Chronic lymphocytic leukemia (CLL) is the most prevalent lymphoid malignancy in the elderly of the Western world. Although treatment options have improved over the past two decades, 10–15% of patients still have a poor prognosis and are often resistant to therapy. Aberrations in the p53 pathway, such as a deleted (del17p13) or mutated p53 gene, are highly enriched in this class of patients. In an extensive screen for p53-independent apoptosis inducers, actinomycin D was identified from 1496 substances and shown to induce apoptosis in primary CLL cells derived from high-risk patients including those with aberrant p53, revealing a novel p53-independent mechanism of action. Both pro-survival genes BCL2 and MCL1 are targeted by actinomycin D, in contrast to fludarabine the backbone of current treatment schedules. In the well-established TCL1 transgenic mouse model for high-risk CLL, actinomycin D treatment was more effective in reducing tumor load than fludarabine, with no evidence of resistance after three treatment cycles and an overall survival increase of over 300%. Tumor load reduction was coupled to BCL2 downregulation. Our results identify the clinically approved compound actinomycin D as a potentially valuable treatment option for CLL high-risk patients.
Chronic lymphocytic leukemia (CLL) is the most common type of leukemia in the elderly. It is characterized by the accumulation of CD5/CD19/CD23 positive B-lymphocytes in the blood, bone marrow, lymph nodes and spleen. The clinical course of the disease is highly variable, ranging from patients who remain asymptomatic over 10 years to others who require aggressive therapy immediately after diagnosis.1, 2 The chromosomal aberrations del17p13 and del11q22, found in over 10% of patients with early-stage disease, are both associated with inferior outcome and in most cases lead to abrogation of the genes encoding the tumor suppressor p53 and the DNA damage response protein ATM, respectively.3, 4, 5, 6, 7, 8 It was recently shown that p53 point mutations have the same negative prognostic power as del17p13.9, 10 About 10–15% of CLL patients are ‘ultra high risk’, with a median survival of <2 years; however, not all of them have p53 aberrations. In these patients, current whole-genome or exome sequencing efforts have revealed mutations within NOTCH1 and BIRC3 as possible reasons for therapy resistance.11, 12 ‘Ultra-high-risk’ patients show primary resistance to purine analog-based therapy or relapse within 24 months of first treatment with modern chemoimmunotherapy regimens such as fludarabine, cyclophosphamide, rituximab (FCR) or bendamustine, rituximab (BR).13, 14, 15 Treatment options for ‘ultra-high-risk’ patients are limited and mainly consist of allogeneic stem cell transplantation, the anti-CD52 antibody alemtuzumab or agents still in clinical trials, such as flavopiridol. Alemtuzumab-based therapy has emerged as the preferred treatment strategy for del17p13 and/or fludarabine-resistant patients, although problems like short duration of response and lack of efficacy in many patients remain.8, 16 Two recent studies have also shown that flavopiridol can induce partial remission in fludarabine-resistant and del17p13 patients with an average of four prior therapies.17, 18 However, these current treatments are still far from satisfactory, with <50% response rates recently reported from ongoing clinical trials. Additionally, given the early relapse of these patients, there is an urgent need for the identification of new, more potent alternatives. We performed a high throughput screen of 1496 substances, applying different hierarchic filters such as apoptosis induction, p53 independence, toxicity and activity in primary CLL cells to identify compounds that could induce apoptosis in cells with high-risk features such as del17p13 and/or unmutated B-cell receptor. We identified actinomycin D among nine other compounds, as being a highly effective apoptosis inducer. Actinomycin D inhibits DNA-dependent RNA synthesis by binding to guanine residues, and is clinically approved for treating rare tumors such as Wilms tumor and rhabdomyosarcoma.19, 20 In CLL, it has been compared with fludarabine as a general inhibitor of RNA synthesis; however, its differential effect on individual mRNAs has not been analyzed.21
In this study, we demonstrate that actinomycin D induced apoptosis in CLL is independent of del17p status. On the basis of transcriptome profiling, protein quantification and intracellular-staining using fluorescence-activated cell sorting, we find that in CLL cells actinomycin D targets both survival proteins MCL1 and BCL2. Furthermore, the efficacy of actinomycin D is tested in two different mouse models for high-risk CLL.22, 23, 24, 25
