Chronic Lymphocytic Leukemia

Chaetoglobosin A preferentially induces apoptosis in chronic lymphocytic leukemia cells by targeting the cytoskeleton

Abstract

Chronic lymphocytic leukemia (CLL) is an incurable malignancy of mature B cells. One of the major challenges in treatment of CLL is the achievement of a complete remission to prevent relapse of disease originating from cells within lymphoid tissues and subsequent chemoresistance. In search for novel drugs that target CLL cells in protective microenvironments, we performed a fungal extract screen using cocultures of primary CLL cells with bone marrow-derived stromal cells. A secondary metabolite produced by Penicillium aquamarinium was identified as Chaetoglobosin A (ChA), a member of the cytochalasan family that showed preferential induction of apoptosis in CLL cells, even under culture conditions that mimic lymphoid tissues. In vitro testing of 89 CLL cases revealed effective targeting of CLL cells by ChA, independent of bad prognosis characteristics, like 17p deletion or TP53 mutation. To provide insight into its mechanism of action, we showed that ChA targets filamentous actin in CLL cells and thereby induces cell-cycle arrest and inhibits membrane ruffling and cell migration. Our data further revealed that ChA prevents CLL cell activation and sensitizes them for treatment with PI3K and BTK inhibitors, suggesting this compound as a novel potential drug for CLL.

Introduction

Despite tremendous advances in the treatment, chronic lymphocytic leukemia (CLL) remains an incurable disease and all patients requiring therapy eventually relapse, often resulting in chemoresistance with short overall survival rates.1 Especially patients with poor prognosis factors, like 17p deletion or mutation of TP53, show no benefit from chemoimmunotherapy and still remain a major clinical problem.2, 3 Novel treatment approaches using kinase inhibitors appear to be more effective in this patient population, with the main activity of these drugs being on CLL cell trafficking.4, 5 As eradication of the leukemic population was not achieved with these inhibitors, combination treatment with chemotherapeutics or other targeted drugs might become necessary for curative approaches.

Apoptosis resistance of CLL cells and their accumulation in lymphoid tissues are attributed to the activity of pro-survival signaling pathways mediated by the B-cell receptor (BCR) or cytokine receptors, both in an autocrine manner or induced by stimuli from the microenvironment.6, 7, 8, 9 The importance of external survival factors is exemplified by the fact that CLL cells rapidly undergo spontaneous apoptosis in vitro,10 unless cocultured with bone marrow (BM)-derived stromal cells or other non-malignant cells.11, 12, 13, 14, 15 In accordance with that, the relevance of migration and homing of CLL cells into the secondary lymphatic tissues for disease development and progression has been suggested by several groups.16, 17, 18 In addition, drugs targeting cellular interactions, like Lenalidomide or Plerixafor that are investigated for the treatment of CLL, have been shown to impact on migration and homing of malignant cells.19, 20, 21

CLL cells that are localized in the niche of, for example, BM or lymph nodes (LN) appear to be less effectively targeted by treatment and represent the origin of relapse of the disease. Screening for novel potential drugs that target CLL cells even when protected by the niche is therefore urgently needed. The CLL/stroma coculture model we have established, using the human BM-derived stromal cell line HS-5, provides a microenvironment that supports long-term survival in vitro and is therefore very well suited for the testing of new potential drugs.22 Fungal extracts have been shown to be an important source for biologically active agents, which are of therapeutic use, such as Lovastatin or cyclosporine. By screening extracts of the IBT Fungal Culture Collection (http://www.bio.dtu.dk), griseofulvin was identified as an inhibitor of centrosomal clustering,23 and further developments resulted in semisynthetic griseofulvin derivates with improved activities.24 The aim of our study was to screen for novel potential drugs by using this culture collection in a high-throughput coculture set-up of primary CLL cells and HS-5 stromal cells. Chaetoglobosin A (ChA) was identified as the active metabolite of one of the most promising hits of this screen, as a compound that preferentially induces apoptosis in CLL cells.

Materials and methods

Preparation and fractionation of fungal extracts and identification of ChA by HPLC-DAD-MS

A detailed description of the used Material and Methods is provided as Supplementary Information.

Primary cells and cell lines

Peripheral blood (PB) and serum samples were obtained from CLL patients (Supplementary Table 1) and healthy donors (HD) after informed consent. All CLL cases used in this study matched the standard diagnostic criteria for CLL. PB mononuclear cells (PBMC) as well as B cells of CLL patients or HD were isolated as described before.25 All cell lines used in this study are listed in Supplementary Table 2.

CLL/HS-5 cocultures were established as described.14 Cocultures of CLL cells and irradiated BHK-huCD40L cells were supplemented with 50 U/ml IL-4 (Miltenyi Biotec, Bergisch Gladbach, Germany).

