Concentrations Of Fludarabine Research Articles

Role of STAT1 signaling pathway in osteoblast apoptosis and effect of blocking the path

Objective To investigate the role of signal transducer and activator of transcription (STAT1) signaling pathway in osteoblast apoptosis, and to study the effect of STAT1 inhibitor (fludarabine) on osteoblast. Methods The viability of the osteoblasts in different concentration of fludarabine was detected by cell counting kit-8 (CCK-8) assay. Osteoblasts were divided into 4 groups: control group, dexamethasone (Dex) group, fludarabine (Flu) group, and Dex + Flu group. Enzyme linked immunosorbent (ELISA) was used for determining the protein expression of Bcl-2 Associated X Protein (BAX), STAT1 and phosphorylation-signal transducers and activators of transcription 1 (p-STAT1), and cleaved caspase-3 to reflect the apoptosis in all groups. Results Treatment with Dex increased the protein expression of apoptosis-related proteins and p-STAT1 in time-dependent and dose-dependent manner. Exposure of the cells to Flu, which was a selective STAT1 inhibitor, resulted in a decreased the protein levels of apoptosis-related proteins. Conclusions Our results suggest that fludarabine could suppress Dex-induced apoptosis through the inhibition of STAT1-mediated up-regulation cleaved caspase-3 expression in osteoblasts. Key words: Osteoblasts/DE; Vidarabine/AA; Vidarabine/PD; Apoptosis; STAT1 transcription factor

An ultrasonic nanobubble-mediated PNP/fludarabine suicide gene system: A new approach for the treatment of hepatocellular carcinoma.

ObjectiveThe purpose of this study is to generate an ultrasonic nanobubble (NB)-mediated purine nucleoside phosphorylase (PNP)/fludarabine suicide gene system for the treatment of human hepatocellular carcinoma (HCC).MethodsNBs were prepared from a mixture the phospholipids 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA), perfluoropropane gas and other materials using the high shear dispersion method. NBs treated with ultrasound irradiation functioned as a gene-transfer system, and a self-constructed suicide gene expression plasmid, pcDNA3.1(+)/PNP, treated with fludarabine functioned as a therapeutic gene. This system was used to determine the cytotoxic effects of PNP/fludarabine on HepG2 cells and SMMC7721 cells.Results1. NBs with a small diameter (208–416 nm) and at a high concentration and fine homogeneity were prepared under the optimal method. 2. The pcDNA3.1(+)/PNP plasmid was efficiently transfected into HCC cells using ultrasonic NBs. 3. At 0.75μg/ml fludarabine, PNP/fludarabine showed marked cytotoxic effects toward HepG2 and SMMC7721 cells. PNP/fludarabine achieved the same effect against both SMMC7721 and HepG2 cells but at a lower concentration of fludarabine for the latter. 4. Bystander effects: a 10–20% decrease in the cell survival rate was observed when only 5–10% of transfected cells were PNP positive.ConclusionsNBs constitute a non-toxic, stable and effective gene-delivery platform. The PNP/fludarabine suicide gene system inhibited the growth of HCC cells, induced HCC cell apoptosis, and caused a notable bystander effect at a low fludarabine concentration. This study establishes an important new method for miniaturizing microbubbles and improving a new NB-mediated approach for gene therapy of HCC.

Abstract A24: The dual PI3K δ/γ inhibitor, RP6530, in combination with ibrutinib or fludarabine, synergistically enhances cytotoxicity in primary CLL cells in vitro.

