Cytotoxicity In Chronic Lymphocytic Leukemia Cells Research Articles

Lysosome-Disrupting Agents in Combination with Venetoclax Increase Apoptotic Response in Primary Chronic Lymphocytic Leukemia (CLL) Cells Mediated by Lysosomal Cathepsin D Release and Inhibition of Autophagy.

Venetoclax and obinutuzumab are becoming frontline therapies for chronic lymphocytic leukemia (CLL) patients. Unfortunately, drug resistance still occurs, and the combination could be immunosuppressive. Lysosomes have previously been identified as a target for obinutuzumab cytotoxicity in CLL cells, but the mechanism remains unclear. In addition, studies have shown that lysosomotropic agents can cause synergistic cell death in vitro when combined with the BTK inhibitor, ibrutinib, in primary CLL cells. This indicates that targeting lysosomes could be a treatment strategy for CLL. In this study, we have shown that obinutuzumab induces lysosome membrane permeabilization (LMP) and cathepsin D release in CLL cells. Inhibition of cathepsins reduced obinutuzumab-induced cell death in CLL cells. We further determined that the lysosomotropic agent siramesine in combination with venetoclax increased cell death in primary CLL cells through an increase in reactive oxygen species (ROS) and cathepsin release. Siramesine treatment also induced synergistic cytotoxicity when combined with venetoclax. Microenvironmental factors IL4 and CD40L or incubation with HS-5 stromal cells failed to significantly protect CLL cells from siramesine- and venetoclax-induced apoptosis. We also found that siramesine treatment inhibited autophagy through reduced autolysosomes. Finally, the autophagy inhibitor chloroquine failed to further increase siramesine-induced cell death. Taken together, lysosome-targeting drugs could be an effective strategy in combination with venetoclax to overcome drug resistance in CLL.

NUSAP1 Enhances Drug Resistance By Binding to RAD51 through DNA Damage Repair Pathway in Chronic Lymphocytic Leukemia

Introduction: Enhanced DNA damage repair effect is an important mechanism for drug-resistance in chronic lymphocytic leukemia (CLL). Moreover, the ability of cancer cells to repair under radiation or chemotherapy drug induced DNA damage also serves as one of the mechanisms for therapy resistance. It is reported that nucleolar and spindle associated protein 1 (NUSAP1), a microtubule binding protein, has been involved in DNA damage repair process and plays important roles in the development, progression, and metastasis in several types of cancer. However, its role and mechanism in the development of CLL are still unclear. Methods: Expression levels of NUSAP1 mRNA and protein in CLL cell lines and patient specimens were detected by qRT-PCR and Western blot, and Kaplan-Meier survival curve and overall survival were analyzed by log-rank test. Peripheral blood samples from de novo CLL patients and healthy volunteers were collected with informed consents at the Department of Hematology in Shandong Provincial Hospital Affiliated to Shandong University (SPHASU). Microarray datasets GSE22762 were obtained from Gene Expression Omnibus. With altering NUSAP1 expression by lentivirus-transfected cells in vitro, the effects of NUSAP1 on cell proliferation, apoptosis and cycle were detected by CCK8, Annexin V-PE /7AAD staining and PI/RNase staining respectively. Bioinformatics analysis, luciferase reporter analysis, immunoprecipitation and were applied to discern and examine the relationship between NUSAP1 and its potential targets. Results: According to clinical specimens and bioinformatics analysis, the expression level of NUSAP1 gene in samples of CLL patients was significantly increased than that of healthy donors (P<0.05) (Figure A). Besides, the results indicated that the OS of patients with highly expressed NUSAP1 was significantly worse than in patients with low expression with the statistical analysis database GSE22762. mRNA and protein expression levels of NUSAP1 were significantly higher in CLL cell lines than in PBMCs from healthy donors (Figure C). Our findings indicated that NUSAP1 knockdown notably inhibited cell proliferation when compared with the Scramble group (Figure D). Moreover, the amounts of DNA fragmentation of the apoptotic cells were remarkably increased by NUSAP1 shRNA in MEC-1 and EHEB cells when compared with the Scramble group (Figure E). In addition, after knocking down NUSAP1, MEC-1 and EHEB cells were blocked in G0/G1 phase (Figure F). Moreover, addition to fludarabine or ibrutinib with shNUSAP1 group showed enhanced cytotoxicity in CLL cells (Figure G). The differential genes were analyzed via RNA-seq between Scramble and ShNUSAP1 group. Intriguingly, annotations of gene ontology (GO) analysis indicated that NUSAP1 was closely related to biological processes including cell cycle and response to drug. Kyoto encyclopedia of genes and genomes (KEGG) analysis showed that NUSAP1 were enriched in pathways in cancer, DNA replication and cell cycle. Gene set enrichment analysis (GSEA) implicated that NUSAP1 was functionally enriched in DNA replication, cell cycle and proteasome (Figure H). Immunofluorescence showed that NUSAP1 was mainly distributed in the cell nucleus, and the expression level of RAD51 was positively correlated with the change of NUSAP1 expression (Figure I). Surppression of NUSAP1 inhibited the action of proteins in DNA damage repair pathway (Figure J). Through COIP, NUSAP1 was identified to bind with RAD51 and play an important role in DNA damage repair pathway (Figure K). Hence, NUSAP1 participates in the DNA damage repair process and enhances the drug resistance in CLL. Conclusions: This study first demonstrated that the high expression of NUSAP1 in CLL patients is associated with poor prognosis through database analysis and experiments in vitro. Interference of NUSAP1 expression led to a slower CLL cell proliferation and a higher apoptosis rate, meanwhile induced the G1 phase arrest. Collectively, our findings demonstrated that NUSAP1 contributes to DNA damage repairing by binding to RAD51 and enhances drug resistance in CLL. Therefore, NUSAP1 is expected to be a potential target for the treatment of CLL with drug-resistance. Figure 1 Disclosures No relevant conflicts of interest to declare.