Fungal extract screen

CLL/HS-5 cocultures were set-up in 96-well plates by seeding 4 × 104 HS-5 cells per well and 4 h later 1 × 105 CLL cells in 100 μl DMEM+10% FCS+1% Pen/Strep. Raw extracts were applied in a series of eight 1:4 dilutions in DMSO. After 2 days of culture, cells in suspension were transferred to 96-well round-bottom plates, stained with 5 μg/ml propidium iodide in PBS and analyzed by FACSArray (BD Biosciences, Heidelberg, Germany).

Cell viability and apoptosis assays

Cell viability was assessed using CellTiter-Glo assay (Promega, Madison, WI, USA) according to the manufacturers protocol. Luminescence signals were recorded using a Mithras LB940 plate reader (Berthold Technologies, Bad Wildbad, Germany). Drugs used for treatment were ChA, etoposide and fludarabine (Enzo Life Sciences, Lörrach, Germany), as well as CAL-101/GS-1101 and PCI-32765/Ibrutinib (Selleckchem, Munich, Germany).

Apoptotic cell death was detected by Flow cytometry using Annexin V-PE and 7-amino-actinomycin staining kit (BD Biosciences) as described.22 To distinguish effects of ChA on B cells and T cells, PBMC were in addition stained with CD19-APC.

Staining for active caspase 3 was performed after fixation and permeabilization of cells using BD Cytofix/Cytoperm solution as suggested by the manufacturer by using PE-conjugated anti-active caspase 3 antibodies. Apoptosis was inhibited by applying 0.1 mM of the pan-caspase inhibitor Z-VAD-FMK (Promega).

All antibodies used for Flow cytometry are listed in Supplementary Table 3 and analyses were carried out using a FACSCanto II Flow cytometer equipped with FACSDiva software (BD Biosciences). FlowJo software (TreeStar Inc., Ashland, OR, USA) was used for data analysis.

Proliferation assays

DNA replication in CLL cells was assessed using Click-iT EdU kit (Invitrogen, Darmstadt, Germany) according to the manufacturers protocol. CLL cells were seeded on irradiated BHK-huCD40L cells and 24 h later ChA was added for 96 h. 10 μM EdU was applied for the last 48 h. After staining with Alexa Fluor 488 dye azide, cells were analyzed by Flow cytometry.

To analyze cell-cycle distribution, MEC-1 cells were fixed in 70% ethanol on ice, washed and stained with 2.5 μg/ml propidium iodide in PBS containing 0.5 mg/ml RNase A for 5 min before analysis by Flow cytometry.

Actin polymerization assay

Actin polymerization was tested as previously described.26 After 2 h of serum starvation, CLL cells were incubated for 30 min with DMSO or ChA in a medium containing 0.5% BSA at 37 °C. Cells were stimulated with 200 ng/ml SDF1α (USBiological, Swampscott, MA, USA) between 15 and 300 s, stained with Alexa Fluor 488-phalloidin (Invitrogen) in a solution containing 0.5 mg/ml l-α-Lysophosphatidylcholine (Sigma-Aldrich, Munich, Germany) and 18% formaldehyde, and analyzed by Flow cytometry. Changes in the mean fluorescence intensity were recorded and normalized to the values of unstimulated cells.

Confocal microscopy

JEKO-1 cells were treated for 30 min with 1 μM ChA or 0.1% DMSO. After washing, 4 × 105 cells per ml RPMI containing 0.5% BSA were stimulated with 200 ng/ml SDF1α. One-step cell fixation, permeabilization and staining with phalloidin were performed. Thereafter, cells were mounted on glass slides and scanned using a Zeiss LSM780 confocal microscope. Image analysis was performed with ImageJ software.

Chemotaxis assay

Chemotaxis assays were performed using transwells for 24-well plates with 5 μM pore size (Corning, New York, NY, USA) applying 3 × 106 CLL cells per well. After 3 h, migrated cells were washed off the lower side of the membrane and counted using a ViCell counter XR 2.03 (Beckmann Coulter, Krefeld, Germany). Complete medium containing 200 ng/ml SDF1α was used as a chemoattractant.