Abstract Background: The PI3K pathway is a central pro-survival mechanism in chronic lymphocytic leukemia (CLL). While the delta isoform of PI3K is largely associated with B-cell signaling, gamma is involved in cytokine trafficking and modulation of the tumor microenvironment. Selective pharmacologic targeting of PI3K δ/γ may therefore have therapeutic potential. RP6530 is a novel, potent, and selective PI3K δ/γ inhibitor. RP6530 demonstrates high potency against PI3Kδ (IC50=25 nM) and γ (IC50=33 nM) enzymes with selectivity over α (>300-fold) and β (>100-fold) isoforms. In this study, the potential of RP6530 in inducing primary CLL cell cytotoxicity, both as a single agent and in combination with standard CLL drugs, was assessed. Methods: Blood from treatment-naïve CLL patients seen at the Duke Center for CLL and enrolled in IRB approved protocols at the Duke University and Durham VA Medical Centers was collected. Cells were enriched using Rosette-Sep B cells from Stem Cell Technology, generally giving purity of > 97% of B cells/CLL cells. 2.5 x 105 primary CLL cells were incubated in serum free medium (SFM) with serial dilutions of RP6530, ibrutinib, fludarabine, bendamustine, and chlorambucil, either alone or in combination, in 96 well plates in triplicate. After three days, cytotoxicity was determined using the MTS assay. Fractional cytotoxicity was calculated by comparing absorbance for each experimental condition to absorbance from wells containing CLL cells cultured in SFM alone. For cytotoxicity results of single drugs, a 2-parameter log-logistic model was used to plot the data. For cytotoxicity results of drugs in combination, a 4-parameter log-logistic model was used to plot the data. Modeling and graphing was performed using the drc package in the statistical environment R. Results: The efficacy of RP6530 in CLL lymphocytes collected from five patients was evaluated; 4 with normal FISH cytogenetics, and 1 with 13q deletion. IGHV was mutated in three of the five samples including the one with 13q deletion. Data demonstrate that RP6530 was highly effective at killing primary CLL cells in vitro. Calculated IC50 for RP6530 (362 nM) was lower that the Burton tyrosine kinase inhibitor, Ibrutinib (567 nM), as well the other conventional chemotherapy agents such as fludarabine (14.8 μM), bendamustine (382 μM), and chlorambucil (152 μM). Combination with RP6530 with any of the four other drugs demonstrated potentiation of cytotoxicity. Combination of very low concentrations of ibrutinib and RP6530 (3 nM of each) resulted in a >50% cell kill indicating a dramatic synergistic effect. Likewise, the addition of low concentrations of fludarabine (3 μM) potentiated the cytotoxic effect by causing a 10-fold increase in potency of RP6530 (IC50 = 32 nM). On the other hand, addition of only the higher concentrations of bendamustine (500 μM) or chlorambucil (60 μM) potentiated the cytotoxicity due to RP6530 (IC50 of <3 nM and 60 nM respectively), indicating that the combinatorial effect is most likely to be additive. Conclusions: RP6530 is a potent PI3K δ/γ inhibitor that induces concentration-dependent cytotoxicity in primary CLL lymphocytes both as a single agent as well as in combination with standard CLL therapy. The extreme synergism demonstrated by RP6530 and Ibrutinib even at the lowest concentrations tested warrant evaluation of the efficacy of the combination in CLL patients. Citation Format: Swaroop Vakkalanka, Kumar V. Penmetsa, Dawn Chasse, Youwei Chen, Srikant Viswanadha, Daphne R. Friedman, J Brice Weinberg. The dual PI3K δ/γ inhibitor, RP6530, in combination with ibrutinib or fludarabine, synergistically enhances cytotoxicity in primary CLL cells in vitro. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr A24.

P38 MAPK Controls Sensitivity Towards BH3 Mimetics By Regulating Mcl-1 Expression of Chronic Lymphocytic Leukemia in Hypoxia and Acquired Resistance

Background: CLL patients frequently suffer relapse after an initially successful chemotherapy. This distinct resistance towards chemotherapy is thought to be caused by microenvironmental stimulation. Within the tumor microenvironment (TME) cells are not only stimulated by well-known external stimuli like CD40 ligand (CD40L) or activation of the B cell receptor (BCR), but are also exposed to hypoxia, which was found in the bone marrow and lymphatic tissue. Despite the known importance of hypoxia in solid tumors, its impact on survival and treatment response in CLL is still poorly understood.Methods: We have established a novel in vitro model for the CLL microenvironment, which considers both the external stimulation by CD40L and the hypoxic oxygen levels (1% O2). Treatment efficacy of different drugs in normoxia (21% O2) and hypoxia were determined by AnnexinV/7-AAD staining and subsequent FACS analysis. The underlying molecular mechanisms were analyzed via qRT-PCR and immunoblot. Furthermore B-cell lines Raji, Ramos and Mec-1 were continuously exposed to increasing concentrations of fludarabine or the BH3 mimetic ABT-737. After establishment of resistance the molecular adaptation was assessed and correlated to the changes induced by hypoxia.Results: Hypoxia is known to protect solid cancers from chemotherapy. In our model we made similar observations for CLL, since sensitivity to the classical DNA-targeting drugs fludarabine and bendamustine was reduced under hypoxic conditions. Interestingly, the tyrosine kinase inhibitor ibrutinib did not benefit from hypoxia either. However, this resistance was overcome by the mitochondria-targeting BH3 mimetics ABT-199 and ABT-737, whose effect was pronounced under hypoxia.We reveal that this effect was caused by an uncoupling of major signaling pathways. Under hypoxic conditions the activity of Akt, ERK1/2 and NFκB was reduced, while p38 MAPK became hyperphosphorylated. Phospho-p38 (pp38) downregulated Mcl-1 levels, which are the main regulator of sensitivity towards BH3 mimetics. Despite the known heterogeneity in between CLL patients this effect was found in most samples analyzed. The functional importance was underlined by the observation that pharmacological inhibition of p38 MAPK could reconstitute Mcl-1 levels and thereby resistance in hypoxia.The relevance of the pp38-Mcl-1 axis for ABT efficacy was emphasized by findings in B-cell lines with acquired resistance. Each ABT-resistant clone of the three tested cell lines induced p38 activity and decreased Mcl-1 levels. In contrast, in the fludarabine-resistant clones the pp38-Mcl-1 axis was not altered.Conclusion: These are the first experiments providing evidence that hypoxia has a crucial impact on survival and response to chemotherapy in CLL. We show that hypoxia renders CLL cells resistant to classical DNA-targeting agents, while the small molecules ABT-199 and ABT-737, which specifically target mitochondria, efficiently eradicate CLL cells within the microenvironment. Furthermore, we identified the pp38-Mcl-1 axis to be a major determinant of sensitivity to these BH3 mimetics, which warrants further evaluation of p38 as a novel biomarker for prediction of sensitivity to BH3 mimetics. DisclosuresNo relevant conflicts of interest to declare.

