develop more physiologically relevant ex vivo models to support therapeutic discovery. A "functional proteomics" screen was undertaken to measure the specific activity of a set of protein kinases in a two-step cell-free biochemical assay to define dominant kinase activities to identify potentially novel drug targets that may have been overlooked in studies interrogating GBM-derived cell lines. A dominant kinase activity derived from the tumour tissue, but not patient-derived GBM stem-like cell lines, was Bruton tyrosine kinase (BTK). We demonstrate that BTK is expressed in more than one cell type within GBM tissue; SOX2-positive cells, CD163-positive cells, CD68-positive cells, and an unidentified cell population which is SOX2-negative CD163-negative and/or CD68-negative. The data provide a strategy to better mimic GBM tissue ex vivo by reconstituting more physiologically heterogeneous cell co-culture models including BTK-positive/negative cancer and immune cells. These data also have implications for the design and/or interpretation of emerging clinical trials using BTK inhibitors because BTK expression within GBM tissue was linked to longer patient survival.

• MeSH
• Glioblastoma enzymology   mortality   pathology   MeSH
• Coculture Techniques methods   MeSH
• Humans MeSH
• Survival Rate MeSH
• Cell Line, Tumor MeSH
• Neoplastic Stem Cells enzymology   MeSH
• Brain Neoplasms enzymology   mortality   pathology   MeSH
• Agammaglobulinaemia Tyrosine Kinase metabolism   MeSH
• Proteome metabolism   MeSH
• Proteomics methods   MeSH
• Signal Transduction * MeSH
• SOXB1 Transcription Factors metabolism   MeSH
• Cell Survival MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• BTK protein, human MeSH Browser
• Agammaglobulinaemia Tyrosine Kinase MeSH
• Proteome MeSH
• SOX2 protein, human MeSH Browser
• SOXB1 Transcription Factors MeSH

The approval of BTK and PI3K inhibitors (ibrutinib, idelalisib) represents a revolution in the therapy of B cell malignancies such as chronic lymphocytic leukemia (CLL), mantle-cell lymphoma (MCL), diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), or Waldenström's macroglobulinemia (WM). However, these "BCR inhibitors" function by interfering with B cell pathophysiology in a more complex way than anticipated, and resistance develops through multiple mechanisms. In ibrutinib treated patients, the most commonly described resistance-mechanism is a mutation in BTK itself, which prevents the covalent binding of ibrutinib, or a mutation in PLCG2, which acts to bypass the dependency on BTK at the BCR signalosome. However, additional genetic aberrations leading to resistance are being described (such as mutations in the CARD11, CCND1, BIRC3, TRAF2, TRAF3, TNFAIP3, loss of chromosomal region 6q or 8p, a gain of Toll-like receptor (TLR)/MYD88 signaling or gain of 2p chromosomal region). Furthermore, relative resistance to BTK inhibitors can be caused by non-genetic adaptive mechanisms leading to compensatory pro-survival pathway activation. For instance, PI3K/mTOR/Akt, NFkB and MAPK activation, BCL2, MYC, and XPO1 upregulation or PTEN downregulation lead to B cell survival despite BTK inhibition. Resistance could also arise from activating microenvironmental pathways such as chemokine or integrin signaling via CXCR4 or VLA4 upregulation, respectively. Defining these compensatory pro-survival mechanisms can help to develop novel therapeutic combinations of BTK inhibitors with other inhibitors (such as BH3-mimetic venetoclax, XPO1 inhibitor selinexor, mTOR, or MEK inhibitors). The mechanisms of resistance to PI3K inhibitors remain relatively unclear, but some studies point to MAPK signaling upregulation via both genetic and non-genetic changes, which could be co-targeted therapeutically. Alternatively, drugs mimicking the BTK/PI3K inhibition effect can be used to prevent adhesion and/or malignant B cell migration (chemokine and integrin inhibitors) or to block the pro-proliferative T cell signals in the microenvironment (such as IL4/STAT signaling inhibitors). Here we review the genetic and non-genetic mechanisms of resistance and adaptation to the first generation of BTK and PI3K inhibitors (ibrutinib and idelalisib, respectively), and discuss possible combinatorial therapeutic strategies to overcome resistance or to increase clinical efficacy.