Materials and methods
Patient samples, cell lines and chemotherapeutics
Peripheral blood samples of CLL patients were collected during routine examinations at the outpatient clinic of the third medical department, University Hospital Salzburg, after obtaining informed consent and in agreement with the declaration of Helsinki. CLL was defined according to the latest iwCLL workshop criteria.26 The clinical characteristics of CLL patients are shown in Supplementary Table 1. Control blood was derived from buffy coats (age matched) of healthy volunteers. Peripheral blood mononuclear cells were isolated by density centrifugation using Biocoll (d=1.077 g/ml, Biochrom AG, Berlin, Germany). ABT-737 was a generous gift from Abbot Laboratories (Abbott Park, IL, USA). Actinomycin D and 2-fluoroadenine-9-β-D-arabinofuranoside (F-ara-A) were obtained from Sigma Aldrich (St Louis, MO, USA). F-ara-A, the active metabolite of fludarabine, was used for in vitro experiments, and fludarabine was used for in vivo experiments (Bayer Schering Pharma, Vienna, Austria). The compound set was derived from plant and microbial sources (MEGx and NATx from Analyticon Discovery, Potsdam, Germany) and was selected for high diversity containing >250 different chemotypes. A list of cell lines is given in Supplementary Materials and Methods (see Supplementary Table S2).
XTT metabolic assay
Metabolic activity was measured using the XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) assay system according to the manufacturer’s protocol (Sigma Aldrich, St Louis, MO, USA).27 As positive controls, arsenic trioxide (4 μM), which we have previously shown to be preferentially active in del17p13 CLL cells, and the BCL2-family inhibitor ABT-737 (0.1 μM), which is highly potent in CLL cells, were used.28, 29
Peripheral blood mononuclear cells of CLL patients were incubated for the indicated times in the absence or presence of actinomycin D or fludarabine. CD19-positive cells were isolated to over 98% purity using CD19-coupled magnetic beads from Miltenyi Biotec (Bergisch-Gladbach, Germany), and used for RNA extraction (RNAeasy kit, Qiagen, Hilden, Germany). Reverse transcription and qRT-PCR was performed using pre-designed fluorescently labeled primer sets from Applied Biosystems (Carlsbad, CA, USA). As standard the 18S ribosomal subunit RNA was employed and fold regulation calculated using the 2−ΔΔCt formula. For mRNA arrays total RNA from CD19-purified peripheral blood mononuclear cells of four CLL patients was isolated after 15 h incubation with or without 20 nM actinomycin D using the RNAeasy kit. mRNA microarray hybridization, statistics and western blots are described in Supplementary Materials and Methods.
CLL mouse model
Approval from the Austrian Animal Ethics committee was obtained before performing the experiments. The original Eμ-TCL1a transgenic mice (C. Croce, Columbus, OH, USA) have been backcrossed to C57BL/6 mice for >9 generations. In vivo experiments were performed in C57BL/6 wild-type mice, which were engrafted with tumor cells from Eμ-TCL-1 transgenic mice as described earlier.22, 30 The percentage of CD5+/CD19+ cells in the peripheral blood was routinely checked in mice by taking blood from the tail vein and analyzing it via flow cytometry. When the percentage of tumor cells in the peripheral blood reached 40–60%, treatment was started. Drugs were applied daily via i.p. injections. The p53-deficient CLL mouse model is described in Supplementary Materials and Methods.
Cell viability was assessed by staining with an Annexin V-FITC conjugate (Alexis Biochemicals, San Diego, CA, USA) for apoptosis and 7-AAD (Beckman Coulter, Miami, FL, USA) for cell permeability. The CLL cells were stained with anti-human CD19-PE and CD5-Cy5 (all from Beckman Coulter). To measure intracellular BCL2 levels from the mice, blood samples were taken weekly. The whole blood was first stained with anti-murine CD19-PE (BioLegend, San Diego, CA, USA) and CD5-Cy5 (BioLegend), and was then lysed via FACS Lysing Solution (Becton-Dickinson, Franklin Lakes, NJ, USA). For the intracellular staining an unlabeled BCL2 rabbit polyclonal IgG antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and as secondary antibody a goat anti-rabbit IgG labeled with pacific blue (Invitrogen, Carlsbad, CA, USA) were employed. The fixation/permeabilization kit was used (Beckman Coulter).