In VitroSensitivity of CLL Cells to Fludarabine May Be Modulated by the Stimulation of Toll-like Receptors

The emerging role of Toll-like receptors (TLR) in the pathogenesis of chronic lymphocytic leukemia (CLL) led us to ask whether TLR stimulation may protect CLL cells from drug-induced apoptosis. We cultured in vitro malignant B cells freshly isolated from 44 patients with CLLs in the presence or the absence of different concentrations of fludarabine before or after 24-hour TLR stimulation with specific ligands and evaluated cell viability, apoptosis, and molecular pathways involved. Heterogeneity was observed among samples. In leukemic cells from patients bearing adverse prognostic factors, TLR stimulation caused a significant increase of protection to fludarabine treatment, whereas this did not occur in the cells from patients with good prognosis. To identify novel molecular mechanisms accounting for the dichotomy of response between the two groups of patients, we conducted an apoptosis gene expression profile on leukemic cells either unstimulated or stimulated with TLR9 ligand. Strikingly, TLR9 stimulation specifically upregulated the expression of lymphotoxin-α in cells where an increased protection to fludarabine treatment was observed. Also, the expression of miR-155-3p was significantly increased after stimulation of distinct TLR in cells where fludarabine treatment was less effective. These results suggest that at least in a proportion of patients, in vitro sensitivity to fludarabine may be modulated by the stimulation of TLR, likely mimicking microenvironmental signals occurring in vivo.

P B S-1086, a “Pan-Rel” Inhibitor, Decreases Viability of Chronic Lymphocytic Leukemia Cells

AbstractAbstract 867The NF-κB signaling pathway is constitutively activated in chronic lymphocytic leukemia (CLL). Aberrant activation of the p65 (RelA) subunit of NF-κB is associated with chemoresistance, a shorter lymphocyte doubling time and poor overall survival (OS) in CLL. The transcription factor, NF-κB, regulates anti-apoptotic and pro-survival genes, and therefore represents a drug target for therapeutic intervention. Several studies have shown that targeting NF-κB decreases CLL cell survival, induces apoptosis and inhibits transcription of key pro-survival genes. In CLL, poor response to therapy and poor outcome are closely related to cytogenetic abnormalities. Loss of p53 function (del(17p) and/or TP53 mutation) confers chemo-resistance due to a defective DNA damage response pathway. Loss of the ATM (ataxia telangiectasia mutated) kinase (del(11q) and/or mutation of ATM) is also associated with chemo-resistance. We hypothesized that blocking NF-κB may circumvent the requirement for an active DNA damage response pathway, and that NF-κB inhibition may be efficacious independent of cytogenetic abnormalities in CLL. We tested the hypothesis with PBS-1086, a novel small molecule “pan-rel” inhibitor that has shown activity against multiple myeloma in vivo.For target validation, we analyzed NF-κB activity by quantifying NF-κB subunit DNA binding and rel expression in nuclear extracts prepared from patient-derived CLL cells. Levels of activated NF-κB binding correlated with rel protein levels. We confirmed that constitutive activation of the p65 subunit predicted shorter overall survival (p= 0.03, n=80, HR (hazard ratio) 2.4). We also found that the p50 and RelB subunits were associated with poor survival (p = 0.04, n = 80, HR = 2.5 and p=0.17, n=50, respectively). Whereas p65 and p50 (activated by the canonical pathway through IKKβ), were present at high levels in the majority of cases, RelB (activated by the non-canonical pathway) was found to be variable among patients, with levels ranging from very high to undetectable. We found that patients with del(17p) had higher levels of p65 and p50 DNA binding activity, whereas patients with del(11q) had higher levels of RelB activity. We examined the effect of PBS-1086, a “pan-rel” inhibitor, on viability of patient-derived CLL cells and compared it with BAY 11–7082, an IKKβ inhibitor that affects p65 and p50. PBS-1086 was more potent at decreasing CLL viability in a concentration-dependent manner than BAY 11–7082 (mean LC50 6.4 +/− 1.9 μM, and 14.7 +/− 1.7 μM, respectively, n=20). LC50 values of PBS-1086 and BAY 11–7082 derived from individual CLL samples correlated significantly, suggesting that they acted by inhibiting the same pathway (p=0.02, n=20). PBS-1086 LC50 values also correlated inversely with the DNA binding activity of p65 (p=0.01, n=17), suggesting that these CLL cells were heavily reliant on p65 for survival, and were therefore exquisitely vulnerable to compounds inhibiting p65. There was no significant difference in sensitivity to PBS-1086 or BAY 11–7082 among patients with different cytogenetic abnormalities, or Binet stage A or stage B/C cases, or between cases with or without IGVH gene mutations. Patients who had received prior chemotherapies at the time of sample collection showed a trend to be more resistant to ex vivo treatment with Fludarabine than those naïve to chemotherapies, however treatment status had no impact on ex vivo sensitivity to PBS-1086. Submaximal concentrations of Fludarabine and PBS-1086 were additive in decreasing CLL cell viability (n=3). Mechanistic studies showed that PBS-1086 inhibited p65 and p50 DNA binding activity in CLL cells (n=5). In one example case that had constitutive expression of all of the NF-κB subunits, PBS 1086 inhibited the DNA binding activity of all rel subunits. Both PBS-1086 and BAY 11–7082 induced apoptosis in a concentration-dependent manner in CLL cells (Annexin V staining, n=4) that paralled the inhibitor-induced loss of viability.These data demonstrate that PBS-1086 reduced survival of CLL cells ex vivo through inhibiting the DNA binding activity of rel subunits concomitantly with induction of apoptosis. Such effects of PBS-1086 were independent of poor prognosis cytogenetics, and of Binet stage or IGVH status. Thus, PBS-1086 is promising as a therapeutic agent by itself and also as a chemo-sensitizer to overcome NF-κB-mediated chemo-resistance in CLL. Disclosures:No relevant conflicts of interest to declare.

