Combined chemosensitivity and chromatin profiling prioritizes drug combinations in CLL.

The Bruton tyrosine kinase (BTK) inhibitor ibrutinib has substantially improved therapeutic options for chronic lymphocytic leukemia (CLL). Although ibrutinib is not curative, it has a profound effect on CLL cells and may create new pharmacologically exploitable vulnerabilities. To identify such vulnerabilities, we developed a systematic approach that combines epigenome profiling (charting the gene-regulatory basis of cell state) with single-cell chemosensitivity profiling (quantifying cell-type-specific drug response) and bioinformatic data integration. By applying our method to a cohort of matched patient samples collected before and during ibrutinib therapy, we identified characteristic ibrutinib-induced changes that provide a starting point for the rational design of ibrutinib combination therapies. Specifically, we observed and validated preferential sensitivity to proteasome, PLK1, and mTOR inhibitors during ibrutinib treatment. More generally, our study establishes a broadly applicable method for investigating treatment-specific vulnerabilities by integrating the complementary perspectives of epigenetic cell states and phenotypic drug responses in primary patient samples.

Gliotoxin is a potent NOTCH2 transactivation inhibitor and efficiently induces apoptosis in chronic lymphocytic leukaemia (CLL) cells.

Chronic lymphocytic leukaemia (CLL) cells express constitutively activated NOTCH2 in a protein kinase C (PKC)- dependent manner. The transcriptional activity of NOTCH2 correlates not only with the expression of its target gene FCER2 (CD23) but is also functionally linked with CLL cell viability. In the majority of CLL cases, DNA-bound NOTCH2 complexes are less sensitive to the γ-secretase inhibitor (GSI) DAPT. Therefore, we searched for compounds that interfere with NOTCH2 signalling at the transcription factor level. Using electrophoretic mobility shift assays (EMSA), we identified the Aspergillum-derived secondary metabolite gliotoxin as a potent NOTCH2 transactivation inhibitor. Gliotoxin completely blocked the formation of DNA-bound NOTCH2 complexes in CLL cells independent of their sensitivity to DAPT. The inhibition of NOTCH2 signalling by gliotoxin was associated with down regulation of CD23 (FCER) expression and induction of apoptosis. Short time exposure of CLL cells indicated that the early apoptotic effect of gliotoxin is independent of proteasome regulated nuclear factor κB activity, and is associated with up regulation of NOTCH3 and NR4A1 expression. Gliotoxin could overcome the supportive effect of primary bone marrow stromal cells in an ex vivo CLL microenvironment model. In conclusion, we identified gliotoxin as a potent NOTCH2 inhibitor with a promising therapeutic potential in CLL.

Reconstitution of PTEN activity by CK2 inhibitors and interference with the PI3-K/Akt cascade counteract the antiapoptotic effect of human stromal cells in chronic lymphocytic leukemia.

Evidence suggests that tumor microenvironment is critically involved in supporting survival of chronic lymphocytic leukemia (CLL) cells. However, the molecular mechanisms of this effect and the clinical significance are not fully understood. We applied a microenvironment model to explore the interaction between CLL cells and stromal cells and to elucidate the role of phosphatidylinositol 3 kinase (PI3-K)/Akt/phosphatase and tensin homolog detected on chromosome 10 (PTEN) cascade in this process and its in vivo relevance. Primary human stromal cells from bone marrow, lymph nodes, and spleen significantly inhibited spontaneous apoptosis of CLL cells. Pan-PI3-K inhibitors (LY294002, wortmannin, PI-103), isotype-specific inhibitors of p110α, p110ß, p110γ, and small interfering RNA against PI3-K and Akt1 counteracted the antiapoptotic effect of the stromal cells. Induction of apoptosis was associated with a decrease in phosphatidylinositol-3,4,5-triphosphate, PI3-K-p85, and dephosphorylation of phosphatidylinositol-dependent kinase-1 (PDK-1), Akt1, and PTEN. Freshly isolated peripheral blood mononuclear cells from patients with CLL (n = 44) showed significantly higher levels of phosphorylated Akt1, PDK-1, PTEN, and CK2 than healthy persons (n = 8). CK2 inhibitors (4,5,6,7-tetrabromo-1H-benzotriazole, apigenin, and 5,6-dichloro-1-ß-D-ribofuranosylbenzimidazol) decreased phosphorylation of PTEN and Akt, induced apoptosis in CLL cells, and enhanced the response to fludarabine. In conclusion, bone marrow microenvironment modulates the PI3-K/Akt/PTEN cascade and prevents apoptosis of CLL cells. Combined inhibition of PI3-K/Akt and recovery of PTEN activity may represent a novel therapeutic concept for CLL.

NOTCH2 links protein kinase C delta to the expression of CD23 in chronic lymphocytic leukaemia (CLL) cells.