Identification of p53-independent apoptosis inducers
To identify new compounds that could be used to treat high-risk CLL patients, we screened 1496 structurally diverse, chemically pure substances with an XTT-based cell proliferation assay using two different cell lines: the p53-deficient T-cell acute lymphoblastic leukemia cell line CEM-C7H2, which would reveal compounds that can induce apoptosis independently of the p53 pathway, and cells from a CLL patient with wild-type p53 who had high lymphocyte counts of >1.5 × 105 cells/μl, to identify compounds specific for CLL. From these two screens, 14 substances were identified that induced apoptosis only in CEM-C7H2 cells, 20 induced apoptosis only in CLL cells and 33 substances induced apoptosis in both cell types. To determine which of these substances had the greatest potential to become an ultimately successful therapy for ultra-high-risk patients, the two latter groups were combined and used to test for apoptosis induction in CLL cells from 14 patients including three with chromosomal aberration del17p13 (Supplementary Table 1). Of the 53 tested compounds, 26 induced apoptosis in more than 11 of these 14 CLL cell samples (that is, >80%) and were selected for further analysis. To isolate compounds that would be more likely to progress to the clinic, toxicity screens were performed in neoplastic and non-neoplastic cell lines derived from liver (Hep-G2), skin (WS1), lung (SHP-77) and kidney (HEK-293). As an additional control, substances were also tested in human umbilical cord venous endothelial cells (HUVEC) derived from four different donors. The nine compounds (actinomycin D, cephaeline, ochratoxin, lasalocid A, neosolaniol, oligomycin A, fumonisin B1, substance 13, rotenolone) with the lowest toxicity to the cells described above were tested for their half maximal inhibitory concentration at 48 h (IC5048 h) in CLL cells. The compound with the lowest IC5048 h was actinomycin D (0.01 μM, n=7), an anticancer drug in clinical use (Figure 1 and Supplementary Figures 1–4). Its activity in p53-aberrant CLL cells, known clinical properties, low IC50 and moderate toxicity prompted us to further investigate actinomycin D as treatment option for CLL patients with high-risk features.
Role of p53 in actinomycin D-induced apoptosis
To study the role of del17p13 on the apoptosis-inducing effect of actinomycin D, CLL cells with and without del17p13 were coincubated for 48 h with different concentrations of actinomycin D or fludarabine, the backbone of current CLL treatment regimens. As expected, 5 μM fludarabine induced apoptosis in a mean of 52±32% of CLL cells of patients without del17p13 (n=24), but was unable to induce apoptosis in del17p13 (n=8) patient samples (0±15%; P<0.001), which are known to be resistant. In contrast, 0.02 μM actinomycin D effectively induced apoptosis in >60% of CLL cells irrespective of their del17p13 status (67±22%, no del17p; 62±24%, del17p; P=0.565), suggesting that its main route of apoptosis induction is independent of p53 (Figure 2a). To corroborate this, the effect of actinomycin D was also assessed in p53 wild-type (DOHH-2) and p53-mutated B-cell lymphoma cell lines (WSU-NHL, SU-DHL-4, MEC1). The p53 wild-type DOHH-2 cell line responded equally well to actinomycin D and fludarabine. In contrast, the three p53-mutated cell lines displayed resistance toward fludarabine-induced apoptosis, but were all susceptible towards actinomycin D (Supplementary Figure 5).