P505-15, a Highly Selective SYK Inhibitor, Shows Significant Activity In Primary CLL Cells and Is Synergistic with Fludarabine at Low Concentrations

AbstractAbstract 2839 Background:Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western World. Despite significant progress, CLL remains incurable so novel therapies are needed. Recent studies have highlighted the importance of B-cell receptor (BCR) associated kinases in the pathogenesis of B cell malignancies and as potential therapeutic targets. Spleen tyrosine kinase (SYK) is of particular interest as its activation results in enhanced proliferation and survival of B-cells. Analysis of non-Hodgkins lymphoma (NHL) cell lines and primary chronic lymphocytic leukemia (CLL) samples have shown that SYK is persistently phosphorylated and that SYK inhibition results in abrogation of downstream kinase activity, leading to apoptosis. The kinase inhibitor FosD (R788), which has shown clinical activity in heavily pre-treated NHL and CLL patients, exhibits inhibitory activity against SYK (IC50 = 41nM) but also inhibits a broad spectrum of other kinase targets. By contrast, P505-15 is a novel small-molecule SYK inhibitor (SYK IC50 = 1nM) that is far more selective than previously described SYK inhibitory compounds with anti SYK activity that is at least 80-fold greater than its affinity for other kinases. We applied P505-15 to primary CLL cells to evaluate the effect of P505-15 on cell viability alone or in combination with fludarabine. Methods:Fresh primary CLL cells from 42 CLL patients were purified using a Ficoll gradient. Purified cells were then added to wells (5 × 104 per well) containing four serial dilutions of P505-15 (ranging from 10 nM to 10 μM) with or without different concentrations of fludarabine (also ranging from 10nM to 10 μM). Three days after adding primary CLL cells to each well, we performed a tetrazolium-based cell viability assay (MTS) to evaluate the effect of P505-15 on CLL cells. The viability data are normalized to untreated controls and used to calculate IC50 values. Synergy (P505-15 plus fludarabine) was evaluated using the Calcusyn program. Results:We saw significantly decreased cell viability (IC50 < 3 μM) in 15/42 (36%) of primary CLL samples. Twelve (29%) of these samples had IC50 drug concentrations < 1 μM (median 393.6 nM). One of seven samples with the 17p deletion exhibited significant sensitivity (IC50 = 37.5 nM). In the presence of P505-15, significant decreases in cell viability were also seen in samples from relapsed patients and in those with additional poor risk features such as ZAP70 and/or CD38 expression, chromosome 11q deletion, and/or an unmutated IgVh. When P505-15 was combined with fludarabine (n=15), synergy was observed in 13 (87%) of samples including at the lowest concentrations of P505-15 (10 nM) and fludarabine (10 nM)-See Figure 1. [Display omitted] Conclusions:These data demonstrate single agent activity of the highly specific SYK inhibitor P505-15 in CLL. Combination therapy with fludarabine is synergistic at very low concentrations of both P505-15 and fludarabine. Thus, P505-15 appears to be an attractive compound for clinical development especially given its high selectivity for SYK. Our results validate the rationale of targeting SYK in CLL. An initial human dose finding study in normal healthy volunteers is ongoing. Disclosures:Druker:Molecular MD: Equity Ownership. Pandey:Portola Pharmaceuticals: Employment. Sinha:Portola Pharmaceuticals: Employment.