PIM kinase inhibitor, AZD1208, inhibits protein translation and induces autophagy in primary chronic lymphocytic leukemia cells.

The PIM1, PIM2, and PIM3 serine/threonine kinases play a role in the proliferation and survival of cancer cells. Mice lacking these three kinases were viable. Further, in human hematological malignancies, these proteins are overexpressed making them suitable targets. Several small molecule inhibitors against this enzyme were synthesized and tested. AZD1208, an orally available small-molecule drug, inhibits all three PIM kinases at a low nanomolar range. AZD1208 has been tested in clinical trials for patients with solid tumors and hematological malignancies, especially acute myelogenous leukemia. The present study evaluated the efficacy and biological actions of AZD1208 in chronic lymphocytic leukemia (CLL) cells. CLL cells had higher levels of PIM2 protein and mRNAs than did normal lymphocytes from healthy donors. Treatment of CLL lymphocytes with AZD1208 resulted in modest cell death, whereas practically no cytotoxicity was observed in healthy lymphocytes. To determine the mechanism by which AZD1208 inhibits PIM kinase function, we evaluated PIM kinase pathway and downstream substrates. Because peripheral blood CLL cells are replicationally quiescent, we analyzed substrates involved in apoptosis, transcription, and translation but not cell cycle targets. AZD1208 inhibited protein translation by decreasing phosphorylation levels of 4E-binding protein 1 (4E-BP1). AZD1208 induced autophagy in replicationally-quiescent CLL cells, which is consistent with protein translation inhibition. These data suggest that AZD1208 may elicit cytotoxicity in CLL cells through inhibiting translation and autophagy induction.

Indole-3-Carbinol Synergizes with and Restores Fludarabine Sensitivity in Chronic Lymphocytic Leukemia Cells Irrespective of p53 Activity and Treatment Resistances.