To test the efficacy of actinomycin D in conditions where CLL cells are supported by their microenvironment, we co-cultured CLL cell samples from different patients (n=12, including two del17p cases) with the murine bone marrow fibroblast cell line M2-10B4. SDF-1-CXCR4 and VCAM-1-VLA-4 interactions, delivering a major part of cell-adhesion-based protection to CLL cells, are highly conserved between the human and murine system, and thus CLL cell co-culture with murine M2-10B4 has been shown to be a reliable in vitro model to study complex microenvironmental interactions.31, 32 The viability of fludarabine-treated CLL cells was enhanced by co-culturing with M2-10B4 (from 52±23% to 83±38%; P>0.001). In contrast, the viability after actinomycin D treatment was similar in the absence (26±21%) or presence (25±22%) of M2-10B4 stromal cells (P=0.367, Figure 2b). To exclude direct effects of the substances on the stroma, we then pre-incubated the CLL cells with the respective substance, washed them and then added them to the stroma cells. Under these conditions the actinomycin D-treated cells were protected by the microenvironment, suggesting that the presence of actinomycin D prevents the release of protective factors from the stroma cells (Figure 2c).
Actinomycin D suppresses BCL2 and MCL1
At micromolar concentrations actinomycin D is a global inhibitor of DNA-dependent transcription.19, 20 In contrast, the transcriptional response to actinomycin D in the nanomolar range in CLL cells, as used in this study, is unknown. Therefore, we treated CLL cells without del17p from four different patients with and without 20 nM actinomycin D, and after 14 h collected isolated mRNA from CD19-sorted cells, which was then used to generate cDNA. The cDNA was hybridized to a microarray containing 30 000 different probes. Strikingly, only 20 of 10 861 detectable mRNAs (0.18%) were deregulated by actinomycin D at the nanomolar range. Within this small group of repressed genes we found pro-survival genes BCL2 , LYN and TOSO, known to be overrepresented in CLL cells (Supplementary Figure 6). This has also been confirmed using qRT-PCR (Supplementary Figure 7 and Figure 3a). BCL2 is a key survival factor in CLL cells, whose overexpression is caused by the depletion of inhibitory miR-15 and miR-16.33 LYN is a tyrosine kinase that links the B-cell receptor to its downstream pathways and is constitutively activated in CLL cells.34 High expression of TOSO, also called Fas inhibitory molecule 3, is associated with unfavorable prognosis and may explain resistance of CLL cells to Fas-induced apoptosis.35
In addition, expression of BCL2 family genes (BCL2, MCL1, BIM, BMF and NOXA) and p53-dependent genes (p53, p21 and PUMA) in CD19-purified CLL cells after 24 h actinomycin D treatment was assessed using RT-PCR. No influence of the CD19-purification process on the expression of the tested genes could be observed (Supplementary Figure 8). A greater than 70-fold suppression of BCL2 mRNA by actinomycin D in CLL cells was observed, ranging from 40 to 150-fold depending on the patient (Figure 3a). In contrast, no BCL2 deregulation could be observed in fludarabine-treated cells. In the latter, p53 downstream targets p21 and PUMA were induced 30- and 27-fold, respectively, in accordance with the notion that fludarabine-induced cell death is largely p53 dependent. Thus, BCL2 mRNA downregulation in CLL seems to be specific for actinomycin D treatment (Figure 3a).
We then analyzed the effect of actinomycin D treatment on the protein levels of pro-survival genes BCL2 and MCL1, as well as on the cell-cycle regulator p21. BCL2 protein levels were not affected, despite the strong mRNA repression, in contrast we observed degradation of MCL1. The stability of BCL2 is not surprising given the reported 20–38 h half-life of the protein (Figure 3b).36
Treatment of overt CLL with actinomycin D
Next, we set out to test actinomycin D’s ability to counteract overt leukemia in one of the best-characterized mouse models for CLL, the Eμ-TCL1 mouse.22 The TCL1 mouse was constructed to specifically overexpress the TCL1 gene in B cells using the Eμ-enhancer promoter system.22, 23, 24, 30 The mean difference between the expressed Ig V(H) genes of these mice and germ line is only 0.5%, thus the TCL1 mouse is thought to model the more aggressive, IgV(H) unmutated form of the disease in humans.37 Lymphocytes of TCL1-mice with overt leukemia were engrafted in 10 C57BL/6 mice, as previously described.22, 23, 24, 30 However, two of them died before the start of treatment, indicating that they had advanced tumor progression. The remaining eight mice were equally divided between control (phosphate-buffered saline) and treatment arms (0.06 mg/kg actinomycin D for 14 consecutive days i.p.). Groups were selected for similar mean tumor burden. By day 67 post engraftment, all of the mice in the control arm had died. In contrast, all mice in the actinomycin D arm were still alive. At day 78 after engraftment, one mouse of the actinomycin D treatment group died. This mouse had the highest initial tumor burden in the treatment group, suggesting that the point of no return may have already been reached before the start of treatment (Figures 4a and b). Blood tests still showed low percentages of tumor cells at day 108 after engraftment in the remaining three mice. In contrast, at day 129 post engraftment, about 11 weeks after treatment start, mice showed increasing levels of CD5/CD19 positive tumor cells. These mice were retreated with actinomycin D, leading to a second remission that lasted a further 11 weeks in mouse M2 and M4, and 15 weeks in mouse M6. A third treatment cycle was started at day 211 post engraftment for mice M2 and M4, and at day 242 post engraftment for mouse M6. Again tumor load was severely reduced in all treated mice (Figures 4c and d).