ATM Mutant Chronic Lymphocytic Leukaemia Cells are Chemosensitized by Inhibition of DNA-Dependent Protein Kinase

AbstractAbstract 433Defects in ataxia telangiectasia mutated kinase (ATM) confer poor survival in B-cell chronic lymphocytic leukaemia (CLL). ATM gene deletion (11q23) occurs in approximately 20% of cases, and of these, 38% harbor an ATM mutation on the second allele, resulting in complete loss of ATM function. This prevents signaling of DNA double strand break (DSB) damage and compromises homologous recombinational (HR) repair. Importantly, del(11q) cases without corresponding ATM mutation display wild type ATM activity, with an intact DNA damage response and better outcome than ATM mutated cases1. Thus, in terms of chemotherapeutic resistance, it has been established that not all del(11q) cases should be placed in one group, since those with wild type ATM function have a distinct response. DNA-dependent protein kinase (DNA-PK) is also important for DSB repair and mediates the alternative non homologous end-joining (NHEJ) pathway. We previously showed that increased DNA-PK activity and expression is frequent in poor prognosis CLL and is associated with shorter survival2. We hypothesized that targeting the NHEJ pathway (via DNA-PK inhibition), in ATM deficient CLL cells that already have a defective DNA damage response, would completely compromise the ability of these cells to perform DSB repair, resulting in selective killing of ATM mutant CLL cells.The ATM status of CLL cases was determined using denaturing HPLC followed by confirmatory direct sequencing. Only those cases with pathogenic mutations (e.g. predicted to cause truncations or change in protein structure, or previously reported in A-T patients) were classed as ‘ATM mutant’. The resulting defects in ATM activity in these cases was confirmed by quantifying phosphorylation of downstream ATM targets (e.g autophosphorylation of ATM (ser1981) or phosphorylation of SMC-1) in lysates of ionizing-radiation-treated cells. Ex vivo cytotoxicity using freshly isolated CLL cells was possible in 77 cases of known ATM status. Cells were exposed to increasing concentrations of Fludarabine in combination with the DNA-PK inhibitor, NU7441 (1μ M), for 72 hr, and viability was assessed using an XTT kit. NU7441 potentiated the cytotoxicity of Fludarabine in 31 cases, including 8 cases with a known ATM mutation. As expected, ATM mutant CLL cells were more drug-resistant than those with wild type ATM, but even highly resistant (>20μ M) ATM mutant cases were resensitized up to 20-fold by NU7441. Pilot data using a similar approach showed ATM mutant cases to be 5-fold more resistant to mitoxantrone, but NU7441 potentiated the cytotoxicity of mitoxantrone in ATM mutant cases (n = 3) and ATM wild type cases (n = 5) by up to 6-fold or 3-fold respectively.Preliminary data (n =18) using an ex vivo assay dot blot assay to measure DNA-PK activity correlated DNA-PK activity to expression of the DNA-PK catalytic subunit as determined by Western blotting. Furthermore, DNA-PK activity was significantly higher in ATM mutant, compared to wild type CLL cells (p = 0.008). Current studies are exploring the formation and longevity of γH2AX foci in ATM mutant CLL cells treated with DNA damaging agents, and the role of DNA-PK in repair of DSB in these cells. Also, the link between DNA-PK activity and ATM mutation will be further examined as it provides a possible mechanism and proof of concept of increased sensitization by DNA-PK inhibitors. These results suggest that DNA-PK inhibition can sensitize poor prognosis, ATM mutant CLL cells to chemotherapeutics. Our data are consistent with the concept of synthetic lethality, where tumor cells harboring a DNA repair defect can be killed by targeting the compensatory DNA repair pathway, and suggest a group of patients that may benefit from this combination.1 Austen et al., Journal of Clin. Oncol. 20082 Willmore et al., Clin. Can. Res. 2008 Disclosures:No relevant conflicts of interest to declare.

Low-dose fludarabine increases rituximab cytotoxicity in B-CLL cells by triggering caspases activation in vitro

Rituximab maintenance therapy provides a significant benefit in patients with indolent B-cell non-Hodgkin lymphoma (NHL). Based on its efficacy in improving response to chemotherapy, the anti-CD20 antibody is currently under evaluation as maintenance therapy also in patients with B-CLL. We evaluated rituximab-mediated cytotoxicity in 10 B-CLL cases pretreated in vitro with non-cytotoxic concentrations of fludarabine. This combination induced a synergic cytotoxic effect in 8 out of 10 patients at a mean level of 26.15 ± 13.9%, compared to 8.05 ± 5.3% cytotoxicity observed with rituximab alone. Consistent with the viability assay, we found an increased caspase-3 activity together with activation of caspase-9 in B-CLL cells sensitive to sequential non-cytotoxic fludarabine and rituximab exposure. Non-cytotoxic fludarabine concentrations may sensitize B-CLL cells to rituximab-mediated cytotoxicity via caspase-3 activation.