Chronic lymphocytic leukemia (CLL) still is lacking a cure. Relapse and development of refractoriness to current treatments are common. New therapies are needed to improve patient prognosis and survival. Indole-3-carbinol (I3C) is a natural product with antitumor properties already clinically tested. The effect of I3C, F-ara-A, and combinations of both drugs on CLL cells from patients representing different Rai stages, IGHV mutation status, cytogenetic alterations, p53 functionality, and treatment resistances was tested, as well as the toxicity of these treatments in mice. I3C induces cytotoxicity in CLL cells but not in normal lymphocytes. I3C strongly synergized with F-ara-A in all CLL cells tested, including those with p53 deficiency and/or F-ara-A resistance. The mechanism of cell death involved p53-dependent and -independent apoptosis. The combination of I3C + F-ara-A was equally effective in CLL cells irrespective of IGHV mutation stage and patient refractoriness. Moreover, CLL survival and treatment resistance induced by co-culturing CLL cells on stroma cells were overcome by the combinatory I3C + F-ara-A treatment. No toxicity was associated with the combined I3C + fludarabine treatment in mice. I3C in combination with F-ara-A is highly cytotoxic in CLL cells from refractory patients and those with p53 deficiency. The striking dose reduction index for F-ara-A in combination with I3C would reduce fludarabine toxicity while having a similar or better anti-CLL effectiveness. Moreover, the low toxicity of I3C, already clinically tested, supports its use as adjuvant and combinatory therapy in CLL, particularly for patients with relapsed or refractory disease.

OSU-2S: a novel non-immunosuppressive FTY720 derivative mediates potent cytotoxic activity through PKC dependent phosphorylation of tumor suppressor SHP1 and down modulation of Tcl1 oncogene in chronic lymphocytic leukemia (P2120)

Abstract We have recently demonstrated cytotoxic activity of FTY720, a sphingosine analogue, against several B cell malignancies including chronic lymphocytic leukemia (CLL).To overcome the limitations associated with immunosuppressive properties of FTY720, we have developed OSU-2S, a synthetic FTY720 derivative with cytotoxic potency and negated immunosuppressive property. Treatment of primary CLL cells with OSU-2S resulted in potent cytotoxicity associated with PKC mediated phosphorylation of Serine591 (S591) of SHP1 and its nuclear translocation consistent with a potential role for S591 phosphorylation. Concentrations of bisindolylmaleimide that inhibited PKC and thus SHP1S591 phosphorylation abrogated OSU-2S mediated cytotoxicity in CLL cells. Gene expression profiling (GEP) revealed OSU-2S mediated down regulation of TCL1A. Consistent with this OSU-2S induced down regulation of TCL1A at mRNA and protein levels in primary CLL cells. The therapeutic efficacy of OSU-2S was demonstrated in Eµ-Tcl1 transgenic CLL mouse model and SCID-Raji xenograft models with significant reduction in leukemic B cells. Further, administration of OSU-2S immunoliposome targeting receptor tyrosine kinase-like orphan receptor (ROR1) expressed in lymphoid malignancies including CLL in Eμ-human ROR1 transgenic mice resulted in selective depletion of ROR1 positive B cells. In summary, we have identified OSU-2S as a modulator of tumor suppressor and oncogene targets in CLL resulting in potent cytotoxicity.

Abstract 2057: In vitro effect of AZD1208 (Pim Kinase Inhibitor) and 17-AAG (HSP90 Inhibitor) combination in CLL.