In the same mouse model, it has been shown that treatment with 34 mg/kg fludarabine for 5 days in three cycles resulted in the development of resistance after the third treatment cycle.24 In contrast, in actinomycin D-treated mice, no resistance development could be observed. The remaining three mice of the actinomycin D group lived until day 295, 338 and 388, nearly reaching their natural life span. The mean survival time after engraftment of the four mice from the actinomycin D group was fourfold longer than that of the mock-treated mice (P=0.008, Figure 4b). When we analyzed the three mice at the time of death, the relative amount of tumor cells in the lymphocytes of the peripheral blood was 4% for mouse M4, 13% for M6 and 44% for M2. These values were reflected in the absolute cell count, revealing neoplastic cells/μl blood of (0.6±0.18) × 103 for M4, (0.9±0.24) × 103 for M6 and (14.0±4.2) × 103 for M2. Thus, in two of three mice tumor cell count was well below 5 × 103 cell/μl, which is one of the leukemia-defining criteria in humans.26 This was in concordance with the values measured for spleen weight (0.34±0.03, 0.33±0.03 and 1.9±0.2 g) and complete spleen cell count ((0.8±0.24) × 108, (1.8±0.18) × 108 and (9.9±2.97) × 108 cells, for mouse M4, M6, and M2 respectively). The values for mouse M4 and M6 were closer to those found in a non-neoplastic mouse (Figures 4e and f). Mouse M2 had an increasing CD5+/CD19+ cell count shortly before death (Figures 4c and d).
Mouse M2, M4 and M6 all received three treatment cycles of actinomycin D, and there were no visible signs of toxicity, such as microvesicular steatosis, cholestasis, fibrosis or acute inflammation by neutrophils observed by histologic examination (Supplementary Figure 9). Thus, actinomycin D leads to tumor regression in this mouse model of CLL, and in two of four mice renewed lymphoma formation was prevented, strongly suggesting a potent role for actinomycin D in CLL treatment in humans.
Direct comparison of fludarabine and actinomycin D
Next, we directly compared actinomycin D with fludarabine treatment, which is the backbone of most current first-line regimens for CLL patients. Fludarabine was applied in cycles of 34 mg/kg for 5 days, i.p., a dose that has been described to prolong overall survival in TCL1 mice.24 Mice were split into three groups of comparable mean tumor load and treated with one cycle either actinomycin D (0.06 mg/kg/14 days i.p., n=9), fludarabine (34 mg/kg/5 days i.p., n=6) or phosphate-buffered saline (14 days i.p., n=6). The median overall survival from time of engraftment was 49 days for the control, 80 days for fludarabine and 135 days for actinomycin D, revealing a strong life-prolonging effect of actinomycin D and, to a lesser degree, of fludarabine (log-rank test; P<0.001 for actinomycin D and P=0.007 for fludarabine, respectively). It is noteworthy that actinomycin D also had a significant life-prolonging effect when directly compared with the fludarabine group (P=0.026), indicating the superiority of actinomycin D (Figures 5a and b). At day 57 all mice of the control and fludarabine group were above the 5 × 103 neoplastic cells/μl threshold known as defining feature of CLL in humans.26 In contrast, in the actinomycin D group 89% of the mice (8/9) were below that level. Taken together, these data indicate that actinomycin D is more effective than fludarabine in reducing tumor load in the TCL1-dependent CLL mouse model (Figure 5c).