Presence of heterozygous ATM deletion may not be critical in the primary response of chronic lymphocytic leukemia cells to fludarabine

Abnormalities of the TP53 or ATM, cooperating tumor-suppressor genes, significantly worsen the treatment options for chronic lymphocytic leukemia (CLL) patients. Although the aberrations seem to be mutually exclusive in this leukemia, inactivation of the former gene leads to worse prognosis. We tested the in vitro sensitivity of the CLL samples with heterozygous ATM deletion to fludarabine and combination of fludarabine and rituximab; the responses were compared with the TP53-abnormal and wild-type (wt) cells to delimitate relative significance of ATM deletion. In vitro analysis was performed on fifty-nine characterized CLL samples using viability assay WST-1. Western blot and real-time RT-PCR were used to monitor the activation of the ATM/p53 pathway. At the clinically relevant concentration of fludarabine, TP53-abnormal samples exhibited markedly higher resistance to fludarabine than the remaining CLL samples (P = 0.012); cohort with ATM deletion was not more resistant than wt cells. A similar induction of the p53 protein and its downstream target genes PUMA and BAX in ATM-deleted and wt cells confirmed that the former subgroup has preserved a critical pro-apoptotic response. Proportions of the samples, which had been sensitized to fludarabine by rituximab pretreatment, were insignificantly lower (P = 0.22) in the TP53-abnormal and ATM-deleted subgroups compared to the wt cases (30%; 29%; 50%, respectively). The presence of ATM (11q22-23) deletion in the CLL cells should not be considered an indication of resistance to fludarabine or its combination with rituximab.

Impaired up-Regulation of Polo-Like Kinase 2 (PLK2) during Fludarabine- and 2-Chlorodeoxyadenosine (CdA)-Induced Cell Killing in B-Cell Chronic Lymphocytic Leukemia (B-CLL) Lymphocytes: A Potential Marker of Defective Damage Response

Patients with B-cell chronic lymphocytic leukemia (B-CLL) carrying alterations in the p53 pathway respond poorly to most of the existing conventional treatments, presumably because an intact p53 pathway is required for cell killing induced by nucleoside analogs (NA) and alkylators. It is thus critical to identify these patients in order to orient them toward alternative non-genotoxic approaches. One promising way to select these patients is to use p53 functional assays which rely on the detection of an impaired up-regulation of p21 (a transcriptional target of p53) in response to ionizing radiations (IR). Since IR is not used therapeutically in B-CLL, we hypothesized that it would be sensible to investigate p53 dysfunction after incubation with NA, which are standard first line approach in B-CLL patients. We firstly performed microarrays in B-CLL samples (n=4) incubated with clinically achievable concentrations of fludarabine and CdA, and selected for sensitivity to these drugs (i.e. low half maximal inhibitory concentration (IC50) by MTT assay). We observed thatNA increased expression of genes which were predominantly p53- dependent,among the latter genes, PLK2 was the most significantly activated at earliest time points (8h) after NA exposure, andthere was no major difference between fludarabine and CdA at equitoxic concentration.Conversely, in highly resistant samples (i.e. high IC50 by MTT assay, n=3), p53 and PLK2 response analyzed by microarray was abolished. We confirmed a significant dose- and time-dependent PLK2 mRNA upregulation after CdA and fludarabine using quantitative real-time PCR (qPCR), and also showed that PLK2 was increased in response to other DNA damaging conditions (UV light), and in response to p53 activation by Nutlin-3. Based on these results, we sought to compare the levels of PLK2 mRNA induction with in vitro chemosensitivity to NA in a large number of individual B-CLL patient's samples (n=18). We observed that cytotoxicity induced by a fixed concentration of fludarabine (3μM) or CdA (0.3μM), measured by MTT at 96h, was well correlated (P<0.01) with PLK2 mRNA induction, measured by qPCR at 24h. In particular, in 12 patients sensitive to fludarabine (median IC50=0.7 μM, range 0.12–2.21), increase in PLK2 mRNA (after normalization with an housekeeping gene) was 10.3 fold (arbitrary units, range 3.6–22.4, P<0.0001) as compared to untreated control. In comparison, in 6 patients with high level of in vitro refractoriness to fludarabine (median IC50=24.6 μM, range 13.6->100), PLK2 increase was only 1.4 fold above untreated control. In parallel experiments, we had verified that chemoresistance to NA was not due to deficiency of deoxycytidine kinase, the limiting enzyme in the conversion of NA into phosphorylated derivatives. Interestingly, lack of PLK2 activation and chemoresistance to NA were observed in patient with normal 17p by FISH, showing that TP53 deletion assessment is only partially overlapping with p53 functional tests. In conclusion, our results show that PLK2 RNA activation after a 24-h incubation with NA is a potential tool to investigate p53 functional integrity in B-CLL cells, and might represent an alternative to p21 protein measurement after IR. Because of the tight correlation with in vitro toxicity of NA, PLK2 testing might constitute a predictor of clinical sensitivity to these drugs which are broadly used in B-CLL

Polydeoxyribonucleotides Antagonize Nucleoside Analogues in Cancer Therapy.