Abstract Heat shock protein 90 (HSP90) is a molecular chaperone that regulates the functional stability of client oncoproteins, which is driven by the binding and hydrolysis of ATP. The geldanamycin analog, 17-AAG, binds to the ATP pocket of HSP90 leading to the degradation of client proteins such as Akt, p53, STAT3, Bcr-Abl, and Pim-1. Pim (Pim-1, -2, -3) kinases are constitutively active serine/threonine kinases overexpressed in hematological malignancies such as chronic lymphocytic leukemia (CLL). Pim kinases have several substrates which are involved in gene transcription, protein translation, cell proliferation, and apoptosis. AZD1208 is a small molecule Pim kinase inhibitor currently in clinical trials. The PI3K-dependent activation of AKT (a client protein of HSP90) and the JAK/STAT-dependent induction of PIM are partially overlapping kinase pathways. Therefore, we hypothesized that the mechanism-based combination of 17-AAG (to decrease AKT levels) with AZD1208 (to inhibit Pim kinases) will result in an increase in cytotoxicity in CLL cells. An AZD1208 dose-response experiment was performed in 4 CLL patient samples to determine the optimal cytotoxic concentrations. AZD1208 concentrations of 3 μM and 10 μM were selected to treat 22 additional CLL patient samples for 24 hours. Cell death ranged from 0-30% and 2-45%, respectively, as determined by Annexin-V/PI staining after subtraction of endogenous cell death. All CLL patient samples were sensitive to AZD1208 regardless of their prognostic markers such as IgVH mutation and ZAP70 positivity or cytogenetics such as 17p del (p53 locus) or 11q del (ATM locus). Similarly, prior treatment history or Rai stage did not influence the biological response of AZD1208 in CLL cells. Based on published data, 0.5 μM and 2 μM 17-AAG were selected for the combination treatments with AZD1208 for 24 and 48 hours. Cytotoxicity for these combinations was assessed in 13 CLL blood samples, resulting in additive (n=8) or more than additive (n=3) cell death when using the fractional two-drug combination analysis. Immunoblot analysis was performed in 3 CLL samples probing for HSP90, HSP70, Akt, Pim-1, Mcl-1, Bcl-2, Phospho-4E-BP1 (Thr37/46), 4E-BP1, and β-actin. In agreement with previous work, 17-AAG treatment resulted in an increase of HSP90 and HSP70 protein levels while Akt levels were markedly decreased. In these preliminary immunoblots, Pim-1 and Bcl-2 levels remained unchanged; however, Mcl-1 protein levels decreased in the combination treatments at 24 and 48 hours. Levels for total and phospho-4E-BP1 also declined. Taken together, these preliminary data indicate that AZD1208 (at 3 and 10 μM) has a modest cytotoxic effect in CLL cells. However, when combined with 17-AAG for 24 and 48 hours there was an additive or more than additive effect in majority of CLL samples, with the possible mechanisms of action being through the Akt and 4E-BP1 pathways. Citation Format: Fabiola Gomez, Lisa Chen, William Wierda, Varsha Gandhi. In vitro effect of AZD1208 (Pim Kinase Inhibitor) and 17-AAG (HSP90 Inhibitor) combination in CLL. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2057. doi:10.1158/1538-7445.AM2013-2057

Inhibition of Fatty Acid Oxidation Leads to Apoptosis of Resting and Proliferating Chronic Lymphocytic Leukemia Cells in Vitro

AbstractAbstract 3879Tumor-specific metabolic changes could serve as new therapeutic targets. While much attention has been given to the metabolism of glucose in cancer cells and intervening therapies, relatively little attention has been given to fatty acids and fatty acid β-oxidation (FAO) in promoting cancer cell survival. Here, we reveal new findings implicating fatty acid metabolism in Chronic lymphocytic leukemia (CLL) and have developed a small molecule inhibitor of this pathway as a novel therapeutic approach to treat CLL. We have found that B cells isolated from CLL patients show increased levels of b-oxidation as compared to B cells isolated from PBMCs of healthy donor controls. NXT629, a small molecule inhibitor of the FAO pathway, dose-dependently reduced b-oxidation in CLL cells but did not reduce b-oxidation in B cells isolated from healthy volunteers. A negative control compound (NXT962) that has a similar chemical structure to NXT629, but does not significantly bind to the relevant target, did not inhibit FAO. NXT629 exerted dose-dependent cytotoxicity in CLL cells (CC50=10 mM, n=5 different patient samples) whereas NXT962 was inactive. Since the cellular microenvironment can protect CLL cells from spontaneous and drug-induced apoptosis, it is pivotal to evaluate novel drugs in this context. As expected, we found that macrophages protected CLL cells from spontaneous and fludarabine-induced apoptosis in vitro. However, NXT629 significantly reduced CLL cells viablity in the co-cultures. NXT629 (at 10 mM, n=2 different patient samples) induced apoptosis in 80% of cells after 7 days of co-culture in a setting where fludarabine was inactive.Though most CLL cells in the periphery are in a resting state, CLL cells proliferate in lymphoid organs. To mimic these conditions, we examined the activity of FAO inhibition on CLL cells that were induced to proliferate in vitro. To induce proliferation, CLL cells were cultured in media containing human interleukin (IL)-4, IL-10, and allogeneic T blasts. Labeling CLL cells with carboxyfluorescein succinimidyl ester, allowed us to monitor several rounds of induced CLL-cell division via flow cytometry, and within 5 days the CLL cells had expanded 3-fold. We found that addition of NXT629 to such cultures resulted in a dose-dependent reduction in the number of leukemia cells induced to undergo cell-division (IC50=3.7 mM, n=6), whereas the negative control compound showed no activity. Furthermore, when used in combination with dexamethasone (0.3 mM), NXT629 (3 mM) showed a significant decrease in the number of viable cells (38% +/−3%) over either dexamethasone (65% +/−12%) or NXT629 (74%+/−15%) alone. The additivity of FAO inhibition and dexamethasone in vitro suggests that a combination warrants further testing and might yield improved clinical responses. Overall, these results suggest that fatty acids promote primary CLL cell survival and proliferation, and that targeting the FAO pathway could be a new therapeutic approach to treating CLL. Disclosures:Messmer:Inception Sciences: Employment, Equity Ownership. Lorrain:Inception Sciences: Employment, Equity Ownership. Bravo:Inception Sciences: Employment, Equity Ownership. Stock:Inception Sciences: Employment, Equity Ownership. Bundey:Inception Sciences: Employment, Equity Ownership. Correa:Inception Sciences: Employment, Equity Ownership. Poon:Inception Sciences: Employment, Equity Ownership. Stebbins:Inception Sciencees: Employment, Equity Ownership. Cabrera:Inception Sciences: Employment, Equity Ownership. Chen:Inception Sciences: Employment, Equity Ownership. Jacintho:Inception Sciences: Employment, Equity Ownership. Spaner:Inception Sciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding. Prasit:Inception Sciences: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Lorrain:Inception Sciences: Employment, Equity Ownership.