Results

Fungal extract screen identifies ChA with cytotoxic activity for primary CLL cells

The IBT Fungal Culture Collection consisting of more than 30 000 strains with characteristic metabolite patterns was used to screen for compounds with cytotoxic activity for CLL cells. The fungal extracts were selected based on a chemotaxonomic screening approach,27 so as to maximize the chemodiversity to be tested. A high-throughput screen was established using 96-well plate-based cocultures of primary CLL cells and the human BM-derived stromal cell line HS-5 that maintains survival of CLL cells. To screen for potential drugs, viability of CLL cells and HS-5 cells after treatment with eight step-wise dilutions of fungal extracts were evaluated simultaneously by FACS array analysis after propidium iodide staining. Out of 289 extracts tested, 61 induced cell death in a concentration-dependent manner in CLL cells, but not in HS-5 cells. Fractionation and retesting of the twelve most interesting extracts lead to the subsequent identification of bio-active compounds within eight fungal strains,28 of which Penicillium aquamarinium showed the highest preference for CLL cells (Figure 1a). Chromatographic fractionation of this extract and retesting of all fractions in CLL/HS-5 cocultures resulted in the identification of one fraction with major activity, which was subsequently analyzed by LC-DAD mass spectrometry to identify the active compound (Figure 1b). A comparison of the extracted mass and UV spectra with the natural product database, Antibase,29 resulted in the tentative identification of ChA as the most abundant compound within this active fraction. The compound exhibited a UV absorption pattern at wavelengths identical with those reported for the pure ChA30 (Figures 1c and d). Subsequent testing of several commercially available cytochalasan family members (ChA, Cytochalasin A, B, C, E, J and 19,20-Epoxycytochalasin C) in CLL/HS-5 cocultures revealed a preferential cytotoxic activity for CLL cells for ChA (Supplementary Table 4 and Supplementary Figures 1 and 2).

Figure 1
figure1

Identification of ChA as a fungal metabolite with cytotoxic activity for CLL cells. (a) Overview of results of a fungal extract screen using CLL cells in coculture with HS-5 stromal cells. (b) The UV spectrum of the active fraction of Penicillium aquamarinium was obtained and ChA tentatively identified as one of the compounds within this fraction. (c) The total ion chromatogram of the active fraction of Penicillium aquamarinium revealed two peaks that correspond to the compounds ChA and U. The chromatogram was examined for ion patterns corresponding to the known adduct formation of the positive electro spray ionization mode. (d) Chemical structure of ChA.

ChA induces apoptosis preferentially in primary CLL cells

To evaluate the potential of ChA as a novel drug for CLL, dose-response curves of PBMC isolated from CLL patients or HD were obtained using purified ChA in a concentration range from 1–10 μM. Analysis of cell viability after 24 h of treatment using CellTiter-Glo assay revealed a higher sensitivity of CLL cells with a median LD50 value of 2.8 μM (n=8) compared with 9.5 μM (n=4) for HD PBMC (Figure 2a). Dose-response curves of CLL cases carrying mutated or unmutated IGHV genes were similar. Treatment of HS-5 stromal cells or the primary human lung fibroblast cell line Wi-38 with ChA revealed no major impact on cell viability up to a concentration of 10 μM (Supplementary Figure 3). Testing of several leukemia and carcinoma cell lines showed a general better response of the suspension cells compared with the adherent cell types (data not shown).

Figure 2
figure2

ChA preferentially kills CLL cells via induction of apoptosis. (a) PBMC (3 × 105 cells/well) isolated from CLL or HD samples were seeded in 96-well plates and treated for 24 h with ChA at the indicated concentrations. Cell viability was analyzed by the CellTiter-Glo assay measuring each data point as duplicate. Relative cell viability compared with DMSO control (0.1%) is depicted as mean values +s.d. of eight CLL cases and four HD samples. (b) CLL or HD PBMC were treated with ChA for 24 h. Thereafter, cells were labeled with CD19-APC antibodies, followed by Annexin V-PE/7-amino-actinomycin staining and analysis by Flow cytometry, either gating on CD19-positive or CD19-negative lymphocytes. Annexin V-PE/7-amino-actinomycin-negative cells were counted as viable cells. Relative cell viability compared with DMSO control (0.1%) is depicted as mean values +s.d. of 3 CLL and HD samples each. (c) CLL PBMC were treated with ChA at the indicated concentrations for 48 h and the percentage of apoptotic cells was determined by Flow cytometry after Annexin V-PE/7-amino-actinomycin staining or intracellular staining of activated caspase 3. One representative result as well as mean values +s.d. of nine independently analyzed samples are depicted. (d) CLL cell viability after 24 h of ChA treatment in the presence or absence of 0.1 mM of the pan-caspase inhibitor Z-VAD-FMK was analyzed by the CellTiter-Glo assay. One representative example out of two independently performed experiments measured in duplicates is depicted.