Background: In the past years, an increasing number of nucleic acid based drug candidates (e.g. antisense oligonucleotides, aptamers, ribozymes, RNA interference) have been identified and already tested in clinical trials. But until now, no information is available whether these treatment approaches might interfere with chemically related drugs, e.g. nucleoside analogues used in cancer or viral therapy.Objective: In the current survey we investigated, whether randomly synthesized single-stranded polydeoxyribonucleotides of different length could interfere with the cytotoxic effects of nucleoside analogues (e.g. fludarabine, cytarabine) on lymphocytes or myeloid blasts.Methods: For this purpose, T-lymphocytes or CD33 positive myeloid blasts (purity >95%) were labelled with [1μM] carboxyfluorescein diacetate, succinimidyl ester (5(6)-CFDA-SE) and incubated with different concentrations of fludarabine or cytarabine (200μM, 20μM und 2μM). Cellular proliferation was induced by addition of CD3/CD28 Dynabeads for the T-cells, or a cytokine cocktail containing 50ng/mL of SCF, 25ng/mL of IL-3, 100ng/mL of GM-CSF, 100ng/mL of G-CSF, 2 U/mL of EPO, 0.47g/L of transferrin, and 5×10-5mmol/L of 2-ME for the myeloid blasts. Random polydeoxribonucleotides of different length (20–80b) and concentrations were added immediately or with a delay of 24, 48 or 72 hours to the assay. After 5 days, quantitative CFDA distribution was measured by flow cytometry to assess the cellular proliferation of the T cells and the myeloid blasts. Moreover, cell viability was measured by trypan blue exclusion. Each experiment was performed at least three times.Results: Polydeoxyribonucleotides of different length and composition could antagonize the cytotoxic effects of nucleoside analogues (fludarabine, cytarabine) in both, T-cells and myeloid blasts. Thereby, the antagonistic effects of polydeoxyribonucleotides revealed to be concentration- and time dependent. Moreover interference with a equimolar concentrations of polydeoxyribonucletides could be detected even with a delay of 48 hours. Of importance, the concentration of nucleoside analogues used in these experiments, referred to concentrations applied in standard clinical treatment protocols.Conclusion: Treatment approaches using polydeoxyribonucleotides, might be critical in context with nucleoside analogues, like fludarabine and cytarabine and diminish the efficacy of these drugs. Further experiments are necessary to elucidate the precise mechanism of these effects.

Rituximab Sensitizes B-CLL Cells with Inactivated p53 to Fludarabine.

Background: Defects in the p53 gene predispose B-CLL patients for an inferior outcome, particularly a resistance to treatment by conventional chemotherapeutics. Very little data exist, however, about the efficacy of monoclonal antibody rituximab on B-CLL cells bearing p53 abnormalities. One reason for that might be a methodological - rituximab alone does not have virtually any effect on the viability of B-CLL cells when cultivated in vitro, unless an active human plasma is added. After that, however, the cells are quickly lysed by complement, what is a process independent on p53.Aims: We used an in vitro system (not containing the active human plasma) to monitor a rituximab activity on B-CLL cells with p53 inactivation in relation to subsequently used nucleoside analog fludarabine, which demonstrably acts through the p53 in B-CLL cells.Methods: The p53 abnormalities in blood samples of B-CLL patients were detected by FISH and by functional yeast analysis coupled to sequencing, as described previously (Trbusek et al., Leukemia 2006, 20: 1159–1161). Vitally frozen samples were used in all cases, after 24h pre-cultivation. Mononuclear cells were cultivated for 72h with or without 20μg/ml of rituximab and subsequently for another 48h with four different concentrations of fludarabine (40μg/ml–0,625μg/ml). The cell viability was determined by a WST-1 assay. A sensitization effect of rituximab pretreatment was determined by an ANOVA analysis, with the value p=0,05 being a threshold for a statistical significance. To monitor an apoptosis (a suppossed mechanism of fludarabine action), the Western blotting was used for the caspase-3 cleavage, which was proved previously to occur in drug-treated B-CLL cells.Results: In the subgroup of eleven p53-wt samples the three cases manifested sensitization by rituximab for fludarabine activity, one case showed an oposite (antagonistic) effect, while there was no significant difference for another seven samples. Among ten p53-mutated samples there was just one case exhibiting no influence of rituximab pretreatment (with the p53 alleles being deletion / 281 Asp→Glu), one sample manifested with antagonistic effect (del / 220 Tyr→Cys), while the remaining eight cases showed a statistically significant sensitization by rituximab (del / truncated protein aa 314, del / no protein, del/ wt protein, del / 248 Arg→Gln, del / 249 Arg→Gly, del / del aa 252, del / del aa 252–254, and a composed mutant 281Asp→Asn / 254 Ile→Thr). We noticed a statistically significant potentiation also in three out of four ATM-deleted samples (ATM is the p53-regulatory kinase). An apoptosis occured after fludarabine addition both in pretreated and control cells, as evidenced by the caspase-3 cleavage in some (but not all) samples.Conclusions: We show, to our konwledge for the first time, that rituximab can significantly sensitize the B-CLL cells bearing different types of p53 mutations to fludarabine. This result warrants further investigation of the mechanism behind.