Pre-Clinical and Interim Results of a Phase II Trial of Perifosine In Patients with Relapsed or Refractory Chronic Lymphocytic Leukemia (CLL)

AbstractAbstract 1842Perifosine (Keryx Biopharmaceuticals) is an oral alkylphospholipid that has been shown to have clinical activity in multiple myeloma and Waldenstrom's macroglobulinemia. Pre-clinical data suggest that its activity is due to inhibition of the Akt signal transduction pathway. The Akt pathway is known to be important for viability in CLL, another B-cell malignancy. Therefore, we investigated the in vitro activity of perifosine in freshly isolated primary CLL cells. CLL patients at the Duke University and Durham VA Medical Centers were enrolled in an IRB-approved research protocol for blood sample collection. CLL cells were negatively selected using the RosetteSep B-cell enrichment cocktail (StemCell Technologies) and a ficoll-Hypaque gradient. This method yields greater than 95% purity of malignant lymphocytes, determined by flow cytometry. Prognostic markers such as IgVH mutation status, CD38 and ZAP70 expression, and interphase cytogenetics were determined. We found the 50% effective dose (ED50) of perifosine for inducing cytotoxicity in CLL cells after a three-day incubation using the MTS colorimetric assay to be 510 nM (n = 29, range 120 – 1540 nM). CLL cells were obtained from patients with generally poor prognostic markers: 52% CD38+, 93% ZAP70+, 78% IgVH unmutated, 42% 17p deletion, 8% 11q deletion, 27% trisomy 12, 12% normal, and 12% 13q deletion as a sole abnormality. There were no statistical differences in ED50 between cells obtained from patients in high or low risk prognostic groups. Perifosine induced apoptosis in a dose- and time-dependent manner, measured by Annexin V and PI staining (n = 4). Based upon these pre-clinical results, we initiated a phase II study of perifosine (50 mg orally, twice daily) in relapsed or refractory CLL/small lymphocytic lymphoma (SLL) (NCT00873457). The primary objective of this study is to assess the response rate at 3 and 6 months of perifosine treatment in patients with relapsed or refractory CLL/SLL. Secondary objectives are to monitor toxicity, evaluate overall survival, progression-free survival and response duration, and perform laboratory correlates. Early interim results of this study are presented. Since trial initiation in September 2009, 13 patients have been enrolled. Nine patients had Rai stage III/IV disease at the time of therapy, and 4 patients were fludarabine-refractory. Patients had extensive prior treatment (median 4, range 1 – 11). Many patients had high-risk prognostic features: 9/11 IgVH unmutated, 10/13 CD38+, and 11/13 ZAP70+. Evaluation of interphase and metaphase cytogenetics demonstrated 4 patients with 17p deletion, two with 11q deletion, 2 with trisomy 12, 4 normal, and 4 with other complex cytogenetic anomalies. Of 12 patients who began therapy, 5 patients withdrew from the study prior to 3 months, 6 patients received at least 3 months of therapy, and 1 patient completed 6 months of therapy. Of the patients who received at least 3 months of therapy, there were 5 patients with stable disease, and one patient with partial response, using iwCLL response criteria. Grade 3/4 toxicities included anemia (n=2), fatigue (n=2), dehydration (n=1), febrile neutropenia (n=1), hyperbilirubinemia (n=1), hyponatremia (n=1), cough (n=1), and dyspnea (n=1). One patient required a dose reduction to 50 mg daily and two patients required dose delays due to toxicities. In conclusion, perifosine has potent in vitro activity against primary CLL cells. Preliminary results of this ongoing phase II study of oral perifosine in relapsed or refractory CLL/SLL demonstrate mostly disease stabilization in a group of very high-risk patients and an acceptable toxicity profile. Completion of this clinical study is necessary to determine if perifosine monotherapy has a potential role in the treatment of CLL/SLL. Disclosures:Sportelli:Keryx Biopharmaceutical: Employment, Equity Ownership. Weinberg:Keryx Biopharmaceuticals: Research Funding.