One characteristic of chronic lymphocytic leukaemia (CLL) lymphocytes is high expression of CD23, which has previously been identified as a downstream target for NOTCH2 signalling. The mechanisms regulating NOTCH2-dependent CD23 expression, however, are largely unknown. This study showed that peripheral CLL cells overexpressed transcriptionally active NOTCH2 (N2(IC)), irrespective of their prognostic marker profile. When placed in culture, NOTCH2 activity was spontaneously decreased in 25 out of 31 CLL cases (81%) within 24 h. DNA-bound N2(IC) complexes could be maintained by the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) or by gamma-interferon (IFN-gamma), two CLL characteristic inducers of CD23 expression. Inhibition of PKC-delta by RNA interference or by rottlerin antagonised PMA-induced NOTCH2 activation and also suppressed NOTCH2 activity in CLL cases with constitutively activated NOTCH2 signalling. In 23 out of 29 CLL cases tested (79%), DNA-bound N2(IC) complexes were found to be resistant to the gamma-secretase inhibitor (GSI) DAPT, suggesting that GSIs will be only effective in a subset of CLL cases. These data suggest that deregulation of NOTCH2 signalling is critically involved in maintaining the malignant phenotype of CLL lymphocytes and point to a link between PKC-delta and NOTCH2 signalling in the leukemic cells.

Targeting Nuclear NOTCH2 by Gliotoxin Recovers a Tumor-Suppressor NOTCH3 Activity in CLL.

NOTCH signaling represents a promising therapeutic target in chronic lymphocytic leukemia (CLL). We compared the anti-neoplastic effects of the nuclear NOTCH2 inhibitor gliotoxin and the pan-NOTCH γ-secretase inhibitor RO4929097 in primary CLL cells with special emphasis on the individual roles of the different NOTCH receptors. Gliotoxin rapidly induced apoptosis in all CLL cases tested, whereas RO4929097 exerted a variable and delayed effect on CLL cell viability. Gliotoxin-induced apoptosis was associated with inhibition of the NOTCH2/FCER2 (CD23) axis together with concomitant upregulation of the NOTCH3/NR4A1 axis. In contrast, RO4929097 downregulated the NOTCH3/NR4A1 axis and counteracted the spontaneous and gliotoxin-induced apoptosis. On the cell surface, NOTCH3 and CD23 expression were mutually exclusive, suggesting that downregulation of NOTCH2 signaling is a prerequisite for NOTCH3 expression in CLL cells. ATAC-seq confirmed that gliotoxin targeted the canonical NOTCH signaling, as indicated by the loss of chromatin accessibility at the potential NOTCH/CSL site containing the gene regulatory elements. This was accompanied by a gain in accessibility at the NR4A1, NFκB, and ATF3 motifs close to the genes involved in B-cell activation, differentiation, and apoptosis. In summary, these data show that gliotoxin recovers a non-canonical tumor-suppressing NOTCH3 activity, indicating that nuclear NOTCH2 inhibitors might be beneficial compared to pan-NOTCH inhibitors in the treatment of CLL.

Combined approach for characterization and quality assessment of rabbit bone marrow-derived mesenchymal stem cells intended for gene banking.

Rabbit mesenchymal stem cells (rMSCs) are promising agents for the preservation of genetic biodiversity in domestic rabbit breeds. However, rMSCs must meet certain requirements to be used for cryopreservation in animal gene banks. Currently, there are numerous discrepancies in the published data regarding the rMSC phenotype, which may complicate efforts to evaluate their purity and suitability for reuse after cryopreservation in gene and tissue banks. We propose a combined approach (flow cytometry, PCR, differentiation and ultrastructure studies) for the characterization and recovery of rMSCs after cryopreservation. Flow cytometric analyses of rMSCs confirmed the expression of CD29, CD44, vimentin, desmin and α-SMA. RT-PCR revealed the expression of other markers at the mRNA level (SSEA-4, CD73, CD90, CD105, CD146 and CD166). rMSCs showed efficient multilineage differentiation into adipo-, chondro- and osteogenic lineages, SOX2 expression (pluripotency) and typical MSC morphology and ultrastructure. The confirmed rMSCs were subsequently used for cryopreservation. Efficient recovery of rMSCs after cryogenic freezing was demonstrated by high cell viability, normal ultrastructure of reseeded rMSCs, high expression of CD29 and CD44 and lineage differentiation capacity. The proposed combined approach could be used for characterization, cryopreservation and recovery of rMSCs as genetic resources for native rabbit breeds.

Critical assessment of the efficiency of CD34 and CD133 antibodies for enrichment of rabbit hematopoietic stem cells.