The Histone Deacetylase Inhibitor MS-275 Interacts Synergistically with Fludarabine to Induce Apoptosis in Human Leukemia Cells

Interactions between the novel benzamide histone deacetylase (HDAC) inhibitor MS-275 and fludarabine were examined in lymphoid and myeloid human leukemia cells in relation to mitochondrial injury, signal transduction events, and apoptosis. Prior exposure of Jurkat lymphoblastic leukemia cells to a marginally toxic concentration of MS-275 (e.g., 500 nM) for 24 h sharply increased mitochondrial injury, caspase activation, and apoptosis in response to a minimally toxic concentration of fludarabine (500 nM), resulting in highly synergistic antileukemic interactions and loss of clonogenic survival. Simultaneous exposure to MS-275 and fludarabine also led to synergistic effects, but these were not as pronounced as observed with sequential treatment. Similar interactions were noted in the case of (a) other human leukemia cell lines (e.g., U937, CCRF-CEM); (b) other HDAC inhibitors (e.g., sodium butyrate); and (c) other nucleoside analogues (e.g., 1-beta-D-arabinofuranosylcytosine, gemcitabine). Potentiation of fludarabine lethality by MS-275 was associated with acetylation of histones H3 and H4, down-regulation of the antiapoptotic proteins XIAP and Mcl-1, enhanced cytosolic release of proapoptotic mitochondrial proteins (e.g., cytochrome c, Smac/DIABLO, and apoptosis-inducing factor), and caspase activation. It was also accompanied by the caspase-dependent down-regulation of p27(KIP1), cyclins A, E, and D(1), and cleavage and diminished phosphorylation of retinoblastoma protein. However, increased lethality of the combination was not associated with enhanced fludarabine triphosphate formation or DNA incorporation and occurred despite a slight reduction in the S-phase fraction. Prior exposure to MS-275 attenuated fludarabine-mediated activation of MEK1/2, extracellular signal-regulated kinase, and Akt, and enhanced c-Jun NH(2)-terminal kinase phosphorylation; furthermore, inducible expression of constitutively active MEK1/2 or Akt significantly diminished MS-275/fludarabine-induced lethality. Combined exposure of cells to MS-275 and fludarabine was associated with a significant increase in generation of reactive oxygen species; moreover, both the increase in reactive oxygen species and apoptosis were largely attenuated by coadministration of the free radical scavenger L-N-acetylcysteine. Finally, prior administration of MS-275 markedly potentiated fludarabine-mediated generation of the proapoptotic lipid second messenger ceramide. Taken together, these findings indicate that the HDAC inhibitor MS-275 induces multiple perturbations in signal transduction, survival, and cell cycle regulatory pathways that lower the threshold for fludarabine-mediated mitochondrial injury and apoptosis in human leukemia cells. They also provide insights into possible mechanisms by which novel, clinically relevant HDAC inhibitors might be used to enhance the antileukemic activity of established nucleoside analogues such as fludarabine.

In vitro sensitivity of B-CLL cells to fludarabine and interferons.

In this study we evaluated the cytotoxicity of Fludarabine (FAMP) both alone and in combination with alpha and beta interferon (IFN) against B-cells from patients affected by chronic lymphocytic leukemia (CLL). We used an in vitro colorimetric assay based on the bioreduction of the tetrazolium salt XTT by viable cells. Fludarabine concentrations ranging from 0.03 to 30 microM were tested on cells collected from 22 B-CLL patients. For each fludarabine concentration, 800 I.U. of either alpha or beta IFN were added. Interferon alone did not exert any cytotoxic effect, while Fludarabine showed a strong cytotoxicity against B-CLL cells. The concentration of Fludarabine required to induce a 50% cytotoxicity (IC50) was below 3 microM (the achievable serum level after standard dose in vivo administration) for 19 out of 22 patients. After IFNs supplementation to Fludarabine, it was possible to identify three groups of samples. The first in which IFNs addition did not produce almost any significant change in Fludarabine cytotoxicity (13/22), the second in which there was an improvement in FAMP IC50 (6/22), and finally the third group in which IFNs worsened it (3/22). Stage of disease was the only identified factor accounting for these different results. The second group included samples from 5 patients at stage A and one at stage B, while in the third group all three samples were from patients at stage C. Interferon-alpha and -beta induced the same degree of FAMP IC50 variation.(ABSTRACT TRUNCATED AT 250 WORDS)