To study the effect of actinomycin D on BCL2 within the living mouse, an intracellular staining protocol for murine BCL2 within the CD5/CD19+/+ tumor cell population was established. The mice were treated with 0.06 mg/kg/day actinomycin D, i.p., for 2 weeks. Relative tumor load and BCL2 levels were measured at the start, during and after actinomycin D treatment. During and after actinomycin D treatment we measured a more than 10-fold tumor load reduction, in accordance with our previous experiments (Figure 5c). Concomitantly with the reduction in tumor load, a 4-fold decrease of intracellular BCL2 levels during actinomycin D treatment was observed in both mice (Figure 5d).
Finally, we combined all TCL1 mice analyzed in the course of this study and stratified them according to their treatment (phosphate-buffered saline, n=15; fludarabine, n=12 and actinomycin D, n=22). These mice received three or less treatment cycles of the respective substance. The mean survival of the control, fludarabine and actinomycin D group was 24, 42 and 96.5 days, respectively. Kaplan–Meier log-rank analysis revealed a highly significant advantage for actinomycin D over the fludarabine group (P<0.001, Figure 6).
p53-deficient engraftment model of human CLL
Moreover, we have also modeled p53 deletion in a murine system by engrafting the human p53-aberrant MEC1 cells into Rag2−/− gamma c −/− mice as has been recently described.25 When these mice developed a tumor they were assigned to three groups and treated with actinomycin D (n=4), fludarabine (n=5) or phosphate-buffered saline (n=4) in the schedules described above. Three weeks after treatment start mice were killed. The tumor volume was comparable in the control and fludarabine groups (2.1±0.4 and 2.1±0.7 cm3, respectively), but significantly reduced in the actinomycin D group (0.9±0.5 cm3). This was also mirrored in the tumor weight (1.2±0.5, 1.0±0.3 and 0.6±0.2 g; control, fludarabine and actinomycin D, respectively). In short, actinomycin D, in contrast to fludarabine, was able to reduce tumor growth in this p53-aberrant CLL model system (P=0.026, see Figure 7). These data together with the experiments in primary CLL cells and p53-mutated B-cell lymphoma cell lines suggest that actinomycin D is indeed able to overcome therapy resistance mediated by p53 aberrations in CLL.
Modern chemoimmunotherapy schedules (for example, fludarabine, cyclophosphamide and rituximab) have improved the outcome for CLL patients except the p53-aberrant high-risk group.13, 15, 38 It is now clear that patients with p53 deletion or mutation should be considered for alternative treatment options up front.13, 14 Despite the increased response rates in p53-aberrant patients with clinically approved alemtuzumab, more than half display no response to therapy and relapse early, stressing an urgent need for alternative treatment options for this patient subgroup.39, 40 From a screen of 1496 highly diverse substances, we identified actinomycin D as a potent inhibitor of viability in CLL cells with del17p and/or unmutated IgVH status. Moreover, we show tumor regression in two highly diverse mouse models of CLL—the well-characterized transgenic TCL1 mice22 as well as the recently published xenograft model using Rag2 −/− gamma c −/− mice engrafted with human p53-deficient MEC1 cells.25 Actinomycin D is currently used as a highly effective chemotherapeutic approved for rare tumor entities occurring mostly in children, such as Wilms tumor, rhabdomysarcoma and Ewing sarcoma.41 Its known risk profile and its current use in the clinic would allow for testing of actinomycin D in CLL high-risk patients without further delay.
Actinomycin D in low nanomolar concentrations has been described as a potent activator of the p53 pathway and in this respect has similar features to the MDM2 inhibitor Nutlin-3a, which shows prominent activity in CLL.42, 43, 44 However, Nutlin-3a has been shown to be inactive in CLL cells of del17p13 patients.45 In contrast, we show here that actinomycin D induces apoptosis in primary CLL cells and other B-cell lymphoma cell lines irrespective of p53 status (Figure 2 and Supplementary Figure 5). This argues for an important p53-independent component of actinomycin D induced apoptosis.