To verify that normal B cells are less sensitive to ChA than CLL cells, we treated three samples of CLL and HD PBMC and 24 h later analyzed the viability of B cells by Flow cytometry after staining with CD19-APC and Annexin V-PE/7-amino-actinomycin. Thereby we observed a significantly stronger cytotoxic activity of ChA for CLL compared with normal B cells. Further, CD19-negative lymphocytes that are mainly T cells, both of patients or HD, were barely affected by the maximal concentration of 10 μM tested in this assay (Figure 2b).

To further extend activity testing of ChA on other non-Hodgkin lymphoma, we performed CellTiter-Glo assay applying 1–10 μM ChA to three diffuse large B-cell lymphoma (DLBCL) and three mantle cell lymphoma cell lines. The two DLBCL cell lines SuDHL-5 and SuDHL-6, as well as the EBV-negative mantle cell lymphoma line JEKO-1, were effectively killed by ChA with LD50 values between 2 and 3 μM (Supplementary Figure 4). MC116, with a LD50 value of 5 μM and the two mantle cell lymphoma lines JVM-2 and Granta-519, with LD50 values of 7 μM and 9 μM, respectively, were less effectively targeted by the treatment, suggesting varying susceptibilities of different malignant B cells to this drug.

To evaluate whether ChA induces apoptosis, treated CLL cells of nine cases were either stained with antibodies specific for activated caspase 3, or with Annexin V-PE/7-amino-actinomycin and analyzed by Flow cytometry. This showed that ChA treatment strongly enhanced the percentage of CLL cells with an active caspase 3, as well as Annexin V-PE positive cells, suggesting apoptosis induction in CLL cells (n=9; Figure 2c). The higher apoptotic rates obtained by Annexin V compared with caspase 3 staining are most likely due to the higher sensitivity of this assay. Apoptosis as a mode of cell death was further verified by applying the pan-caspase inhibitor Z-VAD-FMK, which completely rescued CLL cells from ChA-induced cell death (Figure 2d; n=2).

ChA overcomes microenvironmental protection

To test the efficacy of ChA in microenvironmental settings that mimic the protective conditions in the BM and PB, we established cocultures of either unsorted CLL or HD PBMC with HS-5 cells, and applied CD19-sorted CLL or normal B cells to the stromal cells. In addition, we cultured CLL or HD PBMC in a medium containing 10% autologous blood plasma. The results confirmed a general increase in cell survival in these cultures, as described before,22, 31 and more importantly showed that response to ChA treatment remained more pronounced for CLL cells (n=3) compared with HD PBMC (n=3–4) with LD50 values of 5 μM compared with >10 μM in HS-5 cocultures (Figure 3a), and 7 μM compared with 13 μM in cultures containing autologous serum, respectively (Figure 3b). To mimic the LN microenvironment, cocultures of CLL cells with irradiated BHK cells expressing membrane-bound human CD40L in IL-4-containing medium were established from ten CLL cases. Under these conditions, spontaneous apoptosis of primary CLL cells was prevented and DNA replication was induced as measured by a Flow cytometry-based EdU incorporation assay, with an average percentage of cycling cells of 16%, ranging from 2 to 35% (Figure 3c). As a result of the protective milieu in these cultures, a reduction in CLL cell viability of 50% was only obtained when applying 10 μM ChA (n=10; Figure 3d), recapitulating the increased apoptosis resistance within the LN microenvironment. In addition, we observed a significant drop in EdU-positive CLL cells at 10 μM of the compound (n=9; Figure 3c). In comparison to that, fludarabine completely inhibited DNA replication, but did not decrease viability of CLL cells within this protective culture condition.

Figure 3
figure3

ChA overcomes microenvironmental protection. (a) Cocultures of HS-5 cells and CLL or HD PBMC, as well as CD19-sorted CLL or normal B cells were treated for 24 h with ChA. CLL or B-cell viability was determined after Annexin V-PE/7-amino-actinomycin staining by Flow cytometry. Unsorted PBMC were further stained with CD19-APC to enable gating on B cells (CLL gated and HD gated). Mean values +s.d. of three independently performed experiments are depicted. (b) CLL or HD PBMC were cultured in a medium containing 20% autologous serum and treated with the indicated concentrations of ChA for 24 h. Cell viability was measured by the CellTiter-Glo assay in duplicates and relative mean values +SEM compared with DMSO control (0.1%) of three CLL and four HD samples are depicted. (c) CLL cells were seeded on irradiated BHK-huCD40L cells and 24 h later ChA or 10 μM fludarabine was added for 96 h. For the last 48 h of the experiment 10 μM EdU was applied to the cultures and the percentage of cells that entered the cell cycle was determined by Click-iT assay via Flow cytometry using Alexa Fluor 488 dye azide. (d) CLL cells were cocultured with BHK-huCD40L cells and proliferation was determined as described in (c). Cell viability was analyzed by Annexin V-PE/7-amino-actinomycin staining. Relative proliferation and viability rates compared with DMSO-treated control cultures were calculated and are depicted as mean values +s.d. of 9 and 10 independently performed experiments, respectively. Statistical significance was calculated using student’s t test: **P-value<0.01; ***P-value<0.001.