• Keywords
• B cell malignancies, B cell receptor, BCR inhibitor, adaptation, ibrutinib, resistance, targeted therapy,
• Publication type
• Journal Article MeSH

The introduction of anti-CD20 monoclonal antibodies such as rituximab, ofatumumab, or obinutuzumab improved the therapy of B-cell malignancies even though the precise physiological role and regulation of CD20 remains unclear. Furthermore, CD20 expression is highly variable between different B-cell malignancies, patients with the same malignancy, and even between intraclonal subpopulations in an individual patient. Several epigenetic (EZH2, HDAC1/2, HDAC1/4, HDAC6, complex Sin3A-HDAC1) and transcription factors (USF, OCT1/2, PU.1, PiP, ELK1, ETS1, SP1, NFκB, FOXO1, CREM, SMAD2/3) regulating CD20 expression (encoded by MS4A1) have been characterized. CD20 is induced in the context of microenvironmental interactions by CXCR4/SDF1 (CXCL12) chemokine signaling and the molecular function of CD20 has been linked to the signaling propensity of B-cell receptor (BCR). CD20 has also been shown to interact with multiple other surface proteins on B cells (such as CD40, MHCII, CD53, CD81, CD82, and CBP). Current efforts to combine anti-CD20 monoclonal antibodies with BCR signaling inhibitors targeting BTK or PI3K (ibrutinib, acalabrutinib, idelalisib, duvelisib) or BH3-mimetics (venetoclax) lead to the necessity to better understand both the mechanisms of regulation and the biological functions of CD20. This is underscored by the observation that CD20 is decreased in response to the "BCR inhibitor" ibrutinib which largely prevents its successful combination with rituximab. Several small molecules (such as histone deacetylase inhibitors, DNA methyl-transferase inhibitors, aurora kinase A/B inhibitors, farnesyltransferase inhibitors, FOXO1 inhibitors, and bryostatin-1) are being tested to upregulate cell-surface CD20 levels and increase the efficacy of anti-CD20 monoclonal antibodies. Herein, we review the current understanding of CD20 function, and the mechanisms of its regulation in normal and malignant B cells, highlighting the therapeutic implications.

• MeSH
• Antigens, CD20 * MeSH
• B-Lymphocytes MeSH
• Leukemia, Lymphocytic, Chronic, B-Cell * MeSH
• Humans MeSH
• Antibodies, Monoclonal MeSH
• Pyrimidines MeSH
• Rituximab MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Antigens, CD20 * MeSH
• Antibodies, Monoclonal MeSH
• Pyrimidines MeSH
• Rituximab MeSH

Introduction of small-molecule inhibitors of B-cell receptor signaling and BCL2 protein significantly improves therapeutic options in chronic lymphocytic leukemia. However, some patients suffer from adverse effects mandating treatment discontinuation, and cases with TP53 defects more frequently experience early progression of the disease. Development of alternative therapeutic approaches is, therefore, of critical importance. Here we report details of the anti-chronic lymphocytic leukemia single-agent activity of MU380, our recently identified potent, selective, and metabolically robust inhibitor of checkpoint kinase 1. We also describe a newly developed enantioselective synthesis of MU380, which allows preparation of gram quantities of the substance. Checkpoint kinase 1 is a master regulator of replication operating primarily in intra-S and G2/M cell cycle checkpoints. Initially tested in leukemia and lymphoma cell lines, MU380 significantly potentiated efficacy of gemcitabine, a clinically used inducer of replication stress. Moreover, MU380 manifested substantial single-agent activity in both TP53-wild type and TP53-mutated leukemia and lymphoma cell lines. In chronic lymphocytic leukemia-derived cell lines MEC-1, MEC-2 (both TP53-mut), and OSU-CLL (TP53-wt) the inhibitor impaired cell cycle progression and induced apoptosis. In primary clinical samples, MU380 used as a single-agent noticeably reduced the viability of unstimulated chronic lymphocytic leukemia cells as well as those induced to proliferate by anti-CD40/IL-4 stimuli. In both cases, effects were comparable in samples harboring p53 pathway dysfunction (TP53 mutations or ATM mutations) and TP53-wt/ATM-wt cells. Lastly, MU380 also exhibited significant in vivo activity in a xenotransplant mouse model (immunodeficient strain NOD-scid IL2Rγnull ) where it efficiently suppressed growth of subcutaneous tumors generated from MEC-1 cells.