Rabbits have many hereditary diseases common to humans and are therefore a valuable model for regenerative disease and hematopoietic stem cell (HSC) therapies. Currently, there is no substantial data on the isolation and/or enrichment of rabbit HSCs. This study was initiated to evaluate the efficiency of the commercially available anti-CD34 and anti-CD133 antibodies for the detection and potential enrichment of rabbit HSCs from peripheral blood. PBMCs from rabbit and human blood were labelled with different clones of anti-human CD34 monoclonal antibodies (AC136, 581, and 8G12) and rabbit polyclonal CD34 antibody (pCD34) and anti-human CD133 monoclonal antibodies (AC133 and 293C3). Flow cytometry showed a higher percentage of rabbit CD34+ cells labelled by AC136 in comparison to the clone 581 and pCD34 (P < 0.01). A higher percentage of rabbit CD133+ cells were also detected by 293C3 compared to the AC133 clone (P < 0.01). Therefore, AC136 clone was used for the indirect immunomagnetic enrichment of rabbit CD34+ cells using magnetic-activated cell sorting (MACS). The enrichment of the rabbit CD34+ cells after sorting was low in comparison to human samples (2.4% vs. 39.6%). PCR analyses confirmed the efficient enrichment of human CD34+ cells and the low expression of CD34 mRNA in rabbit positive fraction. In conclusion, the tested antibodies might be suitable for detection, but not for sorting the rabbit CD34+ HSCs and new specific anti-rabbit CD34 antibodies are needed for efficient enrichment of rabbit HSCs. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018 © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1278-1289, 2018.

TGF-beta1 induces bone marrow reticulin fibrosis in hairy cell leukemia.

The mechanisms that lead to reticulin fibrosis of bone marrow (BM) in hairy cell leukemia (HCL) are not fully understood. We therefore investigated the involvement of TGF-beta1, a potent fibrogenic cytokine, in this process. Immunoassays revealed that TGF-beta1 is present at higher concentrations in BM, serum, and plasma of HCL patients in comparison with healthy donors (P < 0.001). RT-PCR and immunofluorescence studies showed that TGF-beta1 is overexpressed at the mRNA and protein levels in peripheral blood, spleen, and BM mononuclear cells and that hairy cells (HCs) are the main source of TGF-beta1. Active TGF-beta1 correlated significantly with grades of BM fibrosis, infiltration with HCs, and serum procollagen type III aminoterminal propeptide (PIIINP). Ex vivo studies demonstrated that TGF-beta1 significantly enhances the production and deposition of reticulin and collagen fibers by BM fibroblasts. In addition, BM plasma of HCL patients increased the synthesis of type I and type III procollagens, the main components of reticulin fibers, at the mRNA and protein levels. This fibrogenic activity of BM plasma was abolished by neutralizing anti-TGF-beta1 antibodies. These results show, for the first time to our knowledge, that TGF-beta1 is highly expressed in HCs and is directly involved in the pathogenesis of BM reticulin fibrosis in HCL.

Gliotoxin Targets Nuclear NOTCH2 in Human Solid Tumor Derived Cell Lines In Vitro and Inhibits Melanoma Growth in Xenograft Mouse Model.

Deregulation of NOTCH2 signaling is implicated in a wide variety of human neoplasias. The current concept of targeting NOTCH is based on using gamma secretase inhibitors (GSI) to regulate the release of the active NOTCH intracellular domain. However, the clinical outcome of GSI remains unsatisfactory. Therefore we analyzed human solid tumor derived cell lines for their nuclear NOTCH activity and evaluated the therapeutic potential of the NOTCH2 transactivation inhibitor gliotoxin in comparison to the representative GSI DAPT. Electrophoretic mobility shift assays (EMSA) were used as a surrogate method for the detection of NOTCH/CSL transcription factor complexes. The effect of gliotoxin on cell viability and its clinical relevance was evaluated in vitro and in a melanoma xenograft mouse model. Cell lines derived from melanoma (518A2), hepatocellular carcinoma (SNU398, HCC-3, Hep3B), and pancreas carcinoma (PANC1) express high amounts of nuclear NOTCH2. Gliotoxin efficiently induced apoptosis in these cell lines whereas the GSI DAPT was ineffective. The specificity of gliotoxin was demonstrated in the well differentiated nuclear NOTCH negative cell line Huh7, which was resistant to gliotoxin treatment in vitro. In xenotransplanted 518A2 melanomas, a single day dosing schedule of gliotoxin was well tolerated without any study limiting side effects. Gliotoxin significantly reduced the tumor volume in early (83 mm3 vs. 115 mm3, p = 0.008) as well as in late stage (218 mm3 vs. 576 mm3, p = 0.005) tumor models. In conclusion, NOTCH2 appears to be a key target of gliotoxin in human neoplasias and gliotoxin deserves further evaluation as a potential therapeutic agent in cancer management.

Effect of combined spa-exercise therapy on circulating TGF-beta1 levels in patients with ankylosing spondylitis.