ChA targets CLL cells independently of their genetic aberrations

To evaluate the efficacy of ChA for CLL cells harboring different genetic aberrations, a cohort of 89 CLL cases with the following characteristics were included in our studies: del11q: n=14; del13q: n=44; del17p: n=12; trisomy12: n=11; and mutTP53: n=14. Viably frozen CLL PBMC were thawed and cultured in a medium containing 10% human serum and treated with either 8 μM ChA or 1 μM fludarabine. After 48 h, cell viability was analyzed by the CellTiter-Glo assay and calculated as percentage compared with 0.1% DMSO-treated control samples. ChA treatment reduced viability of CLL cells to an average of 49% and no statistical difference between the genetic subgroups was observed. Of interest, patients harboring a deletion on chromosome 17p13 or a mutation of TP53, both genetic aberrations with the worst prognosis in CLL, responded equally well to treatment with ChA as patients without these genetic abnormalities (Figure 4; del17p13: 49.64±24.90 vs 43.42±18.05; P=0.47; mutTP53: 50.18±24.76 vs 43.43±20.49; P=0.25). As expected, fludarabine treatment resulted in significantly higher cell viability rates in cases with 17p13 deletion (67.14±18.40 vs 88.25±15.35; P<0.001) or TP53 mutation (66.63±18.36 vs 85.43±16.87; P<0.001).

Figure 4
figure4

ChA kills CLL cells independently of genetic abnormalities. CLL PBMC (5 × 104 cells/well) were cultured in medium containing 10% human serum in 384-well plates and treated for 48 h with either 8 μM ChA or 1 μM fludarabine. Cell viability was analyzed by the CellTiter-Glo assay and is given as percentage of DMSO-treated control samples. Mean values +s.d. of cases with or without del17p13 or mutTP53 are depicted in the plots. Statistical significance was calculated using student’s t test: ***P-value<0.001.

ChA targets the cytoskeleton in CLL cells and thereby impairs cell migration and cytokinesis

Most cytochalasan family members bind to filamentous actin and slow down or block polymerization or depolymerization of actin filaments. To test whether ChA effects the assembly or disassembly of filamentous actin in CLL cells, we used Alexa Fluor 488-phalloidin staining after applying SDF1α as stimulus and analyzed cells by Flow cytometry as described.26 The results of five CLL cases showed a clear induction of actin polymerization 15 s after addition of SDF1α in control cells, which slowly declined during the observation period of five minutes (n=5; Figure 5a). In the presence of AMD3100 (Plerixafor), an inhibitor of SDF1α receptor (CXCR4), we observed a complete inhibition of actin polymerization, as expected. Treatment of cells with 1, 5 or 10 μM ChA also prevented actin polymerization upon SDF1α stimulation, indicating that ChA targets the cytoskeleton in CLL cells and therefore renders them unresponsive. Interestingly, the fluorescence intensity detected with phalloidin in ChA-treated cells was on average 1.8-fold higher compared with untreated or AMD3100-treated cells (Supplementary Figure 5), suggesting that ChA enhances the presence of filamentous actin in the cells. This was confirmed by phalloidin staining of CLL cells treated for 30 min with increasing concentrations of ChA that resulted in a significant increase in filamentous actin at 10 and 20 μM (n=2–5; Figure 5b). In concordance, an increase in Alexa Fluor 488-phalloidin staining of the B-cell line JEKO-1 upon treatment with 1 μM ChA was observed by confocal microscopy (Figure 5c, left). Compared with the control cells, which showed phalloidin-stained microvilli extruding from the cell membrane, ChA-treated cells lacked these structures. Instead, a rather diffuse staining with patches of increased signal intensities were observed on the surface and within the cytoplasm of the cells. Stimulation of control cells with SDF1α induced membrane ruffling and the formation of a flattened leading-edge extension at the front of the cells. However, this formation was completely inhibited by pre treatment with 1 μM ChA (Figure 5c, right and Supplementary Figure 6), which is in accordance with findings of other cytochalasan family members that are known to prevent membrane ruffles at concentrations below 2 μM.32