The Novel Tubulin-Targeting Agent Pyrrolo-1,5-Benzoxazepine-15 Induces Apoptosis in Poor Prognostic Subgroups of Chronic Lymphocytic Leukemia

Pyrrolo-1,5-benzoxazepine-15 (PBOX-15) is a novel microtubule depolymerization agent that induces cell cycle arrest and subsequent apoptosis in a number of cancer cell lines. Chronic lymphocytic leukemia (CLL) is characterized by clonal expansion of predominately nonproliferating mature B cells. Here, we present data suggesting PBOX-15 is a potential therapeutic agent for CLL. We show activity of PBOX-15 in samples taken from a cohort of CLL patients (n = 55) representing both high-risk and low-risk disease. PBOX-15 exhibited cytotoxicity in CLL cells (n = 19) in a dose-dependent manner, with mean IC(50) of 0.55 micromol/L. PBOX-15 significantly induced apoptosis in CLL cells (n = 46) including cells with poor prognostic markers: unmutated IgV(H) genes, CD38 and zeta-associated protein 70 (ZAP-70) expression, and fludarabine-resistant cells with chromosomal deletions in 17p. In addition, PBOX-15 was more potent than fludarabine in inducing apoptosis in fludarabine-sensitive cells. Pharmacologic inhibition and small interfering RNA knockdown of caspase-8 significantly inhibited PBOX-15-induced apoptosis. Pharmacologic inhibition of c-jun NH(2)-terminal kinase inhibited PBOX-15-induced apoptosis in mutated IgV(H) and ZAP-70(-) CLL cells but not in unmutated IgV(H) and ZAP-70(+) cells. PBOX-15 exhibited selective cytotoxicity in CLL cells compared with normal hematopoietic cells. Our data suggest that PBOX-15 represents a novel class of agents that are toxic toward both high-risk and low-risk CLL cells. The need for novel treatments is acute in CLL, especially for the subgroup of patients with poor clinical outcome and drug-resistant disease. This study identifies a novel agent with significant clinical potential.

Direct and complement dependent cytotoxicity in CLL cells from patients with high-risk early–intermediate stage chronic lymphocytic leukemia (CLL) treated with alemtuzumab and rituximab

The mechanism of cytotoxicity of alemtuzumab and rituximab in chronic lymphocytic leukemia (CLL) is not well understood. We obtained fresh CLL cells from early–intermediate stage high-risk patients just prior to treatment with alemtuzumab and rituximab to study mechanisms of action and resistance. Alemtuzumab had minimal direct cytotoxicity but caused significant complement dependent cytotoxicity (CDC) although a subpopulation of CLL cells had intrinsic resistance. Rituximab had no direct cytotoxicity and caused minimal CDC in cells from most patients. These data suggest that CDC has a therapeutic role in patients treated with alemtuzumab and that measures to decrease resistance to CDC could increase efficacy.