The earliest description of actinomycin D in CLL compared its mechanism of action with fludarabine and α-amanitin, the specific RNA-polymerase II blocker.21 The degree of apoptosis induction of fludarabine and α-amanitin was directly correlated to reduction of [5-3H]uridine incorporation, suggesting that the inhibition of RNA synthesis, rather than DNA synthesis is an important mechanism of fludarabine toxicity. However, it was also noted that fludarabine, when reducing RNA synthesis to the same degree as actinomycin D, had a much stronger apoptosis-inducing effect, thereby implicating additional mechanisms of cell death induction. It was hypothesized that blocking the background DNA repair might activate the p53 pathway in CLL cells.21 This is consistent with resistance of del17p13 CLL cells to fludarabine and the strong induction of p21 and PUMA mRNA observed in this study. Moreover, we observed that α-amanitin can induce apoptosis in CLL cells irrespective of p53 status similar to actinomycin D, suggesting that the p53-dependent component in fludarabine induced apoptosis is not caused by transcriptional repression (Supplementary Figure 10). In contrast to α-amanitin that inhibits synthesis of all mRNAs, we show here that nanomolar concentrations of actinomycin D, suppress only a small set of mRNAs including BCL2, LYN and TOSO, all of which have been shown to be important for CLL cell survival. Interestingly, actinomycin D caused a >70-fold suppression of the BCL2-mRNA, which was unique to actinomycin D (Figure 3a). In accordance with this, in the TCL1-mouse model BCL2 protein levels were strongly suppressed in the neoplastic compartment by actinomycin D treatment (Figure 5d). In vitro, we observed degradation of MCL1 protein after actinomycin D challenge despite no deregulation of its mRNA. This may be explained by the fact that MCL1 in contrast to BCL2 is easily proteolytically degraded.46 Recently it was demonstrated that under conditions of stress, glycogen synthase kinase-3 (GSK-3) phosphorylates MCL1 in that way providing a docking site for FBW7, the substrate binding component of the ubiquitin ligase complex, finally leading to MCL1 degradation.47, 48 In CLL, the simultaneous deactivation of MCL1 and BCL2 is likely to exert a strong pro-apoptotic signal.
We show in this study that actinomycin D targets survival proteins TOSO, BCL2 and MCL1 and is active in two different mouse models that are characterized by either unmutated B-cell receptor or inactive p53 function, both of which are known negative prognostic factors in CLL. Therefore, we want to stress the need to test actinomycin D in clinical trials for ‘ultra-high-risk‘ patients as a single agent or in combination with other drugs established for CLL.
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This work was supported by Klinische Malignom und Zytokinforschung Salzburg-Innsbruck GmbH (RG), the Jubiläumsfond der Österreichischen Nationalbank (Grant 12170 to RG), Spezialforschungsprogramm P021 (RG), Fonds zur Förderung der wissenschaftlichen Forschung P-18478-B12 (LK), L488-B13 (AE), P19481-B12 (AE), the Genome Austria Research project ‘Inflammobiota’ (LK) and the Novus Sanguis Consortium, LeJeune Foundation (LK). We are grateful to Richard Moriggl for critical discussion of the manuscript.
OM and NW designed the research. NW, ES, TM, FH and TK performed the experimental work; M Schl and LK performed H&E stains of FFPE tissues; MS provided patient material; M Sche produced mRNA arrays; UD, JPH, AE performed murine experiments; OM and NW wrote the paper; OM, LK and RG did the final approval and correction of the manuscript. OM, LK and RG contributed to project development and obtained funding.
The authors declare no conflict of interest.
Supplementary Information accompanies the paper on the Leukemia website
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Merkel, O., Wacht, N., Sifft, E. et al. Actinomycin D induces p53-independent cell death and prolongs survival in high-risk chronic lymphocytic leukemia. Leukemia 26, 2508–2516 (2012). https://doi.org/10.1038/leu.2012.147
- actinomycin D
- mouse model
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