Figure 5
figure5

ChA targets filamentous actin. (a) Serum starved CLL cells were treated for 30 min with 1 μM ChA, 40 μM AMD3100, or 0.1% DMSO as control. Thereafter, cells were stimulated with 200 ng/ml SDF1α for the indicated time points and stained with Alexa Fluor 488-phalloidin for Flow cytometry analysis. Mean fluorescence intensities were normalized to unstimulated cells and mean values +s.d. of 5 independently performed experiments are depicted. (b) CLL cells were treated for 30 min with ChA, 40 μM AMD3100, or 0.1% DMSO, and filamentous actin was quantified using Alexa Fluor 488-phalloidin by Flow cytometry. Mean fluorescence intensities relative to DMSO-treated cells of two to five independently performed experiments per data point are depicted. Error bars represent standard deviations. Statistical significance was calculated using student’s paired t test: **P-value<0.01. (c) JEKO-1 cells were treated for 30 min with 1 μM ChA or DMSO (0.1%) as a control. Thereafter, 200 ng/ml SDF1α was added for 60 s and filamentous actin was stained with Alexa Fluor 488-phalloidin and cells were analyzed by confocal microscopy (n=2).

As ChA disturbs actin filaments and prevents membrane ruffling, we investigated its effect on the migratory potential of CLL cells. Therefore, CLL cells pre treated for 30 min with 1, 5 or 10 μM ChA, DMSO, or the CXCR4 inhibitor AMD3100 were tested in migration assays using 200 ng/ml SDF1α as the chemoattractant. Quantification of migrated cells after 3 h of incubation showed that ChA treatment inhibited SDF1α-induced migration of CLL cells (n=3; Figure 6a), which was comparable to the effect of AMD3100 treatment.

Figure 6
figure6

ChA impairs cell migration and cytokinesis. (a) Chemotaxis assays were performed using transwells for 24-well plates with 5 μM pore size applying 3 × 106 CLL cells per well. Complete medium with or without 200 ng/ml SDF1α was used as a chemoattractant. After 3 h, migrated cells were washed off the lower side of the membrane and counted. Mean values +s.d. of cell counts of three independently performed experiments are depicted. (b) MEC-1 cells were treated with 1 or 5 μM ChA or 0.1% DMSO for 12 h and cell-cycle distribution was quantified by Flow cytometry after propidium iodide staining of fixed cells. One representative example out of three samples that were analyzed in triplicates is depicted.

Cytochalasan family members are known to block cytokinesis, a process that is highly dependent on actin filament reorganization. As expected, treatment of the CLL cell line MEC-1 with 1 μM ChA resulted in accumulation of cells in the G2-phase, confirming inhibition of cell division by this compound (n=3; Figure 6b). Upon 5 μM ChA treatment, an increase in sub-G1 cells was observed, confirming induction of apoptosis.

ChA reduces activation of CLL cells and sensitizes them to PI3K and BTK inhibitors

CLL cells in the LN display gene signatures that indicate BCR and nuclear factor-κB activation, which are believed to contribute to the increased therapy resistance of these cells.33 This activation phenotype is recapitulated in CD40L/IL-4 culture conditions, in which we observed an increase in cell size and upregulation of costimulatory proteins, like CD80 and CD86 (Figure 7a). Interestingly, treatment with ChA reduces this activation phenotype (Figures 7a and b). To investigate whether this results in an increased sensitivity of CLL cells to drugs, we established cocultures of five CLL cases with BHK-huCD40L. After pre treatment with 10 μM ChA or DMSO, we applied the PI3K inhibitor CAL-101 (GS-1101), the BTK inhibitor PCI-32765 (Ibrutinib), or fludarabine to the cultures. CellTiter-Glo measurements revealed that viability of CD40L-stimulated CLL cells was barely affected by a 48 h treatment with the inhibitors alone (n=4 to 5; Figure 7c). However, pre treatment of cells for 48 h with ChA resulted in a significant reduction of cell viability in the presence of PI3K and BTK inhibitors, but not in cultures where fludarabine was applied. This suggests that ChA sensitizes CLL cells to treatment with PI3K and BTK inhibitors.

Figure 7
figure7

ChA reduces activation and sensitizes CLL cells to treatment with PI3K and BTK inhibitors. Cocultures of five CLL samples with irradiated BHK-huCD40L cells were established and after 24 h ChA or 0.1% DMSO was added for 48 h. (a) To determine the activation status of the cells, Flow cytometry analyses were carried out using CD80-PE or CD86-PE before (d0) and after culture (d3). One representative example of CD86 staining is presented with the following histograms: gray filled=isotype control at d0; green line=CD86 at d0; blue line=CD86 after 48 h of 5 μM ChA; red line=CD86 after 48 h of DMSO. (b) Relative mean fluorescence intensity values of CD80 and CD86 were normalized to DMSO control samples at d3. Mean values +s.d. of 4 CLL samples treated with 1 μM or 5 μM ChA are depicted. (c) After 48 h of cultivation with ChA or DMSO, 10 μM of either fludarabine, the PI3K inhibitor CAL-101 or the BTK inhibitor PCI-32765 were added for further 48 h to the cultures. CLL cell viability was analyzed via CellTiter-Glo assay. Mean values +s.d. of four to five independently performed experiments are depicted. Statistical significance was calculated with student’s paired t test: *P-value<0.05; **P-value<0.01.