• MeSH
• Apoptosis MeSH
• Cell Cycle MeSH
• Checkpoint Kinase 1 antagonists & inhibitors   MeSH
• Drug Resistance, Neoplasm drug effects   MeSH
• Leukemia, Lymphocytic, Chronic, B-Cell drug therapy   genetics   pathology   MeSH
• Deoxycytidine analogs & derivatives   pharmacology   MeSH
• Gemcitabine MeSH
• Protein Kinase Inhibitors pharmacology   MeSH
• Humans MeSH
• Mutation * MeSH
• Mice, Inbred NOD MeSH
• Mice, SCID MeSH
• Mice MeSH
• Biomarkers, Tumor genetics   MeSH
• Tumor Cells, Cultured MeSH
• Tumor Suppressor Protein p53 genetics   MeSH
• Piperidines pharmacology   MeSH
• Cell Proliferation MeSH
• Antimetabolites, Antineoplastic pharmacology   MeSH
• Pyrazoles pharmacology   MeSH
• Pyrimidines pharmacology   MeSH
• Gene Expression Regulation, Neoplastic drug effects   MeSH
• Drug Synergism * MeSH
• Xenograft Model Antitumor Assays MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Checkpoint Kinase 1 MeSH
• CHEK1 protein, human MeSH Browser
• Deoxycytidine MeSH
• Gemcitabine MeSH
• Protein Kinase Inhibitors MeSH
• MK-8776 MeSH Browser
• MU380 MeSH Browser
• Biomarkers, Tumor MeSH
• Tumor Suppressor Protein p53 MeSH
• Piperidines MeSH
• Antimetabolites, Antineoplastic MeSH
• Pyrazoles MeSH
• Pyrimidines MeSH
• TP53 protein, human MeSH Browser

Agents targeting B-cell receptor (BCR) signaling-associated kinases such as Bruton tyrosine kinase (BTK) or phosphatidylinositol 3-kinase can induce mobilization of neoplastic B cells from the lymphoid tissues into the blood, which makes them potentially ideal to combine with anti-CD20 monoclonal antibodies (such as rituximab, obinutuzumab, or ofatumumab) for treatment of B-cell lymphomas and chronic lymphocytic leukemia (CLL). Here we show that interactions between leukemia cells and stromal cells (HS-5) upregulate CD20 on CLL cells and that administering ibrutinib downmodulates CD20 (MS4A1) expression in vivo. We observed that CLL cells that have recently exited the lymph node microenvironment and moved into the peripheral blood (CXCR4(dim)CD5(bright) subpopulation) have higher cell surface levels of CD20 than the cells circulating in the bloodstream for a longer time (CXCR4(bright)CD5(dim) cells). We found that CD20 is directly upregulated by CXCR4 ligand stromal cell-derived factor 1 (SDF-1α, CXCL12) produced by stromal cells, and BTK-inhibitor ibrutinib and CXCR4-inhibitor plerixafor block SDF-1α-mediated CD20 upregulation. Ibrutinib also downmodulated Mcl1 levels in CLL cells in vivo and in coculture with stromal cells. Overall, our study provides a first detailed mechanistic explanation of CD20 expression regulation in the context of chemokine signaling and microenvironmental interactions, which may have important implications for microenvironment-targeting therapies.

• MeSH
• Adenine analogs & derivatives   MeSH
• Antigens, CD20 chemistry   genetics   metabolism   MeSH
• Chemokine CXCL12 genetics   metabolism   MeSH
• Leukemia, Lymphocytic, Chronic, B-Cell drug therapy   metabolism   pathology   MeSH
• Humans MeSH
• Tumor Cells, Cultured MeSH
• Piperidines MeSH
• Pyrazoles pharmacology   MeSH
• Pyrimidines pharmacology   MeSH
• Receptors, CXCR4 genetics   metabolism   MeSH
• Gene Expression Regulation, Neoplastic drug effects   MeSH
• Signal Transduction MeSH
• Up-Regulation MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Adenine MeSH
• Antigens, CD20 MeSH
• Chemokine CXCL12 MeSH
• CXCL12 protein, human MeSH Browser
• CXCR4 protein, human MeSH Browser
• ibrutinib MeSH Browser
• Piperidines MeSH
• Pyrazoles MeSH
• Pyrimidines MeSH
• Receptors, CXCR4 MeSH