Flavopiridol Induces Apoptosis in Chronic Lymphocytic Leukemia Cells Via Activation of Caspase-3 Without Evidence of bcl-2 Modulation or Dependence on Functional p53

Flavopiridol has been reported to induce apoptosis in lymphoid cell lines via downregulation of bcl-2. The in vitro activity of flavopiridol against human chronic lymphocytic leukemia (CLL) cells and potential mechanisms of action for inducing cytotoxicity were studied. The in vitro viability of mononuclear cells from CLL patients (n = 11) was reduced by 50% at 4 hours, 24 hours, and 4 days at a flavopiridol concentration of 1.15 micromol/L (95% confidence interval [CI] +/-0.31), 0.18 micromol/L (95% CI +/-0.04), and 0.16 micromol/L (95% CI +/-0.04), respectively. Loss of viability in human CLL cells correlated with early induction of apoptosis. Exposure of CLL cells to 0.18 micromol/L of flavopiridol resulted in both decreased expression of p53 protein and cleavage of the caspase-3 zymogen 32-kD protein with the appearance of its 20-kD subunit. Contrasting observations of others in tumor cell lines, flavopiridol cytotoxicity in CLL cells did not correlate with changes in bcl-2 protein expression alterations. We evaluated flavopiridol's dependence on intact p53 by exposing splenocytes from wild-type (p53(+/+)) and p53 null (p53(-/-)) mice that demonstrated no preferential cytotoxicity as compared with a marked differential with F-ara-a and radiation. Incubation of CLL cells with antiapoptotic cytokine interleukin-4 (IL-4) did not alter the LC50 of flavopiridol, as compared with a marked elevation noted with F-ara-a in the majority of patients tested. These data demonstrate that flavopiridol has significant in vitro activity against human CLL cells through activation of caspase-3, which appears to occur independently of bcl-2 modulation, the presence of IL-4, or p53 status. Such findings strongly support the early introduction of flavopiridol into clinical trials for patients with B-CLL.

Flavopiridol Induces Apoptosis in Chronic Lymphocytic Leukemia Cells Via Activation of Caspase-3 Without Evidence of bcl-2 Modulation or Dependence on Functional p53

Flavopiridol has been reported to induce apoptosis in lymphoid cell lines via downregulation of bcl-2. The in vitro activity of flavopiridol against human chronic lymphocytic leukemia (CLL) cells and potential mechanisms of action for inducing cytotoxicity were studied. The in vitro viability of mononuclear cells from CLL patients (n = 11) was reduced by 50% at 4 hours, 24 hours, and 4 days at a flavopiridol concentration of 1.15 μmol/L (95% confidence interval [CI] ±0.31), 0.18 μmol/L (95% CI ±0.04), and 0.16 μmol/L (95% CI ±0.04), respectively. Loss of viability in human CLL cells correlated with early induction of apoptosis. Exposure of CLL cells to 0.18 μmol/L of flavopiridol resulted in both decreased expression of p53 protein and cleavage of the caspase-3 zymogen 32-kD protein with the appearance of its 20-kD subunit. Contrasting observations of others in tumor cell lines, flavopiridol cytotoxicity in CLL cells did not correlate with changes in bcl-2 protein expression alterations. We evaluated flavopiridol’s dependence on intact p53 by exposing splenocytes from wild-type (p53+/+) and p53 null (p53−/−) mice that demonstrated no preferential cytotoxicity as compared with a marked differential with F-ara-a and radiation. Incubation of CLL cells with antiapoptotic cytokine interleukin-4 (IL-4) did not alter the LC50 of flavopiridol, as compared with a marked elevation noted with F-ara-a in the majority of patients tested. These data demonstrate that flavopiridol has significant in vitro activity against human CLL cells through activation of caspase-3, which appears to occur independently of bcl-2 modulation, the presence of IL-4, or p53 status. Such findings strongly support the early introduction of flavopiridol into clinical trials for patients with B-CLL.