Discussion

Even though most CLL patients respond to currently available chemo- or antibody-based therapies with a profound reduction in tumor burden, relapse of disease and subsequent resistance to therapy is frequent and often associated with clonal evolution of CLL cells.34, 35, 36 This is mainly due to the fact that CLL cells within protective microenvironments in the LN or BM are less effectively targeted by the therapy. Therefore, novel drug screening approaches using CLL coculture systems that mimic the protective in vivo microenvironment are necessary to identify compounds that are able to overcome this protection. In previous studies, we and others have shown that CLL/HS-5 cocultures provide long-term survival support and chemoprotection for CLL cells,22 indicating that they mimic the in vivo situation. In this study we have used such cocultures to screen for novel drugs. In addition to the identification of compounds that overcome chemoprotection by the microenvironment, this system also allowed for exclusion of compounds that showed general cytotoxic activity, which was monitored via the viability of HS-5 stromal cells. The screen was performed with a fungal extract library selected to achieve a maximal variety of different biologically active compounds. This approach resulted in the identification of eight compounds that were evaluated for their potential as novel therapeutics.28 The identification of ChA as the active principle within the extract of Penicillium aquamarinium that shows preferential cytotoxic activity for CLL cells even in protective microenvironments mimicking the BM or LN situation represents a proof of principle for this screening approach, and at the same time revealed a novel potential drug for CLL.

ChA belongs to the family of cytochalasans, which are known to interact with the actin filament network by capping their growing ends, and thereby blocking the assembly or disassembly of the microfilaments resulting in altered dynamic properties.37 The concentration of the cytochalasan as well as of ADP/ATP, Ca2+ and Mg2+ were shown to determine its specific activity.38 As a result of its action, a variety of cellular phenotypes like inhibition of cell adhesion or movement, changes in cellular morphology, inhibition of cytokinesis, extrusion of the nucleus, or induction of apoptosis were observed. Low concentrations of cytochalasan (0.2 μM) were shown to prevent membrane ruffling, but higher concentrations (2–20 μM) were needed to remove stress fibers.32 As alterations in the organization of the cytoskeleton and changes in cellular morphology and motility are characteristic features of transformed cancer cells,39 cytochalasan family members have raised considerable interest as anti-cancer drug candidates.40, 41, 42, 43 In vitro and in vivo studies with a panel of murine and human cancer cell lines confirmed cytotoxic effects of several cytochalasan family members for these tumor cells.41, 44, 45 Chaetoglobosins A–G and J further displayed cytotoxicity against HeLa cells with IC50 values ranging from 3–20 μg/ml.30 However, to date no actin-targeting drugs have entered clinical trials because their mechanism of action interfering with vital cellular processes suggests severe and non-selective cytotoxicity.46 Of interest, recently liposomal cytochalasin D successfully inhibited tumor growth in mice without significant side effects.47

By analyzing the effects of seven different cytochalasan family members on CLL cell viability, we showed that ChA was the compound with the highest preference for CLL cells. Detailed in vitro testing of ChA suggested that, for so far unknown reasons, primary blood cells and leukemic cell lines that grow in suspension are more effectively targeted by this compound compared with fibroblasts or adherent cell lines of different carcinomas. Of major interest is our observation that in a direct comparison of normal B cells and CLL B cells under comparable culture conditions, ChA showed lower LD50 values for CLL cells. Significantly, different effects on B or CLL cells were observed between 2 and 10 μM ChA, suggesting a therapeutic window. Importantly, T cells and primary fibroblasts were not or only barely affected at these drug concentrations. To induce apoptosis in CLL cells in the BHK-CD40L coculture set-up, a microenvironment that mimics proliferation centers in the LN, higher concentrations of ChA are required. But even though cells are hardly killed under these conditions, we showed that 1 μM ChA is sufficient to induce a G2/M cell-cycle arrest and a complete inhibition of CLL cell migration. This suggests that the expansion and recirculation of CLL cells into protective lymphatic niches is impaired at non-toxic ChA concentrations. The successful development of drugs, such as Plerixafor, that target cell migration in CLL, further highlights the relevance of cell motility in the pathogenesis of this malignancy.20

Even though in vitro, a preferential cytotoxic activity of ChA for CLL cells was identified, it is not clear whether the local concentrations of the compound when administered in vivo will be suitable to eradicate the leukemic cells without damaging the normal tissue. So far there are no pharmacodynamics/kinetics data available for the substance, but as its main acute toxicity was observed to be in the spleen and thymus, it is expected that ChA reaches those niches. As CLL cells from patients with poor prognosis, harboring del17p or mutTP53, or showing refractoriness to currently available treatments, responded well to ChA, it seems to be especially of interest for these patients, as it has a distinct mode of action independent from the mechanism of any other therapeutics.