Mantle cell lymphoma (MCL) is a chronically relapsing aggressive type of B-cell non-Hodgkin lymphoma considered incurable by currently used treatment approaches. Fludarabine is a purine analog clinically still widely used in the therapy of relapsed MCL. Molecular mechanisms of fludarabine resistance have not, however, been studied in the setting of MCL so far. We therefore derived fludarabine-resistant MCL cells (Mino/FR) and performed their detailed functional and proteomic characterization compared to the original fludarabine sensitive cells (Mino). We demonstrated that Mino/FR were highly cross-resistant to other antinucleosides (cytarabine, cladribine, gemcitabine) and to an inhibitor of Bruton tyrosine kinase (BTK) ibrutinib. Sensitivity to other types of anti-lymphoma agents was altered only mildly (methotrexate, doxorubicin, bortezomib) or remained unaffacted (cisplatin, bendamustine). The detailed proteomic analysis of Mino/FR compared to Mino cells unveiled over 300 differentially expressed proteins. Mino/FR were characterized by the marked downregulation of deoxycytidine kinase (dCK) and BTK (thus explaining the observed crossresistance to antinucleosides and ibrutinib), but also by the upregulation of several enzymes of de novo nucleotide synthesis, as well as the up-regulation of the numerous proteins of DNA repair and replication. The significant upregulation of the key antiapoptotic protein Bcl-2 in Mino/FR cells was associated with the markedly increased sensitivity of the fludarabine-resistant MCL cells to Bcl-2-specific inhibitor ABT199 compared to fludarabine-sensitive cells. Our data thus demonstrate that a detailed molecular analysis of drug-resistant tumor cells can indeed open a way to personalized therapy of resistant malignancies.

• MeSH
• Drug Resistance, Neoplasm * MeSH
• Chromatography, Liquid methods   MeSH
• Isotope Labeling methods   MeSH
• Humans MeSH
• Lymphoma, Mantle-Cell drug therapy   metabolism   MeSH
• Biomarkers, Tumor metabolism   MeSH
• Tumor Cells, Cultured MeSH
• Proteomics methods   MeSH
• Antineoplastic Agents pharmacology   MeSH
• Tandem Mass Spectrometry methods   MeSH
• Vidarabine analogs & derivatives   pharmacology   MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• fludarabine MeSH Browser
• Biomarkers, Tumor MeSH
• Antineoplastic Agents MeSH
• Vidarabine MeSH

MicroRNAs (miRNAs) represent important regulators of gene expression besides transcriptional control. miRNA regulation can be involved in the cell developmental fate decisions, but can also have more subtle roles in buffering stochastic fluctuations in gene expression. They participate in pathways fundamental to B-cell development like B-cell receptor (BCR) signalling, B-cell migration/adhesion, cell-cell interactions in immune niches, and the production and class-switching of immunoglobulins. miRNAs influence B-cell maturation, generation of pre-, marginal zone, follicular, B1, plasma and memory B cells. In this review, we discuss miRNAs with essential functions in malignant B-cell development (such as miR-150, miR-155, miR-21, miR-34a, miR-17-92 and miR-15-16). We also put these miRNAs in the context of normal B-cell differentiation, as this is intimately connected to neoplastic B-cell development. We review miRNAs' role in the most common B-cell malignancies, including chronic lymphocytic leukaemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL) and mantle cell lymphoma (MCL). We focus on miR-contribution to the regulation of important signalling pathways (such as NF-κB, PI3K/AKT and TGF-β), BCR signalling and its modulators (such as PTEN, SHIP-1, ZAP-70, GAB1 and BTK), anti- and pro-apoptotic proteins (such as BCL2, MCL1, TCL1, BIM, p53 and SIRT1) and transcription factors (such as MYC, MYB, PU.1, FOXP1 and BCL6). We also discuss the association of miRNAs' expression levels with the patients' survival and response to therapy, summarizing their potential use as predictive and prognostic markers. Importantly, the targeting of miRNAs (like use of anti-miR-155 or miR-34a mimic) could provide a novel therapeutic approach as evidenced by tumour regression in xenograft mouse models and initial promising data from clinical trials.

• MeSH
• Apoptosis MeSH
• Lymphoma, B-Cell genetics   metabolism   MeSH
• Gene Deletion MeSH
• Humans MeSH
• MicroRNAs metabolism   MeSH
• Mice MeSH
• NF-kappa B p50 Subunit metabolism   MeSH
• DNA Damage MeSH
• Receptors, Antigen, B-Cell metabolism   MeSH
• Gene Expression Regulation, Neoplastic * MeSH
• Signal Transduction MeSH
• Gene Expression Profiling MeSH
• Neoplasm Transplantation MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• MicroRNAs MeSH
• MIRN150 microRNA, human MeSH Browser
• MIRN155 microRNA, human MeSH Browser
• MIRN21 microRNA, human MeSH Browser
• MIRN34 microRNA, human MeSH Browser
• NF-kappa B p50 Subunit MeSH
• NFKB1 protein, human MeSH Browser
• Receptors, Antigen, B-Cell MeSH