BACKGROUND: Ankylosing spondylitis (AS) is a chronic inflammatory disease of the axial joints with no satisfactory therapy. Reduction of joint pain has been reported after a course of therapy at a spa, Gasteiner Heilstollen, in Badgastein in Austria. The mechanism underlying this beneficial effect is not clearly understood and objective evidence for the biological response to therapy is lacking. The aim of this study was to find evidence for a biological response to speleotherapy in patients with AS and to study the involvement of the antiinflammatory cytokine TGF-beta1 in this response. PATIENTS AND METHODS: 83 patients with AS were treated in Badgastein for 3-4 weeks. Therapy included active exercises, hyperthermia and exposure to low doses of radon in a former mine. Response to therapy was assessed from measurement of morning pain and immunoassay of serum levels of TGF-beta1 before and after therapy. Ten AS patients who received conventional therapy and 10 patients with low back pain (LBP) served as controls. RESULTS: A significant increase in TGF-beta1 (total and active) was found in AS patients after spa therapy. Mean concentration of total TGF-beta1 increased from 28,715 pg/ml to 43,136 pg/ml, (P<0.01) and active TGF-beta1 increased from 77 pg/ml to 1096 pg/ml (P<0.001). When the AS patients were divided into two groups according to pain reduction, group 1 (decrease in morning pain, responders: n=46) exhibited a 17-fold increase of active TGF-beta1 levels (96 pg/ml to 1654 pg/ml, P<0.0001) whereas group 2 (no change or an increase in morning pain: nonresponders: n=37), showed only 7-fold increase (53 pg/ml to 402 pg/ml, P<0.01). There was a moderate increase in active TGF-beta1 from 31 pg/ml to 42 pg/ml (P<0.05) in patients with LBP and no significant change was observed in the patients treated with conventional therapy. CONCLUSION: These results demonstrate a significant increase in circulating TGF-beta1 in patients with AS after the combined spa-exercise therapy in Badgastein. The results also provide evidence for a biological response to speleotherapy and suggest that TGF-beta, through its antiinflammatory function, may play a role in this response.

The role of soluble CD23 in distinguishing stable and progressive forms of B-chronic lymphocytic leukemia.

Soluble CD23 (sCD23) has been recognized as an important prognostic parameter in patients with chronic lymphocytic leukemia (B-CLL) at early clinical stages. There is, however, no clear information on its prognostic significance in advanced stages and on its role as an indicator for aggressive or indolent courses of disease. Therefore, sCD23 was measured in the serum of 145 patients at diagnosis and serial determinations were carried out for 8 years in 38 patients. The results indicate that in patients with identical clinical stages at first presentation the disease could take different courses depending on initial sCD23 concentrations below or above specific threshold levels (860 and 5900U/ml). sCD23 higher than these thresholds was associated with faster progression into upper clinical stages. Furthermore, sCD23-doubling time (sCD23-DT) indicated that patients with long DT progressed slowly, while those with short DT had more aggressive disease. Particularly in patients with advanced disease stages, long sCD23-DT indicated development of smoldering disease. Since sCD23 levels reflect total tumor mass, determination of sCD23-DT has probably a better predictive value than lymphocyte doubling time. It appears that B-CLL patients can be divided into different risk categories according to initial determinations of sCD23 and that sCD23-DT is an additional important parameter in predicting disease progression.

Notch2 is involved in the overexpression of CD23 in B-cell chronic lymphocytic leukemia.

Members of the Notch family encode transmembrane receptors that modulate differentiation, proliferation, and apoptotic programs of many precursor cells, including hematopoietic progenitors. Stimulation of Notch causes cleavage followed by translocation of the intracellular domain (NotchIC) to the nucleus, where it activates transcription of CBF1 responsive genes. The aim of this study was to elucidate the mechanisms leading to the overexpression of CD23, a striking feature of B-cell chronic lymphocytic leukemia (B-CLL) cells. By electrophoretic mobility shift assays, we identified a transcription factor complex (C1) that binds sequence specific to one known and 4 newly identified putative CBF1 recognition sites in the CD23a core promoter region. With the use of Epstein-Barr virus (EBV)-infected B cells as a model for CBF1 mediated CD23a expression, C1 was found to be EBV inducible. Supershift assays revealed that the nuclear form of Notch2 is a component of C1 in B-CLL cells, supporting a model in which NotchIC activates transcription by binding to CBF1 tethered to DNA. Transient transfection of REH pre-B cells with an activated form of Notch2 induced endogenous CD23a, confirming that CD23a is a target gene of Notch2 signaling. Finally, reverse transcription-polymerase chain reaction and kinetic analysis demonstrated that the Notch2 oncogene is not only overexpressed in B-CLL cells but might also be related to the failure of apoptosis characteristic for this disease. In conclusion, these data suggest that deregulation of Notch2 signaling is involved in the aberrant expression of CD23 in B-CLL.