The presence of so called ‘smudge’ cells in routine blood smears is a well-known characteristic of leukemic non-Hodgkin lymphoma including CLL and is seen in virtually all patients. Smudge cells are ruptured malignant B cells due to cell fragility, and their percentage in blood smears was shown to be a predictor of survival in CLL.48 Interestingly, smudge-cell formation has been related to the content of the cytoskeletal protein vimentin that shows aberrantly low expression levels in CLL cells.49 Vimentin is an intermediate filament protein critical for lymphocyte rigidity and integrity, and rearrangement of vimentin filaments was shown to participate in cell activation and signal transduction.50 In addition, lymphocytes of CLL patients contain abnormally low levels of total actin,51 and harbor aberrant rearrangements of the cytoskeleton.52 The relevance of the actin binding protein HS1 in CLL, concerning cytoskeleton regulation, trafficking and homing of CLL cells, as well as disease progression and lymphoid organ infiltration has been recently shown.53, 54 Altogether, these observations suggest that abnormalities within the cytoskeleton might contribute to the preferential killing of CLL cells by ChA.

In recent years, it has become clear that the cytoskeleton has a crucial role in controlling BCR dynamics and signaling. Treanor et al.55 showed that an ezrin- and actin-defined network influences steady-state BCR horizontal diffusion within the cell membrane. As CLL cell survival depends on signaling mediated via the BCR or cytokine receptors, the disruption of signaling cascades by ChA most likely leads to apoptosis induction. In addition, our data show that ChA reduces CLL cell activation and sensitizes them for treatment with kinase inhibitors that impair BCR signaling. These kinase inhibitors are currently tested with great success for treatment of CLL patients.4, 5 Therapeutic approaches that target the cytoskeleton and BCR signaling molecules might therefore improve therapy response in CLL. Clinical observations and in vitro data revealed that these kinase inhibitors impact on the cytoskeleton of CLL cells and therefore inhibit cell migration, circulation and homing of malignant cells into lymphatic tissues.56, 57, 58 In summary, our results show that by targeting the cytoskeleton, ChA might hit CLL at different angles: (i) it inhibits cell migration, which is essential for circulation of CLL cells to lymphatic tissues; (ii) it reduces activation of CLL cells and sensitizes them to kinase inhibitors, suggesting that drug combinations with this compound might improve treatment results in CLL patients. In addition, the disruption of pro-survival signaling cascades that are dependent on a dynamic actin filament network contributes presumably to the cytotoxic activity of ChA for CLL cells. Altogether, these findings suggest this compound as a novel potential drug for CLL, especially for cases with chemorefractory disease that still represent the most challenging clinical problem. As actin filament reorganization is involved in many vital cellular processes, the question of general toxicity in vivo, which might result in severe side effects remains to be answered. Injection of ChA in mice and rats resulted in necrosis of the spleen and thymus, as well as degeneration of spermatocytes in the testicles,59 indicating that in vivo lymphocytes and highly proliferating cells are targeted by the compound. Future studies using murine models of CLL will evaluate the potential of ChA as a novel drug for this malignancy.

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Acknowledgements

This study was supported by a grant from the German José Carreras Leukemia-foundation (DJCLS R 10/04), by the Helmholtz Virtual Institute ‘Understanding and overcoming resistance to apoptosis and therapy in leukemia’ and by the research project of the German Federal Ministry of Education and Research ‘CancerEpiSys’. We acknowledge the support of the Danish Cancer Society (R20-A1157-10-S2).

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Hereby all authors declare, that they have approved the manuscript, they concur with the submission and that the material submitted for publication has not been previously reported and is not under consideration for publication elsewhere.

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Correspondence to T O Larsen or M Seiffert.

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Supplementary Information accompanies this paper on the Leukemia website

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Knudsen, P., Hanna, B., Ohl, S. et al. Chaetoglobosin A preferentially induces apoptosis in chronic lymphocytic leukemia cells by targeting the cytoskeleton. Leukemia 28, 1289–1298 (2014) doi:10.1038/leu.2013.360

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Keywords

  • chronic lymphocytic leukemia
  • cytoskeleton
  • migration
  • drug screening
  • cytochalasan
  • microenvironment

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